ovalbumin and Pulmonary-Eosinophilia

Antigen-presenting dendritic cells are essential for the recognition and presentation of allergens to the cells of the immune system. Airway dendritic cells capture allergen in the mucosa and present it to naive T cells after migration into the draining lymph nodes. In this review article, we discuss the most recent findings from animal models of asthma, which highlight an essential role for these cells in the induction and maintenance of eosinophilic airway inflammation. This increasing knowledge might lead to the identification of new targets for the prevention and therapy of asthma.

Topics: Administration, Inhalation; Allergens; Animals; Antigen Presentation; Asthma; Dendritic Cells; Disease Models, Animal; Humans; Lung; Mice; Models, Immunological; Ovalbumin; Pulmonary Eosinophilia; Rats; T-Lymphocytes

Topics: Allergens; Animals; Asthma; Biopsy; Blood Proteins; Bronchi; Cell Adhesion; Crosses, Genetic; Dendritic Cells; Eosinophil Granule Proteins; Eosinophils; Epithelial Cells; Fibroblasts; Humans; Inflammation Mediators; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Muscle, Smooth; Ovalbumin; Pulmonary Eosinophilia; Respiratory Tract Infections; Ribonucleases; Th1 Cells; Th2 Cells; Tissue Inhibitor of Metalloproteinase-1; Transgenes; Virus Diseases

Traditionally, the roots of Angelica reflexa B.Y.Lee (AR) have been used to treat cough, phlegm, neuralgia, and arthralgia in Northeast Asia.. The anti-asthmatic effect of AR root extract (ARE) was determined using a murine airway allergic inflammation model and the primary T cell polarization assay.. To evaluate the anti-asthmatic effect of ARE, inflammatory cell infiltration was determined histologically and inflammatory mediators were measured in bronchoalveolar lavage fluid (BALF). Furthermore, the effects of AREs on Th2 cell differentiation and activation were determined by western blotting and flow cytometry.. Asthmatic phenotypes were alleviated by ARE treatment, which reduced mucus production, inflammatory cell infiltration (especially eosinophilia), and type 2 cytokine levels in BALF. ARE administration to mice reduced the number of activated Th2 (CD4. Our findings indicate that the anti-asthmatic effect of AREs is mediated by the reduction in Th2 cell activation by regulating IRF4.

Topics: Angelica; Animals; Anti-Asthmatic Agents; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Female; GATA3 Transcription Factor; Hypersensitivity; Interferon Regulatory Factors; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Ovalbumin; Plant Extracts; Plant Roots; Pneumonia; Pulmonary Eosinophilia; RAW 264.7 Cells; Th2 Cells

Studying the proteomes of tissue-derived extracellular vesicles (EVs) can lead to the identification of biomarkers of disease and can provide a better understanding of cell-to-cell communication in both healthy and diseased tissue. The aim of this study was to apply our previously established tissue-derived EV isolation protocol to mouse lungs in order to determine the changes in the proteomes of lung tissue-derived EVs during allergen-induced eosinophilic airway inflammation. A mouse model for allergic airway inflammation was used by sensitizing the mice intraperitoneal with ovalbumin (OVA), and one week after the final sensitization, the mice were challenged intranasal with OVA or PBS. The animals were sacrificed 24 h after the final challenge, and their lungs were removed and sliced into smaller pieces that were incubated in culture media with DNase I and Collagenase D for 30 min at 37 °C. Vesicles were isolated from the medium by ultracentrifugation and bottom-loaded iodixanol density cushions, and the proteomes were determined using quantitative mass spectrometry. More EVs were present in the lungs of the OVA-challenged mice compared to the PBS-challenged control mice. In total, 4510 proteins were quantified in all samples. Among them, over 1000 proteins were significantly altered (fold change >2), with 614 proteins being increased and 425 proteins being decreased in the EVs from OVA-challenged mice compared to EVs from PBS-challenged animals. The associated cellular components and biological processes were analyzed for the altered EV proteins, and the proteins enriched during allergen-induced airway inflammation were mainly associated with gene ontology (GO) terms related to immune responses. In conclusion, EVs can be isolated from mouse lung tissue, and the EVs' proteomes undergo changes in response to allergen-induced airway inflammation. This suggests that the composition of lung-derived EVs is altered in diseases associated with inflammation of the lung, which may have implications in type-2 driven eosinophilic asthma pathogenesis.

Topics: Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Extracellular Vesicles; Gene Ontology; Lung; Male; Mice; Mice, Inbred C57BL; Mitochondria; Nanoparticles; Ovalbumin; Proteome; Pulmonary Eosinophilia; Respiratory Hypersensitivity

Air pollution events frequently occur in China during the winter. Most investigations of pollution studies have focused on the physical and chemical properties of PM2.5. Many of these studies have indicated that PM2.5 exacerbates asthma or eosinophil inflammation. However, few studies have evaluated the relationship between bacterial loads in PM2.5, and especially pathogenic bacteria and childhood asthma. Airborne PM2.5 samples from heavily polluted air were collected in Hangzhou, China between December 2014 and January 2015. PM2.5 and ovalbumin (OVA) were intratracheally administered twice in 4-week intervals to induce the allergic pulmonary inflammation in adolescent C57/BL6 mice. PM2.5 exposure caused neutrophilic alveolitis and bronchitis. In the presence of OVA, the levels of the Th2 cytokines IL-4, IL-12, and IL-17 were significantly increased in bronchoalveolar lavage fluids (BALF) after PM2.5 exposure, while eosinophil infiltration and mucin secretion were also induced. In addition to adjuvant effects on OVA-induced allergic inflammation, PM2.5 exposure also led to the maturation of dendritic cells. These results suggest that PM2.5 exposure may aggravate lung eosinophilia and that PM2.5-bound microbial can exacerbate allergic and inflammatory lung diseases.

Topics: Age Factors; Air Microbiology; Animals; Bacterial Load; Cells, Cultured; Coculture Techniques; Cytokines; Dendritic Cells; Disease Models, Animal; Lung; Male; Mice, Inbred C57BL; Mice, Inbred ICR; Ovalbumin; Particle Size; Particulate Matter; Pneumonia; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Th2 Cells

Asthma is a common chronic inflammatory airway disease. Recent studies have reported that interleukin (IL)‑33 is a potential link between the airway epithelium and Th2‑type inflammatory responses, which are closely related to the progression of asthma. The IL‑33 receptor, ST2, is highly expressed in group 2 innate lymphoid cells (ILC2s), Th2 cells, mast cells, eosinophils and natural killer (NK) cells. Cnidii Fructus is a Chinese herb with a long history of use in the treatment of asthma in China. Osthole is one of the major components of Cnidii Fructus. The present study examined the anti‑asthmatic effects of osthole in mice and aimed to elucidate the underlying mechanisms involving the IL‑33/ST2 pathway. BALB/c mice were sensitized and challenged with ovalbumin and then treated with an intraperitoneal injection of osthole (25 and 50 mg/kg). Subsequently, the airway hyper‑responsiveness (AHR) and inflammation of the lungs were evaluated. The amounts of IL‑4, IL‑5, IL‑13, interferon (IFN)‑γ and IL‑33 in the bronchoalveolar lavage fluid (BALF) were measured by Luminex assay and their mRNA levels in the lungs were measured by reverse transcription‑quantitative PCR. The histopathology of the lungs was performed with H&E, PAS and Masson's staining. The expression of ST2 in the lungs was evaluated by immunohistochemistry. The data demonstrated that osthole markedly reduced AHR and decreased the number of eosinophils and lymphocytes in BALF. It was also observed that osthole significantly inhibited the release of Th2‑type cytokines (IL‑4, IL‑5 and IL‑13) and upregulated the IFN‑γ level in BALF. Moreover, osthole significantly attenuated the IL‑33 and ST2 expression in the lungs of asthmatic mice. On the whole, osthole attenuated ovalbumin‑induced lung inflammation through the inhibition of IL‑33/ST2 signaling in an asthmatic mouse model. These results suggest that osthole is a promising target for the development of an asthma medication.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Coumarins; Disease Models, Animal; Drugs, Chinese Herbal; Female; Gene Expression Regulation; Inflammation; Interferon-gamma; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Interleukins; Lung; Lymphocyte Count; Mice; Mice, Inbred BALB C; Ovalbumin; Phytotherapy; Pulmonary Eosinophilia; Random Allocation; RNA, Messenger; Signal Transduction

Silica crystals (silica), which are a major mineral component of volcanic ash and desert dust, contribute to the pathogenesis of pulmonary disorders such as asthma and fibrosis. Although administration of silica or sand dust to rodents exacerbates development of ovalbumin-induced or house dust mite-induced asthma-like airway inflammation, the detailed mechanisms remain unclear. Here, using murine models, we found that silica can induce IL-33 expression in pulmonary epithelial cells. IL-33, but not IL-25 or TSLP, and type 2 cytokines such as IL-5 and IL-13 were critically involved in silica's exacerbation of OVA-induced airway eosinophilia in mice. Innate lymphoid cells (ILCs), but not T, B or NKT cells, were also involved in the setting. Moreover, a scavenger receptor that recognized silica was important for silica's exacerbating effect. These observations suggest that IL-33 induced in epithelial cells by silica activates ILCs to produce IL-5 and/or IL-13, contributing to silica's exacerbation of OVA-induced airway eosinophilia in mice. Our findings provide new insight into the underlying mechanisms of exacerbation of pulmonary disorders such as asthma following inhalation of silica-containing materials such as volcanic ash and desert dust.

Topics: Animals; Asthma; Cytokines; Interleukin-13; Interleukin-33; Interleukin-5; Interleukins; Lung; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Receptors, Scavenger; Silicon Dioxide; Thymic Stromal Lymphopoietin

Eosinophilia (EOS) is an important component of airway inflammation and hyperresponsiveness in allergic reactions including those leading to asthma. Although cigarette smoking (CS) is a significant contributor to long-term adverse outcomes in these lung disorders, there are also the curious reports of its ability to produce acute suppression of inflammatory responses including EOS through poorly understood mechanisms. One possibility is that proinflammatory processes are suppressed by nicotine in CS acting through nicotinic receptor α7 (α7). Here we addressed the role of α7 in modulating EOS with two mouse models of an allergic response: house dust mites (HDM; Dermatophagoides sp.) and ovalbumin (OVA). The influence of α7 on EOS was experimentally resolved in wild-type mice or in mice in which a point mutation of the α7 receptor (α7

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Cigarette Smoking; Cytokines; Female; Lung; Macrophages, Alveolar; Male; Mice; Ovalbumin; Pulmonary Eosinophilia; Pyroglyphidae

To find bioactive medicinal herbs exerting anti-asthmatic activity, we investigated the effect of an aqueous extract of Urtica dioica L. (Urticaceae) leaves (UD), the closest extract to the Algerian traditional use.. In this study, we investigated the in vivo anti-asthmatic and antioxidant activities of nettle extract.. Adult male Wistar rats were divided into four groups: Group I: negative control; group II: Ovalbumin sensitized/challenged rats (positive control); group III: received UD extract (1.5 g/kg/day) orally along the experimental protocol; group IV: received UD extract (1.5 g/kg/day) orally along the experimental protocol and sensitized/challenged with ovalbumin. After 25 days, blood and tissue samples were collected for haematological and histopathological analysis, respectively. The oxidative stress parameters were evaluated in the lungs, liver and erythrocytes. Then, correlations between markers of airway inflammation and markers of oxidative stress were explored.. UD extract significantly (p < 0.01) inhibited eosinophilia increases in BALF (-60%) and the levels of leucocytes (-32.75%) and lymphocytes (-29.22%) in serum, and effectively suppressed inflammatory cells recruitment in the asthmatic rat model. Besides, the lipid peroxidation generated by allergen administration was significantly (p < 0.05) diminished by UD treatment in lung tissue (-48.58%). The nettle extract was also investigated for the total phenolic content (30.79 ± 0.96 mg gallic acid/g dry extract) and shows DPPH radical scavenging activity with 152.34 ± 0.37 μg/mL IC. The results confirmed that UD administration might be responsible for the protective effects of this extract against airway inflammation.

Topics: Animals; Anti-Asthmatic Agents; Anti-Inflammatory Agents; Antioxidants; Asthma; Biphenyl Compounds; Disease Models, Animal; Lipid Peroxidation; Lung; Male; Ovalbumin; Oxidative Stress; Phenols; Phytotherapy; Picrates; Plant Extracts; Plant Leaves; Plants, Medicinal; Pneumonia; Pulmonary Eosinophilia; Rats, Wistar; Urtica dioica

Exposure to ozone induces deleterious responses\ in the airways that include shortness of breath,\ inflammation, and bronchoconstriction. People\ with asthma have increased airway sensitivity to\ ozone and other irritants. Dr. Allison Fryer and\ colleagues addressed how exposure to ozone affects\ the immune and physiological responses in\ guinea pigs. Guinea pigs are considered a useful\ animal model for studies of respiratory and physiological\ responses in humans; their response to\ airborne allergens is similar to that in humans and\ shares some features of allergic asthma.\ Fryer and colleagues had previously observed\ that within 24 hours of exposure, ozone not only\ induced bronchoconstriction but also stimulated\ the production of new cells in the bone marrow,\ where all white blood cells develop. As a result\ of ozone exposure, increased numbers of newly\ synthesized white blood cells, particularly eosinophils,\ moved into the blood and lungs.\ The central hypothesis of the current study was\ that newly synthesized eosinophils recruited to\ the lungs 3 days after ozone exposure were beneficial\ to the animals because they reduced ozoneinduced\ bronchoconstriction. The investigators\ also hypothesized that the beneficial effect seen\ in normal (nonsensitized) animals was lost in animals\ that had been injected with an allergen, ovalbumin\ (sensitized). They also planned to explore\ the effects of inhibitors of certain cytokines (cellsignaling\ molecules).\ Immune responses in sensitized animals are\ dominated by a Th2 pattern, which is characterized\ by the synthesis of cytokines (interleukin\ [IL]-4, IL-5, and IL-13) and the Th2 subset of CD4+\ T lymphocytes and the cells they activate (predominantly\ eosinophils, and B lymphocytes that\ switch to making immunoglobulin E [IgE]). Thus,\ sensitized animals were used as a model of allergic\ humans, whose immune responses tend to be\ dominated by IgE.. Fryer and colleagues exposed normal and sensitized\ (allergic) guinea pigs to 2 ppm ozone or filtered\ air for 4 hours and measured changes in cell\ numbers and airway responses 1 or 3 days later.\ They counted the numbers of eosinophils and other\ white blood cells (macrophages, neutrophils, and\ lymphocytes) in bone marrow, blood, and bronchoalveolar\ lung lavage fluid. The investigators\ also measured important physiological responses,\ including bronchoconstriction. Some animals were\ pretreated with etanercept and monoclonal anti-IL-5,\ which block tumor necrosis factor-a (TNFa) and IL-5,\ respectively. TNFa and IL-5 blockers have been used\ to treat patients with asthma.\ A key feature of the study was a technique to distinguish\ which white blood cells were synthesized after\ exposure from those that already existed, by injecting\ animals with bromodeoxyuridine (BrdU). BrdU\ is a thymidine analogue that is incorporated into the\ DNA of dividing cells, serving as a marker of newly\ produced cells. Therefore, a snapshot can be obtained\ of the proportion of newly synthesized (BrdU-positive)\ versus pre-existing (BrdU-negative) cell types.. 1. Allergic and normal animals differed in the time\ course of bronchoconstriction and changes in cell\ types after ozone exposure. In normal animals,\ bronchoconstriction increased substantially at\ day 1 but decreased by day 3 after ozone exposure.\ In contrast, in allergic animals bronchoconstriction\ remained high at day 3. Ozone also increased\ the percentage of newly formed, BrdU2\ positive eosinophils in the bone marrow and\ lungs of normal but not allergic animals.\ 2. Pretreatment with the TNFa blocker etanercept\ had complex effects, which differed between\ normal and allergic animals. In normal animals,\ etanercept decreased ozone-induced new synthesis\ of eosinophils in the bone marrow and\ blocked eosinophil migration to the lung; it also\ increased bronchoconstriction at day 3 (relative\ to day 1 without etanercept). In allergic animals,\ etanercept had no effect on any cell type in the\ bone marrow or lung after exposure to ozone and\ did not change bronchoconstriction compared\ with allergic animals not treated with etanercept.\ Etanercept tended to increase the numbers of\ blood monocytes and lymphocytes in air- and\ ozone-exposed normal and allergic animals at\ day 3, but had no effect on eosinophils in blood\ at this time point. This was one of the few statistically\ significant findings in the blood of exposed\ animals in the study.\ 3. Anti-IL-5 reduced bronchoconstriction at day 3 after\ exposure of allergic animals to ozone. In contrast,\ bronchoconstriction was greatly increased\ in normal animals treated with anti-IL-5.. Fryer and colleagues explored the airway and cellular\ responses in guinea pigs exposed to ozone. The\ HEI Review Committee, which conducted an independent\ review of the study, agreed that the findings\ supported the authors’ hypothesis (1) that exposure\ to ozone stimulates production of eosinophils in bone\ marrow, (2) that these newly formed eosinophils migrate\ to the lungs, and (3) that those eosinophils play\ a delayed but potentially beneficial role in reducing\ ozone-induced inflammation in the airways of healthy\ normal animals, but not in allergen-sensitized animals.\ The Committee also agreed that guinea pigs\ were a good model for studying responses to an allergen,\ because a major subtype of asthma (the high\ Th2 or allergic type) is associated with high levels of\ eosinophils in the blood.\ A novel finding was that the TNFa blocker etanercept\ decreased ozone-induced formation of eosinophils\ in the bone marrow and blocked eosinophil\ migration to the lung in normal animals. However,\ because injecting etanercept had little effect on eosinophils\ and did not decrease bronchoconstriction in\ allergic guinea pigs, the potential for treating patients\ with allergic asthma with TNFa blockers is uncertain.\ This is consistent with the poor performance of TNFa\ blockers in clinical studies of asthma treatment.\ Blocking the cytokine IL-5 with an anti-IL-5 antibody\ substantially decreased bronchoconstriction in\ sensitized animals. This suggests that therapies targeting\ IL-5 and eosinophils would be promising in at\ least some types of asthma. The Committee expressed\ caution toward experiments with cytokine blockers,\ both in animal models and humans, because such\ blockers are often not specific to a particular cell type\ and may differ at different sites in the body. Without\ further detailed confirmation of the effects of the\ blockers, interpreting these experiments can be challenging.\ The Committee concluded that the study by Fryer\ and colleagues raises several intriguing directions for\ future research, including exploring ways in which\ newly formed eosinophils differ from pre-existing\ ones, and how such findings apply to humans with\ allergy or asthma.

Topics: Administration, Inhalation; Animals; Bronchoconstriction; Cytokines; Eosinophils; Guinea Pigs; Ovalbumin; Ozone; Pulmonary Eosinophilia; Tumor Necrosis Factor-alpha

The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response.. We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF(-/-) mice and mixed wild-type:PLZF(-/-) bone marrow chimeras.. PLZF(-/-) mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive TH2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated ex vivo for their capacity to release type 2 cytokines.. PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired.. PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice.

Topics: Adaptive Immunity; Adoptive Transfer; Allergens; Animals; Antigens, Surface; Biomarkers; Bone Marrow Transplantation; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Helminthiasis; Helminths; Hypersensitivity; Immunity, Innate; Immunophenotyping; Interleukin-33; Interleukins; Kruppel-Like Transcription Factors; Lymphocyte Activation; Lymphocyte Subsets; Mice; Mice, Knockout; Ovalbumin; Papain; Promyelocytic Leukemia Zinc Finger Protein; Pulmonary Eosinophilia; Th2 Cells

Asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Peroxisome proliferator-activated receptors have been reported to regulate inflammatory responses in many cells. In this study, we examined the effects of intranasal rosiglitazone on airway remodeling in a chronic asthma model.. We developed a mouse model of airway remodeling, including smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice per week for 3 months. Mice were treated intranasally with rosiglitazone with or without an antagonist during OVA challenge. We determined airway inflammation and the degree of airway remodeling by smooth muscle actin area and collagen deposition.. Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, compared with control mice. Additionally, the mice developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of rosiglitazone intranasally inhibited the eosinophilic inflammation significantly, and, importantly, airway smooth muscle remodeling in mice chronically exposed to OVA. Expression of Toll-like receptor (TLR)-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was increased in the OVA group and decreased in the rosiglitazone group. Co-treatment with GW9660 (a rosiglitazone antagonist) and rosiglitazone increased the expression of TLR-4 and NF-κB.. These results suggest that intranasal administration of rosiglitazone can prevent not only air way inf lammation but also air way remodeling associated with chronic allergen challenge. This beneficial effect is mediated by inhibition of TLR-4 and NF-κB pathways.

Topics: Actins; Administration, Inhalation; Airway Remodeling; Animals; Anti-Asthmatic Agents; Asthma; Chronic Disease; Collagen; Disease Models, Animal; Female; Lung; Mice, Inbred BALB C; NF-kappa B; Ovalbumin; Pneumonia; PPAR gamma; Pulmonary Eosinophilia; Rosiglitazone; Signal Transduction; Thiazolidinediones; Toll-Like Receptor 4

The biological and chemical natures of materials adsorbed onto fine particulate matter (PM2.5) vary by origin and passage routes. The exacerbating effects of the two samples-urban PM2.5 (U-PM2.5) collected during the hazy weather in a Chinese city and fine particles (ASD-PM2.5) collected during Asian sand dust (ASD) storm event days in Japan-on murine lung eosinophilia were compared to clarify the role of toxic materials in PM2.5. The amounts of β-glucan and mineral components were higher in ASD-PM2.5 than in U-PM2.5. On the other hand, organic chemicals, including polycyclic aromatic hydrocarbons (PAHs), were higher in U-PM2.5 than in ASD-PM2.5. When BALB/c mice were intratracheally instilled with U-PM2.5 and ASD-PM2.5 (total 0.4 mg/mouse) with or without ovalbumin (OVA), various biological effects were observed, including enhancement of eosinophil recruitment induced by OVA in the submucosa of the airway, goblet cell proliferation in the bronchial epithelium, synergic increase of OVA-induced eosinophil-relevant cytokines and a chemokine in bronchoalveolar lavage fluid, and increase of serum OVA-specific IgG1 and IgE. Data demonstrate that U-PM2.5 and ASD-PM2.5 induced allergic inflammatory changes and caused lung pathology. U-PM2.5 and ASD-PM2.5 increased F4/80(+) CD11b(+) cells, indicating that an influx of inflammatory and exudative macrophages in lung tissue had occurred. The ratio of CD206 positive F4/80(+) CD11b(+) cells (M2 macrophages) in lung tissue was higher in the OVA+ASD-PM2.5 treated mice than in the OVA+U-PM2.5 treated mice. These results suggest that the lung eosinophilia exacerbated by both PM2.5 is due to activation of a Th2-associated immune response along with induced M2 macrophages and the exacerbating effect is greater in microbial element (β-glucan)-rich ASD-PM2.5 than in organic chemical-rich U-PM2.5.

Topics: Air Pollutants; Allergens; Animals; beta-Glucans; Bronchoalveolar Lavage Fluid; Cell Count; China; Cytokines; Immunoglobulin E; Immunoglobulin G; Japan; Lung; Male; Metals; Mice, Inbred BALB C; Ovalbumin; Particulate Matter; Pulmonary Eosinophilia

Urban particulate matter (PM) is associated with an increase in asthma. PM2.5 (

Topics: Air Pollutants; Allergens; Animals; beta-Glucans; Bronchoalveolar Lavage Fluid; Cell Count; China; Cities; Cytokines; Heme Oxygenase-1; Immunoglobulin E; Immunoglobulin G; Lipopolysaccharides; Lung; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase Type II; Ovalbumin; Particle Size; Particulate Matter; Pulmonary Eosinophilia; RAW 264.7 Cells; Respiratory Hypersensitivity

Asthma is an inflammatory disease of the lung characterized by airways hyper-responsiveness (AHR), inflammation, and mucus hyperproduction. Current mainstream therapies include bronchodilators that relieve bronchoconstriction and inhaled glucocorticoids to reduce inflammation. The small molecule hormone and neurotransmitter serotonin has long been known to be involved in inflammatory processes; however, its precise role in asthma is unknown. We have previously established that activation of serotonin 5-hydroxytryptamine (5-HT)(2A) receptors has potent anti-inflammatory activity in primary cultures of vascular tissues and in the whole animal in vasculature and gut tissues. The 5-HT(2A) receptor agonist, (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI] is especially potent. In this work, we have examined the effect of (R)-DOI in an established mouse model of allergic asthma. In the ovalbumin mouse model of allergic inflammation, we demonstrate that inhalation of (R)-DOI prevents the development of many key features of allergic asthma, including AHR, mucus hyperproduction, airways inflammation, and pulmonary eosinophil recruitment. Our results highlight a likely role of the 5-HT2 receptors in allergic airways disease and suggest that 5-HT2 receptor agonists may represent an effective and novel small molecule-based therapy for asthma.

Topics: 5-Hydroxytryptophan; Amphetamines; Animals; Anti-Inflammatory Agents; Asthma; Bronchial Hyperreactivity; Disease Models, Animal; Enzyme Activation; Eosinophils; Immunoglobulin E; Inflammation; Lung; Male; Mice; Mice, Inbred BALB C; Mucus; Ovalbumin; Pulmonary Eosinophilia; Receptors, Serotonin, 5-HT2; Serotonin 5-HT2 Receptor Agonists

Type-2 innate lymphoid cells (ILC2s) and the acquired CD4(+) Th2 and Th17 cells contribute to the pathogenesis of experimental asthma; however, their roles in Ag-driven exacerbation of chronic murine allergic airway diseases remain elusive. In this study, we report that repeated intranasal rechallenges with only OVA Ag were sufficient to trigger airway hyperresponsiveness, prominent eosinophilic inflammation, and significantly increased serum OVA-specific IgG1 and IgE in rested mice that previously developed murine allergic airway diseases. The recall response to repeated OVA inoculation preferentially triggered a further increase of lung OVA-specific CD4(+) Th2 cells, whereas CD4(+) Th17 and ILC2 cell numbers remained constant. Furthermore, the acquired CD4(+) Th17 cells in Stat6(-/-)/IL-17-GFP mice, or innate ILC2s in CD4(+) T cell-ablated mice, failed to mount an allergic recall response to OVA Ag. After repeated OVA rechallenge or CD4(+) T cell ablation, the increase or loss of CD4(+) Th2 cells resulted in an enhanced or reduced IL-13 production by lung ILC2s in response to IL-25 and IL-33 stimulation, respectively. In return, ILC2s enhanced Ag-mediated proliferation of cocultured CD4(+) Th2 cells and their cytokine production, and promoted eosinophilic airway inflammation and goblet cell hyperplasia driven by adoptively transferred Ag-specific CD4(+) Th2 cells. Thus, these results suggest that an allergic recall response to recurring Ag exposures preferentially triggers an increase of Ag-specific CD4(+) Th2 cells, which facilitates the collaborative interactions between acquired CD4(+) Th2 cells and innate ILC2s to drive the exacerbation of a murine allergic airway diseases with an eosinophilic phenotype.

Topics: Animals; Asthma; Cell Communication; Immunity, Innate; Inflammation; Interleukin-33; Interleukins; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; STAT6 Transcription Factor; Th17 Cells; Th2 Cells

Asthma is associated with reversible airway obstruction, leucocyte infiltration, airways hyperresponsiveness (AHR), and airways remodeling. Fluid accumulation causes pulmonary edema contributing to airways obstruction. We examined the temporal relationship between the late asthmatic response (LAR) following allergen challenge of sensitized guinea-pigs and pulmonary edema measured by magnetic resonance imaging (MRI).. Ovalbumin (OVA) sensitized guinea-pigs received either a single OVA inhalation (acute) or nine OVA inhalations at 48 h intervals (chronic). Airways obstruction was measured as specific airways conductance (sG(aw)) by whole body plethysmography. AHR to inhaled histamine and bronchoalveolar lavage for leucocyte counts were measured 24 h after a single or the final chronic ovalbumin challenges. MRI was performed at intervals after OVA challenge and high-intensity edemic signals were quantified.. Ovalbumin caused early bronchoconstriction, followed at 7 h by an LAR and at 24 h AHR and leucocyte influx. The bright-intensity MRI edema signal, peaking at 7 h, was significantly (P < .05) greater after chronic (9.0 ± 0.7 × 10(3) mm(3)) than acute OVA (7.6 ± 0.2 × 10(3) mm(3)). Dexamethasone treatment before acute OVA abolished the AHR and LAR and significantly reduced eosinophils and the bright-intensity MRI edema from 9.1 ± 1.0 to 6.4 ± 0.3 × 10(3) mm(3).. We show a temporal relationship between edema and the LAR and their parallel reduction, along with eosinophils and AHR, by dexamethasone. This suggests a close causative association between pulmonary edema and impaired airways function.

Topics: Allergens; Animals; Anti-Asthmatic Agents; Asthma; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Chemotaxis, Leukocyte; Dexamethasone; Disease Models, Animal; Guinea Pigs; Lung; Magnetic Resonance Imaging; Male; Ovalbumin; Predictive Value of Tests; Pulmonary Edema; Pulmonary Eosinophilia; Time Factors

Paeoniflorin has been demonstrated to exert anti-inflammatory and immunomodulatory effects in the animal study. In this study, we investigated immunoregulatory effects of paeoniflorin on anti-asthmatic effects and underlying mechanisms. Asthma model was established by ovalbumin-induced. A total of 50 mice were randomly assigned to five experimental groups: control, model, dexamethasone (2 mg/kg), and paeoniflorin (10 and 20 mg/kg). Airway resistance (Raw) were measured by the forced oscillation technique; histological studies were evaluated by the hematoxylin and eosin (HE) staining; Th1/Th2 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA); Th1/Th2 cells were evaluated by flow cytometry (FCM); and GATA3 and T-bet were evaluated by Western blot. Our study demonstrated that, compared with model group, paeoniflorin inhibited ovalbumin (OVA)-induced increases in Raw and eosinophil count; interleukin (IL)-4, IgE levels were recovered in bronchoalveolar lavage fluid compared; increased IFN-γ level in bronchoalveolar lavage fluid; histological studies demonstrated that paeoniflorin substantially inhibited OVA-induced eosinophilia in lung tissue and lung tissue compared with model group. Flow cytometry studies demonstrated that paeoniflorin can regulate Th1/Th2 balance. These findings suggest that paeoniflorin may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.

Topics: Airway Resistance; Animals; Anti-Asthmatic Agents; Asthma; Bronchoalveolar Lavage Fluid; Dexamethasone; Disease Models, Animal; Dose-Response Relationship, Drug; Female; GATA3 Transcription Factor; Glucosides; Immunoglobulin E; Interferon-gamma; Interleukin-4; Lung; Mice, Inbred BALB C; Monoterpenes; Ovalbumin; Phytotherapy; Plants, Medicinal; Pulmonary Eosinophilia; T-Box Domain Proteins; Th1 Cells; Th1-Th2 Balance; Th2 Cells

The aim of the study was to investigate the anti-asthmatic effects of oxymatrine (OXY) and the possible underlying mechanisms. The mouse asthma model was established by ovalbumin (OVA) intraperitoneal injection. A total of fifty mice were randomly assigned to five groups: control, OVA, OVA + dexamethasone (Dex, 2 mg · kg(-1)), and OVA + OXY (40 mg · kg(-1)), and OVA + OXY (80 mg · kg(-1)), respectively. Histological studies were conducted by the hematoxylin and eosin (HE) staining, the levels of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13, and IgE were evaluated by enzyme-linked immunosorbent assay (ELISA), and the protein level of CD40 was analyzed by Western blotting. OXY inhibited OVA-induced increases in eosinophil count; the levels of IL-4, IL-5, IgE, and IL-13 were recovered. It also substantially inhibited OVA-induced eosinophilia in lung tissues and the expression of CD40 protein. These findings suggest that OXY may effectively ameliorate the progression of asthma and could be explored as a possible therapy for patients with allergic asthma.

Topics: Alkaloids; Animals; Anti-Asthmatic Agents; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; CD40 Antigens; Dexamethasone; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Immunoglobulin E; Interleukins; Irritants; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Quinolizines; Random Allocation; Signal Transduction

Immune system dysfunctions including the increased Th1/Th2 ratio are common in chronic kidney disease (CKD) patients, and a wide variety of skin diseases including Th1-mediated uremic pruritis are associated with CKD. Although there are more than 90 uremic toxins reported, it is yet to be known which uremic solute is associated with the unbalanced Th1/Th2 ratio and how it works. Indoxyl 3-sulfate (I3S), one of uremic toxins and a potent aryl hydrocarbon receptor (AhR) ligand, accumulates in blood and tissues, increasing up to 81.04 μM in CKD patients, compared with 1.03 μM in healthy subjects. I3S activates NF-κB and AhR. Thus, we investigated roles of I3S in the differentiation of Th1 and Th2 cells. I3S inhibited Th2 differentiation but showed little or no effect on Th1 differentiation. I3S suppressed Th2-mediated ovalbumin-induced allergic asthma in mice and decreased the frequency of IL-4 producing CD4 T cells in the lungs. I3S inhibited phosphorylation of STAT5 and STAT6, transcription factors associated with Th2 differentiation. Effects of I3S on Th2 differentiation were suppressed by α-naphtoflavone, an AhR antagonist, indicating that I3S regulates Th2 differentiation AhR-dependently.

Topics: Animals; Asthma; Basic Helix-Loop-Helix Transcription Factors; Benzoflavones; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Immunoglobulin E; Indican; Interleukin-4; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphorylation; Pulmonary Eosinophilia; Receptors, Aryl Hydrocarbon; STAT5 Transcription Factor; STAT6 Transcription Factor; Th1 Cells; Th2 Cells; Uremia

Perillyl alcohol (POH) is an isoprenoid which inhibits farnesyl transferase and geranylgeranyl transferase, key enzymes that induce conformational and functional changes in small G proteins to conduct signal production for cell proliferation. Thus, it has been tried for the treatment of cancers. However, although it affects the proliferation of immunocytes, its influence on immune responses has been examined in only a few studies. Notably, its effect on antigen-induced immune responses has not been studied. In this study, we examined whether POH suppresses Ag-induced immune responses with a mouse model of allergic airway inflammation. POH treatment of sensitized mice suppressed proliferation and cytokine production in Ag-stimulated spleen cells or CD4(+) T cells. Further, sensitized mice received aerosolized OVA to induce allergic airway inflammation, and some mice received POH treatment. POH significantly suppressed indicators of allergic airway inflammation such as airway eosinophilia. Cytokine production in thoracic lymph nodes was also significantly suppressed. These results demonstrate that POH suppresses antigen-induced immune responses in the lung. Considering that it exists naturally, POH could be a novel preventive or therapeutic option for immunologic lung disorders such as asthma with minimal side effects.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Hypersensitivity; Immunosuppression Therapy; Immunosuppressive Agents; Inflammation; Lung; Male; Mice; Mice, Inbred BALB C; Monoterpenes; Ovalbumin; Pulmonary Eosinophilia

Aspergillus fumigatus is often associated in asthmatic patients with the exacerbation of asthma symptoms. The pathomechanism of this phenomenon has not been fully understood. Here, we evaluated the immunological mechanisms and the role of the prostaglandin D2 / Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells (CRTH2) pathway in the development of Aspergillus-associated asthma exacerbation. We studied the effects of A. fumigatus on airway inflammation and bronchial hyper-responsiveness in a rat model of chronic asthma. Inhalation delivery of A. fumigatus conidia increased the airway eosinophilia and bronchial hyper-responsiveness in ovalbumin-sensitized, challenged rats. These changes were associated with prostaglandin D2 synthesis and CRTH2 expression in the lungs. Direct inflammation occurred in ovalbumin-sensitized, challenged animals, whereas pre-treatment with an antagonist against CRTH2 nearly completely eliminated the A. fumigatus-induced worsening of airway eosinophilia and bronchial hyper-responsiveness. Our data demonstrate that production of prostaglandin D2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenic factors responsible for the A. fumigatus-induced enhancement of airway inflammation and responsiveness.

Topics: Animals; Anti-Inflammatory Agents; Aspergillus fumigatus; Asthma; Bronchial Hyperreactivity; Disease Models, Animal; Eosinophils; Lung; Male; Ovalbumin; Prostaglandin D2; Pulmonary Aspergillosis; Pulmonary Eosinophilia; Rats; Rats, Wistar; Receptors, Immunologic; Receptors, Prostaglandin; Signal Transduction

Allergic asthma is a complex disease characterized by acute inflammation of the airways that over time leads to the development of significant structural changes termed remodeling. TNF-related apoptosis-inducing ligand (TRAIL) has an important regulatory role in acute allergic airways inflammation through up-regulation of the E3 ubiquitin ligase Midline-1 (MID-1), which limits protein phosphatase 2A (PP2A) activity and downstream dephosphorylation of proinflammatory signaling molecules. The relevance of TRAIL in the development of airways remodeling has yet to be determined. In this study, the lungs of wild-type (WT) BALB/c and Tnfsf10 knockout (TRAIL-/-) mice were chronically exposed to ovalbumin (OVA) for 12 weeks to induce hallmark features of chronic allergic airways disease, including airways hyperreactivity (AHR), subepithelial collagen deposition, goblet cell hyperplasia, and smooth muscle hypertrophy. TRAIL-/- mice were largely protected from the development of AHR and peribronchial eosinophilia and had reduced levels of mast cells in the airways. This correlated with lower levels of cytokines, including IL-4, -5, -10, and -13, and with lower levels of proinflammatory chemokines from cultured cells isolated from the draining lymph nodes. TRAIL-/- mice were also protected from the characteristic features of airways remodeling, including peribronchial fibrosis, smooth muscle hypertrophy, and mucus hypersecretion, which correlated with reduced TGF-β1 levels in the lungs. MID-1 expression was reduced in TRAIL-/- mice and up-regulated in allergic WT mice. Raising PP2A activity using 2-amino-4-(4-heptyloyphenol)-2-methylbutan-1-ol in allergic WT mice reduced eosinophilia, TGF-β1, and peribronchial fibrosis. This study shows that TRAIL promotes airways remodeling in an OVA-induced model of chronic allergic airways disease. Targeting TRAIL and its downstream proinflammatory signaling pathway involving PP2A may be of therapeutic benefit in reducing the hallmark features of airways remodeling observed in chronic allergic airways inflammation.

Topics: Airway Remodeling; Animals; Apoptosis; Blotting, Western; Bronchial Hyperreactivity; Cytokines; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Mice, Knockout; Mucus; Muscle, Smooth; Ovalbumin; Protein Phosphatase 2; Proteins; Pulmonary Eosinophilia; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta1; Ubiquitin-Protein Ligases

Mangiferin is a major bioactive ingredient in Mangifera indica Linn. (Anacardiaceae) leaves. Aqueous extract of such leaves have been used as an indigenous remedy for respiratory diseases like asthma and coughing in traditional Chinese medicine. However, underlying molecular mechanisms of mangiferin on anti-asthma remain unclear. In our present study, we investigated the anti-asthmatic effect of mangiferin on Th1/Th2 cytokine profiles and explored its underlying immunoregulatory mechanism in mouse model of allergic asthma. Mangiferin significantly reduced the total inflammatory cell counts and eosinophil infiltration, decreased the production of ovalbumin-specific IgE in serum and PGD2 in BALF. The antibody array analysis showed that mangiferin down-regulated the levels of one group of cytokines/chemokines including Th2-related IL-4, IL-5, IL-13, and others IL-3, IL-9, IL-17, RANTES, TNF-α, but simultaneously up-regulated Th1-related IFN-γ, IL-2 and IL-10 and IL-12 expression in serum. Thus it attenuates the imbalance of Th1/Th2 cells ratio by diminishing the abnormal mRNA levels of Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-4, IL-5 and IL-13). Finally, mangiferin substantially inhibited the activation and expression of STAT-6 and GATA-3 in excised lung tissues. Our results suggest that mangiferin can exert anti-asthmatic effect. The underlying mechanism may attribute to the modulation of Th1/Th2 cytokine imbalance via inhibiting the STAT6 signaling pathway.

Topics: Animals; Asthma; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Th1 Cells; Th2 Cells; Xanthones

Asthma is associated with a loss of the structural integrity of airway epithelium and dysfunction of the physical barrier, which protects airways from external harmful factors. Granulocyte activation causes the formation of extracellular traps, releasing web-like structures of DNA and proteins, being important to kill pathogens extracellularly. We investigated whether eosinophils infiltrating airways in an experimental model of asthma would induce eosinophil extracellular traps (EETs) in bronchoalveolar lavage fluid and lung tissue. We showed that an ovalbumin (OVA) asthma protocol presented a significant increase in eosinophil counts with increased extracellular DNA in bronchoalveolar lavage fluid as well as in lung tissue, confirming the presence of DNA traps colocalized with eosinophil peroxidase. EETs formation was reversed by DNase treatment. With these approaches, we demonstrated for the first time that OVA-challenged mice release extracellular DNA traps, which could aggravate pulmonary dysfunction.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; DNA; Extracellular Traps; Inflammation; Mice; Ovalbumin; Pulmonary Eosinophilia

The organic chemicals present in Asian sand dust (ASD) might contribute to the aggravation of lung eosinophila. Therefore, the aggravating effects of the Tar fraction from ASD on ovalbumin (OVA)-induced lung eosinophilia were investigated.. The Tar fraction was extracted from ASD collected from the atmosphere in Fukuoka, Japan. ASD collected from the Gobi desert was heated at 360°C to inactivate toxic organic substances (H-ASD). ICR mice were instilled intratracheally with 12 different test samples prepared with Tar (1 μg and 5 μg), H-ASD, and OVA in a normal saline solution containing 0.02% Tween 80. The lung pathology, cytological profiles in the bronchoalveolar lavage fluid (BALF), inflammatory cytokines/chemokines in BALF and OVA-specific immunoglobulin in serum were investigated.. Several kinds of polycyclic aromatic hydrocarbons (PAHs) were detected in the Tar sample. H-ASD + Tar 5 μg induced slight neutrophilic lung inflammation. In the presence of OVA, Tar 5 μg increased the level of eosinophils slightly and induced trace levels of Th2 cytokines IL-5 and IL-13 in BALF. Also mild to moderate goblet cell proliferation and mild infiltration of eosinophils in the submucosa of airway were observed. These pathological changes caused by H-ASD + OVA were relatively small. However, in the presence of OVA and H-ASD, Tar, at as low a level as 1 μg, induced severe eosinophil infiltration and proliferation of goblet cells in the airways and significantly increased Th2 cytokines IL-5 and IL-13 in BALF. The mixture showed an adjuvant effect on OVA-specific IgG1 production.. These results indicate that H-ASD with even low levels of Tar exacerbates OVA-induced lung eosinophilia via increases of Th2-mediated cytokines. These results suggest that ASD-bound PAHs might contribute to the aggravation of lung eosinophila.

Topics: Air Pollutants; Animals; Asthma; Disease Models, Animal; Dust; Lung; Male; Mice; Mice, Inbred ICR; Ovalbumin; Pulmonary Eosinophilia; Specific Pathogen-Free Organisms; Tars

CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.. We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.. Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs' ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.. These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.

Topics: Animals; Asthma; B7-2 Antigen; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Cells, Cultured; Coculture Techniques; Cytokines; Dendritic Cells; Disease Models, Animal; Immunoglobulin E; Lung; Lymphocyte Activation; Mice, Inbred BALB C; Ovalbumin; Phenotype; Pulmonary Eosinophilia; RNA Interference; RNAi Therapeutics; Th2 Cells; Transfection

Atopic march refers to the typical transition from a food allergy in early childhood to allergic asthma in older children and adults. However the precise interplay of events involving gut, skin and pulmonary inflammation in this process is not completely understood.. To develop a mouse model of mixed food and respiratory allergy mimicking the atopic march and better understand the impact of food allergies on asthma.. Food allergy to ovalbumin (OVA) was induced through intra-peritoneal sensitization and intra-gastric challenge, and/or a respiratory allergy to house dust mite (HDM) was obtained through percutaneous sensitization and intra-nasal challenges with dermatophagoides farinae (Der f) extract. Digestive, respiratory and systemic parameters were analyzed.. OVA-mediated gut allergy was associated with an increase in jejunum permeability, and a worsening of Der f-induced asthma with stronger airway hyperresponsiveness and pulmonary cell infiltration, notably eosinophils. There was overproduction of the pro-eosinophil chemokine RANTES in broncho-alveolar lavages associated with an enhanced Th2 cytokine secretion and increased total and Der f-specific IgE when the two allergies were present. Both AHR and lung inflammation increased after a second pulmonary challenge.. Gut sensitization to OVA amplifies Der f-induced asthma in mice.

Topics: Animals; Antigens, Dermatophagoides; Arthropod Proteins; Asthma; Bronchial Hyperreactivity; Bronchoconstriction; Chemokine CCL5; Disease Models, Animal; Female; Food Hypersensitivity; Immunoglobulin E; Intestinal Mucosa; Intestines; Lung; Mice, Inbred BALB C; Ovalbumin; Permeability; Pneumonia; Pulmonary Eosinophilia; Th2 Cells; Time Factors

This study examines the immunological responses in rats following inhalation to titanium dioxide nanoparticles (TiO2 NPs), in naïve rats and in rats with induced allergic airway disease. The responses of two different inbred rat strains were compared: the Dark Aguoti (DA), susceptible to chronic inflammatory disorders, and the Brown Norwegian (BN), susceptible to atopic allergic inflammation. Naïve rats were exposed to an aerosol of TiO2 NPs once daily for 10 days. Another subset of rats was sensitized to the allergen ovalbumin (OVA) in order to induce airway inflammation. These sensitized rats were exposed to TiO2 NPs before and during the allergen challenge. Naïve rats exposed to TiO2 NPs developed an increase of neutrophils and lymphocytes in both rat strains. Airway hyperreactivity and production of inflammatory mediators typical of a T helper 1 type immune response were significantly increased, only in DA rats. Sensitization of the rats induced a prominent OVA-specific-IgE and IgG response in the BN rat while DA rats only showed an increased IgG response. Sensitized rats of both strains developed airway eosinophilia following allergen challenge, which declined upon exposure to TiO2 NPs. The level of neutrophils and lymphocytes increased upon exposure to TiO2 NPs in the airways of DA rats but remained unchanged in the airways of BN rats. In conclusion, the responses to TiO2 NPs were strain-dependent, indicating that genetics play a role in both immune and airway reactivity. DA rats were found to be higher responder compared to BN rats, both when it comes to responses in naïve and sensitized rats. The impact of genetically determined factors influencing the inflammatory reactions pinpoints the complexity of assessing health risks associated with nanoparticle exposures.

Topics: Aerosols; Animals; Bronchial Hyperreactivity; Cytokines; Disease Models, Animal; Genetic Variation; Genotype; Immunoglobulin E; Immunoglobulin G; Inflammation Mediators; Inhalation Exposure; Lung; Male; Metal Nanoparticles; Neutrophils; Ovalbumin; Phenotype; Pneumonia; Pulmonary Eosinophilia; Rats, Inbred BN; Risk Factors; Species Specificity; Th1 Cells; Titanium

Native phosphatidylinositol mannosides (PIMs), isolated from the cell wall of Mycobacterium bovis, and synthetic PIM analogues have been reported to offer a variety of immunomodulating properties, including both suppressive and stimulatory activity. While numerous studies have examined the biological activity of these molecules, the aim of this research was to assess the physicochemical properties at a molecular level and correlate these characteristics with biological activity in a mouse model of airway eosinophilia. To accomplish this, we varied the flexibility and lipophilicity of synthetic PIMs by changing the polar headgroup (inositol- vs glycerol-based core) and the length of the acyl chains of the fatty acid residues (C0, C10, C16, and C18). A series of six phosphatidylinositol dimannosides (PIM2s) and phosphatidylglycerol dimannosides (PGM2s) were synthesized and characterized in this study. Langmuir monolayer studies showed that surface pressure-area (π-A) isotherms were greatly influenced by the length of the lipid acyl chains as well as the steric hindrance and volume of the headgroups. In aqueous solution, lipidated PIM2 and PGM2 compounds were observed to self-assemble into circular aggregates, as confirmed by dynamic light scattering and transmission electron microscopic investigations. Removal of the inositol ring but retention of the three-carbon glycerol unit maintained biological activity. We found that the deacylated PGM2, which did not show self-organization, had no effect on the eosinophil numbers but did have an impact on the expansion of OVA-specific CD4(+) Vα2Vβ5 T cells.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Wall; Chemical Phenomena; Cytokines; Disease Models, Animal; Immunologic Factors; Male; Mannosides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Molecular Structure; Mycobacterium bovis; Ovalbumin; Particle Size; Phosphatidylinositols; Pulmonary Eosinophilia

With the increase in production and use of engineered nanoparticles (NP; ≤ 100 nm), safety concerns have risen about the potential health effects of occupational or environmental NP exposure. Results of animal toxicology studies suggest that inhalation of NP may cause pulmonary injury with subsequent acute or chronic inflammation. People with chronic respiratory diseases like asthma or allergic rhinitis may be even more susceptible to toxic effects of inhaled NP. Few studies, however, have investigated adverse effects of inhaled NP that may enhance the development of allergic airway disease.. We investigated the potential of polyethylene glycol coated amorphous silica NP (SNP; 90 nm diameter) to promote allergic airway disease when co-exposed during sensitization with an allergen. BALB/c mice were sensitized by intranasal instillation with 0.02% ovalbumin (OVA; allergen) or saline (control), and co-exposed to 0, 10, 100, or 400 μg of SNP. OVA-sensitized mice were then challenged intranasally with 0.5% OVA 14 and 15 days after sensitization, and all animals were sacrificed a day after the last OVA challenge. Blood and bronchoalveolar lavage fluid (BALF) were collected, and pulmonary tissue was processed for histopathology and biochemical and molecular analyses.. Co-exposure to SNP during OVA sensitization caused a dose-dependent enhancement of allergic airway disease upon challenge with OVA alone. This adjuvant-like effect was manifested by significantly greater OVA-specific serum IgE, airway eosinophil infiltration, mucous cell metaplasia, and Th2 and Th17 cytokine gene and protein expression, as compared to mice that were sensitized to OVA without SNP. In saline controls, SNP exposure did cause a moderate increase in airway neutrophils at the highest doses.. These results suggest that airway exposure to engineered SNP could enhance allergen sensitization and foster greater manifestation of allergic airway disease upon secondary allergen exposures. Whereas SNP caused innate immune responses at high doses in non-allergic mice, the adjuvant effects of SNP were found at lower doses in allergic mice and were Th2/Th17 related. In conclusion, these findings in mice suggest that individuals exposed to SNP might be more prone to manifest allergic airway disease, due to adjuvant-like properties of SNP.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Immunoglobulin E; Inflammation Mediators; Inhalation Exposure; Lung; Lymph Nodes; Mice, Inbred BALB C; Nanoparticles; Neutrophil Infiltration; Ovalbumin; Particle Size; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Risk Assessment; Silicon Dioxide; Th17 Cells; Th2 Cells; Time Factors

STAT6 plays a central role in IL-4-mediated allergic responses. Several studies indicate that regulatory T cells (Tregs) can be modulated by IL-4 in vitro. We previously showed that STAT6(-/-) mice are highly resistant to allergic lung inflammation even when wild-type Th2 effectors were provided and that they have increased numbers of Tregs. However, the role of STAT6 in modulating Tregs in vivo during allergic lung inflammation has not been thoroughly investigated. To examine Treg and STAT6 interaction during allergic inflammation, STAT6(-/-), STAT6xRAG2(-/-), and RAG2(-/-) mice were subjected to OVA sensitization and challenge following adoptive transfer of OVA-specific, wild-type Th2 effectors with or without prior Treg depletion/inactivation, using anti-CD25 (PC61). As expected, STAT6(-/-) mice were highly resistant to airway inflammation and remodeling. In contrast, allergic lung inflammation was partially restored in STAT6(-/-) mice treated with PC61 to levels observed in STAT6xRAG2(-/-) mice. In some cases, STAT6xRAG2(-/-) mice were also given natural Tregs along with Th2 effectors. Adoptive transfer of natural Tregs caused a substantial reduction in bronchoalveolar lavage eosinophil composition and suppressed airway remodeling and T cell migration into the lung in STAT6xRAG2(-/-) mice to levels comparable to those in STAT6(-/-) mice. These results demonstrate the STAT6-dependent suppression of Tregs in vivo to promote allergic airway inflammation.

Topics: Administration, Intranasal; Adoptive Transfer; Airway Remodeling; Allergens; Animals; Bronchoalveolar Lavage Fluid; DNA-Binding Proteins; Forkhead Transcription Factors; Immune Tolerance; Interleukin-2 Receptor alpha Subunit; Interleukin-5; Lung; Lymphocyte Count; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; STAT6 Transcription Factor; T-Lymphocytes, Regulatory; Th2 Cells

Epidemiological studies suggest that maternal exposure to environmental hazards, such as particulate matter, is associated with increased incidence of asthma in childhood. We hypothesized that maternal exposure to combustion derived ultrafine particles containing persistent free radicals (MCP230) disrupts the development of the infant immune system and results in aberrant immune responses to allergens and enhances asthma severity.. Pregnant C57/BL6 mice received MCP230 or saline by oropharyngeal aspiration on gestational days 10 and 17. Three days after the second administration, blood was collected from MCP230 or saline treated dams and 8-isoprostanes in the serum were measured to assess maternal oxidative stress. Pulmonary T cell populations were assayed in the infant mice at six days, three and six weeks of postnatal age. When the infant mice matured to adults (i.e. six weeks of age), an asthma model was established with ovalbumin (OVA). Airway inflammation, mucus production and airway hyperresponsiveness were then examined.. Maternal exposure to MCP230 induced systemic oxidative stress. The development of pulmonary T helper (Th1/Th2/Th17) and T regulatory (Treg) cells were inhibited in the infant offspring from MCP230-exposed dams. As the offspring matured, the development of Th2 and Treg cells recovered and eventually became equivalent to that of offspring from non-exposed dams. However, Th1 and Th17 cells remained attenuated through 6 weeks of age. Following OVA sensitization and challenge, mice from MCP230-exposed dams exhibited greater airway hyperresponsiveness, eosinophilia and pulmonary Th2 responses compared to offspring from non-exposed dams.. Our data suggest that maternal exposure to MCP230 enhances postnatal asthma development in mice, which might be related to the inhibition of pulmonary Th1 maturation and systemic oxidative stress in the dams.

Topics: Age Factors; Animals; Asthma; Bronchial Hyperreactivity; Cytokines; Dinoprost; Female; Gestational Age; Inflammation Mediators; Inhalation Exposure; Lung; Maternal Exposure; Mice, Inbred C57BL; Ovalbumin; Oxidative Stress; Particulate Matter; Pregnancy; Prenatal Exposure Delayed Effects; Pulmonary Eosinophilia; Severity of Illness Index; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Th2 Cells

Neuroimmune semaphorin 4D (Sema4D) was found to be expressed and function in the nervous and immune systems. In the immune system, Sema4D is constitutively expressed on T cells and regulates T cell priming. In addition, it displays a stimulatory function on macrophages, DC, NK cells, and neutrophils. As all these cells are deeply involved in asthma pathology, we hypothesized that Sema4D plays a critical non-redundant regulatory role in allergic airway response. To test our hypothesis, we exposed Sema4D(-/-) and WT mice to OVA injections and challenges in the well-defined mouse model of OVA-induced experimental asthma. We observed a significant decrease in eosinophilic airway infiltration in allergen-treated Sema4D(-/-) mice relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL IL-5, IL-13, TGFβ1, IL-6, and IL-17A levels. In addition, T cell proliferation in OVA₃₂₃₋₃₃₉-restimulated Sema4D(-/-) cell cultures was downregulated. We also found increased Treg numbers in spleens of Sema4D(-/-) mice. However, airway hyperreactivity (AHR) to methacholine challenges was not affected by Sema4D deficiency in either acute or chronic experimental disease setting. Surprisingly, lung DC number and activation were not affected by Sema4D deficiency. These data provide a new insight into Sema4D biology and define Sema4D as an important regulator of Th2-driven lung pathophysiology and as a potential target for a combinatory disease immunotherapy.

Topics: Animals; Antigens, CD; Asthma; Bronchoalveolar Lavage Fluid; Cell Proliferation; Cells, Cultured; Cytokines; Flow Cytometry; Humans; Hypersensitivity; Lung; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Semaphorins; Th1 Cells; Th17 Cells; Th2 Cells

To investigate whether eosinophilic bronchitis without airway hyperresponsiveness will develop bronchial asthma in allergic mice.. Mice were sensitized with OVA on days 0, 7, and 14, challenged on days 21 to 23 (1(st) OVA challenge), and re-challenged on days 46 to 48 (2(nd) OVA challenge), intranasally with 10 (the EB group) and 200 (the AS group) μg OVA. Lung resistance (RL) was assessed 24 h after each challenge and on day 45 followed by analysis of leukocyte distribution in the bronchoalveolar lavage (BAL) fluid and histological examination.. Twenty-four hours after the 1(st) OVA challenge, aerosolized methacholine caused a dose-dependent increase in RL in all groups. At doses ≥1.56 mg/mL, RL in the AS group was significantly higher than that of the NS-1 group (P<0.01 or 0.05) and at doses ≥12.5 mg/mL, RL was markedly higher in the AS group than that of the EB group (P<0.01). The percentage of eosinophils in both the EB group and the AS group was markedly higher than that of the control group. Twenty-four hours after the 2(nd) OVA challenge, at doses ≤12.5 mg/mL, there was no significant difference in RL among all groups (P>0.05). At doses ≥12.5 mg/mL, RL in the AS group was significantly higher than that of the control group and EB group (P<0.01 or 0.05). The percentage of eosinophils in the AS group was noticeably higher than that of the EB group(P<0.05). Furthermore, there was apparent infiltration by inflammatory cells, predominantly eosinophils, into the sub-epithelial region of the bronchus and the bronchioles and around the vessels in the EB and AS group.. Re-challenge with low doses of ovalbumin did not increase airway reactivity and failed to induce bronchial asthma in mice with ovalbumin-induced EB.

Topics: Administration, Intranasal; Aerosols; Animals; Asthma; Bronchial Hyperreactivity; Bronchitis; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Eosinophils; Female; Lung; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia

Leukotriene C4 (LTC4) and its extracellular metabolites, LTD4 and LTE4, mediate airway inflammation. They signal through three specific receptors (type 1 cys-LT receptor [CysLT1R], CysLT2R, and GPR99) with overlapping ligand preferences. In this article, we demonstrate that LTC4, but not LTD4 or LTE4, activates mouse platelets exclusively through CysLT2R. Platelets expressed CysLT1R and CysLT2R proteins. LTC4 induced surface expression of CD62P by wild-type mouse platelets in platelet-rich plasma (PRP) and caused their secretion of thromboxane A2 and CXCL4. LTC4 was fully active on PRP from mice lacking either CysLT1R or GPR99, but completely inactive on PRP from CysLT2R-null (Cysltr2(-/-)) mice. LTC4/CysLT2R signaling required an autocrine ADP-mediated response through P2Y12 receptors. LTC4 potentiated airway inflammation in a platelet- and CysLT2R-dependent manner. Thus, CysLT2R on platelets recognizes LTC4 with unexpected selectivity. Nascent LTC4 may activate platelets at a synapse with granulocytes before it is converted to LTD4, promoting mediator generation and the formation of leukocyte-platelet complexes that facilitate inflammation.

Topics: Adenosine Diphosphate; Animals; Autocrine Communication; Blood Platelets; Leukotriene C4; Leukotriene D4; Leukotriene E4; Mice; Mice, Knockout; Ovalbumin; P-Selectin; Platelet Activation; Platelet Factor 4; Platelet-Rich Plasma; Pulmonary Eosinophilia; Receptors, Leukotriene; Receptors, Purinergic P2; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

Ovarian cancer G protein-coupled receptor 1 (OGR1) stimulation by extracellular protons causes the activation of G proteins and subsequent cellular functions. However, the physiological and pathophysiological roles of OGR1 in airway responses remain largely unknown. In the present study, we show that OGR1-deficient mice are resistant to the cardinal features of asthma, including airway eosinophilia, airway hyperresponsiveness (AHR), and goblet cell metaplasia, in association with a remarkable inhibition of Th2 cytokine and IgE production, in an ovalbumin (OVA)-induced asthma model. Intratracheal transfer to wild-type mice of OVA-primed bone marrow-derived dendritic cells (DCs) from OGR1-deficient mice developed lower AHR and eosinophilia after OVA inhalation compared with the transfer of those from wild-type mice. Migration of OVA-pulsed DCs to peribronchial lymph nodes was also inhibited by OGR1 deficiency in the adoption experiments. The presence of functional OGR1 in DCs was confirmed by the expression of OGR1 mRNA and the OGR1-sensitive Ca(2+) response. OVA-induced expression of CCR7, a mature DC chemokine receptor, and migration response to CCR7 ligands in an in vitro Transwell assay were attenuated by OGR1 deficiency. We conclude that OGR1 on DCs is critical for migration to draining lymph nodes, which, in turn, stimulates Th2 phenotype change and subsequent induction of airway inflammation and AHR.

Topics: Adoptive Transfer; Animals; Asthma; Bronchial Hyperreactivity; Calcium; Cell Movement; Dendritic Cells; Female; Gene Expression Regulation; Goblet Cells; Immunoglobulin E; Lung; Lymph Nodes; Mice; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Receptors, CCR7; Receptors, G-Protein-Coupled; Signal Transduction; Th1-Th2 Balance; Th2 Cells

The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4(+) splenocytes and subsequently production of GATA3-dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.

Topics: Animals; CARD Signaling Adaptor Proteins; GATA3 Transcription Factor; Gene Expression Regulation; Genetic Vectors; Humans; Interleukin-4; Interleukins; Lymphocyte Activation; Mice; Mice, Knockout; Ovalbumin; Proto-Oncogene Proteins c-jun; Pulmonary Eosinophilia; Radiation Chimera; Recombinant Fusion Proteins; Sequence Deletion; Specific Pathogen-Free Organisms; Th2 Cells; Transfection

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

Topics: Animals; Antigens; Asthma; Chemokine CCL11; Disease Models, Animal; Drug Evaluation, Preclinical; Epithelial Cells; Female; Gene Expression Regulation; Interleukin-4; Male; Naphthalenesulfonates; Ovalbumin; Protein-Arginine N-Methyltransferases; Pulmonary Eosinophilia; Rats; Recombinant Proteins; Respiratory System; RNA Interference; RNA, Small Interfering; Specific Pathogen-Free Organisms; Urea

The root of Peucedanum praeruptorum Dunn (PPD) is a commonly used traditional Chinese medicine for the treatment of asthma. Its major constituents, coumarins, were presumed to be responsible for its efficacy.. The potential of coumarins from PPD (CPPD) as anti-asthma agent was investigated.. Female BALB/c mice were sensitized and challenged with ovalbumin (OVA) to induce allergic airway inflammation. CPPD was administered intragastrically before every OVA challenge. Airway reactivity to the intravenous administration of acetylcholine chloride was measured 48h after final OVA inhalation. Airway inflammation was evaluated by leukocyte counts of bronchoalveolar lavage fluid (BALF) and histopathological analysis of lung lesions. Levels of interleukin (IL)-4, IL-5, IL-10, IL-13 and IFN-γ in BALF and OVA-specific immunoglobulin (Ig) E in serum, and activity of eosinophil peroxidase (EPO) in lung was measured. The percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells among CD4(+) T cells in spleen was analyzed by flow cytometry.. Compared with model group, CPPD significantly reduced airway hyperreactivity and airway eosinophilic inflammation, improved pathologic lesion of the lungs, reduced levels of IL-4, IL-5, IL-13 in BALF and OVA-specific IgE in serum, inhibited the activities of EPO in lung, and up-regulated levels of IL-10 and IFN-γ in BALF as well as the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in spleen.. CPPD can significantly suppress OVA-induced airway inflammation, airway hyperreactivity and Th2 predominant response in mice, showing great therapeutic potential for the treatment of allergic asthma.

Topics: Acetylcholine; Animals; Anti-Allergic Agents; Anti-Asthmatic Agents; Antigens; Apiaceae; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Coumarins; Disease Models, Animal; Drugs, Chinese Herbal; Female; Flow Cytometry; Inflammation Mediators; Mice; Mice, Inbred BALB C; Ovalbumin; Phytotherapy; Plant Roots; Plants, Medicinal; Pulmonary Eosinophilia; T-Lymphocytes, Regulatory; Th2 Cells

Most studies about functions of aryl hydrocarbon receptor (AhR) in the pathogenesis of asthma have been carried out with non-physiological industrial by-products such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene. In the present study, effects of 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct postulated as a candidate physiological ligand of AhR, on the pathogenesis of asthma were examined and then underlying mechanisms of its immumodulatory effects were investigated. FICZ significantly reduced pulmonary eosinophilia and Th2 cytokine expression in the lungs. Flow cytometric analysis of mediastinal lymph nodes showed that IL-4 producing cells decreased in FICZ-treated mice compared with PBS control. Next, effects of FICZ on in vitro Th2 differentiation and expression of the Th2 transcription factor GATA-3 were examined. CD4+ T cells were isolated from the spleen and incubated under the Th2 differentiation conditions. FICZ inhibited both Th2 differentiation and the expression of GATA-3. Finally, activation of STAT6, which is necessary for Th2 differentiation, was inhibited by FICZ.

Topics: Animals; Anti-Allergic Agents; Asthma; Carbazoles; Cells, Cultured; Cytokines; Disease Models, Animal; GATA3 Transcription Factor; Gene Expression Regulation; Humans; Immunosuppression Therapy; Ligands; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Receptors, Aryl Hydrocarbon; Th2 Cells; Tryptophan

Allergic airways disease (AAD) is associated with an increased influx of eosinophils to the lungs, mucus hypersecretion and Th2 cytokine production. Dietary antioxidant supplementation may alter cytokine responses and thus allergic inflammation. Lycopene is a potent dietary antioxidant. The objective of this study was to investigate the effects of lycopene, on allergic inflammation, in a mouse model of AAD. BALB/c mice receiving lycopene supplement or control were intraperitoneally sensitised and intranasally challenged with ovalbumin (OVA) to induce AAD. The effect of supplementation on inflammatory cell influx into bronchoalveolar lavage fluid, lung tissue and blood, mucus-secreting cell numbers in the airways, draining lymph node OVA-specific cytokine release, serum IgG1 levels and lung function in AAD was assessed. Supplementation reduced eosinophilic infiltrates in the bronchoalveolar lavage fluid, lung tissue and blood, and mucus-secreting cell numbers in the airways. The OVA-specific release of Th2-associated cytokines IL-4 and IL-5 was also reduced. We conclude that supplementation with lycopene reduces allergic inflammation both in the lungs and systemically, by decreasing Th2 cytokine responses. Thus, lycopene supplementation may have a protective effect against asthma.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Carotenoids; Cell Count; Cells, Cultured; Cytokines; Dietary Supplements; Immunoglobulin G; Inflammation Mediators; Lung; Lycopene; Lymph Nodes; Lymphocyte Count; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Phytotherapy; Pulmonary Eosinophilia; Th2 Cells

Eosinophilic bronchitis (EB) is a useful tool for studying airway hyperresponsiveness (AHR), but an exact EB animal model is lacking. Our objective was to establish an EB mouse model using ovalbumin (OVA). Mice were divided into asthma, normal saline (NS) control and three model groups. Asthma mice were challenged intranasally with 200 μg OVA. Model groups were challenged with one of the three OVA doses (10, 20 or 100 μg), and NS control mice received normal saline. Changes in lung resistance (R(L)), serum OVA-specific immunoglobulin-E (IgE) and differential inflammatory cell counts in bronchoalveolar lavage fluid (BALF) were determined after exposure to increasing doses of methacholine (MCh). Lung histological sections were examined for inflammatory infiltration. R(L) in the 10-μg OVA-challenged model group was not significantly different compared with the NS group at any MCh concentration but was significantly different compared with the asthma group (P < 0.01). R(L) in the other two model groups was intermediate between the asthma and NS groups. Serum OVA-specific IgE and eosinophils in BALF were increased significantly in all model and asthma groups compared with the NS group, but no significant differences were observed between model and asthma groups. Inflammatory cells were seen around bronchioles and capillaries in model and asthma groups but not the NS group. A mouse model of EB without AHR can be established by 10 μg OVA challenge and provides a useful tool for studying AHR mechanisms.

Topics: Animals; Asthma; Bronchitis; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Histocytochemistry; Immunoglobulin E; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Respiratory Function Tests

Clinical and epidemiological studies have shown the contribution of viral infection to the development of allergic asthma. Many RNA viruses, pathogenic for the respiratory tract, generate double-stranded (ds)RNA during their replication. Typical innate immune responses triggered by dsRNA involve the endosomal and cytoplasmic pathways. The former is mediated by Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF), and the latter by IFN-β promoter stimulator 1 (IPS-1). We explored the effect of polyinocinic polycytidilic acid, a synthetic dsRNA, on the development of an asthma phenotype in mice. Administration of dsRNA during ovalbumin sensitization augmented airway eosinophilia and airway hyperresponsiveness after an antigen challenge, which was associated with enhanced induction of IL-13-producing CD8(+) T cells. The augmentation was induced in IPS-1-deficient mice but not in TRIF-deficient mice. The interactions between dendritic cells (DCs) and T cells are regulated by B7-family costimulatory molecules, including B7-H1 (also known as PD-L1), a putative ligand for programmed death-1 (PD-1). Treatment of bone marrow-derived DCs with dsRNA enhanced B7-H1 expression in a TRIF-dependent manner. Additionally, dsRNA increased B7-H1 expression on DCs in the draining lymph nodes of ovalbumin-sensitized mice. The augmentation of the asthma phenotype was prevented by the treatment of mice with anti-B7-H1 mAb but not with anti-PD-1 mAb. The augmentation was not induced in B7-H1-deficient mice. These results suggest that dsRNA-triggered activation of the innate immune system in sensitization leads to augmentation of the asthma phenotype via IL-13 mainly from CD8(+) T cells. B7-H1 plays a crucial role in the process without requiring interaction with PD-1.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Asthma; B7-1 Antigen; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Dendritic Cells; Immunity, Innate; Interleukin-13; Membrane Glycoproteins; Mice; Mice, Knockout; Ovalbumin; Peptides; Phenotype; Pulmonary Eosinophilia; RNA, Double-Stranded

Cathepsin S (Cat S) is predominantly expressed in antigen-presenting cells and is up-regulated in several preclinical models of antigen-induced inflammation, suggesting a role in the allergic response. Prophylactic dosing of an irreversible Cat S inhibitor has been shown to attenuate pulmonary eosinophilia in mice, supporting the hypothesis that Cat S inhibition before the initiation of airway inflammation is beneficial in airway disease. In addition, Cat S has been shown to play a role in more distal events in the allergic response. To determine where Cat S inhibition may affect the allergic response, we used complementary genetic and pharmacological approaches to investigate the role of Cat S in the early and downstream allergic events in a murine model of antigen-induced lung inflammation. Cat S knockout mice did not develop ovalbumin-induced pulmonary inflammation, consistent with a role for Cat S in the development of the allergic response. Alternatively, wild-type mice were treated with a reversible, highly selective Cat S inhibitor in prophylactic and therapeutic dosing paradigms and assessed for changes in airway inflammation. Although both treatment paradigms resulted in potent Cat S inhibition, only prophylactic Cat S inhibitor dosing blocked lung inflammation, consistent with our findings in Cat S knockout mice. The findings indicate that although Cat S is up-regulated in allergic models, it does not appear to play a significant role in the downstream effector inflammatory phase in this model; however, our results demonstrate that Cat S inhibition in a prophylactic paradigm would ameliorate airway inflammation.

Topics: Animals; Asthma; Cathepsins; Disease Models, Animal; Drug Evaluation; Humans; Mice; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Up-Regulation

Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM₂) analogue 4 that mimics the suppressive activity of natural PIMs and also synthetic PIM₂ have been developed. This analogue, where the inositol core was replaced by glycerol, was tested for its ability to suppress cellular inflammation in a mouse model of allergic asthma and shown to be effective in suppressing airway eosinophilia. Suppression of all inflammatory cells monitored was observed, indicating a general blockade of cellular activity. These data indicate that the inositol core is not essential for this suppressive activity.

Topics: Administration, Intranasal; Animals; Anti-Allergic Agents; Asthma; Mice; Ovalbumin; Phosphatidylinositols; Pulmonary Eosinophilia

Tumor Necrosis Factor (TNF) is a pleiotropic cytokine consisting of soluble and transmembrane forms, with distinct roles in inflammation and immunity. TNF is an important factor in allergic airway inflammation. However, the disparate functions of soluble (sol) and transmembrane (tm) TNF in lung pathology are not well understood. Our aim was to assess the activities of solTNF and tmTNF in murine models of allergic airway disease, and to evaluate the efficacy of solTNF-selective inhibition. We used ovalbumin sensitization and challenge of TNF knockout, tmTNF knockin, and wild-type C57BL/6 mice to distinguish differences in airway inflammation and hyperreactivity mediated by solTNF and tmTNF. Functions of solTNF and tmTNF in hyperresponsive, wild-type Balb/c mice were assessed by comparing dominant-negative anti-TNF biologics, which antagonize solTNF yet spare tmTNF, to etanercept, a nonselective inhibitor of both TNF forms. Responses in transgenic C57BL/6 mice demonstrated that solTNF, and not tmTNF, is necessary to drive airway inflammation. In Balb/c mice, dominant-negative TNF biologics administered during immunization decreased the recruitment of eosinophils and lymphocytes into the bronchoalveolar space and lung parenchyma, reduced specific serum IgE, goblet-cell hyperplasia, and eosinophilic inflammation, and suppressed methacholine-induced airway hyperreactivity. Concentrations of IL-5, CCL5/RANTES, CCL11/eotaxin, and CCL17/TARC were also reduced in bronchoalveolar lavage. Dominant-negative TNFs reduced lung eosinophilia, even when given only during antigen challenge. The selective inhibition of soluble TNF suppresses inflammation, hyperreactivity, and remodeling in transgenic and wild-type murine models of allergic airway disease, and may offer safety advantages in therapies that preserve the immunoprotective functions of transmembrane TNF.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biological Products; Bronchial Hyperreactivity; Cytokines; Disease Models, Animal; Etanercept; Goblet Cells; Humans; Hyperplasia; Immunoglobulin E; Immunoglobulin G; Inflammation Mediators; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutation; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Receptors, Tumor Necrosis Factor; Tumor Necrosis Factor-alpha

The role of tumor necrosis factor-α (TNF-α) in contributing to allergen induced airway remodeling in asthma is unknown. In this study we have utilized a mouse model of chronic OVA allergen induced airway remodeling to determine whether TNF p55/p75 receptor deficient mice (abbreviated TNF-R KO) had reduced levels of airway remodeling. Chronic OVA challenged WT mice had significantly increased levels of lung eosinophilic inflammation as well as features of airway remodeling including increased peribronchial fibrosis, thickness of the peribronchial smooth muscle layer, mucus expression, and deposition of extracellular matrix proteins. In contrast, TNF-R KO mice had significantly reduced levels of major basic protein positive peribronchial eosinophils and significantly reduced peribronchial fibrosis assessed by quantitating the area of peribronchial trichrome staining and total lung collagen. In addition, TNF-R KO mice had significantly reduced thickness of the peribronchial smooth muscle layer, area of peribronchial α-smooth muscle actin immunostaining, and levels of the extracellular matrix protein fibronectin. There was a non-significant trend for reduced mucus expression in TNF-R KO mice. Levels of peribronchial cells immunostaining positive for TGF-β1 were significantly reduced in TNF-R KO mice suggesting that reduced levels of TGF-β1 expression in TNF-R KO mice may contribute to reduced airway remodeling. Overall, this study suggests an important role for TNF-α in contributing to many features of allergen induced airway remodeling including changes in levels of peribronchial smooth muscle, subepithelial fibrosis, and deposition of extracellular matrix.

Topics: Airway Remodeling; Allergens; Animals; Asthma; Bronchi; Collagen; Eosinophils; Extracellular Matrix Proteins; Fibronectins; Immunoglobulin E; Interleukin-5; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucus; Muscle, Smooth; Ovalbumin; Pulmonary Eosinophilia; Pulmonary Fibrosis; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Transforming Growth Factor beta1; Tumor Necrosis Factor Decoy Receptors; Tumor Necrosis Factor-alpha

Most of the studies investigating the effectiveness of blocking the leukotriene B4 (LTB4) receptor 1 (BLT1) have been performed in models of primary or acute allergen challenge. The role of the LTB4-BLT1 pathway in secondary challenge models, where airway hyperresponsiveness (AHR) and airway inflammation have been established, has not been defined. We investigated the effects of blocking BLT1 on early- and late-phase development of AHR and airway inflammation in previously sensitized and challenged mice. Female BALB/c mice were sensitized (Days 1 and 14) and challenged (primary, Days 28-30) with ovalbumin. On Day 72, mice were challenged (secondary) with a single OVA aerosol, and the early and late phases of AHR and inflammation were determined. Specific blockade of BLT1 was attained by oral administration of a BLT1 antagonist on Days 70 through 72. Administration of the antagonist inhibited the secondary ovalbumin challenge-induced alterations in airway responses during the late phase but not during the early phase, as demonstrated by decreases in AHR and in bronchoalveolar lavage neutrophilia and eosinophilia 6 and 48 hours after secondary challenge. The latter was associated with decreased levels of KC protein, macrophage inflammatory protein 2, and IL-17 in the airways. These data identify the importance of the LTB4-BLT1 pathway in the development of late-phase, allergen-induced airway responsiveness after secondary airway challenge in mice with established airway disease.

Topics: Animals; Anti-Asthmatic Agents; Antibodies; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Chemokine CXCL1; Chemokine CXCL2; Disease Models, Animal; Female; Inflammation Mediators; Interleukin-17; Leukotriene Antagonists; Lung; Mice; Mice, Inbred BALB C; Neutrophil Infiltration; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Receptors, Leukotriene B4; Time Factors

The prostaglandin D(2) (PGD(2))/CRTH2 pathway is important for eosinophil trafficking in vitro; however, genetic deficiency of CRTH2 does not suppress in vivo eosinophilic airway inflammation in acute models of asthma, and the role of CRTH2 in the pathogenesis of asthma is still ambiguous. Therefore, in the present study we explored whether the PGD(2)/CRTH2 pathway could affect the phenotypes of chronic asthma. Either CRTH2-deficient (CRTH2-/-) or wild-type mice were sensitized and exposed to ovalbumin (OVA) for 3 days (acute model) or 6 weeks (chronic model). While the magnitude of the acute eosinophilic inflammation was equivalent between CRTH2-/- and wild-type mice, the number of inflammatory cells and eosinophils in bronchoalveolar lavage fluid after chronic OVA exposure was significantly reduced in CRTH2-/- mice (18.0 ± 2.6 × 10(4) cells and 2.0 ± 0.5 × 10(4) cells) compared to wild-type mice (27.9 ± 2.5 × 10(4) cells and 6.8 ± 1.1 × 10(4) cells, p < 0.001). On the contrary, no difference was observed between CRTH2-/- and wild-type mice in terms of airway hyperresponsiveness or remodeling (goblet cell hyperplasia) in the chronic model of asthma. In conclusion, CRTH2 that mediates PGD(2) activity is essential for sustained eosinophilic inflammation in the airways, and its antagonists could exert an anti-inflammatory effect in chronic asthma.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Eosinophils; Goblet Cells; Interleukin-13; Interleukin-5; Lung; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Mice, Knockout; Mucins; Ovalbumin; Pulmonary Eosinophilia; Receptors, Immunologic; Receptors, Prostaglandin; Vaccination

Interleukin (IL)-33, a new member of the IL-1 cytokine family, has been recognized as a key cytokine that enhances T helper 2-balanced immune regulation through its receptor ST2; however, the function and relationship of the IL-33 and ST2 pathways in bronchial asthma are still unclear. We investigated the cellular origin and regulation of IL-33 and ST2 in allergic bronchial asthma in vivo and in vitro.. BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Mice were exposed to aerosolized 1% OVA for 30 min a day for 7 days. These mice were then challenged with aerosolized 1% OVA 2 days after the last day of exposure. After the OVA challenge, the mice were sacrificed and their lung tissues were obtained. Mouse lung fibroblasts were cultured and treated with IL-33 or IL-13.. The levels of IL-33 mRNA and IL-33 protein in lung tissue increased after the OVA challenge. Most IL-33-expressing cells were CD11c+ cells and epithelial cells, and many ST2-expressing cells were stained lung fibroblasts and inflammatory cells. IL-33 induced eotaxin/CCL11 production in lung fibroblasts. IL-33 and IL-13 synergistically induced eotaxin expression.. IL-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. IL-33 and ST2 may play important roles in allergic bronchial asthma.

Topics: Animals; Asthma; CD11c Antigen; Cells, Cultured; Chemokine CCL11; Dendritic Cells; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Female; Fibroblasts; Gene Expression; Interleukin-1 Receptor-Like 1 Protein; Interleukin-13; Interleukin-33; Interleukins; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Receptors, Interleukin; Vimentin

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1(-/-) mice. Bone marrow cells from Rcan1(-/-) mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.

Topics: Animals; Apoptosis; Asthma; Bronchoalveolar Lavage Fluid; Calcineurin; Calcium-Binding Proteins; Cell Movement; Cell Proliferation; Eosinophils; Hematopoietic Stem Cells; Interleukin-4; Interleukin-5; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Muscle Proteins; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Stem Cells

There is increasing evidence that inflammation plays a pivotal role in the pathogenesis of some forms of pulmonary hypertension (PH). We recently demonstrated that deficiency of adiponectin (APN) in a mouse model of PH induced by eosinophilic inflammation increases pulmonary arterial remodeling, pulmonary pressures, and the accumulation of eosinophils in the lung. Based on these data, we hypothesized that APN deficiency exacerbates PH indirectly by increasing eosinophil recruitment. Herein, we examined the role of eosinophils in the development of inflammation-induced PH. Elimination of eosinophils in APN-deficient mice by treatment with anti-interleukin-5 antibody attenuated pulmonary arterial muscularization and PH. In addition, we observed that transgenic mice that are devoid of eosinophils also do not develop pulmonary arterial muscularization in eosinophilic inflammation-induced PH. To investigate the mechanism by which APN deficiency increased eosinophil accumulation in response to an allergic inflammatory stimulus, we measured expression levels of the eosinophil-specific chemokines in alveolar macrophages isolated from the lungs of mice with eosinophilic inflammation-induced PH. In these experiments, the levels of CCL11 and CCL24 were higher in macrophages isolated from APN-deficient mice than in macrophages from wild-type mice. Finally, we demonstrate that the extracts of eosinophil granules promoted the proliferation of pulmonary arterial smooth muscle cells in vitro. These data suggest that APN deficiency may exacerbate PH, in part, by increasing eosinophil recruitment into the lung and that eosinophils could play an important role in the pathogenesis of inflammation-induced PH. These results may have implications for the pathogenesis and treatment of PH caused by vascular inflammation.

Topics: Adiponectin; Animals; Antibodies; Cell Extracts; Cell Proliferation; Cells, Cultured; Chemokine CCL11; Chemokine CCL2; Chemokine CCL24; Disease Models, Animal; Eosinophils; Gene Knockout Techniques; Hypertension, Pulmonary; Interleukin-5; Lung; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogens; Myocytes, Smooth Muscle; Ovalbumin; Primary Cell Culture; Pulmonary Artery; Pulmonary Eosinophilia; Transcription, Genetic

New phenoxyacetic acid antagonists of CRTH2 are described. Following the discovery of a hit compound by a focused screening, high protein binding was identified as its main weakness. Optimization aimed at reducing serum protein binding led to the identification of several compounds that showed not only excellent affinities for the receptor (41 compounds with K(i) < 10 nM) but also excellent potencies in a human whole blood assay (IC(50) < 100 nM; PGD2-induced eosinophil shape change). Additional optimization of the PK characteristics led to the identification of several compounds suitable for in vivo testing. Of these, 19k and 19s were tested in two different pharmacological models (acute FITC-mediated contact hypersensitivity and ovalbumin-induced eosinophilia models) and found to be active after oral dosing (10 and 30 mg/kg).

Topics: Acetates; Administration, Oral; Alkynes; Animals; Anti-Allergic Agents; Anti-Inflammatory Agents; Binding, Competitive; Blood Proteins; Caco-2 Cells; Cell Membrane Permeability; Cell Shape; Chemotaxis, Leukocyte; Dermatitis, Contact; Eosinophils; Female; HEK293 Cells; Humans; Mice; Mice, Inbred BALB C; Microsomes, Liver; Ovalbumin; Phenoxyacetates; Protein Binding; Pulmonary Eosinophilia; Radioligand Assay; Rats; Receptors, Immunologic; Receptors, Prostaglandin; Structure-Activity Relationship; Sulfones

Recent studies suggest that plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of the fibrinolytic system, may promote the development of asthma. To further investigate the significance of PAI-1 in the pathogenesis of asthma and determine the possibility that PAI-1 could be a therapeutic target for asthma, this study was conducted. First, PAI-1 levels in induced sputum (IS) from asthmatic subjects and healthy controls were measured. In asthmatic subjects, IS PAI-1 levels were elevated, compared with that of healthy controls, and were significantly higher in patients with long-duration asthma compared with short-duration asthma. PAI-1 levels were also found to correlate with IS transforming growth factor-β levels. Then, acute and chronic asthma models induced by ovalbumin were established in PAI-1-deficient mice and wild-type mice that received intra-airway administrations of small interfering RNA against PAI-1 (PAI-1-siRNA). We could demonstrate that eosinophilic airway inflammation and airway hyperresponsiveness were reduced in an acute asthma model, and airway remodeling was suppressed in a chronic asthma model in both PAI-1-deficient mice and wild-type mice that received intra-airway administration of PAI-1-siRNA. These results indicate that PAI-1 is strongly involved in the pathogenesis of asthma, and intra-airway administration of PAI-1-siRNA may be able to become a new therapeutic approach for asthma.

Topics: Airway Remodeling; Animals; Asthma; Bronchitis; Bronchoalveolar Lavage Fluid; Drug Evaluation, Preclinical; Female; Hepatocyte Growth Factor; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Targeted Therapy; Ovalbumin; Plasminogen Activator Inhibitor 1; Pulmonary Eosinophilia; RNA Interference; RNA, Small Interfering; Sputum

Evidence demonstrating an important role of the intestinal microbiota in the incidence of allergic disorders has led to the concept of using probiotics as possible antiallergic therapy. This study aimed to select a bacterial strain with the best antiallergic treatment effects from a panel of 6 bacterial strains in a mouse model of ovalbumin(OVA)-allergic asthma.. OVA-sensitized BALB/c mice were orally administered the bacterial strains Bifidobacterium breve M-16V, B. infantis NumRes251, B. animalis NumRes252 and NumRes253, Lactobacillus plantarum NumRes8 and L. rhamnosus NumRes6. After challenge by OVA inhalation in the lungs, the response to methacholine was measured. Pulmonary inflammation was assessed by analyzing bronchoalveolar lavage fluid for the presence of eosinophils, neutrophils, macrophages and lymphocytes and for interleukin 4, interleukin 5, interleukin 10 and interferon-gamma. OVA-specific IgE, IgG1 and IgG2a were measured in serum. Next, the effect on acute allergic skin reaction was measured after treatment with B. breve M-16V and L. plantarum NumRes8.. Of the panel of 6 strains, B. breve M-16V and L. plantarum NumRes8 inhibited (1) the response to methacholine, (2) reduced the number of eosinophils in the bronchoalveolar lavage fluid, (3) reduced both OVA-specific IgE and (4) OVA-specific IgG1, whereas the other strains did not affect all these parameters simultaneously. B. breve M-16V but not L. plantarum NumRes8 reduced interleukin 4, interleukin 5 and interleukin 10. Furthermore, B. breve M-16V but not L. plantarum NumRes8 reduced acute allergic skin reactions to OVA.. B. breve M-16V was identified as the most potent antiallergic strain.

Topics: Administration, Oral; Animals; Asthma; Bifidobacterium; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Cell Count; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukin-5; Lacticaseibacillus rhamnosus; Lactobacillus plantarum; Lymphocytes; Macrophages, Alveolar; Male; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Probiotics; Pulmonary Eosinophilia; Skin Tests; Specific Pathogen-Free Organisms; Vaccination

Asthma is a chronic disease characterized by airway inflammation caused by the dysregulated production of cytokines secreted by allergen-specific type 2 T helper (Th2) cells. Antrodia camphorata is a commonly used fungus in Asian folk medicine, and A. camphorata polysaccharides are reported to possess anti-cancer activities. In this study, the immunomodulatory effects of purified fractionated polysaccharides (GF2) from A. camphorata on dendritic cells (DCs) and their potential preventive effects against ovalbumin (OVA) -induced asthma were investigated. In the presence of GF2, lipopolysaccharide (LPS) -activated DCs exhibited up-regulated expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules, as well as enhanced interleukin-10 (IL-10) and IL-12 production. GF2 treatment on LPS-activated DCs suppressed naïve CD4(+) T-cell proliferation and Th2 cell polarization with IL-10 production in an allogeneic mixed lymphocyte reaction. In animal experiments, a high dose of GF2 efficiently reduced expression levels of OVA-specific immunoglobulin G1 (IgG1) and IgE. However, lower doses of GF2 significantly enhanced OVA-specific IgG2a production. Our data also showed that administration of GF2 dose-dependently inhibited the development of airway hyperresponsiveness, airway eosinophilia and Th2 responses. OVA-specific CD4(+) T cells from higher doses of GF2-treated mice had significantly lower proliferative capacities compared with control mice. Moreover, treatment with GF2 significantly increased the high levels of IL-10 and low levels of interferon-gamma produced by T cells. Taken together, these data indicate that administration of A. camphorata polysaccharides (GF2) may have therapeutic potential when used as an adjuvant for the immunomodulatory treatment of allergic asthma.

Topics: Allergens; Animals; Antrodia; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cell Proliferation; Cell Survival; Cytokines; Dendritic Cells; Female; Gene Expression; Immunoglobulin E; Immunoglobulin G; Interleukin-10; Interleukin-12; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Polysaccharides; Pulmonary Eosinophilia; Spleen; T-Lymphocytes; Th2 Cells; Vaccination

Schistosoma mansoni infection has been associated with protection against allergies. The mechanisms underlying this association may involve regulatory cells and cytokines. We evaluated the immune response induced by the S. mansoni antigens Sm22.6, PIII and Sm29 in a murine model of ovalbumin (OVA)-induced airway inflammation. BALB/c mice were sensitized with subcutaneously injected OVA-alum and challenged with aerolized OVA. Mice were given three doses of the different S. mansoni antigens. Lung histopathology, cellularity of bronchoalveolar lavage (BAL) and eosinophil peroxidase activity in lung were evaluated. Immunoglobulin (Ig)E levels in serum and cytokines in BAL were also measured. Additionally, we evaluated the frequency of CD4+forkhead box P3 (FoxP3)+ T cells in cultures stimulated with OVA and the expression of interleukin (IL)-10 by these cells. The number of total cells and eosinophils in BAL and the levels of OVA-specific IgE were reduced in the immunized mice. Also, the levels of IL-4 and IL-5 in the BAL of mice immunized with PIII and Sm22.6 were decreased, while the levels of IL-10 were higher in mice immunized with Sm22.6 compared to the non-immunized mice. The frequency of CD4+FoxP3+ T cells was higher in the groups of mice who received Sm22.6, Sm29 and PIII, being the expression of IL-10 by these cells only higher in mice immunized with Sm22.6. We concluded that the S. mansoni antigens used in this study are able to down-modulate allergic inflammatory mediators in a murine model of airway inflammation and that the CD4+FoxP3+ T cells, even in the absence of IL-10 expression, might play an important role in this process.

Topics: Alveolitis, Extrinsic Allergic; Animals; Antigens, Helminth; Asthma; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Female; Forkhead Transcription Factors; Immunization; Interleukins; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Schistosoma mansoni; T-Lymphocyte Subsets

Eosinophilic inflammation is closely related to angiogenesis in asthmatic airway remodeling. In ovalbumin (OVA)-sensitized mice bone marrow-derived, proangiogenic endothelial progenitor cells (EPCs) are rapidly recruited into the lungs after OVA aerosol challenge and promptly followed by mobilization and recruitment of eosinophils.. We hypothesized that bone marrow-derived EPCs initiate the recruitment of eosinophils through expression of the eosinophil chemoattractant eotaxin-1.. EPCs were isolated from an OVA murine model of allergic airway inflammation and from asthmatic patients. Endothelial and smooth muscle cells were isolated from mice. Eotaxin-1 expression was analyzed by means of immunofluorescence, real-time PCR, or ELISA. In vivo recruitment of eosinophils by EPCs was analyzed in mice.. Circulating EPCs of asthmatic patients had higher levels of eotaxin-1 compared with those seen in control subjects. In the murine model OVA allergen exposure augmented eotaxin-1 mRNA and protein levels in EPCs. The EPCs from OVA-sensitized and OVA-challenged mice released high levels of eotaxin-1 on contact with lung endothelial cells from sensitized and challenged mice but not from control animals and not on contact with cardiac or hepatic endothelial cells from sensitized and challenged mice. Intranasal administration of the eotaxin-rich media overlying cultures of EPCs caused recruitment into the lungs, confirming functional chemoattractant activity.. Bone marrow-derived EPCs are early responders to environmental allergen exposures and initiate a parallel switch to a proangiogenic and proeosinophilic environment in the lungs of asthmatic patients.

Topics: Adult; Airway Remodeling; Allergens; Animals; Asthma; Bone Marrow Cells; Chemokine CCL11; Endothelium; Eosinophils; Female; Humans; Male; Mice; Mice, Inbred BALB C; Neovascularization, Physiologic; Ovalbumin; Pulmonary Eosinophilia; Stem Cells

Basic and clinical studies suggest that hypothalamic-pituitary-adrenal (HPA) axis is the neuroendocrine-immune pathway that functionally regulates the chronic inflammatory disease including asthma. Our previous studies showed corresponding changes of cytokines and leukotriene B4 (LTB4) between brain and lung tissues in antigen-challenged asthmatic rats. Here, we investigated how the increased LTB4 level in brain interacts with HPA axis in regulating antigen-induced asthmatic response in sensitized rats.. Ovalbumin-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v) 30 min before challenge. Lung resistance (RL) and dynamic lung compliance (Cdyn) were measured before and after antigen challenge. Inflammatory response in lung tissue was assessed 24 h after challenge. Expression of CRH mRNA and protein in hypothalamus were evaluated by RT-PCR and Western Blot, and plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using the ELISA kits.. Antigen challenge decreased pulmonary function and induced airway inflammation, evoked HPA axis response in sensitized rats. Administration of LTB4 via i.c.v markedly attenuated airway contraction and inflammation. Meanwhile, LTB4 via i.c.v markedly increased CORT and ACTH level in plasma before antigen challenge, and followed by further increases in CORT and ACTH levels in plasma after antigen challenge in sensitized rats. Expression of CRH mRNA and protein in hypothalamus were also significantly increased by LTB4 via i.c.v in sensitized rats after antigen challenge. These effect were completely blocked by pre-treatment with BLT1 receptor antagonist U75302 (10 ng), but not by BLT2 antagonist LY255283.. LTB4 administered via i.c.v down-regulates the airway contraction response and inflammation through activation of the HPA axis via its BLT1 receptor. This study expands our concept of the regulatory role of intracranial inflammatory mediators in inflammatory diseases including asthma. The favourable effects of LTB4 on the HPA axis may help to explain the phenomenon of self-relief after an asthmatic attack.

Topics: Adrenocorticotropic Hormone; Airway Resistance; Animals; Asthma; Blotting, Western; Corticotropin-Releasing Hormone; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fatty Alcohols; Female; Glycols; Hypothalamo-Hypophyseal System; Hypothalamus; Inflammation Mediators; Injections, Intraventricular; Leukotriene B4; Lung; Lung Compliance; Male; Ovalbumin; Pituitary-Adrenal System; Pulmonary Eosinophilia; Rats; Rats, Sprague-Dawley; Receptors, Leukotriene B4; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Allergic asthma is associated with eosinophilic inflammation in the airways. Animal models commonly used to elucidate allergic inflammation mechanisms include BALB/c and C57BL/6 mice. Our aim was to evaluate lung eosinophilia and the corresponding Th1/Th2 balance in the two strains after allergen exposure. BALB/c and C57BL/6 mice were subjected to ovalbumin-induced allergic airway inflammation using BrdU to label newly produced cells. The numbers of new eosinophils were evaluated by differential cell count and immunocytochemistry (MBP+BrdU+). Proliferation rate of lung eosinophils was measured by analysis of CD45+CCR3+BrdU+ cells by FACS. Distribution of newly produced eosinophils in the lung and the Th1/Th2 (CD4+T-bet+/CD4+GATA-3+) balance was evaluated by immunohistochemistry. Allergen challenge with ovalbumin induced comparable eosinophilia in bone marrow (BM), blood and lung tissue in both strains of mice compared to phosphate-buffered saline controls, which was confirmed by immunocytochemistry. There was a small increase in the number of lung MBP+BrdU(-) eosinophils in C57BL/6 mice compared to BALB/c mice, which suggests a basal increase in this strain following sensitization. While there was no difference in eosinophilic proliferation in the lung, the distribution of the newly produced eosinophils differs between the two strains. BALB/c mice showed staining primarily around vessels and airways, whereas C57BL/6 mice showed a more even distribution in the lung tissue. No difference in the Th1/Th2 balance was observed between two strains. This study shows that there is a difference in the distribution of eosinophils in the lung between the C57BL/6 and BALB/c mice, but no difference in eosinophil production or Th1/Th2 balance.

Topics: Allergens; Animals; Asthma; CD4 Antigens; Eosinophils; GATA3 Transcription Factor; Leukocyte Common Antigens; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Receptors, CCR3; T-Box Domain Proteins; Th1 Cells; Th2 Cells

The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol.. This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses.. Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 microg of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 microg of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE.. Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 microg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses.. The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.

Topics: Administration, Intranasal; Allergens; Animals; Antigens, Plant; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Dose-Response Relationship, Immunologic; Immunoglobulin E; Injections, Subcutaneous; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Ovalbumin; Pulmonary Eosinophilia; Pyroglyphidae; Rats; Rats, Wistar; Species Specificity; Time Factors

Natural killer T (NK T) cells have been shown to play an essential role in the development of allergen-induced airway hyperresponsiveness (AHR) and/or airway inflammation in mouse models of acute asthma. Recently, NK T cells have been reported to be required for the development of AHR in a virus induced chronic asthma model. We investigated whether NK T cells were required for the development of allergen-induced AHR, airway inflammation and airway remodelling in a mouse model of chronic asthma. CD1d-/- mice that lack NK T cells were used for the experiments. In the chronic model, AHR, eosinophilic inflammation, remodelling characteristics including mucus metaplasia, subepithelial fibrosis and increased mass of the airway smooth muscle, T helper type 2 (Th2) immune response and immunoglobulin (Ig)E production were equally increased in both CD1d-/- mice and wild-type mice. However, in the acute model, AHR, eosinophilic inflammation, Th2 immune response and IgE production were significantly decreased in the CD1d-/- mice compared to wild-type. CD1d-dependent NK T cells may not be required for the development of allergen-induced AHR, eosinophilic airway inflammation and airway remodelling in chronic asthma model, although they play a role in the development of AHR and eosinophilic inflammation in acute asthma model.

Topics: Acute Disease; Airway Remodeling; Airway Resistance; Allergens; Animals; Antigens, CD1d; Asthma; Bronchial Hyperreactivity; Bronchitis; Chronic Disease; Disease Models, Animal; Female; Fibrosis; Immunoglobulin E; Male; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Knockout; Muscle, Smooth; Natural Killer T-Cells; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

As genetically engineered mutant mice deficient in single genes are usually generated on a C57BL/6 background, to study mast cell trafficking in mutant mice, we initially investigated whether mast cells accumulated in bronchi in C57BL/6 mice challenged with OVA allergen acutely or chronically for 1 to 3 months. The total number of bronchial mast cells were quantitated using toluidine blue staining in airways of different sizes, i.e. , small (<90 microm), medium (90-155 microm), or large (>150 microm) airways. Non-OVA challenged and acute OVA challenged mice (C57BL/6 and BALB/c) had no detectable bronchial mast cells. Chronic OVA challenge in BALB/c mice for 1 or 3 months induced a significant increase in the number of bronchial mast cells in small-, medium-, and large-sized airways but minimal change in the number of bronchial mast cells in C57BL/6 mice. Both BALB/c and C57BL/6 mice developed significant lung eosinophilia following acute or chronic OVA challenge. Studies of IL-9-deficient mice on a BALB/c background demonstrated a significant increase in the number of bronchial mast cells in IL-9-deficient mice suggesting that IL-9 was not required for the bronchial accumulation of mast cells. Overall, these studies demonstrate that the chronic OVA challenge protocol we have utilized in BALB/c mice provides a model to study the mechanism of bronchial mast cell accumulation and that bronchial mast cell accumulation in chronic OVA challenged mice is independent of IL-9 in this model.

Topics: Allergens; Animals; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Interleukin-9; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Species Specificity

It has been reported that ambient particulate matter (PM) in some large cities, such as Beijing, China, causes adverse respiratory health effects. However, there is currently no experimental report on the relationship between bronchial asthma and urban PM (UPM) in northeast Asia. In this study, the microbial and chemical substances adsorbed onto UPM collected in Beijing were excluded by heat-treatment at 360 degrees C for 30 min. The effects of UPM or heated UPM (H-UPM) toward allergic lung inflammation were compared in murine lungs to investigate the role of organic substances. ICR mice were administrated intratracheally with the two kinds of UPM and/or ovalbumin (OVA) 4 times at 2-week intervals. UPM and H-UPM enhanced eosinophil recruitment induced by OVA in the alveoli and in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. UPM and H-UPM synergistically increased Th-2 cytokines--interleukin (IL)-4 and IL-13, eosinophil-relevant cytokines and chemokines, such as IL-5 and monocyte chemotactic protein-3 (MCP-3), induced by OVA in bronchoalveolar lavage fluid (BALF). The enhancing effects were much greater in UPM than in H-UPM. UPM induced adjuvant effects on specific immunoglobulin E (IgE) and IgG1 production by OVA. In an in vitro study using RAW264.7 cells, UPM increased the expression of Toll-like receptor 2 (TLR2) mRNA, but not TLR4 mRNA. H-UPM caused no expression of both TLR mRNAs. These results suggest that the aggravated lung eosinophilia in UPM was due to activation of a Th2-associated immune response via the activation of TLR2 by microbial materials. Chemical materials of air pollutant origin contained in UPM, and inorganic components (elemental carbon, mineral elements) in H-UPM, could also cause the aggravation.

Topics: Air Pollutants; Animals; Bronchoalveolar Lavage Fluid; Cell Line; Chemokines; China; Drug Synergism; Eosinophils; Gene Expression; Hot Temperature; Intubation, Intratracheal; Lung; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Ovalbumin; Particulate Matter; Pulmonary Alveoli; Pulmonary Eosinophilia; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 4

Chronic allergic asthma is the result of a T-helper type 2 (Th2)-biased immune status. Current asthma therapies control symptoms in some patients, but a long-lasting therapy has not been established. Anti-Asthma Simplified Herbal Medicine Intervention (ASHMI™), a Chinese herbal formula, improved symptoms and lung function, and reduced Th2 responses in a controlled trial of patients with persistent moderate to severe asthma.. We evaluated the persistence of ASHMI™ beneficial effects following therapy in a murine model of chronic asthma and the immunological mechanisms underlying such effects. Methods BALB/c mice sensitized intraperitoneally with ovalbumin (OVA) received 3 weekly intratracheal OVA challenges to induce airway hyper-reactivity (AHR) and inflammation (OVA mice). Additionally, OVA mice were treated with ASHMI™ (OVA/ASHMI™) or water (OVA/sham) for 4 weeks, and then challenged immediately and 8 weeks post-therapy. In other experiments, OVA mice received ASHMI™ treatment with concomitant neutralization of IFN-γ or TGF-β. Effects on airway responses, cytokine- and OVA-specific IgE levels were determined 8 weeks post-therapy.. Before treatment, OVA mice exhibited AHR and pulmonary eosinophilic inflammation following OVA challenge, which was almost completely resolved immediately after completing treatment with ASHMI™ and did not re-occur following OVA re-challenge up to 8 weeks post-therapy. Decreased allergen-specific IgE and Th2 cytokine levels, and increased IFN-γ levels also persisted at least 8 weeks post-therapy. ASHMI™ effects were eliminated by the neutralization of IFN-γ, but not TGF-β, during therapy.. ASHMI™ induced long-lasting post-therapy tolerance to antigen-induced inflammation and AHR. IFN-γ is a critical factor in ASHMI™ effects.

Topics: Animals; Anti-Asthmatic Agents; Antibodies; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Cells, Cultured; Disease Models, Animal; Drugs, Chinese Herbal; Female; Immunoglobulin E; Interferon-gamma; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Th2 Cells; Time Factors; Transforming Growth Factor beta

Sanguisorba officinalis L. is known to have anti-inflammatory properties. However, the potential effects of S. officinalis against asthma have not been reported. In the present study, we investigated the protective effects and underlying mechanisms of S. officinalis in a murine ovalbumin (OVA)-induced asthma model. Mice were sensitized and challenged by OVA inhalation to induce airway inflammation and remodeling. S. officinalis ethanolic extract (SOEE) markedly decreased the number of infiltrated inflammatory cells, together with a reduction in the levels of T-helper type 2 cytokines and immunoglobulin E levels. Histopathological studies showed that inflammatory cell infiltration and mucus hypersecretion were inhibited by SOEE. In addition, OVA-induced increases in reactive oxygen species were attenuated by SOEE. All these effects were correlated with heme oxygenase-1 (HO-1) induction by SOEE. These results indicate that SOEE has therapeutic potential against bronchial asthma associated with allergic diseases that is due, at least in part, to HO-1 upregulation.

Topics: Analysis of Variance; Animals; Anti-Asthmatic Agents; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Female; Heme Oxygenase-1; Histocytochemistry; Immunoglobulin E; Lung; Mice; Mice, Inbred BALB C; Mucus; Neutrophil Infiltration; Ovalbumin; Oxidative Stress; Plant Extracts; Plant Roots; Pulmonary Eosinophilia; Reactive Oxygen Species; Sanguisorba; Up-Regulation

CD27 is a costimulatory molecule of the TNFR family strongly expressed on activated CD4(+) and CD8(+) T lymphocytes. Binding with its ligand CD70, present on lymphocytes and DCs, leads to enhanced T cell activation and proliferation. Several other costimulatory molecules of the TNFR family like CD30, CD134 (OX40) or CD137 (4-1BB) have been shown to be critically involved in the development of asthma and/or respiratory tolerance. However, the role of CD27/CD70 signalling in these disease models has not been studied intensively. The aim of this study was to directly investigate the role of CD27 for the development of asthma and respiratory tolerance by comparative analysis of wild type (WT) and CD27(-/-) mice in the corresponding murine models. Ovalbumin (OVA)-sensitized and challenged CD27(-/-) mice developed comparably increased airway hyperreactivity (AHR), eosinophilic airway inflammation, mucus hypersecretion and elevated OVA-specific serum IgE levels in response to OVA sensitization as WT mice. In addition, Th2 cytokine production in spleen cell culture supernatants and proliferation of splenocytes after in vitro OVA restimulation was equally enhanced when derived from WT and CD27(-/-) mice. Furthermore, the absence of CD27 had no decisive impact on tolerance induction, so that WT and CD27(-/-) mice were comparably protected from asthma development by mucosal antigen application before sensitization. Our results suggest that CD27 costimulation is dispensable for a Th2 cell mediated allergic asthma response and respiratory tolerance induction in murine models.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Humans; Immune Tolerance; Immunization; Immunoglobulin E; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; T-Lymphocytes; Tumor Necrosis Factor Receptor Superfamily, Member 7

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. Chlorogenic acid (CGA), an ester of caffeic acid with quinic acid, is one of the most abundant polyphenol compounds in various agricultural products. CGA shows various biological properties, such as anti-oxidant, anti-viral, anti-carcinogenic and anti-inflammatory activities. We investigated suppressive effects of CGA on ovalbumin (OVA)-induced allergic asthma in mice and underlying mechanisms of them. CGA significantly reduced pulmonary eosinophilia and expression of IL-4, IL-5 and TNF-α in the lung as well as the serum levels of total and OVA-specific IgE, while CGA enhanced those of total and OVA-specific IgG3, of which isotype switching is down-regulated by IL-4. In vitro IgE production from LPS/IL-4-stimulated splenocytes was remarkably reduced by CGA, while that of IgG3 was enhanced. The Cε germ line transcription, which is necessary for IL-4 mediated IgE isotype switching, was reduced by CGA in LPS/IL-4-stimulated splenocytes. IgE isotype switching is mediated via several transduction pathways, activating several molecules including STAT-6, NF-κB, ERK1/2, and JNK. Among the molecules, which were activated by IL-4/LPS, activation of STAT-6 and JNK was inhibited by CGA.

Topics: Animals; Artemisia; Asthma; Chlorogenic Acid; Cytokines; Hypersensitivity; Immunoglobulin E; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; STAT6 Transcription Factor; Th2 Cells

There is no experimental study demonstrating the effects of airborne Asian sand dust (AASD) on allergic lung eosinophilia. The organic substances adsorbed onto AASD collected from the atmosphere of Iki-island in Japan were excluded by heat treatment at 360°C for 30 min. The effects of AASD or heated-AASD (H-AASD) towards allergic lung inflammation were compared in murine lungs to investigate the role of organic substances. ICR mice were administrated with the two kinds of AASD and/or ovalbumin (OVA) intratracheally four times at 2-week intervals. AASD and H-AASD enhanced eosinophil recruitment induced by OVA in the alveoli and in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. AASD and H-AASD synergistically increased Th2 cytokines-interleukin-13 (IL-13), eosinophil-relevant cytokine and chemokine, such as IL-5, and monocyte chemotactic protein-3 (MCP-3) induced by OVA in whole lung lavage fluid. The enhancing effects were much greater in AASD than in H-AASD. AASD induced adjuvant effects on OVA-specific immunoglobulin E (IgE) and IgG1 production. In an in vitro study using RAW264.7 cells, AASD increased the expression of Toll-like receptors 2 (TLR2) mRNA, but not TLR4 mRNA. AASD increased mRNA expression of NALP3, ASC, and IL-1ß compared with the control. H-AASD caused no expression of either mRNA. These results suggest that the aggravated lung eosinophilia in AASD is due to activation of a Th2-associated immune response and that the activation of TLR2 and NALP3 inflammasome by microbial materials could be participating in this phenomenon.

Topics: Air Pollutants; Air Pollution; Animals; Carrier Proteins; Cell Line; Cytokines; Drug Synergism; Dust; Eosinophils; Gene Expression; Immunoglobulins; Inhalation Exposure; Macrophages; Male; Mice; Mice, Inbred ICR; NLR Family, Pyrin Domain-Containing 3 Protein; Ovalbumin; Particulate Matter; Pulmonary Alveoli; Pulmonary Eosinophilia; Silicon Dioxide; Toll-Like Receptor 2

A very low dose of dexamethasone (DEX) was as equally as sufficient as a pharmacological dose to decrease eosinophil inflammation in airways and bone marrow. The timing of DEX treatment in relation to allergen challenge was strongly decisive for the outcome of the inflammatory response.. We aimed to study compartmental allergic airway inflammatory responses to classic pharmacological and also extremely low physiological DEX dosage, given at different time points close to allergen challenge in a murine model.. Ovalbumin-sensitized BALB/c-mice were exposed to intra-nasal ovalbumin. DEX was given i.p. as 1 microg/kg low-dose or 500 microg/kg pharmacological single-dose 2 h before, immediately before or 7 h after each of three challenges. Inflammatory cells were evaluated in bronchoalveolar lavage (BAL), lungs, nasal mucosa, and bone marrow.. Groups treated with low-dose DEX decreased eosinophilia in BAL to the same extent as the pharmacological dose, but only when administered before challenge. The most prominent decrease of eosinophils in BAL was seen in mice treated with the low dose 2 h before challenge. A similar response pattern as in BAL eosinophilia was detected in lung histopathology. DEX treatments had no obvious effects on inflammation in nasal mucosa.

Topics: Animals; Anti-Inflammatory Agents; Bone Marrow; Bronchoalveolar Lavage Fluid; Dexamethasone; Dose-Response Relationship, Drug; Eosinophilia; Eosinophils; Injections, Intraperitoneal; Leukocyte Count; Leukotriene B4; Lung; Male; Mice; Mice, Inbred BALB C; Nasal Mucosa; Ovalbumin; Premedication; Pulmonary Eosinophilia; Respiratory Hypersensitivity

The contribution of CD4 T cells and other CD4+ cells to lung inflammation and airway remodeling remains unclear during bouts of chronic exposure to airborne allergen. Previously, murine models have shown that CD4 T cells are required for initiation of acute inflammation and the remodeling process. However, it is unknown whether CD4 T cells or other CD4+ cells continue to be required for remodeling during ongoing allergen challenges after the development of acute eosinophilic lung inflammation. To test this, mice were sensitized and challenged with ovalbumin (OVA). After acute airway inflammation was established, a CD4 depleting antibody was administered for 4 wk during a period of chronic exposure to intranasal OVA, resulting in effective depletion of CD4+ cells from all organs, including the lung, lung-draining lymph nodes, and spleen. In these mice, levels of peribronchial inflammation, bronchoalveolar (BAL) eosinophils, and lung CD11c+, CD8+, and Siglec-F+CD11c- cells were significantly reduced. However, mucus metaplasia, peribronchial subepithelial fibrosis, and smooth muscle mass were not affected. Additionally, depletion of CD4+ cells before the last week of chronic allergen challenges also led to significant reductions in BAL eosinophils, peribronchial inflammation, and lung CD11c+, CD8+, and Siglec-F+CD11c- cells. These results show that CD4 T cells, and other CD4+ cells including subsets of dendritic cells, iNKT cells, and LTi cells, play a role in ongoing eosinophilic lung inflammation during periods of chronic allergen challenge, but are not required for progressive airway remodeling that develops after initial acute inflammation.

Topics: Allergens; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Chronic Disease; Class Ib Phosphatidylinositol 3-Kinase; Dendritic Cells; Flow Cytometry; Immunoblotting; Isoenzymes; Lung; Lymph Nodes; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Ovalbumin; Phosphatidylinositol 3-Kinases; Pulmonary Eosinophilia; Respiratory System

To identify asthma-susceptibility genes, we did proteome analyses of the lung from control and ovalbumin-sensitized BALB/c mice. Among the 6 up-regulated proteins is alpha(1)-protease inhibitor (alpha(1)-PI) type 2, which is a member of the serine protease inhibitor superfamily of protease inhibitors that participate in a variety of physiological functions, including extracellular matrix remodeling and inflammation. The up-regulated expression of alpha(1)-PI type 2 was confirmed by real-time PCR. Then we examined mRNA expression of five members of the alpha(1)-PI family genes (alpha(1)-PI types 1-5) in several organs of BALB/c mice and found that in addition to the liver, all the organs tested also expressed different isoforms of alpha(1)-PI in a tissue-specific manner, albeit to a lesser extent compared with the liver. When a similar study was performed with C57BL/6 mice, which have been shown to be more susceptible to ovalbumin-induced asthma than BALB/c mice, a pair of remarkable differences between the mouse strains were revealed: (1) the magnitude of alpha(1)-PI type 2 mRNA in all the organs was much higher in BALB/c than in C57BL/6 mice and (2) alpha(1)-PI type 2 is the only isoform expressed in the lung of BALB/c, but not of C57BL/c mice. Using the antisense oligonucleotide technology to specifically down-regulate expression of alpha(1)-PI type 2, we demonstrated that pulmonary infiltration of eosinophils was significantly increased by intranasal administration of alpha(1)-PI type 2 antisense oligonucleotides in OVA-sensitized mice, suggesting that alpha(1)-PI type 2 may suppress the progress of asthma, probably by acting on neutrophil elastase, which can produce many of the pathological features of asthma.

Topics: Administration, Intranasal; alpha 1-Antitrypsin; Animals; Cell Movement; Eosinophils; Gene Expression; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oligonucleotides, Antisense; Organ Specificity; Ovalbumin; Proteomics; Pulmonary Eosinophilia; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

There is growing evidence that hypoxia-inducible transcription factors are involved in the pathophysiology of asthma. Hypoxia-inducible factor-1alpha (HIF-1alpha) in particular controls the expression of many hypoxia regulated genes, but whether HIF-1alpha directly contributes to allergen-driven immune responses is not known.. Partially HIF-1alpha-deficient mice (HIF-1alpha(+/-)) or wild-type littermate controls were used in all experiments. Spleen CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 antibodies and cytokine secretion was measured in vitro. Mice were sensitized by intraperitoneal injection of ovalbumin (Ova) plus alum, and then challenged by intranasal Ova followed by bronchoalveolar lavage (BAL) and isolation of spleen cells. BAL cells were counted and the differential determined using cytospin, and splenocytes were incubated with Ova to measure recall cytokine production.. Interferon-gamma secretion was significantly higher in anti-CD3 plus anti-CD28 stimulated CD4+ T cells obtained from HIF-1alpha(+/-) mice compared to wild-type controls. HIF-1alpha(+/-) mice were protected from lung eosinophilia 72 h after allergen challenge, in association with enhanced secretion of interferon-gamma in recall responses of splenocytes.. HIF-1alpha contributes to allergic immune responses and lung eosinophilia in a mouse model of asthma.

Topics: Animals; Antibodies, Monoclonal; Asthma; Bronchoalveolar Lavage Fluid; CD28 Antigens; CD3 Complex; CD4-Positive T-Lymphocytes; Disease Models, Animal; Female; GATA3 Transcription Factor; Gene Expression; Hypoxia-Inducible Factor 1, alpha Subunit; Interferon-gamma; Interleukin-4; Mice; Ovalbumin; Pulmonary Eosinophilia

Obesity is associated with an increased incidence and severity of asthma, as well as other lung disorders, such as pulmonary hypertension. Adiponectin (APN), an antiinflammatory adipocytokine, circulates at lower levels in the obese, which is thought to contribute to obesity-related inflammatory diseases. We sought to determine the effects of APN deficiency in a murine model of chronic asthma. Allergic airway inflammation was induced in APN-deficient mice (APN(-/-)) using sensitization without adjuvant followed by airway challenge with ovalbumin. The mice were then analyzed for changes in inflammation and lung remodeling. APN(-/-) mice in this model develop increased allergic airway inflammation compared with wild-type mice, with greater accumulation of eosinophils and monocytes in the airways associated with elevated lung chemokine levels. Surprisingly, APN(-/-) mice developed severe pulmonary arterial muscularization and pulmonary arterial hypertension in this model, whereas wild-type mice had only mild vascular remodeling and comparatively less pulmonary arterial hypertension. Our findings demonstrate that APN modulates allergic inflammation and pulmonary vascular remodeling in a model of chronic asthma. These data provide a possible mechanism for the association between obesity and asthma, and suggest a potential novel link between obesity, inflammatory lung disease, and pulmonary hypertension.

Topics: Adiponectin; Airway Resistance; Animals; Asthma; Chemokines; Disease Models, Animal; Disease Susceptibility; Female; Hyperplasia; Hypertension, Pulmonary; Hypoxia; Inflammation; Lung Compliance; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Muscle, Smooth, Vascular; Obesity; Ovalbumin; Pulmonary Artery; Pulmonary Eosinophilia

Dietary fat intake has been associated with obesity and obesity in its turn with attenuated airway function and asthma, but it is unclear whether or how high-fat intake per se alters immune function relevant to development of allergic asthma.. To use a non-obese mouse model of mild to moderate allergic asthma to compare effects of high-fat with isocaloric control-diet on allergic immune responses.. C57BL/6 mice weaned and maintained on control (11% fat calories) or isocaloric high-fat diet (58% fat calories) were systemically sensitized with ovalbumin and challenged in the lungs. Allergic airway inflammation was assessed by measuring lung inflammation; serum antibodies; and, cytokines in serum, bronchoalveolar lavage (BAL) fluid and in supernatants of in vitro stimulated lung draining lymph node and spleen lymphocytes.. There was a significant reduction in lung eosinophilia and IL-5 in high-fat fed mice. Lung draining lymph node cells from these mice showed reduced pro-inflammatory cytokine (MCP-1 and TNF-alpha) release after ovalbumin re-stimulation and reduced release of IL-13 after concanavalin-A stimulation, indicating a general rather than just an antigen-specific change. There was no difference in IFN-gamma release. In contrast, pro-inflammatory cytokine release was increased from splenocytes. Decreased eosinophilia was not due to increased regulatory T cell or IL-10 induction in draining lymph nodes or spleen, nor to changes in antibody response to ovalbumin. However, decreased levels of serum and BAL eotaxin were found in high-fat fed animals.. The data indicate that high-fat dietary content redirects local immune responses to allergen in the lungs and systemic responses in the spleen and serum. These effects are not due to changes in regulatory T cell populations but may reflect a failure to mobilize eosinophils in response to allergic challenge.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Concanavalin A; Cytokines; Dietary Fats; Disease Models, Animal; Lung; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; T-Lymphocytes, Regulatory

Phosphodiesterase 4 selective inhibitor may prevent airway inflammation and remodeling.. The aim of this study was to investigate the effects of KF19514, a phosphodiesterase 4 and 1 dual inhibitor, on chronic airway inflammation and remodeling following chronic exposure to aerosolized antigen in mice.. Ovalbumin (OVA) was administered intraperitoneally to BALB/c mice on days 0 and 14, and the mice were then exposed to aerosolized OVA daily for 4 weeks. Twenty-four hours following the final inhalation, bronchial responsiveness to acetylcholine was measured, and histologic examination and hydroxyproline content of the lung were evaluated.. Bronchial responsiveness to acetylcholine, number of inflammatory cells and eosinophils in the lamina propria, thickness of epithelial and subepithelial collagen layers, and hydroxyproline content of the lung increased following chronic exposure to OVA for 7 weeks. KF19514 significantly prevented all of these changes.. Phosphodiesterase 4 and 1 inhibitors such as KF19514 may help prevent bronchial hyperresponsiveness and chronic asthma-induced airway remodeling.

Topics: Acetylcholine; Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoconstriction; Cyclic Nucleotide Phosphodiesterases, Type 1; Female; Hydroxyproline; Inflammation; Lung; Mice; Mice, Inbred BALB C; Mucous Membrane; Naphthyridines; Ovalbumin; Phosphodiesterase 4 Inhibitors; Phosphodiesterase Inhibitors; Pulmonary Eosinophilia; Respiratory Mucosa; Specific Pathogen-Free Organisms; Trachea; Vaccination

Allergic asthma is characterized by airway hyperresponsiveness (AHR) and allergic inflammation of the airways, driven by allergen-specific Th2 cells. The asthma phenotypes and especially AHR are sensitive to the presence and activity of regulatory T (Treg) cells in the lung. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is known to have a co-stimulatory function on effector CD4+ T cells, rendering these cells insensitive to Treg suppression. However, the effects of GITR signaling on polarized Th1 and Th2 cell effector functions are not well-established. We sought to evaluate the effect of GITR signaling on fully differentiated Th1 and Th2 cells and to determine the effects of GITR activation at the time of allergen provocation on AHR and airway inflammation in a Th2-driven mouse model of asthma.. CD4+CD25- cells were polarized in vitro into Th1 and Th2 effector cells, and re-stimulated in the presence of GITR agonistic antibodies to assess the effect on IFNgamma and IL-4 production. To evaluate the effects of GITR stimulation on AHR and allergic inflammation in a mouse asthma model, BALB/c mice were sensitized to OVA followed by airway challenges in the presence or absence of GITR agonist antibodies.. GITR engagement potentiated cytokine release from CD3/CD28-stimulated Th2 but not Th1 cells in vitro. In the mouse asthma model, GITR triggering at the time of challenge induced enhanced airway hyperresponsiveness, serum IgE and ex vivo Th2 cytokine release, but did not increase BAL eosinophilia.. GITR exerts a differential effect on cytokine release of fully differentiated Th1 and Th2 cells in vitro, potentiating Th2 but not Th1 cytokine production. This effect on Th2 effector functions was also observed in vivo in our mouse model of asthma, resulting in enhanced AHR, serum IgE responses and Th2 cytokine production. This is the first report showing the effects of GITR activation on cytokine production by polarized primary Th1 and Th2 populations and the relevance of this pathway for AHR in mouse models for asthma. Our data provides crucial information on the mode of action of the GITR signaling, a pathway which is currently being considered for therapeutic intervention.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstriction; CD28 Antigens; CD3 Complex; Cells, Cultured; Disease Models, Animal; Glucocorticoid-Induced TNFR-Related Protein; Humans; Immunoglobulin E; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Interleukins; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Rats; Receptors, Nerve Growth Factor; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Signal Transduction; Th1 Cells; Th2 Cells

Formaldehyde (FA) is a common indoor air pollutant that can cause asthma in people experiencing long-term exposure. While FA and other man-made chemicals contribute to the stimulation of asthma in the general population, the underlying molecular pathogenesis of this relationship is not yet well understood.. To explore FA as an irritant for the onset of asthma and as an adjuvant for the induction of allergy.. In the present study, 40 Wistar rats in five experimental groups were exposed to: (i) saline; (ii) ovalbumin (OVA); (iii) OVA + FA at 417 ppb; (iv) OVA + FA at 2500 ppb; and (v) FA at 2500 ppb. Current and prior occupational exposure limits in China were established at 417 ppb and 2500 ppb, respectively. Gaseous FA was administrated to the animals for 6 h/day before and during OVA immunization or saline treatment. Measured outcomes included in situ lung function analysis, cytokine measurement, and histological changes in the rat lungs.. The airway reactivity, lung histological changes, pulmonary interleukin-4 secretion, and eosinophil infiltration in the OVA and FA exposed rats were significantly higher after gaseous FA exposures of 417 and 2500 ppb. While FA exposure alone did not induce significant structural changes to the airway, and the rate of inflammatory cell infiltration was the same as for the control group, pulmonary levels of interferon-gamma were significantly elevated in the exposed rats.. FA may be an irritant as well as serve as an adjuvant for the onset of asthma or asthma-like symptoms.

Topics: Air Pollutants; Air Pollution, Indoor; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Dose-Response Relationship, Drug; Formaldehyde; Gases; Inhalation Exposure; Interferon-gamma; Interleukin-4; Irritants; Male; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Rats; Rats, Wistar; Time Factors

Eosinophilic infiltration into the airways is frequently associated with allergic asthma; however, the role of antigen deposition in mediating this phenomenon has not been studied in detail.. Using a murine model of ovalbumin (OVA) allergy, we examined how differential deposition of OVA during antigen challenge affects pulmonary eosinophilia, immune response and airway hyper-reactivity (AHR).. Differential allergen deposition to the upper respiratory tract (URT) alone or combined upper and lower respiratory tract (ULRT) was accomplished by administering OVA intranasally to either anaesthetized or unanaesthetized mice, respectively. BALB/c mice (6-7 weeks old) were sensitized with OVA-alum via the intraperitoneal route, and then challenged intranasally using OVA, with or without anaesthesia. AHR, enumeration of inflammatory cells and quantitative measurement of inflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF), lung histopathology and immune responses were subsequently assessed.. In sensitized animals challenged via the ULRT route, a profound eosinophilia and goblet cell hyperplasia was observed in lung tissue. Conversely, sensitized mice receiving an identical challenge dose via the URT route alone exhibited only negligible levels of inflammation. Interestingly, AHR and OVA-specific IgG(1) and IgE systemic responses were comparable between the two groups.. This study indicates that direct exposure of allergen in the deep lung is highly correlated with airway eosinophilia and lung inflammation, but does not correlate with AHR or immune response.

Topics: Administration, Intranasal; Allergens; Alum Compounds; Animals; Asthma; Bronchi; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Inflammation; Mice; Mice, Inbred BALB C; Ovalbumin; Plethysmography, Whole Body; Pulmonary Eosinophilia; Trachea

Epidemiological studies have identified childhood exposure to environmental tobacco smoke as a significant risk factor for the onset and exacerbation of asthma, but studies of smoking in adults are less conclusive, and mainstream cigarette smoke (MCS) has been reported to both enhance and attenuate allergic airway inflammation in animal models. We sensitized mice to ovalbumin (OVA) and exposed them to MCS in a well-characterized exposure system. Exposure to MCS (600 mg/m(3) total suspended particulates, TSP) for 1 h/day suppresses the allergic airway response, with reductions in eosinophilia, tissue inflammation, goblet cell metaplasia, IL-4 and IL-5 in bronchoalveolar lavage (BAL) fluid, and OVA-specific antibodies. Suppression is associated with a loss of antigen-specific proliferation and cytokine production by T cells. However, exposure to a lower dose of MCS (77 mg/m(3) TSP) had no effect on the number of BAL eosinophils or OVA-specific antibodies. This is the first report to demonstrate, using identical smoking methodologies, that MCS inhibits immune responses in a dose-dependent manner and may explain the observation that, although smoking provokes a systemic inflammatory response, it also inhibits T cell-mediated responses involved in a number of diseases.

Topics: Allergens; Animals; Cytokines; Female; Goblet Cells; Hyperplasia; Immune Tolerance; Immunization; Immunoglobulin E; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Respiratory System; Smoking; T-Lymphocytes

Non-selective cation influx through canonical transient receptor potential channels (TRPCs) is thought to be an important event leading to airway inflammation. TRPC6 is highly expressed in the lung, but its role in allergic processes is still poorly understood.. The purpose of this study was to evaluate the role of TRPC6 in airway hyperresponsiveness (AHR) and allergic inflammation of the lung.. Methacholine-induced AHR was assessed by head-out body plethysmography of wild type (WT) and TRPC6(-/-) mice. Experimental airway inflammation was induced by intraperitoneal ovalbumin (OVA) sensitization, followed by OVA aerosol challenges. Allergic inflammation and mucus production were analysed 24 h after the last allergen challenge.. Methacholine-induced AHR and agonist-induced contractility of tracheal rings were increased in TRPC6(-/-) mice compared with WT mice, most probably due to compensatory up-regulation of TRPC3 in airway smooth muscle cells. Most interestingly, when compared with WT mice, TRPC6(-/-) mice exhibited reduced allergic responses after allergen challenge as evidenced by a decrease in airway eosinophilia and blood IgE levels, as well as decreased levels of T-helper type 2 (Th2) cytokines (IL-5, IL-13) in the bronchoalveolar lavage. However, lung mucus production after allergen challenge was not altered by TRPC6 deficiency.. TRPC6 deficiency inhibits specific allergic immune responses, pointing to an important immunological function of this cation channel in Th2 cells, eosinophils, mast cells and B cells.

Topics: Animals; Bronchial Hyperreactivity; Cells, Cultured; Epithelial Cells; Guinea Pigs; Hypersensitivity; Immunoglobulin E; In Vitro Techniques; Interleukin-13; Interleukin-5; Leukocytes; Lung; Mice; Mice, Knockout; Mucus; Muscle Contraction; Muscle, Smooth; Ovalbumin; Pulmonary Eosinophilia; Trachea; TRPC Cation Channels; TRPC6 Cation Channel

The Helicobacter pylori neutrophil-activating protein (HP-NAP) is able in vitro to elicit IL-12 and IL-23 production via agonistic interaction with toll-like receptor 2, and to promote Th1 polarization of allergen-specific T-cell responses. This study was aimed to assess whether systemic/intraperitoneal and/or mucosal HP-NAP administration inhibited the Th2-mediated bronchial inflammation using a mouse model of allergic asthma induced by inhaled ovalbumin (OVA). Systemic HP-NAP delivery markedly reduced the lung eosinophilia in response to repeated challenge with aerosolized OVA. Likewise, the production of IL-4, IL-5 and GM-CSF was significantly lower in the bronchoalveolar lavage of animals treated with systemic HP-NAP plus OVA than that of animals treated with OVA alone. Systemic HP-NAP also significantly resulted in both reduction of total serum IgE and increase of IL-12 plasma levels. Mucosal administration of HP-NAP was equally successful as the systemic delivery in reducing eosinophilia, IgE and Th2 cytokine levels in bronchoalveolar lavage. However, no suppression of lung eosinophilia and bronchial Th2 cytokines was observed in toll-like receptor 2-knock-out mice following HP-NAP treatment. These results identify HP-NAP as a candidate for novel strategies of prevention and treatment of allergic diseases.

Topics: Animals; Antigens, Bacterial; Asthma; Bacterial Proteins; Bronchoalveolar Lavage Fluid; Cytokines; Humans; Immunoglobulin E; Inflammation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

Chronic stress has been proposed to aggravate allergic inflammation, whereas acute stress may have functional beneficial effects. The aim of this study was to investigate the influence of timing of single short restraint stress (RST) in a model of eosinophilic airway inflammation.. The airways of ovalbumin (OVA)-sensitized mice were exposed to an intranasal OVA challenge. RST was applied in two different ways; either 2 h before (pre-stress) or after (post-stress) the OVA challenge, respectively, or as a combination of stress before and after (double-stress) the OVA challenge. One group of mice was also treated with metyrapone (ME) prior to a pre-stress challenge. The inflammatory cell response was evaluated in bronchoalveolar lavage fluid (BALF), lung and nasal tissue, as well as bone marrow.. RST applied prior to the OVA challenge (pre-stress) inhibited OVA-induced airway inflammation in BALF and lung tissue, and reduced nasal histopathology compared to unstressed mice. Given as post-stress or double-stress, RST did not affect the inflammation in BALF, lungs or nasal tissue. Pre-treatment with ME prevented the pre-challenge stress evoked decrease in inflammation in BALF and lungs.. Effects of RST on eosinophilic airway inflammation appear to be strongly dependent on timing and, as could be judged from the ME inhibition pattern, also corticosterone dependent. Hypothalamic-pituitary-adrenal axis activation probably influences eosinophilic inflammation through specific sequences of compartmental activation and thereby timing effects are evident on cellular recruitment pattern during the allergic reaction.

Topics: Animals; Asthma; Bronchi; Corticosterone; Enzyme Inhibitors; Eosinophils; Hypothalamo-Hypophyseal System; Inflammation Mediators; Male; Metyrapone; Mice; Mice, Inbred BALB C; Neuroimmunomodulation; Ovalbumin; Pituitary-Adrenal System; Pulmonary Eosinophilia; Restraint, Physical; Stress, Psychological; Time Factors

We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantly increased airway hyperresponsiveness, pulmonary eosinophilia, and enhanced chemokine and Th2 cytokine responses in lung tissue and in lung-derived CD4(+) lymphocytes. Although several TFs have been associated with enhanced inflammatory responses in the lung, ATF3 attenuates the inflammatory responses associated with allergic airway disease.

Topics: Activating Transcription Factor 3; Allergens; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Chemokines; Gene Expression Regulation; Humans; Interleukin-13; Interleukin-4; Interleukin-5; Lung; Mice; Mice, Knockout; Ovalbumin; Pneumonia; Promoter Regions, Genetic; Pulmonary Eosinophilia; Th2 Cells

Eotaxin-1/CCL11 is important for early eosinophil recruitment to the airways of asthmatics. In order to clarify whether eotaxin-2/CCL24 accounts for prolonged airway eosinophilia, the authors determined the expression of CCL11 and CCL24 in lung tissue and bronchoalveolar lavage (BAL) as well as eosinophil infiltration over 14 days in BALB/c mice sensitised (intraperitonealy) and challenged (inhalations) with ovalbumin (OVA). Allergen exposure induced perivascular, peribronchial, and BAL eosinophilia for up to 7 days. CCL11 and CCL24 were highly expressed in lung tissue from 6 and up to 72 hours. Peak expression of CCL11 protein was 1557 +/- 109 pg/mL for OVA (mean +/- SEM) versus 404 +/- 73 pg/mL in controls (SAL) (P < .001) and 1690 +/- 54 versus 455 +/- 165 pg/mL for CCL24 (P < .01). In BAL, only eotaxin-2/CCL24 was significantly increased (1623 +/- 85 pg/mL for OVA versus 157 +/- 22 pg/mL for SAL, P < .01). Peak eosinophilia and CCL24 expression occurred later in BAL than in lung tissue. These data suggest that both CCL11 and CCL24 are important for recruitment of eosinophils to perivascular and peribronchial tissue seen up to 72 hours. This finding implies redundancy between these chemokines rather than differentially regulated expression over time. In contrast, only CCL24 seems important for recruitment of eosinophils into BAL. Specific inhibition of CCL11 alone is therefore unlikely to inhibit eosinophil recruitment to the airways.

Topics: Allergens; Animals; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokine CCL24; Eosinophils; Female; Gene Expression Regulation; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; RNA, Messenger

A correlation between stressful events experienced by the mother during pregnancy and progression of respiratory disease in descendants has been reported. Prenatal exposure to lipopolyssacharide (LPS) reduces allergic airway inflammation in mice offspring. In this study we investigated whether reduction of airways inflammation by maternal LPS exposure involves activation of inducible nitric oxide synthase (iNOS) at prenatal life. Since LPS also induces the release of TNF-alpha, and that this cytokine has been implicated in pathogenesis of asthma, we also evaluated whether TNF-alpha plays a role in the allergic airways inflammation by maternal LPS exposure. Pregnant rats were pretreated with the iNOS inhibitor aminoguanidine (50mg/rat per day; given from day 13 of gestation up to delivery) before exposure to LPS (7mug/kg, given at day 17 of gestation). At adult ages, female and male offspring were sensitized with ovalbumin (OVA), and 14 days later they were challenged with this allergen. OVA challenge in sensitized offspring increased markedly the eosinophil number in bronchoalveolar lavage (BAL) fluid at 48h compared with the non-sensitized group. However, the eosinophil number was largely reduced in offspring from maternal LPS exposure, irrespective of offspring gender. Maternal pretreatment with aminoguanidine fully reversed the eosinophil suppression by LPS. The maternal LPS exposure also reduced the eosinophil number in bone marrow and peripheral blood of offspring, but this was not affected by aminoguanidine. No differences in TNF-alpha levels in BAL fluid were found. In conclusion, our study shows that maternal LPS exposure markedly reduces allergic airways eosinophil recruitment in adult offspring by mechanisms possibly involving iNOS activation.

Topics: Animals; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Enzyme Activation; Eosinophils; Female; Guanidines; Leukocyte Count; Lipopolysaccharides; Male; Maternal Exposure; Maternal-Fetal Exchange; Nitric Oxide Synthase Type II; Ovalbumin; Pregnancy; Prenatal Exposure Delayed Effects; Pulmonary Eosinophilia; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

We developed a model of severe allergic inflammation and investigated the impact of airway and lung parenchyma remodelling on in vivo and in vitro respiratory mechanics. BALB/c mice were sensitized and challenged with ovalbumin in severe allergic inflammation (SA) group. The control group (C) received saline using the same protocol. Light and electron microscopy showed eosinophil and neutrophil infiltration and fibrosis in airway and lung parenchyma, mucus gland hyperplasia, and airway smooth muscle hypertrophy and hyperplasia in SA group. These morphological changes led to in vivo (resistive and viscoelastic pressures, and static elastance) and in vitro (tissue elastance and resistance) lung mechanical alterations. Airway responsiveness to methacholine was markedly enhanced in SA as compared with C group. Additionally, IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid were higher in SA group. In conclusion, this model of severe allergic lung inflammation enabled us to directly assess the role of airway and lung parenchyma inflammation and remodelling on respiratory mechanics.

Topics: Animals; Asthma; Disease Models, Animal; Dose-Response Relationship, Drug; Hyperplasia; Hypertrophy; Methacholine Chloride; Mice; Mice, Inbred BALB C; Microscopy, Electron; Muscarinic Agonists; Muscle, Smooth; Ovalbumin; Pulmonary Alveoli; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Respiratory Mechanics

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. The incidence of asthma is on the rise despite ongoing intensive asthma research. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found around Korea and has been used as a traditional anti-inflammatory medicine in liver diseases. We investigated suppressive effects of AIP1, a water-soluble carbohydrate fraction from A. iwayomogi on ovalbumin-induced allergic asthma in BALB/c mice and studied the possible mechanisms of its anti-allergic action. AIP1 significantly reduced pulmonary eosinophilia and Th2 cytokine expression in the lungs as well as serum IgE levels. Flow cytometric analysis of lung-infiltrating cells showed that the surface levels of CD11c and MHC II in CD11c+MHC II+ cells, potent dendritic cells, decreased in animals treated with AIP1. Expression of TNF-alpha, one of several proinflammatory cytokines released into the airway during episodes of asthma, was down-regulated by AIP1 injection, suggesting that reduced expression of TNF-alpha could account for the suppression of pulmonary eosinophilia and Th2-type cytokine production by AIP1.

Topics: Allergens; Animals; Artemisia; Asthma; Carbohydrates; Cytokines; Down-Regulation; Drugs, Chinese Herbal; Immunosuppressive Agents; Injections, Intraperitoneal; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells; Tumor Necrosis Factor-alpha

The Th1/Th2 paradigm has become an important issue in the pathogenesis of asthma, characterized by normal Th1 and elevated Th2 cytokine expression. Vitamin A deficiency (VAD) can produce a Th1 bias, whereas high-level dietary vitamin A can promote a Th2 bias. We used the OVA exposure mouse model to determine the contributions of vitamin A-deficient, control (4IU/g), and high-level vitamin A (250-IU/g) diets to the development of allergic airway inflammation and hyperresponsiveness. VAD reduced serum IgE and IgG1 responses, pulmonary eosinophilia, and the levels of IL-4 and IL-5 in bronchoalveolar lavage specimens, whereas the 250-IU/g diet increased serum IgE. Also, VAD blocked pulmonary hyperresponsiveness following methacholine challenge while the 250-IU/g diet exacerbated pulmonary hyperresponsiveness. In conclusion, VAD diminished and high-level dietary vitamin A enhanced the development of experimental asthma in this model system. These data suggest that excessive intake of vitamin A may increase the risk or severity of asthma in industrialized countries whereas vitamin A deficiency continues to increase mortality from infectious diseases in developing countries.

Topics: Animals; Asthma; Body Weight; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Diet; Disease Models, Animal; Female; Immunoglobulin E; Immunoglobulin G; Interleukin-4; Interleukin-5; Liver; Lung; Macrophages; Male; Mice; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Vitamin A; Vitamin A Deficiency

Janus kinase 3 (JAK-3) is a tyrosine kinase that has been shown to participate in the signaling of several cytokines that are believed to play a role in allergic airway disease, e.g. IL-2, 4 and 9. The current study describes the immunosuppressive effects of CP-690550, a novel, small molecule inhibitor of JAK-3, in a murine model of allergic pulmonary inflammation. In vitro, CP-690550 potently inhibited IL-4 induced upregulation of CD23 (IC(50)=57 nM) and class II major histocompatibility complex (MHCII) expression (IC(50)=71 nM) on murine B cells. Repeat aerosol exposure to ovalbumin in wild-type mice sensitized to the antigen resulted in preferential recruitment of Th2-like cells (IL-4+ and IL-5+) into bronchoalveolar lavage fluid (BAL). The importance of IL-4 in the development of pulmonary eosinophilia was supported by a marked (90%) reduction in the influx of these cells in IL-4KO mice similarly sensitized and ovalbumin exposed. Animals dosed with CP-690550 (15 mg/kg/d) during the period of antigen sensitization and boost demonstrated marked reductions in BAL eosinophils and levels of IL-13 and eotaxin following ovalbumin aerosol exposure. The JAK-3 inhibitor (1.5-15 mg/kg/d) also effectively reduced the same parameters when administered during the period of antigen challenge. In contrast, the calcineurin inhibitor tacrolimus (10 mg/kg) was effective only when administered during the period of ovalbumin aerosol exposure. These data support the participation of JAK-3 in processes that contribute to pulmonary eosinophilia in the allergic mouse model. CP-690550 represents an intriguing novel therapy for treatment of allergic conditions associated with airway eosinophilia including asthma and rhinitis.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; B-Lymphocytes; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Dose-Response Relationship, Drug; Eosinophils; Female; Flow Cytometry; Histocompatibility Antigens Class II; In Vitro Techniques; Interleukin-4; Janus Kinase 3; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Piperidines; Pulmonary Eosinophilia; Pyrimidines; Pyrroles; Receptors, IgE; Th2 Cells

Interleukin-5 (IL-5) has been suggested to be involved in the development of airway hyper-responsiveness (AHR). Both clinical and experimental investigations have shown strong correlation between the presence of eosinophils and AHR. In this study, we used small interfering RNA (siRNA) as an approach to inhibiting the expression of IL-5 and reducing AHR. siRNAs targeting IL-5 were characterized in vitro, and siRNA-expressing lentiviruses were administered intratracheally to OVA-sensitized BALB/c mice. AHR, cytokine levels, serum levels of OVA-specific antibodies and infiltration of inflammatory cells were analyzed to investigate the effects of siRNA in an OVA-induced murine model of asthma. Lentivirus-delivered siRNA targeting IL-5 efficiently moderated the characteristics of asthma, including AHR, cellular infiltration of lung tissues, eotaxin levels in the bronchoalveolar lavage fluid and IL-5 mRNA levels in lungs in the mouse model of asthma. However, there was no effect on OVA-specific IgE level. These data demonstrate that siRNA delivered by the lentiviral system is an efficacious therapeutic strategy for asthma.

Topics: 5' Untranslated Regions; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Line, Tumor; Chemokine CCL11; Genetic Therapy; Interleukin-5; Lentivirus; Lung; Mice; Mice, Inbred BALB C; Models, Animal; Ovalbumin; Pulmonary Eosinophilia; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

To experimentally examine the hygiene hypothesis, here we studied the effect of chlamydial infection on the development of allergic responses induced by OVA and the involvement of NK cells in this process using a mouse model of airway inflammation. We found that prior Chlamydia muridarum infection can inhibit airway eosinophilic inflammation and mucus production induced by allergen sensitization and challenge. The inhibition was correlated with an alteration of allergen-driven cytokine-producing patterns of T cells. We demonstrated that NK cells were activated following chlamydial infection, showing both cell expansion and cytokine secretion. The in vivo depletion of NK cells using anti-NK Ab before OVA sensitization and challenge partially abolished the inhibitory effect of chlamydial infection, which was associated with a partial restoration of Th2 cytokine production. In contrast, the adoptive transfer of NK cells that were isolated from infected mice showed a significant inhibitory effect on allergic responses, similar to that observed in natural infection. The data suggest that the innate immune cells such as NK cells may play an important role in infection-mediated inhibition of allergic responses.

Topics: Allergens; Animals; Cell Separation; Chlamydia muridarum; Cytokines; Female; Hypersensitivity; Killer Cells, Natural; Mice; Mice, Knockout; Mucus; Organic Anion Transport Protein 1; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells; Vascular Cell Adhesion Molecule-1

The present study is performed to investigate the inhibitory effects of Radix Adenophorae extract (RAE) on ovalbumin-induced asthma murine model. To study the anti-inflammatory and antiasthmatic effects of RAE, we examined the development of pulmonary eosinophilic inflammation and inhibitory effects of T cells in murine by RAE and cyclosporine A (CsA). We examined determination of airway hyperresponsiveness, flow cytometric analysis (FACS), enzyme-linked immunosorbent assay (ELISA), quantitative real time (PCR), hematoxylin-eosin staining, and Masson trichrome staining in lung tissue, lung weight, total cells, and eosinophil numbers in lung tissue. We demonstrated how RAE suppressed development on inflammation and decreased airway damage.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Cyclosporine; Cytokines; Female; Humans; Immunoglobulin E; Immunosuppressive Agents; Lung; Mice; Mice, Inbred C57BL; Ovalbumin; Plant Extracts; Pulmonary Eosinophilia

Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.

Topics: Administration, Inhalation; Administration, Oral; Airway Resistance; Animals; Asthma; Chronic Disease; Collagen; Disease Models, Animal; Elastic Tissue; Extracellular Matrix; Guinea Pigs; Immune Tolerance; Lung; Lung Compliance; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Time Factors

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a potent inhibitor of NF-kappaB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)(2)D(3) potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-beta are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 microg. Interestingly, coadministration of 10 ng of 1,25(OH)(2)D(3) with 100 microg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-beta and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-kappaB pathway using the selective IkappaB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-beta before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)(2)D(3) potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-beta play a crucial role in the effector phase of this mouse model.

Topics: Animals; Asthma; Cytokines; Desensitization, Immunologic; Disease Models, Animal; Immunoglobulin E; Immunoglobulin G; Interleukin-10; Lung; Male; Mice; Mice, Inbred BALB C; NF-kappaB-Inducing Kinase; Ovalbumin; Protein Serine-Threonine Kinases; Pulmonary Eosinophilia; Transforming Growth Factor beta; Vitamin D

Epidemiological studies suggest that infections with helminths protect from the development of asthma. Supporting this view is our published finding that infection with Nippostrongylus brasiliensis decreased ovalbumin-induced Th2 responses in the lung of mice.. To evaluate if N. brasiliensis excretory-secretory products also prevent the development of asthma.. Mice were immunized with ovalbumin/alum intraperitoneally in the absence or presence of helminthic products and then challenged intranasally with ovalbumin. Six days later, we analyzed if the mice developed Th2 responses in the lung.. The application of the helminthic products together with ovalbumin/alum during the sensitization period totally inhibited the development of eosinophilia and goblet cell metaplasia in the airways and also strongly reduced the development of airway hyperreactivity. Allergen-specific IgG1 and IgE serum levels were also strongly reduced. These findings correlated with decreased levels of IL-4 and IL-5 in the airways in product-treated animals. The suppressive effects on the development of allergic responses were independent of the presence of Toll-like receptors 2 and 4, IFN-gamma, and most important, IL-10. Interestingly, suppression was still observed when the helminthic products were heated or treated with proteinase K. Paradoxically, we found that strong helminth product-specific Th2 responses were induced in parallel with the inhibition of ovalbumin-specific responses.. Our results suggest that helminths suppress the development of asthma by secreting substances that modulate allergic responses without affecting the generation of helminth-specific Th2 immunity. The identification of these products may lead to the design of novel therapeutic intervention strategies for the treatment of asthma.

Topics: Animals; Bronchial Provocation Tests; Endopeptidase K; Female; Goblet Cells; Helminthiasis, Animal; Immunoglobulin E; Immunoglobulin G; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Nippostrongylus; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Th2 Cells

Pulmonary eosinophilia is one of the most consistent hallmarks of asthma. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell NADPH oxidase, which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether endothelial-derived NADPH oxidase modulates eosinophil recruitment in vivo, mice deficient in NADPH oxidase (CYBB mice) were irradiated and received wild-type hematopoietic cells to generate chimeric CYBB mice. In response to ovalbumin (OVA) challenge, the chimeric CYBB mice had increased numbers of eosinophils bound to the endothelium as well as reduced eosinophilia in the lung tissue and bronchoalveolar lavage. This occurred independent of changes in VCAM-1 expression, cytokine/chemokine levels (IL-5, IL-10, IL-13, IFNgamma, or eotaxin), or numbers of T cells, neutrophils, or mononuclear cells in the lavage fluids or lung tissue of OVA-challenged mice. Importantly, the OVA-challenged chimeric CYBB mice had reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice was restored by bypassing the endothelium with intratracheal administration of eosinophils. These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary for the eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in asthma therapies.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Chemokine CCL11; Chemokines, CC; Cytokines; Disease Models, Animal; Endothelium, Vascular; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; NADPH Oxidases; Ovalbumin; Pulmonary Eosinophilia; Vascular Cell Adhesion Molecule-1

Eosinophilic chemokines and histamine play distinct but important roles in allergic diseases. Inhibition of both eosinophilic chemokines and histamine, therefore, is an ideal strategy for the treatment of allergic inflammation, such as asthma, allergic rhinitis, and atopic dermatitis. YM-344484 was found to potently inhibit both the CCL11-induced Ca2+ influx in human CCR3-expressing cells (Kb=1.8 nM) and histamine-induced Ca2+ influx in histamine H1 receptor-expressing PC3 cells (Kb=47 nM). YM-344484 also inhibited the CCL11-induced chemotaxis of human CCR3-expressing cells (IC50=6.2 nM) and CCL11-induced eosinophil-derived neurotoxin release from human eosinophils (IC50=19 nM). Orally administered YM-344484 inhibited the increase in histamine-induced vascular permeability in mice (82% inhibition at a dose of 10 mg/kg) and the accumulation of eosinophils in a mouse asthma model (74% at a dose of 300 mg/kg). These results indicate that YM-344484, a novel and functional dual antagonist for chemokine CCR3 receptor and histamine H1 receptor, is an attractive candidate for development as a novel anti-allergic inflammation drug.

Topics: Animals; Anti-Allergic Agents; Anti-Inflammatory Agents; Asthma; Calcium Signaling; Capillary Permeability; Cell Line, Tumor; Chemotaxis; Disease Models, Animal; Dose-Response Relationship, Drug; Eosinophil-Derived Neurotoxin; Eosinophils; Female; Histamine; Histamine Antagonists; Humans; Mice; Mice, Inbred BALB C; Ovalbumin; Piperidines; Pneumonia; Pulmonary Eosinophilia; Pyridazines; Rats; Receptors, CCR3; Receptors, Chemokine; Receptors, Histamine H1; Skin; Transfection

In asthmatic mice, dexamethasone (30.0 mg/kg) was administered orally once daily on Days 24-27. One hour after dexamethasone on Day 25-27, the mice were exposed to ovalbumin aerosols. Twenty-eight days after the initial ovalbumin immunization, we found that dexamethasone reduced methacholine-induced pulmonary gas trapping and inhibited bronchoalveolar lavage eosinophils and neutrophils. However, five days after the last dose of dexamethasone and last ovalbumin aerosol exposure in other asthmatic mice, the airway obstructive response to methacholine was exacerbated in dexamethasone-treated mice compared to vehicle-treated mice on Day 32. Further, eosinophils, but not neutrophils, were still inhibited after cessation of dexamethasone. Thus, discontinuing dexamethasone worsened methacholine-induced pulmonary gas trapping of asthmatic mice in the absence of eosinophilic airway inflammation.

Topics: Administration, Oral; Airway Resistance; Animals; Anti-Asthmatic Agents; Anti-Inflammatory Agents; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Dexamethasone; Disease Models, Animal; Drug Administration Schedule; Eosinophils; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Pulmonary Eosinophilia; Recurrence

Intracellular signaling pathways that converge on Smad 3 are used by both TGF-beta and activin A, key cytokines implicated in the process of fibrogenesis. To determine the role of Smad 3 in allergen-induced airway remodeling, Smad 3-deficient and wild-type (WT) mice were sensitized to OVA and challenged by repetitive administration of OVA for 1 mo. Increased levels of activin A and increased numbers of peribronchial TGF-beta1(+) cells were detected in WT and Smad 3-deficient mice following repetitive OVA challenge. Smad 3-deficient mice challenged with OVA had significantly less peribronchial fibrosis (total lung collagen content and trichrome staining), reduced thickness of the peribronchial smooth muscle layer, and reduced epithelial mucus production compared with WT mice. As TGF-beta and Smad 3 signaling are hypothesized to mediate differentiation of fibroblasts to myofibroblasts in vivo, we determined the number of peribronchial myofibroblasts (Col-1(+) and alpha-smooth muscle actin(+)) as assessed by double-label immunofluorescence microscopy. Although the number of peribronchial myofibroblasts increased significantly in WT mice following OVA challenge, there was a significant reduction in the number of peribronchial myofibroblasts in OVA-challenged Smad 3-deficient mice. There was no difference in levels of eosinophilic airway inflammation or airway responsiveness in Smad 3-deficient compared with WT mice. These results suggest that Smad 3 signaling is required for allergen-induced airway remodeling, as well as allergen-induced accumulation of myofibroblasts in the airway. However, Smad 3 signaling does not contribute significantly to airway responsiveness.

Topics: Activins; Allergens; Animals; Azo Compounds; Bronchial Hyperreactivity; Cell Movement; Collagen; Eosine Yellowish-(YS); Fibroblasts; Lung; Methyl Green; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucus; Muscle, Smooth; Ovalbumin; Pulmonary Eosinophilia; Respiratory Mucosa; Signal Transduction; Smad3 Protein

There is little information about the involvement of galectin-9 (Gal-9) in allergic inflammation. Thus, we investigated the role of Gal-9 in asthma model guinea pigs.. Airway resistance (R(aw)) was measured using a double-flow plethysmograph system. Gal-9 expression in the lung was assessed by Western blot and immunohistochemistry. Eosinophil chemotactic activity was evaluated in a chamber containing a polyvinylpyrolidone-free membrane. Cell apoptosis was analyzed on a flowcytometry with propidium iodide.. In cloning guinea pig Gal-9 we identified three isoforms that differ only in the length of their linker peptides, just as with human Gal-9. Guinea pig Gal-9 was found to be a chemoattractant for eosinophils and to promote induction of apoptosis in sensitized but not non-sensitized T lymphocytes. In allergic airway hypersensitivity model, a low level of Gal-9 expression was observed in the nonsensitized/nonchallenged group, but upregulation was detected at 7 h after challenge and sustained up to 24 h. Such upregulation correlated with elevation of eosinophil peroxidase activity but not with increased R(aw).. The present results provide evidence that Gal-9 is not involved in airway hypersensitivity, but is partly involved in prolonged eosinophil accumulation in the lung.

Topics: Airway Resistance; Alternative Splicing; Amino Acid Sequence; Animals; Apoptosis; Asthma; Base Sequence; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Chemotaxis, Leukocyte; Disease Models, Animal; DNA, Complementary; Eosinophil Peroxidase; Eosinophils; Exons; Galectins; Guinea Pigs; Humans; Immunization; Lung; Male; Molecular Sequence Data; Open Reading Frames; Ovalbumin; Protein Isoforms; Pulmonary Eosinophilia; Recombinant Fusion Proteins; Respiratory Hypersensitivity; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity

1. Flavonoids are naturally occurring compounds that possess anti-allergic, anti-inflammatory, antiproliferative and anti-oxidant properties. In the present study, we investigated whether the flavonoid narirutin could reduce airway inflammation in ovalbumin (OVA)-sensitized/challenged NC/Nga mice, a model of allergic eosinophilic airway inflammation. 2. Mice were initially immunized intraperitoneally with OVA on Days 0 and 7 and then challenged with inhaled OVA on Days 14, 15 and 16. In addition, some mice received narirutin orally at doses of 0.1, 1 or 10 mg/kg bodyweight daily on Days 7-16. 3. At 10 mg/kg, but not 0.1 or 1 mg/kg, narirutin significantly diminished OVA-induced airway inflammation caused by infiltration of lung tissue with inflammatory and mucus-producing cells, as well as reduced eosinophil counts in the peripheral blood and bronchoalveolar lavage fluid (BALF), interleukin (IL)-4 levels in BALF and IgE levels in serum. 4. The mechanism of the anti-inflammatory effect of narirutin are likely to be associated with a reduction in the OVA-induced increases of IL-4 and IgE in a murine model of allergic eosinophilic airway inflammation. These findings suggest that narirutin may be an effective new tool in the treatment of bronchial asthma.

Topics: Aluminum Hydroxide; Animals; Anti-Asthmatic Agents; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; Disaccharides; Disease Models, Animal; Dose-Response Relationship, Drug; Eosinophils; Female; Flavanones; Immunoglobulin E; Inflammation; Interleukin-4; Interleukin-5; Leukocyte Count; Lung; Mice; Ovalbumin; Pulmonary Eosinophilia

Simendans are novel agents used in the treatment of decompensated heart failure. They sensitize troponin C to calcium and open ATP-sensitive potassium channels and have been shown to reduce cardiac myocyte apoptosis. The aim of the present study was to evaluate whether simendans reduce pulmonary eosinophilia and regulate eosinophil apoptosis. Bronchoalveolar lavage (BAL) eosinophilia was evaluated in ovalbumin-sensitized mice. Effects of simendans on apoptosis in isolated human eosinophils were assessed by relative DNA fragmentation assay, annexin V-binding, and morphological analysis. Dextrosimendan [(+)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl)hydrazono]propanedinitrile] reduced ovalbumin-induced BAL-eosinophilia in sensitized mice. Levosimendan [(-)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propanedinitrile] and dextrosimendan reversed interleukin (IL)-5-afforded survival of human eosinophils by inducing apoptosis in vitro. Even high concentrations of IL-5 were not able to overcome the effect of dextrosimendan. Dextrosimendan further enhanced spontaneous apoptosis as well as that induced by CD95 ligation, without inducing primary necrosis. Dextrosimendan-induced DNA fragmentation was shown to be dependent on caspase and c-Jun NH2-terminal kinase activation, whereas extracellular signal-regulated kinase, p38 mitogen-activated kinase, and ATP-sensitive potassium channels seemed to play no role in its actions. Taken together, our results show that simendans possess antieosinophilic activity and may be useful for the treatment of eosinophilic inflammation.

Topics: Animals; Apoptosis; Bronchoalveolar Lavage Fluid; Caspases; Cell Survival; Cells, Cultured; Disease Models, Animal; DNA Fragmentation; Enzyme Inhibitors; Eosinophils; fas Receptor; Humans; Hydrazones; Interleukin-5; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred BALB C; Ovalbumin; Potassium Channels; Pulmonary Eosinophilia; Pyridazines; Simendan

Allergic rhinitis is one of the most common causes of chronic cough. The characteristic feature of allergic rhinitis is eosinophilic nasal inflammation. This study was determined to find the relation between airway eosinophils and chemically-induced cough in guinea pigs with antigen-induced rhinitis at the early and late allergic phases. Forty animals were sensitized with ovalbumin (OVA) and divided into four separated groups. Four weeks later, the sensitized animals were either once or repeatedly (6 times at 7-day intervals) intranasally challenged with OVA to develop experimental allergic rhinitis. The control group was given saline. Cough was elicited by inhalation of citric acid aerosols and evaluated at 30 min (early phase) or 24 h (late phase) after the 1st or 6th nasal challenge (NC) in the sensitized animals. The citric acid-induced cough was significantly increased in the sensitized animals in the early allergic phase after the first and repeated NC compared with the control values [14(9-19) vs. 16(10-17) vs. 8(6-10); P=0.049], whereas there was no significant increase in the cough response tested in the late allergic phase. A correlation between the cough intensity and the number of eosinophils from nasal mucosa only (P=0.008) was found.

Topics: Aerosols; Animals; Bronchi; Citric Acid; Cough; Disease Models, Animal; Eosinophilia; Guinea Pigs; Larynx; Lung; Mice; Nasal Mucosa; Ovalbumin; Pulmonary Eosinophilia; Rhinitis, Allergic, Perennial; Severity of Illness Index; Time Factors; Trachea

Epidemiological and experimental studies have demonstrated an association between parasitic infections and the allergic diseases. A protective effect in asthma was shown in animals infected with helminths. The aim of this study was to determine the effect of Angiostrongylus costaricensis extract on inflammatory lung response to ovalbumin (OVA) in mice. Four BALB/c mice received A. costaricensis extract by intraperitoneal (i.p.) injection on the first day. Mice were immunised against OVA by i.p. injection on day (D) 5 and D12 and received a daily intranasal OVA challenge (40 microl) between the D19 and D21. On D23, we performed a bronchoalveolar lavage (BAL) on the mice. Four BALB/c mice (control group) were immunised against OVA using the same protocol, but did not receive parasite extract. Total cell counts (TCC) and differential cell counts were performed in BAL fluid samples. Eosinophil cell counts in BAL fluid were lower in the group that received A. costaricensis extract when compared with the control group (0.04 x 10(6) cells/ml and 0.01 x 10(6) cells/ml, respectively; p=0.04). TCC were not different between the groups studied. A. costaricensis extract in mice decreases eosinophilic response to OVA in BAL fluid.

Topics: Angiostrongylus; Animals; Antigens, Helminth; Bronchoalveolar Lavage Fluid; Eosinophils; Leukocyte Count; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Tissue Extracts

Mucosal tolerance can be induced by oral or nasal administration of soluble proteins and results in the suppression of cellular and/or humoral immune responses to the specific antigen.. To compare the effect of oral or nasal ovalbumin administration before, during or after immunization on the development of cellular and humoral immune responses by using a murine asthma model.. To induce lung allergic inflammation, animals were immunized twice with ovalbumin/aluminum hydroxide gel and challenged twice with ovalbumin. To induce tolerance, BALB/c mice received ovalbumin by the oral or nasal routes for 3 consecutive days. The ovalbumin administration was initiated before (day -7), during (day 0), or after immunization (day 7).. Airway eosinophilia, airway hyperreactivity, mucus hypersecretion, and cytokine production were suppressed when oral or nasal ovalbumin administration was initiated before immunization. Oral but not nasal ovalbumin exposure suppressed ovalbumin-specific nonanaphylactic IgG(1) antibodies, whereas both routes suppressed the production of anaphylactic IgG(1) and IgE antibodies. Mucosal ovalbumin administration at day 0 inhibited all T(H)2-mediated allergic parameters but not nonanaphylactic IgG(1) antibodies. Finally, ovalbumin exposure 7 days after immunization was still effective in suppressing lung allergy but not ovalbumin-specific anaphylactic IgG(1) and IgE antibodies.. We show that the effectiveness of mucosal tolerance depends on route and time and presents a hierarchical pattern of suppression in the following order: lung allergic responses > anaphylactic antibodies > ovalbumin-specific IgG(1).

Topics: Administration, Intranasal; Administration, Oral; Anaphylaxis; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Immune Tolerance; Immunization; Immunoglobulin E; Immunoglobulin G; Male; Mice; Mice, Inbred BALB C; Mucous Membrane; Ovalbumin; Pulmonary Eosinophilia; Time Factors

Allergic inflammation of the airways has a critical role in asthma development. We investigated a suppressive effect of verproside (3,4-dihydroxy catalpol) isolated from the extract of Pseudolysimachion longifolium on asthmatic parameters--such as immunoglobulin E (IgE) level, cytokine release, eosinophilia, airway hyperresponsiveness and mucus hypersecretion--in an OVA-sensitized/challenged mouse model. Verproside significantly inhibited the increase of total IgE and the cytokines IL-4 and IL-13 in plasma and bronchoalveolar lavage fluid, and also effectively suppressed airway hyperresponsiveness, eosinophilia and mucus hypersecretion in OVA-induced asthmatic mice. The efficacy of verproside was comparable to montelukast, an anti-asthmatic drug that is currently available. These results suggest that verproside could be a major marker in herbal medicines that are used for asthma treatment, and could also act as a lead for anti-asthmatic drugs.

Topics: Acetates; Animals; Anti-Asthmatic Agents; Asthma; Bronchoalveolar Lavage Fluid; Cyclopropanes; Disease Models, Animal; Female; Glucosides; Immunoglobulin E; Interleukin-13; Interleukin-4; Iridoid Glucosides; Iridoids; Lung; Methacholine Chloride; Mice; Mice, Inbred BALB C; Molecular Structure; Mucus; Ovalbumin; Plant Extracts; Pneumonia; Pulmonary Eosinophilia; Quinolines; Sulfides; Veronica

An increasing prevalence of allergic diseases, such as atopic dermatitis, allergic rhinitis and bronchial asthma, has been noted worldwide. Allergic asthma strongly correlates with airway inflammation caused by the unregulated production of cytokines secreted by allergen-specific type-2 T helper (Th2) cells. This study aims to explore the therapeutic effect of the airway gene transfer of IL-12, IL-10 and TGF-beta on airway inflammation in a mouse model of allergic asthma.. BALB/c mice were sensitized to ovalbumin (OVA) by intraperitoneal injections with OVA and challenged by nebulized OVA. Different cytokine gene plasmids or non-coding vector plasmids were instilled daily into the trachea up to one day before the inhalatory OVA challenge phase.. Intratracheal administration of IL-10, IL-12 or TGF-beta can efficiently inhibit antigen-induced airway hyper-responsiveness and is able to largely significantly lower the number of eosinophils and neutrophils in bronchoalveolar lavage fluid of ovalbumin (OVA) sensitized and challenged mice during the effector phase. Furthermore, the effect of IL-10 plasmids is more remarkable than any other cytokine gene plasmid. On the other hand, local administration of IL-4 gene plasmids before antigen challenge can induce severe airway hyper-responsiveness (AHR) and airway eosinophilia.. Our data demonstrated that anti-inflammatory cytokines, particularly IL-10, have the therapeutic potential for the alleviation of airway inflammation in murine model of asthma.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokines, CC; Disease Models, Animal; Female; Gene Transfer Techniques; Interleukin-10; Interleukin-12; Interleukin-4; Leukotriene B4; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Transforming Growth Factor beta

The effects of nitric oxide (NO) on allergic inflammation are controversial. In particular, the role of endothelial nitric oxide synthase (eNOS) in asthma remains uncertain. In the present study, we examined the effects of overexpression of eNOS on allergic inflammation using eNOS transgenic (eNOS-Tg) mice, in which eNOS protein is overexpressed in the vascular endothelium and airway epithelium. We found that eNOS-Tg mice showed a reduction of the asthmatic response to allergen challenge. Eosinophilic accumulation in the airspaces, eosinophilic activity, and bronchial responsiveness to acetylcholine were significantly attenuated in eNOS-Tg mice, as compared with wild-type mice following ovalbumin sensitization/challenge, even though the levels of circulating eosinophils were comparable in the wild-type and eNOS-Tg mice. The concentrations of eotaxin in the bronchoalveolar lavage fluid were significantly less in eNOS-Tg mice than in the wild-type mice. In addition, immunohistochemical analysis showed that the expressions of both intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 on the pulmonary endothelium of eNOS-Tg mice was decreased compared with the controls. These results suggest that chronic eNOS overexpression contributes to the suppression of allergic inflammation by reducing the production of eotaxin in the airspaces and/or the expression of adhesion molecules in the vascular endothelium.

Topics: Acetylcholine; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokines, CC; Enzyme Inhibitors; Eosinophil Peroxidase; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-4; Leukocyte Count; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Vascular Cell Adhesion Molecule-1

Development of new agents capable of regulating eosinophilic inflammation can uncover novel therapeutic approaches for the treatment of allergic diseases, such as asthma. Here, we evaluated the anti-allergic properties of an extract of the Brazilian Menispermaceae Cissampelos sympodialis, focusing on its effects on allergic eosinophilia. By studying two models of allergic inflammation, an asthma model and the allergic pleurisy in actively sensitized Balb/c mice, we observed that the oral pre-treatment with C. sympodialis reduced pleural eosinophil influx triggered by allergen challenge in a dose-dependent manner. The mechanism involved in C. sympodialis inhibitory effect appeared to be independent of a direct effect on eosinophil locomotory machinery, but depend on a blockage of eotaxin production, a key eosinophil chemoattractant with important roles in allergic reactions. C. sympodialis was also able to affect eosinophil activation, as attested by its ability of inhibiting formation of new cytoplasmic lipid bodies and the secretion of cysteinyl leukotrienes. The alkaloid warifteine isolated from the C. sympodialis extract represents an active component responsible for the anti-eosinophilic effects of the extract, since warifteine was able to reproduce C. sympodialis inhibitory effects on allergic eosinophilia and cysteinyl leukotrienes production. Of interest, C. sympodialis and warifteine post-treatments also effectively inhibited eosinophilic reaction observed after allergic challenge. Therefore, C. sympodialis/warifteine may be a promising anti-allergic therapy, inasmuch as it presents potent anti-eosinophil and anti-leukotrienes activities.

Topics: Alkaloids; Allergens; Animals; Anti-Allergic Agents; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokine CCL11; Chemokines, CC; Cissampelos; Disease Models, Animal; Eosinophils; Female; Humans; Leukocyte Count; Leukotrienes; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Plant Extracts; Plant Leaves; Pleurisy; Pulmonary Eosinophilia

Death receptor-6 (DR6), a member of the death domain-containing TNFR superfamily, is highly expressed in lymphoid tissues and regulated upon lymphocyte activation. Targeted disruption of DR6 results in enhanced CD4(+) T cell proliferation and T helper 2 (Th2) differentiation in vitro, whereas the in vivo role of DR6 in regulating Th2 cell differentiation and effector function remains largely unknown. In the current study, we used a Th2-skewed allergic airway inflammation model induced by ovalbumin (OVA) sensitization and challenge to compare the inflammatory response in the lung of both wild type (WT) and DR6(-/-) mice. DR6(-/-) mice were protected from the development of airway inflammation as evidenced by attenuated eosinophil accumulation and reduced mucus-producing cells in the lining airways of allergen-challenged animals. Consistent with these observations, a profound reduction of Th2 cytokine production (IL-5 and IL-13) was detected in the bronchoalveolar lavage fluid (BAL). Furthermore, a significant increase in the frequency of IFN-gamma secreting cells was observed in the DR6(-/-) mouse lungs after OVA challenge, which may account for the reduced pulmonary Th2 cytokine production. These data point to a critical role of DR6 in regulating airway inflammation in the OVA-induced mouse model of asthma.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Cytokines; Disease Models, Animal; Interferon-gamma; Mice; Mice, Knockout; Mucus; Ovalbumin; Pulmonary Eosinophilia; Receptors, Tumor Necrosis Factor; Th2 Cells

There is a growing body of evidence to suggest that CD8+ T lymphocytes contribute to local allergen-induced eosinophilic inflammation. Since bone marrow (BM) responses are intricately involved in the induction of airway eosinophilia, we hypothesized that CD8+ T lymphocytes, as well as CD4+ T lymphocytes, may be involved in this process.. Several approaches were utilized. Firstly, mice overexpressing interleukin-5 (IL-5) in CD3+ T lymphocytes (NJ.1638; CD3IL-5+ mice) were bred with gene knockout mice lacking either CD4+ T lymphocytes (CD4-/-) or CD8+ T lymphocytes (CD8-/-) to produce CD3IL-5+ knockout mice deficient in CD4+ T lymphocytes (CD3IL-5+/CD4-/-) and CD8+ T lymphocytes (CD3IL-5+/CD8-/-), respectively. Secondly, CD3+, CD4+ and CD8+ T lymphocytes from naïve CD3IL-5+ and C57BL/6 mice were adoptively transferred to immunodeficient SCID-bg mice to determine their effect on BM eosinophilia. Thirdly, CD3IL-5+, CD3IL-5+/CD8-/- and CD3IL-5+/CD4-/- mice were sensitized and allergen challenged. Bone marrow and blood samples were collected in all experiments.. The number of BM eosinophils was significantly reduced in CD3IL-5+/CD8-/- mice compared to CD3IL-5+ mice and CD3IL-5+/CD4-/- mice. Serum IL-5 was significantly higher in CD3IL-5+/CD4-/- mice compared to CD3IL-5+ mice but there was no difference in serum IL-5 between CD3IL-5+/CD4-/- and CD3IL-5+/CD8-/- mice. Adoptive transfer of CD8+, but not CD4+ T lymphocytes from naïve CD3IL-5+ and C57BL/6 mice restored BM eosinophilia in immunodeficient SCID-bg mice. Additionally, allergen challenged CD3IL-5+/CD8-/- mice developed lower numbers of BM eosinophils compared to CD3IL-5+ mice and CD3IL-5+/CD4-/- mice.. This study shows that CD8+ T lymphocytes are intricately involved in the regulation of BM eosinophilopoiesis, both in non-sensitized as well as sensitized and allergen challenged mice.

Topics: Adoptive Transfer; Animals; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Eosinophil Major Basic Protein; Eosinophils; Interleukin-5; Leukocyte Count; Leukopoiesis; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; T-Lymphocyte Subsets

Allergic airway disease can be refractory to anti-inflammatory treatment, whose cause is unclarified. Therefore, in the present experiment, we have tested the hypothesis that co-exposure to lipopolysacharide (Lps) and allergen results in glucocorticoid-resistant eosinophil airway inflammation and hyper-responsiveness (AHR). Ovalbumin (Ova)-sensitized BALB/c mice were primed with 10 microg intranasal Lps 24 h before the start of Ova challenges (20 min on 3 consecutive days). Dexamethasone (5 mg/kg/day) was given on the last 2 days of Ova challenges. AHR, cellular build-up, cytokine and nitrite concentrations of bronchoalveolar lavage fluid (BALF) and lung histology were examined. To assess the role of iNOS-derived NO in airway responsiveness, mice were treated with a selective inhibitor of this enzyme (1400W) 2 h before AHR measurements. More severe eosinophil inflammation and higher nitrite formation were found in Lps-primed than in non-primed allergized mice. After Lps priming, AHR and concentrations of T-helper type 2 cytokines in BALF were decreased, but still remained significantly higher than in controls. Eosinophil inflammation was partially, while nitrite production and AHR were observed to be largely dexamethasone resistant in Lps-primed allergized animals. 1400W effectively and rapidly diminished the AHR in Ova-sensitized and challenged mice, but failed to affect it after Lps priming plus allergization. In conclusion, Lps inhalation may exaggerate eosinophil inflammation and reduce responsiveness to anti-inflammatory treatment in allergic airway disease.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Cytokines; Dexamethasone; Drug Resistance; Female; Glucocorticoids; Imines; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Ovalbumin; Pulmonary Eosinophilia

Extracellular cyclophilins have been well described as chemotactic factors for various leukocyte subsets. This chemotactic capacity is dependent upon interaction of cyclophilins with the cell surface signaling receptor CD147. Elevated levels of extracellular cyclophilins have been documented in several inflammatory diseases. We propose that extracellular cyclophilins, via interaction with CD147, may contribute to the recruitment of leukocytes from the periphery into tissues during inflammatory responses. In this study, we examined whether extracellular cyclophilin-CD147 interactions might influence leukocyte recruitment in the inflammatory disease allergic asthma. Using a mouse model of asthmatic inflammation, we show that 1) extracellular cyclophilins are elevated in the airways of asthmatic mice; 2) mouse eosinophils and CD4+ T cells express CD147, which is up-regulated on CD4+ T cells upon activation; 3) cyclophilins induce CD147-dependent chemotaxis of activated CD4+ T cells in vitro; 4) in vivo treatment with anti-CD147 mAb significantly reduces (by up to 50%) the accumulation of eosinophils and effector/memory CD4+ T lymphocytes, as well as Ag-specific Th2 cytokine secretion, in lung tissues; and 5) anti-CD147 treatment significantly reduces airway epithelial mucin production and bronchial hyperreactivity to methacholine challenge. These findings provide a novel mechanism whereby asthmatic lung inflammation may be reduced by targeting cyclophilin-CD147 interactions.

Topics: Animals; Antibodies, Monoclonal; Asthma; Basigin; Blotting, Western; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage; CD4-Positive T-Lymphocytes; Cyclophilins; Disease Models, Animal; Eosinophils; Extracellular Fluid; Female; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mucins; Neutrophil Infiltration; Ovalbumin; Pneumonia; Pulmonary Eosinophilia

Overactivation of nuclear factor kappaB (NF-kappaB) orchestrates airway eosinophilia, but does not dampen airway hyperresponsiveness in asthma. NF-kappaB repression by arsenic trioxide (As2O3) contributes to apoptosis of eosinophils (EOS) in airways. Here we provide evidence that As2O3 abrogates allergen (OVA)-induced airway eosinophilia by modulating the expression of IkappaBalpha, an NF-kappaB inhibitory protein, and decreases the airway hyperresponsiveness.. Using a murine model of asthma, the airway hyperresponsiveness was conducted by barometric whole-body plethysmography. Airway eosinophilia, OVA-specific IgE in serum, and chemokine eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid were measured by lung histology, Diff-Quick staining, and ELISA. Chemokine-induced EOS chemotactic activity was evaluated using EOS chemotaxis assay. Electrophoretic mobility shift assay and Western blot analysis were performed to assess pulmonary NF-kappaB activation and IkappaBalpha expression, respectively.. As2O3 attenuated the allergen-induced serum IgE, chemokine expression of eotaxin and RANTES, and the EOS recruitment in bronchoalveolar lavage fluid, which is associated with an increased IkappaBalpha expression as well as a decreased NF-kappaB activation. Also, As2O3 suppressed the chemotaxis of EOS dose-dependently in vitro. Additionally, As2O3 significantly ameliorated the allergen-driven airway hyperresponsiveness, the cardinal feature underlying asthma.. These findings demonstrate an essential role of NF-kappaB in airway eosinophilia, and illustrate a potential dissociation between airway inflammation and hyperresponsiveness. As2O3 likely exerts its broad anti-inflammatory effects by suppression of NF-kappaB activation through augmentation of IkappaBalpha expression in asthma.

Topics: Allergens; Animals; Arsenic Trioxide; Arsenicals; Asthma; Bronchitis; Female; Mice; Mice, Inbred BALB C; NF-kappa B; Ovalbumin; Oxides; Pulmonary Eosinophilia; Respiratory Hypersensitivity

The p38 mitogen-activated protein kinase (MAPK) plays a critical role in the activation of inflammatory cells. Therefore, we investigated the antiinflammatory effects of a respirable p38alpha MAPK antisense oligonucleotide (p38alpha-ASO) in a mouse asthma model. A potent and selective p38alpha-ASO was characterized in vitro. Inhalation of aerosolized p38alpha-ASO using an aerosol chamber dosing system produced measurable lung deposition of ASO and significant reduction of ovalbumin (OVA-)-induced increases in total cells, eosinophils, and interleukin 4 (IL-4), IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and dose-dependent inhibition of airway hyperresponsiveness in allergen-challenged mice. Furthermore, inhaled p38alpha-ASO markedly inhibited OVA-induced lung tissue eosinophilia and airway mucus hypersecretion. Quantitative polymerase chain reaction analysis of bronchoalveolar lavage fluid cells and peribronchial lymph node cells showed that p38alpha-ASO significantly reduced p38alpha MAPK mRNA expression. Nose-only aerosol exposure of mice verified the p38alpha-ASO-induced inhibition of OVA-induced pulmonary eosinophilia, mucus hypersecretion, and airway hyperresponsiveness. None of the effects of the p38alpha-ASO were produced by a six-base mismatched control oligonucleotide. These findings demonstrate antisense pharmacodynamic activity in the airways after aerosol delivery and suggest that a p38alpha MAPK ASO approach may have therapeutic potential for asthma and other inflammatory lung diseases.

Topics: Administration, Inhalation; Aerosols; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 14; Mucus; Oligonucleotides, Antisense; Ovalbumin; Polymerase Chain Reaction; Pulmonary Eosinophilia

Asthma is a complex inflammatory disorder involving obstruction, constriction, oedema, remodelling and hyperresponsiveness of the airways. These effects are induced by a raft of mediators, many of which exert their actions by stimulating specific G-protein-coupled receptors linked to a signal transduction pathway involving the monomeric GTPase; rho, and a downstream effector; rho kinase. The aim of this study was to determine whether administration of a selective inhibitor of rho kinase, Y-27632, attenuates airway inflammation, bronchoconstriction and hyperresponsiveness in a murine model of acute allergic inflammation. Intranasal administration of Y-27632 caused a dose-dependent inhibition in the number of eosinophils recovered from bronchoalveolar lavage fluid of ovalbumin-sensitised and challenged (allergic) mice. These inhibitory effects of intranasal Y-27632 on pulmonary eosinophilia were accompanied by a significant inhibition of the development of airways hyperresponsiveness in allergic mice. In additional studies, intranasal Y-27632 inhibited methacholine-induced increases in airways resistance in a time-dependent manner. In conclusion, these findings indicate that activation of rho kinase contributes to bronchoconstriction and eosinophil trafficking in murine models of acute allergic airway inflammation and to the development of airway hyperresponsiveness.

Topics: Administration, Intranasal; Aluminum Hydroxide; Amides; Animals; Australia; Bronchial Hyperreactivity; Bronchoalveolar Lavage; Bronchoconstriction; Disease Models, Animal; Female; Hypersensitivity; Injections, Intraperitoneal; Intracellular Signaling Peptides and Proteins; Lung; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Protein Serine-Threonine Kinases; Pulmonary Eosinophilia; Pyridines; rho-Associated Kinases

Phosphoinositide 3-kinase (PI3K) exhibits broad functional effects in immune cells. We investigated the role of PI3K in allergic airway inflammation using LY294002, a specific PI3K inhibitor, in a mouse asthma model. BALB/c mice were sensitized and challenged with ovalbumin (OVA), and developed airway eosinophilia, mucus hypersecretion, elevation in cytokine levels, and airway hyperresponsiveness. Intratracheal administration of LY294002 significantly inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-5, IL-13, and eotaxin levels in bronchoalveolar lavage fluid. Histological studies show that LY294002 dramatically inhibited OVA-induced lung tissue eosinophilia and airway mucus production. In addition, LY294002 significantly suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. Western blot analysis of whole lung lysates shows that LY294002 markedly attenuated OVA-induced serine phosphorylation of Akt, a direct downstream substrate of PI3K. Taken together, our findings suggest that inhibition of PI3K signaling pathway can suppress T-helper type 2 (Th2) cytokine production, eosinophil infiltration, mucus production, and airway hyperresponsiveness in a mouse asthma model and may have therapeutic potential for the treatment of allergic airway inflammation.

Topics: Animals; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; Cell Movement; Chromones; Cytokines; Disease Models, Animal; Eosinophils; Lung; Male; Mice; Mice, Inbred BALB C; Morpholines; Mucus; Ovalbumin; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pulmonary Eosinophilia; Respiratory Hypersensitivity

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.. OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 microg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 microg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2'-deoxyuridine (BrdU). BrdU+ eosinophils and CD34+ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.. Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU+) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU+ eosinophils and CD34+ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4+ cells after allergen exposure.. Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.

Topics: Animals; Bone Marrow Cells; Bone Marrow Diseases; Interleukin-5; Interleukin-9; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity

The eotaxin chemokines have been implicated in allergen-induced eosinophil responses in the lung. However, the individual and combined contribution of each of the individual eotaxins is not well defined. We aimed to examine the consequences of genetically ablating eotaxin-1 or eotaxin-2 alone, eotaxin-1 and eotaxin-2 together, and CCR3. Mice carrying targeted deletions of these individual or combined genes were subjected to an OVA-induced experimental asthma model. Analysis of airway (luminal) eosinophilia revealed a dominant role for eotaxin-2 and a synergistic reduction in eotaxin-1/2 double-deficient (DKO) and CCR3-deficient mice. Examination of pulmonary tissue eosinophilia revealed a modest role for individually ablated eotaxin-1 or eotaxin-2. However, eotaxin-1/2 DKO mice had a marked decrease in tissue eosinophilia approaching the low levels seen in CCR3-deficient mice. Notably, the organized accumulation of eosinophils in the peribronchial and perivascular regions of allergen-challenged wild-type mice was lost in eotaxin-1/2 DKO and CCR3-deficient mice. Mechanistic analysis revealed distinct expression of eotaxin-2 in bronchoalveolar lavage fluid cells consistent with macrophages. Taken together, these results provide definitive evidence for a fundamental role of the eotaxin/CCR3 pathway in eosinophil recruitment in experimental asthma. These results imply that successful blockade of Ag-induced pulmonary eosinophilia will require antagonism of multiple CCR3 ligands.

Topics: Allergens; Animals; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokine CCL24; Chemokines, CC; Chemotaxis, Leukocyte; Leukocytes, Mononuclear; Lung; Macrophages; Mice; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Receptors, CCR3; Receptors, Chemokine

New therapeutic approaches for the treatment of allergic diseases can be aided by the development of agents capable of regulating eosinophilic leukocytes. Here, we evaluated the anti-allergic properties of a crude extract of the Brazilian bromeliaceae Nidularium procerum, focusing on its effects on allergic eosinophilia. By studying allergic pleurisy in actively sensitized C57Bl/6 mice, we observed that pretreatment with N. procerum (2 mg/kg; i.p.) reduced pleural eosinophil influx triggered by allergen challenge. N. procerum was also able to reduce lipid body numbers found within infiltrating eosinophils, indicating that N. procerum in vivo is able to affect both migration and activation of eosinophils. Consistently, pretreatment with N. procerum blocked pleural eosinophil influx triggered by PAF or eotaxin, key mediators of the development of allergic pleural eosinophilia. The effect of N. procerum was not restricted to eosinophils, since N. procerum also inhibited pleural neutrophil and mononuclear cell influx. Of note, N. procerum failed to alter the acute allergic reaction, characterized by mast cell degranulation, oedema, and cysteinyl leukotriene release. N. procerum also had direct effects on murine eosinophils, since it inhibited both PAF- and eotaxin-induced eosinophil chemotaxis on an in vitro chemotactic assay. Therefore, N. procerum may be a promising anti-allergic therapy, inasmuch as it presents potent anti-eosinophil activity.

Topics: Animals; Anti-Allergic Agents; Asthma; Bromeliaceae; Bronchoalveolar Lavage Fluid; Cell Movement; Cells, Cultured; Chemokine CCL11; Chemokines, CC; Disease Models, Animal; Dose-Response Relationship, Drug; Eosinophils; Female; Inclusion Bodies; Inflammation Mediators; Interleukin-13; Lipid Metabolism; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Ovalbumin; Plant Extracts; Plant Leaves; Platelet Activating Factor; Pleurisy; Pulmonary Eosinophilia; Spleen; Time Factors

Cytokines play an integral role in the coordination and persistence of allergic inflammatory processes and therefore represent prime targets for novel therapies in diseases such as asthma. Multiple attempts to generate low-molecular-weight cytokine inhibitors have failed, and the main attention has focused on the development of biological agents such as neutralizing antibodies. The present work describes a simple and effective method to induce the production of therapeutic anti-cytokine autoantibodies by active immunization against a modified endogenous cytokine.. Balb/c mice were subcutaneously injected with AutoVac TNF106, a recombinant murine TNF-alpha molecule containing a foreign immunogenic T helper epitope, and the induction of neutralizing anti-TNF-alpha autoantibodies was analysed. These mice were then sensitized with ovalbumin (OVA), and the effect of neutralizing anti-TNF-alpha autoantibodies on the allergen-induced airway inflammation was analysed.. AutoVac TNF106-immunized mice developed high titres of neutralizing anti-TNF-alpha autoantibodies, which were maintained for at least 4 weeks after the last booster injection. Mice vaccinated with AutoVac TNF106 and further immunized against OVA showed diminished TNF-alpha levels in the bronchoalveolar lavage (BAL) fluid after OVA challenge. Moreover, pretreatment with AutoVac TNF106 resulted in significantly reduced numbers of eosinophils and neutrophils in BAL fluid in response to single or multiple allergen exposure.. The induction of anti-TNF-alpha autoantibody production by the AutoVac TNF106 technology not only confirmed the role of TNF-alpha in the induction of allergic inflammation but also offers a novel approach to block the activity of cytokines in order to treat allergic inflammatory conditions.

Topics: Animals; Autoantibodies; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Cell Count; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Immunization; Immunoglobulin E; Immunotherapy; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Recombinant Proteins; Tumor Necrosis Factor-alpha

The NOD mouse has proved to be a relevant model of insulin-dependent diabetes mellitus, closely resembling the human disease. However, it is unknown whether this strain presents a general biastoward Th1-mediated autoimmunity or remains capable of mounting complete Th2-mediated responses. Here, we show that NOD mice have the capacity to develop a typical Th2-mediated disease, namely experimental allergic asthma. In contrast to what might have been expected, they even developed a stronger Th2-mediated pulmonary inflammatory response than BALB/c mice, a strain that shows a typical Th2 bias in this model. Thus, after allergen sensitization and intra-nasal challenge, the typical features of experimental asthma were exacerbated in NOD mice, including enhanced bronchopulmonary responsiveness, mucus production and eosinophilic inflammation in the lungs as well as specific IgE titers in serum. These hallmarks of allergic asthma were associated with increased IL-4, IL-5, IL-13 and eotaxin production in the lungs, as compared with BALB/c mice. Notwithstanding their quantitative and functional defect in NOD mice, CD1d-dependent NKT cells contribute to aggravate the disease, since in OVA-immunized CD1d(-/-) NOD mice, which are deficient in this particular T cell subset, airway eosinophilia was clearly diminished relative to NOD littermates. This is the first evidence that autoimmune diabetes-prone NOD mice can also give rise to enhanced Th2-mediated responses and might thus provide a useful model for the study of common genetic and cellular components, including NKT cells that contribute to both asthma and type 1 diabetes.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD1; Antigens, CD1d; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokines, CC; Cytokines; Diabetes Mellitus, Type 1; Immunoglobulin E; Interleukin-5; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Knockout; Mucus; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

We used a TCR-transgenic mouse to investigate whether Th2-mediated airway inflammation is influenced by Ag-specific CD4+CD25+ regulatory T cells. CD4+CD25+ T cells from DO11.10 mice expressed the transgenic TCR and mediated regulatory activity. Unexpectedly, depletion of CD4+CD25+ T cells before Th2 differentiation markedly reduced the expression of IL-4, IL-5, and IL-13 mRNA and protein when compared with unfractionated (total) CD4+ Th2 cells. The CD4+CD25--derived Th2 cells also expressed decreased levels of IL-10 but were clearly Th2 polarized since they did not produce any IFN-gamma. Paradoxically, adoptive transfer of CD4+CD25--derived Th2 cells into BALB/c mice induced an elevated airway eosinophilic inflammation in response to OVA inhalation compared with recipients of total CD4+ Th2 cells. The pronounced eosinophilia was associated with reduced levels of IL-10 and increased amounts of eotaxin in the bronchoalveolar lavage fluid. This Th2 phenotype characterized by reduced Th2 cytokine expression appeared to remain stable in vivo, even after repeated exposure of the animals to OVA aerosols. Our results demonstrate that the immunoregulatory properties of CD4+CD25+ T cells do extend to Th2 responses. Specifically, CD4+CD25+ T cells play a key role in modulating Th2-mediated pulmonary inflammation by suppressing the development of a Th2 phenotype that is highly effective in vivo at promoting airway eosinophilia. Conceivably, this is partly a consequence of regulatory T cells facilitating the production of IL-10.

Topics: Administration, Inhalation; Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; Chemokines; Cytokines; Immunophenotyping; Inflammation Mediators; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; Receptors, Interleukin-2; T-Lymphocyte Subsets; Th2 Cells

Respiratory viral infections in early childhood may interact with the immune system and modify allergen sensitization and/or allergic manifestations. In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic T helper (Th) 2 immune response, characterized by the production of IL-4, IL-5, and IL-13, and inflammatory infiltrates. However, it is unclear whether the RSV-enhanced respiratory allergic response is a result of non-specific virus-induced damage of the lung, or virus-specific immune responses.. In the present study we investigated whether RSV, pneumonia virus of mice (PVM) and influenza A virus similarly affect the allergic response.. BALB/c mice were sensitized and challenged with ovalbumin (OVA), and inoculated with virus during the challenge period. Pulmonary inflammation, lung cytokine mRNA responses, and IgE production in serum were assessed after the last OVA-challenge.. Like RSV, PVM enhanced the OVA-induced pulmonary IL-4, IL-5, and IL-13 mRNA expression, which was associated with enhanced perivascular inflammation. In addition, PVM increased the influx of eosinophils in lung tissue. In contrast, influenza virus decreased the Th2 cytokine mRNA expression in the lungs. However, like PVM, influenza virus enhanced the pulmonary eosinophilic infiltration in OVA-allergic mice.. The Paramyxoviruses RSV and PVM both are able to enhance the allergic Th2 cytokine response and perivascular inflammation in BALB/c mice, while the Orthomyxovirus influenza A is not.

Topics: Animals; Female; Hypersensitivity; Immunoglobulin E; Influenza A virus; Interleukin-13; Interleukin-4; Interleukin-5; Lung; Mice; Mice, Inbred BALB C; Murine pneumonia virus; Orthomyxoviridae Infections; Ovalbumin; Pneumovirus Infections; Pulmonary Eosinophilia; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; RNA, Messenger; Virus Diseases

Inflammatory infiltrates, airway hyper-responsiveness, goblet cell hyperplasia and subepithelial thickening are characteristic of chronic asthma. Current animal models of allergen-induced airway inflammation generally concentrate on the acute inflammation following allergen exposure and fail to mimic all of these features.. The aim of this study was to use a murine model of prolonged allergen-induced airway inflammation in order to characterize the cells and molecules involved in the ensuing airway remodelling. Moreover, we investigated whether remodelling persists in the absence of continued allergen challenge.. Acute pulmonary eosinophilia and airways hyper-reactivity were induced after six serial allergen challenges in sensitized mice (acute phase). Mice were subsequently challenged three times a week with ovalbumin (OVA) (chronic phase) up to day 55. To investigate the persistence of pathology, one group of mice were left for another 4 weeks without further allergen challenge (day 80).. The extended OVA challenge protocol caused significant airway remodelling, which was absent in the acute phase. Specifically, remodelling was characterized by deposition of collagen as well as airway smooth muscle and goblet cell hyperplasia. Importantly, these airway changes, together with tissue eosinophilia were sustained in the absence of further allergen challenge. Examination of cytokines revealed a dramatic up-regulation of IL-4 and tumour growth factor-beta1 during the chronic phase. Interestingly, while IL-4 levels were significantly increased during the chronic phase, levels of IL-13 fell. Levels of the Th1-associated cytokine IFN-gamma also increased during the chronic phase.. In conclusion, we have demonstrated that prolonged allergen challenge results in persistent airway wall remodelling.

Topics: Acute Disease; Allergens; Animals; Asthma; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Chronic Disease; Collagen; Female; Goblet Cells; Hyperplasia; Interferon-gamma; Interleukin-13; Interleukin-4; Mice; Mice, Inbred BALB C; Models, Animal; Muscle, Smooth; Ovalbumin; Pulmonary Eosinophilia; Respiratory System; Time Factors; Transforming Growth Factor beta

Whole-cell pertussis vaccines have been shown to selectively induce T helper 1 (Th1)-type responses in human and animals. In this study, we investigated whether whole-cell B. pertussis could inhibit allergic airway reactions in a murine model of asthma induced by ovalbumin (OVA). Systemic administration of whole-cell B. pertussis strongly inhibited allergic airway reactions such as eosinophil recruitment into the airway, lung inflammation, and airway hyperresponsiveness to methacholine. The inhibitory effect of whole-cell B. pertussis was mediated by chromosomal DNA and pretreatment of DNA with CpG methylase or DNase I resulted in a loss of the inhibitory effect. Treatment of animals with B. pertussis DNA significantly decreased the Th2 cytokine (interleukins IL-4 and IL-5) concentrations in the airways without increasing Th1 cytokines. These results suggest that B. pertussis DNA containing unmethylated CpG appears to be responsible for the inhibitory effect of whole cell B. pertussis on the allergic airway reactions through inhibition of the Th2 response.

Topics: Animals; Asthma; Bordetella pertussis; Bronchoconstrictor Agents; DNA, Bacterial; Down-Regulation; Male; Methacholine Chloride; Mice; Mice, Inbred C57BL; Ovalbumin; Pertussis Vaccine; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Th1 Cells; Th2 Cells

Histamine has a variety of airway actions and is considered to be an important mediator in asthma. This study examined the role of endogenous histamine in allergic airway eosinophil recruitment and hyperresponsiveness using L-histidine decarboxylase gene knockout mice. Histamine levels of the airways in L-histidine decarboxylase knockout mice were largely diminished compared with wild-type mice. Inhalation challenge with ovalbumin (OVA) in OVA-sensitized wild-type mice caused eosinophil accumulation in the lung as well as airway hyperresponsiveness to methacholine 3 days after the challenge. The eosinophil recruitment was significantly reduced in the knockout mice. In the bone marrow, the proliferation of eosinophils was enhanced after OVA challenge in the wild-type mice; however, the proliferation was significantly reduced in the knockout mice. The induction of P-selectin in the lung after OVA challenge was also inhibited in the knockout mice. In contrast, airway hyperresponsiveness was not suppressed in the knockout mice. These results suggest that endogenous histamine is involved in the accumulation of eosinophils into the airways after allergic challenge, possibly acting in the bone marrow and producing P-selectin in the airways. Furthermore, allergen-induced airway hyperresponsiveness appeared to occur independently of airway eosinophilia in our present model.

Topics: Administration, Inhalation; Airway Resistance; Animals; Asthma; Bone Marrow Cells; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eosinophilia; Eosinophils; Histamine; Histidine Decarboxylase; Immunoblotting; Interleukin-5; Leukocyte Count; Lung; Male; Methacholine Chloride; Mice; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Selectins; Time Factors

Asthma, a chronic inflammatory disease of the lung, is characterized by reversible airway obstruction and airway hyperresponsiveness (AHR), and is associated with increased production of IgE and Th2-type cytokines (IL-4, IL-5, and IL-13). Development of inflammation within the asthmatic lung depends on MHC class II-restricted Ag presentation, leading to stimulation of CD4(+) T cells and cytokine generation. Conventional MHC class II pathways require both MHC-associated invariant chain (Ii) and HLA-DM (H2-M in mice) chaperone activities, but alternative modes of Ag presentation may also promote in vivo immunity. In this study, we demonstrate that Ii(-/-) and H2-M(-/-) mice fail to develop lung inflammation or AHR following sensitization and challenge with OVA in a mouse model of allergic inflammation. To assess potentially distinct contributions by Ii chain isoforms to lung immunity, we also compared allergen-induced lung inflammation, eosinophilia, IgE production, and AHR in mice genetically altered to express either p31 Ii or p41 Ii isoform alone. Sole expression of either Ii isoform alone facilitates development of allergen-induced lung inflammation and eosinophilia. However, animals expressing only the p31 Ii isoform exhibit abrogated IgE and AHR responses as compared with p41 Ii mice in this model of allergen-induced lung inflammation, suggesting that realization of complete immunity within the lung requires expression of p41 Ii. These findings reveal a crucial role of Ii and H2-M in controlling the immune response within the lung, and suggest that p31 Ii and p41 Ii manifest nonredundant roles in development of immunity.

Topics: Adjuvants, Immunologic; Animals; Antigens, Differentiation, B-Lymphocyte; Bronchial Hyperreactivity; Cytokines; Histocompatibility Antigens Class II; Immunoglobulin E; Inflammation; Injections, Intraperitoneal; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Protein Isoforms; Pulmonary Eosinophilia

Allergic asthma, a chronic inflammatory disease of the airways, is characterized by the presence of T helper 2 cells and eosinophils in sputum, bronchoalveolar lavage, and mucosal biopsy specimens. Although the T helper 1-promoting cytokine, interleukin-12, is capable of inhibiting the T helper 2-driven asthma symptoms and bronchial responsiveness, the specific mechanisms underlying these interleukin-12 actions are unclear. The anti-allergic response to interleukin-12 is only partially dependent on interferon-gamma, which induces apoptosis by enhancing expression of Fas antigen. We therefore investigated in vivo whether the anti-allergic action of interleukin-12 is mediated through induction of apoptosis. C57BL/6 mice immunized to ovalbumin by intraperitoneal injection were challenged three times with an ovalbumin aerosol every second day for 7 days. Recombinant interleukin-12 was administered intravenously after the final challenge. After the last ovalbumin challenge, mice were examined for effects of interleukin-12 on inflammatory cell infiltration and apoptosis in the lung as detected by terminal deoxynucleotidyl transferase-mediated deoxyribonucleoside triphosphate nick end-labelling. Administration of interleukin-12 reduced ovalbumin-induced pulmonary eosinophilia (P < 0.01) and CD4+ T cell infiltration (P < 0.01). Moreover, treatment with interleukin-12 shortly after ovalbumin inhalation resulted in both increased interferon-gamma production (P < 0.01) and enhanced apoptosis of CD4+ T cells in allergic airway infiltrates (P < 0.05). These results suggest that the beneficial effects of interleukin-12 in asthma may include enhancement of apoptosis of CD4+ T cells in airways.

Topics: Animals; Apoptosis; Asthma; CD4-Positive T-Lymphocytes; Disease Models, Animal; Interferon-gamma; Interleukin-12; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Recombinant Proteins

Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5(-/-) mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5(-/-) mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5(-/-) mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5(-/-) mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4(+) T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.

Topics: Adoptive Transfer; Aerosols; Allergens; Animals; Bronchial Hyperreactivity; CD4-Positive T-Lymphocytes; Disease Models, Animal; Eosinophils; Interleukin-5; Intubation, Intratracheal; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mucus; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Respiratory Mucosa

IFN-beta has been shown to be effective as therapy for multiple sclerosis. Some reports attributed its beneficial effects to the capacity to induce a T(H)2 response. However, other studies have suggested that endogenous type I IFN might downregulate the allergic response in mice.. We sought to define the differential role of endogenous IFN-beta in controlling the development of allergic inflammation.. We assessed whether deletion of the gene encoding IFN-beta (IFNB) with knockout mice participated in the development of allergic response in ovalbumin (OVA)-sensitized and OVA-challenged mice.. OVA-sensitized and OVA-challenged mice with lack of the IFNB gene had more severe pulmonary inflammation with increased lung local response, including IL-4, IL-5, IL-13, IgE, eosinophilia, and goblet cells, than their litter mates (IFN-beta+/-), whereas no differences were observed in regard to local levels of IFN-gamma. Moreover, systemic response with IgE production is also enhanced. Lack of IFN-beta also results in significantly higher antigen-specific T cells, with higher levels of IL-4, IL-5, and IL-13, whereas no significant differences in IFN-gamma response could be observed. We have also detected a higher ratio of CD4+/CD8+ T cells and increased expression of B7.1/B7.2 on B cells and antigen-presenting cells in IFNB knockout mice.. These results demonstrate that IFN-beta plays an important role in immunoregulation of allergic response in mice. The stronger pulmonary inflammation could be a consequence of significantly expanded antigen-specific CD4+ T(H)2 cells as a result of efficient antigen presentation by antigen-presenting cells and hence increased production of IgE by B cells.

Topics: Animals; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Blood Cells; Bronchoalveolar Lavage Fluid; Epitopes; Immune System; Immunoglobulin E; Interferon-beta; Interleukins; Membrane Glycoproteins; Mice; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells; Up-Regulation

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.

Topics: Allergens; Animals; Bronchial Hyperreactivity; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Down-Regulation; Epitopes, T-Lymphocyte; Goblet Cells; Influenza A virus; Interferon-gamma; Interleukin-5; Lung; Lymphocyte Count; Lymphopenia; Metaplasia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nippostrongylus; Orthomyxoviridae Infections; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; Strongylida Infections; Th2 Cells; Up-Regulation

One of the characteristic features of allergic asthma is recruitment of large numbers of inflammatory cells including eosinophils and Th2 lymphocytes to the lung. This influx of inflammatory cells is thought to be a controlled and coordinated process mediated by chemokines and their receptors. It is thought that distinct, differential expression of chemokine receptors allows selective migration of T cell subtypes in response to the chemokines that bind these receptors. Th2 cells preferentially express CCR8 and migrate selectively to its ligand, CC chemokine ligand (CCL)1. We studied the role of the CCR8 ligand, CCL1, in the specific recruitment of Th2 cells and eosinophils to the lung in a murine model of allergic airway disease. We have demonstrated for the first time that CCL1 is up-regulated in the lung following allergen challenge. Moreover, a neutralizing Ab to CCL1 reduced eosinophil migration to the lung, but had no effect on recruitment of Th2 cells following allergen challenge. In addition, there was no change in airway hyperresponsiveness or levels of Th2 cytokines. In a Th2 cell transfer system of pulmonary inflammation, anti-CCL1 also failed to affect recruitment of Th2 cells to the lung following allergen challenge. Significantly, intratracheal instillation of rCCL1 increased recruitment of eosinophils but not Th2 cells to the lung in allergen-sensitized and -challenged mice. In summary, our results indicate that CCL1 is important for the pulmonary recruitment of eosinophils, rather than allergen-specific Th2 cells, following allergen challenge.

Topics: Adoptive Transfer; Allergens; Animals; Bronchial Hyperreactivity; Chemokine CCL1; Chemokines, CC; Chemotaxis, Leukocyte; Cytokines; Female; Immune Sera; Injections, Intravenous; Intubation, Intratracheal; Ligands; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Plethysmography, Whole Body; Pulmonary Eosinophilia; Respiratory Hypersensitivity; T-Lymphocyte Subsets; Th2 Cells

Tissue eosinophilia probably plays important roles in the pathophysiology of bronchial asthma and allergic disorders; however, this concept was challenged recently by controversial results in mouse models of bronchial asthma treated with anti-IL-5 Ab and the failure of anti-IL-5 therapy in humans. We have now used a unique model, IL-5 transgenic (TG) mice, to address a fundamental question: is airway eosinophilia beneficial or detrimental in the allergic response? After sensitization and challenge with OVA, IL-5 TG mice showed a marked airway eosinophilia. Surprisingly, these IL-5 TG mice showed lower airway reactivity to methacholine. Immunohistochemical analysis of the lungs revealed a marked peribronchial infiltration of eosinophils, but no eosinophil degranulation. In vitro, mouse eosinophils from peritoneal lavage fluids did not produce superoxide anion, but did produce an anti-inflammatory and fibrotic cytokine, TGF-beta 1. Indeed, the TGF-beta 1 levels in bronchoalveolar lavage fluid specimens from IL-5 TG mice directly correlated with airway eosinophilia (r = 0.755). Furthermore, anti-IL-5 treatment of IL-5 TG mice decreased both airway eosinophilia and TGF-beta 1 levels in bronchoalveolar lavage fluids and increased airway reactivity. Thus, in mice, marked eosinophilia prevents the development of airway hyper-reactivity during an allergic response. Overall, the roles of eosinophils in asthma and in animal models need to be addressed carefully for their potentially detrimental and beneficial effects.

Topics: Allergens; Animals; Antibodies, Monoclonal; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Eosinophils; Humans; Inflammation; Interleukin-5; Male; Mice; Mice, Inbred C3H; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes

In this study we have compared the therapeutic effect of the administration of immunostimulatory DNA sequences (ISS) with that of corticosteroids on the resolution of airway inflammation and airway hyperreactivity (AHR) in a mouse model. Mice which had already developed significant levels of eosinophilic airway inflammation 24 h after allergen challenge were then treated with either ISS or corticosteroids, and the effect on AHR and airway inflammation assessed 6 d later. ISS inhibited AHR as effectively as corticosteroids. Combination therapy with ISS and corticosteroids was more effective than monotherapy with either ISS or corticosteroids in inhibiting AHR. In ovalbumin-challenged mice, levels of bronchoalveolar lavage (BAL) eosinophils were significantly reduced with either ISS or corticosteroids. ISS induced significant levels of BAL interferon-gamma, whereas corticosteroids did not induce expression of BAL interferon-gamma. Both ISS and corticosteroids significantly reduced levels of interleukin-5 in BAL, as well as the number of Periodic Acid Schiff-positive airway epithelial cells. Corticosteroids, but not ISS, increased the number of eosinophils in regional mediastinal lymph nodes. Very few apoptotic peribronchial cells were noted following ovalbumin challenge as assessed by TUNEL assay. Corticosteroids, but not ISS, induced an increase in the small number of apoptotic peribronchial cells. The mechanism by which either ISS or corticosteroids inhibit AHR is likely to be mediated by distinct and shared cellular pathways. The combination of the shared and distinct anti-inflammatory pathways may account for the additive effect of ISS and corticosteroids on inhibiting AHR.

Topics: Administration, Inhalation; Adrenal Cortex Hormones; Animals; Anti-Inflammatory Agents; Apoptosis; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Dexamethasone; Disease Models, Animal; DNA; Drug Therapy, Combination; Eosinophils; Female; Inflammation; Injections, Intraperitoneal; Interferon-gamma; Interleukin-5; Lymph Nodes; Lymphocytes; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Treatment Outcome

We found that continuous eosinophilic inflammation after repeated antigen instillation into the nose was observed only in A/J mice, not in three other strains. Histologic analysis of tissues from A/J mice revealed features typical of airway remodeling, i.e., airway wall thickening and increased collagen depositions were observed after 12 weeks' antigen exposure. Persistent airway hyperresponsiveness (AHR) was observed in chronically antigen-exposed A/J mice. Eosinophilic inflammation, collagen deposition, and airway wall thickening were all less marked in BALB/c mice than in A/J mice, and no AHR was observed in the former strain. In C57BL/6 and C3H/HeJ mice, eosinophilic inflammation, airway wall thickening, and AHR were not observed at all, although slightly increased collagen deposition was observed. Thus, we found that these changes were strain-dependent. On the other hand, in A/J mice inhalational antigen challenge after ovalbumin/alum immunization led only to a transient increase in eosinophils and to less airway wall thickening, indicating the importance of the protocol used. Use of A/J mice and giving antigen by instillation via the nose is to be recommended for studies of the mechanisms underlying asthma. In particular, useful qualitative and quantitative information relating to the structural and histologic changes in the lungs may be obtainable using this model.

Topics: Administration, Intranasal; Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Eosinophils; Immunoglobulin E; Inflammation; Inhalation Exposure; Instillation, Drug; Lymphocytes; Mice; Mice, Inbred A; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Time Factors

The hygiene hypothesis suggests that a lack of bacterial infections would favour the development of allergic disease. For this reason, bacteria or their components can be used as potential treatment for allergic asthma. We investigated whether heat-killed Mycobacterium vaccae is either able to suppress the induction of allergic asthma or able to suppress already established allergic asthma.. Mice were sensitized with ovalbumin (OVA)/alum on days 0 and 14. Thereafter, mice were challenged on days 35, 39 and 42 by inhalation of either OVA or saline aerosols. M. vaccae-treated mice received an injection with 106, 107 or 108 CFU heat-killed M. vaccae on days 0 and 14 or 107 CFU on days 35 and 39. On day 43, the airway responsiveness of the mice to increasing concentrations of methacholine was assessed, blood was withdrawn to measure serum parameters, and lung lavage was performed to detect cytokines and inflammatory cell number.. Treatment of OVA-sensitized mice with 107 CFU M. vaccae either during sensitization or challenge suppresses airway hyper-responsiveness, airway eosinophilia and IL-5 production after OVA challenge. The increases in OVA-specific serum IgE and in IL-4 by respiratory challenges with OVA were only diminished after M. vaccae treatment (107 CFU) during sensitization.. Heat-killed M. vaccae prevents allergic and asthmatic manifestations in a mouse model and, more importantly, M. vaccae treatment during challenge suppresses features of asthma, which opens up possibilities for new therapeutic interventions.

Topics: Allergens; Animals; Asthma; Bacterial Vaccines; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Cytokines; Disease Models, Animal; Immunoglobulins; Leukocyte Count; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Mycobacterium; Ovalbumin; Pulmonary Eosinophilia

The Vgamma4(+) pulmonary subset of gammadelta T cells regulates innate airway responsiveness in the absence of alphabeta T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vgamma4(+) cells preferentially increased in number following airway challenge. Depletion of Vgamma4(+) cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vgamma1(+) cells had no effect on AHR, and depletion of all TCR-delta(+) cells was no more effective than depletion of Vgamma4(+) cells alone. Adoptively transferred pulmonary lymphocytes containing Vgamma4(+) cells inhibited AHR, but lost this ability when Vgamma4(+) cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vgamma4(+) cells, although cytokine-producing alphabeta T cells in the lung increased. These findings establish Vgamma4(+) gammadelta T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.

Topics: Adoptive Transfer; Animals; Bronchial Hyperreactivity; Cytokines; Female; Goblet Cells; Hyperplasia; Immunization; Injections, Intraperitoneal; Lymphocyte Count; Lymphocyte Depletion; Methacholine Chloride; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pulmonary Alveoli; Pulmonary Eosinophilia; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets; Up-Regulation

The prevalence of atopic asthma, a Th2-dependent disease, is reaching epidemic proportions partly due to improved hygiene in industrialized countries. There is an inverse correlation between the level of environmental endotoxin exposure and the prevalence of atopic sensitization. As dendritic cells (DC) have been implicated in causing sensitization to inhaled Ag, we studied the effect of endotoxin on Th2 development induced by bone marrow DC in vitro and by intratracheal injection in vivo, with particular emphasis on the role played by the polarizing cytokine IL-12. Bone marrow-derived DC stimulated with Escherichia coli O26:B6 LPS produced IL-12p70 for a limited period of time, after which production became refractory to further stimulation with CD40 ligand, a phenomenon previously called "exhaustion." The level of IL-12 production of DC did not correlate with Th1 development, as exhausted OVA-pulsed DC were still capable of shifting the cytokine pattern of responding OVA-specific Th cells toward Th1 in vitro and in vivo. When mice were first immunized by intratracheal injection of OVA-DC and subsequently challenged with OVA aerosol, prior in vitro stimulation of DC with LPS reduced the development of airway eosinophilia and Th2 cytokine production. Most surprisingly, the capacity of LPS to reduce Th2-dependent eosinophilic airway inflammation was IL-12-independent altogether, as IL-12p40 knockout DC had a similar reduced capacity to prime for Th2 responses. These results suggest that LPS reduces sensitization to inhaled Ag by reducing DC-driven Th2 development, but that IL-12 is not necessary for this effect.

Topics: Adoptive Transfer; Animals; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Division; Cell Movement; Cells, Cultured; Dendritic Cells; Epitopes, T-Lymphocyte; Female; Growth Inhibitors; Immunosuppressive Agents; Interleukin-12; Lipopolysaccharides; Lung; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Transgenic; Ovalbumin; Protein Subunits; Pulmonary Eosinophilia; T-Lymphocyte Subsets; Th2 Cells

To examine the influence of genetics on the OVA-induced allergic inflammatory response in lungs we compared rats that are genetically Th2-predisposed (Brown Norway, inbred) or not genetically predisposed (Sprague Dawley, outbred). Rats were sensitized with ovalbumin (OVA) and challenged four weeks later with OVA aerosol. Eighteen hours after challenge, lung tissue was studied for evaluation of numbers of eosinophils, neutrophils, macrophages and mast cells, as well as for expression of P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. From a separate portion of the pulmonary tissue, leucocytes were isolated to analyse numbers of IFNgamma and IL-4 producing cells (ELISPOT assay) and frequencies of T-cell subsets and B cells. We found increased numbers of eosinophils and neutrophils in the lung, an increased number of IL-4 producing cells in lung cell isolates and increased levels of serum (OVA- specific)-IgE in both rat strains. In addition, expression of E-selectin and ICAM-1 was up regulated in both rat strains whereas expression of VCAM-1 was only up regulated in the BN rat. Although the 'allergic' Th2 response to OVA was detectable in both rat strains, it was more pronounced in the BN rat than in the SD rat. However, the SD rat, which is not predisposed to respond in either a Th2 or Th1-like way, appeared capable of mounting an allergic response to OVA. This suggests that other factors than genetic contribute to allergic disease.

Topics: Animals; Asthma; Cell Adhesion Molecules; Cells, Cultured; Cytokines; Genetic Predisposition to Disease; Immunoglobulin E; Inflammation; Kinetics; Leukocytes; Lung; Lymphocyte Subsets; Male; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; Rats, Sprague-Dawley; Species Specificity

T(H)2 cells play a critical role in the pathogenesis of asthma, but the precise immunologic mechanisms that inhibit T(H)2 cell function in vivo are not well understood.. The purpose of our studies was to determine whether T cells producing IL-10 regulate the development of asthma.. We used gene therapy to generate ovalbumin-specific CD4 T-helper cells to express IL-10, and we examined their capacity to regulate allergen-induced airway hyperreactivity.. We demonstrated that the CD4 T-helper cells engineered to express IL-10 abolished airway hyperreactivity and airway eosinophilia in BALB/c mice sensitized and challenged with ovalbumin and in SCID mice reconstituted with ovalbumin-specific T(H)2 effector cells. The inhibitory effect of the IL-10-secreting T-helper cells was accompanied by the presence of increased quantities of IL-10 in the bronchoalveolar lavage fluid, was antigen-specific, and was reversed by neutralization of IL-10. Moreover, neutralization of IL-10 by administration of anti-IL-10 mAb in mice sensitized and challenged with ovalbumin seriously exacerbated airway hyperreactivity and airway inflammation.. Our results demonstrate that T cells secreting IL-10 in the respiratory mucosa can indeed regulate T(H)2-induced airway hyperreactivity and inflammation, and they strongly suggest that IL-10 plays an important inhibitory role in allergic asthma.

Topics: Allergens; Animals; Asthma; Bronchial Hyperreactivity; Cell Line; Cytokines; Dose-Response Relationship, Drug; Genetic Therapy; Inflammation; Interleukin-10; Methacholine Chloride; Mice; Mice, Inbred BALB C; Mice, SCID; Ovalbumin; Protein Engineering; Pulmonary Eosinophilia; T-Lymphocytes, Helper-Inducer; Th2 Cells; Transduction, Genetic

In this study we investigated the impact of chronic allergen exposure on airway inflammation and humoral responses in sensitized mice. We observed marked eosinophilia in the bronchoalveolar lavage, lung tissue, and peripheral blood after 2 wk of exposure. In contrast, eosinophilia was markedly reduced by 3 wk and completely resolved by 4 wk of exposure, despite the continued presence of Ag. Decreases in airway eosinophilia were associated with a robust humoral response. We observed that levels of OVA-specific IgE, IgG1, and IgG2a increased during the course of exposure. To assess whether continuous exposure to Ag impacts the ability of the lung to respond to subsequent Ag challenge, mice were exposed to either 2 or 4 wk of OVA in the context of GM-CSF. All groups were then rested for 28 days and exposed to OVA on three consecutive days. We observed a significant decrease in airway eosinophilia and IL-5 expression in the bronchoalveolar lavage and serum in mice initially exposed to 4 wk of OVA, compared with animals exposed to 2 wk only. However, in both groups expression of B7.2 on dendritic cells as well as CD25, CD69, and T1/ST2 on CD4(+) T cells was enhanced, suggesting immune activation. Delivery of rGM-CSF fully restored airway eosinophilia. This study shows that exposure to innocuous Ag alone does not lead to persistent eosinophilic airway inflammation, but rather to abrogated eosinophilia. This suppression can be reversed by GM-CSF.

Topics: Administration, Intranasal; Animals; Antigen-Presenting Cells; Antigens; Bronchoalveolar Lavage Fluid; Dose-Response Relationship, Immunologic; Drug Administration Schedule; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Immune Tolerance; Immunization; Immunization, Secondary; Immunoglobulin E; Immunoglobulin G; Immunologic Memory; Immunophenotyping; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Recombinant Proteins; T-Lymphocyte Subsets

Microbial heat shock proteins (hsp) have been associated with the generation and induction of Th1-type immune responses. We tested the effects of treatment with five different microbial hsp (Mycobacterium leprae, Streptococcus pneumoniae, Helicobacter pylori, bacillus Calmette-Guérin, and Mycobacterium tuberculosis) in a murine model of allergic airway inflammation and airway hyperresponsiveness (AHR). Mice were sensitized to OVA by i.p. injection and then challenged by OVA inhalation. Hsp were administered to each group by i.p. injection before sensitization and challenge. Sensitized and challenged mice developed increased serum levels of OVA-specific IgE with significant airway eosinophilia and heightened responsiveness to methacholine when compared with nonsensitized animals. Administration of M. leprae hsp prevented both development of AHR as well as bronchoalveolar lavage fluid eosinophilia in a dose-dependent manner. Treatment with M. leprae hsp also resulted in suppression of IL-4 and IL-5 production in bronchoalveolar lavage fluid, while IL-10 and IFN-gamma production were increased. Furthermore, M. leprae hsp treatment significantly suppressed OVA-specific IgE production and goblet cell hyperplasia/mucin hyperproduction. In contrast, treatment with the other hsp failed to prevent changes in airway responsiveness, lung eosinophilia, or cytokine production. Depletion of gamma/delta T lymphocytes before sensitization and challenge abolished the effect of M. leprae hsp treatment on AHR. These results indicate selective and distinctive properties among the hsp, and that M. leprae hsp may have a potential therapeutic role in the treatment of allergic airway inflammation and altered airway function.

Topics: Animals; Bacterial Proteins; Bronchi; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Movement; Cytokines; Down-Regulation; Epitopes; Female; Goblet Cells; Heat-Shock Proteins; Hyperplasia; Immunoglobulin E; Inflammation; Interleukin-4; Interleukin-5; Lung; Lymph Nodes; Lymphocyte Depletion; Mice; Mucins; Mycobacterium leprae; Ovalbumin; Pulmonary Eosinophilia; Receptors, Antigen, T-Cell, gamma-delta; Respiratory Mucosa; T-Lymphocyte Subsets

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

Topics: Allergens; Animals; Bronchial Hyperreactivity; Cell Line; Cell Movement; Crosses, Genetic; Female; Humans; Immunoglobulin E; Inflammation; Injections, Intraperitoneal; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; Receptor, PAR-2; Receptors, Thrombin

The mechanisms of airway hyperresponsiveness (AHR) are still poorly understood. In this study we have established a model of persistent AHR and eosinophilia and evaluated the prophylactic vs. therapeutic effects of dexamethasone on these parameters. Mice were immunised with ovalbumin (OVA) on day 0 and challenged intranasally on days 10, 11, 12 and 13 with OVA or phosphate buffer saline (PBS). Airway responsiveness to methacholine, measured 24-h post multiple intranasal OVA challenges, was significantly increased compared to time matched PBS-controls (P<0.05). AHR could be detected for up to 14 days after the last OVA challenge although the magnitude of the AHR had diminished by day 14 compared to day 1. OVA challenge of mice induced a significant airway eosinophilia at 24h (P<0.05); this persisted for 2 weeks after the challenge. Prophylactic treatment with dexamethasone (1mg x kg (-1)) reduced the OVA induced AHR, eosinophilia and mucus cell hyperplasia/metaplasia measured 24h post challenge. Therapeutic treatment, with dexamethasone (2 mg x kg(-1)), significantly inhibited established airway eosinophilia, measured at 72 h post OVA challenge, only when treatment was initiated at 24h but not 48 h after challenge. In contrast, AHR measured at 72 h post OVA challenge was significantly reduced when treatment was started at either 24 or 48 h post challenge. Our data shows that the immunization and challenge procedures employed resulted in a persistent type of AHR. Prophylactic intervention with steroids almost completely inhibited its development; however therapeutic intervention only partially resolved AHR.

Topics: Animals; Anti-Inflammatory Agents; Bronchial Hyperreactivity; Dexamethasone; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Time Factors

Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction and mortality. Th2 lymphocytes and eosinophils dominate the airway inflammatory infiltrate. We investigated the role of different lymphocyte subsets and their cytokines in the stimulation of mucus production using a system in which T cell receptor (TCR) transgenic CD4+ Th cells were generated in vitro, transferred into recipient mice and activated in the respiratory tract with inhaled antigen. Th2 cells induced mucus production and eosinophilic inflammation, while mice that received Th1 cells exhibited airway inflammation without mucus. Th1 cells failed to stimulate mucus due to the inhibitory effects of interferon (IFN)gamma. Mucus was induced by Th2 cells in the absence of interleukin (IL)4, IL5, eosinophils and mast cells, but not without IL4R alpha signalling. Th2 cells lacking IL13 could not stimulate mucus production, despite the presence of airway inflammation. IL9 also stimulates mucus through an IL13-mediated pathway. Using bone marrow chimeras we show that IL13 acts on structural cells in the lung, most likely by direct stimulation of epithelial cells, and not through intermediate inflammatory cells. In asthma, airway inflammation with CD4+ Th2 cells stimulates mucus production by a single pathway mediated by IL13.

Topics: Administration, Inhalation; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cells, Cultured; Exocytosis; Gene Expression Regulation; Immunization; Interferon-gamma; Interferons; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Interleukin-4; Interleukin-5; Interleukin-9; Mast Cells; Mice; Mice, Transgenic; Models, Animal; Mucins; Mucus; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; Radiation Chimera; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin; Receptors, Interleukin-13; Receptors, Interleukin-4; Respiratory System; Signal Transduction; Th1 Cells; Th2 Cells

CD4(+) T cells have a critical role in the development of allergic pulmonary inflammation, including the recruitment of eosinophils to the airway lumen and interstitium. The expression of interleukin (IL)-5 by CD4(+) cells has, in particular, often been lionized as the central link between allergic inflammation and the concomitant expansion or recruitment of eosinophils. The mechanism(s) by which CD4(+) T cells mediates eosinophil recruitment was assessed with gene knockout mice deficient for T cells or T cell subtypes and a unique IL-5 transgenic mouse (line NJ.1726) that constitutively overexpresses this cytokine in the lung epithelium. Pulmonary IL-5 expression is significantly attenuated in T cell- and CD4(+) but not CD8(+) cell-deficient animals, suggesting an obvious explanation for the lack of eosinophils in the lungs of T cell-deficient and CD4(-/-) mice. However, although the constitutive expression of IL-5 in the lung epithelium of NJ.1726 mice elicited an eosinophilia in the airway lumen of both naive and ovalbumin-treated mice, in the absence of CD4(+) cells, allergen-mediated eosinophil recruitment to the bronchoalveolar lavage fluid was abolished. Moreover, intranasal instillation of the potent eosinophil-specific chemokine eotaxin-2 was incapable of eliciting eosinophil recruitment in naive and ovalbumin-treated NJ.1726 CD4(-/-) mice, suggesting that eosinophil trafficking during allergic inflammatory responses is a consequence of a CD4(+) cell-mediated event(s) in addition to IL-5 expression and the establishment of a pulmonary chemokine gradient.

Topics: Administration, Intranasal; Allergens; Animals; CD4-Positive T-Lymphocytes; Chemokine CCL24; Chemokines, CC; Chemotaxis, Leukocyte; Eosinophils; Epithelium; Immunization; Interleukin-5; Lung; Mice; Mice, Knockout; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia

PGD(2) is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD(2) in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD(2), but not PGE(2), substantially increased in the lungs of WT and TG mice with greater PGD(2) increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-gamma in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-gamma than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD(2) caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD(2) plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Chemokines; Cytokines; Humans; Immunoenzyme Techniques; Intramolecular Oxidoreductases; Leukocyte Count; Lipocalins; Lung; Mice; Mice, Transgenic; Ovalbumin; Prostaglandin D2; Pulmonary Eosinophilia; Receptors, Immunologic; Receptors, Prostaglandin; RNA, Messenger; Th2 Cells; Up-Regulation

Release of human lung mast cell tryptase may be important in the pathophysiology of asthma. We examined the effect of the reversible, nonelectrophilic tryptase inhibitor MOL 6131 on airway inflammation and hyper-reactivity in a murine model of asthma. MOL 6131 is a potent selective nonpeptide inhibitor of human lung mast cell tryptase based upon a beta-strand template (K(i) = 45 nM) that does not inhibit trypsin (K(i) = 1,061 nM), thrombin (K(i) = 23, 640 nM), or other serine proteases. BALB/c mice after i.p. OVA sensitization (day 0) were challenged intratracheally with OVA on days 8, 15, 18, and 21. MOL 6131, administered days 18-21, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 22; intranasal delivery (10 mg/kg) had a greater anti-inflammatory effect than oral delivery (10 or 25 mg/kg) of MOL 6131. MOL 6131 reduced total cells and eosinophils in bronchoalveolar lavage fluid, airway tissue eosinophilia, goblet cell hyperplasia, mucus secretion, and peribronchial edema and also inhibited the release of IL-4 and IL-13 in bronchoalveolar lavage fluid. However, tryptase inhibition did not alter airway hyper-reactivity to methacholine in vivo. These results support tryptase as a therapeutic target in asthma and indicate that selective tryptase inhibitors can reduce allergic airway inflammation.

Topics: Animals; Asthma; Bridged Bicyclo Compounds, Heterocyclic; Bronchial Diseases; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Movement; Cytokines; Eosinophils; Humans; Inflammation; Lung; Mice; Mice, Inbred BALB C; Models, Molecular; Mucus; Ovalbumin; Piperidines; Pulmonary Edema; Pulmonary Eosinophilia; Serine Endopeptidases; Serine Proteinase Inhibitors; Tryptases; Vascular Cell Adhesion Molecule-1

Tyrosine kinases of the Janus kinase (Jak) family transduce signals from the type I and type II cytokine receptors. Jak3 is unique in this family because its expression must be induced and is predominantly limited to cells of the lymphoid and myeloid lineages. Deficient expression of Jak3 interferes with normal development and function of T, B, and NK cells. Using irradiated Jak3-deficient (Jak3-/-) mice reconstituted with normal bone marrow (Jak3-/-chimeric mice), we have investigated possible actions of Jak3 outside of the hematopoietic system. We show that efficient recruitment of inflammatory cells to the airways of OVA-sensitized mice challenged with aerosolized OVA requires the expression of Jak3 in radioresistant nonhematopoietic cells. Failure to develop eosinophil-predominant airway inflammation in Jak3-/- chimeric mice is not due to failure of T cell sensitization, because Jak3-/- chimeric mice showed delayed-type hypersensitivity responses indistinguishable from wild-type chimeric mice. Jak3-/- chimeric mice, however, express less endothelial-associated VCAM-1 after airway Ag challenge. Given the key role of VCAM-1 in recruitment of Th2 cells and eosinophils, our data suggest that Jak3 in airway-associated endothelial cells is required for the expression of eosinophilic airway inflammation. This requirement for nonhematopoietic expression of Jak3 represents the first demonstration of a physiological function of Jak3 outside of the lymphoid lineages.

Topics: Animals; Cell Movement; Chimera; Eosinophils; Hematopoietic Stem Cells; Hypersensitivity, Delayed; Immunoglobulins; Immunohistochemistry; Inflammation; Janus Kinase 3; Lung; Mice; Mice, Knockout; Microscopy, Fluorescence; Ovalbumin; Protein-Tyrosine Kinases; Pulmonary Eosinophilia; Th2 Cells; Vascular Cell Adhesion Molecule-1

Pretreatment with mycobacterial Ags has been shown to be effective in preventing allergic airway inflammation from occurring in a mouse model. Because most asthmatics are treated after the development of asthma, it is crucial to determine whether mycobacterial Ags can reverse established allergic airway inflammation in the presensitized state. Our hypothesis, based upon our previous findings, is that mycobacteria treatment in presensitized mice will suppress the allergic airway inflammation with associated clinical correlates of established asthma, with the noted exception of factors associated with the early allergic response (EAR). BALB/c mice sensitized and challenged with OVA were evaluated for pulmonary functions during both the EAR and late allergic response, and airway hyperresponsiveness to methacholine. Following this, sensitized mice were randomized and treated with placebo or a single dose (1 x 10(5) CFUs) of bacillus Calmette-Guérin (BCG) or Mycobacterium vaccae via nasal or peritoneal injection. One week later, the mice were rechallenged with OVA and methacholine, followed by bronchoalveolar lavage (BAL) and tissue collection. Mice treated with intranasal BCG were most significantly protected from the late allergic response (p < 0.02), airway hypersensitivity (p < 0.001) and hyperreactivity (p < 0.05) to methacholine, BAL (p < 0.05) and peribronchial (p < 0.01) eosinophilia, and BAL fluid IL-5 levels (p < 0.01) as compared with vehicle-treated, sensitized controls. Intranasal M. vaccae treatment was less effective, suppressing airway hypersensitivity (p < 0.01) and BAL eosinophilia (p < 0.05). No changes were observed in the EAR, BAL fluid IL-4 levels, or serum total and Ag-specific IgE. These data suggest that mycobacterial Ags (BCG>>M. vaccae) are effective in attenuating allergic airway inflammation and associated changes in pulmonary functions in an allergen-presensitized state.

Topics: Administration, Intranasal; Airway Resistance; Animals; Antigens, Bacterial; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Female; Hypersensitivity, Immediate; Immunoglobulin E; Interferon-gamma; Interleukin-5; Kinetics; Leukocyte Count; Lung; Mice; Mice, Inbred BALB C; Mycobacterium; Mycobacterium bovis; Ovalbumin; Pulmonary Eosinophilia

A detailed analysis has been carried out of the correlation between the signals detected by MRI in the rat lung after allergen or endotoxin challenge and parameters of inflammation determined in the broncho-alveolar lavage (BAL) fluid. MRI signals after allergen correlated highly significantly with the BAL fluid eosinophil number, eosinophil peroxidase activity and protein concentration. Similar highly significant correlations were seen when the anti-inflammatory glucocorticosteroid, budesonide, manifested against allergen. In contrast, following endotoxin challenge, mucus was the sole BAL fluid parameter that correlated significantly with the long lasting signal detected by MRI. Since edema is an integral component of pulmonary inflammation, MRI provides a noninvasive means of monitoring the course of the inflammatory response and should prove invaluable in profiling anti-inflammatory drugs in vivo. Further, the prospect of noninvasively detecting a sustained mucus hypersecretory phenotype in the lung brings an important new perspective to models of chronic obstructive pulmonary diseases.

Topics: Allergens; Animals; Anti-Inflammatory Agents; Asthma; Bronchoalveolar Lavage Fluid; Budesonide; Eosinophil Peroxidase; Inflammation; Lipopolysaccharides; Lung; Magnetic Resonance Imaging; Male; Mucus; Ovalbumin; Peroxidases; Pulmonary Disease, Chronic Obstructive; Pulmonary Edema; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; Respiration

The complexity and magnitude of interactions leading to the selective infiltration of eosinophils in response to inhaled allergens are formidable obstacles to a larger understanding of the pulmonary pathology associated with allergic asthma. This study uses knockout mice to demonstrate a novel function for the heterotrimeric G protein, G(q), in the regulation of pulmonary eosinophil recruitment. In the absence of G(q) signaling, eosinophils failed to accumulate in the lungs following allergen challenge. These studies demonstrate that the inhibition of eosinophil accumulation in the airways is attributed to the failure of hemopoietically derived cells to elaborate GM-CSF in the airways. The data suggest that activation of a G(q)-coupled receptor(s) on resident leukocytes in the lung elicits expression of GM-CSF, which, in turn, is required for allergen-induced pulmonary eosinophilia, identifying a novel pathway of eosinophil-associated effector functions leading to pulmonary pathology in diseases such as asthma.

Topics: Aerosols; Allergens; Animals; Bone Marrow Cells; Chemotactic Factors, Eosinophil; Chemotaxis, Leukocyte; Cytokines; Female; Granulocyte-Macrophage Colony-Stimulating Factor; GTP-Binding Protein alpha Subunits, Gq-G11; Heterotrimeric GTP-Binding Proteins; Injections, Intraperitoneal; Intubation, Intratracheal; Leukocyte Count; Lymphocyte Activation; Mice; Mice, Knockout; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; Signal Transduction; T-Lymphocyte Subsets; Th2 Cells

Our previous studies, as well as those of others, have demonstrated that local or systemic Mycobacterium bovis bacille Calmette-Guérin (BCG) infection can inhibit de novo allergen-induced asthma-like reactions, but the effect of this infection on established allergic responses is unknown. The aim of this study was therefore to examine the effect of mycobacterial infection on established allergy in a murine model of asthma-like reaction. Mice were sensitized with ovalbumin (OVA) in alum followed by infection with BCG and subsequent intranasal challenge with the same allergen. In some experiments, mice were sensitized with OVA followed by intranasal challenge with OVA and then given BCG infection with subsequent rechallenge with OVA. Mice without BCG infection but treated with OVA in the same manner, were used as a control. The mice were examined for immunoglobulin E (IgE) response and eosinophilic inflammation, mucus production, cytokine/chemokine patterns and adhesion molecule expression in the lung. The results showed that postallergen BCG infection suppressed the established airway eosinophilia and mucus overproduction, but not IgE responses. The inhibition of asthma-like reactions by BCG infection was correlated with a shift of allergen-driven cytokine production pattern and, more interestingly, with a dramatic decrease of vascular cell adhesion molecule-1 (VCAM-1) expression in the lung. These findings suggest that intracellular bacterial infection can inhibit established allergic responses via alteration of local cytokine production and the expression of adhesion molecules.

Topics: Animals; Antigens; Asthma; Cytokines; Endothelium, Vascular; Female; Immune Tolerance; Immunoglobulin E; Mice; Mice, Inbred C57BL; Mucus; Mycobacterium bovis; Mycobacterium Infections; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells; Vascular Cell Adhesion Molecule-1

LPS potently induces dendritic cell maturation and the production of proinflammatory cytokines, such as IL-12, by activation of Toll-like receptor 4 (TLR4). Since IL-12 is important for the generation and maintenance of Th1 responses and may also inhibit Th2 cell generation from naive CD4 T cell precursors, it has been inferred that TLR4 signaling would have similar effects via the induction of IL-12 secretion. Surprisingly, we found that TLR4-defective mice subjected to sensitization and pulmonary challenge with a protein allergen had reductions in airway inflammation with eosinophils, allergen-specific IgE levels, and Th2 cytokine production, compared with wild-type mice. These reduced responses were attributable, at least in part, to decreased dendritic cell function: Dendritic cells from TLR4-defective mice expressed lower levels of CD86, a costimulatory molecule important for Th2 responses. They also induced less Th2 cytokine production by antigenically naive CD4 T cells in vitro and mediated diminished CD4 T cell Ag-specific pulmonary inflammation in vivo. These results indicate that TLR4 is required for optimal Th2 responses to Ags from nonpathogenic sources and suggest a role for TLR4 ligands, such as LPS derived from commensal bacteria or endogenously derived ligands, in maturation of the innate immune system before pathogen exposure.

Topics: Allergens; Animals; Antigens, CD; Asthma; Cells, Cultured; Cytokines; Dendritic Cells; Drosophila Proteins; Female; Histocompatibility Antigens Class II; Immunoglobulin E; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mutation; Ovalbumin; Pulmonary Eosinophilia; Receptors, Cell Surface; Th2 Cells; Toll-Like Receptor 4; Toll-Like Receptors

Allergic airway eosinophilia is suppressed by cysteinyl leukotriene (CysLT) receptor (CysLT1 receptor) antagonists in several species including humans and guinea-pigs, suggesting that CysLTs are directly or indirectly involved in induction of the response.. We examined the effect of CysLT antagonists (pranlukast and MCI-826) on antigen inhalation-induced eosinophilia in peripheral blood and lung, and on IL-5 activity in serum during late increase of airway resistance (late asthmatic response, LAR) in sensitized guinea-pigs.. Guinea-pigs inhaled ovalbumin (OVA) + Al(OH)3 and OVA mists alternately for sensitization and challenge, respectively, once every 2 weeks. At the fifth challenge, the effects of CysLT antagonists and an anti-IL-5 antibody (TRFK-5) on the occurrence of LAR, and blood and lung eosinophilia, which appeared at 5 h after challenge, were examined. The time-course of IL-5 activity in the serum after the challenge was evaluated by measuring in vitro 'eosinophil survival prolongation activity'. The influence of CysLT antagonists on IL-5 activity was assessed.. CysLT antagonists and TRFK-5 completely abolished blood and lung eosinophilia. LAR was suppressed by both MCI-826 and TRFK-5 by 40-50%. Sera obtained from sensitized, challenged animals 3 h and 4 h after challenge induced an obvious prolongation of eosinophil survival. The activity of the sera was completely neutralized by prior exposure to TRFK-5, suggesting that it reflected IL-5 activity. Increased IL-5 activity in the serum was inhibited by both pranlukast and MCI-826 by over 90%.. CysLTs produced after antigen provocation sequentially induced IL-5 production from some immune component cells via CysLT1 receptor activation. Thus, it is likely that CysLTs indirectly cause antigen-induced eosinophilia through IL-5 production.

Topics: Animals; Asthma; Cell Survival; Chromones; Cysteine; Eosinophilia; Guinea Pigs; Interleukin-5; Kinetics; Leukotriene Antagonists; Leukotrienes; Male; Membrane Proteins; Ovalbumin; Pulmonary Eosinophilia; Receptors, Leukotriene; Thiazoles

Correcting Th1/Th2 imbalance with administration of IL-12 before and during antigen challenge holds therapeutic promise in asthma. However, the effects of IL-12 on the established asthmatic responses have not fully been examined.. We investigated whether IL-12 administered after antigen challenge could diminish airway hyper-reactivity (AHR) and eosinophilia in mice actively sensitized to ovalbumin. We also have investigated the ability of administered IL-12 to induce IL-18 receptor (IL-18R) expression that may lead possible synergic action of IL-12 with endogenous IL-18.. C57BL/6 mice immunized to ovalbumin (OVA) by intraperitoneal (i.p.) injection, were challenged three times with an aerosol of OVA every second day for 8 days. Recombinant IL-12 (500 ng) was intravenously administered on a single occasion 1 h after the final challenge of mice. Mice were analysed for effects of IL-12 on AHR, inflammatory cell infiltration and cytokine levels in lung tissue as well as serum immunoglobulin (Ig) E levels. Immunohistochemistry for IL-18R was performed using rat monoclonal antibody specific for murine IL-18Ralpha (IL-1 receptor related protein; IL-1Rrp).. An intravenous IL-12 administration diminished AHR, pulmonary eosinophilia and T lymphocyte infiltration, serum IgE, IL-4 and IL-13 in lung tissue. Expression of IL-18R was induced in the mononuclear cells in the lung of mice exposed to OVA. IL-12 administration enhanced the IL-18R expression compared with the control.. These data indicate that IL-12 can attenuate established antigen-induced AHR and inflammation. In this mechanism it would be interpreted as follows: IL-12 administration in OVA-challenged mice decreased IL-4 production and IgE production thereafter through direct effect on inhibiting the activation of established Th2 cells response and also combined effect with up-regulation of IL-18R expression by inflammatory cells in the lung.

Topics: Administration, Inhalation; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cytokines; Immunoglobulin E; Interleukin-12; Interleukin-18 Receptor alpha Subunit; Lung; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Receptors, Interleukin; Receptors, Interleukin-18

Since antigen-specific IgE and eosinophils are major inducing factors of allergic inflammation of the airways, both factors are therapeutic targets of asthma. We investigated the effects of ONO-4007, a nontoxic lipid A analogue, on antigen-specific antibody response and the recruitment of eosinophils into airways in murine systems.. BALB/c mice were injected ONO-4007 intraperitoneally during sensitization with ovalbumin (OVA) and aluminium hydroxide to determine its effects on the antigen-specific antibody response. ONO-4007 was also injected intravenously during either systemic sensitization and inhalation with OVA, or sensitization or inhalation alone to determine its effects on antigen-induced airway inflammation. In vitro effects of ONO-4007 on the functional differentiation of naive CD4+ T cells were investigated by culturing naive CD4+ T cells derived from DO11.10 mice and OVA-pulsed dendritic cells (CDCs) with ONO-4007.. ONO-4007 inhibited antigen-specific IgE and IgG1, but not IgG2a responses. ONO-4007 decreased the recruitment of eosinophils and the levels of IL-5 in bronchoalveolar lavage fluid, not only when it was injected during systemic sensitization and inhalation with OVA, but also during inhalation alone. ONO-4007 inhibited the differentiation of IL-4- and IL-13-producing CD4+ T cells in vitro, which was partly mediated by DCs.. ONO-4007 inhibited antigen-specific IgE and IgG1 responses and antigen-induced eosinophil recruitment into the airways in BALB/c mice. These effects were mediated, at least partly, by the modulation of DCs, although there may also be other mechanisms.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Antigens; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Movement; Cells, Cultured; Cytokines; Dendritic Cells; Eosinophils; Immunoglobulin E; Immunoglobulin G; Lipid A; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia

Th2 cells are recruited to the lung where they mediate the asthma phenotype. Since the molecular mechanisms regulating Th2 cell trafficking remain unknown, we sought to determine whether trafficking of Th2 cells into the lung is mediated by G alpha i-coupled chemoattractant receptors. We show here that in contrast to untreated Th2 cells, pertussis toxin-treated Th2 cells were unable to traffic into the lung, airways, or lymph nodes following Ag challenge and therefore were unable to induce allergic inflammation in vivo. Pertussis toxin-treated Th2 cells were functional cells, however, and when directly instilled into the airways of mice, bypassing their need to traffic to the lung, were able to induce airway eosinophilic inflammation. These studies conclusively demonstrate that trafficking of Th2 cells into the lung is an active process dependent on chemoattractant receptors.

Topics: Adoptive Transfer; Aerosols; Animals; Bronchoalveolar Lavage Fluid; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Chemotaxis, Leukocyte; Cytokines; Epitopes, T-Lymphocyte; Inflammation; Integrins; Intubation, Intratracheal; Lymph Nodes; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, Transgenic; Mucus; Nebulizers and Vaporizers; Ovalbumin; Pertussis Toxin; Pulmonary Eosinophilia; Receptors, Chemokine; Respiratory Hypersensitivity; Th2 Cells; Virulence Factors, Bordetella

Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.

Topics: Administration, Inhalation; Adoptive Transfer; Allergens; Animals; Antibodies, Monoclonal; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Line; Epitopes, T-Lymphocyte; Inflammation; Injections, Intravenous; Interferon-gamma; Interleukin-4; Lung; Male; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; RNA, Messenger; Th1 Cells; Th2 Cells

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA(323-339) and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA(323-339)-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.

Topics: Animals; Antibody Specificity; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cell Movement; Cells, Cultured; Cytokines; Dendritic Cells; Female; Goblet Cells; Hyperplasia; Immunodominant Epitopes; Inflammation; Intubation, Intratracheal; Lung; Lymph Nodes; Mice; Mice, Inbred BALB C; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; T-Lymphocytes; Th1 Cells; Th2 Cells

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Specificity; Asthma; Bronchial Hyperreactivity; CD4-Positive T-Lymphocytes; Cell Movement; Chemokine CCL17; Chemokines, CC; Cytokines; Disease Models, Animal; Immune Sera; Immunohistochemistry; Immunosuppressive Agents; Injections, Intraperitoneal; Lung; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; RNA, Messenger; Th2 Cells; Thymus Gland

Bronchial-alveolar eosinophilic inflammation is among the characteristic pathological changes in asthma, which has been shown to be correlated with type 2 cytokine and chemokine production. Exogenous IL-12 has been found to be inhibitory for pulmonary eosinophilia in reported studies. Using a murine asthma-like model induced by OVA, we found in the present study that IL-12 gene knockout (KO) mice showed substantially reduced airway recruitment of eosinophils compared with wild-type control mice following OVA sensitization/challenge, although the levels of circulating eosinophils were comparable in these two groups of mice. Cytokine analysis showed Ag-driven Th1 (IFN-gamma) and Th2 (IL-4, IL-5, IL-10, and IL-13) cytokine production by CD4 T cells from local draining lymph nodes and spleen. Similarly, local eotaxin production was comparable in wild-type and IL-12 KO mice. In contrast, immunohistochemical analysis showed that the expression of VCAM-1 on the lung endothelium of IL-12 KO mice was dramatically less than that in wild-type mice. Furthermore, administration of rIL-12 at the stage of sensitization and challenge with OVA restored airway eosinophilia and VCAM-1 expression in IL-12 KO mice. The results suggest that endogenous IL-12 contributes to the recruitment of eosinophils into airways observed in asthma, possibly via enhancement of the expression of VCAM-1 on local vascular endothelial cells.

Topics: Administration, Intranasal; Animals; Asthma; Bronchi; Cytokines; Disease Models, Animal; Down-Regulation; Endothelium, Vascular; Female; Immunoglobulin E; Immunoglobulin G; Injections, Intraperitoneal; Interleukin-12; Intracellular Fluid; Mice; Mice, Inbred C57BL; Mice, Knockout; Mycobacterium bovis; Ovalbumin; Pulmonary Eosinophilia; Recombinant Proteins; Th1 Cells; Th2 Cells; Tuberculosis, Pulmonary; Vascular Cell Adhesion Molecule-1

Although the preliminary characterization of chemokines and their receptors has been prolific, comparatively little is known about the role of chemokines in the evolution of immune responses. We speculate that the preferential recruitment of a particular immune cell population has implications for the short- and long-term features of an adaptive response. To test this hypothesis, we employed adenovirus-mediated gene transfer to express the Th1-affiliated, CXC chemokine IFN-gamma-inducible protein (IP) 10 in the airways of mice undergoing a mucosal sensitization regimen known to result in a Th2-polarized allergic response. This resulted in a approximately 60-75% inhibition of eosinophils in the bronchoalveolar lavage (BAL); these inflammatory changes were accompanied by enhanced IFN-gamma, ablated IL-4, and, peculiarly, unaltered IL-5 and eotaxin levels in the BAL. The effect of IP-10 expression was shown to be dependent on IFN-gamma, as there was no statistically significant reduction in BAL eosinophilia in IFN-gamma knockout mice subjected to the IP-10 intervention. Flow cytometric analysis of mononuclear cells in the lung revealed a approximately 60% reduction in the fraction of CD4(+) cells expressing T1/ST2, a putative Th2 marker, and a parallel increase in the proportion expressing intracellular IFN-gamma following IP-10 treatment. The effect of IP-10 expression at the time of initial Ag encounter is persistent, as mice rechallenged with OVA following the resolution of acute inflammation exhibited reduced eosinophilia and IL-4 in the BAL. Collectively, these data illustrate that local expression of the chemokine IP-10 can introduce Th1 phenomena to a Th2-predisposed context and subvert the development of a Th2 response.

Topics: Adenoviruses, Human; Adjuvants, Immunologic; Administration, Intranasal; Allergens; Animals; Bronchoalveolar Lavage Fluid; Chemokine CXCL10; Chemokines; Chemokines, CXC; Cytokines; Female; Flow Cytometry; Genetic Vectors; Humans; Immunization; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Lung; Mice; Mice, Inbred BALB C; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Respiratory Mucosa; T-Lymphocyte Subsets; Th1 Cells

Airway eosinophilia in asthma is dependent on cytokines secreted by Th2 cells, including IL-5 and IL-4. In these studies we investigated why the absence of IL-4 led to a reduction in airway, but not lung tissue, eosinophils. Using adoptively transferred, in vitro-generated TCR-transgenic Th2 cells deficient in IL-4, we show that this effect is independent of IL-5 and Th2 cell generation. Airway eosinophilia was no longer inhibited when IL-4(-/-) Th2 cells were transferred into IFN-gammaR(-/-) mice, indicating that IFN-gamma was responsible for reducing airway eosinophils in the absence of IL-4. Intranasal administration of IFN-gamma to mice after IL-4(+/+) Th2 cell transfer also caused a reduction in airway, but not lung parenchymal, eosinophils. These studies show that IL-4 indirectly promotes airway eosinophilia by suppressing the production of IFN-gamma. IFN-gamma reduces airway eosinophils by engaging its receptor on hemopoietic cells, possibly the eosinophil itself. These studies capitalize on the complex counterregulatory effects of Th1 and Th2 cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We define a new regulatory role for IFN-gamma, demonstrating that eosinophilic inflammation is differentially regulated at distinct sites within the respiratory tract.

Topics: Administration, Cutaneous; Administration, Intranasal; Adoptive Transfer; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Bronchi; Cell Movement; Down-Regulation; Eosinophils; Female; Hematopoietic Stem Cells; Inflammation; Interferon gamma Receptor; Interferon-gamma; Interleukin-13; Interleukin-4; Interleukin-5; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; Receptors, Interferon; Respiratory Hypersensitivity; Th2 Cells; Up-Regulation

The relationship between CD4(+) T cell-mediated airway eosinophilic inflammation and bronchial hyper-responsiveness (BHR) was investigated. Ovalbumin-reactive T(h)0 clones were adoptively transferred to unprimed BALB/c mice and then the mice were challenged by inhalation of the relevant antigen. Upon antigen provocation, infused T(h) clones infiltrated into the airways, followed by the accumulation and degranulation of eosinophils, goblet cell hyperplasia, edema and increase in bronchial responsiveness to acetylcholine. Transfer of several clones that differed in the levels of IL-5 production revealed that the magnitude of in vivo eosinophilia strongly correlated with the IL-5-producing capacity of the infused T(h) clones. Administration of anti-IL-5 mAb almost completely suppressed antigen-induced eosinophilic inflammation and BHR. Administration of anti-IL-4 mAb or anti-IFN-gamma mAb enhanced the eosinophilia and BHR, whereas anti-IL-2 mAb did not affect them. The number of accumulated eosinophils significantly correlated with the intensity of BHR. Our present results clearly demonstrated that CD4(+) T cells induced BHR as a result of eosinophilic inflammation. IL-5 totally regulated both responses.

Topics: Acetylcholine; Animals; Antibodies, Monoclonal; Bronchial Diseases; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Clone Cells; Cytokines; Eosinophil Peroxidase; Eosinophilia; Interferon-gamma; Interleukin-4; Interleukin-5; Leukocyte Count; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Peroxidases; Pulmonary Eosinophilia; Specific Pathogen-Free Organisms

The closely related Th2 cytokines, IL-4 and IL-13, share many biological functions that are considered important in the development of allergic airway inflammation and airway hyperresponsiveness (AHR). The overlap of their functions results from the IL-4R alpha-chain forming an important functional signaling component of both the IL-4 and IL-13 receptors. Mutations in the C terminus region of the IL-4 protein produce IL-4 mutants that bind to the IL-4R alpha-chain with high affinity, but do not induce cellular responses. A murine IL-4 mutant (C118 deletion) protein (IL-4R antagonist) inhibited IL-4- and IL-13-induced STAT6 phosphorylation as well as IL-4- and IL-13-induced IgE production in vitro. Administration of murine IL-4R antagonist during allergen (OVA) challenge inhibited the development of allergic airway eosinophilia and AHR in mice previously sensitized with OVA. The inhibitory effect on airway eosinophilia and AHR was associated with reduced levels of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid as well as reduced serum levels of OVA-IGE: These observations demonstrate the therapeutic potential of IL-4 mutant protein receptor antagonists that inhibit both IL-4 and IL-13 in the treatment of allergic asthma.

Topics: Animals; Antibody Specificity; B-Lymphocyte Subsets; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Cells, Cultured; Cytokines; Down-Regulation; Eosinophils; Epithelium; Female; Humans; Immunoglobulin E; Immunophenotyping; Immunosuppressive Agents; Injections, Intraperitoneal; Injections, Subcutaneous; Interleukin-13; Interleukin-4; Leukocyte Count; Lung; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Mucus; Ovalbumin; Phosphorylation; Pulmonary Eosinophilia; Receptors, Interleukin-4; Recombinant Proteins; Spleen; STAT6 Transcription Factor; T-Lymphocyte Subsets; Trans-Activators

Knowledge of the factors which control IgE production is essential in order to understand the pathogenesis of immediate hypersensitivity reactions. We have studied the extent to which the route and frequency of antigen application as well as different antigen amounts may influence IgE synthesis.. We established sensitisation protocols in BALB/c mice, in which various doses of ovalbumin (Ova) were applied via intranasal, epicutaneous or intraperitoneal routes. Ova-specific antibodies were measured by ELISA. After 6 weeks of sensitisation, anaphylactic shock was measured following intravenous challenge with Ova. In addition, bronchoalveolar lavages were performed in intranasally sensitised mice.. We were able to show that the most efficient IgE production was achieved by long-term antigen application via the airways, leading to local allergic airway pathology. The epicutaneous route of antigen application also induced very high IgE titres, while intraperitoneal sensitisation led to significantly lower IgE levels. After intraperitoneal sensitisation, IgE synthesis was best induced by increasing the frequency of antigen application, but not by increasing the amount of antigen. In all groups of mice, Ova-specific IgE antibodies were high enough to induce systemic allergic symptoms leading to anaphylactic shock. The severity of shock correlated with the amount of specific IgE.. Taken together, our results demonstrate that antigen application via the airways or skin induces IgE synthesis more efficiently than via the intraperitoneal route. Few exposures with high-dose antigen are less efficient than multiple exposures with low doses. Our finding that both the route and the frequency of antigen application strongly influence IgE synthesis may help to understand how environmental antigens lead to allergic sensitisation.

Topics: Administration, Cutaneous; Administration, Intranasal; Allergens; Anaphylaxis; Animals; Antigens; Female; Hypersensitivity, Immediate; Immunoglobulin E; Immunoglobulin G; Injections; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Peritoneum; Pulmonary Eosinophilia

In this study we examined the effect of oral antigen (Ag) administration on the development of experimental asthma in different mouse strains. We selected BALB/c, BP2, CBA/Ca interleukin (IL)-5 transgenic, and BALB/c T-cell receptor-delta-deficient mouse strains because they exhibit different aspects of the asthma syndrome. Mice exposed to 1% ovalbumin (OVA), dissolved in the drinking water for 5 consecutive days, became unresponsive to subsequent immunogenic OVA challenges. This regimen of OVA administration induced Ag-specific unresponsiveness in all mouse strains tested, including gammadelta-deficient mice that are said to be resistant to tolerance induction. The Ag-specific unresponsiveness was characterized by reduced (almost absent) airway eosinophilic inflammation, airway hyperreactivity, and mucus production; also by low levels of T helper (Th) 2-type cytokines in bronchoalveolar lavage fluid, and decreased immunoglobulin (Ig) G1 and IgE OVA-specific antibody production. The unresponsive state was not associated with increased levels of the suppressive cytokines IL-10 and transforming growth factor (TGF)-beta or with immune deviation toward the Th1 pathway due to increased levels of interferon-gamma and IL-12. Moreover, treatment with anti- TGF-beta antibodies did not abrogate oral tolerance. Oral Ag administration was quite effective in suppressing the development of key features of asthma when initiated after primary immunization (Day 0) or after booster (Day 7), but not after challenge (Day 14) when it increased allergic responses. Collectively, our findings show for the first time the beneficial and detrimental effects of oral Ag administration on the development of experimental asthma.

Topics: Administration, Inhalation; Administration, Oral; Animals; Antibodies; Antigens; Asthma; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Drug Administration Schedule; Immune Tolerance; Immunoglobulin E; Immunoglobulin G; Immunosuppression Therapy; Interleukin-5; Mice; Mice, Inbred Strains; Mice, Transgenic; Mucus; Ovalbumin; Pulmonary Eosinophilia; Receptors, Antigen, T-Cell, gamma-delta; Th2 Cells

In antigen-challenged guinea pigs, airway hyperreactivity is due to recruitment of eosinophils to the airway nerves and dysfunction of M(2) muscarinic receptors. M(2) receptor dysfunction is caused by eosinophil major basic protein, which is an allosteric antagonist at the receptor. Because glucocorticoids inhibit airway hyperreactivity in humans and in animal models of asthma, we tested whether dexamethasone treatment (6 microg. kg(-)(1). d(-)(1) for 3 d, intraperitoneal) before antigen challenge prevents M(2) receptor dysfunction and airway hyperreactivity. Guinea pigs were sensitized to ovalbumin via intraperitoneal injections, and were challenged with ovalbumin via inhalation. Twenty-four hours later, hyperreactivity and M(2) receptor function were tested. Antigen-challenged animals were hyperreactive to vagal stimulation, and demonstrated loss of M(2) receptor function. Dexamethasone pretreatment prevented hyperreactivity and M(2) receptor dysfunction in antigen-challenged guinea pigs. Antigen challenge resulted in recruitment of eosinophils to the airways and to the airway nerves. Dexamethasone prevented recruitment of eosinophils to the airway nerves but did not affect total eosinophil influx into the airways. These results demonstrate that dexamethasone prevents antigen-induced hyperreactivity by protecting neuronal M(2) muscarinic receptors from antagonism by eosinophil major basic protein, and this protective mechanism appears to be by specifically inhibiting eosinophil recruitment to the airway nerves.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Antigens; Balantidiasis; Biopsy; Bronchial Hyperreactivity; Dexamethasone; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Glucocorticoids; Guinea Pigs; Immunization; Ovalbumin; Pulmonary Eosinophilia; Receptor, Muscarinic M2; Receptors, Muscarinic

Topics: Adoptive Transfer; Animals; Asthma; Cell Movement; Clone Cells; Hypersensitivity, Immediate; Immunoglobulins; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

Asthma is characterized by airway inflammation with prominent eosinophil infiltrates. In a murine model of asthma, antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4+ T cells and their cytokines, especially IL-5. In the present study, using ovalbumin-specific T cell receptor transgenic mice, we found that T cell vaccination, which was the administration of preactivated and attenuated antigen-specific T cells by the intraperitoneal route, prevented antigen-induced eosinophil recruitment into the airways. This effect was antigen-specific because ovalbumin-specific T cell vaccination did not affect BSA-induced eosinophil recruitment into the airways. We also found that antigen-specific IgE production as well as antigen-induced proliferation and cytokine production of splenocytes were diminished by T cell vaccination. Moreover, flow-cytometric analyses revealed that T cell vaccination eliminated antigen-specific T cells in the periphery. Together, these results indicate that T cell vaccination prevents antigen-induced eosinophil recruitment into the airways presumably by eliminating antigen-specific T cells.

Topics: Adoptive Transfer; Animals; Asthma; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Female; Genes, T-Cell Receptor; Immunoglobulin E; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ovalbumin; Peptide Fragments; Pulmonary Eosinophilia; Spleen; Vaccination

The objective of this study was to investigate the contribution of secondary lymphoid organs in the generation and maintenance of experimental allergic airway inflammation. We employed a previously reported murine model of respiratory mucosal allergic sensitization, induced by repeated aerosolizations of ovalbumin in the context of a GM-CSF airway environment. We executed this protocol in wild-type (WT) and lymphotoxin-alpha-deficient mice (LTalpha-KO) mice, which are devoid of lymph nodes (LNs) and possess rudimentary spleen structures. Despite the lack of pulmonary LNs draining the airway compartment, LTalpha-KO mice were fully capable of mounting a robust inflammatory response in the airways, consisting of Th2 polarized CD4+ T cells and eosinophils. This was accompanied by IL-5, IL-13, and IFN-gamma production by splenocytes and generation of ovalbumin-specific serum IgE. Exposure to the same antigen 7 weeks after complete resolution of airway inflammation once again induced a Th2 polarized infiltrate, demonstrating intact immunological memory. To investigate inherent plasticity in establishing antigen-specific immunity, mice were splenectomized before sensitization. Allergic sensitization was completely abrogated in splenectomized LTalpha-KO mice, compared with eusplenic LTalpha-KO controls. These data demonstrate that secondary lymphoid organs, either LN or spleen, are essential for the generation of allergic airway responses.

Topics: Administration, Intranasal; Animals; Bronchoalveolar Lavage Fluid; CD8-Positive T-Lymphocytes; Cells, Cultured; Disease Models, Animal; Eosinophils; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Immunoglobulin E; Immunologic Memory; Inflammation; Interferon-gamma; Interleukin-13; Interleukin-5; Lymph Nodes; Lymphotoxin-alpha; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia; Recombinant Fusion Proteins; Respiratory Hypersensitivity; Specific Pathogen-Free Organisms; Spleen; Splenectomy; Th2 Cells

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa in which Th2 cells play crucial roles. Ovalbumin-reactive Th2 clones were labeled with a fluorescent-probe then infused into unprimed mice to elucidate the dynamics of antigen-specific T cells involved in allergic inflammation. Infiltration of not only labeled antigen-specific T cells, but also unlabeled nonspecific CD4(+) T cells into the bronchial mucosa following inhaled antigen challenge was detectable under confocal microscopy and flow cytometry. Accordingly, labeled T cells in the spleen were decreased, whereas those in hilar lymph nodes were increased upon antigen challenge. Approximately 45% of antigen-specific T cells that migrated into the lungs bore CD25, while another early activation marker, CD69, was expressed on 80% of the migrated T cells. Accordingly, antigen challenge to the mice induced in situ proliferation of antigen-specific T cells as well as bronchial epithelial cells in the lungs. Expression of vascular cell adhesion molecule (VCAM)-1, but not intercellular adhesion molecule (ICAM)-1, on the vascular endothelium in the lungs was enhanced following antigen challenge. Nevertheless, treatment with anti-VCAM-1 antibody, and also anti-ICAM-1 antibody strongly suppressed the accumulation of T cells, suggesting that both VCAM-1 and ICAM-1 are essential for antigen-stimulated T cell mobilization into peripheral tissues. Our current study visualized the kinetics and the mechanism of antigen-specific T cell migration in response to local challenge with a protein antigen.

Topics: Adoptive Transfer; Animals; Asthma; Cell Movement; Clone Cells; Inflammation; Intercellular Adhesion Molecule-1; Kinetics; Lung; Lymphocyte Activation; Lymphoid Tissue; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; T-Lymphocytes, Helper-Inducer; Th2 Cells; Vascular Cell Adhesion Molecule-1

Clara cell secretory protein (CC10) is a steroid-inducible protein, and its in vivo function is currently unclear. The role of CC10 in modulation of pulmonary allergic inflammation was examined in mice deficient for the CC10 gene. Wild-type and homozygous CC10-deficient mice were sensitized with an Ag, OVA, and challenged with either OVA or saline. When compared with that seen in wild-type mice, a significantly higher level of pulmonary eosinophilia was found in Ag-sensitized and challenged CC10-deficient mice. Significantly increased levels of Th2 cytokines IL-4, IL-5, IL-9, and IL-13 were also found in CC10-deficient mice. In addition, an increased level of eotaxin, but not RANTES, was also seen in CC10-deficient mice. No significant difference was observed in the level of a Th1 cytokine, IFN-gamma, between different groups of mice. These results provided the first in vivo evidence that CC10 plays a role in the modulation of pulmonary allergic inflammation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokine CCL5; Chemokines, CC; Cytokines; Eosinophils; Gene Expression Profiling; Immunization; Immunoglobulin E; Interferon-gamma; Interleukin-13; Interleukin-4; Interleukin-5; Interleukin-9; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Proteins; Pulmonary Eosinophilia; Th2 Cells; Uteroglobin

Eosinophils are primarily thought of as terminal effectors of allergic responses and of parasite elimination. However, limited studies suggest a more discrete immunomodulatory role for this leukocyte during these inflammatory responses. In this investigation, we highlight the potential of eosinophils to act as APCs and thus modulators of allergic responses by influencing Th2 cell function. In response to Ag provocation of the allergic lung, eosinophils rapidly trafficked to sites of Ag deposition (airways lumen) and presentation (lung-associated lymph nodes and T cell-rich paracortical zones). Eosinophils from the allergic lung expressed class II MHC peptides, T cell costimulatory molecules (CD80 and CD86), and rapidly internalized and processed Ag that was sampled from within the airway lumen. Ag-loaded eosinophils promoted the production of IL-4, IL-5, and IL-13 in cocultures with in vitro-polarized Th2 cells and induced IL-5 production in a dose-dependent manner from Ag-specific CD4(+) T cells isolated from allergic mice. In addition, Ag-loaded eosinophils primed for Th2 cell-driven allergic disease of the lung when transferred to naive mice. Thus, eosinophils have the potential to not only activate Th2 cells to release disease-modulating cytokines but also to assist in priming the immune system for allergic responses. This investigation highlights the potential of eosinophils to not only act as terminal effector cells but also to actively modulate allergic inflammation by amplifying Th2 cell responses.

Topics: Administration, Inhalation; Allergens; Animals; Antigen Presentation; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Bronchoalveolar Lavage Fluid; Chemotaxis; Cytokines; Eosinophils; Histocompatibility Antigens Class II; Immunization; Lung; Lymph Nodes; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Specific Pathogen-Free Organisms; Th2 Cells

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by approximately 50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to beta-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.

Topics: Animals; Asthma; B-Lymphocytes; Bronchial Hyperreactivity; Cells, Cultured; Cytokines; Immunoglobulins; Inflammation; Interleukin-5; Mice; Mice, Inbred C3H; Ovalbumin; Pulmonary Eosinophilia; Self Tolerance; Th2 Cells; Vaccines, DNA

Airway hyperresponsiveness to a variety of specific and nonspecific stimuli is a cardinal feature of asthma, which affects nearly 10% of the population in industrialized countries. Eosinophilic pulmonary inflammation, eosinophil-derived products, as well as Th2 cytokines IL-13, IL-4, and IL-5, have been associated with the development of airway hyperreactivity (AHR), but the specific immunological basis underlying the development of AHR remains controversial. Herein we show that mice with targeted deletion of IL-13 failed to develop allergen-induced AHR, despite the presence of vigorous Th2-biased, eosinophilic pulmonary inflammation. However, AHR was restored in IL-13(-/-) mice by the administration of recombinant IL-13. Moreover, adoptive transfer of OVA-specific Th2 cells generated from TCR-transgenic IL-13(-/-) mice failed to induce AHR in recipient SCID mice, although such IL-13(-/-) Th2 cells produced high levels of IL-4 and IL-5 and induced significant airway inflammation. These studies definitively demonstrate that IL-13 is necessary and sufficient for the induction of AHR and that eosinophilic airway inflammation in the absence of IL-13 is inadequate for the induction of AHR. Therefore, treatment of human asthma with antagonists of IL-13 may be very effective.

Topics: Adoptive Transfer; Allergens; Animals; Immunity, Innate; Interferon-gamma; Interleukin-13; Interleukin-4; Interleukin-5; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Mucus; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Th2 Cells

Epidemiological studies have suggested increased prevalence of atopy in children of maternal smokers. Although secondhand smoke or environmental tobacco smoke (ETS) has been shown to augment allergic responses, its role in atopic sensitization is still controversial. We studied whether ETS could initiate a Th2 response and thus induce primary allergic sensitization. Mice were exposed for 10 consecutive days to either 1% aerosolized OVA, ETS (5 cigarettes), or both ETS and OVA. C57BL/6 mice receiving both ETS and OVA developed OVA-specific IgE and IgG1, 12, 14, and 25 days after the initial exposure, whereas those receiving OVA alone did not. Thirty days after the initial challenge (20 days after its completion), mice were re-exposed to OVA. Bronchoalveolar lavage performed 24 h later revealed an influx of eosinophils in the group initially challenged with both ETS and OVA, but not in those exposed to ETS alone or OVA alone. Increases in IL-5, GM-CSF, and IL-2 were observed in bronchoalveolar lavage from this OVA/ETS-exposed group, whereas IFN-gamma levels were significantly inhibited. These results suggest that ETS can induce allergic sensitization to a normally harmless Ag, and they may explain why secondhand smoke is a major risk factor for the development of allergy in children.

Topics: Allergens; Animals; Bronchoalveolar Lavage Fluid; Cytokines; Female; Hypersensitivity; Immunization; Immunoglobulin E; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Species Specificity; Tobacco Smoke Pollution

1. We examined the effect of SP100030, a novel inhibitor of activator protein-1 (AP-1) and nuclear factor (NF)-kappa B transcription factors, in a rat model of asthma. 2. Sensitized Brown-Norway rats were treated with SP100030 (20 mg kg(-1) day(-1) for 3 days) intraperitoneally prior to allergen challenge. Allergen exposure of sensitized rats induced bronchial hyperresponsiveness (BHR), accumulation of inflammatory cells in bronchoalveolar lavage (BAL) fluid, and also an increase in eosinophils and CD2(+), CD4(+) and CD8(+) T-cells in the airways together with mRNA expression for IL-2, IL-4, IL-5, IL-10, and IFN-gamma. 3. Pre-treatment with SP100030 inhibited BAL lymphocyte influx (P<0.03), specifically reduced CD8(+) T-cell infiltration in the airway submucosa (P<0.03), and mRNA expression for IL-2, IL-5, and IL-10 (P<0.05). Neutrophil, eosinophil, and CD4(+) T-cells accumulation in the airways and BHR were not affected by SP100030. 4. Our results indicate that suppression of IL-2 and IL-5 mRNA expression may not necessarily lead to suppression of BHR. The expression of IL-5 mRNA may contribute to the airway accumulation of eosinophils, but does not correlate with the extent of eosinophilia. 5. The joint AP-1 and NF-kappa B inhibitor, SP100030, selectively inhibits CD8(+) T-cells, and mRNA expression of both Th1 and Th2 cytokines in vivo, but does not inhibit allergen-induced airway eosinophilia and BHR.

Topics: Allergens; Animals; Blotting, Western; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; CD2 Antigens; CD4 Antigens; CD8 Antigens; Cytokines; Eosinophils; Gene Expression Regulation; Immunohistochemistry; Immunosuppressive Agents; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-4; Interleukin-5; Lung; Lymphocytes; Macrophages; Male; Neutrophils; NF-kappa B; Organic Chemicals; Ovalbumin; Proto-Oncogene Proteins c-jun; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; Respiratory Mucosa; RNA, Messenger; Specific Pathogen-Free Organisms; Transcription Factor AP-1; Transcription Factors

Immunostimulatory DNA sequences (ISS) inhibit eosinophilic inflammation and airway hyperreactivity in mouse models of asthma. In vitro ISS activate natural killer (NK) cells to secrete IFN-gamma, and this cytokine is hypothesized to contribute to the antiallergic effect of ISS in vivo.. We investigated whether ISS activation of NK cells is important in mediating the reduction in airway hyperreactivity and the antieosinophilic effect of ISS in vivo.. We assessed whether ISS modulated the development of eosinophilic airway inflammation and airway hyperreactivity to methacholine in ovalbumin (OVA)-sensitized and OVA allergen-challenged mice pretreated with an antibody to deplete NK cells.. Mice sensitized and challenged with OVA had significant bronchoalveolar lavage and lung eosinophilia, as well as airway hyperresponsiveness. ISS induced significant inhibition of bronchoalveolar lavage and lung eosinophilia, as well as airway hyperresponsiveness, in OVA-sensitized mice pretreated before OVA challenge with an NK cell-depleting antibody (NK(-) mice), as well as in mice pretreated with a control non-NK cell-depleting antibody (NK(+) mice). The NK cell-depleting antibody inhibited ISS-induced IFN-gamma production by spleen cells.. These studies demonstrate that depletion of NK cells has no significant effect on ISS-mediated inhibition of airway eosinophilia and airway hyperresponsiveness in vivo, suggesting that non-NK cells and cytokines other than IFN-gamma derived from NK cells mediate the majority of the ISS-inhibitory effect on eosinophilic inflammation and airway hyperresponsiveness in vivo.

Topics: Adjuvants, Immunologic; Animals; Asthma; Bone Marrow Diseases; Bronchial Hyperreactivity; Bronchoconstrictor Agents; Cells, Cultured; DNA; Eosinophilia; Female; Interferon-gamma; Killer Cells, Natural; Lymphocyte Depletion; Methacholine Chloride; Mice; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia

Severe respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans have been inconclusive. By use of a well-characterized murine model of RSV infection and allergic sensitization with ovalbumin, the effect of a preceding severe RSV infection on the development of the pulmonary allergic inflammatory response and airway hyperresponsiveness (AHR) was tested. The impact of prior allergic sensitization on RSV-induced illness, as measured by weight loss, also was evaluated. RSV infection before allergic sensitization decreased allergen-induced AHR, production of interleukin-13 in lung tissue, and lung eosinophilia. In contrast, allergic sensitization before RSV infection increased AHR and decreased RSV-related weight loss and lung levels of interferon-gamma but did not alter viral clearance. These data provide evidence that RSV-associated AHR occurs in hosts with allergic responses and that allergic inflammation is diminished when preceded by RSV infection.

Topics: Allergens; Animals; Bronchial Hyperreactivity; Bronchoconstrictor Agents; Female; Hypersensitivity; Interferon-gamma; Interleukin-13; Lung; Lymphocyte Count; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Time Factors; Virus Replication; Weight Loss

Flt-3 ligand (FL), a recently described growth factor affecting early hematopoietic progenitor cells, can also support the expansion of dendritic cells secreting IL-12. Since type 2 T cells predominate in asthma and IL-12 prevents the differentiation of naive T lymphocytes to a type 2 phenotype, we hypothesized that FL could prevent the development of asthma-like conditions in the ovalbumin mouse model. We found that co-administration of FL during ovalbumin sensitization abrogated late allergic responses, but had no effect on early allergic responses. Airway hyperresponsiveness to methacholine was also blocked by FL treatment. Analysis of bronchoalveolar lavage (BAL) fluid demonstrated a significant reduction in eosinophils, with concomitant decreases in IL-5 and increases in IFN-gamma levels. However, there was no change in BAL fluid IL-4 and serum IgE levels. These data suggest that FL treatment prevents ovalbumin-induced asthma in the mouse and may provide a useful adjuvant in the treatment of human asthma.

Topics: Airway Resistance; Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cell Differentiation; Dendritic Cells; Drug Administration Schedule; Female; Immunization; Immunoglobulin E; Interferon-gamma; Interleukin-12; Interleukin-4; Interleukin-5; Membrane Proteins; Methacholine Chloride; Mice; Mice, Inbred BALB C; Models, Animal; Ovalbumin; Pulmonary Eosinophilia; Th2 Cells

The accumulation of eosinophils in the lung is a hallmark of asthma. In addition to cytokines such as IL-5 which are essential, chemokines have been implicated in the recruitment of eosinophils to the airway. In particular, eotaxin has been shown to be a selective and potent eosinophil chemoattractant, important in the pathogenesis of allergic disease. The goal of the present study was to define the role of eotaxin-1 in the development of allergen-induced eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to inhaled methacholine (MCh).. Eotaxin-1-deficient mice were sensitized and exposed to a single challenge with allergen. Airway function and airway and tissue as well as peripheral blood and bone marrow eosinophilia were examined 18 and 48 h after the last challenge.. Following allergen sensitization and challenge, eotaxin-1-deficient mice developed levels of AHR to inhaled MCh at 18 and 48 h comparable to controls. Further, levels of bronchoalveolar lavage (BAL) and tissue eosinophilia at the same time points were comparable in the two strains of mice. Tissue eosinophilia, assessed by quantitating major basic protein staining cells, preceded BAL eosinophilia in a similar manner. Bone marrow and peripheral blood eosinophilia were unimpaired in deficient mice.. The results demonstrate that the major eotaxin, eotaxin-1 is not essential for the development of airway eosinophilia or AHR, implying that other chemokines, alone or in combination, can overcome this deficiency.

Topics: Allergens; Animals; Blood Cell Count; Bone Marrow; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemokine CCL11; Chemokines, CC; Female; Leukocyte Count; Male; Methacholine Chloride; Mice; Ovalbumin; Pulmonary Eosinophilia

Respiratory allergies represent a failure to generate nonpathogenic responses to innocuous foreign materials. Herein we assessed the role of the sensitizing dose of allergen in this response/nonresponse paradigm, sensitizing BALB/c mice with 5 ng-2 microg of OVA-alum and assessing their responses to repeated OVA aerosol challenge. Mice sensitized with < or = 25 ng of OVA-alum did not develop atopic antibodies, airway hyperresponsiveness (AHR), eosinophilia, or pulmonary Th2 responses, but the 25-ng group animals did develop significant IgA responses. The mice sensitized with 100 ng of OVA-alum developed AHR in the absence of detectable allergic disease, while the mice sensitized with 250 ng-2 microg of OVA/alum developed full-spectrum allergic disease (i.e., eosinophilia, IgE, IgG1, pulmonary Th2 cytokine responses, and AHR). These data indicate that limiting doses of allergen can differentially induce IgA or AHR in the absence of atopic disease in mice.

Topics: Allergens; Alum Compounds; Animals; Antibody Specificity; Bronchial Hyperreactivity; Cytokines; Disease Models, Animal; Dose-Response Relationship, Immunologic; Immunization; Immunoglobulin A; Immunoglobulin E; Immunoglobulin G; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Plethysmography, Whole Body; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Th2 Cells

Cysteinyl leukotrienes are potent inflammatory molecules playing a major role in asthma. The involvement of these mediators in hypersensitivity in mice is not well known. This study aimed at elucidating their implication by using MK-571, a cysLT(1)receptor antagonist. Mice were sensitized with a suspension of ovalbumin (8 microg) adsorbed to alum (2 mg) and were challenged with an aerosolized ovalbumin solution (0.5%). Inflammatory cell infiltration in the bronchoalveolar lavage (mostly eosinophils) following antigen challenge was inhibited by dexamethasone (0.1, 1 and 5 mg kg(-1)s.c.) and MK-571 (1, 10, 100 mg kg(-1)i.v.) in a dose-dependent manner. Maximal inhibition was 95% with 5 mg kg(-1)dexamethasone and 90% with 100 mg kg(-1)MK-571. When injected together they showed an additive inhibitory effect on eosinophil infiltration. Bronchial hyperreactivity, measured by the increased pulmonary insufflation pressure to carbachol injections, was also inhibited dose-dependently by MK-571. The EC(50)values for carbachol were of 22.39+/-1.12 microg kg(-1)in sensitized and challenged animals that did not receive MK-571 and increased to 43.65+/-1.10, 50.12+/-1.15 and 83.18+/-1.16 microg kg(-1)in animals treated with 1, 10 and 100 mg kg(-1)MK-571 respectively. Lung microvascular leakage (as measured by Evans blue extravasation) induced by antigen bronchoprovocation was reduced by 22% after treatment with 10 mg kg(-1)MK-571. All these inhibitory effects of MK-571 suggest a role for leukotriene D(4)in this animal model of allergic asthma.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Capillary Permeability; Dexamethasone; Disease Models, Animal; Drug Therapy, Combination; Leukotriene Antagonists; Leukotriene D4; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Ovalbumin; Propionates; Pulmonary Eosinophilia; Quinolines; Receptors, Leukotriene

IL-11 is a pleiotropic cytokine that induces tissue remodeling with subepithelial fibrosis when expressed in the airway. Its effects on the Th2-dominated airway inflammation that is characteristic of asthma, however, are poorly understood. To characterize the effects of IL-11 on Th2 tissue inflammation, we compared the inflammatory responses elicited by OVA in sensitized mice in which IL-11 is overexpressed in a lung-specific fashion (CC10-IL-11) with that in transgene- wild-type littermate controls. Transgene- and CC10-IL-11 transgene+ mice had comparable levels of circulating Ag-specific IgE after sensitization. OVA challenge of sensitized transgene- mice caused airway and parenchymal eosinophilic inflammation, Th2 cell accumulation, and mucus hypersecretion with mucus metaplasia. Exaggerated levels of immunoreactive endothelial cell VCAM-1, mucin (Muc) 5ac gene expression and bronchoalveolar lavage and lung IL-4, IL-5, and IL-13 protein and mRNA were also noted. In contrast, OVA challenge in CC10-IL-11 animals elicited impressively lower levels of tissue and bronchoalveolar lavage inflammation, eosinophilia, and Th2 cell accumulation, and significantly lower levels of VCAM-1 and IL-4, IL-5, and IL-13 mRNA and protein. IL-11 did not cause a comparable decrease in mucus hypersecretion, Muc 5ac gene expression, or the level of expression of RANTES, monocyte chemoattractant protein-2, or monocyte chemoattractant protein-3. In addition, IL-11 did not augment IFN-gamma production demonstrating that the inhibitory effects of IL-11 were not due to a shift toward Th1 inflammation. These studies demonstrate that IL-11 selectively inhibits Ag-induced eosinophilia, Th2 inflammation, and VCAM-1 gene expression in pulmonary tissues.

Topics: Administration, Inhalation; Aerosols; Allergens; Animals; Bronchoalveolar Lavage Fluid; Cytokines; Gene Expression Regulation; Humans; Immunization; Interleukin-11; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Mucins; Mucus; Ovalbumin; Pulmonary Eosinophilia; Recombinant Proteins; Respiratory Mucosa; Species Specificity; Th2 Cells; Turkeys; Vascular Cell Adhesion Molecule-1

In mice, respiratory syncytial virus (RSV) infection can enhance the consequences of allergic airway sensitization, resulting in lung eosinophilia and the development of airway hyperresponsiveness (AHR) to inhaled methacholine (MCh). To delineate a role for interleukin-5 (IL-5), interleukin-4 (IL-4), and interferon gamma (IFN-gamma) in mediating the effects of RSV infection on subsequent allergic sensitization, we treated BALB/c mice with anti-IL-5 during acute RSV infection but not during subsequent exposure to ovalbumin (OVA). IL-5-deficient and IL-4-deficient mice were also treated with IL-5 either during acute RSV infection or during the sensitization period. Airway responsiveness to inhaled MCh was assessed and numbers of lung eosinophils were monitored. Anti-IL-5 treatment during RSV infection reduced AHR and lung eosinophilia after subsequent exposure to allergen. In IL-5-deficient or IL-4-deficient mice lung eosinophilia and AHR after RSV infection and allergen exposure were also markedly reduced. IL-5 administration during RSV infection restored the responses to allergen in both IL-5- and IL-4-deficient mice. However, IL-5 administration only during sensitization restored these responses in IL-4-deficient but not in IL-5-deficient animals. IFN-gamma-deficient mice developed AHR and some lung eosinophilia after allergen exposure alone and when RSV infection preceded allergen, these responses were enhanced. We conclude that both IL-5, particularly during acute infection, and IL-4 are critical in mediating the effects of RSV infection on allergic airway sensitization, resulting in the development of AHR and lung eosinophilia.

Topics: Animals; Female; Immunization; Interferon-gamma; Interleukin-4; Interleukin-5; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; Respiratory Syncytial Virus Infections

The aim of this study was to investigate whether dendritic cells (DCs) can induce sensitization to aeroallergen in a mouse model of allergic asthma. Ovalbumin-pulsed (OVA-pulsed) or unpulsed myeloid DCs that were injected into the airways of naive mice migrated into the mediastinal lymph nodes. When challenged 2 weeks later with an aerosol of OVA, activated CD4 and CD8 lymphocytes, eosinophils, and neutrophils were recruited to the lungs of actively immunized mice. These CD4(+) lymphocytes produced predominantly IL-4 and IL-5 but also IFN-gamma, whereas CD8(+) lymphocytes produced predominantly IFN-gamma. Histological analysis revealed perivascular and peribronchial eosinophilic infiltrates and goblet cell hyperplasia. Studies in IL-4(-/-) and CD28(-/-) mice revealed that production of IL-4 by host cells and provision of costimulation to T cells by DCs were critical for inducing the response. Lung CD4(+) T cells strongly expressed the Th2 marker T1/ST2, and signaling through this molecule via a ligand expressed on DCs was essential for the establishment of airway eosinophilia. These data demonstrate that DCs in the airways induce sensitization to inhaled antigen and that molecules expressed on the surface of these cells are critical for the development of Th2-dependent airway eosinophilia.

Topics: Administration, Inhalation; Allergens; Animals; Antigens; Asthma; CD28 Antigens; Cytokines; Dendritic Cells; Disease Models, Animal; Interleukin-4; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Ovalbumin; Pulmonary Eosinophilia; Signal Transduction; Th2 Cells

Airway allergen exposure causes local eosinophilic cell infiltration. This cellular inflammatory response is likely to involve the release of eosinophils from peripheral storage pools, and possibly also regeneration of eosinophils in the bone marrow. IL-12 is an inhibitory cytokine in allergic inflammation, shown to reduce eosinophilic cell infiltration. The aim of the present study was to determine whether airway allergen exposure increases bone marrow eosinophil production, and, if so, whether IL-12 modulates this effect.. Ovalbumin-sensitized C57BL/6 mice and IL-12 knockout (KO) mice were exposed to allergen via the airway route, and the inflammatory cell response was evaluated in bronchoalveolar lavage fluid, blood, and bone marrow.. Allergen instillation intranasally produced a dose-dependent inflammatory response in the lower airways of sensitized mice. This inflammatory response was dominated by eosinophils, but there were also increases of both lymphocytes and neutrophils. Sensitization and airway allergen exposure also increased the IL-5-dependent growth of bone marrow cells in vitro. The enhanced bone marrow responsiveness in vitro was paralleled by an increased number of bone marrow eosinophils in vivo. After sensitization and repeated allergen exposure, IL-12 KO mice showed higher eosinophil levels in both BAL and bone marrow than parallel wild-type control mice. Furthermore, BAL-eotaxin levels were increased in IL-12 KO mice as opposed to parallel wild-type controls after allergen exposure.. Airway allergen exposure induced systemic immunologic responses, including increased eosinophil numbers in both airways and bone marrow, and also enhanced IL-5 responsiveness in bone marrow cells. IL-12 may regulate airway eosinophilia at both the level of eosinophilopoiesis and the level of local recruitment of eosinophils into the airways.

Topics: Allergens; Animals; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Cell Culture Techniques; Chemokine CCL11; Chemokines, CC; Colony-Forming Units Assay; Cytokines; Dose-Response Relationship, Immunologic; Interleukin-12; Leukocyte Count; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia

The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVA-sensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2'-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3+ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2'-deoxyuridine positive. After three allergen exposures, both CD3+ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R alpha-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment.

Topics: Administration, Intranasal; Allergens; Animals; Antibodies, Monoclonal; Bone Marrow Cells; Bromodeoxyuridine; Bronchoalveolar Lavage Fluid; Cell Movement; Coloring Agents; Disease Models, Animal; Eosinophils; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Inflammation; Injections, Intraperitoneal; Interleukin-5; Leukocyte Count; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Receptors, Interleukin; Receptors, Interleukin-5; Time Factors

Chronic Th2-dominated inflammation and exaggerated IL-6 production are characteristic features of the asthmatic airway. To understand the processes that are responsible for the chronicity of this response and the role(s) of IL-6 in the regulation of airway Th2 inflammation, we compared the responses induced by OVA in sensitized wild-type mice, IL-6 deficient (-/-) mice, and transgenic mice in which IL-6 was overexpressed in the airway (CC10-IL-6 mice). When compared with wild-type mice, IL-6-/- mice manifest exaggerated inflammation and eosinophilia, increased levels of IL-4, IL-5, and IL-13 protein and mRNA, exaggerated levels of eotaxin, JE/monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha and -2, and mRNA, increased bronchoalveolar lavage (BAL) TGF-beta1, and exaggerated airway responses to aerosolized methacholine. In contrast, CC10-IL-6 mice, on both C57BL/6 and BALB/c backgrounds, manifest diminished inflammation and eosinophilia, decreased levels of IL-4, IL-5, and IL-13 protein and mRNA, and decreased levels of bronchoalveolar lavage TGF-beta1. IL-6 also decreased the expression of endothelial VCAM-1 and airway responsiveness to methacholine in these animals. These alterations in the IL-6-/- and CC10-IL-6 mice were not associated with significant decreases or increases in the levels of IFN-gamma, respectively. These studies demonstrate that endogenous and exogenous IL-6 inhibit aeroallergen-induced Th2 inflammation and that this inhibition is not mediated by regulatory effects of IFN-gamma. IL-6 may be an important anti-inflammatory, counterregulatory, and healing cytokine in the airway.

Topics: Administration, Intranasal; Aerosols; Allergens; Animals; Bronchial Hyperreactivity; Chemokines; Cytokines; Gene Expression Regulation; Inflammation; Interleukin-6; Lung; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Mice, Transgenic; Ovalbumin; Plethysmography, Whole Body; Pulmonary Eosinophilia; RNA, Messenger; Th2 Cells; Vascular Cell Adhesion Molecule-1

Monocyte chemoattractant proteins-1 and -5 have been implicated as important mediators of allergic pulmonary inflammation in murine models of asthma. The only identified receptor for these two chemokines to date is the CCR2. To study the role of CCR2 in a murine model of Ag-induced asthma, we compared the pathologic and physiological responses of CCR2(-/-) mice with those of wild-type (WT) littermates following immunization and challenge with OVA. OVA-immunized/OVA-challenged (OVA/OVA) WT and CCR2(-/-) mice developed significant increases in total cells recovered by bronchoalveolar lavage (BAL) compared with their respective OVA-immunized/PBS-challenged (OVA/PBS) control groups. There were no significant differences in BAL cell counts and differentials (i.e., macrophages, PMNs, lymphocytes, and eosinophils) between OVA/OVA WT and CCR2(-/-) mice. Serologic evaluation revealed no significant difference in total IgE and OVA-specific IgE between OVA/OVA WT mice and CCR2(-/-) mice. Lung mRNA expression and BAL cytokine protein levels of IL-4, IL-5, and IFN-gamma were also similar in WT and CCR2(-/-) mice. Finally, OVA/OVA CCR2(-/-) mice developed increased airway hyper-responsiveness to a degree similar to that in WT mice. We conclude that following repeated airway challenges with Ag in sensitized mice, the development of Th2 responses (elevated IgE, pulmonary eosinophilia, and lung cytokine levels of IL-4 and IL5) and the development of airway hyper-responsiveness are not diminished by a deficiency in CCR2.

Topics: Animals; Antibody Specificity; Antigens; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Chemotaxis, Leukocyte; Cytokines; Eosinophil Peroxidase; Eosinophils; Immunoglobulin E; Injections, Intraperitoneal; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Peroxidases; Pulmonary Eosinophilia; Receptors, CCR2; Receptors, Chemokine; Ribonucleases; RNA, Messenger

The earliest contact between antigen and the innate immune system is thought to direct the subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune system, such as natural killer (NK) cells, NK1.1(+) T cells (NKT cells), and gamma/delta T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of NK1.1(+) cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil and CD3(+) T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon gamma-producing splenic cells were diminished in mice depleted of NK1.1(+) cells before the priming regime. Depletion of NK1.1(+) cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different from those observed in wild-type mice. Mice deficient in gamma/delta T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a pathogenic role for the increased NK cell activity observed in human asthma.

Topics: Allergens; Animals; Antigens; Antigens, Ly; Antigens, Surface; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Killer Cells, Natural; Lectins, C-Type; Lymphocyte Depletion; Male; Mice; Mice, Inbred C57BL; NK Cell Lectin-Like Receptor Subfamily B; Ovalbumin; Proteins; Pulmonary Eosinophilia; Receptors, Antigen, T-Cell, gamma-delta; Spleen; T-Lymphocytes; Time Factors; Vaccination

Murine models of allergen-induced pulmonary inflammation share many features with human asthma, including the development of antigen-induced pulmonary eosinophilia, airway hyperresponsiveness, antigen-specific cellular and antibody responses, the elaboration of Th2 cytokines (interleukin [IL]-4 and IL-5), and the expression of chemokines with activity for eosinophils. We examined the role of B cells and antigen-specific antibody responses in such a model by studying the histopathologic and physiologic responses of B cell-deficient mice compared with wild-type controls, following systemic immunization and airway challenge with ovalbumin (OVA). Both OVA-challenged wild-type and B cell-deficient mice developed (1) airway hyperresponsiveness, (2) pulmonary inflammation with activated T cells and eosinophils, (3) IL-4 and IL-5 secretion into the airway lumen, and (4) increased expression of the eosinophil active chemokines eotaxin and monocyte chemotactic protein-3. There were no significant differences in either the pathologic or physiologic responses in the B cell-deficient mice compared with wild-type mice. These data indicate that B cells and antigen-specific antibodies are not required for the development of airway hyperresponsiveness, eosinophilic pulmonary inflammation, and chemokine expression in sensitized mice following aerosol challenge with antigen.

Topics: Animals; Antibody Specificity; Asthma; B-Lymphocytes; Bronchoalveolar Lavage Fluid; Chemokines; Immunoglobulin E; Lung; Mice; Mice, Mutant Strains; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity; RNA, Messenger

The aim of this study was to examine antigen-induced lung cell migration, eosinophil activation, and pulmonary reactivity of Wistar rats exposed to a new sensitization technique. The animals were sensitized with a single subcutaneous implant of a fragment of heat coagulated hen egg white and challenged 21 days later with an intratracheal injection of heat-aggregated ovalbumin (EWI). For comparison, another group of rats were sensitized by an intraperitoneal injection of ovalbumin in alum as adjuvant, with one booster on day 14 and challenge on day 21 post immunization (OVA/AL). Twenty-four hours after antigen challenge, the EWI group presented a higher number of eosinophils in the bronchoalveolar lavage (BAL) (4.85 +/- 1.43 x 10(6)) than the OVA/AL group (0.2 +/- 0.06 x 10(6)) or the control group, where the level of eosinophils were essentially undetectable. Levels of eosinophil peroxidase activity were increased in the cell-free BAL and homogenates of lung tissue in the EWI group (12.10 +/- 2.97 mg/mL and 36.14 +/- 7.21 ng/mg, respectively), but not in the OVA/AL group (4.83 +/- 1.4 ng/mL and 11.95 +/- 2.54 ng/mg, respectively), as compared with controls (5.16 +/- 1.65 ng/mL and 12.13 +/- 1.74 ng/mg, respectively). Thromboxane B2 levels were also increased in the BAL of EWI group (2.89 +/- 0.54 ng/mL) but not the OVA/AL group (1.13 +/- 0.23 ng/mL) as compared with controls (1.14 +/- 0.19 ng/mL). In contrast, the levels of prostaglandin E2 in the BAL were increased in both groups (456.4 +/- 11.8 pg/mL in the EWI group and 303.5 +/- 31.7 pg/mL in the OVA/AL group) as compared with controls (205.7 +/- 29.7 ng/mL). Moreover, only the EWI group developed increased pulmonary reactivity to serotonin (around two-fold), 24 hours after antigen challenge. The extent of lung eosinophil migration and activation and the pulmonary hyperreactivity induced by this novel sensitization procedure without adjuvants represents a significant improvement over existing experimental models of asthma.

Topics: Animals; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Dinoprostone; Disease Models, Animal; Eosinophil Peroxidase; Eosinophils; Immunization; Ovalbumin; Peroxidases; Pulmonary Eosinophilia; Rats; Rats, Wistar; Respiratory Function Tests; Respiratory Hypersensitivity; Serotonin; Thromboxane B2

We have shown previously that the late airways response (LAR) can be transferred by ovalbumin-primed CD4(+) T lymphocytes in Brown Norway rats. This response is associated with an increase of eosinophils and high expression of TH2 cytokines (IL-4 and IL-5) in bronchoalveolar lavage (BAL) fluid.. In this study we hypothesized that the inhibition of IL-4 or IL-5 production in the CD4(+) cells transferred to a naive animal could decrease the LAR and prevent airway eosinophilia in response to antigen challenge.. CD4(+) cells, purified from the cervical lymph nodes of ovalbumin-sensitized rats, were maintained in culture for 6 hours with medium alone or with 10 microgram/mL IL-4 antisense (AS), IL-5 AS, or control AS oligodeoxynucleotide. Then the cells were administrated intraperitoneally to naive rats, which were challenged 2 days later by a 5% ovalbumin aerosol. The lung resistance was measured for 8 hours, and then BAL was performed. Cytospin preparations from BAL cells were assessed for the presence of eosinophils by immunocytochemistry for major basic protein and for IL-4, IL-5, and IFN-gamma expression.. In rats injected with IL-4 AS-treated T cells, LAR, eosinophils, and IL-4 and IL-5 expression were significantly decreased compared with the other groups. Only IL-5 expression in BAL fluid was slightly decreased consequent to the transfer of IL-5 AS-treated T cells.. This study demonstrates that, in the CD4(+) T cell-driven LAR, the early production of IL-4, but not IL-5, by the transferred CD4(+) cells is essential for the development of the LAR.

Topics: Adoptive Transfer; Animals; Asthma; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Cell Survival; Cells, Cultured; Cytokines; Disease Models, Animal; Immunohistochemistry; Interleukin-4; Interleukin-5; Male; Oligonucleotides, Antisense; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; Th2 Cells

To test the effect of genotype on immune response, C57BL/6 and DBA/2 mice were sensitized with aluminum hydroxide gel (alum)-precipitated ovalbumin (OVA) and challenged with aerosolized OVA. The serum immunoglobulin (Ig) E and IgG1 levels in C57BL/6 mice were higher than those in DBA/2 mice. In contrast, IgG2a levels in C57BL/6 mice were lower than that in DBA/2 mice. C57BL/6 mice were also much more susceptible than DBA/2 mice to OVA-induced pulmonary eosinophilia. Furthermore, patterns of cytokine generation in lung tissue were different between C57BL/6 and DBA/2 mice after OVA challenge. Th2-type cytokine interleukin (IL-) 4 and IL-5 generation in C57BL/6 mice was higher than that in DBA/2 mice, while Thl-type cytokine interferon-gamma (IFN-gamma) generation in C57BL/6 mice was lower than that in DBA/2 mice. Similar patterns of IL-4 and IL-5, and IFN-gamma production in splenocytes from both strains after OVA stimulation in vitro were also observed. The participation of IL-4 and IL-5, and IFN-gamma in the regulation of eosinophil infiltration into the lung was confirmed by injection of anti-IL-5, -IL-4 and -IFN-gamma monoclonal antibodies. These results indicate that C57BL/6 mice preferentially induce IL-4 and IL-5-mediated Th2-type response, while DBA/2 mice induce IFN-gamma-mediated Thl-type response. Thus, the genotype of laboratory strains partially determines whether Th1- or Th2-type immune responses are elicited.

Topics: Animals; Cytokines; Genotype; Immunoglobulin E; Immunoglobulin G; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Ovalbumin; Pulmonary Eosinophilia; Spleen; Th1 Cells; Th2 Cells

As the burden of infectious diseases becomes reduced in many countries, a remarkable increase in the incidence of allergies has occurred. The basis for the rise in atopic disorders as a correlate of the decline in infectious diseases has not been defined. In the present study, we tested experimentally whether prior systemic infection with Mycobacterium bovis bacillus Calmette Guérin (BCG) had any effect on ovalbumin (OVA) Al(OH)3 (alum)-induced immunoglobulin E (IgE) production, airway mucus production and eosinophilic inflammation. The data showed that allergen-specific IgE production and OVA-induced eosinophilia and goblet cell development were significantly inhibited by prior infection with BCG. Correspondingly, following immunization with OVA alum, BCG-infected mice exhibited significantly higher levels of allergen-driven interferon-gamma (IFN-gamma) production than the mice without infection. The ratio of IFN-gamma: interleukin (IL)-4 production was higher in OVA-sensitized mice with prior BCG infection than in those without infection. The abrogation of OVA-induced mucus production and pulmonary eosinophilia in BCG-infected mice correlated with significantly decreased IL-5 production and increased IFN-gamma and IL-12 production. These data provide direct evidence that intracellular bacterial infection (i.e. BCG) can inhibit antigen-specific IgE and airway reactivity induced by environmental allergen. Furthermore, the results suggest that changes in cytokine-producing patterns of T lymphocytes and other cells may be the mechanism by which infections influence allergies.

Topics: Allergens; Animals; Bronchi; Female; Goblet Cells; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Interleukin-12; Interleukin-4; Interleukin-5; Mice; Mice, Inbred C57BL; Mucus; Mycobacterium bovis; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Sprague-Dawley; Tuberculosis

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)-type cytokines interleukin (IL)-4 and IL-5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL-4 and IL-5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti-IL-4 and anti-IL-5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL-4 and IL-5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2-type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)-type responses in the lungs, despite considerable production of IL-4 and IL-5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.

Topics: Animals; Antibodies, Monoclonal; Antigens; Asthma; Cells, Cultured; Disease Models, Animal; Genetic Predisposition to Disease; Immunoglobulin E; Immunoglobulin G; Interleukin-4; Interleukin-5; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Spleen; T-Lymphocytes, Helper-Inducer

In this study, we examined the effects of fluticasone propionate (FP) and pentamidine isethionate (PI) on antigen-induced lung inflammation and airway hyperreactivity in guinea pigs. Male guinea pigs were sensitized on days 0 and 14 with 10 micrograms of ovalbumin (OVA) plus 1 mg of Al(OH)3. On day 21, animals were challenged with a 2% OVA aerosol inhalation until they developed pulmonary obstruction. Animals were treated with aerosol inhalation of FP (2 ml of 0.5 mg/ml, five consecutive doses at 12-hr intervals with the last dose given 6 hr before OVA challenge) or PI (30 mg/ml for 30 min 1 hr before OVA challenge), and control animals received no drug before OVA challenge. Airway reactivity to methacholine (MCh) was assessed before sensitization and 18 hr after OVA challenge. At 18 hr after challenge, histological sections of trachea and lung were examined for eosinophil, dendritic cell (DC) and macrophage cell densities in the airways. In control animals, OVA evoked airway hyperreactivity to MCh in conjunction with pulmonary eosinophilia and increases in DC prevalence in the trachea and bronchi. Treatment with FP or PI abolished the OVA-induced hyperresponsiveness and significantly reduced the OVA-induced increases in eosinophils and DCs in the airways. FP and PI had no effect on saline-treated animals. Our study indicates that both inhaled FP and inhaled PI reduce antigen-induced airway hyperreactivity and pulmonary inflammation in guinea pigs. The results also suggest that the DC is a target of the anti-inflammatory effects of these drugs in the airways.

Topics: Androstadienes; Animals; Anti-Asthmatic Agents; Asthma; Bronchial Hyperreactivity; Dendritic Cells; Fluticasone; Guinea Pigs; Male; Ovalbumin; Pentamidine; Pulmonary Eosinophilia

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.

Topics: Animals; Antibodies, Monoclonal; Antigens; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Chemotaxis, Leukocyte; Disease Models, Animal; Eosinophils; Female; Immunoglobulin A; Intercellular Adhesion Molecule-1; Interleukin-5; Leukocytes; Lung; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; Rats; T-Lymphocytes

Adhesion molecule expression by pulmonary endothelial cells is considered to play an important role in the recruitment of circulating leukocytes to sites of inflammation in the lung. We have used P-selectin- and intercellular adhesion molecule type 1 (ICAM-1)-deficient mice to determine whether these adhesion molecules are important to pulmonary eosinophil recruitment after allergen challenge. There was a significant inhibition of lung tissue eosinophil recruitment in ICAM-1-deficient mice (approximately 84% inhibition compared to wild-type mice) and P-selectin-deficient mice (approximately 67% inhibition compared to wild-type mice) 3 h after allergen challenge. The number of bronchoalveolar lavage (BAL) eosinophils in P-selectin-deficient and ICAM-1-deficient mice was also significantly reduced compared with wild-type mice. Levels of BAL eosinophil peroxidase (EPO) were significantly lower in ICAM-1-deficient mice (0.21 +/- 0.03 EPO units) compared with wild-type mice (3.34 +/- 0.65 EPO units). There was no significant difference in the degree of inhibition of eosinophil recruitment in ICAM-1-deficient mice at the three time points (3, 12, and 24 h) of study after allergen challenge. However, in P-selectin-deficient mice there was a decline in the degree of inhibition of eosinophil recruitment from 3 h (67% inhibition) and 12 h (72% inhibition) postchallenge, to 24 h postchallenge (38% inhibition), suggesting that other adhesion molecules may be playing a more prominent role than P-selectin at later time points. These studies suggest an important role for ICAM-1 and P-selectin in eosinophil recruitment to the lung after allergen challenge.

Topics: Animals; Antigens; Bronchoalveolar Lavage Fluid; Chemotaxis, Leukocyte; Disease Models, Animal; Eosinophil Peroxidase; Eosinophils; Female; Hypersensitivity, Immediate; Intercellular Adhesion Molecule-1; Lung; Mice; Mice, Mutant Strains; Ovalbumin; P-Selectin; Peroxidases; Pulmonary Eosinophilia; Skin

Topics: Animals; Asthma; Disease Models, Animal; Humans; Immunoglobulin E; Interleukin-4; Interleukin-5; Lung; Mice; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity

Airway inflammation plays a major role in human asthma. Increasing evidence points to a close correlation between eosinophil infiltration and allergic lung disease. A new murine model of eosinophilic lung inflammation has recently been developed; it consists of immunizing mice with small fragments of solidified hen egg white implanted (EWI) into the subcutaneous tissue. In this model, which is further characterized here, mice challenged with ovalbumin (OVA) present an intense and persistent lung eosinophilia, as well as histopathological findings that resemble human asthma. In the present work, the effect of oral tolerance on the development of allergic lung inflammation in B6 mice immunized with antigen plus adjuvant or with EWI is investigated. It was found that in mice rendered orally tolerant by previous exposure to antigen in the drinking water, the T-helper type 2 cell (Th2)-associated allergic responses in both protocols of immunization were almost completely abolished. The allergic responses were assessed by pulmonary and bone marrow eosinophilia, lung histopathology and antigen-specific IgE and IgG1 production. These findings provide the first indication that Th2-associated lung pathology can be prevented by oral tolerance.

Topics: Administration, Oral; Adsorption; Aluminum Hydroxide; Animals; Egg White; Eosinophil Peroxidase; Eosinophils; Female; Hypersensitivity; Immune Tolerance; Immunization; Immunoglobulin Isotypes; Immunotherapy; Mice; Mice, Inbred C57BL; Ovalbumin; Peroxidases; Pulmonary Eosinophilia; Th2 Cells

Recently, we reported a reproducible model of asthma in guinea-pigs in vivo, which developed a late asthmatic response (LAR) as well as an early response. In this study, time-related changes in the occurrence of the LAR and leucocyte kinesis were assessed. Furthermore, the state of the activation of eosinophils that migrated into the lower airways was characterized in vitro. Guinea-pigs were alternately sensitized/challenged by inhalation with aerosolized ovalbumin adsorbed on aluminium hydroxide and ovalbumin alone, once every 2 weeks. At defined times before and after the fifth challenge, airway resistance was measured, blood was drawn and bronchoalveolar lavage (BAL) and nasal cavity lavage (NCL) were performed. Superoxide anion (.O2-) production of eosinophils was measured with cytochrome c. Occurrence of LAR and considerable increases in circulating eosinophils coincided with each other 5-7 h after the challenge. After 7 h, eosinophil infiltrations into bronchoalveolar spaces were observed. The capacity of eosinophils from the sensitized animals to produce .O2- was higher than those from the non-sensitized ones, when eosinophils were stimulated by platelet-activating factor. Although an increased number of eosinophils in the NCL fluid was observed, it was much less than that in the BAL fluid. Thus, it has been concluded that eosinophilia in the blood and the lung may participate in the occurrence of the late asthmatic response, which is thought to be preferentially evoked in the lower airways in guinea-pigs in vivo.

Topics: Animals; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Cell Movement; Eosinophilia; Guinea Pigs; Leukocyte Count; Leukocytes; Male; Nasal Lavage Fluid; Ovalbumin; Pulmonary Alveoli; Pulmonary Eosinophilia; Superoxides; Time Factors

IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5(+/+) counterparts, IL-5(-/-) mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1alpha in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5(+/+) mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5(-/-) mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5(-/-) mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia.

Topics: Adenoviridae; Animals; Asthma; Blood; Bone Marrow; Chemokine CCL11; Chemokine CCL3; Chemokine CCL4; Chemokines, CC; Cytokines; Eosinophilia; Gene Transfer Techniques; Genetic Vectors; Interleukin-5; Lung; Macrophage Inflammatory Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Pulmonary Eosinophilia

In the present study, we investigated whether allergen immunotherapy is effective in a murine model with immunologic and pathophysiologic features reminiscent of allergic asthma. Ovalbumin-sensitized mice received increasing (1 microgram to 1 mg) subcutaneous doses of ovalbumin twice a week for 8 wk according to a semirush immunotherapy protocol as used in allergic patients. During immunotherapy, an initial rise in serum levels of ovalbumin-specific antibodies (immunoglobulin [Ig]G1, IgE, IgG2a) occurred, after which IgE levels decreased sharply concomitant with an increase in IgG2a levels. The increase in IgG2a levels, with the decline in IgE levels, suggests that during immunotherapy interferon-gamma production is increased or interleukin (IL)-4 production is decreased. After immunotherapy, inhalation challenge of the mice with ovalbumin revealed almost complete inhibition (98%, P < 0.01) of eosinophil infiltration into bronchoalveolar lavage and airway hyperresponsiveness (100% at 320 microgram/kg methacholine, P < 0.05) compared with sham-treated animals. In addition, IL-4 production of thoracic lymph node cells stimulated with ovalbumin in vitro was largely reduced (60%, P < 0.05) after immunotherapy. Thus, effective immunotherapy in this animal model appears to be due to modulation of antigen-specific T cells. Similar effects on airway symptoms and IL-4 production can be obtained within 1 wk by three injections of the highest dose of ovalbumin (1 mg). This animal model will be used as a preclinical model to improve allergen immunotherapy and to gain more insight into the mechanisms involved.

Topics: Alveolitis, Extrinsic Allergic; Animals; Asthma; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Desensitization, Immunologic; Disease Models, Animal; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Interleukin-4; Lymph Nodes; Male; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; Specific Pathogen-Free Organisms; T-Lymphocytes

The mechanisms that regulate the selective infiltration of eosinophils in certain allergic diseases are still poorly understood. The CC chemokine eotaxin is a potent chemoattractant, highly specific for eosinophils. Recent studies have implicated that eotaxin plays an important role in the recruitment of eosinophils in different inflammation processes. A number of other chemokines, cytokines, and chemoattractants also have chemotactic activities for eosinophils and some of them present high selectivity for eosinophils. To further study the role of eotaxin in inflammation, we generated mutant mice with the eotaxin gene disrupted and replaced by the Escherichia coli beta-galactosidase gene. These mice developed normally and had no histologic or hematopoietic abnormalities. Furthermore, our studies showed that the lack of eotaxin did not affect the recruitment of eosinophils in the inflammation models induced by Sephadex beads and thioglycollate, as well as in an experimental lung eosinophilia model induced by ovalbumin aerosol challenge, even at the onset of the inflammatory response. The replacement of the eotaxin gene by the beta-galactosidase gene provided a useful marker to monitor the activity of the eotaxin promoter under normal conditions and after antigen challenges. Immunohistochemical staining suggested that endothelial cells were the major sources of eotaxin expression.

Topics: Animals; Antigens; Bacterial Proteins; beta-Galactosidase; Chemokine CCL11; Chemokines, CC; Chemotaxis, Leukocyte; Cytokines; Dextrans; Escherichia coli; Gene Targeting; Genes, Reporter; Mice; Mice, Knockout; Mice, Transgenic; Microspheres; Organ Specificity; Ovalbumin; Phenotype; Pulmonary Eosinophilia; Recombinant Fusion Proteins; Thioglycolates

We have used a mouse model of allergen-induced airway hyperresponsiveness to demonstrate that immunostimulatory DNA sequences (ISS) containing a CpG DNA motif significantly inhibit airway eosinophilia and reduce responsiveness to inhaled methacholine. ISS not only inhibited eosinophilia of the airway (by 93%) and lung parenchyma (91%), but also significantly inhibited blood eosinophilia (86%), suggesting that ISS was exerting a significant effect on the bone marrow production of eosinophils. The inhibition of the bone marrow production of eosinophils by 58% was associated with a significant inhibition of T cell-derived cytokine generation (IL-5, granulocyte-macrophage CSF, and IL-3). ISS exerted this inhibitory effect on T cell cytokine production indirectly by stimulating monocytes/macrophages and NK cells to generate IL-12 and IFNs. The onset of the ISS effect on reducing the number of tissue eosinophils was both immediate (within 1 day of administration) and sustained (lasted 6 days), and was not due to ISS directly inducing eosinophil apoptosis. ISS was effective in inhibiting eosinophilic airway inflammation when administered either systemically (i.p.), or mucosally (i.e., intranasally or intratracheally). Interestingly, a single dose of ISS inhibited airway eosinophilia as effectively as daily injections of corticosteroids for 7 days. Moreover, while both ISS and corticosteroids inhibited IL-5 generation, only ISS was able to induce allergen-specific IFN-gamma production and redirect the immune system toward a Th1 response. Thus, systemic or mucosal administration of ISS before allergen exposure could provide a novel form of active immunotherapy in allergic diseases.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Allergens; Animals; Anti-Inflammatory Agents; Bone Marrow; Bronchial Hyperreactivity; Bronchial Provocation Tests; CpG Islands; Desensitization, Immunologic; Dexamethasone; Disease Models, Animal; Drug Evaluation, Preclinical; Eosinophils; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Hypereosinophilic Syndrome; Interferon-gamma; Interleukin-3; Interleukin-5; Methacholine Chloride; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Ovalbumin; Plethysmography, Whole Body; Pulmonary Eosinophilia; Th2 Cells; Trachea

We summarize here the main characteristics of a novel model of pulmonary hypersensitivity. Mice were immunized with a subcutaneous implant of a fragment of heat solidified chicken egg white and 14 days later challenged with ovalbumin given either by aerosol or by intratracheal instillation. This procedure induces a persistent eosinophilic lung inflammation, a marked bone marrow eosinophilia, and Th2-type isotypic profile with histopathological findings that resemble human asthma. Further, this model is simple to perform, reproducible in different strains of mice, does not require adjuvants nor multiple boosters. Based on these characteristics we propose it as a suitable murine model of allergic eosinophilic lung inflammation.

Topics: Animals; Asthma; Disease Models, Animal; Eosinophils; Hypersensitivity; Inflammation; Lung; Mice; Ovalbumin; Pulmonary Eosinophilia

The effects of CS-518, a thromboxane A2 synthase inhibitor, on antigen-induced dual bronchial responses, airway hyperresponsiveness (AHR) and airway eosinophilia were investigated in an experimental guinea pig model of the late asthmatic response. Oral CS-518 (1 and 10 mg/kg) inhibited immediate and late asthmatic responses dose-dependently. It also inhibited AHR and eosinophil accumulation after antigen challenge. Therefore, thromboxane A2 is possibly involved in development of the late asthmatic response and AHR, and CS-518 was inferred to inhibit these via inhibition of eosinophil accumulation and thromboxane production.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Chemotaxis, Leukocyte; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Eosinophils; Guinea Pigs; Male; Methacholine Chloride; Ovalbumin; Pulmonary Eosinophilia; Thiophenes; Thromboxane-A Synthase

To elucidate the mechanism of development of asthma, we tried to develop a model which elicited a late asthmatic response by a combination of systemic and inhaled sensitization with ovalbumin in guinea pigs. Eighty-seven percent of animals elicited both an immediate and late asthmatic response after the third antigen inhalation. Airway eosinophilia and airway hyperresponsiveness (AHR) induced after the third challenge were more severe than those after the first challenge. There was a good correlation between airway eosinophilia and AHR in this model under experimental modulation of the number of eosinophils, such as by interleukin 5 or antieosinophil antibody injection. These results demonstrate that eosinophils play an important role in the development of late asthmatic response and AHR.

Topics: Animals; Antibodies; Asthma; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Disease Models, Animal; Eosinophils; Guinea Pigs; Histamine; Interleukin-5; Leukocyte Count; Leukotriene C4; Male; Methacholine Chloride; Ovalbumin; Passive Cutaneous Anaphylaxis; Pulmonary Eosinophilia; Thromboxane B2; Time Factors

We applied anti-ICAM-1 and anti-LFA-1 monoclonal antibody (mAb) to ovalbumin-sensitized rats and examined the effects on nasal eosinophilia and nasal symptoms following topical antigen challenge. In a general and local immunization (GLI) group of rats, the mAbs were applied during the booster topical immunization period. In a general immunization group and a local immunization (LI) group of rats, the mAbs were applied during the immunization period. The number of sneezes and nasal scratching movements occurring soon after topical antigen (Ag) challenge was significantly suppressed in the GLI and LI group rats. Eosinophil infiltration into nasal mucosa 24 h after Ag challenge was also significantly suppressed in GLI and LI group rats. These findings suggest that the ICAM-1/LFA-1 system is important in topical allergic inflammation in rats.

Topics: Animals; Antibodies, Monoclonal; Intercellular Adhesion Molecule-1; Lymphocyte Function-Associated Antigen-1; Male; Nasal Cavity; Nasal Provocation Tests; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Wistar; Rhinitis, Allergic, Perennial; Vaccination

T lymphocytes have been implicated in controlling the recruitment of eosinophils into the lung in murine models of allergic asthma. The mechanism by which T cells assist in the recruitment of eosinophils to the lung in these models is not completely understood. We hypothesized that eosinophil-active chemokines might be regulated by antigen (Ag)-induced T cell activation in vivo and thereby mediate T cell-dependent eosinophil recruitment. To test this hypothesis, we examined the effect of an anti-CD3 mAb on Ag-induced pulmonary eosinophilia and correlated this with the expression of three eosinophil-active chemokines: eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and RANTES. We found that Ag-induced pulmonary eosinophilia was associated with the induction of eotaxin and MIP-1 alpha, but not RANTES mRNA. Prechallenge treatment with anti-CD3 mAb inhibited eotaxin, but not MIP-1 alpha and RANTES mRNA induction, and significantly reduced eosinophil accumulation in the lung. In addition, Ag-specific antibody responses and mast cell degranulation after Ag challenge in sensitized mice were not affected by T cell elimination, and were not sufficient to induce the expression of eotaxin and cause pulmonary eosinophilia. These findings suggest that eotaxin is one of the molecular links between Ag-specific T cell activation and the recruitment of eosinophils into the airways.

Topics: Animals; Antibodies; Bronchoalveolar Lavage; CD3 Complex; Chemokine CCL11; Chemokine CCL4; Chemokine CCL5; Chemokines, CC; Chemotactic Factors, Eosinophil; Cytokines; Histamine; Immunoglobulin E; Immunoglobulin G; Lymphocyte Activation; Macrophage Inflammatory Proteins; Mice; Mice, Inbred BALB C; Ovalbumin; Pulmonary Eosinophilia; RNA, Messenger; T-Lymphocytes

Inhalation of antigen in immunized mice induces an infiltration of eosinophils into the airways and increased bronchial hyperreactivity as are observed in human asthma. We employed a model of late-phase allergic pulmonary inflammation in mice to address the role of leukotrienes (LT) in mediating airway eosinophilia and hyperreactivity to methacholine. Allergen intranasal challenge in OVA-sensitized mice induced LTB4 and LTC4 release into the airspace, widespread mucus occlusion of the airways, leukocytic infiltration of the airway tissue and broncho-alveolar lavage fluid that was predominantly eosinophils, and bronchial hyperreactivity to methacholine. Specific inhibitors of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) blocked airway mucus release and infiltration by eosinophils indicating a key role for leukotrienes in these features of allergic pulmonary inflammation. The role of leukotrienes or eosinophils in mediating airway hyperresponsiveness to aeroallergen could not be established, however, in this murine model.

Topics: 5-Lipoxygenase-Activating Proteins; Allergens; Animals; Asthma; Bronchial Provocation Tests; Bronchoconstrictor Agents; Carrier Proteins; Disease Models, Animal; Female; Immunoglobulin E; Inflammation; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; Membrane Proteins; Methacholine Chloride; Mice; Mice, Inbred BALB C; Mucus; Ovalbumin; Pulmonary Eosinophilia; Respiratory Function Tests; Respiratory System

CD4+ and CD8+ alpha/beta+ T cells of the T helper cell (Th)2 phenotype produce the cytokines IL-4, IL-5, and IL-13 that promote IgE production and eosinophilic inflammation. IL-4 may play an important role in mediating the differentiation of antigenically naive alpha/beta+ T cells into Th2 cells. Murine NK1.1+ (CD4+ or CD4-CD8-) alpha/beta+ T cells comprise a beta 2-microglobulin (beta 2m)-dependent cell population that rapidly produces IL-4 after cell activation in vitro and in vivo and has been proposed as a source of IL-4 for Th2 cell differentiation. alpha/beta+ CD8+ T cells, most of which require beta 2m for their development, have also been proposed as positive regulators of allergen-induced Th2 responses. We tested whether beta 2m-dependent T cells were essential for Th2 cell-mediated allergic reactions by treating wild-type, beta 2m-deficient (beta 2m -/-), and IL-4-deficient (IL-4 -/-) mice of the C57BL/6 genetic background with ovalbumin (OVA), using a protocol that induces robust allergic pulmonary disease in wild-type mice. OVA-treated beta 2m -/- mice had circulating levels of total and OVA-specific IgE, pulmonary eosinophilia, and expression of IL-4, IL-5, and IL-13 mRNA in bronchial lymph node tissue similar to that of OVA-treated wild-type mice. In contrast, these responses in OVA-treated IL-4 -/- mice were all either undetectable or markedly reduced compared with wild-type mice, confirming that IL-4 was required in this allergic model. These results indicate that the NK1.1+ alpha/beta+ T cell population, as well as other beta 2m-dependent populations, such as most peripheral alpha/beta+ CD8+ T cells, are dispensable for the Th2 pulmonary response to protein allergens.

Topics: Allergens; Animals; Antigens; Antigens, Ly; Antigens, Surface; beta 2-Microglobulin; Bronchoalveolar Lavage Fluid; Immunoglobulin E; Interleukin-13; Interleukin-4; Interleukin-5; Lectins, C-Type; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; NK Cell Lectin-Like Receptor Subfamily B; Ovalbumin; Proteins; Pulmonary Eosinophilia; Respiratory Hypersensitivity; T-Lymphocyte Subsets; Th2 Cells

A model of allergic pulmonary inflammation is described in which the intraperitoneal injection of antigen (Ag)-pulsed cells resulted in T cell priming. Mice received two injections of 10(6) elicited peritoneal macrophages, which had been incubated with Ag for 48 hr, on Days 0 and 6, followed by an aerosol Ag challenge on Day 19. Bronchoalveolar lavage fluid harvested on Day 21 contained increased eosinophil numbers and resembled the cell influx observed following immunization with Ag in alum. Incubation of Ag-presenting cells with interferon-gamma resulted in increased expression of the costimulator molecule B7-2 and of MHC Ags, but did not enhance priming capacity. Using this system, antibodies to CD4 and CD8 were tested for their ability to block sensitization by Ag-pulsed cells. Both anti-CD4 and anti-CD8 antibodies completely blocked the airway eosinophil response following aerosol Ag challenge. This model will be very useful for characterization of the interactions between Ag-presenting cells and T cells which ultimately result in the induction of pulmonary eosinophilia.

Topics: Animals; Antigen-Presenting Cells; Asthma; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Disease Models, Animal; Female; Flow Cytometry; Mice; Mice, Inbred C57BL; Ovalbumin; Pulmonary Eosinophilia; T-Lymphocytes

We examined the effect of a type IV (rolipram) and a combined type III and IV (Org 20421) isoenzyme-selective phosphodiesterase inhibitor upon allergen-induced pulmonary eosinophil recruitment in sensitised Brown Norway rats. Rats were sensitised with ovalbumin intraperitoneally and later challenged with ovalbumin aerosol which induced a significant increase in the total eosinophil and neutrophil count in bronchovalveolar lavage fluid at 24 hours (from 0.38 +/- 0.12 to 1.36 +/- 0.18 x 10(6), p < 0.01 and from 0.06 +/- 0.01 to 0.33 +/- 0.07 x 10(6), p < 0.01) respectively. Pretreatment with rolipram (30 mumol/kg) and Org 20421 (30 mumol/kg) abolished the eosinophilia and neutrophilia evoked by ovalbumin. We conclude that type IV and possibly type III isozyme phosphodiesterase inhibitors may regulate, directly or indirectly, eosinophil and neutrophil activity and/or those cells responsible for attracting them into the lung.

Topics: Administration, Inhalation; Aerosols; Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Cell Count; Disease Models, Animal; Eosinophils; Female; Injections, Intraperitoneal; Neutrophils; Ovalbumin; Phosphodiesterase Inhibitors; Pulmonary Eosinophilia; Pyrrolidinones; Rats; Rats, Inbred BN; Rolipram; Thiazoles

The aim of the study was to evaluate the effects of blocking the integrins VLA-4 and LFA-1 on allergen-induced airway eosinophilia and responsiveness in Brown-Norway rats. Ovalbumin-sensitized rats were exposed to either aerosols of ovalbumin or saline. Airway responsiveness to methacholine (MCh) was determined 8 and 32 h after challenge. Cellular populations in the lung lavage and lung tissues were determined 32 h after allergen challenge. Total numbers of eosinophils were increased in the lung lavage (25 ml) and the small airways/parenchyma in the ovalbumin (OA)-challenged rats (4.37 x 10(6) +/- 0.71 and 15.54 x 10(6) +/- 1.99, respectively) compared with the saline-challenged rats (0.99 x 10(6) +/- 0.81 and 4.84 x 10(6) +/- 2.27; p < 0.05). Animals treated with both anti-VLA-4 and anti-LFA-1 mAbs and with anti-LFA-1 mAb alone had reduced numbers of eosinophils in the lung lavage (0.76 x 10(6) +/- 0.80 and 0.40 x 10(6) +/- 1.14, respectively; p < 0.05) and in the small airways/parenchyma (8.64 x 10(6) +/- 2.07 and 4.44 x 10(6) +/- 3.20; p < 0.05). Anti-VLA-4 mAb treatment alone did not alter the eosinophils recovered from the lung. Airway responsiveness to methacholine increased from 8 to 32 h in all ovalbumin-challenged rats, but treatment with anti-VLA-4, anti-LFA-1, or both mAbs prevented the increase in responsiveness. In conclusion, allergen-induced airway hyperresponsiveness is inhibitable by blocking either VLA-4 or LFA-1 integrins and is associated with a lung eosinophilia that is LFA-1 dependent and VLA-4 independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Bronchial Hyperreactivity; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Leukocyte Count; Lymphocyte Function-Associated Antigen-1; Lymphocyte Subsets; Male; Methacholine Chloride; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; Receptors, Very Late Antigen; Respiratory Hypersensitivity

Topics: Animals; Bone Marrow; Hypersensitivity; Immunotherapy; Interferon-gamma; Interferons; Interleukin-4; Interleukin-5; Mice; Mice, Inbred Strains; Ovalbumin; Pulmonary Eosinophilia; Recombinant Proteins

One hundred twenty male Sprague-Dawley rats (3 weeks old) were given biotin-deficient diets containing ovalbumin as the protein source. Ten control rats of the same origin were fed a commercially available purified diet that used casein as a protein source. Eosinophils and histiocytes were observed at a higher frequency in lungs of rats fed the purified diets containing ovalbumin than in the controls. Foam cells were confined to subpleural and peribronchial regions, reacting positively to anti-lysozyme antibody. The incidence of pulmonary histiocytosis was 76/120 rats (63.3%) in the groups fed the ovalbumin-containing diets as compared with 1/10 (10.0%) in the controls. The accumulation of eosinophils in lung was highest (6/24 rats, 25%) at 3 months. This lesion was not seen in the controls. Eosinophils were first observed in the perivascular and peribronchiolar regions. In advanced lesions, macrophages and mast cells also appeared in the lesions, which at this stage resembled so-called idiopathic chronic eosinophilic pneumonia of human beings. Neither foam cells nor eosinophils were present in any of the other organs. Because there was no difference in the composition of the diets with the exception of the protein source, these lung lesions may be due to biotin deficiency resulting from the use of ovalbumin as the protein source.

Topics: Animals; Biotin; Body Weight; Diet; Disease Models, Animal; Eosinophils; Foam Cells; Lung; Male; Microscopy, Electron; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Sprague-Dawley; Staining and Labeling

The effect of a novel leuktriene B4 receptor antagonist N-[5[[8-(1-hydroxy-2- phenyl)ethyl]dibenzofuran-2yl]5-hydroxypentanoyl]pyrrolidine (PF 10042) has been evaluated in comparison with 2-[3(1-hydroxyhexyl)phenoxymethyl]quinoline hydrochloride (PF 5901), a specific inhibitor of the 5-lipoxygenase pathway of arachidonic acid metabolism, against platelet activating factor (PAF) and allergen induced bronchial hyperresponsiveness and pulmonary eosinophil infiltration in the guinea pig. PF 10042 significantly displaced radiolabelled [3H]leukotriene B4 from binding sites on human neutrophils with an EC50 of 3 muM. PF 10042 (100 mg/kg, i.p.) significantly inhibited PAF and allergen induced bronchial hyperresponsiveness without reducing the concomitant eosinophil infiltration, whereas PF 5901 (100 mg/kg, p.o.) significantly inhibited both PAF and allergen induced bronchial hyperresponsiveness and eosinophil infiltration. We suggest from these results that PAF and allergen induced bronchial hyperresponsiveness may be secondary to the release of leukotriene B4, but this lipoxygenase metabolite does not contribute significantly to the observed eosinophil infiltration.

Topics: Animals; Benzofurans; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Guinea Pigs; Histamine; Humans; Leukotriene Antagonists; Lipoxygenase; Lipoxygenase Inhibitors; Male; Ovalbumin; Platelet Activating Factor; Pulmonary Eosinophilia; Quinolines

Pulmonary inflammation with eosinophil (EOs) infiltration is a prominent feature of allergic respiratory diseases such as asthma. In order to study the cellular response during the disease development, an animal model of IgE-mediated pulmonary inflammation with characteristic eosinophilia is needed. We developed a method for inducing severe pulmonary eosinophilia in the mouse and also studied the numbers of EOs in blood and bone marrow and the response to corticosteroid treatment. Animals were sensitized with alum-precipitated ovalbumin (OVA) and challenged with aerosolized OVA 12 days later when serum IgE levels were significantly elevated. Four to eight hours after challenge there were moderate increases in the number of EOs in the bone marrow and peripheral blood, but only a few EOs were observed in the lung tissue and in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, there was a marked reduction of EOs in bone marrow, while the number of EOs peaked in the perivascular and peribronchial regions of the lung. Forty-eight hours after challenge, the highest number of EOs was found in the BAL fluid, making up > 80% of all cells in that compartment. The high levels of EOs in the lung tissue and BAL fluid lasted for 2-3 days and was followed by a more moderate but persistent eosinophilia for another 10 days. Nonsensitized animals showed no significant changes in the number of EOs in BAL fluid, lungs, blood or bone marrow. Histopathological evaluation also revealed epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenal Cortex Hormones; Animals; Asthma; Betamethasone; Blood Cells; Bone Marrow Cells; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Eosinophils; Immunization; Leukocyte Count; Male; Mice; Mice, Inbred Strains; Neutrophils; Ovalbumin; Pneumonia; Pulmonary Eosinophilia; Time Factors

Even though the eosinophil is potentially an important contributor to airway narrowing during the late allergic airway response, direct evidence of its participation is lacking. Therefore, we examined the effects of eosinophilia induced by Sephadex on the magnitude of the late airway response of sensitized rats following allergen challenge. Brown Norway rats were actively sensitized to ovalbumin (OA). At the same time and 14 days later, a test group was administered Sephadex G200 (0.5 mg intravenously). The animals were challenged with an aerosol of OA and pulmonary resistance (RL) was measured over 6 h. The early response to OA reached a peak more rapidly and the magnitude of the late response, measured as the area under the curve of RL against time, was significantly greater in the Sephadex-treated group (48.3; geometric mean) compared to the control animals (18.9; p < 0.02). The percentage of eosinophils was increased in the bronchoalveolar lavage of Sephadex-treated animals (4%) compared to the controls (0.9%; p < 0.02) following OA challenge. These results demonstrate that Sephadex induces eosinophilia in Brown Norway rats and is associated with an increase in the late allergic airway response. This is consistent with the hypothesis that the eosinophil is an important determinant of the late response.

Topics: Administration, Inhalation; Airway Resistance; Allergens; Animals; Bronchoalveolar Lavage Fluid; Dextrans; Eosinophils; Injections, Intravenous; Leukocyte Count; Male; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Inbred BN; Rats, Sprague-Dawley; Respiratory System; Serotonin

Eosinophil peroxidase (EPO) has been used previously to detect the number of eosinophils in the peritoneal exudate and bone marrow of mice. The present study was undertaken to determine 1) whether EPO activity may provide a measure of a change in eosinophils in bronchoalveolar lavage fluid (BALF) of guinea pigs, 2) whether immunoglobulin (Ig)G1 could play a role in pulmonary eosinophilia and 3) effects of pharmacological agents on the EPO response in an IgG1 passively sensitized animal model. The activity of EPO was assessed by the ability of cell lysates (0.1% Triton-100 treatment) to oxidize 1 mM o-phenylenediamine in the presence of 1 mM H2O2 for 5 min at 22 degrees C. The enzyme activity was found to be eosinophil dependent, inhibited by the EPO inhibitor 3-amino-1,2,4-triazole (IC50 = approximately 0.1 mM) and relatively resistant to heat treatment (no loss of activity after 2-hr preincubation at 56 degrees C). To determine antigen-dependent eosinophil and EPO responses, guinea pigs were passively sensitized i.p. with 0.5 mg/kg of an affinity-purified antiovalbumin (OA) IgG1. Two to 3 days later, the sensitized animals were injected with pyrilamine (5 mg/kg, i.p.) before OA aerosol challenge. Aerosolized OA (0.1%) caused a significant increase in both eosinophil number and EPO activity in BALF of sensitized guinea pigs at 18 to 24 hr post-challenge. At a given concentration of aerosolized OA, the enzyme activity increased as a function of the antibody dose and time post-OA challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antigens; Biomarkers; Bronchoalveolar Lavage Fluid; Eosinophil Peroxidase; Eosinophils; Female; Guinea Pigs; Immunoglobulin G; Leukocytes; Ovalbumin; Peroxidases; Platelet Activating Factor; Pulmonary Eosinophilia

Peptide leukotrienes have been suggested to play an important role in bronchial asthma. As antigen-induced bronchoconstrictions, airway hyperreactivity, and pulmonary eosinophil accumulation are characteristics of the pathology of asthma, we investigated the effect of a peptide leukotriene receptor antagonist, ONO-1078, on these responses using guinea-pig models of asthma. Oral administration of ONO-1078 (3 mg/kg) significantly inhibited slow-reacting substance of anaphylaxis-mediated bronchoconstriction induced by i.v. administered ovalbumin. ONO-1078 (30-100 mg/kg), when administered orally both 1 h before and 4 h after ovalbumin challenge, significantly reduced immediate- and late-phase asthmatic responses, with peak responses occurring immediately and 5-11 h after challenge with inhaled ovalbumin. Oral administration of ONO-1078 significantly reduced the airway hyperreactivity (10-30 mg/kg) and the pulmonary eosinophil accumulation (30-100 mg/kg) observed 4 and 24 h after ovalbumin challenge, respectively. These results suggest that ONO-1078 may be of therapeutic use for bronchial asthma.

Topics: Acetylcholine; Acute-Phase Reaction; Animals; Asthma; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Bronchodilator Agents; Chromones; Disease Models, Animal; Guinea Pigs; Hypersensitivity; Immunization; Male; Ovalbumin; Phthalazines; Pulmonary Eosinophilia; Pyrilamine; SRS-A

Antigen inhalation in sensitized dogs, guinea pigs and rats resulted in a marked, late-phase, eosinophil-rich, influx of inflammatory cells into the bronchial lumen. Attempts to demonstrate an associated late-phase bronchoconstriction were disappointing. We were unable to demonstrate a late-phase bronchoconstriction in either rats or dogs, even when dogs were pretreated with metyrapone to reduce blood cortisol levels. In ovalbumin-sensitized guinea pigs, challenged with low doses of ovalbumin, we observed an immediate bronchoconstriction, a late-phase bronchopulmonary eosinophilia but no late-phase bronchoconstriction. However, inhalation of very high doses of antigen in mepyramine-treated sensitized guinea pigs did induce a moderate late-phase bronchoconstriction.

Topics: Animals; Ascaris; Bronchi; Bronchial Hyperreactivity; Disease Models, Animal; Dogs; Guinea Pigs; Metyrapone; Neutrophils; Ovalbumin; Pulmonary Eosinophilia; Pyrilamine; Rats; Respiratory Function Tests

Exposure of actively sensitized boosted guinea pigs to aerosolized antigen, 3 times on alternate days, produced pulmonary eosinophilia but not pulmonary hyperresponsiveness to methacholine Cl measured 3 days after the last antigen challenge. These data suggest that the presence of large numbers of eosinophils in the airways and tissues of the lungs is not sufficient to produce nonspecific pulmonary hyperresponsiveness. These data also suggest that actively sensitized and boosted guinea pigs respond differently to repeated antigen exposure than do asthmatics or wild caught allergic cynomolgus monkeys.

Topics: Aerosols; Animals; Antigens; Bronchoalveolar Lavage Fluid; Guinea Pigs; Immunoglobulin E; Immunoglobulin G; Lung Diseases, Obstructive; Lung Volume Measurements; Male; Methacholine Compounds; Ovalbumin; Pulmonary Eosinophilia; Respiratory Hypersensitivity

1. We examined effects of bradykinin (BK) receptor antagonists on airway hyperresponsiveness and eosinophilia in sensitized guinea-pigs that had been administered single, as well as repeated (chronic) challenges with inhaled ovalbumin. In addition, the effects of BK antagonists on antigen-induced respiratory distress during the chronic study were noted. 2. At 24 h following single antigen challenge, guinea-pigs exhibited airway hyperresponsiveness to the bronchoconstrictor effect of i.v. histamine, characterized by a left shift in the dose-response curve. In addition, responses to the maximum dose of histamine that could be used were significantly increased in hyperresponsive guinea-pigs. The percentages of bronchoalveolar fluid, eosinophil and neutrophils also increased. 3. A BK B1 receptor antagonist, desArg9-[Leu8]-BK, significantly inhibited airway hyperresponsiveness induced by single antigen challenge. A B2 receptor antagonist, D-Arg-[Hyp3, Thi5,8,D-Phe7]-BK (NPC 349) had a small, but statistically significant inhibitory effect on responsiveness to the highest histamine dose in challenged animals. DesArg9-[Leu8]-BK significantly inhibited the neutrophilia, whereas NPC 349 inhibited infiltration by both cell types. 4. Chronic antigen challenge also caused airway hyperresponsiveness to i.v. acetylcholine (ACh), distinguished by an increase in the slope of the dose-response curve. Thus, the magnitude of the bronchoconstrictor responses to the maximum dose of ACh that could be used was significantly increased. No change in sensitivity to ACh was evident. Marked eosinophilia was also noted in the trachea, bronchi and lung parenchyma. 5. Airway hyperresponsiveness and eosinophilia, induced by chronic antigen challenge, were markedly inhibited by the B2 antagonists, D-Arg-[Hyp3,D-Phe7]-BK (NPC 567) or D-Arg-[Hyp3,Thi5d-Tic7,Tic8]-BK (NPC 16731).NPC 16731 also abolished antigen-induced cyanosis, and delayed the onset of dyspnoea,doubling the time taken for animals to exhibit respiratory distress.6. The ability of BK receptor antagonists to inhibit antigen-induced airway hyperresponsiveness, in addition to eosinophilia, indicates an important role for endogenous kinins. Moreover, the abrogation of eosinophil infiltration suggests that BK has a significant function in maintaining allergic inflammation of the airways.

Topics: Acetylcholine; Animals; Bradykinin; Bronchial Hyperreactivity; Dose-Response Relationship, Drug; Female; Guinea Pigs; Humans; Infant, Newborn; Male; Ovalbumin; Pulmonary Eosinophilia; Receptors, Bradykinin; Receptors, Neurotransmitter; Respiratory Distress Syndrome, Newborn

Described is a guinea pig model of allergic asthma, in which animals are sensitized by intraperitoneal injections of ovalbumin complexed with aluminum hydroxide to elicit an IgE response. Aerosol exposure to ovalbumin induces acute bronchoconstriction and nonspecific airways hyperreactivity, evaluated by intravenous challenges of histamine or acetylcholine. This model mimics the hallmark features of human asthma, is induced by respired allergen and is pharmacologically modulated by prophylactic drugs (methyl prednisolone, ketotifen, theophylline) and the competitive PAF receptor antagonist SDZ 264-412.

Topics: Acetylcholine; Aerosols; Allergens; Animals; Asthma; Bronchial Hyperreactivity; Diphenhydramine; Guinea Pigs; Histamine; Hormone Antagonists; Immunoglobulin E; Ketotifen; Organic Chemicals; Ovalbumin; Platelet Membrane Glycoproteins; Prednisolone; Pulmonary Eosinophilia; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Theophylline

A close association has been recognized between activated T cells and eosinophils in asthma, albeit circumstantial. The present study attempted to investigate this relationship in an animal model of lung eosinophilia using the new generation of T cell-selective immunosuppressants, cyclosporin A and FK506, compared with the myelotoxic immunosuppressive agent cyclophosphamide. Antigen challenge of ovalbumin-sensitized guinea-pigs resulted in a lung eosinophilia which was assessed by bronchoalveolar lavage. All three agents caused a marked suppression of lung eosinophilia at 24 h post-challenge when the compounds were administered at the time of sensitization but not when administered for 3 days before lavage. However, the lung eosinophilia at 72 h post-challenge was reduced significantly by FK506 and by cyclophosphamide, but not by cyclosporin A, when the drugs were administered for 3 days, before lavage. These results strongly suggest the involvement of T cells in antigen-induced late phase (72 h) eosinophilia in guinea-pigs but not at 24 h. The effects of cyclophosphamide were always associated with a reduction in circulating white cell counts, whereas cyclosporin A and FK506 showed no myelotoxic properties. These results suggest the potential therapeutic use of selective, non-cytotoxic immunosuppressive agents in asthma.

Topics: Animals; Antigens; Bronchoalveolar Lavage Fluid; Cyclophosphamide; Cyclosporine; Guinea Pigs; Leukocyte Count; Male; Ovalbumin; Pulmonary Eosinophilia; Tacrolimus

Sensitised guinea-pigs were exposed to aerosolised antigen. The resultant cellular infiltration into the lung was assessed in lung tissue and bronchoalveolar lavage fluid 6, 24, 72 h and 7 days later. An early neutrophil infiltration peaking at 6 h was succeeded by eosinophil migration which persisted for 7 days, at which time some of the eosinophils appeared immature. The lung eosinophilia was accompanied by an initial fall in eosinophilic cells in the bone marrow, followed by an increase in this population. Treatment with dexamethasone (25 mg/kg i.p.) given daily for 7 days after antigen challenge reduced the lung eosinophilia and observed bone marrow changes.

Topics: Administration, Inhalation; Animals; Bone Marrow; Bronchial Provocation Tests; Bronchoalveolar Lavage Fluid; Dexamethasone; Disease Models, Animal; Eosinophils; Guinea Pigs; Immunophenotyping; Leukocyte Count; Male; Neutrophils; Ovalbumin; Pulmonary Eosinophilia

Guinea pigs were sensitized by intraperitoneal injection of ovalbumin, 10 micrograms mixed with 100 mg A1(OH)3 in saline. On days 15-30 sensitized guinea pigs were challenged with ovalbumin aerosol (0.5 mg/ml, 30 s, 15 psi) which produced immediate asthmatic responses characterized by dyspnea, convulsions, and some deaths during the first 14 min. Twenty to 24 h later the animals were sacrificed with an overdose of pentobarbital, and lungs, bronchi, and lower trachea were dissected and fixed in 10% neutral buffered formalin. Histopathological examination of randomly coded tissues of the respiratory tract revealed a pulmonary eosinophilic cellular infiltrate in the epithelium/subepithelium of trachea, bronchi, and bronchioles as well as the peribronchial, peribronchiolar, and perivascular areas of the lungs. Oral administration of mepyramine (10 mg/kg) 2 h before aeroallergen challenge provided complete protection against immediate asthmatic responses and prevented deaths during the first 14 min without influencing the late phase associated lung eosinophilic cellular infiltrate. The immediate asthmatic responses were not influenced by methylprednisolone (30 mg/kg) administered orally 24 and 2 h before aeroallergen challenge. Following an additional dose of methylprednisolone 4 h after challenge, there was a significant inhibition of pulmonary eosinophilia (30 mg/kg; -24 h, -2 h, and +4 h). These observations suggest that histamine is the principal mediator of immediate asthma attacks in guinea pigs. Methylprednisolone may be acting by inhibiting the production of eosinophil chemotactic factor of anaphylaxis (platelet-activating factor or leukotriene B4) from the alveolar macrophages, T lymphocytes, and perhaps other cells, thus preventing pulmonary eosinophilia.

Topics: Administration, Oral; Aerosols; Aminopyridines; Animals; Asthma; Drug Administration Schedule; Guinea Pigs; Methylprednisolone; Ovalbumin; Premedication; Pulmonary Eosinophilia; Pyrilamine

The selective leukotriene B4 (LTB4) antagonist, U-75302, 6-(6-(3-hydroxy-1E,5Z-undecadien-1-yl)-2-pyridinyl)-1,5-hexa nediol) was examined for its ability to inhibit the "late-phase" bronchopulmonary eosinophilia that occurs 6 to 24 h after inhalation of specific antigen in sensitized guinea pigs. Groups of 6 male guinea pigs, sensitized with ovalbumin, were pretreated with U-75302, 1.0, 10.0, or 30.0 mg/kg, or vehicle 1 h before and 7 h after antigen inhalation. Twenty-four hours after antigen provocation, the lungs were lavaged for the enumeration of inflammatory cell populations. Doses of U-75302 (1.0, 10.0 and 30.0 mg/kg) administered orally produced 12.2%, (p greater than 0.05), 43.2% (p less than 0.05), and 61.1% (p less than 0.05) inhibition, respectively, of the antigen-induced influx of eosinophils into the bronchial lumen. Neutrophil populations were not significantly affected by treatment with U-75302. In a separate study, we compared the histopathological changes that occurred following antigen challenge in U-75302-treated or vehicle-treated guinea pigs. Vehicle-treated, sensitized animals exhibited marked changes in the airway at 8 min, 6 h, and 24 h after antigen challenge. U-75302 treatment produced a significant reduction in eosinophil adherence to peribronchial/peribronchiolar capillaries followed by a dramatic and specific reduction of peribronchial eosinophil infiltration (81% reduction at 6 h and 79% reduction at 24 h). Neutrophil migration appeared unaffected. These data implicate LTB4 as a mediator of antigen-induced bronchopulmonary eosinophilia in the guinea pig.

Topics: Animals; Antigens; Bronchoalveolar Lavage Fluid; Eosinophils; Fatty Alcohols; Glycols; Guinea Pigs; Immunization; Leukocytes; Leukotriene B4; Lung; Male; Neutrophils; Ovalbumin; Pulmonary Eosinophilia

Considerable attention has recently focused on the role of inflammation in the pathophysiology of asthma, with special emphasis on "late-phase" bronchoconstriction and increased airway hyperreactivity after antigen challenge in sensitized subjects. The present report describes the histopathologic changes in guinea-pig lung and trachea at various time intervals after ovalbumin inhalation in nonsensitized (control) and sensitized animals. Bronchoalveolar lavage (BAL) was also used to assess the accompanying accumulation of intraluminal leukocytes. A distinct leukocyte margination, consisting of neutrophils and eosinophils, was observed in the peribronchial vasculature as early as 8 min postchallenge in sensitized guinea pigs. At 6 h, the eosinophils predominated and migrated to the peribronchiolar smooth muscle layer. Between 6 h and 18 h, eosinophils were seen in tracts between the smooth muscle cell layers, accumulating in large numbers in the bronchial mucosal epithelium. This pattern persisted for at least 7 days postchallenge during which eosinophils remained the dominant cell type present. Peribronchiolar accumulation of neutrophils and mononuclear cells was minimal at all time points studied. Intraluminal mucus eosinophilia developed between 18 h and 7 days. A similar pattern of eosinophil infiltration was observed in the tracheal epithelium. Control, nonsensitized, guinea-pig lungs showed minor changes with little or no eosinophil infiltration at any time after antigen challenge. These findings correlated well with the BAL study in which sensitized guinea pigs exhibited a marked delayed increase in eosinophil counts between 18 h and 7 days compared with that in nonsensitized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Eosinophils; Guinea Pigs; Leukocyte Count; Lung; Male; Neutrophils; Ovalbumin; Pulmonary Eosinophilia; Time Factors; Trachea