oxazolone and Dermatitis--Contact

Immune contact dermatitis is an inflammation of the skin resulting from exposure to allergens in the environment. The aim of this study was to compare the actions of lactoferrin (LF), a natural immunomodulator, on the elicitation phases of the cellular and humoral, cutaneous immune responses to oxazolone and toluene diisocyanate (TDI), respectively. LF was given i.v. in a 10 mg/mouse dose, together with the eliciting doses of the antigens. The ear edema and the number of lymphocytes in the draining lymph nodes were measured. In addition, the production of IL-2 in the cultures of lymph node cells and the content of IL-4 in lymph node cells were determined. LF had a profound inhibitory effect on the eliciting phase of the immune response to oxazolone as measured by the ear edema and lymph node cell number. The suppressive effect of LF on the effector phase of the immune response to TDI was moderate. LF had some stimulatory effect on the ex vivo content of IL-4 in lymphocytes in the immune response to TDI. On the other hand, it significantly inhibited IL-2 in vitro production in the immune response to oxazolone. The data strongly suggest that LF exerted differential actions on the activities of antigen-specific Th1 and Th2 cells involved in respective types of the cutaneous immune responses.

Topics: Adjuvants, Immunologic; Allergens; Animals; Dermatitis, Contact; Immunity, Cellular; Immunity, Humoral; Interleukin-2; Interleukin-4; Lactoferrin; Lymph Nodes; Mice; Oxazolone; Skin; Th2 Cells; Toluene 2,4-Diisocyanate

The gray, short-tailed opossum, Monodelphis domestica, has been used for photobiologic studies since 1984. The presence of a light-activated DNA repair pathway in the tissues of Monodelphis has been used to identify pyrimidine dimers in DNA as initiating events for a number of ultraviolet radiation (UVR)-induced pathologies of the skin and cornea. Furthermore, Monodelphis, unlike common laboratory rodents, is susceptible to the induction of melanoma by UVR alone.

Topics: Animals; Cornea; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; DNA Repair; Eye Neoplasms; Melanoma; Mice; Mice, Nude; Opossums; Oxazolone; Photobiology; Pyrimidine Dimers; Skin; Skin Neoplasms; Ultraviolet Rays; Urocanic Acid

To compare previously used protocols for ultraviolet (UV)-induced suppression of contact hypersensitivity in mice, and to develop an optimized protocol for C3H mice, the effect of 3 different allergens, varying allergen concentrations in the induction or challenge phase, local and distant sites of allergen application in respect to irradiation site, 2 mouse substrains and 2 different light sources was studied. A concentration of 0.5% of oxazolone (OXA) gave a slightly better contact sensitization than a 1% concentration of trinitrochlorobenzene (TNCB). Titration experiments revealed that for both OXA and TNCB, a 1% sensitization concentration was optimal, while the optimal challenge concentration was 0.5% for OXA and 1% for TNCB. The magnitude of the resulting contact sensitization was not influenced by either the mouse substrain (C3H/HeJ or C3H/HeN) or the site of allergen application (back or belly), but application of fluorescein isothiocyanate to the ears only produced weak sensitization. A standard UVB dose of 1.3 kJ/m2 suppressed TNCB contact sensitivity to a greater extent than that of OXA. A similar degree of UV-induced suppression was obtained with a given UVB dose, irrespective of a 50-fold difference in the concomitant UVA dose. Based on our results, a proper protocol of contact sensitization for UV-induced immunosuppression in C3H mice includes sensitization with 0.5% OXA on either the mouse back or belly, ear challenge with 0.5% OXA and ear swelling reading 24 h after challenge.

Topics: Allergens; Animals; Dermatitis, Contact; Fluorescein-5-isothiocyanate; Immune Tolerance; Immunization; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Oxazolone; Picryl Chloride; Radiation Dosage; Ultraviolet Rays

A guinea pig experimental model which allows comparison of the macroscopic appearance of skin tests with the nature and degree of the dermal inflammatory cell infiltrate has been used to study the effects of cyclophosphamide, methotrexate, azathioprine and cyclosporin A on contact reactions. The immunomodulating capabilities of the agents tested are assessed by their effects on the allergic contact reaction to oxazolone, a cell-mediated delayed hypersensitivity reaction. Non-specific, anti-inflammatory actions are assessed by effects on the toxic contact reaction to croton oil. Changes are compared to findings in reference animals for each reaction. The reference materials and the uses and limitations of the experimental model are evaluated. When administered prior to sensitization, all agents enhanced the macroscopic appearance of the allergic contact reactions. Changes in the dermal cellular infiltrates were not pronounced. When administered prior to testing, cyclophosphamide, methotrexate and azathioprine caused changes compared to controls which varied in direction and degree both macroscopically and microscopically. Cyclophosphamide which was the most active agent showed non-specific, anti-inflammatory effects and caused a peripheral blood leukopenia, the level and character of which was essentially independent of the dermal cellular infiltrate of tests. Cyclosporin A demonstrated no non-specific, anti-inflammatory activity on the toxic reaction, but had by far the most pronounced immunosuppressant effect of the agents tested, with virtual quenching of both the macroscopic appearance and all aspects of the dermal cellular infiltrates of the allergic contact reaction.

Topics: Animals; Azathioprine; Basophils; Cell Division; Croton Oil; Cyclophosphamide; Cyclosporins; Dermatitis, Contact; Disease Models, Animal; Drug Administration Schedule; Female; Guinea Pigs; Hypersensitivity, Delayed; Immunity, Cellular; Immunosuppression Therapy; Mast Cells; Methotrexate; Oxazolone

Topics: Animals; Cell Count; Chemical Phenomena; Chemistry; Dermatitis, Contact; Epitopes; Genes, MHC Class II; Histocompatibility Antigens Class II; Immune Tolerance; Lymphocyte Cooperation; Lymphokines; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Oxazolone; Phenotype; Receptors, Antigen, T-Cell; Spleen; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory; Trinitrobenzenes

Dermal regulatory T cells (Tregs) are essential for maintenance of skin homeostasis and control of skin inflammatory responses. In mice, Tregs in the skin are characterized by high expression of CD103, the αE integrin. Evidence indicates that CD103 promotes Treg retention within the skin, although the mechanism underlying this effect is unknown. The main ligand of CD103, E-cadherin, is predominantly expressed by cells in the epidermis. However, because Tregs are predominantly located within the dermis, the nature of the interactions between E-cadherin and CD103-expressing Tregs is unclear. In this study, we used multiphoton intravital microscopy to examine the contribution of CD103 to Treg behavior in resting and inflamed skin of mice undergoing oxazolone-induced contact hypersensitivity. Inhibition of CD103 in uninflamed skin did not alter Treg behavior, whereas 48 h after inducing contact hypersensitivity by oxazolone challenge, CD103 inhibition increased Treg migration. This coincided with E-cadherin upregulation on infiltrating myeloid leukocytes in the dermis. Using CD11c-enhanced yellow fluorescent protein (EYFP) × Foxp3-GFP dual-reporter mice, inhibition of CD103 was found to reduce Treg interactions with dermal dendritic cells. CD103 inhibition also resulted in increased recruitment of effector CD4+ T cells and IFN-γ expression in challenged skin and resulted in reduced glucocorticoid-induced TNFR-related protein expression on Tregs. These results demonstrate that CD103 controls intradermal Treg migration, but only at later stages in the inflammatory response, when E-cadherin expression in the dermis is increased, and provide evidence that CD103-mediated interactions between Tregs and dermal dendritic cells support regulation of skin inflammation.

Topics: Animals; Cadherins; Dermatitis, Contact; Inflammation; Integrin alpha Chains; Mice; Oxazolone; T-Lymphocytes, Regulatory

Topics: Animals; Cytokines; Dermatitis, Atopic; Dermatitis, Contact; Haptens; Interleukin-13; Interleukin-17; Interleukin-4; Interleukin-5; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oxazolone; RNA, Messenger; Skin

Chronic dermatitis is a hallmark of Dedicator of cytokinesis 8 (DOCK8) deficiency. The migration of DOCK8-deficient T cells to the skin and their survival there have been reported to be defective. Surprisingly, we found that Dock8

Topics: Animals; Dermatitis, Contact; Disease Models, Animal; Female; Guanine Nucleotide Exchange Factors; Humans; Immune Tolerance; Interferon-gamma; Male; Mice; Mice, Knockout; Oxazolone; Skin; T-Lymphocytes, Regulatory

Allergic contact dermatitis (ACD) is a common skin disorder affecting an estimated 15-20% of the general population. The mouse model of ACD is contact hypersensitivity (CHS), which consists of two phases: induction and elicitation. Although neutrophils are required for both CHS disease phases their mechanisms of action are poorly understood. Neutrophils release myeloperoxidase (MPO) that through oxidation of biomolecules leads to cellular damage.. This study investigated mechanisms whereby MPO contributes to CHS pathogenesis.. CHS was induced in mice using oxazolone (OX) as the initiating hapten applied to the skin. After 7 days, CHS was elicited by application of OX to the ear and disease severity was measured by ear thickness and vascular permeability in the ear. The role of MPO in the two phases of CHS was determined utilizing MPO-deficient mice and a specific MPO inhibitor.. During the CHS induction phase MPO-deficiency lead to a reduction in IL-1β production in the skin and a subsequent reduction in migratory dendritic cells (DC) and effector T cells in the draining lymph node. During the elicitation phase, inhibition of MPO significantly reduced both ear swelling and vascular permeability.. MPO plays dual roles in CHS pathogenesis. In the initiation phase MPO promotes IL-1β production in the skin and activation of migratory DC that promote effector T cell priming. In the elicitation phase MPO drives vascular permeability contributing to inflammation. These results indicate that MPO it could be a potential therapeutic target for the treatment of ACD in humans.

Topics: Animals; Cell Movement; Dendritic Cells; Dermatitis, Allergic Contact; Dermatitis, Contact; Haptens; Inflammation; Interleukin-1beta; Lymph Nodes; Mice; Mice, Inbred C57BL; Neutrophils; Oxazolone; Peroxidase; Skin; T-Lymphocytes

A number of different B cell subsets have been shown to exhibit regulatory activity using a variety of mechanisms to attenuate inflammatory diseases. Here we show, using anti-CD20-mediated partial B cell depletion in mice, that a population of mature B cells distinguishable by IgD

Topics: Animals; B-Lymphocyte Subsets; Cell Separation; Cells, Cultured; Coculture Techniques; Dermatitis, Contact; Disease Models, Animal; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Developmental; Healthy Volunteers; Humans; Immune Tolerance; Immunoglobulin D; Leukocytes, Mononuclear; Mice; Mice, Inbred C57BL; Oxazolone; Spleen; T-Lymphocytes, Regulatory; Tumor Necrosis Factors

Contact dermatitis and psoriasis are skin disorders caused by immune dysregulation, yet much remains unknown about their underlying mechanisms. Ghrelin, a recently discovered novel peptide and potential endogenous anti-inflammatory factor expressed in the epidermis, is involved in skin repair and disease. In this study, we investigated the expression pattern and therapeutic effect of ghrelin in both contact dermatitis and psoriasis mouse models induced by oxazolone (OXA) and imiquimod (IMQ), respectively, and in TNF-α-stimulated RAW264.7 macrophages, NHEKs and skin fibroblasts. Ghrelin expression was reduced in both the OXA-induced contact dermatitis and IMQ-induced psoriasis mouse models. Furthermore, treatment with ghrelin attenuated skin inflammation in both the contact dermatitis and psoriasis mouse models. Mice administered PBS after OXA- or IMQ-induced model generation exhibited typical skin inflammation, whereas ghrelin treatment in these mouse models substantially decreased the dermatitis phenotype. In addition, exogenous ghrelin attenuated the inflammatory reaction induced by TNF-α in RAW264.7 cells. Moreover, ghrelin administration limited activation of NF-κB signaling. In summary, ghrelin may represent a potential molecular target for the prevention and treatment of inflammatory skin diseases, including contact dermatitis and psoriasis.

Topics: Animals; Dermatitis, Contact; Disease Models, Animal; Fibroblasts; Gene Expression Regulation; Ghrelin; Humans; Imiquimod; Immune System Diseases; Inflammation; Mice; NF-kappa B; Oxazolone; Psoriasis; RAW 264.7 Cells; Signal Transduction; Skin; Tumor Necrosis Factor-alpha

Constantly increasing prevalence of allergic diseases determines the attempts to elaborate the therapeutic strategies activating immune tolerance to particular allergen. Our current research focuses on the antigen-specific action of CD8+ suppressor T (Ts) lymphocytes induced in mice by intravenous administration of a high dose of haptenated syngeneic erythrocytes. While the regulatory activity of Ts cells mediated by exosome-delivered miRNA-150 is well de ned, the mechanism of their induction remained unclear. Therefore, the current studies investigated the immune e ects induced in mice by intravenous administration of contact allergens coupled to syngeneic erythrocytes. In mouse models of hapten-induced contact hypersensitivity (CHS) and delayed-type hypersensitivity to ovalbumin, we have shown that intravenous administration of hapten-coupled erythrocytes failed to induce CHS effector cells. Moreover, hapten-induced CHS reaction occurred to be suppressed in mice intravenously administered with syngeneic erythrocytes coupled with protein allergen. Finally, we have demonstrated that intravenously administered allergen induces immune tolerance only when bound to syngeneic erythrocytes, proving that intravenously delivered allergens are deprived of their immunizing properties when coupled with membrane of self cells. Altogether, our current studies suggest that alteration of self cell membrane by allergen binding is enough to induce Ts cell-mediated immune tolerance to nonpathogenic agents, which express a great translational potential in such conditions as allergies and hypersensitivity-related autoimmune disorders.

Topics: Allergens; Animals; Dermatitis, Contact; Erythrocyte Transfusion; Haptens; Hypersensitivity; Immune Tolerance; Mice; Mice, Inbred CBA; Oxazolone; T-Lymphocyte Subsets; Transplantation, Isogeneic; Trinitrobenzenes

The function of regulatory immune cells in peripheral tissues is crucial to the onset and severity of various diseases. Interleukin-10 (IL-10)-producing regulatory B (IL-10

Topics: Animals; B-Lymphocyte Subsets; B-Lymphocytes, Regulatory; Cytokines; Dermatitis, Contact; Disease Models, Animal; Fluorescent Antibody Technique; Immunoglobulin Isotypes; Interleukin-5; Male; Mast Cells; Mice; Mice, Knockout; Oxazolone; Peripheral Tolerance

In this work we investigated the efficacy of two topically applied azaphenothiazine derivatives, 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5), in the amelioration of inflammatory symptoms of contact sensitivity (CS) to oxazolone in mice, in relation to the commercial ointment Protopic® (tacrolimus), the reference drug. The compounds were administered 24 h following elicitation of CS and, 24 h later, the parameters of inflammation, such as ear edema, blood composition, leukocyte level, numbers of cells in the draining lymph nodes, histological picture of the inflamed tissue, and the morphometric analysis, were analyzed. The study showed that the effectiveness of the studied azaphenothiazines applied as a 0.1% ointment was comparable to the reference drug regarding suppression of the inflammatory process, when all the investigated histological parameters are taken into account.

Topics: Adjuvants, Immunologic; Administration, Topical; Animals; Dermatitis, Contact; Female; Mice; Mice, Inbred BALB C; Ointments; Oxazolone; Phenothiazines

Topics: Administration, Cutaneous; Animals; Aprepitant; Behavior, Animal; Calcineurin Inhibitors; Dermatitis, Contact; Disease Models, Animal; Filaggrin Proteins; Ganglia, Spinal; Humans; Intermediate Filament Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Transgenic; Ointments; Oxazolone; Oximes; Pruritus; Skin; Substance P; Tacrolimus; TRPA1 Cation Channel

It remains unclear how monocytes are mobilized to amplify inflammatory reactions in T cell-mediated adaptive immunity. Here, we investigate dynamic cellular events in the cascade of inflammatory responses through intravital imaging of a multicolor-labeled murine contact hypersensitivity model. We found that monocytes formed clusters around hair follicles in the contact hypersensitivity model. In this process, effector T cells encountered dendritic cells under regions of monocyte clusters and secreted IFN-γ, which mobilizes CCR2-dependent monocyte interstitial migration and CXCR2-dependent monocyte cluster formation. We showed that hair follicles shaped the inflammatory microenvironment for communication among the monocytes, keratinocytes, and effector T cells. After disrupting the T cell-mobilized monocyte clusters through CXCR2 antagonization, monocyte activation and keratinocyte apoptosis were significantly inhibited. Our study provides a new perspective on effector T cell-regulated monocyte behavior, which amplifies the inflammatory reaction in acquired cutaneous immunity.

Topics: Adaptive Immunity; Animals; Apoptosis; CD11c Antigen; Cell Communication; Cells, Cultured; CX3C Chemokine Receptor 1; Dendritic Cells; Dermatitis, Contact; Disease Models, Animal; Female; Genes, Reporter; Humans; Intravital Microscopy; Keratinocytes; Luminescent Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Monocytes; Oxazolone; Skin; T-Lymphocyte Subsets; Time-Lapse Imaging

Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.

Topics: Adaptive Immunity; Amino Acid Sequence; Animals; Antigen Presentation; Cancer Vaccines; Dermatitis, Contact; Dinitrofluorobenzene; Female; Homeodomain Proteins; Immunologic Memory; Killer Cells, Natural; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; NK Cell Lectin-Like Receptor Subfamily A; Oxazoles; Peptides; Vaccination

Topics: Aminoquinolines; Animals; Case-Control Studies; Dermatitis, Contact; Female; Humans; Imiquimod; Male; Mice; Oxazolone; Psoriasis; Receptors, Interleukin

DNA methylation and specifically the DNA methyltransferase enzyme DNMT3A are involved in the pathogenesis of a variety of hematological diseases and in regulating the function of immune cells. Although altered DNA methylation patterns and mutations in

Topics: Animals; Azacitidine; Cell Degranulation; Cell Proliferation; Cells, Cultured; Decitabine; Dermatitis, Contact; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Epigenesis, Genetic; Female; Fluorescent Antibody Technique; Immunoglobulin E; Interleukin-3; Mast Cells; Mastocytosis, Systemic; Mice; Mice, Knockout; Mutation; Oxazolone; Passive Cutaneous Anaphylaxis; ras GTPase-Activating Proteins; RNA Interference; RNA, Small Interfering

This study aimed to investigate whether mangiferin played a protective role in a well-established dermatitis mouse model and tumor necrosis factor alpha (TNF-α)-induced RAW264.7 macrophages. Contact dermatitis is an inflammatory skin disease in the clinic, while its underlying mechanism still remains to be elucidated. Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-β-d-glucoside (C-glucosyl xanthone), a natural antioxidant that was reported to inhibit inflammatory reactions, has been recently proved to be a potential therapy for inflammation. As a result, the oxazolone-induced dermatitis mice models were established to explore whether mangiferin has an anti-inflammatory role in vivo. The phosphate-buffered saline treatment groups showed emblematic skin inflammation, whereas the administration of mangiferin obviously inhibited dermatitis in the mice models. Furthermore, exogenous mangiferin alleviated the inflammatory reaction in TNF-α-induced macrophages by suppressing the production of inflammation- and oxidative stress-associated molecules. Also, mangiferin treatment repressed the activation of nuclear factor-kappaB signaling pathway. To sum up, mangiferin could provide a new target for the therapy and prevention of skin inflammation.

Topics: Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Disease Models, Animal; Humans; Inflammation; Macrophages; Male; Mice; Mice, Inbred C57BL; Oxazolone; RAW 264.7 Cells; Signal Transduction; Skin; Tumor Necrosis Factor-alpha; Xanthones

Anti-oxidant coenzyme Q10 (Co-Q10) is commonly used in clinic. Recently, Co-Q10 was reported to antagonize TNF-α-induced inflammation and play a protective role in various inflammatory conditions. However, its role in dermatitis is unknown. Herein, RAW264.7 macrophage cell line was cultured with stimulation of TNF-α, and administration of Co-Q10 alleviated TNF-α-mediated inflammatory reaction in vitro. Furthermore, oxazolone-induced dermatitis mice model was established, and treatment of Co-Q10 markedly attenuated dermatitis phenotype in this mice model. Moreover, the protective role of Co-Q10 in vitro and in dermatitis was probably due to its repression on NF-κB signaling. Collectively, Co-Q10 may represent a potential molecular target for prevention and treatment of inflammatory skin diseases.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cell Line; Dermatitis, Contact; Disease Models, Animal; Inflammation; Interleukin-1beta; Interleukin-6; Macrophages; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Oxazolone; Skin; Tumor Necrosis Factor-alpha; Ubiquinone

Allergic contact dermatitis is a chronic T cell-driven inflammatory skin disease that is caused by repeated exposure to contact allergens. Based on murine studies of acute contact hypersensitivity, mast cells (MCs) are believed to play a role in its pathogenesis. The role of MCs in chronic allergic contact dermatitis has not been investigated, in part because of the lack of murine models for chronic contact hypersensitivity. We developed and used a chronic contact hypersensitivity model in wild-type and MC-deficient mice and assessed skin inflammatory responses to identify and characterize the role of MCs in chronic allergic contact dermatitis. Ear swelling chronic contact hypersensitivity responses increased markedly, up to 4-fold, in MC-deficient Kit

Topics: Allergens; Animals; CD8-Positive T-Lymphocytes; Chronic Disease; Cytokines; Dermatitis, Contact; Disease Models, Animal; Immunologic Memory; Mast Cells; Mice; Mice, Transgenic; Oxazolone; Proto-Oncogene Proteins c-kit; Skin

The process of sensitisation by specific contact allergens is indispensable for the induction of allergic contact dermatitis. Oxazolone is a well-characterised contact allergen. Previous studies suggested that immune cells bearing the FcRγ subunit are essential for oxazolone-induced contact hypersensitivity, but the biological functions of the FcRγ subunit in the process of sensitisation to oxazolone remain unknown. In this study, we show that FcRγ deficiency decreases ear-swelling responses to oxazolone in mice. However, we found that oxazolone-sensitised FcRγ(-/-) mice and oxazolone-sensitised wild-type (WT) mice have comparable numbers of CD11c(+) MHCII(hi) dendritic cells (DCs) in their draining lymph nodes (LNs). In addition, oxazolone-sensitised LN cells from both FcRγ(-/-) and WT mice showed considerable production of interferon-gamma (IFNγ), interleukin-4 (IL-4) and IL-17A upon oxazolone-keyhole limpet haemocyanin loading. Consistent with these data, oxazolone-sensitised FcRγ(-/-) and FcRγ(+/+) LN cells conferred contact hypersensitivity to WT naïve mice challenged with the hapten. Our findings clearly indicate that, in an experimental mouse model, the FcRγ subunit positively regulates contact hypersensitivity to oxazolone without affecting the contact sensitisation process.

Topics: Adjuvants, Immunologic; Animals; Dendritic Cells; Dermatitis, Allergic Contact; Dermatitis, Contact; Dinitrochlorobenzene; Immunoglobulin E; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Receptors, IgE; Receptors, IgG

Congenitally or early impaired skin barrier as the first event starting the 'atopic march' in atopic dermatitis (AD) patients can increase allergen penetration that results in sensitization, even in the airways, followed by asthma and allergic rhinitis. Thymic stromal lymphopoietin (TSLP) is a cytokine existing in high levels in AD skin and is considered as a novel therapeutic target for atopic disease. We generated oxazolone (Ox)-induced AD-like (Ox-AD) hairless mice and divided them into four groups according to the therapeutic challenges: topical glucocorticoid, pimecrolimus, emollient, and control (acetone-only treated). We assessed the functional studies of skin barrier, epidermal expressions of differentiation markers, IL-1α, TNF-α, proteinase-activated receptor-2 (PAR-2), TSLP and antimicrobial peptides (AMP), and serum IgE in each group. Topical glucocorticoid or pimecrolimus treatment improved AD-like skin lesions and barrier functions, and restored the epidermal expression of differentiation markers, IL-1α, TNF-α, PAR-2, and TSLP, in Ox-AD mice. The improvement was relatively better with the glucocorticoid than pimecrolimus. Epidermal AMP expression was restored by topical glucocorticoid, but not pimecrolimus. Our result showed that topical glucocorticoid or pimecrolimus improved the AD-like skin lesions and barrier impairment by suppressing TSLP-related allergic inflammation.

Topics: Adjuvants, Immunologic; Administration, Topical; Animals; Anti-Inflammatory Agents; Biomarkers; Cytokines; Dermatitis, Contact; Epidermis; Female; Gene Expression Regulation; Methylprednisolone; Mice; Mice, Hairless; Oxazolone; Permeability; Protein Precursors; RNA, Messenger; Tacrolimus; Thymic Stromal Lymphopoietin

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis.

Topics: Animals; Arachidonate 5-Lipoxygenase; CD8-Positive T-Lymphocytes; Chemokine CXCL1; Chemokine CXCL2; Dermatitis, Contact; Epoxide Hydrolases; Fatty Alcohols; Female; Glycols; Inflammation; Interferon-gamma; Interleukin-1beta; Leucine; Leukotriene B4; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Oxazolone; Receptors, Leukotriene B4; Skin

Even with the widespread clinical use of cannabinoid receptor (CBR) stimulating compounds, such as palmitoylethanolamine, the role of CBR agonists on inflammatory skin diseases is not yet fully understood. This study was performed to investigate the effects of CBR agonists on skin inflammation, using acute and chronic inflammation animal models.. The effectiveness of the newly synthesized cannabinoid receptor 1 (CB1R) agonists was determined using in vitro assays. Markers for epidermal permeability barrier function and skin inflammation were measured, and histological assessments were performed for evaluation.. Topical application of CB1R-specific agonist significantly accelerated the recovery of epidermal permeability barrier function and showed anti-inflammatory activity in both acute and chronic inflammation models. Histological assessments also confirmed the anti-inflammatory effects, which is consistent with previous reports.. All of the results suggest that topical application of CB1R-specific agonist can be beneficial for alleviating the inflammatory symptoms in chronic skin diseases, including atopic dermatitis.

Topics: Acute Disease; Administration, Cutaneous; Animals; Cannabinoid Receptor Agonists; Chronic Disease; Dermatitis, Atopic; Dermatitis, Contact; Disease Models, Animal; Fatty Acids, Unsaturated; Female; Mice, Inbred BALB C; Oxazolone; Permeability; Propanolamines; Receptor, Cannabinoid, CB1; Skin; Skin Physiological Phenomena; Tetradecanoylphorbol Acetate; Water Loss, Insensible

Changes in the stratum corneum extracellular matrix impair epidermal barrier function and may cause dermatoses. The aim of this study was to examine the effect of exogenous cholesterol application on skin barrier function and cutaneous inflammation. Skin barrier-disrupted or hapten-stimulated mice were treated with topical cholesterol. The effect of topical cholesterol application on an oxazolone (OXA)-induced hypersensitivity reaction was evaluated. Topical application of cholesterol efficiently decreased transepidermal water loss in areas of barrier-disrupted skin and ameliorated OXA-induced cutaneous hypersensitivity. These favourable effects may have resulted from sustained expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the cholesterol-treated skin. As 11β-HSD1 is known to produce active cortisol, topical cholesterol may attenuate contact hypersensitivity by normalizing secretion of hormonally active cortisol from the skin.

Topics: 11-beta-Hydroxysteroid Dehydrogenase Type 1; Administration, Topical; Animals; Body Water; Cholesterol; Dermatitis, Contact; Epidermis; Gene Expression; Haptens; Hydrocortisone; Mice; Mice, Inbred BALB C; Oxazolone

Contact hypersensitivity (CHS) induced by topical application of haptens is a commonly used model to study dermal inflammatory responses in mice. Several recent studies have indicated that CHS-induced skin inflammation triggers lymphangiogenesis but may negatively impact the immune-function of lymphatic vessels, namely fluid drainage and dendritic cell (DC) migration to draining lymph nodes (dLNs). On the other hand, haptens have been shown to exert immune-stimulatory activity by inducing DC maturation. In this study we investigated how the presence of pre-established CHS-induced skin inflammation affects the induction of adaptive immunity in dLNs. Using a mouse model of oxazolone-induced skin inflammation we observed that lymphatic drainage was reduced and DC migration from skin to dLNs was partially compromised. At the same time, a significantly stronger adaptive immune response towards ovalbumin (OVA) was induced when immunization had occurred in CHS-inflamed skin as compared to uninflamed control skin. In fact, immunization with sterile OVA in CHS-inflamed skin evoked a delayed-type hypersensitivity (DTH) response comparable to the one induced by conventional immunization with OVA and adjuvant in uninflamed skin. Striking phenotypic and functional differences were observed when comparing DCs from LNs draining uninflamed or CHS-inflamed skin. DCs from LNs draining CHS-inflamed skin expressed higher levels of co-stimulatory molecules and MHC molecules, produced higher levels of the interleukin-12/23 p40 subunit (IL-12/23-p40) and more potently induced T cell activation in vitro. Immunization experiments revealed that blockade of IL-12/23-p40 during the priming phase partially reverted the CHS-induced enhancement of the adaptive immune response. Collectively, our findings indicate that CHS-induced skin inflammation generates an overall immune-stimulatory milieu, which outweighs the potentially suppressive effect of reduced lymphatic vessel function.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; Cell Movement; Cytokines; Dendritic Cells; Dermatitis, Contact; Disease Models, Animal; Interleukin-12; Lymph Nodes; Lymphangiogenesis; Lymphocyte Activation; Mice; Mice, Transgenic; Oxazolone; T-Lymphocytes; Vascular Endothelial Growth Factor A

PGRN and its derived engineered protein, Atsttrin, were reported to antagonize TNFα and protect against inflammatory arthritis [Tang, W. et al. (2011) The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332 (6028) 478-484]. Here we found that PGRN level was also significantly elevated in skin inflammation. PGRN-/- mice exhibited more severe inflammation following induction of oxazolone (OXA). In contrast, recombinant Atsttrin protein effectively attenuated inflammation in mice dermatitis model. In addition, the protective role of PGRN and Atsttrin in dermatitis was probably due to their inhibition on NF-κB signaling. Collectively, PGRN, especially its derived engineered protein, Atsttrin, may represent a potential molecular target for prevention and treatment of inflammatory skin diseases.

Topics: Animals; Dermatitis, Contact; Granulins; Intercellular Signaling Peptides and Proteins; Mice; NF-kappa B; Oxazolone; Progranulins; Recombinant Fusion Proteins; Signal Transduction; Skin; Up-Regulation

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition.

Topics: Allergens; Animals; Dermatitis, Contact; Female; Hyperalgesia; Mice; Neutrophils; Oxazolone; Pain; Receptors, Calcitonin Gene-Related Peptide; Skin; Ubiquitin Thiolesterase; Up-Regulation; Vulva; Vulvodynia

Short dimeric or mulitmeric peptides derived from a highly conserved stretch of amino acids from gammaretroviral envelope proteins has been found to have immunosuppressive properties in vitro. Here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders.. The anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of 17 amino acids was tested in two murine skin inflammation models, a TPA-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. Overall, mice (n = 24) treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of 28.8% in ear thickness (range 20.1-42.5), whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect (p = 0.028). Furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mRNA of the pro-inflammatory cytokines TNF-α, IL-17C, and IL-6 as well as the chemokine CXCL2 compared to mice treated with control peptides.. Using two murine skin inflammation models, we show that an immunosuppressive retroviral peptide is capable of reducing inflammatory disorders. The results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation.

Topics: Amino Acid Sequence; Animals; Chemokine CXCL2; Dermatitis, Contact; Dermatitis, Irritant; Dimerization; Disease Models, Animal; Gene Expression; Humans; Immunosuppressive Agents; Interleukin-17; Interleukin-6; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Oxazolone; Peptides; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-immunoglobulin (Ig) has immunosuppressive properties both in vivo and in vitro, but much is still unknown about the mechanisms by which CTLA-4-Ig exerts its immunosuppressive activities in vivo. The aim of this study was to investigate the effect of CTLA-4-Ig in a mouse model of contact hypersensitivity (CHS). The inflammatory response in the presence or absence of CTLA-4-Ig was evaluated by measuring the increase in ear thickness in sensitized animals after challenge. We observed a dose-dependent suppression of the ear swelling in both dinitrofluorobenzene (DNFB)- and oxazolone-induced CHS. The suppressive effect was still present 3 weeks after administration, even in the absence of circulating levels of CTLA-4-Ig. It was further shown that CTLA-4-Ig inhibits activation of T cells in the draining lymph node after sensitization and affects the maturation level of both dendritic cells and B cells. Furthermore, CTLA-4-Ig reduces infiltration of activated CD8(+) T cells into the inflamed ear tissue and suppresses both local and systemic inflammation, as illustrated by reduced expression of cytokines and chemokines in the inflamed ear and a reduced level of acute-phase proteins in circulation. Finally, our results suggest that CTLA-4-Ig has a mainly immunosuppressive effect during the sensitization phase. We conclude that CTLA-4-Ig induces long-term immunosuppression of both DNFB- and oxazolone-induced inflammation and our data are the first to compare the effect of this compound in both DNFB- and oxazolone-induced CHS and to show that CTLA-4-Ig exerts an immunosuppressive effect on both local and systemic inflammatory mediators which is mediated principally during the sensitization phase.

Topics: Abatacept; Animals; Cells, Cultured; Cytokines; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; Female; Humans; Immune Tolerance; Immunization; Immunoconjugates; Immunotherapy; Inflammation Mediators; Mice; Mice, Inbred BALB C; Oxazolone; Protein Binding; Receptor Cross-Talk

Macrolide antibiotics inhibit the secretion of Th1 cytokines while their effects on the release of Th2 cytokines are variable. We investigated molecular and cellular markers of Th1- and Th2-mediated inflammatory mechanisms and the anti-inflammatory activity of azithromycin and clarithromycin in phorbol 12-myristate 13-acetate (PMA) and oxazolone (OXA)-induced skin inflammation. Dexamethasone (50 μg/ear), azithromycin, and clarithromycin (500 μg/ear) reduced TNF-α and interleukin (IL)-1β concentration in ear tissue by inhibiting inflammatory cell accumulation in PMA-induced inflammation. In OXA-induced early delayed-type hypersensitivity (DTH), the macrolides (2 mg/ear) and dexamethasone (25 μg/ear) reduced ear tissue inflammatory cell infiltration and secretion of IL-4 while clarithromycin also decreased IFN-γ concentration. Macrolides showed better activity when administered after the challenge. In OXA-induced chronic DTH, azithromycin (1 mg/ear) reduced the number of ear tissue mast cells and decreased the concentration of IL-4 in ear tissue and of immunoglobulin (Ig)E in serum. Clarithromycin (1 mg/ear) reduced serum IgE concentration, possibly by a mechanism independent of IL-4, while both macrolides attenuated mast cell degranulation. In conclusion, azithromycin and clarithromycin attenuate pro-inflammatory cytokine production and leukocyte infiltration during innate immune reactions, while selectively affecting Th2 rather than Th1 immunity in DTH reactions.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Azithromycin; Cell Degranulation; Clarithromycin; Dermatitis, Contact; Dexamethasone; Ear; Hypersensitivity, Delayed; Immunoglobulin E; Interferon-gamma; Interleukin-1beta; Interleukin-4; Mast Cells; Mice; Oxazolone; Skin; Tetradecanoylphorbol Acetate; Th2 Cells; Tumor Necrosis Factor-alpha

Pyridoxine (vitamin B(6)) is commonly used as a dietary supplement and beneficial effects of it are expected. However, excess ingestion of pyridoxine has been shown to cause a severe sensory neuropathy in humans and experimental animals. We have been studying the linkage between the nervous and immune systems using a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model. We have found that activation of transient receptor potential ankyrin 1 (TRPA1), which is expressed on sensory neurons, enhances skin sensitization to FITC. Another feature of FITC-induced CHS is its dependence on T helper 2 (Th2) type responses. We hypothesized that the excess intake of pyridoxine may affect sensitization to FITC and influence helper T-cell polarization. We examined FITC-induced CHS in BALB/c mice fed a diet containing excess pyridoxine (120 mg/kg diet) for 3 weeks. We found that mice fed on the excess-pyridoxine diet exhibited a lower response as to FITC-induced CHS compared with ones fed on a diet with a standard pyridoxine content (6.0 mg/kg diet). Moreover, the interferon (IFN)-γ/interleukin (IL)-4 ratio produced by draining lymph node cells was significantly higher with the excess-pyridoxine diet. This suggested that the cytokine balance was shifted toward Th1 with the excess-pyridoxine diet. Consistently, Th1-dependent oxazolone-induced CHS was enhanced with the excess-pyridoxine diet. These results suggested that an excess pyridoxine intake actively influences the immune system by altering helper T cell polarization.

Topics: Animals; Cytokines; Dendritic Cells; Dermatitis, Contact; Diet; Disease Models, Animal; Female; Fluorescein-5-isothiocyanate; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Pyridoxine; Th1 Cells; Th2 Cells; Vitamin B Complex

Irritant contact dermatitis (ICD) is an inflammatory skin disease triggered by exposure to a chemical that is toxic or irritating to the skin. A major characteristic of chronic ICD is an inflammatory dry-skin condition with associated itching. Although glucosylceramide (GlcCer) is known to improve the skin barrier function, its mechanism of action is unknown.. Using a mouse model of oxazolone-induced chronic ICD, this study investigated the effects of oral administration of GlcCer on inflammatory dry skin.. Chronic ICD was induced by repeated application of oxazolone in mice. GlcCer was orally administered once daily throughout the elicitation phase. The beneficial efficacy of GlcCer on cutaneous inflammation was evaluated by assessing ear thickness, lymph node weight, histological findings, and mRNA expression of pro-inflammatory cytokines such as IL-1β and IL-6. Additionally, parameters of the itch-associated response, including scratching behavior, water content of the skin, and aquaporin-3 levels in the lesional ear, were measured.. Oral GlcCer administration significantly suppressed mRNA expression of the pro-inflammatory cytokines IL-1β and IL-6. GlcCer also suppressed ear swelling, lymph node weight gains, and infiltration of leukocytes and mast cells in ICD mice. In oxazolone-induced ICD mice, GlcCer significantly inhibited irritant-related scratching behavior and dehydration of the stratum corneum, and decreased aquaporin-3 expression.. Our results indicate that GlcCer suppressed inflammation not only by inhibiting cytokine production but also by repairing the skin barrier function, suggesting a potential beneficial role for GlcCer in the improvement of chronic ICD.

Topics: Administration, Oral; Animals; Aquaporin 3; Dermatitis, Contact; Dermatitis, Irritant; Ear; Glucosylceramides; Inflammation; Interleukin-1beta; Interleukin-6; Lymph Nodes; Male; Mice; Mice, Inbred BALB C; Models, Biological; Oxazolone; RNA, Messenger; Skin

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.

Topics: Animals; B-Lymphocyte Subsets; Biomarkers; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dermatitis, Contact; Immunoglobulin M; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Interleukin-5; Interleukins; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Mice, Nude; Oxazolone; Receptors, Interleukin

Neonates have a developing immune response, with a predisposition towards induction of tolerance. As the immune system develops, immunity rather than tolerance is induced, with this development of immunity occurring in response to external factors such as the environment. As ultraviolet radiation (UVR) suppresses immunity, it is likely that the effect of UVR on the neonatal immune system would be augmentation of the suppressive response. In support, childhood exposure to UVR has been linked with an increased incidence of melanoma; consistent with an increase in suppression. To address this, phenotypic and functional immune system studies were undertaken at 8 weeks after one single exposure of solar-simulated UVR to mice, when mice had reached adulthood. Subtle changes were observed in cell populations resident in the skin-draining lymph nodes (LNs) and there also appeared to be a subtle, but not statistically significant, increase in the production of interleukin-10 and interferon-γ. Importantly, these changes also corresponded with significant suppression of the contact hypersensitivity response in irradiated mice compared with their control counterparts. This suppression was apparent when antigen sensitisation occurred during the neonatal or adult period, and thus did not appear to be analogous to UVR-induced suppression in adults. Although the percentage of T regulatory cells was increased in the skin-draining LNs, they were induced in a different manner to those induced following adult UVR exposure, with no increase in function on a per-cell basis. It therefore appears that one single neonatal exposure to UVR alters development of the immune system, leading to long-term implications for induction of immunity.

Topics: Animals; Animals, Newborn; Cell Proliferation; Cells, Cultured; Dermatitis, Contact; Female; Hypersensitivity; Immune System; Immune Tolerance; Interferon-gamma; Interleukin-10; Lymph Nodes; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Oxazolone; Skin; T-Lymphocytes, Regulatory; Ultraviolet Rays

Inflammatory mediators released from mast cells (MCs) through engagement of the high-affinity receptor for IgE (FcepsilonRI) have pivotal roles in chemical allergen-induced contact hypersensitivity (CHS) reactions, which suggests that the blockade of MC activation through FcepsilonRI stimulation may attenuate allergic contact dermatitis (CD). To address this possibility, we employed the following two approaches: (i) modulation of FcepsilonRI-mediated MC activation by introducing mutations in tyrosine residues of the FcepsilonRI beta-chain immunoreceptor tyrosine-based activation motif (ITAM) and (ii) blockade of FcepsilonRI-mediated MC activation employing a recombinant soluble ecto-domain of the human FcepsilonRIalpha-chain (rsFcepsilonRIalpha). In this study, we show that optimal MC activation through the FcepsilonRI beta-chain ITAM has essential roles in the onset of CHS to oxazolone (Oxa), a well-characterized chemical allergen. In addition, we demonstrate that administration of the rsFcepsilonRIalpha after sensitization successfully prevents murine CHS to Oxa. In a chronic CD model elicited by multiple challenges with low-dose Oxa, application of the rsFcepsilonRIalpha during the course of the challenges showed suppressive effects on CHS to Oxa. Taken together, our data indicate that inhibition of FcepsilonRI-dependent MC activation can suppress allergic CD.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Bone Marrow Cells; CHO Cells; Cricetinae; Cricetulus; Dermatitis, Contact; Female; Immunoglobulin E; Male; Mast Cells; Mice; Mice, Inbred C57BL; Mutagenesis; Oxazolone; Receptors, IgE; Transfection

Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo.. To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy.. A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized.. T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation.. Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; Cell Migration Inhibition; Cell Movement; Chemokines; Dermatitis, Contact; Female; Mice; Mice, Inbred BALB C; Mice, SCID; Models, Animal; Oxazolone; Receptors, CCR10; Receptors, CCR4; Skin; Statistics as Topic

Propolis is a honeybee product that has been used in traditional medicine for antioxidant, immune-stimulating, anti-inflammatory and anti-cancer effects. Here, the potential of the topical application of a crude ethanolic extract of Sydney propolis to protect against UV-radiation-induced impairments associated with an increased risk of photocarcinogenesis has been tested in the hairless mouse.. Solutions providing between 10 and 200 mg/kg propolis were applied to the skin following UV irradiation. The inflammation from exposure to UV (290-400 nm) was quantitated by measurement of increased skinfold thickness; lipid peroxidation was assayed by the induction of thiobarbituric acid reactive species in the skin; immune function was measured by the contact hypersensitivity (CHS) reaction and supported by the changes in epidermal cytokine expression.. Propolis protected significantly and dose-dependently against both sunburn oedema and the suppression of CHS, and (at 100 mg/kg) against lipid peroxidation. The overexpression of IL-10 and the depletion of IL-12 characteristic of photoimmune suppression were markedly reduced by propolis. Further, the upregulation of IL-6 was decreased, and the associated induction of haem oxygenase was shown to play a role in propolis skin protection.. Sydney propolis was able to effectively reduce cutaneous inflammation, immunosuppression and lipid peroxidation induced by UV exposure. It is concluded that Sydney propolis might have strong beneficial protective effects against photodamage and skin cancer development in humans.

Topics: Animals; Cytokines; Dermatitis, Contact; Enzyme Inhibitors; Female; Flavonoids; Heme Oxygenase-1; Immunosuppression Therapy; Lipid Peroxidation; Metalloporphyrins; Mice; Mice, Hairless; Oxazolone; Propolis; Protoporphyrins; Radiodermatitis; Skin; Skinfold Thickness; Sunburn; Terpenes; Thiobarbituric Acid Reactive Substances

Glucocorticoids (GC) are highly potent anti-inflammatory agents frequently administered in clinical medicine. However, even for the most potent GC dexamethasone, only modest effects have been observed in several murine studies. Here we demonstrate that intraperitoneal administration of dexamethasone displays no anti-inflammatory activity in two different mouse models. Low doses of topically applied dexamethasone entirely prevented ear swelling in a contact hypersensitivity model in BALB/c mice, while intraperitoneally injected dexamethasone had no effect on disease progression. Moreover, subcutaneously administered dexamethasone completely inhibited lipopolysaccharide (LPS)-mediated lethality in C57BL/6 mice. In contrast, even ultra-high doses of intraperitoneally injected dexamethasone could not prevent endotoxin-induced death. In conclusion, these results demonstrate that intraperitoneal application of dexamethasone is ineffective in these models of inflammation, which has broad implications for mouse models evaluating the in vivo efficiency of GCs.

Topics: Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Dexamethasone; Disease Models, Animal; Disease Progression; Drug Administration Routes; Endotoxemia; Immunity; Inflammation; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oxazolone

Skin-infiltrating T-cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis, psoriasis and allergic contact dermatitis. These T-cells are attracted by several chemotactic factors including the chemokine CCL5/RANTES, a CC chemokine inducing both the migration and activation of specific leukocyte subsets. CCL5 has been found to be associated with various cell-mediated hypersensitive disorders such as psoriasis, atopic dermatitis and irritant contact dermatitis. We have used two antagonists, the first, Met-CCL5, a dual CCR1/CCR5 antagonist and the second, a variant in which GAG binding is abrogated, (44)AANA(47)-CCL5, which acts as a dominant negative inhibitor of CCL5. The antagonists were tested in two models of contact skin reaction. The first, irritant contact dermatitis (ICD) is a pathological non-specific inflammatory skin condition arising from the release of pro-inflammatory cytokines by keratinocytes in response to haptens, usually chemicals. The second, contact hypersensitivity (CHS) is a T-cell dependent model, mimicking in part the T-cell-mediated skin diseases such as psoriasis. In both models, the CCL5 antagonists showed therapeutic efficacy by reducing swelling by 50% as well as the reduction of soluble mediators in homogenates derived from challenged ears. These results demonstrate that blocking the receptor or the ligand are both effective strategies to inhibit skin inflammation.

Topics: Animals; Cells, Cultured; Chemokine CCL5; Dermatitis, Contact; Disease Models, Animal; Female; Immunohistochemistry; Mice; Oxazolone

IL-1 is a prototypic inflammatory cytokine that has pathogenic roles in various skin disorders. Although Langerhans cells (LCs) have been reported to express IL-1beta mRNA upon application of contact sensitizers, it remains unclear whether other cell types produce IL-1beta in skin. Thus, we sought to directly identify IL-1beta-producing cells in living animals by construction of transgenic mice expressing DsRed fluorescence protein gene under the control of IL-1beta promoter. Little DsRed fluorescence signal was detected in skin under steady-state conditions. Striking increases in DsRed signal were observed after topical application of a contact sensitizer, oxazolone, which also induced markedly elevated IL-1beta mRNA and protein expression. DsRed signal was expressed primarily by CD45(+)/CD11b(+) myeloid leukocytes in both epidermal and dermal compartments and was detected only in small fractions of epidermal LCs. Interestingly, DsRed(+) cells emerged preferentially as clusters around hair follicles. Intravital confocal imaging experiments revealed highly motile potentials of DsRed(+) cells-they constantly crawled around hair follicles via amoeba-like movements with a mean velocity of 1.0+/-0.4 microm min(-1) (epidermis) or 2.7+/-1.4 microm min(-1) (dermis). The newly developed in vivo imaging system represents a useful tool for studying spatial regulation of IL-1beta production in skin.

Topics: Animals; Arthritis; Cells, Cultured; Dermatitis, Contact; Disease Models, Animal; Interleukin-1beta; Lipopolysaccharides; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Oxazolone; Peritonitis; Skin; Zymosan

The anti-inflammatory effect of physalin E, a seco-steroid isolated from Physalis angulata L. was evaluated on acute and chronic models of dermatitis induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and oxazolone, respectively, in mouse ear. The changes in ear edema/thickness, production of pro-inflammatory cytokines (TNF-alpha and IFN-gamma), myeloperoxidase (MPO) activity, and histological and immunohistochemical findings were analysed, as indicators of dermal inflammation. Similar to dexamethasone, topically applied Physalin E (0.125; 0.25 and 0.5 mg/ear) potently inhibited the TPA and oxazolone-induced dermatitis, leading to substantial reductions in ear edema/thickness, pro-inflammatory cytokines, and MPO activity. These effects were reversed by mifepristone, a steroid antagonist and confirmed by immunohistochemical and histopathological analysis. The data suggest that physalin E may be a potent and topically effective anti-inflammatory agent useful to treat the acute and chronic skin inflammatory conditions.

Topics: Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Immunohistochemistry; Male; Mice; Oxazolone; Physalis; Secosteroids; Tetradecanoylphorbol Acetate

Using ELISA, we have quantified the levels of IL-2 and IFN-gamma in the oral mucosa, ear skin and regional and distant lymph nodes in an experimental murine model of contact sensitivity (CS), induced by the hapten oxazolone (OXA). Compared to normal conditions, the levels of IL-2 peaked early (4-6 h) after hapten exposure in the hapten-exposed tissues analysed both during the first hapten exposure (sensitization) and the second (elicitation) phase, thereafter quickly to subside. The oral mucosa displayed maximal 24-fold increase in IL-2 levels after sensitization and 39-fold increase after elicitation. Respective figures for ear skin were x27 and x35 and for regional lymph nodes x8 and x9, respectively. The distant lymph nodes displayed only minor cytokine increases at any time. IFN-gamma-levels did not increase after sensitization with OXA. An increase in IFN-gamma was seen after the second exposure, peaking at 8-24 h, thereafter quickly subsiding. The oral mucosa IFN-gamma increased x14 after elicitation, the ear skin x8 and regional lymph nodes x37. The weight of the four regional lymph nodes increased from 10 to 38 mg, and the total number of cells in these lymph nodes was increased x11, peaking 48 h after the elicitation. We conclude that in CS reactions, tissue levels of IL-2 increased in buccal mucosa, ear skin and in regional lymph nodes after hapten exposure and re-exposure, IFN-gamma appeared only after re-exposure to the hapten. The increased weight of the regional lymph nodes was mainly attributed to cell proliferation. The common ectodermal origin and the similarity of the CS reactions on skin and in buccal mucosa indicate that these tissues share common immunological patterns of Th1 cell reactivity, at least in dealing with haptens like OXA.

Topics: Animals; Dermatitis, Contact; Enzyme-Linked Immunosorbent Assay; Female; Interferon-gamma; Interleukin-2; Kinetics; Lymph Nodes; Male; Mice; Mice, Inbred C3H; Mouth Mucosa; Organ Size; Oxazolone; Skin

Contact hypersensitivity (CHS) reaction is a form of delayed-type of hypersensitivity caused by contact allergens such as oxazolone (OXA). In previous studies it has been shown that poly(ADP-ribose) polymerase (PARP) inhibition reduces the extent of inflammation in CHS. We aimed to shed light on the molecular events causing the protective effect of PARP inhibitors. PARP-1 and -2 knockout mice were sensitized by abdominal delivery of OXA, and a week later CHS was induced by applying OXA on the ears of the mice. PARP-1(-/-) mice were protected against OXA-induced CHS in contrast to PARP-2(-/-) mice. In PARP-1(-/-) mice, neutrophil infiltration was reduced in line with the suppressed expression of proinflammatory cytokines, cell adhesion factors, and matrix metalloproteinase-9, which is likely because of impaired activation of NF-κB p65 and activating transcription factor-2, the two redox-sensitive transcription factors. Moreover, reduced nitrosative and oxidative stress was observed under inflammatory conditions in the PARP-1(-/-) mice when compared with PARP-1(+/+). In conclusion, PARP-1 activation is necessary for proinflammatory gene expression through which PARP-1 enhances neutrophil infiltration and hence oxidative/nitrosative stress, forming a vicious circle, and further aggravating the inflammatory process. Our data identify PARP-1 as a possible target in CHS.

Topics: Adjuvants, Immunologic; Animals; Cell Movement; Dermatitis, Contact; Female; Gene Deletion; Irritants; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophil Activation; Oxazolone; Oxidative Stress; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reactive Nitrogen Species; Reactive Oxygen Species

Chemokine receptor CCR4 is expressed by Th2 cells and is involved in the recruitment of inflammatory cells into the skin. We studied the effects of CCR4 deficiency in the murine model of oxazolone-induced contact hypersensitivity in CCR4-/- and wild-type (WT) mice. The inflammatory response in the skin at 24  hours post-elicitation was stronger in CCR4-/- mice compared with WT, evidenced by increased ear swelling and inflammatory cell infiltration. In addition, the mRNA expression levels of several cytokines, chemokines, chemokine receptors, and selectins in the skin of CCR4-/- mice were significantly elevated compared with WT mice. Time kinetic experiments during the sensitization and elicitation phases revealed that the number of CD3+CD4+ cells in CCR4-/- mice remained high longer during the sensitization phase and increased more rapidly during the elicitation phase compared with WT mice. These data demonstrate that the absence of CCR4 results in enhanced secondary immune response during allergic skin inflammation.

Topics: Adjuvants, Immunologic; Animals; CD3 Complex; CD4 Antigens; Dermatitis; Dermatitis, Contact; Disease Models, Animal; Edema; Interleukin-13; Mice; Mice, Mutant Strains; Oxazolone; Receptors, CCR4; T-Lymphocytes, Regulatory; Up-Regulation

To ascertain the influence of vitamin D3 and its metabolites on the function of the skin immune system and the induction of the contact hypersensitivity (CHS) response, a population of vitamin D3-deficient BALB/c mice was established, through dietary vitamin D3 restriction and limitation of exposure to UVB irradiation. Vitamin D3 normal female mice had higher CHS responses than their male counterparts, and dietary vitamin D3 deficiency significantly increased the CHS responses in male, but not in female, mice. This change in the vitamin D3-deficient male mice was not due to an alteration in skin dendritic cell function including antigen carriage, migration or costimulatory molecule expression. In addition, 18 h after sensitisation, the lymph node populations in the vitamin D3-deficient and normal male mice showed similar proliferation and IFN-gamma production. However, during the sensitisation phase of CHS, there was lower lymphocyte recruitment to the skin draining lymph nodes of the vitamin D3-deficient and normal male mice compared with their female counterparts which could account for the difference between the sexes in the extent of the CHS response. These results indicate the vitamin D system can influence cutaneous immune responses in male mice, but this did not occur through the modulation of the dendritic cell functions analysed.

Topics: Adjuvants, Immunologic; Animals; Antigens; Cell Proliferation; Cells, Cultured; Cholecalciferol; Cytokines; Dermatitis, Contact; Diet; Female; Humans; Interferon-gamma; Lymph Nodes; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Oxazolone; Skin; Ultraviolet Rays; Vitamin D Deficiency

The local lymph node assay (LLNA) is widely used to identify chemicals that are contact sensitizers. The assay involves dosing mice with the chemical on both ears and pooling the superficial parotid lymph nodes for assessment of lymphocyte proliferation as a marker of sensitization. The present study explored potential reduction in animal usage by dosing one ear with the allergen and the other with vehicle-only. The respective draining lymph nodes were processed separately for tritiated thymidine ((3)H-TdR) incorporation. Cell proliferation in proper axillary and renal nodes, as well as in the spleen was also assessed. Cross-contamination of the chemicals from the dosed ears to other parts of the body via preening was prevented by dosing restrained animals and washing off the residual chemical with saline after 4h. Dosing the left ear with 0.02% oxazolone (OX) on unrestrained animals resulted in marked cell proliferation in its draining lymph node (stimulation index, SI=12.8) and in the lymph node draining the contra-lateral vehicle-dosed ear (SI=6), as well as the proper axillary lymph nodes (SI=3.3). Increased (3)H-TdR incorporation was not observed in the renal lymph nodes (SI=1.1). Similar stimulation of cells was observed in the lymph node draining the ear contra-lateral to the 30% hexylcinnamaldehyde (HCA)-dosed ear. Increased proliferative activity was observed in contra-lateral draining lymph nodes of restrained mice demonstrating that these results cannot be attributed to cross-contamination of adjacent skin. A significant increase in proliferation of splenocytes was also observed. It is concluded that dermal application of a contact allergen, as exemplified by OX and HCA, may induce cell proliferation in the neighboring lymph nodes and spleen indicative of hapten and/or haptenated proteins diffusing through the skin to peripheral nodes and the blood to produce systemic sensitization. It is also possible that lymphatic capillaries may communicate between the left and right side of the mouse head. Thus the contra-lateral draining superficial parotid node cannot be used as a control for application of contact allergen to a single ear in a modified LLNA.

Topics: Allergens; Animals; Cell Proliferation; Dermatitis, Contact; Female; Local Lymph Node Assay; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Spleen; Terminology as Topic; Thymidine

Ketoprofen (KP) is a widely used nonsteroidal anti-inflammatory drug that inhibits prostaglandin biosynthesis. We have previously shown that topical KP treatment at the sensitizing site inhibits the development of contact hypersensitivity (CHS) to picryl chloride (PCl).. We investigated the mechanism underlying the KP-induced immunosuppression of CHS by application of KP.. We analyzed the CHS responses to the non-sensitizing site and subsequent sensitization with PCl, and by transfer of the draining lymph node cells (LNCs) from KP-tolerated mice to recipient mice. Changes in the Foxp3 expression of LNCs from KP-phototreated skin were also examined by real-time PCR.. Topical application of KP to not only the sensitizing but also non-sensitizing site suppressed CHS response. The immunosuppression was transferred with LNCs from mice treated with PCl plus KP, but not from mice treated oxazolone plus KP. In this transfer study, the CD4(+) CD25(+) subset of LNCs exerted the suppressive effect, while CD25(+) cell-depleted LNCs lost the inhibitory ability. CTLA-4 blocking with a specific antibody, but not IL-10 blocking, abrogated the activity of CD4(+) CD25(+) cells. Moreover, Foxp3 mRNA expression was remarkably increased in LNCs from PCl and KP-treated mice.. The immunosuppression of CHS by topical application of KP is systemic and haptein-specific. Treg cells play an important role in the suppressive effect by KP.

Topics: Adjuvants, Immunologic; Administration, Topical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; CD4 Antigens; CTLA-4 Antigen; Dermatitis, Contact; Dinoprostone; Female; Forkhead Transcription Factors; Haptens; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Ketoprofen; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Picryl Chloride; T-Lymphocytes, Regulatory

It is often difficult to discriminate between chemically induced skin irritation and sensitization due to their similar clinical, pathological, and immunological responses. More information than that currently available from local lymph node assays (LLNAs), such as data from gene expression and pathway analysis, can provide more insightful data than the assay itself for distinguishing skin sensitization from skin irritation. This study investigated the gene expression profiles and pathways in ear skins of mice topically exposed daily for three consecutive days to the known strong contact sensitizer 1-chloro-2,4-dinitrobenzene, the skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone, the skin or respiratory sensitizer toluene 2,4-diisocyanate, or to the non-sensitizing irritant croton oil. All the sensitizers induced histological changes in ear tissues similar to those induced by the croton oil. In gene expression microarrays, sensitizers up-regulated 193 genes and down-regulated 61 genes in ear skin following chemical exposure. 13 genes whose expression was affected by more than two-fold by all three of the sensitizers, but not by the irritant, were selected by microarray analysis. Microarray and real-time RT-PCR analyses revealed that, of these genes, the allergic inflammation-related genes Oasl2 and Zbp1 were up-regulated in skin inflammation by the sensitizers. In gene expression pathway analysis of all the sensitizers and the croton oil, the top functions of the 48 genes were related to cytokine and cytokine receptors interactions, and only two genes (Cxcl9 and Cxcl10) were specific to skin sensitizer-induced skin inflammation. Thus, although contact sensitizer-induced skin inflammation is similar to irritant-induced responses in terms of histological changes and gene expression profiles, the regulation of allergic inflammation-related gene transcripts, such as those of Oasl2 and Zbp1 or Cxcl9 and Cxcl10, could help to discriminate skin sensitization from chemically induced skin inflammation.

Topics: Allergens; Animals; Cytokines; Dermatitis, Allergic Contact; Dermatitis, Contact; Dinitrochlorobenzene; DNA Primers; Ear, External; Female; Gene Expression Profiling; Irritants; Mice; Mice, Inbred CBA; Oligonucleotide Array Sequence Analysis; Oxazolone; Receptors, Cytokine; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Skin; Toluene 2,4-Diisocyanate

Transforming growth factor (TGF)-beta is an important modulator of immune functions and cellular responses, such as differentiation, proliferation, migration and apoptosis. The Smad proteins, which are intracellular TGF-beta signal transducers, mediate most actions of TGF-beta.. This study examines the role of Smad3 in a murine model of contact hypersensitivity (CHS).. The CHS response to oxazolone was studied in Smad3-deficient mice. The ear swelling response was measured and skin biopsies from oxazolone-sensitized skin areas were obtained for RNA isolation, immunohistochemical analyses and histology. Ear draining lymph nodes were collected for RNA isolation and proliferation tests. Quantitative real-time polymerase chain reaction was used to quantify mRNA expression of cytokines, chemokines and transcription factors. Results The expression of proinflammatory [interleukin (IL)-1beta, tumour necrosis factor-alpha, IL-6], Th2 (IL-4) and Th17 type cytokines (IL-17), as well as regulatory components (TGF-beta, Foxp3) increased significantly at the mRNA level in the skin of oxazolone-treated Smad3-/- mice when compared with wild-type controls. The expression of the Th1 type cytokine IFN-gamma and the chemokines CXCL9 and CXCL10 was, however, unaffected by the lack of Smad3. The number of neutrophils and expression of the chemokines CCL3 and CXCL5, which are both involved in neutrophil recruitment, were increased in mice lacking Smad3. Also Th2 type chemokines CCL24, CCL3 and CXCL5 were increased in the skin of Smad3-/- mice compared with wild-type mice. In the lymph nodes, mRNA of IL-1beta and IL-17, but not IL-4, TGF-beta or Foxp3, was increased in Smad3-/- mice during the CHS response.. The lack of intact TGF-beta signalling via Smad3 results in an increased proinflammatory, Th2 and Th17 type response in the skin, as well as increased expression of regulatory elements such as TGF-beta and Foxp3. Understanding the role of Smad3 in the CHS response may offer treatment and prevention strategies in this often disabling disease.

Topics: Adjuvants, Immunologic; Animals; Biomarkers; Chemokines; Dermatitis, Contact; Female; Gene Expression; Immunohistochemistry; Interleukin-10; Interleukin-17; Interleukin-1beta; Interleukin-4; Interleukin-6; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Neutrophil Infiltration; Oxazolone; RNA, Messenger; Signal Transduction; Skin; Smad3 Protein; Statistics, Nonparametric; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The diterpene compounds, centipedic acid (CA) and 12-acetoxyhawtriwaic acid lactone (AHAL, tanabalin) isolated from the flower buds of Egletes viscosa LESS. (Asteraceae) were evaluated on acute and chronic models of mouse ear dermatitis. A single topical application of CA (0.125; 0.25 and 0.5 mg/ear) or AHAL (0.125, 0.25, 0.5 mg/ear) immediately before 12-O-tetradecanoylphorbol-13-acetate (TPA, 2.5 mug/ear) caused a dose-related significant inhibition of ear inflammatory edema and influx of polymorphonuclear cells, as evidenced by a decrease in ear thickness and reduced myeloperoxidase (MPO) activity and tumor necrosis factor-alpha (TNF-alpha) in ear tissue homogenates. The maximal obtained inhibition for both ear edema and neutrophil influx were almost similar to that of topically applied dexamethasone (0.05 mg/ear). The extent of inhibitions for the respective treatments of CA (0.5 mg/ear), AHAL (0.5 mg/ear), or dexamethasone (0.05 mg/ear) were in the order of 63%, 61% and 81% for the ear edema, and 90%, 95% and 95% for the neutrophil influx. Also, at similar doses, both diterpenes and dexamethasone effectively inhibited the delayed-type hypersensitivity reaction induced by repeated topical application of 1% oxazolone (OXA, 20 microl/ear), as evidenced by significant decreases in ear thickness and interferon-gamma (INF-gamma) levels in ear tissue. Histopathological analysis revealed a marked decrease in epidermal hyperplasia and neutrophil infiltration in animals pretreated with CA or AHAL, in a manner similar to dexamethasone. These data provide evidence for the anti-dermatitis effect of Egletes viscosa diterpenes, by mechanisms that involve a reduced neutrophil influx and decreased production of inflammatory cytokines, TNF-alpha and IFN-gamma.

Topics: Acute Disease; Administration, Topical; Animals; Anti-Inflammatory Agents; Asteraceae; Chronic Disease; Dermatitis, Contact; Diterpenes; Ear, External; Fatty Acids, Unsaturated; Flowers; Furans; Interferon-gamma; Lactones; Male; Mice; Oxazolone; Peroxidase; Skin; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

CCR6 is expressed in a number of dermatological inflammatory diseases. Here, we report that mice sensitized with the hapten oxazolone had increased numbers of CCR6+ T cells in the draining lymph nodes. Using CCR6-/- mice, we assessed the role of CCR6 on the development of contact hypersensitivity. After hapten sensitization and re-challenge, ear swelling in CCR6-/- animals was reduced 80% as compared with wild-type (WT) control mice. This decreased level of inflammation was not related to an inhibition in T-cell activation, because CCR6-/- lymph node cells from sensitized mice produced threefold higher levels of IFN-gamma in culture than cells from sensitized WT mice and, when these cells were directly injected into the site of hapten challenge, induced a robust inflammatory response. However, intravenous injection of CCR6-/- lymph node cells from sensitized mice were unable to prime naive mice to re-challenge whereas cells from primed WT mice were able to sensitize animals. These results suggest that CCR6 plays an important role in directing the trafficking of activated T cells into the skin and suggests that a CCR6 antagonist could be useful to treat skin-mediated inflammatory reactions.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Cell Movement; Dermatitis, Contact; Gene Expression; Haptens; Interferon-gamma; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Receptors, CCR6; T-Lymphocytes

1-methylnicotinamide (MNA) displays anti-inflammatory effects in patients with contact dermatitis, though the mechanisms involved remain unknown. Herein, we examined the anti-inflammatory effects of MNA and its parent molecule, nicotinamide, in the contact hypersensitivity reaction to oxazolone in CBA/J inbred mice. Feeding mice with MNA or nicotinamide (100 mg/kg, 10 days) resulted in the inhibition of the development of contact hypersensitivity reaction by 37% and 35%, respectively, as assessed by the magnitude of ear swelling. This effect was not associated with changes in the expression of adhesion molecules (CD49d(+) and CD54(+)) on CD4(+) and CD8(+) oxazolone-specific T lymphocytes, the major cell component of an inflammatory infiltrate in contact hypersensitivity reaction. Furthermore, in the adoptive transfer model of contact hypersensitivity reaction, pretreatment of mice (recipients of oxazolone-specific T cells), with MNA, resulted in a remarkable anti-inflammatory effect (inhibition of contact hypersensitivity reaction by 66%). Interestingly, in the presence of prostanoid IP receptor antagonist R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO-3244794) (10 mg/kg) the MNA was inactive. In summary, pretreatment with MNA profoundly attenuated contact hypersensitivity reaction in vivo. In particular, the vessel dependent phase of contact hypersensitivity reaction was affected, in spite of the fact that MNA did not alter the expression of adhesive molecules on oxazolone-specific T lymphocytes. However, the anti-inflammatory action of MNA was completely reversed by the antagonist of prostanoid IP receptor. Accordingly, our results demonstrate for the first time that anti-inflammatory properties of MNA are linked to endothelial, PGI(2)-mediated mechanisms.

Topics: Adoptive Transfer; Animals; Anti-Inflammatory Agents; Benzofurans; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dermatitis, Contact; Dermatologic Agents; Disease Models, Animal; Endothelium, Vascular; Epoprostenol; Integrin alpha4; Intercellular Adhesion Molecule-1; Male; Mice; Niacinamide; Oxazolone; Propionates; Receptors, Epoprostenol; Receptors, Prostaglandin; Skin

A previous study in the hairless mouse, in which the photoimmune protective properties of a topical phytoestrogen or 17-beta-estradiol were abrogated by the estrogen receptor antagonist ICI 182,780, revealed that estrogen receptor (Er) signaling is involved in the regulation of the suppression of immune function by UVB (290-320 nm) radiation. Here we identify the expression of Er-beta but not Er-alpha mRNA in hairless mouse skin, whereas Er-alpha and Er-beta mRNA were present in normal haired mouse skin. This suggests that the non-classical estrogen target Er-beta is involved in the photoimmune modulation, and is consistent with Er-alpha being more closely associated with hair growth control, as indicated by other studies. In mice with a null mutation for Er-beta, there was a significant exacerbation of the solar simulated UV (290-400 nm)-induced suppression of contact hypersensitivity. Immunohistochemical analysis revealed that the Er-beta deficiency inhibited the normally immunoprotective upregulation by the UVA (320-400 nm) waveband of the epidermal expression of the cytokines IFN-gamma and IL-12. Er-beta deficiency also significantly increased the UVB-induced expression of the immunosuppressive cytokine IL-10. Thus Er signalling via the Er-beta is evidently a major regulator of the UVA and UVB waveband interactions that determine the skin's immune functional status, and achieves this by normalization of the cutaneous cytokine array in the UV-irradiated skin.

Topics: Animals; Cytokines; Dermatitis, Contact; Epidermis; Estrogen Receptor beta; Female; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; RNA, Messenger; Signal Transduction; Ultraviolet Rays

While psoriasis is one of the most common skin disorders in humans, effective, safe and inexpensive treatments are still largely unavailable. Chinese herbal medicine (CHM) has been used for centuries for treating psoriasis and several reports claim that systemic administration of one such CHM, Tuhuai, mainly composed of flos sophorae, smilax glabra roxb and licorice, is effective in psoriasis. However, the mechanisms by which this CHM improves psoriasis are not yet clear. Two universal features of psoriasis are epidermal hyperplasia and inflammation. Moreover, drugs that specifically inhibit epidermal hyperplasia and/or inflammation are widely used to treat psoriasis. Here, we investigated whether topical applications of Tuhuai extract exhibit anti-proliferative and anti-inflammatory activities in two murine models of inflammatory dermatoses. To assess Tuhuai's potential anti-proliferative effect, we disrupted epidermal barrier function twice-daily for 4 days in normal hairless mice followed by topical applications of either 1% Tuhuai extract or Vehicle to both flanks immediately after each barrier perturbation. Changes in epidermal proliferation and apoptosis were evaluated by immunohistochemistry and TUNEL staining. To assess the anti-inflammatory effects of Tuhuai, both irritant (phorbol ester) and acute allergic contact dermatitis (oxazolone) models were used. Whereas topical Tuhuai extract did not alter epidermal proliferation or induce irritation in normal skin, it both reduced epidermal hyperplasia in the epidermal hyperproliferative model, and reduced inflammation in both irritant and allergic contact dermatitis models. As topical Tuhuai extract exhibits anti-proliferative and anti-inflammatory properties in a variety of human models of inflammatory dermatoses, Tuhuai could provide an effective, relatively safe and inexpensive therapeutic alternative for the treatment of inflammatory dermatoses, including psoriasis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Proliferation; Dermatitis, Allergic Contact; Dermatitis, Contact; Disease Models, Animal; Drugs, Chinese Herbal; Fabaceae; Female; Glycyrrhiza; Humans; Hyperplasia; Male; Mice; Mice, Hairless; Oxazolone; Phytotherapy; Psoriasis; Skin; Smilax; Tetradecanoylphorbol Acetate

Oatmeal has been used for centuries as a soothing agent to relieve itch and irritation associated with various xerotic dermatoses; however few studies have sought to identify the active phytochemical(s) in oat that mediate this anti-inflammatory activity. Avenanthramides are phenolic compounds present in oats at approximately 300 parts per million (ppm) and have been reported to exhibit anti-oxidant activity in various cell-types. In the current study we investigated whether these compounds exert anti-inflammatory activity in the skin. We found that avenanthramides at concentrations as low as 1 parts per billion inhibited the degradation of inhibitor of nuclear factor kappa B-alpha (IkappaB-alpha) in keratinocytes which correlated with decreased phosphorylation of p65 subunit of nuclear factor kappa B (NF-kappaB). Furthermore, cells treated with avenanthramides showed a significant inhibition of tumor necrosis factor-alpha (TNF-alpha) induced NF-kappaB luciferase activity and subsequent reduction of interleukin-8 (IL-8) release. Additionally, topical application of 1-3 ppm avenanthramides mitigated inflammation in murine models of contact hypersensitivity and neurogenic inflammation and reduced pruritogen-induced scratching in a murine itch model. Taken together these results demonstrate that avenanthramides are potent anti-inflammatory agents that appear to mediate the anti-irritant effects of oats.

Topics: Animals; Avena; Cells, Cultured; Dermatitis, Contact; Disease Models, Animal; Diterpenes; Flavonoids; Humans; Inflammation; Interleukin-8; Keratinocytes; Mice; Mice, Inbred ICR; NF-kappa B; ortho-Aminobenzoates; Oxazolone; Phenols; Phytotherapy; Polyphenols; Pruritus; Signal Transduction

The role that mast cells play during contact hypersensitivity (CS) response is unclear because some studies have shown that mast cell-deficient mice have relatively intact CS responses whereas others have shown opposing results. Mast cells secrete a wide range of immunomodulatory mediators and can potentially influence the type of immune response generated in the skin during CS. Therefore, we examined the type of microenvironment generated during CS in both W/Wv mast cell-deficient and wild-type mice in response to different immunizing doses of hapten (oxazolone). The CS response elicited after low-dose oxazolone was significantly diminished in W/Wv mice compared with wild-type mice. Unexpectedly, the CS response elicited in W/Wv mice immunized with high-dose oxazolone was more severe compared with wild-type mice. In addition, after immunization with high-dose oxazolone, the granulocyte infiltrate in W/Wv mice was increased by twofold compared with wild-type mice. A shift in the cytokine milieu toward the expression of type-1 cytokines as well as a significant increase in the local adhesion of neutrophils and CD4 T cells in the microvasculature of the skin was observed after hapten challenge in W/Wv mice immunized with high-dose oxazolone compared with wild-type mice. These results suggest that mast cells can act as regulators and inducers of the inflammatory response depending on immunizing stimulus strength.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Adhesion; Cells, Cultured; Cytokines; Dermatitis, Contact; Haptens; Mast Cells; Mice; Mice, Inbred BALB C; Microcirculation; Oxazolone; Skin

The hyaluronan receptor LYVE-1 is expressed abundantly on the surfaces of lymphatic vessels and lymph node sinus endothelial cells from early development, where it has been suggested to function both in cell adhesion/transmigration and as a scavenger for hyaluronan turnover. To investigate the physiological role(s) of LYVE-1, we generated mice in which the gene for the receptor was inactivated by replacement with a beta-galactosidase reporter. LYVE-1(-/-) mice displayed an apparently normal phenotype, with no obvious alteration in lymphatic vessel ultrastructure or function and no apparent change in secondary lymphoid tissue structure or cellularity. In addition, the levels of hyaluronan in tissue and blood were unchanged. LYVE-1(-/-) mice also displayed normal trafficking of cutaneous CD11c(+) dendritic cells to draining lymph nodes via afferent lymphatics and normal resolution of oxazolone-induced skin inflammation. Finally, LYVE-1(-/-) mice supported normal growth of transplanted B16F10 melanomas and Lewis lung carcinomas. These results indicate that LYVE-1 is not obligatory for normal lymphatic development and function and suggest either the existence of compensatory receptors or a role more specific than that previously envisaged.

Topics: Animals; beta-Galactosidase; Carcinoma, Lewis Lung; CD11c Antigen; Cell Movement; Dendritic Cells; Dermatitis, Contact; Glycoproteins; Hyaluronic Acid; Inflammation; Lymph Nodes; Lymphatic Vessels; Melanoma; Membrane Transport Proteins; Mice; Mice, Knockout; Neoplasm Transplantation; Oxazolone

Chronic contact hypersensitivity (CH) models induced by repeated hapten exposure exhibit chronic dermatitis and immunological abnormalities resembling atopic dermatitis. To assess the contribution of endothelial selectins (P- and E-selectins) to cutaneous chronic inflammation, chronic CH responses were assessed in mice lacking P- or E-selectin. Elicitation with oxazolone on the ears of P-selectin(-/-) mice 7 days after the sensitization induced a typical delayed-type hypersensitivity response similar to that found in wild-type mice. By contrast, a significant increase in ear swelling was observed in E-selectin(-/-) mice 36 to 48 hours after first elicitation. E-selectin(-/-) mice showed augmented P-selectin up-regulation, and administration of anti-P-selectin monoclonal antibody significantly inhibited the enhanced ear response, suggesting that the enhanced ear-swelling response in E-selectin(-/-) mice resulted from compensatory increase in P-selectin expression. In the late phase of chronic CH, acceleration of ear swelling was significantly reduced in both E- and P-selectin(-/-) mice relative to wild-type littermates. Thus, the loss of P- or E-selectin suppressed inflammatory responses during the chronic phase of the chronic models, whereas early-phase inflammatory responses were exacerbated by E-selectin blockade. Collectively, P- and E-selectins cooperatively regulate CH response, although their roles may be different depending on the phase of the reaction.

Topics: Adjuvants, Immunologic; Animals; Chronic Disease; Dermatitis, Atopic; Dermatitis, Contact; E-Selectin; Immunohistochemistry; Inflammation; Mice; Mice, Inbred C57BL; Oxazolone; P-Selectin; Reverse Transcriptase Polymerase Chain Reaction

Chemokine receptor CCR4 and its ligands (CCL17 and CCL22) are important for the recruitment of memory T cells into the skin in various cutaneous immune diseases. However, information on CCR4 and its ligands in contact hypersensitivity is relatively limited. In this study, we investigated the expression of CCR4, CCL17, and CCL22 in a mouse model of contact hypersensitivity to oxazolone. Contact sensitization to oxazolone increased the proportions of memory CD4+ T cells in the draining lymph nodes, spleen, and peripheral blood. Although CCR4+ mRNA and CCR4+ cells were detectable in naive mouse lymph nodes, they significantly increased in the sensitized mice. The majority of CCR4+ cells in both control and sensitized mouse lymph nodes were CD4+ T cells. In the skin of naive mice, the mRNAs for CCR4, CCL17, and CCL22 were detectable, but only CCL17 and CCL22 proteins were constitutively expressed in the skin, particularly in the epidermis. Interestingly, the mRNAs for CCR4 and its two ligands were significantly elevated in the inflamed skin of mice with contact hypersensitivity to oxazolone. Furthermore, a subpopulation of cells that infiltrated the skin was CCR4+ cells. Finally, the expression of CCL17 and CCL22 proteins was significantly enhanced in the epidermis of inflamed skin. Thus, our study provides direct evidence for the presence of CCR4 and its ligands in mouse contact hypersensitivity.

Topics: Animals; CD4-Positive T-Lymphocytes; Chemokine CCL17; Chemokine CCL22; Dermatitis, Contact; Female; Immunologic Memory; Ligands; Mice; Mice, Inbred BALB C; Oxazolone; Receptors, CCR4; Skin

Epicutaneous (EC) immunization with protein antigens has been shown to induce antigen nonspecific suppression of subsequent T cell-dependent contact hypersensitivity (CS) reactions after active immunization. The aim of this work was to test if EC application of Toll-like receptor (TLR) ligands together with protein antigen could reverse suppression of CS.. Mice were EC immunized by applying gauze patches soaked with a solution of protein antigen alone or in the presence of crude bacterial material (bacterial lysates or heat-killed bacteria) or purified TLR ligands and then tested for CS response. To test if reversal of EC-induced suppression is antigen-specific, mice were patched with TNP- or OX-substituted mouse Ig alone or together with LPS and then tested for CS with corresponding or non-cross-reacting hapten. Influence of EC immunization on cytokine production by lymph node cells was measured by ELISA.. EC immunization with protein antigen induces antigen nonspecific suppression that can be reversed by crude bacterial material as well as purified TLR-2, TLR-3, TLR-4, and TLR-9 ligands. The effect of TLR-4 ligand LPS was not observed in the Tlr-4 mutant C3H/HeJ mouse, indicating that this effect was dependent upon intact TLR-4 signaling. Unlike the antigen nonspecific suppression of CS by EC immunization with antigen alone, the reversal of suppression by TLR ligands was specific for the protein antigen applied in the EC protocol.. Our results strongly suggest that EC immunization with protein antigen together with TLR ligands induces a particular antigen-specific cell population, akin to previously described contrasuppressor cells, which protects immune cells against the action of suppressor cells but have no direct influence on antigen nonspecific suppressor cells induced by antigen alone.

Topics: Animals; Antigens; Cytokines; Dermatitis, Contact; Epitopes; Immunization; Ligands; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred CBA; Oxazolone; Skin; T-Lymphocytes; Toll-Like Receptors; Trinitrobenzenes

It is commonly believed that only T lymphocytes and B lymphocytes expressing recombination-dependent antigen-specific receptors mediate contact hypersensitivity responses to haptens. Here we found that mice devoid of T cells and B cells demonstrated substantial contact hypersensitivity responses to 2,4-dinitrofluorobenzene and oxazolone. Those responses were adaptive in nature, as they persisted for at least 4 weeks and were elicited only by haptens to which mice were previously sensitized. No contact hypersensitivity was induced in mice lacking all lymphocytes, including natural killer cells. Contact hypersensitivity responses were acquired by such mice after adoptive transfer of natural killer cells from sensitized donors. Transferable hapten-specific memory resided in a Ly49C-I(+) natural killer subpopulation localized specifically in donor livers. These observations indicate that natural killer cells can mediate long-lived, antigen-specific adaptive recall responses independent of B cells and T cells.

Topics: Adjuvants, Immunologic; Animals; B-Lymphocytes; Dermatitis, Contact; Dinitrofluorobenzene; DNA-Binding Proteins; Immunologic Memory; Killer Cells, Natural; Liver; Mice; Mice, Knockout; Oxazolone; T-Lymphocytes; Urinary Bladder

Elicitation of contact sensitivity (CS) depends on B-1-cell-derived antigen-specific immunoglobulin M (IgM) antibodies that recruit CS effector T cells into the local tissue, which is followed by infiltration of antigen-nonspecific mononuclear cells and polymorphonuclear cells, such as neutrophils and eosinophils. In this study, we investigated the role of interleukin (IL)-5, which has broad effects on both eosinophils and B-1 cells, in elicitation of CS.. IL-5 receptor alpha-chain-deficient (IL-5Ralpha-/-) mice and IL-5Ralpha+/+ mice were contact sensitized with oxazolone hapten. Four days later, mice were challenged with the same hapten, and ear swelling responses were measured at 24 h after challenge. Eosinophil infiltration into the local tissue was determined by examination of skin histology and eosinophil peroxidase activity. To investigate the role of IL-5 in B-1 cell activation, the number of oxazolone-specific IgM-producing cells in the spleen was determined by enzyme-linked immunospot assay.. Ear swelling responses in IL-5Ralpha-/- mice were about half of those in IL-5Ralpha+/+ mice, and nearly no eosinophil infiltration was observed in IL-5Ralpha-/- mouse skin. Eosinophil peroxidase activity in the sensitized and challenged IL-5Ralpha-/- mice was about 11 times less than that in immunized IL-5Ralpha+/+ mice. Contact sensitization significantly increased in numbers of oxazolne-specific IgM-producing cells in IL-5Ralpha+/+ mouse spleen, but not in IL-5Ralpha-/- mouse spleen.. We conclude that IL-5-dependent activation of eosinophils and B-1 cells is necessary for induction and elicitation of CS. These findings provide a new insight into complicated mechanisms of CS elicitation and suggest a novel role of IL-5 in the regulation of immune responses.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; B-Lymphocyte Subsets; Dermatitis, Contact; Enzyme-Linked Immunosorbent Assay; Eosinophil Peroxidase; Eosinophils; Female; Haptens; Immunoglobulin M; Interleukin-5; Male; Mice; Mice, Knockout; Oxazolone; Spleen

The inhibitory effects of the Korean red ginseng (steamed root of Panax ginseng C.A. MEYER, family Araliaceae) saponin fraction (KRGS) and its constituents ginsenosides Rg3, Rf, and Rh2 in mouse passive cutaneous anaphylaxis (PCA) and contact dermatitis models were measured. Orally administered KRGS and its genuine ginsenosides potently inhibited the PCA reaction induced by IgE. However, when these ginsenosides were intraperitoneally administered, ginsenoside Rh2 showed the most potent inhibition. The ginsenoside Rh2 also the most potently inhibited the beta-hexosaminidase release from RBL-2H3 cells induced by IgE with antigen. KRGS administered topically at a dose of 0.1% suppressed ear swelling in an oxazolone-induced mouse contact dermatitis model by 38.8%. Its constituents ginsenosides Rg3, Rf, and Rh2 at a concentration of 0.05% also potently suppressed mouse ear swelling by 47.5%, 34.8%, and 49.9% at 16 d, respectively. These ginsenosides also significantly reduced mRNA expression levels of cyclooxygenase (COX)-2, interleukin (IL)-1beta, tumor necrosis factor-alpha and interferon-gamma induced by oxazolone applied to mouse ears. However, the ginsenosides, except for ginsenoside Rh2, almost did not notably reduce IL-4 levels. The ginsenoside Rh2 also potently inhibited COX-2 and inducible NO synthetase protein expression in liphopolysaccharide-stimulated RAW264.7 cells. Based on these findings, KRGS and its ginsenosides are suggested to improve atopic and contact dermatitis by regulating expression of cytokines.

Topics: Animals; Cytokines; Dermatitis, Contact; Female; Ginsenosides; Male; Mice; NF-kappa B; Oxazolone; Panax; Passive Cutaneous Anaphylaxis; Plant Extracts; Prostaglandin-Endoperoxide Synthases; RNA, Messenger

The effect of a main constituent ginsenoside Rg5 isolated from red ginseng and its metabolite ginsenoside Rh3 in a chronic dermatitis model was investigated. Ginsenosides Rg5 and Rh3 suppressed swelling of oxazolone-induced mouse ear contact dermatitis. These ginsenosides also reduced mRNA expressions of cyclooxygenase-2, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. The inhibition of ginsenoside Rh3 was more potent than that of ginsenoside Rg5. These findings suggest that ginsenoside Rh3 metabolized from ginsenoside Rg5 may improve chronic dermatitis or psoriasis by the regulation of IL-1beta and TNF-alpha produced by macrophage cells and of IFN-gamma produced by Th cells.

Topics: Animals; Chronic Disease; Cyclooxygenase 2; Dermatitis, Contact; Female; Ginsenosides; Interferon-gamma; Interleukin-1beta; Mice; Mice, Inbred ICR; Oxazolone; Tumor Necrosis Factor-alpha

In humans, lesions of contact eczema or atopic dermatitis can exhibit increases in epidermal nerves, but the mechanism resulting in such nerve elongation are not fully understood. We found that contact hypersensitivity reactions to oxazolone in mice were associated with significant increases in the length of nerves in the epidermis and dermis. Using genetically mast cell-deficient c-kit mutant mice selectively repaired of their dermal mast cell deficiency with either wild-type or tumor necrosis factor (TNF)-deficient mast cells, we found that mast cells, and mast cell-derived TNF, significantly contributed to the elongation of epidermal and dermal PGP 9.5+ nerves and dermal CGRP+ nerves, as well as to the inflammation observed at sites of contact hypersensitivity in response to oxazolone. Moreover, the percentage of mast cells in close proximity to dermal PGP 9.5+ nerve fibers was significantly higher in wild-type mice and in c-kit mutant mice repaired of their dermal mast cell deficiency by the adoptive transfer of wild-type mast cells than in TNF-deficient mice or in TNF-/- mast cell-engrafted c-kit mutant mice. These observations show that mast cells, and mast cell-derived TNF, can promote the elongation of cutaneous nerve fibers during contact hypersensitivity in the mouse.

Topics: Animals; Dermatitis, Contact; Ear; Mast Cells; Mice; Mice, Inbred C57BL; Nerve Fibers; Oxazolone; Skin; Tumor Necrosis Factors

The possible involvement of 2-arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), in contact dermatitis in mouse ear was investigated. We found that the level of 2-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR144528, a CB2 receptor antagonist, immediately after sensitization, or the administration of SR144528 upon the challenge. The effect of AM251, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB2 receptor and its endogenous ligand 2-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II(+)) contain a substantial amount of CB2 receptor mRNA, whereas keratinocytes (MHC class II(-)) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR144528. We next examined whether the CB2 receptor and 2-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of 2-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR144528 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB2 receptor and 2-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.

Topics: Animals; Arachidonic Acids; Dermatitis, Contact; Ear; Endocannabinoids; Glycerides; Inflammation; Keratinocytes; Langerhans Cells; Ligands; Male; Mice; Mice, Inbred ICR; Oxazolone; Receptor, Cannabinoid, CB2; RNA, Messenger

Regulated migration and spatial localization of dendritic cells (DCs) are critical events during the initiation of physiologic immune responses and maintenance of tolerance. Here we have used cells deficient in the Wiskott-Aldrich syndrome protein (WASp) to demonstrate the importance of dynamic remodeling of the actin cytoskeleton for these trafficking processes to occur in vitro and in vivo. On fibronectin-coated surfaces, WASp-null immature murine DCs exhibited defects both of attachment and detachment, resulting in impaired net translocation compared with normal cells. The chemokinetic response to CCL21, which is critical for normal lymphatic trafficking, was also abrogated in the absence of WASp. In vivo in both fluorescein isothiocyanate (FITC) and oxazolone contact hypersensitivity models, WASp-null Langerhans cell (LC) migration was compromised, as judged by exit from the skin as well as by homing to the draining lymph node (LN). Furthermore, following systemic challenge with lipopolysaccharide (LPS) or toxoplasma-derived antigen, WASp-null DCs showed incomplete redistribution to T-cell areas in the spleen. Instead, they were retained ectopically in the marginal zone. DC trafficking in vivo is therefore dependent on a normally regulated actin cytoskeleton, which performs an essential function during maintenance of physiologic immunity and when disturbed may contribute significantly to the immunopathology of Wiskott-Aldrich Syndrome.

Topics: Animals; Bone Marrow Cells; Cell Movement; Chemokine CCL21; Chemokines, CC; Dendritic Cells; Dermatitis, Contact; Disease Models, Animal; Langerhans Cells; Lymph Nodes; Mice; Mice, Knockout; Oxazolone; Proteins; Skin; Spleen; T-Lymphocytes; Time Factors; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein

CD1d-restricted T-cells are activated by glycolipids presented by the major histocompatibility complex class-Ib molecule CD1d, found on the surface of antigen-presenting cells (APC). This interaction between APC, most notably dendritic cells (DC), and CD1d-restricted T-cells is an important regulatory step in the initiation of adaptive immune responses. It is well known that DC play a crucial role in the induction of contact hypersensitivity (CHS), a frequently studied form of in vivo T-cell-mediated immunity. In this study, we show that CD1d-restricted T-cells are also necessary for CHS, because both wild-type mice treated systemically or topically with CD1d glycolipid antagonists and CD1d-restricted T-cell-null mice have markedly diminished CHS responses. Thus, pharmacologic antagonists of CD1d can be used as effective inhibitors of CHS, a prototype for a variety of delayed-type tissue hypersensitivity responses.

Topics: Administration, Topical; Animals; Antigen Presentation; Antigens, CD1; Antigens, CD1d; Cell Line; Dendritic Cells; Dermatitis; Dermatitis, Contact; Dose-Response Relationship, Drug; Glycolipids; Hypersensitivity; Killer Cells, Natural; Major Histocompatibility Complex; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Oxazolone; Phosphatidylethanolamines; Polyethylene Glycols; Receptors, Antigen, T-Cell

During the screening program to discover antipsoriatic agents from natural products, ginseng was found to show inhibitory activity in oxazolone-induced mouse ear dermatitis. Therefore, the effects of a main constituent ginsenoside Rb1 isolated from ginseng and its metabolite compound K on oxazolone-induced mouse ear dermatitis were investigated. Compound K at concentrations of 0.02% and 0.05% also potently suppressed mouse ear swelling by 54% and 76% at 16 days, respectively, although ginsenoside Rb1 did not significantly show the inhibitory activity. The compound K also significantly reduced the levels of mRNA of cyclooxygenase (COX)-2, IL-1beta, TNF-alpha, IFN-gamma and IL-4 increased in oxazolone-applied mouse ears. Based on these findings, the compound K may improve contact dermatitis or psoriasis by the regulation of COX-2 produced by macrophage cells and interferon-gamma and IL-4 induced by Th cells.

Topics: Animals; Cell Line; Chronic Disease; Cyclooxygenase 1; Cyclooxygenase 2; Dermatitis, Contact; Dinoprostone; Female; Ginsenosides; Macrophages; Membrane Proteins; Mice; Mice, Inbred ICR; Nitric Oxide; Oxazolone; Prostaglandin-Endoperoxide Synthases; Psoriasis; RNA, Messenger

It is suggested that atopic dermatitis is a skin disease associated with itching as subjective symptoms, and histamine H(1) receptor antagonists are used in order to prevent the itching, and the deterioration for scratch by itching. Histamine H(1) receptor selective anti-histamine olopatadine hydrochloride (olopatadine; Allelock shows consistent efficacy and safety in the treatment of allergic disorders. We investigated the possible efficacy of olopatadine on the number of scratching induced by repeated application of oxazolone in BALB/c mice. The repeated treatment of olopatadine significantly inhibited the ear swelling and the increased number of scratching. It significantly inhibited the increased production of interleukin (IL)-4, IL-1beta and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lesioned ear. Moreover, it significantly inhibited the increased production of nerve growth factor (NGF) and substance P. On the other hand, loratadine, bepotastine and chlorpheniramine did not inhibit the ear swelling and the increased number of scratching. These results indicate that olopatadine inhibited not only the increased production of cytokines but also NGF and substance P unlike other histamine H(1) receptor antagonists. It was suggested that olopatadine suppressed the increased number of scratching by the anti-inflammatory effects. Therefore, olopatadine appears to exert additional biological effects besides its blockade of a histamine H(1) receptor.

Topics: Animals; Anti-Inflammatory Agents; Antipruritics; Chlorpheniramine; Cytokines; Dermatitis, Contact; Dibenzoxepins; Dose-Response Relationship, Drug; Ear; Histamine H1 Antagonists; Immunoglobulin E; Male; Mice; Mice, Inbred BALB C; Nerve Growth Factor; Olopatadine Hydrochloride; Oxazolone; Prednisolone; Pruritus; Severity of Illness Index

Contact sensitivity responses require both effective immune sensitization following cutaneous exposure to chemical haptens and antigen-specific elicitation of inflammation upon subsequent hapten challenge. We have observed that that antigen-independent effects of immunoglobulin E (IgE) antibodies promote immune sensitization to haptens in the skin. Contact sensitivity is markedly impaired in IgE-/- mice but can be restored by either transfer of sensitized cells from wild-type mice or administration of hapten-irrelevant IgE before sensitization. Moreover, IgE-/- mice exhibit impairment in the emigration of dendritic cells from the epidermis after hapten exposure. Monomeric IgE has been reported to influence mast cell function. We observe diminished contact sensitivity in mice lacking FcepsilonRI or mast cells, and mRNA for several mast cell-associated genes is reduced in IgE-/- vs. wild-type skin after hapten exposure. We propose that levels of IgE normally present in mice favour immune sensitization via antigen-independent effects on mast cells.

Topics: Adjuvants, Immunologic; Administration, Cutaneous; Animals; Cytokines; Dermatitis, Contact; Haptens; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Mast Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Oxazolone; Receptors, IgE; Skin; Stem Cell Factor

Epicutaneous application of haptens to UV-exposed skin induces hapten-specific tolerance. This is mediated via regulatory T cells (Tr), as i.v. injection of T cells from UV-tolerized mice into naive animals renders the recipients unresponsive to the respective hapten. However, when UV-induced Tr are injected i.v. into sensitized mice, contact hypersensitivity (CHS) is not suppressed, suggesting that Tr inhibit the induction, but not the elicitation, of CHS and are inferior to T effector cells. As sensitization takes place in the lymph nodes, but elicitation occurs in the area of challenge, we postulated that Tr injected i.v. locate to the lymph nodes and not to the periphery and therefore only suppress the induction, not the elicitation, of CHS. Indeed, i.v. injection of Tr into sensitized mice did not inhibit CHS, although injection of Tr into the ears of sensitized mice suppressed the challenge. Inhibition was hapten specific, as injection of dinitrofluorobenzene (DNFB)-specific Tr into the ears of oxazolone (OXA)-sensitized mice did not affect challenge with OXA. However, when ears of OXA-sensitized mice were injected with DNFB-specific Tr and painted with DNFB before OXA challenge, CHS was suppressed. Inhibition correlated with the local expression of IL-10. Depletion studies and FACS analysis revealed that Tr express the lymph node-homing receptor L-selectin, but not the ligands for the skin-homing receptors E- and P-selectin, suggesting that UV-induced Tr, although able to inhibit T effector cells, do not suppress the elicitation of CHS upon i.v. injection, because they obviously do not migrate into the skin.

Topics: Adoptive Transfer; Animals; Dermatitis, Contact; Dinitrofluorobenzene; Ear, External; Haptens; Immunization; Immunophenotyping; Injections, Intradermal; Injections, Intravenous; L-Selectin; Lymph Nodes; Mice; Mice, Inbred C3H; Oxazolone; Receptors, Interleukin-2; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Ultraviolet Rays

The information gathered by dendritic cells during the innate immune response is determinant for the type and strength of the adaptive response. We showed that the sympathetic neurotransmitter norepinephrine influences dendritic cell migration and T helper priming via alpha- and beta-adrenoceptors. Others have shown that Langerhans cells also express mRNA for beta 1-, beta 2-, and alpha 1A-adrenoceptors and that catecholamines may inhibit the antigen-presenting capability via beta 2-adrenoceptors. Here we report that oxazolone, which induces a predominant T-helper-1-type contact hypersensitivity response, but not fluorescein isothiocyanate, which induces a prevailing T-helper-2-type response, inhibits the local norepinephrine turnover in the skin of mice during the first 8 h of sensitization. Oxazolone also induced higher expression of the inflammatory cytokines interleukin-1 and interleukin-6 mRNA in the skin. Lack or blockade of these cytokines as well as inhibition of prostaglandin synthesis, however, did not influence the oxazolone effect. Only the nonspecific anti-inflammatory steroid dexamethasone could neutralize the effect of oxazolone. Furthermore, fluorescein isothiocyanate but not oxazolone sensitization in the presence of the specific beta 2-adrenoceptor antagonist ICI 118,551 enhanced the consequent contact hypersensitivity response as well as the production of T helper 1 cytokines in draining lymph nodes; conversely T helper 2 cytokines were not affected. Thus, the extent of T helper 1 priming in the adaptive response to a sensitizing agent seems to depend also on its ability to modulate the local sympathetic nervous activity during the innate immune response.

Topics: Adjuvants, Immunologic; Adrenergic beta-2 Receptor Antagonists; Adrenergic beta-Antagonists; Allergens; Animals; Dermatitis, Contact; Female; Gene Expression; Interleukin-1; Interleukin-6; Langerhans Cells; Mice; Mice, Inbred BALB C; Norepinephrine; Oxazolone; Propanolamines; Skin; Sympathetic Nervous System; Th1 Cells; Th2 Cells

Glycyrrhizin (18beta-glycyrrhetinic acid-3-O-beta-D-glucuronopyranosyl-(1 --> 2)-beta-D-glucuronide, GL) was transformed to 18beta-glycyrrhetinic acid-3-O-beta-D-glucuronide (GAMG) by Streptococcus LJ-22. The antiallergic activities of GL and GAMG was measured using a RBL cell assay system and contact hypersensitivity model mice. GAMG exhibited anti-allergic activity with IC50 values of 0.28 mM. GAMG, which is sweeter than GL, and 18beta-glycyrrhetinic acid, which is a GAMG metabolite by human intestinal bacteria, also inhibited the passive cutaneous anaphylaxis and skin contact inflammation. In conclusion, GAMG may be useful as a new sweet food additive and an anti-allergic agent.

Topics: Administration, Oral; Animals; Anti-Allergic Agents; beta-N-Acetylhexosaminidases; Cell Line; Dermatitis, Contact; Dexamethasone; Disease Models, Animal; Drug Evaluation, Preclinical; Glucuronides; Glycyrrhetinic Acid; Glycyrrhizic Acid; Humans; Injections, Intraperitoneal; Intestines; Male; Mice; Mice, Inbred ICR; Nitrites; Oxazolone; Passive Cutaneous Anaphylaxis; Streptococcus

Contact sensitivity responses require both effective immune sensitization following cutaneous exposure to chemical haptens and antigen-specific elicitation of inflammation upon subsequent hapten challenge. We report that antigen-independent effects of IgE antibodies can promote immune sensitization to haptens in the skin. Contact sensitivity was markedly impaired in IgE(-/-) mice but was restored by either transfer of sensitized cells from wild-type mice or administration of hapten-irrelevant IgE before sensitization. Moreover, IgE(-/-) mice exhibited impairment in the reduction of dendritic cell numbers in the epidermis after hapten exposure. Monomeric IgE has been reported to influence mast cell function. We observed diminished contact sensitivity in mice lacking FcepsilonRI or mast cells, and mRNA for several mast cell-associated genes was reduced in IgE(-/-) versus wild-type skin after hapten exposure. We speculate that levels of IgE normally present in mice favor immune sensitization via antigen-independent but FcepsilonRI-dependent effects on mast cells.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; B-Lymphocytes; Cell Movement; Dendritic Cells; Dermatitis, Contact; Haptens; Immunization; Immunoglobulin E; Mast Cells; Mice; Oxazolone; Receptors, IgE; Reverse Transcriptase Polymerase Chain Reaction; Skin

Contact sensitivity (CS) is an inflammatory disorder characterized by early and late phases of leukocyte recruitment. We used a noninvasive intravital microscopy technique allowing for the direct visualization of leukocyte rolling and adhesion on blood vessel endothelium. By blocking specific adhesion molecules, we elucidated the molecular mechanisms mediating early leukocyte recruitment to be E- and P-selectin and demonstrated that leukocyte recruitment in the late phase had a different adhesive profile (mainly alpha(4)-integrin). Complete blockade of E- and P-selectin within the first 2 h of leukocyte-endothelial cell interactions (but not later) eliminated selectin-independent leukocyte recruitment at 24 h. Despite the predominance of neutrophils in the early phase, specific elimination of CD4(+) lymphocytes in the early phase eliminated the late response. CD4(+) lymphocytes homed to skin via E- and P-selectin within the early phase and induced the late phase response. Addition of these same CD4(+) lymphocytes 2 h after antigen challenge was too late for these cells to home to the skin and induce late phase responses. Our data clearly demonstrate that the antigen-challenged microenvironment is only accessible to CD4(+) lymphocytes for the first 2 h, and that this process is essential for the subsequent recruitment of other leukocyte populations in late phase responses.

Topics: Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; Cell Adhesion; Chemotaxis, Leukocyte; Dermatitis, Contact; E-Selectin; Hypersensitivity, Delayed; Inflammation; Leukocytes; Lymphocyte Transfusion; Male; Mice; Mice, Inbred C57BL; Oxazolone; P-Selectin; Receptors, Lymphocyte Homing; Selectins; Skin; Time Factors

The inhibitory effects of C-2 epimeric isomers of (-)-epigallocatechin-3-O-gallate (EGCG) and two O-methylated EGCG derivatives, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG3''Me) and (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4''Me), against oxazolone-induced type IV allergy in male mice were investigated. These compounds exhibited strong antiallergic effects by percutaneous administration at a dose of 0.13 mg/ear. The inhibition rates of (-)-gallocatechin-3-O-gallate (GCG), (-)-gallocatechin-3-O-(3-O-methyl)gallate (GCG3''Me), and (-)-gallocatechin-3-O-(4-O-methyl)gallate (GCG4''Me) on mouse type IV allergy were 52.1, 53.3, and 54.8%, respectively. However, the antiallergic effects were weaker than those of their corresponding original tea catechins (2R,3R type). The inhibition rates of those were 88.0, 73.2, and 77.6%, respectively. For all of the catechins tested, oral administration at a dose of 50 mg/kg body weight significantly suppressed the allergic symptoms. The inhibitory rates varied from 24.0 to 60.6%. No significant differences were observed between the effects of the epimers (2S,3R type) and their corresponding original catechins (2R,3R type). The antiallergic effects of tea catechins and their C-2 epimers observed in this study were dose-dependent. These results suggest that C-2 epimers of tea catechins, which are produced during heat processing at high temperatures, could be disadvantageous for the antiallergic effects on type IV allergy.

Topics: Animals; Anti-Allergic Agents; Catechin; Dermatitis, Contact; Isomerism; Male; Mice; Mice, Inbred ICR; Oxazolone; Tea

Low zone tolerance (LZT) to contact allergens is induced by epicutaneous exposure to haptens in subsensitizing doses resulting in an inhibition of contact hypersensitivity (CHS), which, in contrast, occurs after sensitization with immunogenic doses of allergens. Performing the protocol of tolerance induction resulted in robust LZT to allergens in B cell-deficient mice in vivo, indicating that B cells are not required for the induction and effector phase of LZT. However, CHS reactions in vivo were restricted in B cell-deficient mice as compared to wild-type (WT) mice. In contrast, analysis of hapten-specific T cell activation in vitro revealed a strong proliferative response of T cells derived from both WT and B cell-deficient sensitized mice. Similar to WT animals, T cells obtained from tolerized B cell-deficient mice produced a Tc2 cytokine pattern of LZT with high levels of IL-4 and IL-10, whereas sensitization of B cell-deficient mice resulted in the typical Tc1 cytokine profile of CHS. Adoptive transfer of CD8+ effector T cells from tolerized or sensitized B cell-deficient mice induced significant LZT or CHS reactions, respectively, in WT recipients, demonstrating that the development of hapten-specific effector CD8+ T cells of LZT and CHS is independent of B cells.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Allergens; Animals; B-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Dermatitis, Contact; Immune Tolerance; Interferon-gamma; Interleukins; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Picryl Chloride

Olopatadine hydrochloride (olopatadine) is one of the second-generation antihistamines, which is prescribed for allergic disorders such as rhinitis, urticaria and eczema dermatitis.. To investigate the possible anti-inflammatory effect of olopatadine on the chronic contact hypersensitivity response to repeated topical application of oxazolone in mice.. The preventive and therapeutic effects of oral olopatadine were quantified by measurements of ear swelling, cytokine protein and mRNA expression in the ear lesion, and were compared with those of topical betamethasone 17-valerate (betamethasone).. The ear receiving repeated applications of oxazolone exhibited erythema, oedema and abrasion. Both preventive and therapeutic administration of olopatadine (10 mg kg(-1) day(-1)) significantly inhibited the ear swelling and the increased production of interleukin (IL)-4, IL-1beta, granulocyte-macrophage colony-stimulating factor (GM-CSF) and nerve growth factor. In the histopathological analysis, olopatadine ameliorated epidermal hyperplasia and infiltration of inflammatory cells. Consistent with these results, olopatadine significantly reduced the increased expression of interferon-gamma and IL-4 mRNA. Although betamethasone (0.012 mg ear(-1) day(-1)) showed similar activities to olopatadine against these responses, it caused atrophy of the ear skin.. These results indicate that olopatadine is an antihistamine agent having inhibitory activities against chronic inflammatory dermatitis, possibly resulting from its diminishing effect on elevated cytokines.

Topics: Adjuvants, Immunologic; Animals; Anti-Inflammatory Agents, Non-Steroidal; Chronic Disease; Cytokines; Dermatitis, Contact; Dibenzoxepins; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Histamine H1 Antagonists, Non-Sedating; Immunoglobulin E; Male; Mice; Mice, Inbred BALB C; Nerve Growth Factor; Olopatadine Hydrochloride; Oxazolone; RNA, Messenger

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

Topics: Administration, Topical; Allergens; Animals; Cell Division; Dermatitis, Contact; Dinitrofluorobenzene; DNA Primers; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mucins; Oligonucleotide Array Sequence Analysis; Oxazolone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Repeated Ag exposure results in a shift in the time course of contact hypersensitivity (CH) from a typical delayed-type to an immediate-type response followed by a late phase reaction. Chronic CH responses are clinically relevant to human skin allergic diseases, such as atopic dermatitis, that are usually caused by repeated stimulation with environmental Ags. Chronic inflammatory responses result in part from infiltrating leukocytes. To determine the role of leukocyte adhesion molecules in chronic inflammation, chronic CH responses were assessed in mice lacking L-selectin, ICAM-1, or both adhesion molecules. Following repeated hapten sensitization for 24 days at 2-day intervals, wild-type littermates developed an immediate-type response at 30 min after elicitation, followed by a late phase reaction. By contrast, loss of ICAM-1, L-selectin, or both, eliminated the immediate-type response and inhibited the late phase reaction. Similar results were obtained when wild-type littermates repeatedly exposed to hapten for 22 days were treated with mAbs to L-selectin and/or ICAM-1 before the elicitation on day 24. The lack of an immediate-type response on day 24 paralleled a lack of mast cell accumulation after 30 min of elicitation and decreased serum IgE production. Repeated Ag exposure in wild-type littermates resulted in increased levels of serum L-selectin, a finding also observed in atopic dermatitis patients. The current study demonstrates that L-selectin and ICAM-1 cooperatively regulate the induction of the immediate-type response by mediating mast cell accumulation into inflammatory sites and suggests that L-selectin and ICAM-1 are potential therapeutic targets for regulating human allergic reactions.

Topics: Administration, Cutaneous; Animals; Antibodies, Monoclonal; Antigens; Cell Migration Inhibition; Cell Movement; Dermatitis, Contact; Down-Regulation; Edema; Hypersensitivity, Immediate; Immunoglobulin E; Injections, Intravenous; Intercellular Adhesion Molecule-1; L-Selectin; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone

Apoptosis plays an important role in immune responses, but little is known about its involvement in contact hypersensitivity (CH). In this study, we have investigated the role of Fas/Fas ligand (FasL)-mediated apoptosis in the pathogenesis of CH. Mice were sensitized by one topical application of 100 microl of 3% oxazolone to shaved skin of the abdomen. Six days later, CH was provoked by challenging both sides of sensitized mouse right ear with 15 microl of 1% oxazolone. Using a DNA ladder assay, we found that apoptosis was induced in the skin of oxazolone-sensitized mice 24-96 h after allergen challenge. Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis flow cytometric assay showed that early apoptotic CD4(+) T cells (annexin V-FITC(+)PI(-)), but not late apoptotic CD4(+) T cells (annexin V-FITC(+)PI(+)), increased in the inflamed skin of mice with CH. Moreover, the expressions of mRNAs for T helper (Th2) cytokine (interleukin (IL)-4), Th1 cytokine (interferon (IFN)-gamma) and proapoptotic molecules (Bax, Fas, FasL and IL-1beta-converting enzyme (ICE)/caspase-1) were significantly elevated in the oxazolone-sensitized mouse skin 6-72 h after allergen challenge. Dramatic increase in IL-10 mRNA was only observed in the sensitized mouse skin 6 and 12 h after allergen challenge. Furthermore, CH was significantly inhibited with decreased apoptosis and early apoptotic CD4(+) T cells in inflamed skin in Fas mutant lpr/lpr mice compared to wild-type mice, whereas there were no significant differences in IL-4, IFN-gamma, IL-10, Bax and ICE mRNAs in the inflamed skin of CH between lpr/lpr and wild-type mice. Our results thus suggest that Fas/FasL pathway partially contributes to apoptosis in murine CH and that Fas/FasL-mediated apoptosis plays a partial role in the development of CH. The contribution of Fas/FasL-mediated apoptosis to CH appears independent of Th1 and Th2 cytokines.

Topics: Adjuvants, Immunologic; Administration, Cutaneous; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 1; CD4-Positive T-Lymphocytes; Dermatitis, Contact; DNA Fragmentation; Fas Ligand Protein; fas Receptor; Female; Interferon-gamma; Interleukin-10; Interleukin-4; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Oxazolone; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5-20 micro M inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4(+) T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5-20 micro M inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.

Topics: Allergens; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigen Presentation; Azoles; Clone Cells; Concanavalin A; Dendritic Cells; Dermatitis, Contact; Epitopes, T-Lymphocyte; Female; Irritants; Isoindoles; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Transgenic; Organoselenium Compounds; Oxazolone; Reactive Oxygen Species; Th1 Cells

One of the essential functions of dendritic cells is to take up Ags in peripheral tissues and migrate into secondary lymphoid organs to present Ags to lymphocytes for the induction of immune responses. Although many studies have demonstrated that the migration of dendritic cells is closely associated with the development of immune responses, little is known about factors that inhibit dendritic cell migration and control the extent of immune responses to Ag stimulation. We show that Slit2, a neuronal repellent factor, is up-regulated in the skin by allergen sensitization and down-regulates the migration of Langerhans cells. The effect is mediated by direct interaction of Slit2 with cells that express a Slit-specific receptor, Robo1. Slit2-mediated inhibition of Langerhans cell migration results in suppression of contact hypersensitivity responses. These findings provide insights into a novel mechanism by which Slit2 functions as an anti-inflammatory factor for the initiation of immune responses.

Topics: Animals; Cell Communication; Cell Line; Cell Migration Inhibition; Cell Movement; Dendritic Cells; Dermatitis, Contact; Dinitrofluorobenzene; Female; Haptens; Humans; Immunosuppressive Agents; Injections, Subcutaneous; Intercellular Signaling Peptides and Proteins; Langerhans Cells; Mice; Mice, Inbred A; Mice, Inbred C57BL; Nerve Tissue Proteins; Organ Culture Techniques; Oxazolone; Receptors, Immunologic; Roundabout Proteins; Skin; Solubility

Tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-18 are all known to contribute to the regulation of epidermal Langerhans cells (LC) migration and the subsequent accumulation of dendritic cells (DC) in draining lymph nodes following skin sensitization. However, the cytokine signals that control these responses following skin irritation have yet to be defined. We demonstrate that IL-1alpha, a cytokine associated with skin injury and inflammation, is able to stimulate the activation and migration from the epidermis of LC and their subsequent accumulation in skin-draining lymph nodes. Stimulation of these responses by IL-1alpha required the local availability of TNF-alpha. Using specific neutralizing antibodies, LC migration induced following skin sensitization with oxazolone (Ox) was found to be dependent upon IL-1beta and independent of a requirement for IL-1alpha. However, the converse was true following stimulation of responses with the nonsensitizing skin irritant sodium lauryl sulfate (SLS). Here, the loss of LC from the epidermis and the accumulation of DC in draining lymph nodes required IL-1alpha and not IL-1beta. Despite utilizing different IL-1 isoforms for LC mobilization, the phenotypic characteristics of DC arriving in draining lymph nodes in response to Ox and SLS were similar with respect to the membrane determinants MHC class II, B7-1, B7-2, and intercellular adhesion molecule-1. These data suggest that contact sensitization and skin irritation employ subtly different cytokine networks in the regulation of LC migration, both involving TNF-alpha but demonstrating differential requirements for IL-1 cytokines. The proposal is that different forms of cutaneous trauma may achieve LC migration through distinct molecular mechanisms.

Topics: Adjuvants, Immunologic; Allergens; Animals; Cell Movement; Cell Separation; Dendritic Cells; Dermatitis, Contact; Epidermal Cells; Epidermis; Flow Cytometry; In Vitro Techniques; Interleukin-1; Irritants; Langerhans Cells; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Phenotype; Tumor Necrosis Factor-alpha

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-gamma following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-gamma production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-gamma production in caspase-1(-/-) LN cells. An impaired IFN-gamma production was seen in caspase-1(-/-) LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-gamma production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1(-/-) mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.

Topics: Administration, Cutaneous; Animals; Caspase 1; Cell Line; Cell Movement; Dermatitis, Contact; Haptens; Immune Sera; Immunization; Injections, Intraperitoneal; Interferon-gamma; Interleukin-12; Interleukin-18; Langerhans Cells; Lymph Nodes; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; RNA, Messenger; Up-Regulation

Previous studies in mice have reported a decrease in epidermal Langerhans cell (LC) density in aged skin, however, the impact of this reduction on LC function and cutaneous immune responses is unclear. In the present series of experiments, the frequency of major histocompatibility complex class II+ LC in the epidermis of older (6-month-old) mice was found to be reduced significantly compared with that observed for young (6-8-week-old) mice. LC mobilization and the subsequent accumulation of dendritic cells (DC) in regional lymph nodes in response to topical challenge with a chemical allergen were found to be less vigorous in older mice. Flow cytometric analyses of DC derived from the draining lymph nodes of fluorescein isothiocyanate (FITC)-sensitized mice revealed that the frequency of FITC+-DC arriving in draining lymph nodes was also reduced in older mice but that the fluorescence intensity was comparable. Control and allergen-treated-older mice also displayed decreased total lymph node cellularity. Contact hypersensitivity responses were found not to be compromised in older mice. However, the cytokine regulation of LC migration in the two age groups of mice did differ. LC migration provoked by intradermal injection of tumour necrosis factor-alpha (TNF-alpha) was reduced in older animals, whereas, the percentage of LC that migrated in response to exogenous interleukin-1beta (IL-1beta) was comparable for both young and aged mice. Since both allergen- and TNF-alpha-induced LC responses are known to require receipt by LC of a signal from IL-1beta for effective migration, the suggestion is that impaired LC migration in older mice may be due to a reduced availability of epidermal IL-1beta.

Topics: Aging; Allergens; Animals; Cell Count; Cell Movement; Cells, Cultured; Dendritic Cells; Dermatitis, Contact; Epidermis; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Histocompatibility Antigens Class II; Interleukin-1; Langerhans Cells; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Tumor Necrosis Factor-alpha

Studies of mice rendered deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) have established unique roles for these cytokines in pulmonary homeostasis, resistance to infection, and antigen-specific T- and B-cell responses. In addition to these distinctive properties, however, GM-CSF and IL-3 also stimulate the development and activation of hematopoietic cells in many similar ways, raising the possibility that each factor might partially compensate for the other's absence in singly deficient mice. To test whether endogenous GM-CSF and IL-3 mediate redundant functions in vivo, we generated mice lacking both cytokines through sequential gene targeting experiments in embryonic stem (ES) cells. Surprisingly, doubly deficient animals, but not single knockouts, showed increased numbers of circulating eosinophils. Doubly deficient mice, moreover, developed weaker contact hypersensitivity reactions to haptens applied epicutaneously than mice deficient in either factor alone. Together, these findings delineate overlapping roles for GM-CSF and IL-3 in hematopoiesis and immunity. (Blood. 2001;97:922-928)

Topics: Animals; Dendritic Cells; Dermatitis, Contact; Eosinophilia; Eosinophils; Gene Targeting; Granulocyte-Macrophage Colony-Stimulating Factor; Haptens; Hematopoiesis; Homeostasis; Interleukin-3; Leukocyte Count; Membrane Proteins; Mice; Mice, Knockout; Neoplasm Transplantation; Oxazolone; Skin; Tumor Cells, Cultured

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-gamma, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.

Topics: Abatacept; Adjuvants, Immunologic; Administration, Cutaneous; Animals; Antigens, CD; Antigens, Differentiation; Asthma; B-Lymphocytes; B7-1 Antigen; B7-2 Antigen; Bronchoalveolar Lavage Fluid; CTLA-4 Antigen; Dermatitis, Contact; Drug Administration Schedule; Drug Combinations; Female; Hemocyanins; Humans; Immunoconjugates; Immunoglobulin Fc Fragments; Immunoglobulin G; Inducible T-Cell Co-Stimulator Ligand; Injections, Intraperitoneal; Lymph Nodes; Lymphocyte Count; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Oxazolone; Recombinant Fusion Proteins; Skin; Spleen; T-Lymphocytes

The deleterious effects of ultraviolet B radiation (UVR) on cutaneous immunity are mediated in part by cytokines released from cutaneous cells following radiation exposure. On the one hand, TNF-alpha has been advocated as the primary mediator of failed contact hypersensitivity induction, and, on the other hand, IL-10 has been held responsible for tolerance. While keratinocytes exposed to UVR have been found to produce both TNF-alpha and IL-10, there is reason to question whether these major cellular constituents of the epidermis are the relevant source of immunomodulatory cytokines after UVR. Dermal mast cells also produce TNF-alpha and IL-10, and we have recently reported that mast cell-derived TNF-alpha is required for UVR-induced impairment of CH induction. In this study, we have examined whether mast cells are also a relevant source of IL-10 in UVR-dependent tolerance. We found that (a) UVR fails to induce tolerance in mast cell-deficient mice, and (b) that tolerance occurs if mast cells are triggered to degranulate after ligation of the IgE receptor. Both types of tolerance were neutralized with anti-IL-10 antibodies, are hapten specific, and are associated with regulatory lymphoid cells. We conclude that mast cells are required in UVR-induced tolerance and may be one of the major sources of IL-10 that mediates the tolerance induced by acute, low-dose UVR.

Topics: Adjuvants, Immunologic; Animals; Cell Degranulation; Dermatitis, Contact; Histamine Release; Immune Tolerance; Immunoglobulin E; Interleukin-10; Mast Cells; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Oxazolone; Picryl Chloride; Radiation Dosage; Skin; Ultraviolet Rays

Peroxynitrite-induced poly(ADP-ribose) polymerase activation has been implicated in the pathogenesis of various inflammatory conditions. Here we have investigated whether peroxynitrite and poly(ADP-ribose) polymerase may play a role in the pathophysiology of the elicitation phase of contact hypersensitivity. We have detected nitrotyrosine, DNA breakage, and poly(ADP-ribose) polymerase activation in the epidermis of mice in an oxazolone-induced contact hypersensitivity model. As tyrosine nitration is mostly mediated by peroxynitrite, a nitric-oxide-derived cytotoxic oxidant capable of causing DNA breakage, we have applied peroxynitrite directly on mouse skin and showed poly(ADP-ribose) polymerase activation in keratinocytes and in some scattered dermal cells. We have also investigated the cellular effects of peroxynitrite in HaCaT cells, a human keratinocyte cell line. We found that peroxynitrite inhibited cell proliferation and at higher concentrations also caused cytotoxicity. Peroxynitrite activates poly(ADP-ribose) polymerase in HaCaT cells and poly(ADP-ribose) polymerase activation contributes to peroxynitrite-induced cytotoxicity, as indicated by the cytoprotective effect of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. The cytoprotective effect of 3-aminobenzamide cannot be attributed to inhibition of apoptosis, as apoptotic parameters (caspase activation and DNA fragmentation) were not reduced in the presence of 3-aminobenzamide in peroxynitrite-treated cells. Moreover, poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide dose-dependently reduced interferon-induced intercellular adhesion molecule 1 expression as well as interleukin-1beta-induced interleukin-8 expression. Our results indicate that peroxynitrite and poly(ADP-ribose) polymerase regulate keratinocyte function and death in contact hypersensitivity.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Caspases; Cell Division; Cell Line; Dermatitis, Contact; DNA Damage; DNA Fragmentation; Female; In Situ Nick-End Labeling; Intercellular Adhesion Molecule-1; Interleukin-8; Keratinocytes; Mice; Mice, Inbred Strains; Necrosis; Nitrates; Oxazolone; Poly(ADP-ribose) Polymerases; Skin; Tyrosine

The anti-inflammatory and anti-allergic activity of perilla leaf extract was investigated. The oral administration of perilla leaf extract to mice inhibited two types of acute inflammatory models, arachidonic acid-induced ear edema and 12-o-tetradecanoylphorbol-13-acetate-induced ear edema. Oral administration of perilla leaf extract also inhibited the contact dermatitis model, oxazolone-induced ear edema, by affecting sensitization.

Topics: Administration, Oral; Animals; Anti-Allergic Agents; Anti-Inflammatory Agents; Arachidonic Acid; Dermatitis, Contact; Disease Models, Animal; Edema; Magnoliopsida; Male; Mice; Mice, Inbred ICR; Oxazolone; Plant Extracts; Tetradecanoylphorbol Acetate

Microcirculation is the primary mechanism for delivering lymphocytes to inflammatory tissues. Blood flow within microvessels ensures a supply of lymphocytes at the blood-endothelial interface. Whether the structure of the inflammatory microcirculation facilitates lymphocyte transmigration is less clear. To illuminate the microcirculatory changes associated with lymphocyte transmigration, we used intravital videomicroscopy to examine the dermal microcirculation after application of the epicutaneous antigen oxazolone. Intravascular injection of fluorescein-labeled dextran demonstrated focal topographic changes in the microcirculation. These focal changes had the appearance of loops or hairpin turns in the oxazolone-stimulated skin. Changes were maximal at 96 h and coincided with peak lymphocyte recruitment. To determine whether these changes were associated with lymphocyte transmigration, lymphocytes obtained from efferent lymph of draining lymph nodes at 96 h were fluorescently labeled and reinjected into inflammatory microcirculation. Epifuorescence intravital video microscopy demonstrated focal areas were associated with lymphocyte slowing and occasional transmigration. In contrast, focal loops and lymphocyte slowing were rarely observed in the contralateral control microcirculation. Results suggest that structural adaptations in inflammatory microcirculation represented by focal topographic changes may contribute to regulation of tissue entry by recirculating lymphocytes.

Topics: Adjuvants, Immunologic; Animals; Cell Movement; Dermatitis, Contact; Ear; Lymphocytes; Microcirculation; Oxazolone; Sheep; Skin; Time Factors

Oxidation of lipoprotein-derived lipids is generally accepted to be important in atherogenesis, and lipophilic antioxidants have been suggested as potential antiatherosclerotic agents. The antiatherogenic effects observed by certain antioxidants, especially probucol, in different animal models support this suggestion. There are however also cases where other lipophilic antioxidants have not been able to support this hypothesis. This has raised the question whether the effects of probucol and similar compounds are mainly due to some other property, unrelated to their antioxidant efficacy. For example, probucol is shown to possess immunomodulatory properties. Immune reactions are known to occur during atherogenesis. We therefore tested the dimer of N-acetylcysteine, DiNAC, which is a disulfide with immunomodulating properties and enhances oxazolone-induced contact sensitivity (CS) reactions in mice, for effects on atherosclerosis. When given to male heritable hyperlipidemic rabbit (WHHL) rabbits from 10 to 22 weeks of age, this compound reduced by 50% thoracic aorta atherosclerosis (p < 0.05), without affecting plasma lipid levels. Here we also show that probucol and a close chemical analog, both known to prevent atherosclerosis in WHHL rabbits, enhance the CS reaction in mice, while two other related antioxidants did not affect the CS reaction. At least one of these is also without effect on atherosclerosis in WHHL rabbits. The results show that DiNAC might represent a new treatment modality for atherosclerosis-related disease, and suggest that some antioxidants may have antiatherosclerotic properties more related to "immunomodulatory" properties than to antioxidant properties in general.

Topics: Acetylcysteine; Adjuvants, Immunologic; Animals; Antioxidants; Arteriosclerosis; Cholesterol; Cystine; Dermatitis, Contact; Disulfides; Male; Mice; Mice, Inbred BALB C; Oxazolone; Probucol; Rabbits; Receptors, LDL; Structure-Activity Relationship; Triglycerides

Although the role of T cells in skin contact sensitivity (CS) immune reactions has been intensely studied, much less is known about the regulatory properties of T cells in the oral mucosa. Animal experiments have shown that hapten sensitization of the ectodermal oral mucosa leads to antigen-specific hypersensitivity reactions in the skin. Furthermore, oral mucosa or skin hapten sensitization resulted in CS inflammatory reactions in the oral mucosa on challenge. The oral mucosa CS responses were similar to those found skin with regard to cell phenotypes and cytokines. CS-like reactions were also found in the oral mucosa after exposure to an irritant detergent, sodium lauryl sulfate (SLS). The oral mucosa reacted at smaller SLS doses than did skin. Ions and molecules released fron dental restorative materials (together with saliva and food and/or beverages) expose the gastrointestinal mucosa continuously over long time periods. From animal experiments we have learned that mice given antigen by gastric feeding, subsequently antigen-sensitized on skin, and finally elicited in the oral mucosa and in ear skin, showed tolerance in skin but gave simultaneous CS inflammatory reactions in the oral mucosa. Moreover, exposure of colon mucosa to antigen produced CS reactions in oral mucosa after challenge. Are there CS reactions in the oral mucosa? Clinical and experimental studies indicate that the oral mucosa can function both as induction and expression site of CS. The GI tract may be an important modifier of the CS inflammatory reactions seen in the oral mucosa.

Topics: Adjuvants, Immunologic; Animals; Dermatitis, Contact; Haptens; Humans; Hypersensitivity, Delayed; Irritants; Mice; Models, Animal; Mouth Mucosa; Oxazolone; Rats; Sodium Dodecyl Sulfate; Surface-Active Agents; T-Lymphocytes; Trinitrobenzenesulfonic Acid

We evaluated the importance of IL-2 and IL-6 in primary antigen-induced proliferation of lymph node cells (LNC) and the induction of contact hypersensitivity (CH). These responses were examined in cytokine-deficient mice following application of the contact sensitizer, oxazolone (OX). Proliferation and induction of IL-6 by LNC from IL-2-deficient (IL-2(-/-)) mice were reduced by approximately 95 %, relative to the proliferation of LNC from IL-2(+/+) mice, although induction and elicitation of CH responses was not significantly affected. In contrast, the proliferation of LNC from sensitized IL-6(- /-) mice was reduced by approximately 50% and the CH response was significantly reduced, relative to responses of IL-6(+/+) mice. Although nonspecific inflammatory responses induced by croton oil were similar in IL-6(+/+) and IL-6(-/-) mice, both the acute inflammatory response to OX and the second phase of the inflammatory response were significantly reduced. Thus IL-2 and IL-6 play a significant role in the total proliferative response of LNC following primary contact sensitization. However, the proliferation they promote is not critical for priming the antigen-specific effector cells responsible for eliciting CH responses and IL-6 appears to be more important for expression of the later phases of acute inflammation and the CH induced by OX.

Topics: Animals; Dermatitis, Contact; Interleukin-2; Interleukin-6; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Oxazolone

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.

Topics: Administration, Oral; Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; Dermatitis, Contact; Dinitrofluorobenzene; DNA-Binding Proteins; Epitopes, T-Lymphocyte; Female; Haptens; Immune Tolerance; Interferon-gamma; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Skin; T-Lymphocytes, Cytotoxic; Transposases

Clenoliximab and keliximab are monkey/human chimeric monoclonal antibodies (mAbs) of the immunoglobulin G4 (IgG4) and IgG1 isotypes, respectively, that recognize the same epitope on human CD4. The two mAbs possess identical idiotypes and exhibit equal affinities for CD4. Upon administration of these mAbs to mice that express a human CD4 transgene, but not mouse CD4 (HuCD4/Tg mice), clenoliximab and keliximab exhibited similar kinetics of binding to CD4, and induced the same degree of CD4 modulation from the cell surface, although only keliximab mediated CD4+ T-cell depletion. Epicutaneous sensitization and challenge of HuCD4/Tg mice with the hapten oxazolone resulted in a contact sensitivity response characterized by tissue swelling, and the presence of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the local tissue. Administration of a single 2-mg dose of either clenoliximab or keliximab to HuCD4/Tg mice prior to sensitization significantly reduced post-challenge tissue swelling, and levels of IFN-gamma and IL-4, indicating that CD4+ T-cell depletion is not required for anti-CD4 mAb-mediated inhibition of contact sensitivity. Administration of either mAb prior to challenge failed to inhibit the contact sensitivity response, indicating differential sensitivity of the afferent and efferent phases of the response to inhibition by CD4-specific mAbs. Collectively, these data indicate that CD4 functions as a positive regulatory molecule in the contact sensitivity response.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; CD4 Antigens; CD4-Positive T-Lymphocytes; Dermatitis, Contact; Dose-Response Relationship, Immunologic; Humans; Immune Tolerance; Interferon-gamma; Interleukin-4; Mice; Mice, Transgenic; Oxazolone

Contact hypersensitivity (CHS) is thought to be mainly associated with the activation of T helper type 1 (Th1) cells. However, there is also evidence that Th2 cells or Th2 cytokines play a role in the development of CHS. To analyze the functional contribution of Th2 cytokines interleukin (IL)-4 and IL-13, signal transducer and activator of transcription 6 (STAT6)-deficient (STAT6(-/)-) and wild-type (wt) control C57BL/6 mice were contact sensitized with 5% 2,4,6-trinitrochlorobenzene (TNCB), 0.5% 2,4-dinitrofluorobenzene, or 5% 4-ethoxyl methylene-2-phenyl-2-oxazolin-5-one, and any skin reactions were examined. Ear swelling was significantly reduced with a delayed peak response in STAT6(-/)- mice compared with wt mice.A histological analysis revealed that the infiltration of both eosinophils and neutrophils in the skin challenged after 24 h in STAT6(-/)- mice decreased substantially compared with that in wt mice. The expression of Th2 cytokines (IL-4, IL-5) in TNCB-challenged skin tissues and the supernatants from T cells stimulated by 2,4,6-trinitrobenzene sulfonate-modified spleen cells, as well as the immunoglobulin (Ig)E and IgG1 response after challenge, were also profoundly reduced in STAT6(-/)- mice, whereas the expression of interferon gamma was the same in STAT6(-/)- and wt mice after challenge. Furthermore, adoptive transfer experiments revealed that STAT6(-/)- mice induced CHS after injection of lymph node cells obtained from sensitized wt mice. Our data suggest that the STAT6 signal plays a critical role in the induction phase of CHS.

Topics: Animals; Antigens, Surface; Cell Count; Cytokines; Dendritic Cells; Dermatitis, Contact; Dinitrofluorobenzene; Epidermal Cells; Epidermis; Erythrocytes; Flow Cytometry; Hypersensitivity, Delayed; Immunoglobulin E; Immunoglobulin G; Irritants; Langerhans Cells; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazoles; Picryl Chloride; Sheep; Signal Transduction; STAT6 Transcription Factor; T-Lymphocytes; Thy-1 Antigens; Trans-Activators

Risk assessment of sensitizing chemicals requires, besides hazard identification, the assessment of potency. To examine the sensitizing capacity of low molecular weight chemicals, a murine local lymph node assay (LLNA) was used. The sensitizing capacity of known allergens was quantified by dose-response modeling. At a stimulatory index (SI) of 3, the corresponding estimated concentration was calculated (EC(3)), together with a confidence interval to take account of the quality of the particular data set. We tested ten allergens (ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), 4-ethoxymethylene 2-phenyl oxazol-5-one (oxazolone), phthalic anhydride (PA), toluene diisocyanate (TDI), trimellitic anhydride (TMA), tetramethylthiuramdisulfide (TMTD) and zincdimethyldithiocarbamate (ZDMC)). Oxazolone showed the strongest sensitizing potency followed in this order by DNCB, TDI, TMA, PA, TMTD, ZDMC, MBT, benzocaine and DEA. The approach performed in this study is a way to accurately assess the potency of sensitizing chemicals and thus a possibility for classification.

Topics: Allergens; Animals; Benzocaine; Benzothiazoles; Dermatitis, Contact; Diethylamines; Dinitrochlorobenzene; Dose-Response Relationship, Immunologic; Female; Linear Models; Lymph Nodes; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides; Regression Analysis; Scintillation Counting; Specific Pathogen-Free Organisms; Thiazoles; Thiram; Toluene 2,4-Diisocyanate; Ziram

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Cell Migration Inhibition; Chemokine CCL21; Chemokines, CC; Cytokines; Dermatitis, Contact; Dinitrofluorobenzene; Female; Haptens; Immune Tolerance; Immunization; Injections, Intravenous; Langerhans Cells; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazolone; T-Lymphocytes

Recent reports have suggested that chemical-induced allergic contact dermatitis may not be a traditional type IV hypersensitivity, in part due to the dual irritant and antigenic properties of sensitizing chemicals. In order to investigate the contribution of these properties to the molecular and cellular mechanism underlying allergic contact dermatitis, we evaluated oxazolone-induced changes in cell populations and cytokine production in the dermis of transgenic mice with impaired innate immunity (the FcgammaR subunit knockout mouse), and absent specific immunity (the athymic mouse), and the appropriate B6,129F2 and C57BL/6 control mice. Oxazolone and croton oil were applied in a single sensitizing dose, or in sensitizing and challenge doses, and the dermal response was evaluated by immunohistochemistry. In the wild type mice, with or without sensitization to oxazolone or croton oil, we observed mixed Th1/Th2 cytokine production and both CD4+ and CD8+ T lymphocytes; however, the neutrophil was the predominant cell in the dermis, even 72 h after final chemical application. Athymic mice displayed a similar neutrophil response with moderate Th1/Th2 cytokine production, and FcgammaR subunit knockout mice exhibited very mild dermatitis when treated with either oxazolone or croton oil. These results provide support for the hypothesis that allergic contact dermatitis is not a classic delayed type hypersensitivity, demonstrate the importance of the interaction between the irritant and antigenic properties of sensitizing chemicals in the development of allergic contact dermatitis, and suggest that the irritant effect of chemicals may be mediated through the cutaneous innate immune system.

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Croton Oil; Cytokines; Dermatitis, Contact; Dermatologic Agents; Ear; Immunity; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Nude; Neutrophils; Oxazolone; Receptors, IgG; Reference Values; Skin; Th1 Cells; Th2 Cells

UV radiation causes a number of cellular changes within the skin which play a role in tumor outgrowth, including immunosuppression and production of growth-enhancing cytokines. Both of these enable tumors to grow but their relative importance in carcinogenesis is poorly defined. In this study, C3H/HeN mice were exposed to a single inflammatory dose of 410 mJ/cm(2) UVB radiation (plus 100 mJ/cm(2) UVA radiation) followed by the inoculation of a regressor squamous cell carcinoma into or the painting of oxazolone onto the treated skin. Tumors transplanted 2 or 3 but not 4 days after irradiation had a significantly higher growth rate than tumors inoculated into unirradiated control mice. In contrast, mice failed to respond to hapten when it was applied 2, 3 or 4 days after irradiation. Cytofluorimetric analysis demonstrated that the number of F4/80(+) Langerhans cells was not significantly reduced until 4 days after irradiation, while the number of dendritic epidermal T cells was significantly lower at all time points observed after UV-irradiation. Furthermore, a large cellular infiltration of CD11b(+), Gr-1(+), CD45(+) MHC class II(+) and CD45(+) MHC class II(-) cells into the epidermis was observed 2 and 3 days after irradiation, which corresponded with the enhanced tumor growth. To a lesser extent tumor growth was also associated with CD45(+) MHC class II(hi) cells, possibly the previously described UV-induced macrophage. In contrast, suppression of contact hypersensitivity corresponded with the reduction in dendritic epidermal T cells but not with other cell changes. The results suggest that, in this model, where immunosuppression did not appear to be responsible for enhanced tumor growth, inflammatory infiltrates may contribute to the promotion of skin tumor growth within UV-irradiated skin.

Topics: Adjuvants, Immunologic; Animals; Carcinoma, Squamous Cell; Cell Division; Cell Movement; Dermatitis, Contact; Epidermal Cells; Epidermis; Female; Langerhans Cells; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Oxazolone; Skin Neoplasms; T-Lymphocytes; Ultraviolet Rays

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.

Topics: Administration, Cutaneous; Adoptive Transfer; Animals; CD4 Antigens; CD8 Antigens; Dermatitis, Contact; Dinitrofluorobenzene; Immune Sera; Immune Tolerance; Injections, Intravenous; Interferon-gamma; Interleukin-10; Interleukin-4; Intracellular Fluid; Lymph Nodes; Lymphocyte Depletion; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Th1 Cells; Th2 Cells

Chemical allergens that induce contact sensitivity cause changes in levels of epidermal cytokines. In mice one of the earliest epidermal cytokines to be upregulated following sensitization is interleukin-1 beta (Iota L-1 beta). The present study investigated the kinetics and in situ localization of induced IL-1 beta expression in mouse skin following topical exposure to the contact allergen oxazolone. Mice were exposed topically to 1% oxazolone, with control mice exposed to vehicle (acetone:olive oil 4:1) alone, and at various times thereafter skin was excised for IL-1 beta mRNA and protein determination by in situ hybridization and enzyme-linked immunosorbant assay (ELISA), respectively. IL-1 beta mRNA was found to be expressed constitutively at low levels in skin from naïve (untreated) and vehicle-treated mice, with mRNA localized in some hair follicles and sebaceous glands; no IL-1 beta mRNA was detected in the epidermis of control animals. Following topical exposure of mice to oxazolone for 5-15 min, upregulation of IL-1 beta mRNA was observed in the epidermis, dermis, hair follicles, and sebaceous glands; at 90 min and beyond the pattern of IL-1 beta mRNA expression declined toward control. Analysis of whole skin homogenates by ELISA demonstrated cutaneous IL-1 beta protein to be present constitutively in both vehicle-treated and naïve mice. Following exposure to oxazolone, cutaneous IL-1 beta protein expression was elevated at 30 min, decreased at 1 h, and fell below the limit of detection of the assay at 2 h before returning to constitutive levels at 4 and 24 h. IL-1 beta protein levels in vehicle-treated mice, naïve mice, and mice treated with the respiratory allergen trimellitic anhydride were unchanged over this time period. The present study demonstrated that IL-1 beta mRNA expression was upregulated rapidly and transiently in well-defined regions of mouse epidermis and dermis during contact sensitization, and was succeeded by an elevation in IL-1 beta protein. This early highly localized upregulation of IL-1 beta lends further support to the hypothesis that this cytokine plays a key role in the initial stages of skin sensitization. Such information will enhance our understanding of the molecular processes involved in allergic contact dermatitis and may provide a mechanistic basis for designing refined animal and in vitro alternatives to existing models of skin sensitization.

Topics: Animals; Dermatitis, Contact; Female; Interleukin-1; Mice; Mice, Inbred BALB C; Oxazolone; RNA, Messenger; Skin

The T-cell-mediated contact hypersensitivity reaction (CHR) is thought to be involved in the pathogenesis of clinical cutaneous disorders including atopic dermatitis. A novel diaminouracil derivative, CX-659S, has been reported to have an inhibitory activity against picryl chloride (PC)-induced CHR when administered either orally or percutaneously. The inhibitory effect of topical CX-659S was assessed in three CHR models in the present study. In addition, to elucidate the mechanism of action of this compound, we examined the effect of CX-659S on the expression of messenger RNAs for proinflammatory cytokines after elicitation in PC models.. For the in vivo evaluation of the efficacy of CX-659S, we used PC- or oxazolone-induced CHR in mice and 2,4-dinitrochlorobenzene (DNCB)-induced CHR in guinea pigs. CX-659S was topically applied immediately after the hapten challenge in each model. To assess the effect on gene expression of cytokines, we used the reverse transcriptase-polymerase chain reaction (RT-PCR), a semiquantitative technique with specific primers.. Topical CX-659S dose-dependently inhibited ear swelling at 24 h after the challenge in the two mouse models. This inhibitory effect was histologically confirmed in the PC model. Topically applied CX-659S also inhibited erythema and edema formation 24 h after challenge in the guinea pig model. CX-659S inhibited the expression of mRNA for proinflammatory cytokines IL-1 beta and TNF-alpha in vivo.. Topically applied CX-659S showed significant inhibitory activities against CHR models both in mice and in guinea pigs. Inhibition profiles of CX-659S toward mRNA expression for proinflammatory cytokines corroborated these findings. CX-659S thus could be a useful therapeutic agent for allergic cutaneous disorders such as allergic contact dermatitis and atopic dermatitis.

Topics: Administration, Topical; Animals; Base Sequence; Dermatitis, Contact; Dinitrochlorobenzene; Disease Models, Animal; DNA Primers; Guinea Pigs; Humans; Immunosuppressive Agents; Interleukin-1; Male; Mice; Mice, Inbred ICR; Oxazolone; RNA, Messenger; Tumor Necrosis Factor-alpha; Uracil

We have studied vascular endothelial activation and increased expression of ICAM-1 and VCAM-1 at the onset of the elicitation phase of oxazolone contact hypersensitivity in mice. By measuring the local uptake of i.v. administered radiolabeled anti-ICAM-1 and anti-VCAM-1 mAb, we found that endothelial ICAM-1 and VCAM-1 was increased by 4 h after challenge, 2 h later than the first peak of ear swelling and 125I-labeled human serum albumen uptake. Increased expression of endothelial ICAM-1 and VCAM-1 was significantly greater in sensitized animals than in naive animals. Anti-TNF-alpha antiserum significantly inhibited both the increase in ear thickness (p < 0.01), and the up-regulation of ICAM-1 and VCAM-1 expression (p < 0.01 for both) at 4 h. In contrast, the combination of anti-IL-1alpha and IL-1beta had only a small inhibitory effect on ICAM-1 expression (p < 0.05) and no significant effect on increased ear thickness or on VCAM-1 expression. A mixture of anti-TNF-alpha, anti-IL-1alpha, and IL-1beta was no more inhibitory for endothelial ICAM-1 and VCAM-1 expression than anti-TNF-alpha alone. ICAM-1 and VCAM-1 expression at 4 h was unaffected by a combination of mAb against alpha4 and beta2 integrins, whereas expression at 24 h was significantly inhibited (p < 0.05), suggesting that the release of TNF-alpha and other cytokines involved in the initiation of the response may not require leukocyte traffic or other leukocyte functions involving these integrins. We conclude that the early up-regulation of endothelial ICAM-1 and VCAM-1 during the elicitation of contact hypersensitivity is primarily due to the immune-dependent local release of TNF-alpha.

Topics: Animals; Antibodies, Monoclonal; Dermatitis, Contact; Endothelium, Vascular; Female; Humans; Intercellular Adhesion Molecule-1; Kinetics; Mice; Mice, Inbred BALB C; Oxazolone; Serum Albumin; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Oral N-acetyl-L-cysteine (NAC) is used clinically for treatment of chronic obstructive pulmonary disease. NAC is easily oxidized to its disulfide. We show here that N,N'-diacetyl-L-cystine (DiNAC) is a potent modulator of contact sensitivity (CS)/delayed type hypersensitivity (DTH) reactions in rodents. Oral treatment of BALB/c mice with 0.003 to 30 micromol/kg DiNAC leads to enhancement of a CS reaction to oxazolone; DiNAC is 100 to 1000 times more potent than NAC in this respect, indicating that it does not act as a prodrug of NAC. Structure-activity studies suggest that a stereochemically-defined disulfide element is needed for activity. The DiNAC-induced enhancement of the CS reaction is counteracted by simultaneous NAC-treatment; in contrast, the CS reaction is even more enhanced in animals treated with DiNAC together with the glutathione-depleting agent buthionine sulfoximine. These data suggest that DiNAC acts via redox processes. Immunohistochemically, ear specimens from oxazolone-sensitized and -challenged BALB/c mice treated with DiNAC display increased numbers of CD8(+) cells. DiNAC treatment augments the CS reaction also when fluorescein isothiocyanate is used as a sensitizer in BALB/c mice; this is a purported TH2 type of response. However, when dinitrofluorobenzene is used as a sensitizer, inducing a purported TH1 type of response, DiNAC treatment reduces the reaction. Treatment with DiNAC also reduces a DTH footpad-swelling reaction to methylated BSA. Collectively, these data indicate that DiNAC in vivo acts as a potent and effective immunomodulator that can either enhance or reduce the CS or DTH response depending on the experimental conditions.

Topics: Acetylcysteine; Adjuvants, Immunologic; Animals; CD8-Positive T-Lymphocytes; Cystine; Dermatitis, Contact; Dinitrofluorobenzene; Ear; Female; Fluorescein-5-isothiocyanate; Foot; Granuloma; Hypersensitivity, Delayed; Immunohistochemistry; Lymphocyte Count; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Oxazolone; Rabbits; Serum Albumin, Bovine

Solar radiation contains ultraviolet B (280-315 nm) and ultraviolet A (ultraviolet AII, 315-340 nm; ultraviolet AI, 340-400 nm) wavebands. Ultraviolet B is known to suppress certain aspects of cell mediated immunity. Using three ultraviolet lamps (the broad-band ultraviolet B TL-12, the narrow-band ultraviolet B TL-01 and an ultraviolet AI source), we investigated the dose and waveband dependencies for the suppression of contact hypersensitivity to oxazolone and delayed-type hypersensitivity to herpes simplex virus, plus the formation of cis-urocanic acid in C3H/HeN mice. A single exposure of 1500 J/m2 TL-12 or 10,000 J/m2 TL-01 or 500,000 J/m2 ultraviolet AI corresponded to 1 minimum erythema dose in this mouse strain. The percentage of cis-urocanic acid of the total urocanic acid rose from a background level of 1.7% to 40% with 1000 J/m2 TL-12 or 10,000 J/m2 TL-01, but only 17% cis-urocanic acid was obtained with 500,000 J/m2 ultraviolet AI. The contact hypersensitivity response was significantly suppressed after a minimum dose of 5000 J/m2 TL-12 or 50,000 J/m2 TL-01 or 500,000 J/m2 ultraviolet AI. The delayed-type hypersensitivity response was suppressed by a minimum dose of 100 J/m2 TL-12 or 10,000 J/m2 TL-01 or 1000 J/m2 ultraviolet AI. So, whereas a low dose of ultraviolet AI reduced the delayed-type hypersensitivity response, a 500-fold higher dose was required to suppress contact hypersensitivity. There was no correlation between the suppression of these responses and the concentration of cis-urocanic acid in the skin. Thus different mediators may modulate the various immune responses affected by ultraviolet exposure, depending on the wavelength of the radiation.

Topics: Animals; Dermatitis, Contact; Dose-Response Relationship, Radiation; Erythema; Female; Hypersensitivity, Delayed; Mice; Mice, Inbred C3H; Oxazolone; Simplexvirus; Skin; Ultraviolet Rays; Urocanic Acid

1. Small, N- to C-terminal cyclized peptides containing the leucyl-aspartyl-valine (LDV) motif from fibronectin connecting segment-1 (CS-1) have been investigated for their effects on the adhesion of human T-lymphoblastic leukaemia cells (MOLT-4) to human plasma fibronectin in vitro mediated by the integrin Very Late Antigen (VLA)-4 (alpha4beta1, CD49d/CD29). 2. Cyclo(-isoleucyl-leucyl-aspartyl-valyl-aminohexanoyl-) (c(ILDV-NH(CH2)5CO)) was approximately 5 fold more potent (IC50 3.6+/-0.44 microM) than the 25-amino acid linear CS-1 peptide. Cyclic peptides containing two more or one less methylene groups had similar potency to c(ILDV-NH(CH2)5CO) while a compound containing three less methylene groups, c(ILDV-NH(CH2)2CO), was inactive at 100 microM. 3. c(ILDV-NH(CH2)5CO) had little effect on cell adhesion mediated by two other integrins, VLA-5 (alpha5,beta1, CD49e/CD29) (K562 cell adhesion to fibronectin) or Leukocyte Function Associated molecule-1 (LFA-1, alphabeta2, CD11a/CD18) (U937 cell adhesion to Chinese hamster ovary cells transfected with intercellular adhesion molecule-1) at concentrations up to 300 microM. 4. c(ILDV-NH(CH2)5CO) inhibited ovalbumin delayed-type hypersensitivity or oxazolone contact hypersensitivity in Balb/c mice when dosed continuously from subcutaneous osmotic mini-pumps (0.1-10 mg kg(-1) day(-1)). Maximum inhibition (approximately 40%) was similar to that caused by the monoclonal antibody PS/2 (7.5 mg kg(-1) i.v.) directed against the alpha4 integrin subunit. 5. c(ILDV-NH(CH2)5CO) also inhibited oxazolone contact hypersensitivity when dosed intravenously 20 h after oxazolone challenge (1-10 mg kg(-1)). Ear swelling was reduced at 3 h and 4 h but not at 1 h and 2 h post-dose (10 mg kg(-1)). 6. Small molecule VLA-4 inhibitors derived from c(ILDV-NH(CH2)5CO) may be useful as anti-inflammatory agents.

Topics: Amino Acid Sequence; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Adhesion; CHO Cells; Cricetinae; Dermatitis, Contact; Female; Fibronectins; Humans; Hypersensitivity, Delayed; Inflammation; Integrin alpha4beta1; Integrins; Intercellular Adhesion Molecule-1; Intercellular Signaling Peptides and Proteins; Leukemia, Erythroblastic, Acute; Leukemia, T-Cell; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Ovalbumin; Oxazolone; Peptides; Rats; Receptors, Lymphocyte Homing; T-Lymphocytes; Transfection

We have examined the role of endogenously produced interleukin-4 (IL-4) in the contact hypersensitivity (CH) reaction to the haptene trinitrochlorobenzene (TNCB). The CH reaction was abolished in IL-4 genetically deficient mice (IL-4 KO), when compared to wild-type (wt) mice. The CH reaction was restored by treatment with IL-4 and further analysis revealed that IL-4 exerted its action both at the induction and effector stages of the CH reaction. Despite failure to develop a CH reaction, IL-4 KO mice developed a T helper type 1 (Th1) response to TNCB, in terms of lymphokine production in vitro. Furthermore, the number of Vgamma3+ cells accumulating in the lymph nodes of TNCB-immune IL-4 KO mice was normal. The recruitment of mononuclear cells and vascular leakage at the challenge site were consistently reduced in IL-4 KO mice and were restored by injection of IL-4. This suggests that IL-4 acts as a proinflammatory mediator in CH, perhaps favouring the accumulation of mononuclear cells at the site of inflammation. Among Th2-type cytokines, IL-13, but not IL-10, was shown to restore the CH reaction to TNCB in IL-4 KO mice. However, IL-4 KO mice developed a normal CH response to oxazolone, indicating that IL-4 was required for the CH reaction to TNCB, but not for that to oxazolone.

Topics: Adjuvants, Immunologic; Animals; Capillary Permeability; Dermatitis, Contact; Inflammation Mediators; Interleukin-10; Interleukin-13; Interleukin-4; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxazolone; Picryl Chloride; Th1 Cells

The interleukin-4 transgenic mice investigated here exhibit a ubiquitous expression of interleukin-4 in all organs, including the skin. In this study, the induction phase of oxazolone-induced local primary contact hypersensitivity and croton oil-induced irritant contact dermatitis in transgenic and wild-type mice was analysed. Compared to wild-type mice, the transgenic mice showed a decreased activation of the skin-draining lymph nodes but a strong hyperreactivity in the skin after topical sensitisation. In contrast to this, both the transgenic and the wild-type mice developed a strong and comparable inflammatory skin reaction after topical irritation. A striking increased expression level of tumour necrosis factor-alpha and macrophage inflammatory protein-2 genes were found in the skin of the transgenic mice during primary local contact hypersensitivity, while both the transgenic and the wild-type mice developed comparable expression levels of these cytokines during irritant contact dermatitis. Compared to wild-type mice, a strongly enhanced expression level of interleukin-6 transcripts derived from epidermal antigen presenting cells were detected in the skin of IL-4 transgenic mice, whereas in the skin-draining lymph nodes of transgenic mice significantly lower levels were detected. We conclude that the migration of epidermal antigen-presenting cells towards the skin-draining lymph nodes is reduced in transgenic mice, which could be due to the different cytokine balance in these mice strains. The atypical irritant-like reaction observed in transgenic mice after topical sensitisation is a phenomenon comparable to atopic diseases and therefore this transgenic strain might be a helpful model for investigating the immunopathophysiological features of these diseases.

Topics: Animals; Antigen-Presenting Cells; Cell Movement; Chemokine CXCL2; Croton Oil; Dermatitis, Contact; Ear; Epidermis; Female; Gene Expression; Immunophenotyping; Interleukin-4; Interleukin-6; Lymph Nodes; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Monokines; Oxazolone; RNA, Messenger; Skin; Tumor Necrosis Factor-alpha

An analysis was conducted of the cytokine profile and inflammatory response in oxazolone sensitized mouse skin. Following exposure to oxazolone, the intralesional production of inflammatory cytokines was demonstrable at the levels of both mRNA and protein. An initial challenge led to a transient increase in tumor necrosis factor-alpha production followed predominately by the T helper (Th)1 cytokine, interferon-gamma. There was a minimal production of interleukin-4, a Th2 cytokine. Continued exposure to oxazolone led to a downregulation of interferon-gamma and an upregulation of interleukin-4 production. A strong relationship was found between interleukin-4 and the inflammatory response, as measured by ear thickness. Similar experiments conducted in mast cell-deficient mice revealed reduced neutrophil influx but only minor changes in cytokine profile. An irritant response induced by chronic exposure of mouse skin to phorbol ester did not reveal any significant interferon-gamma or interleukin-4 response but was characterized by a tumor necrosis factor-alpha response that correlated with the inflammatory response. These observations suggest that the major source of interferon-gamma and interleukin-4 in the oxazolone response may be the infiltrating lymphocytes; whereas the tumor necrosis factor-alpha may result from the local irritation seen with both oxazolone and phorbol ester. At the end of 4 wk of chronic exposure to oxazolone, it was found that serum IgE levels had significantly increased. Histologic analysis of the skin lesion revealed that a mixed infiltrate including eosinophils developed upon repeat exposure to oxazolone. These findings are consistent with an early predominate Th1 response that is reduced and largely replaced with a Th2 response upon chronic T cell activation.

Topics: Animals; Chronic Disease; Dermatitis, Contact; Immunoglobulin E; Interferon-gamma; Interleukin-4; Male; Mast Cells; Mice; Mice, Inbred BALB C; Oxazolone; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

The development of contact hypersensitivity (CHS) greatly depends on the allergenicity of the inducing agent. However, various cofactors are known to influence the outcome of the response as well. From this perspective, we have compared the effects of five different vehicles: acetone, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and a 4 to 1 mixture of acetone and olive oil (AOO) on the cellular and humoral immune responses to epicutaneously applied oxazolone in female BALB/c mice. A single application of 0.2% oxazolone dissolved in acetone or ethanol induced stronger proliferative responses and higher lymph node cell numbers than the other three vehicles. Moreover, both vehicles led to higher numbers of oxazolone-specific Ab forming cells in the draining lymph nodes of sensitized animals. When the IgG2a/IgG1 ratios were determined to indicate the type of T helper cell involved, the highest values were obtained with AOO and lowest with DMF and DMSO, while acetone and ethanol were in between. Moreover, no correlation was found between oxazolone-specific antibody production and cellular responses, measured as [3H]thymidine incorporation of draining lymph node cells after sensitization and increased ear thickness after challenge. From this study it can be concluded that cellular and humoral responses in CHS to oxazolone are dissimilarly affected by the vehicles used.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Cell Division; Dermatitis, Contact; Edema; Enzyme-Linked Immunosorbent Assay; Female; Immunity, Cellular; Immunoglobulin G; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Pharmaceutical Vehicles; Serum Albumin, Bovine; Thymidine

Productive interactions between B7-1 and B7-2 costimulatory molecules on dendritic cells (DC) and CD28 on T cells are thought to be critical for successful antigen presentation. Epicutaneous application of haptens induces both contact hypersensitivity (CHS), an inflammatory cutaneous response mediated by CD8+ T cells, and an anti-hapten antibody response mediated by CD4+ helper T cells. The role of B7 costimulation in the immune response to oxazolone (Ox) was analyzed using mice lacking either B7-1 (B7-1-/-), B7-2 (B7-2-/-), or both (Db-/-) of these costimulatory molecules. The absence of both B7-1 and B7-2 results in diminished CHS. This inhibition is largely overcome at higher hapten sensitizing doses indicating the presence of compensatory pathways. In contrast, anti-Ox IgG1 and IgG2a responses were not detected in the absence of both B7-1 and B7-2, even at high sensitizing doses, indicating an obligatory role of B7 costimulation in IgG class switching. B7-1 and B7-2 have overlapping functions in both CHS responses and anti-hapten response. B7-2-/- mice demonstrated a modestly reduced CHS response only at very low doses of Ox (0.05%), but responded normally at higher Ox doses, and B7-1-/- mice had CHS responses indistinguishable from those of wild-type mice. Similarly, anti-Ox IgG responses were comparable in wild-type, B7-1-/- and B7-2-/- mice. Taken together, these studies reveal distinct roles for B7 costimulation in response to epicutaneous antigens with an obligatory role for IgG class switching and an important, but nonessential role for CHS responses.

Topics: Animals; Antibody Formation; Antigen Presentation; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Dendritic Cells; Dermatitis, Contact; Membrane Glycoproteins; Mice; Mice, Knockout; Oxazolone; T-Lymphocytes

In the induction of contact hypersensitivity (CH) to an epicutaneously applied hapten, we have previously proposed that low doses of hapten sensitize primarily through epidermal Langerhans' cells (LC), whereas high doses rely largely on dermal antigen-presenting cells (APC). To examine this issue further, we applied either high or low doses of dinitrofluorobenzene (DNFB) epicutaneously to mice. We observed reduced LC density at the site after 12 h (nadir), which returned to normal levels at 24 h only after a low dose of hapten. When a low dose of an unrelated hapten, oxazolone, was painted on skin that had been painted 12 h previously with high dose of DNFB, oxazolone-specific CH was impaired. When grafts of whole skin, dermis alone, and epidermis alone prepared from skin painted 2 h previously with low or high doses of DNFB were placed onto naive, syngeneic mice, CH was induced by whole skin after both types of doses, by epidermis only after a low dose, and by dermis only after high dose. When epidermal cell suspensions were derivatized in vitro with low or high doses of DNFB, only cells exposed to a low dose induced proliferation of hapten-specific Tcells. Thus, only a low dose of hapten reveals the APC functions of LC without the participation of dermal APC.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; Dermatitis, Contact; Dinitrofluorobenzene; Dose-Response Relationship, Immunologic; Epidermis; Epithelium; Haptens; Mice; Mice, Inbred BALB C; Oxazolone; Skin; Skin Transplantation

The suppression of contact hypersensitivity by UVB (280-320 nm) radiation can be prevented by photoreactivating light (PRL; 320-400 nm) in the opossum Monodelphis domestica, implicating epidermal DNA lesions as the immunosuppressive impairment. However, contact hypersensitivity can also be suppressed in the opossum with exogenous cis-urocanic acid, a molecule which is produced in UVB-irradiated epidermis and is a second potential mediator of photo-immunosuppression apparently independent of UVB-induced DNA damage. Here we demonstrate that irradiation of opossums with PRL either before or following treatment with exogenous cis-urocanic acid, significantly reduced the degree of immunosuppression. This suggests that, in addition to its capacity to initiate post-UVB-exposure epidermal DNA repair, the PRL waveband can induce an immunoprotective product, as yet unidentified, in opossum epidermis.

Topics: Administration, Topical; Animals; Dermatitis, Contact; Dinitrofluorobenzene; Immune Tolerance; Opossums; Oxazolone; Skin; Ultraviolet Rays; Urocanic Acid

Topical glucocorticosteroids represent the mainstay of antiinflammatory therapy in the treatment of inflammatory skin diseases. Their clinical use, however, is limited by local and systemic side-effects. Thus, in dermatopharmacology there is a large demand for alternative non-steroidal antiinflammatories. Other than transplantation models, most of the frequently used in vivo test systems for assessment of drug-induced immunosuppression measure changes in inflammatory skin responses by means of skin erythema and edema after challenge of sensitized animals. The aim of this study was to develop an alternative mouse model to detect and analyse immunosuppressive effects of topically applied drugs. On the basis of a modified local lymph node assay, we analysed effects of topical hydrocortisone, dexamethasone, mometasone furoate and FK506 (tacrolimus) during the induction phase of contact hypersensitivity. On 4 consecutive days, NMRI mice were treated on the dorsal surfaces of both ears with increasing concentrations of test compound. During the last 3 days, the mice received in addition the contact sensitizer, oxazolone (1%). On day 5, draining auricular lymph nodes were removed in order to assess lymph node cell counts and perform flow cytometric analysis of lymph node cell subpopulations (CD4+/CD25+, Ia+/CD69+, Ia+/B220+). All test compounds proved to exert significant immunosuppressive effects after topical application, but showed differences in their immunomodulatory potential. In conclusion, the local lymph node assay serves as an appropriate model to characterize immunosuppressive effects of topically applied drugs by measuring immunologically relevant end-points.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Dexamethasone; Drug Evaluation, Preclinical; Female; Hydrocortisone; Immunosuppressive Agents; Lymph Nodes; Mice; Mometasone Furoate; Oxazolone; Pregnadienediols; Tacrolimus

Topics: Adenosine Triphosphatases; Animals; Dermatitis, Contact; Epidermal Cells; Epidermis; Immunosuppression Therapy; Interleukin-4; Langerhans Cells; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Knockout; Oxazolone; Ultraviolet Rays

Steroid hormones, such as glucocorticoids (GC), influence immune and inflammatory responses through their suppressive actions. Recent evidence suggests that another steroid hormone, dehydroepiandrosterone (DHEA), provides an immunostimulatory influence opposing the effect of GC. DHEA circulates in its inactive sulphated form, DHEAS, requiring conversion to DHEA by a steroid sulphatase (SS) enzyme for biological activity. Therefore, inhibition of SS activity may affect immune responses, allowing endogenous GC effects to predominate. We have shown that administration of DHEA and DHEAS in contact sensitization (CS) augments ear swelling by 39 and 46% respectively (P < 0.001). DHEAS at doses of 0.5, 5 and 50 mg/kg reverses the inhibitory effect of corticosterone (5 mg/kg) (P < 0.01). In CS, CT2251 (SS inhibitor) at 10 and 0.1 mg/kg inhibited ear swelling by 61 and 38% (P < 0.05) respectively. In addition, it inhibited DHEAS-augmented responses by 49 and 35% respectively (P < 0.05), with no effect on DHEA-augmented responses. DHEAS reversed CT2251 inhibition of the CS response with complete reversal at 50 mg/kg (P < 0.05). DHEAS and CT2251 appear to affect cellular infiltration into the ear, since DHEAS increased the number of lymphocytes by 63.8% and macrophages by 107% (P < 0.001), whereas CT2251 at 0.1 mg/kg decreased the number of lymphocytes by 65% (P < 0.001) and macrophages by 80% (P < 0.001). DHEAS, CT2251 and dexamethasone had no effect on oedema in the ear. From our data we have shown that steroid hormones, such as DHEA, have the potential to act as immunostimulatory factors in vivo. Inhibiting the conversion of DHEAS to DHEA by SS enzyme leads to an anti-inflammatory effect.

Topics: Adjuvants, Immunologic; Animals; Arylsulfatases; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Dermatitis, Contact; Dexamethasone; Enzyme Inhibitors; Estrone; Male; Mice; Mice, Inbred BALB C; Oxazolone; Steryl-Sulfatase

Ear edema models are regularly used for topical testing of antiinflammatory compounds. However, test compounds are usually applied simultaneously with proinflammatory agents at the same site which may result in mutual interactions. In order to avoid the occurrence of false antiinflammatory effects, a model of oxazolone-induced contact hypersensitivity has been described in which the hapten and test compound are each applied separately to only one side of the ear. By splitting and weighing the dorsal and ventral cutis of the ears, it was shown that the edemateous response of the control nonhapten side was comparable with the hapten-treated side. Some agents with antiinflammatory properties, as for example, dapsone, cimetidine, cyclosporine A, and budesonide, were tested simultaneously with oxazolone on both sides of the ear or applied separately on the dorsal and ventral ear sides, respectively. When dissolving the compounds in solutions of oxazolone, marked colorations of the test solutions were noted, indicating the occurrence of a chemical interaction. On simultaneous application at the same area, almost complete inhibition of the edemateous response was obtained for all compounds tested. In contrast, when applied separately, only budesonide appeared to exhibit antiinflammatory activity. The results indicate that the proposed model can be used to avoid the occurrence of interactions between oxazolone, and possibly other sensitizers, and substances that are being evaluated for topical antiinflammatory activity. By use of this model spurious antiinflammatory activity can be detected.

Topics: Adjuvants, Immunologic; Animals; Artifacts; Budesonide; Dermatitis, Contact; Disease Models, Animal; Drug Interactions; Ear; Edema; False Positive Reactions; Female; Haptens; Mice; Mice, Inbred BALB C; Organ Size; Oxazolone; Toxicity Tests

A MeOH extract from Z. africana was examined for topical antiinflammatory activity and proved to be active against arachidonic acid (AA) acute edema, 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced chronic inflammation, and oxazolone delayed-type hypersensitivity in mice. The extract also showed significant inhibitory activity of Naja naja phospholipase A2 when a polarographic method was used. Two oleanane-type triterpene saponins, zanhasaponins A (1) and B (2), and the cyclitol pinitol (4), isolated from the extract, were active as inhibitors of PLA2. A further saponin, zanhasaponin C (3) was inactive in this assay.

Topics: Adjuvants, Immunologic; Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Edema; Enzyme Inhibitors; Female; Mice; Oxazolone; Peroxidase; Phospholipases A; Phospholipases A2; Saponins; Skin; Tetradecanoylphorbol Acetate; Triterpenes

The effect of the histamine H2-receptor antagonist, cimetidine, on the cutaneous Arthus-like hypersensitivity to oxazolone elicited injecting subcutaneously oxazolone conjugated to egg-albumin (EA-OX) has been examined in the chicken. Cimetidine had opposite effects on the cutaneous reaction to oxazolone in relation to a different immunization schedule. Cimetidine enhanced the cutaneous reaction to oxazolone obtained immunizing chickens with oxazolone dissolved in ethanol (Eth-OX); instead cimetidine inhibited the cutaneous reaction obtained in chickens immunized with oxazolone dissolved in complete Freund adjuvant (CFA-OX). Optimum enhancement of the cutaneous arthus-like reaction to oxazolone occurred when cimetidine was given for three consecutive days starting at the immunization. The enhancing effect was absent in neonatally bursectomized chickens. Moreover, cimetidine stimulated bursal cell proliferation at day 1 after sensitization. The study of the immunoglobulin class of oxazolone antibodies produced in the immunized chickens demonstrated that cimetidine stimulated the IgM oxazolone antibody synthesis in Eth-OX immunized chickens and inhibited the IgY oxazolone antibody production in Eth-OX and total oxazolone antibody production in CFA-OX immunized chickens. The relationship between increased IgM oxazolone antibody synthesis and enhancement of the cutaneous Arthus reaction is discussed. The role of IgM antibodies in the pathogenesis of Arthus reaction in the chicken is hypothesized.

Topics: Adjuvants, Immunologic; Albumins; Animals; Antibody Formation; Arthus Reaction; B-Lymphocytes; Bursa of Fabricius; Chickens; Cimetidine; Dermatitis, Contact; Ethanol; Freund's Adjuvant; Histamine H2 Antagonists; Lymphocyte Activation; Male; Oxazolone

The induction phase of contact sensitization is associated with the movement of epidermal Langerhans cells (LC) from the skin and their migration, via afferent lymphatics, to draining lymph nodes where they accumulate as immunostimulatory dendritic cells (DC). It has been demonstrated previously that tumour necrosis factor-alpha (TNF-alpha) provides an important signal for LC migration and that in the absence of this cytokine, movement of LC from the epidermis to regional lymph nodes is inhibited. Recent evidence indicates that interleukin-1 beta (IL-1 beta), a cytokine produced in murine epidermis exclusively by LC, may also play a role in LC migration. The purpose of the investigations described here was to clarify, using relevant neutralizing anti-cytokine antibodies, the contributions made by TNF-alpha and IL-1 beta to the migration of LC from the epidermis. It was found that like anti-TNF-alpha, anti-IL-1 beta administered systemically to mice (by intraperitoneal injection), prior to skin sensitization with the contact allergen oxazolone, resulted in a marked inhibition of DC accumulation in draining lymph nodes. It was shown also that anti-IL-1 beta inhibited TNF-alpha-induced LC migration and DC accumulation and that; in similar fashion, the stimulation of LC migration and DC accumulation induced by IL-1 beta was compromised by prior treatment with anti-TNF-alpha. Based upon these data it is proposed that the stimulation of LC migration in response to skin sensitization requires the receipt by LC of two independent signals, one provided by TNF-alpha and the other by IL-1 beta. Morphological analyses of LC in epidermal sheets prepared from animals exposed to these cytokines with or without prior systemic treatment with anti-cytokine antibody suggested that the changes induced in LC by TNF-alpha and IL-1 beta may include the altered expression of adhesion molecules and acquisition of the ability to interact with and pass through the basement membrane.

Topics: Animals; Cell Movement; Dendritic Cells; Dermatitis, Contact; Epidermis; Fluorescent Antibody Technique, Indirect; Interleukin-1; Langerhans Cells; Lymph Nodes; Male; Mice; Mice, Inbred BALB C; Oxazolone; Signal Transduction; Tumor Necrosis Factor-alpha

In the current study, we confirmed previous findings suggesting that gamma delta T cells were involved in the successful adoptive cell transfer of contact sensitivity (CS) by alpha beta CS-effector T cells. In this study, we used hamster anti-mouse gamma delta-TCR mAb treatment of CS-effector T cells, followed by enrichment and removal of the gamma delta T cells with goat anti-hamster Ig-linked magnetic beads, or by addition of hemolytic rabbit C. This removal of gamma delta T cells abrogated adoptive cell transfers of CS, despite the presence of alpha beta T cells that are known to mediate CS. FACS analysis documented enrichment of gamma delta T cells rising from 1 to 2% of the starting cells, to 60 to 95% of the magnetic bead adherent cells. Adoptive cell transfer of CS was reconstituted by adding back to the alpha beta cells, highly enriched gamma delta cells attached to anti-gamma delta-TCR magnetic beads. Not only were gamma delta-enriched T cells from sensitized mice able to assist immune CS-effector alpha beta T cells, but gamma delta T cells from normal nonimmune mice also had CS-assisting activity, and furthermore, neither were MHC-restricted in this function. Thus, CS-assisting gamma delta T cells were present endogenously in normal mice without prior immunization, and acted without Ag specificity and without MHC restriction, to assist CS-effector alpha beta T cells. Similar studies, with hamster mAbs specific for V gamma and V delta portions of gamma delta-TCR, demonstrated that the gamma delta T cells that assisted the CS-effector alpha beta T cells preferentially expressed V gamma 5 and V delta 4 in their TCR. PCR analysis on extracted mRNA showed that V gamma 5 and V delta 4 gene segments indeed were rearranged and expressed in the sensitized and normal lymph nodes; and one-and two-color FACS analysis of magnetic bead-fractionated cells suggested that V gamma 5 and V delta 4 were expressed on the same T cells. In summary, these results demonstrated that V gamma 5+, V delta 4+, gamma delta T cells were needed to assist alpha beta effector T cells in the adoptive cell transfer of CS.

Topics: Animals; Base Sequence; Dermatitis, Contact; Flow Cytometry; Gene Rearrangement, T-Lymphocyte; Immunomagnetic Separation; Immunotherapy, Adoptive; Lymph Nodes; Male; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Molecular Sequence Data; Oxazolone; Picryl Chloride; Polymerase Chain Reaction; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Spleen; T-Lymphocyte Subsets

In mice with hapten-induced CH, T cells of the CD4+ and CD8+ phenotypes activated the gene for JE, whereas CD8+ T cells alone caused activation of the gene for IP-10. In animals tolerized by feeding either TNCB or OX, hapten-induced expression of IP-10 but not JE mRNA was lost. The down-regulation of IP-10 gene activation was adoptively transferred from tolerized mice to naive mice by CD4+ splenic T cells. These findings reflect the differential roles of individual T-cell subsets in both enhancing and diminishing chemokine gene expression in contact hypersensitivity reactions.

Topics: Administration, Oral; Administration, Topical; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chemokines; Dermatitis, Contact; Ear; Gene Expression Regulation; Haptens; Immune Tolerance; Mice; Oxazolone; Picryl Chloride; RNA, Messenger; Skin; Spleen; T-Lymphocytes; Transcriptional Activation

Allergic contact dermatitis (contact sensitivity) may be caused by a wide variety of chemicals. In addition, some chemical allergens may also induce respiratory sensitization. It has been demonstrated previously that topical exposure of mice to chemical contact and respiratory sensitizers stimulates divergent immune responses consistent with the selective activation of T helper 1 (Th1)- and Th2-type cells, respectively. Thus, exposure to trimellitic: anhydride (TMA) induces hapten-specific IgE antibody and a substantial increase in the total serum concentration of IgE. Conversely, oxazolone fails to provoke IgE production. In addition, lymph node cells (LNC) isolated following repeated topical exposure of mice to oxazolone or TMA display cytokine secretion profiles characteristic of Th1- and Th2-type cell stimulation. The purpose of the present investigations was to determine whether chemical allergens other than TMA and oxazolone, the respiratory allergen toluene diisocyanate (TDI) and the skin sensitizer dinitrofluorobenzene (DNFB), provoke differential cytokine expression. The production of the Th1-type product interferon gamma (IFN-gamma) and the Th2-type cytokines interleukins 4 and 10 (IL-4 and IL-10) by TDI- and DNFB-activated LNC has been measured. LNC derived from DNFB-exposed animals expressed substantial amounts of IFN-gamma, but only low levels of IL-10 and mitogen-inducible IL-4; exposure to TDI resulted in the converse profile of cytokine secretion. These data demonstrate that repeated topical administration of chemical allergen of different classes elicits in mice divergent cytokine secretion patterns consistent with the selective stimulation of distinct Th subsets. Analysis of such cytokine production profiles may permit in a single integrated assay the simultaneous identification and classification of chemical allergens.

Topics: Allergens; Animals; Cytokines; Dermatitis, Contact; Dinitrofluorobenzene; Female; Interferon-gamma; Interleukin-10; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides; Th1 Cells; Th2 Cells; Toluene 2,4-Diisocyanate

Cis-urocanic acid (cUCA) has been suggested as a mediator of impairment of contact hypersensitivity induction by ultraviolet B (UVB) irradiation. We ascertained whether topical cUCA influences local lymph node activation during induction of contact hypersensitivity. Topical cUCA or vehicle was applied during the local lymph node assay to oxazolone. Local lymph node weight and cell number were assessed in all animals. Additionally, cell proliferation rate was studied in Hartley guinea-pigs and CBA/Ca mice, whereas activation of antigen-presenting cells was quantified in NMRI mice and Wistar rats. Topical cUCA suppressed all parameters of local lymph node activation due to oxazolone application in guinea-pigs. No effect, with the exception of a suppression of antigen-presenting cell activity, was seen in mice. No effect was seen in rats. The study shows that topical cUCA may suppress local lymph node activation during contact sensitization and suggests that differences between the effect of cUCA in different animal species may exist.

Topics: Animals; Cell Count; Cell Division; Dermatitis, Contact; Female; Guinea Pigs; Immune Tolerance; Immunosuppressive Agents; Lymph Nodes; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Organ Size; Oxazolone; Rats; Rats, Wistar; Species Specificity; Urocanic Acid

To investigate the potential roles of CD4+ and CD8+ T cells during contact hypersensitivity, we examined the T-cell-dependent expression of proinflammatory cytokine genes in the responses to dinitrofluorobenzene and oxazolone. Whole cell RNA was isolated from challenged ear tissue and analyzed for level of cytokine gene expression by Northern blot and densitometry analysis. Expression of interleukin 1 beta and the three chemokine genes (IP-10, JE, and KC) examined was dependent on the hapten dose used for sensitization and correlated with the immune response, i.e., ear swelling, elicited. Antibody-mediated depletion of CD8+ T cells before sensitization resulted in the absence of IP-10 expression following hapten challenge, indicating the ability of immune CD8+ T cells to mediate IP-10 expression. Depletion of CD4+ T cells resulted in higher levels of IP-10 and KC expression during elicitation of contact sensitivity, suggesting CD4+ T cells inhibit the expression of these proinflammatory genes. Depletion of CD4+ T cells resulted in contact hypersensitivity responses of higher magnitude and depletion of CD8+ T cells resulted in responses of lower magnitude. Transfer of CD8+ T-cell-depleted immune cells resulted in low, but detectable levels of IP-10 expression, indicating the ability of some oxazolone-immune CD4+ T cells to mediate IP-10 expression. These results indicate the differential induction of proinflammatory cytokine gene expression during elicitation of contact hypersensitivity in which expression of IP-10 is primarily mediated by immune CD8+ T cells and inhibited by immune CD4+ T cells.

Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Transplantation; Chemokine CXCL10; Chemokines; Chemokines, CXC; Cytokines; Dermatitis, Contact; Dinitrofluorobenzene; Gene Expression; Mice; Mice, Inbred BALB C; Osmolar Concentration; Oxazolone

In a hamster model, we compared contact sensitivity to the metal salt, potassium dichromate, to that of oxazolone, a well-known strong sensitizing agent. Using the ear swelling test, originally developed in mice, no significant differences could be observed between animals treated with potassium dichromate and controls, although oxazolone-treated animals showed a significant increase in ear thickness compared to controls. These observations were confirmed using the local lymph node assay (LLNA) where oxazolone proved to be a strong sensitizing agent, and potassium dichromate only resulted in a weak response. When the draining auricular lymph nodes were compared with the inguinal lymph nodes in the LLNA, more pronounced effects were obtained with the auricular lymph nodes. This study indicates that, also in hamsters, the LLNA is a feasible sensitization test system.

Topics: Animals; Cricetinae; Dermatitis, Contact; Ear, External; Edema; Female; Immunologic Tests; Lymph Nodes; Male; Mesocricetus; Mice; Mice, Inbred BALB C; Oxazolone; Potassium Dichromate

Topical exposure of mice to the contact allergen oxazolone induces both 4 persistent antigen-specific down-regulation of subsequent lymph node cell (LNC) proliferative responses stimulated by the same chemical and a more transient depression of LNC proliferative responses provoked by exposure to unrelated chemical sensitizers: the latter being associated with antigenic competition in contact sensitivity. In this paper a relationship between reduced LNC proliferative activity and the secretion of interleukin 6 (IL-6) is described. Pretreatment of mice with oxazolone caused a persistent, dose-dependent inhibition of LNC proliferative activity and a parallel reduction of IL-6 secretion when mice were re-exposed, at a different site, to the same chemical. Consistent with dendritic cells (DC) being the major source of IL-6 within allergen-activated lymph nodes, depletion of Thy-lt T lymphocytes did not compromise production of this cytokine. Although in mice pretreated with oxazolone IL-6 secretion by cultured LNC was impaired markedly, the initial IL-6 content of freshly isolated LNC was apparently normal. These data suggest that the down-regulation of lymphocyte proliferative responses induced by exposure of mice to oxazolone, and the consequential impaired responsiveness, is associated with, and possibly secondary to, the reduced secretion by lymph node DC of IL-6, a cytokine that is a costimulator of T lymphocyte activation and the production of which correlates closely with the vigour of LNC proliferative activity.

Topics: Adjuvants, Immunologic; Animals; Clonal Anergy; Dermatitis, Contact; Down-Regulation; Immunosuppression Therapy; Interleukin-6; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazolone; T-Lymphocytes

Contact dermatitis is a cutaneous inflammatory reaction mediated by hapten-specific T cells. We used a murine model to investigate the contact sensitivity reaction elicited by different concentrations (optimal and suboptimal) of the haptens DNFB and oxazolone applied singly or in combination. The simultaneous application of DNFB and oxazolone at optimal concentrations (0.2% and 0.4% respectively) did not significantly increase the ear swelling response induced by each of the allergens when applied singly. No contact sensitivity response was observed when the haptens were tested individually at subthreshold concentrations (0.05% and 0.1% respectively). However, mixing the 2 molecules at the same concentrations gave rise to a clinical contact sensitivity reaction. The simultaneous application of the haptens at a 2 x higher, but still suboptimal concentrations (0.1% and 0.2% respectively), elicited an inflammatory response that was significantly greater than the responses elicited by either of the haptens when applied separately. These results suggest that a "false-positive" reaction to a mix may reveal a genuine sensitization to the constituents.

Topics: Animals; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; Dose-Response Relationship, Drug; False Positive Reactions; Female; Haptens; Mice; Mice, Inbred BALB C; Oxazolone; Patch Tests

The role of interferon-gamma (IFN gamma) in contact hypersensitivity induced by the haptens, oxazolone and 2,4,6-trinitrochlorobenzene (TNCB), was investigated in mice with a targeted disruption of the IFN gamma receptor (IFN gamma-R-/-). The 24-h ear-swelling response to oxazolone or TNCB in sensitized animals was not significantly reduced by the disruption of IFN gamma signalling. Dermal mononuclear infiltrates (MN) and epidermal microabscesses, however, were clearly diminished in the mutant mice. The hapten-induced upregulation of intercellular-adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I in IFN gamma-R-/- mice was smaller when compared to wild-type mice. It is concluded that oxazolone- and TNCB-induced contact hypersensitivity is partially dependent on a functional IFN gamma system. While the cutaneous oedema is IFN gamma-independent, the mononuclear cell infiltration and epidermal microabscess formation are at least partly IFN gamma-dependent. Therefore, reduced cellular infiltrates are likely due to a reduced upregulation of ICAM-1 and class I antigen expression in IFN gamma-R-/- mice.

Topics: Adjuvants, Immunologic; Animals; Dermatitis, Contact; Female; Genotype; Haptens; Histocompatibility Antigens Class II; Interferon-gamma; Major Histocompatibility Complex; Male; Mice; Mice, Inbred Strains; Oxazolone; Picryl Chloride; Receptors, Interferon; Up-Regulation

We conducted a study on the primary in vitro activation of T cells from non-sensitized mice by using hapten-conjugated Pam 212 cells (keratinocyte cell line). Furthermore, we attempted to develop a simple, quantitative in vitro test to assess the sensitizing potency of contact allergens and applied it to determine the stimulation index (SI) of various chemicals with known degrees of sensitizing potency. Monolayered Pam 212 cells were incubated with a variety of chemicals exhibiting allergenic potential. Washed and fixed T cells depleted of autoreactive T cells and macrophages from spleens of nonsensitized Balb/c mice were cocultured for 5 days with those monolayered Pam cells conjugated with chemicals. They were then harvested and restimulated with mitomycin-C-treated spleen cells conjugated with chemicals in 96-well culture plates to inhibit the proliferation of stimulator cells. We evaluated the sensitizing potency of the following chemicals: oxazolone, TNBS, DNFB and FITC (strong sensitizers); p-phenylendiamine (p-PD), nickel chloride and potassium dichromate (potent sensitizers); betamethasone and budesonide (corticosteroids), and methyl salicylate (MS) as an irritant. T cells sensitized in vitro with TNP-Pam cells and macrophages demonstrated antigen-specific proliferation when restimulated in vitro with mitomycin-C-treated TNP-spleen cells. Subcutaneous injection of these T cells induced contact sensitivity in vivo in an antigen-specific fashion. While T cells cocultured with TNP-3T3 could not be activated even in the presence of macrophages. The SI of strong sensitizers was about 4.00 and that of p-PD was 2.36. The SIs of budesonide, betamethasone and MS were 1.62, 0.98 and 1.21, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenal Cortex Hormones; Allergens; Animals; Antigen Presentation; Cell Division; Cell Line; Chloroquine; Dermatitis, Contact; Dinitrofluorobenzene; Epitopes; Female; Haptens; Immunization, Passive; Irritants; Keratinocytes; Lymphocyte Activation; Macrophages; Methods; Mice; Nickel; Oxazolone; T-Lymphocytes; Trinitrobenzenesulfonic Acid

The inflammatory response at sites of contact hypersensitivity induced by oxazolone was examined in the ears of P-selectin-deficient and wild-type mice. Accumulation of CD4+ T lymphocytes, monocytes, and neutrophils was reduced significantly in the mutant mice, as well as mast cell degranulation. In contrast, there was no significant difference in vascular permeability or edema between the two genotypes. The results demonstrate a role for P-selectin in recruitment of CD4+ T lymphocytes and show that P-selectin plays a role in long-term inflammation as well as in acute responses.

Topics: Animals; Dermatitis, Contact; Female; Inflammation; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Mutant Strains; Neutrophils; Oxazolone; P-Selectin; Platelet Membrane Glycoproteins; Skin

L-selectin, a cell surface adhesion molecule that is expressed by most leukocytes, mediates leukocyte rolling along vascular endothelium at sites of inflammation. The contribution of L-selectin to leukocyte migration in models of chronic inflammation was assessed by using mice that lack cell surface L-selectin expression. Significant inhibition of neutrophil (56-62%), lymphocyte (70-75%), and monocyte (72-78%) migration into an inflamed peritoneum was observed 24 and 48 h after administration of thioglycollate, an inflammatory stimulus. L-selectin-deficient mice were also significantly impaired in delayed-type hypersensitivity reactions. Footpad swelling in response to sheep red blood cell challenge was reduced 75% in L-selectin-deficient mice compared with wild-type mice. Ear swelling in a model of contact hypersensitivity induced by oxazolone challenge was also reduced by 69% compared to wild-type mice. Consistent with L-selectin-mediating leukocyte migration into diverse vascular beds during inflammation, L-selectin-deficient mice were significantly resistant to death resulting from lipopolysaccharide (LPS)-induced toxic shock. LPS administration resulted in a 90% mortality rate in control mice after 24 h, while there was a 90% survival rate in L-selectin-deficient mice. These results demonstrate that L-selectin plays a prominent role in leukocyte homing to nonlymphoid tissues during inflammation and that blocking this process can be beneficial during pathological inflammatory responses.

Topics: Animals; Cell Adhesion; Cell Adhesion Molecules; Crosses, Genetic; Dermatitis, Contact; Endothelium, Vascular; Inflammation; L-Selectin; Leukocytes; Lymphocytes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neutrophils; Oxazolone

Topics: Animals; Dermatitis, Contact; Dinitrochlorobenzene; Disease Models, Animal; Guinea Pigs; Humans; Immunoassay; Immunosuppressive Agents; Lymph Nodes; Male; Oxazolone; Tacrolimus

Ultraviolet B (UVB) irradiation of C3H mice causes suppression of delayed hypersensitivity and contact hypersensitivity (CH) to antigens encountered following exposure, and is accompanied by a reduction in Langerhans cell (LC) numbers in the epidermis, loss of epidermal antigen-presenting cell function, and accumulation of dendritic cells in lymph nodes draining the site of irradiation. Various photoreceptors and mediators of these changes have been proposed, one of which is cis-urocanic acid (cis-UCA) formed from the naturally occurring trans-UCA in the epidermis on UV irradiation. A monoclonal antibody that reacts with cis-UCA has become available recently and has been used in this study to clarify the role of UCA. Pretreatment of C3H mice with the monoclonal antibody abrogated the UVB-induced and cis-UCA-induced reduction in epidermal LC numbers. It also prevented the UV-induced suppression of epidermal antigen-presenting cell ability as measured by the mixed skin lymphocyte response. However, it had no effect on the accumulation of dendritic cells in lymph nodes draining the site of UV exposure. With regard to hypersensitivity responses, it did not prevent UV-induced suppression of CH to oxazolone at a range of concentrations but it restored to normal the UV-suppressed delayed hypersensitivity to herpes simplex virus, if administered before exposure. Thus cis-UCA is involved in some UV-induced changes in murine skin but not in others, where alternative mediators, such as tumor necrosis factor-alpha, may be more important.

Topics: Animals; Antibodies, Monoclonal; Cell Count; Dendritic Cells; Dermatitis, Contact; Female; Hypersensitivity, Delayed; Langerhans Cells; Lymph Nodes; Mice; Oxazolone; Simplexvirus; Stereoisomerism; Ultraviolet Rays; Urocanic Acid

Following skin sensitization epidermal Langerhans' cells (LC), many of which bear antigen, are stimulated to migrate from the skin and traffic via afferent lymphatics to lymph nodes draining the site of exposure. It has been proposed previously that tumour necrosis factor-alpha (TNF-alpha), a keratinocyte-derived epidermal cytokine (the expression of which is augmented following cutaneous sensitization), provides one signal for LC migration. In the experiments described here the influence of systemically administered neutralizing anti-TNF-alpha antibody on dendritic cell (DC) accumulation in draining lymph nodes has been investigated. Treatment with anti-TNF-alpha inhibited markedly the frequency of DC in draining nodes measured 18 hr following exposure to the skin allergens oxazolone and fluorescein isothiocyanate or to the non-sensitizing skin irritant sodium lauryl sulphate. Similar treatment with anti-TNF-alpha 2 hr prior to primary exposure to oxazolone impaired significantly the efficiency of skin sensitization measured 5 days later as a function of challenge-induced increases in ear thickness. The same antibody administered 18 hr following initial exposure to oxazolone was without effect on skin sensitization. These data confirm the importance of TNF-alpha for the migration of LC from the skin to draining lymph nodes and demonstrate that this cytokine is required for optimal contact sensitization.

Topics: Animals; Antibodies, Monoclonal; Cell Movement; Dendritic Cells; Dermatitis, Contact; Epidermis; Fluorescein-5-isothiocyanate; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Signal Transduction; Sodium Dodecyl Sulfate; Tumor Necrosis Factor-alpha

Previous studies have shown that prenatal exposure to the organochlorine pesticide chlordane significantly decreases the ear swelling response to the contact allergen oxazolone in BALB/c mice. Alterations of macrophage function in the efferent arm of the contact hypersensitivity response have also been reported. In the current study, chlordane was applied topically and the effects of oxazolone-induced contact hypersensitivity were determined. Initially, the reduction in oxazolone-induced ear swelling in topically-exposed female BALB/c mice was compared to 30-day-old BALB/c female mice exposed prenatally to chlordane. Prenatal chlordane exposure induced a 36% reduction in ear swelling compared to a 60% reduction following topical treatment at the challenge phase. Topically-applied chlordane also reduced the oxazolone-induced ear swelling by 40% when applied at sensitization. When applied at both sensitization and challenge, ear swelling was reduced by 71%. In a time-course study, it was determined that chlordane must be applied at the time of sensitization, challenge or both or within 1 h post-challenge to significantly reduce ear swelling. A dose-response study showed that the lowest concentration of chlordane resulting in a significantly reduced ear swelling response was 20 micrograms per ear.

Topics: Administration, Topical; Animals; Chlordan; Dermatitis, Contact; Dose-Response Relationship, Drug; Ear, External; Edema; Female; Immunity, Cellular; Insecticides; Male; Mice; Mice, Inbred BALB C; Oxazolone; Pregnancy; Prenatal Exposure Delayed Effects; Random Allocation

Cis-urocanic acid, converted from trans-urocanic acid in stratum corneum by ultraviolet B irradiation, has been shown to impair contact hypersensitivity induction. To study whether topical cis-urocanic acid also alters contact hypersensitivity elicitation, as well as immediate hypersensitivity and acute irritation, we treated mice with 1% topical cis-urocanic acid or vehicle prior to induction or elicitation of hypersensitivity to contact allergen oxazolone or respiratory allergen trimellitic anhydride or prior to acute irritation from croton oil. Topical cis-urocanic acid suppressed both induction and elicitation of contact hypersensitivity to oxazolone. However, no effect by cis-urocanic acid on induction or elicitation of trimellitic anhydride allergy or croton oil irritation was seen. The possible efficacy of topical cis-urocanic acid as a treatment of inflammatory skin diseases responsive to ultraviolet B irradiation may be worthwhile to investigate.

Topics: Administration, Topical; Allergens; Animals; Croton Oil; Dermatitis, Contact; Female; Irritants; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides; Urocanic Acid

Induction of contact hypersensitivity (CH) by molybdenum chloride (MoCl5) was determined by auricular lymph node (ALN) test in C57B1/6 mice. The ALN test was further improved by immunophenotyping and cytometric analysis of subset-specific cell in the draining node. Skin sensitization was induced by topical ear exposure to 1.0-50% oxazolone and resulted in a strong dose-related ALN reaction. Analogous exposure to MoCl5 resulted in a weaker but marked dose-related reaction, also manifested as an increase in cell number/ALN. Other differences between the oxazolone-induced strong sensitization and the MoCl5-related ALN reaction were: (1) an increase in the total number of Ig+ cells, which was, however, unchanged in the MoCl5-exposed mice; (2) a significant increase in the total number of large/activated T-cell subsets; and (3) a marked shift in the relative percentage of gated large/activated subsets of ALN cells, which was not observed in the MoCl5-exposed animals. Thus, it appeared that the molybdenum exposure induced a nonspecific increase in the cell number/ALN and was not accompanied by any marked activation of the T-cell subsets. Immunotoxicity of a 14 day subchronic exposure to MoCl5 at 1-100 ppm in food was studied by quantification of splenic humoral IgM response to sheep erythrocytes (SRBC). Plaque-forming cells (PFC) and enzyme-linked immunosorbent assay (ELISA) revealed unchanged humoral exposure in MoCl5-exposed mice. Cytometric assay of fluorescent beads uptake showed unchanged phagocytic activity of peritoneal macrophages from the MoCl5-exposed mice. Immunophenotyping of CD4+, CD8+, Thy 1.2+ and Ig+ cells revealed no effect of MoCl5 exposure on the total count of cell subsets in the ungated populations of spleen, lymph nodes and peripheral blood cells. Molybdenum chloride should thus be considered as a non-immunotoxic and a weak, nonspecific contact irritant.

Topics: Adjuvants, Immunologic; Allergens; Animals; Antibody Formation; B-Lymphocytes; Dermatitis, Contact; Ear, External; Enzyme-Linked Immunosorbent Assay; Female; Lymph Nodes; Mice; Mice, Inbred C57BL; Molybdenum; Oxazolone; Phagocytosis; Phenotype; Sheep; Spleen; T-Lymphocytes

Topics: Administration, Cutaneous; Administration, Sublingual; Animals; Antigen Presentation; Dendritic Cells; Dermatitis, Contact; Ear, External; Hindlimb; Immunotherapy, Adoptive; Inflammation; Langerhans Cells; Lymph Nodes; Mice; Mice, Inbred BALB C; Mouth Mucosa; Organ Specificity; Oxazolone; Picryl Chloride; Skin

Cytokines produced by keratinocytes play an essential role in the induction of immune suppression following ultraviolet (UV) exposure. Using antibodies specific for either interleukin-10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), we present evidence indicating that IL-10 suppresses delayed-type hypersensitivity (DTH) but not contact hypersensitivity (CHS), whereas TNF-alpha suppresses CHS but not DTH following UV exposure. UV exposure also activates antigen-specific suppressor T cells. To determine whether the antigen-specific CD4+ T cells that transfer suppression in this system mediate their suppressive effect by releasing IL-4 or IL-10, we transferred UV Ts into normal mice that were then injected with either anti-IL-4 or anti-IL-10 antibody. Both anti-IL-4 and anti-IL-10 blocked the ability of UV Ts cells to suppress DTH in the recipient animals. When UV Ts that suppress CHS were transferred into normal recipients, however, neither antibody was able to block the UV Ts activity. These findings suggest that UV Ts suppress DTH by secreting IL-4 and IL-10 and appear to act like Th2 cells. Because anti-IL-4 and anti-IL-10 did not block the activity of the UV Ts that regulate contact hypersensitivity, their effects appear to be mediated by a different mechanism.

Topics: Animals; Antibodies; Dermatitis, Contact; Down-Regulation; Female; Hypersensitivity, Delayed; Immune System; Immune Tolerance; Immunization; Interleukin-10; Interleukin-4; Isoantigens; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Oxazolone; Radiation Injuries, Experimental; Rats; Spleen; Tumor Necrosis Factor-alpha; Ultraviolet Rays

In response to exogenous stimuli such as phorbol-12-myristate 13-acetate, ultraviolet B radiation, and lipopolysaccharide, human keratinocytes produce soluble mediators that are important in primary contact irritancy including cytokines that are associated with proinflammatory properties (interleukin-1 alpha [IL-1 alpha], tumor necrosis factor alpha), chemotaxis (IL-8), and growth activation (granulocyte/macrophage colony stimulating factor, IL-6, transforming growth factor alpha). We examined qualitative and quantitative changes in selected intracellular and secreted cytokines in human keratinocyte cultures in response to non-sensitizing contact irritants (croton oil, sodium lauryl sulfate, methyl salicylate, ethyl phenylpropiolate), sensitizing irritants (oxazolone, dinitrofluorobenzene), and ulcerative agents (phenol, benzalkonium chloride, chromium trioxide). The chemicals were also applied to mouse skin to assess whether the chemical-specific pattern of inflammation correlated with the in vitro production of keratinocyte-derived cytokines. Although all agents elicited neutrophils to the site of chemical application, time dependent and chemical-specific patterns of inflammation could be detected. Sodium lauryl sulfate, phenol, and croton oil induced increases in IL-8 production at non-cytotoxic concentrations in semi-confluent human keratinocyte cultures. Phenol and croton oil stimulated tumor necrosis factor alpha production, whereas croton oil was the only agent found to induce granulocyte/macrophage colony-stimulating factor production. Croton oil, phenol, benzalkonium chloride, and dinitrofluorobenzene induced the intracellular production of IL-1 alpha without a concomitant release into the medium. The release of cytokines occurred in parallel with a relative increase in cytokine-specific mRNA transcripts. Studies using neutralizing antibodies to tumor necrosis factor alpha and IL-1 alpha demonstrated that IL-8 induction by croton oil and phenol occurred directly rather than through autocrine circuits. These data suggest that a given pattern of cytokine production is chemical-specific and may predict the contribution of keratinocytes to skin inflammation.

Topics: Animals; Cells, Cultured; Croton Oil; Cytokines; Dermatitis, Contact; Female; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-8; Keratinocytes; Mice; Mice, Inbred BALB C; Oxazolone; Phenols; RNA, Messenger; Skin; Time Factors; Tumor Necrosis Factor-alpha

Interleukin-6 (IL-6) production and proliferative responses by draining lymph node cells were studied in mice exposed topically to a series of chemicals. Chemicals with the capacity to induce sensitisation, but not non-sensitisers, promoted both IL-6 production and lymph node cell proliferation ex vivo. The responses exhibited similar kinetics, were dependent upon the dose of topically applied allergen, and correlated significantly. We demonstrate that the main source of IL-6 within draining lymph nodes is not proliferating T lymphocytes. The induction of a strong IL-6 response, and the relationship of this to cellular proliferation indicate that production of this cytokine within the lymph node is closely associated with the induction of contact sensitivity in mice.

Topics: Animals; Dermatitis, Contact; Dose-Response Relationship, Immunologic; Female; Interleukin-6; Lymph Nodes; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Oxazolone; T-Lymphocytes; Time Factors

Irradiation with ultraviolet B light (UVB) is known to suppress contact and delayed hypersensitivity response to a variety of antigens encountered within a short period following exposure. Such irradiation results in loss of Langerhans' cells and in synthesis of tumour necrosis factor-alpha (TNF-alpha) in the epidermis. In the present study the effect of broad-band (270-350 nm) and narrow-band (311-312 nm) UVB on the induction of contact hypersensitivity (CH) and on dendritic cell (DC) numbers in draining lymph nodes (DLN) of mice was examined. Broad-band UVB induced the accumulation of DC in DLN and this increase was substantially abrogated by treatment of mice with neutralizing antibody to TNF-alpha before irradiation. In addition, irradiation before sensitization with oxazolone resulted in a suppressed CH response. The suppression was negated to a considerable extent by TNF-alpha antibodies, administered before irradiation. Thus, one of the major effects of broad-band UVB is likely to be the synthesis of epidermal TNF-alpha which, in turn induces the migration of Langerhans' cells to DLN and leads to an impairment of their activity or function. Conversely narrow-band UVB did not result in an accumulation of DC in DLN or in a suppressed CH response. Such irradiation does, however, cause the isomerization from trans to cis-UCA in the epidermis. Cis-UCA has been proposed as a photoreceptor for UV and suppresses immune responses in a variety of experimental systems. Thus cis-UCA does not act through TNF-alpha induction or by influencing DC migration, and other studies indicate that histamine-like receptors in the skin may be involved.

Topics: Animals; Antibodies; Cell Movement; Dendritic Cells; Dermatitis, Contact; Female; Immune Tolerance; Lymph Nodes; Mice; Mice, Inbred C3H; Oxazolone; Tumor Necrosis Factor-alpha; Ultraviolet Rays

The synthesis and role of several lymphokines were examined during contact sensitization to oxazolone (OX). Application of OX to the skin of mice increased the delayed-type hypersensitivity (DTH) response to challenge, serum titres of OX-specific IgG1 and IgG2a, and draining lymph node cell (LNC) numbers. At day 3, LN contained detectable interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-2 or IL-3 mRNAs; IL-3 and higher levels of IL-4, IFN-gamma and GM-CSF mRNAs were measured after 24 hr culture with anti-CD3 antibody in OX-primed but not unprimed LNC. As a result of sensitization, LNC secreted IL-3 constitutively and produced elevated levels of IL-2, IL-3, IL-4 and IFN-gamma in response to anti-CD3 antibody; a similar but weaker lymphokine response was recalled by OX-protein conjugate. CD4+ cells were the major source of the anti-CD3-induced lymphokines except IFN-gamma, which was derived mainly from CD8+ cells. Since both IL-4 and IFN-gamma were synthesized by OX-primed LNC in vivo and in vitro, their role was investigated by administering anti-lymphokine antibodies at the time of sensitization. Anti-IL-4 treatment reduced OX-specific serum IgG1 titres without affecting IgG2a titres, whereas anti-IFN-gamma treatment reduced IgG2a but not IgG1 titres. Although neither antibody altered DTH responsiveness, anti-IFN-gamma treatment markedly increased IL-4 production by CD4+ LNC and reduced IFN-gamma production in vitro, particularly by CD4+ cells. We conclude that endogenous IL-4 and IFN-gamma reciprocally influence the isotype of the Ig response to OX and that IFN-gamma also affects the relative levels of IL-4 and IFN-gamma synthesis by CD4+ LNC.

Topics: Animals; Dermatitis, Contact; Immunoglobulin G; Interferon-gamma; Interleukin-4; Lymphocyte Activation; Lymphokines; Mice; Mice, Inbred Strains; Oxazolone; RNA, Messenger; T-Lymphocytes

It is known that the release of granule-associated inflammatory amines by isolated mouse tissue-type mast cells is subject to regulation in vitro by a number of cytokines that are produced during the immune response. In this study we investigated whether mast cell secretory function might also be subject to regulation in vivo during active sensitization. Mice were sensitized with one of three chemical allergens (trimellitic anhydride, TMA; 2,4-dinitrochlorobenzene, DNCB; or oxazolone) all of which induce contact sensitization and one of which (TMA) in addition causes immediate hypersensitivity. Peritoneal mast cells isolated from treated mice and sensitized passively with IgE released a greater proportion of cellular serotonin (5-HT) on stimulation either by anti-IgE or by specific antigen than did cells isolated from vehicle-treated controls. These results show that the function of mast cells is susceptible in vivo to regulatory influences that result from induction of an immune response.

Topics: Allergens; Animals; Dermatitis, Contact; Dinitrochlorobenzene; Female; Immunoglobulin E; Mast Cells; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides; Serotonin

Phosphatidylserine (PS) modulates several immune functions in vitro, including T cell activation, antibody and cytokine production, and macrophage growth. In the present work we studied the effects of PS on the induction of contact hypersensitivity (CH) in mice. BALB/c mice painted with PS (9.4-75 mg/kg) and with a sensitizing dose of DNFB or oxazolone on the same skin site exhibited a dose-dependent augmentation of CH reactions to either DNFB (> 60%) or oxazolone (> 35%), respectively. Bovine brain PS-enriched phospholipid mixture, lyso-PS, and dipalmitoyl-PS also induced similar enhanced CH responses, whereas phosphatidylglycerols had no effect. Increased CH was observed only when PS was applied from 2 days before to 12 h after DNFB. Immunization of naive syngeneic mice with skin grafts that were treated with PS and DNFB also led to enhanced (> 50%) CH responses. In addition, immunization by iv injection of epidermal cell suspensions enriched for Langerhans cells (LC) or of purified LC that were treated with PS (1-100 microM, 30 min, 37 degrees C), and then modified in vitro with DNBS (1 mg/ml, 30 min, 37 degrees C) led to increased (> 30-75%) CH responses in recipient syngeneic animals. Finally, adoptive transfer of DNFB-immune lymph node cells obtained from mice that were treated with PS induced augmented CH responses in recipient animals. The results suggest that PS is capable of up-regulating the induction of CH in mice by stimulating the APC function of epidermal LC.

Topics: Animals; Antigen-Presenting Cells; Dermatitis, Contact; Dinitrofluorobenzene; Dose-Response Relationship, Drug; Epidermal Cells; Epidermis; Female; Haptens; Immunization, Passive; Langerhans Cells; Mice; Mice, Inbred BALB C; Oxazolone; Phosphatidylserines; Time Factors

Specific immediate and delayed (24-hour) local dermal reactivity to oxazolone and dinitrochlorobenzene (DNCB) was passively transferred to naive guinea pigs by the intradermal injection of immune guinea pig serum and its IgG1 fraction. In both actively sensitized and passively sensitized animals, neutrophils were the major cells present in the immediate reaction to the specific contactant. Basophils and mononuclear cells were the major cells present in the delayed reaction to the contactant. This late-phase reaction is, therefore, a cutaneous basophil hypersensitivity (CBH) response. The serum factor that passively transferred this CBH response was stable when heated at 56 degrees C for 1-4 h. Since transfer factor, cutaneous basophil hypersensitivity factor and guinea pig IgE are heat-sensitive, these factors probably made little or no contribution to this response. Because proteinase inhibitors are known to inhibit mast-cell degranulation in vitro, we tested the effect of soybean proteinase inhibitor in vivo. This inhibitor suppressed both the immediate and the delayed skin reactivities mediated by intradermal contactant-specific IgG1. These studies support the following concept: IgG1 in guinea pigs (and IgE in human beings) sensitize mast cells to specific antigens. Such antigens degranulate mast cells, releasing histamine and other mediators for the immediate hypersensitivity reaction, and cause mast cells to produce cytokines that recruit basophils, eosinophils and mononuclear cells for the late-phase (CBH) reaction.

Topics: Animals; Basophils; Cell Degranulation; Cell Movement; Dermatitis, Contact; Female; Glycine max; Guinea Pigs; Hypersensitivity, Delayed; Hypersensitivity, Immediate; Immunity, Active; Immunization, Passive; Immunoglobulin G; Intradermal Tests; Mast Cells; Oxazolone; Protease Inhibitors; Skin

Environmental influences are increasingly recognized as nonspecific modulators triggering and aggravating allergic diseases. To obtain better insight into the pathomechanisms of these nonantigen-specific phenomena, we studied the augmenting and inhibitory effects of croton oil and glucocorticosteroid (GC) on the induction of murine allergic contact dermatitis. Application of the irritant croton oil simultaneously with the sensitization dose of the contact allergen oxazolone strongly amplified the inflammatory reaction (measured as ear swelling) during the effector phase. Immunohistologically, this effect correlated with an increased percentage of MRP8- and MRP14-positive phagocytes in the infiltrate of the early inflammatory reaction 8 h after sensitization with oxazolone plus croton oil as compared to oxazolone alone (62 vs. 27%; p < 0.05). Intravenously administered GC was used as an inhibitor of contact sensitization. The suppressive effect of GC on sensitization was dependent on the time of its application: suppression of the inflammatory reaction during the effector phase was clearly more pronounced when GC had been injected 1 day as compared to 1 h before sensitization. Correspondingly, the percentage of BM8-positive macrophages in the infiltrate of the early inflammatory reaction 8 h after sensitization was differentially decreased depending on the time of GC application: suppression of the percentage of BM8-positive macrophages was clearly more pronounced when GC had been injected 1 day as compared to 1 h before sensitization (17 vs. 25%; base value without GC 35%; p < 0.05). Furthermore, the percentage of BM8-positive macrophages in the inflammatory infiltrate of the effector phase was increased when sensitization had been enhanced by concomitant irritant contact dermatitis (47 vs. 59%; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Croton Oil; Dermatitis, Contact; Disease Models, Animal; Female; Immunization; Immunoenzyme Techniques; Immunophenotyping; Macrophages; Mice; Mice, Inbred BALB C; Oxazolone; Phagocytes; Triamcinolone Acetonide

A wide variety of chemicals may induce allergic contact dermatitis (contact sensitivity). Some chemical allergens may, in addition, cause respiratory sensitization. Topical exposure of mice to contact and respiratory chemical sensitizers results in the initiation of divergent immune responses characteristic of preferential activation of different functional subpopulations of T helper (TH) cells. In the present study we have sought to make use of these differences, particularly differences in the ability of contact and respiratory allergens to provoke IgE responses, and to question whether opportunities exist for the identification of chemicals with the potential for respiratory sensitization. We have examined alterations in the serum concentration of IgE following topical exposure of mice to seven chemical allergens; trimellitic anhydride (TMA), phthalic anhydride, diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), oxazolone and 2,4-dinitrochlorobenzene (DNCB). Three of these--TMA, phthalic anhydride and MDI--are known human respiratory sensitizers. The other four--HMDI, IPDI, oxazolone and DNCB--appear not to cause respiratory allergy, or at least have a very limited potential to do so. At the concentrations tested, exposure to all chemicals caused a lymphocyte proliferative response in lymph nodes draining the site of application. However, exposure only to TMA, phthalic anhydride and MDI resulted in a substantial increase in the concentration of serum IgE. Treatment with HMDI and IPDI failed to induce any change in serum IgE concentration. DNCB and oxazolone caused only small and transient elevations of IgE that were considerably less marked than those observed with respiratory sensitizers.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allergens; Animals; Cyanates; Dermatitis, Contact; Dinitrochlorobenzene; Female; Humans; Immunoglobulin E; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides; Respiratory Hypersensitivity

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (OX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.

Topics: Anaphylaxis; Animals; Arthus Reaction; Chickens; Dermatitis, Contact; Freund's Adjuvant; Immune Sera; Male; Oxazolone; Skin

In this study, we investigated whether mice given ultraviolet (UV)-B (280-320 nm) radiation in doses sufficient to alter cutaneous immune cells and impair the induction of contact hypersensitivity would also have impaired resistance to infectious agents administered at the site of UV irradiation. C3H mice were exposed to 400 J/m2 UVR from FS40 sunlamps on four consecutive days. Immediately after the last UV treatment, groups of mice were injected subcutaneously with Candida albicans, injected intradermally (ID) with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or infected percutaneously with Schistosoma mansoni in UV-irradiated skin. The induction of the delayed hypersensitivity response to C. albicans and BCG, as assessed by footpad swelling, was unaffected by UV irradiation. However, the number of viable mycobacteria recovered from the lymphoid organs of BCG-infected mice was increased significantly in the UV-irradiated animals for a period of more than 2 months. Low-dose UV irradiation of the skin at the site of infection did not influence the number of S. mansoni parasites recoverable from the internal organs of mice that had been infected with cercariae percutaneously 6 weeks earlier. We conclude that the ability of UV radiation to impair the development of cell-mediated immunity to antigens introduced in a UV-irradiated site is not universal and depends on the particular antigen administered. We hypothesize that the involvement of epidermal Langerhans cells as the primary antigen-presenting cells in the induction of cell-mediated immunity may be the critical factor in determining whether a particular immune response will be affected by local UV irradiation.

Topics: Animals; Candida albicans; Candidiasis; Cell Count; Dendritic Cells; Dermatitis, Contact; Disease Models, Animal; Drug Eruptions; Female; Hypersensitivity, Delayed; Mice; Mice, Inbred C3H; Mycobacterium bovis; Oxazolone; Schistosoma mansoni; Schistosomiasis mansoni; Tuberculosis; Ultraviolet Rays

In previous studies we observed that exposure of mice to the contact allergen 2,4-dinitrochlorobenzene (DNCB) and the respiratory allergen trimellitic anhydride (TMA) resulted in qualitatively different immune responses characteristic of selective Th1- and Th2-type T helper cell activation respectively. The purpose of the present investigation was to determine whether the effects recorded with DNCB and TMA are characteristic of immune responses to contact and respiratory chemical allergens in general. Experiments have been performed with phthalic anhydride, a known human respiratory sensitizer, and with oxazolone, a potent contact allergen. Under conditions of exposure where both chemicals elicited an IgG anti-hapten antibody response, only phthalic anhydride caused an increase in the serum concentration of IgE. Furthermore, like TMA, phthalic anhydride preferentially induced IgG2b rather than IgG2a antibody. In contrast, oxazolone, like DNCB, induced a markedly stronger IgG2a than IgG2b antibody response. These data provide confirmatory evidence that chemical respiratory allergens and chemical contact allergens elicit qualitatively different immune responses which reflect their clinical effects in man.

Topics: Allergens; Animals; Antibodies; Antibody Formation; Dermatitis, Contact; Dinitrochlorobenzene; Female; Haptens; Immunoglobulin E; Immunoglobulin G; Immunoglobulin Isotypes; Mice; Mice, Inbred BALB C; Oxazolone; Phthalic Anhydrides; Respiratory Hypersensitivity

Guinea pigs were sensitized by the topical application of either dinitrochlorobenzene (DNCB) or oxazolone on days 1, 2, 3, and 10. Seventeen days after the first treatment with the sensitizer, full-thickness 1.0-cm2 explants of untreated areas of skin were topically exposed in vitro to these contactants. Compared to the response of skin from control guinea pigs, skin from specifically sensitized animals showed a dose-related increase in the number of epidermal cells containing vacuoles. A specific increase in epidermal microblistering paralleled the increase in epidermal vacuolization. In addition, skin explants from sensitized animals (exposed to the contactant) showed a specific decrease in the incorporation of [14C]leucine. Full-thickness skin explants from unsensitized guinea pigs were sensitized in vitro by the intradermal injection of serum IgG1 fraction from oxazolone-sensitized guinea pigs. In such passively sensitized explants, the specific contactant produced an increase in the number of epidermal vacuoles, an increase in the amount of microblistering, and a decrease in the number of mast cells detectable by Giemsa staining. To elicit this specific response, the concentration of the specific contactant had to be mildly injurious, as well as antigenic. This requirement for nonspecific injury could be met by topically exposing skin explants to a nonspecific irritant followed by a sub-threshold concentration of the specific contactant. In contrast to vacuole formation and blistering, contactant-specific degranulation of mast cells (measured by the decrease in their number) did not require irritant levels of the contactant. These studies show that several components of contact sensitivity reactions can be reproduced in vitro by the passive transfer of sera containing antigen-specific immunoglobulins. Banks of such sera might, therefore, be useful in identifying (in human populations) many pre-existing sensitivities to chemical compounds.

Topics: Animals; Antigens; Cell Degranulation; Dermatitis, Contact; Dinitrochlorobenzene; Epidermis; Female; Guinea Pigs; Immunoglobulin G; Leucine; Mast Cells; Oxazolone; Vacuoles

We investigated the effectiveness of very low doses of the contact sensitizer dinitrofluorobenzene in sensitizing BALB/cJ mice. Surprisingly, the ear swelling reactions were greater with lower dinitrofluorobenzene doses, down to one-twentieth of doses commonly used. Although it is common practice to use much lower doses at challenge than at sensitization, we found greater reactions with lower doses at sensitization than at challenge. We also studied the timing of the development and waning of reactivity to dinitrofluorobenzene, dinitrochlorobenzene and oxazolone. Reactivity peaked at day 5 for dinitrofluorobenzene and dinitrochlorobenzene, and at day 3 for oxazolone. Reactivity waned by 3 weeks with dinitrofluorobenzene and oxazolone, and by day 7 with dinitrochlorobenzene. Pretreatment with cyclophosphamide caused a delay in the development and waning of reactivity.

Topics: Animals; Cyclophosphamide; Dermatitis, Contact; Dinitrochlorobenzene; Dinitrofluorobenzene; Dose-Response Relationship, Drug; Female; Mice; Mice, Inbred BALB C; Oxazolone; Skin; Time Factors

It is supposed that neuropeptides participate in the regulation of delayed-type hypersensitivity (DTH) reactions. However, their function in this kind of immune response is not known presently. Therefore, in vivo studies were initiated to test the effect on allergic (ACD) and irritant contact dermatitis (ICD) of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin (SOM), which are released from afferent neurons in the skin. Each neuropeptide was applied topically at the site of contact with the allergen (oxazolone) or irritant (croton oil) during the challenge and sensitization phase of contact dermatitis. The intensity of the inflammation was measured as an increase of ear-swelling response, which represents the degree of plasma extravasation in the early phase of inflammation. Neuropeptides alone led only to a distinct vasodilation. All three neuropeptides were equally able to increase allergic and irritant inflammation. Even minor irritant stimuli were enhanced. Beyond that, CGRP was able to boost sensitization, whereas SOM and SP did not show any effects on the sensitization process. The results presented demonstrate that neuropeptides increase plasma extravasation independent of the pathogenesis of inflammation and may act as priming substances for other mediators of increased vascular permeability. In addition, CGRP enhances the sensitization process.

Topics: Animals; Calcitonin Gene-Related Peptide; Croton Oil; Dermatitis, Contact; Histamine Release; Irritants; Mice; Mice, Inbred BALB C; Neuropeptides; Oxazolone; Somatostatin; Substance P

The expression of MHC class II (Ia) antigens on mouse keratinocytes was studied following both the induction and elicitation of contact sensitivity, and after primary irritant reactions. IA+ and IE+ keratinocytes were detected, using an indirect immunofluorescence assay on epidermal sheets, only after the induction and elicitation of contact sensitivity with the sensitizers oxazalone, picryl chloride and 2,4-dinitrochlorobenzene but not with formaldehyde. Ia+ keratinocytes were not detected after epicutaneous application of the non-sensitizing irritants croton oil, SDS and anthralin, or following attempted sensitization of nude mice, suggesting that the expression of Ia antigen on keratinocytes during contact sensitivity reactions is T-cell mediated. Because Ia antigen expression on keratinocytes could be detected only several days after induction or elicitation of contact sensitivity, and contact sensitization could also be demonstrated to occur independently of aberrant Ia expression, Ia+ keratinocytes cannot be involved in the initiation of these reactions. However, they might be important in exerting an immunomodulatory influence during the later stages of the responses to certain sensitizers.

Topics: Animals; Dermatitis, Contact; Dinitrochlorobenzene; Female; Fluorescent Antibody Technique; Histocompatibility Antigens Class II; Irritants; Keratinocytes; Mice; Mice, Inbred Strains; Mice, Nude; Oxazolone; Picryl Chloride; Time Factors

We report that challenge of previously contact-sensitized mice results in a significant increase in the concentration of serum histamine. In an attempt to determine whether this phenomenon might form the basis of an alternative method for the evaluation of elicitation reactions in experimental contact sensitivity, we have compared challenge-induced increases in ear thickness with elevations in serum histamine. Challenge of sensitized mice revealed that both ear thickness changes and increases in the serum level of histamine were dependent upon the concentration of oxazolone used for sensitization. The kinetics of changes in serum histamine concentration were found to be biphasic, with a small increase measurable 2 h following challenge and the maximal response at 24 or 48 h. In contrast, increases in ear thickness were monophasic, although maximum responses were also observed at 24 h. It is concluded that, although they do not exactly parallel increases in ear thickness, changes in histamine concentration may provide a useful serological correlate of the challenge reaction in contact sensitivity.

Topics: Animals; Dermatitis, Contact; Histamine; Kinetics; Mice; Mice, Inbred BALB C; Oxazolone

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.

Topics: Animals; Antigens, Ly; Dermatitis, Contact; Lymphocyte Activation; Male; Mice; Mice, Inbred CBA; Oxazolone; T-Lymphocytes, Regulatory; Trinitrobenzenes

The mechanism of action of pentamidine isethionate, a diamidino compound used in the treatment of Pneumocystis carinii pneumonia, is unknown. We recently reported that this drug may inhibit the release of inflammatory mediators from alveolar macrophages, which may be associated with its antiparasite activity. As a potential anti-inflammatory agent, we report that topically applied pentamidine reduces ear swelling in the contact hypersensitivity reaction to oxazolone in B6C3F1 mice. The application of pentamidine must occur within 1 h, at the challenge site, to be effective. Topical application appears necessary, because i.v. injection had no effect on reduction of ear swelling. In dose-response studies, a 50% reduction in ear swelling was achieved with as little as 20 micrograms of pentamidine. Pentamidine did not affect Ag transport from the challenge site to the draining lymph nodes, as measured by FITC transport. However, there was a 30 to 40% reduction in epidermal cells expressing Ia Ag from pentamidine-treated mouse ears, compared with control. Ia expression is almost exclusively limited to Langerhans cells in the normal epidermis. This reduction in Ia expression was not due to simple depletion of Langerhans cells by pentamidine, because CD45 expression was unaffected. Concurrent with reduced Ia expression, Ag presentation by pentamidine-treated Langerhans cells was also reduced. Taken together, a mechanism of action for pentamidine in inhibition of the contact hypersensitivity reaction appears to be via a reduction in Ag presentation by decreasing Ia+ Langerhans cells.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD; CD4 Antigens; Dermatitis, Contact; Female; Histocompatibility Antigens; Histocompatibility Antigens Class II; Langerhans Cells; Leukocyte Common Antigens; Mice; Mice, Inbred BALB C; Oxazolone; Pentamidine

Monoclonal and conventional cryptococcal-specific T suppressor factors (TsF) (also called TsFmp) depress phagocytosis by a subset of macrophages, while picryl- and oxazolone-specific TsF depress the passive transfer of contact sensitivity. This paper shows that these haptene-specific TsF also inhibit phagocytosis by a subset of macrophages and, using this assay, that the anti-haptene TsF resemble the anti-cryptococcal TsF in five respects: (i) the need for reexposure to specific antigen to trigger the release of TsF; (ii) genetic restriction in action; (iii) possession of an antigen-binding site; (iv) expression of I-J determinants; and (v) inactivation by reduction and alkylation. Purification of the anti-picryl TsF by sequential affinity chromatography indicates that the inhibition of phagocytosis is due to the TsF itself and not to a TsF-antigen complex. The TsF inhibits phagocytosis by a direct action as macrophages treated with TsF and exposed to antigen do not release a second factor which inhibits phagocytosis. These results and those of the accompanying paper indicate that the anti-cryptococcal and anti-haptene TsF are functionally equivalent, antigen-specific suppressor factors.

Topics: Alkylation; Animals; Antigens, Bacterial; Cryptococcus; Dermatitis, Contact; H-2 Antigens; Haptens; Immunization, Passive; Macrophages; Mice; Mice, Inbred Strains; Oxazolone; Oxidation-Reduction; Phagocytosis; Picrates; Polysaccharides, Bacterial; Suppressor Factors, Immunologic

Exposure of the flank of mice to either oxazolone or trinitrochlorobenzene (TNCB) 5 days prior to the application of oxazolone on the ear resulted in a reduced capacity of oxazolone-induced draining lymph node cells to express IL-2 receptors, produce IL-2, protein, RNA and DNA. However, histological examination of the draining lymph node suggest that antigen-specific and antigen-non-specific influences differ with respect to the frequency of pyroninophilic cells. Pre-exposure to oxazolone suppressed the number of oxazolone-induced pyroninophilic T cell blasts, whereas draining lymph nodes from TNCB-pretreated mice contained significantly more pyroninophilic cells than from oxazolone-pretreated mice. However, the majority of these cells were incorporating little or no thymidine. Thus exposure to certain contact sensitizers induces at least two systemic control mechanisms which serve to regulate subsequent lymphoproliferative responses. These mechanisms appear to exert their influences at different stages of in-vivo T cell activation.

Topics: Animals; Antigens; Cell Division; Dermatitis, Contact; Haptens; Interleukin-2; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Picryl Chloride; T-Lymphocytes

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different, antigen-specific, Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This cell clone, WP-3.27, releases an antigen-specific factor (OX-F) that sensitizes mast cells such that specific antigen challenge will induce serotonin release which mediates the early phase of DTH. In normal mice contact sensitized with picryl chloride (PCl), a similar polyclonal factor (PCl-F) has a similar activity and is also known to bind to macrophages. Thus, we measured macrophage production of TNF alpha, IL-1, IL-6, and PGE2 in response to the hapten affinity-purified DTH-initiating factors OX-F and PCl-F. Both factors induced significant release of each cytokine and PGE2. The production of TNF alpha, IL-1, and IL-6 was measured by bioassays. Northern blot analysis showed rapid accumulation of cytokine mRNA (2-4 hr), while maximal production of PGE2 occurred at approximately 8 hr. These macrophage activating properties of OX-F and PCl-F were not due to contamination with LPS as determined by the low levels of LPS present in OX-F and PCl-F and by the failure of polymyxin B to inhibit factor-induced PGE2 and TNF alpha production. Also, macrophage activation was shown not to be due to the action of several lymphokines known to be produced by WP3.27. Separation of OX-F and PCl-F by preparative isoelectric focusing showed a similar pattern: there were two major peaks of PGE2-inducing activity observed for both factors (for PCl-F at pI of 2-3 and 5.0, and for OX-F at pI of 3.5-4 and 5.0), but not for a sham factor produced by WEHI-3 cells. The ability of DTH-initiating factors to rapidly induce macrophage cytokine release and PGE2 synthesis 4-6 hr later may suggest a role for these mediators during the respective early vascular and late cellular phases of inflammation in DTH.

Topics: Animals; Blotting, Northern; Cytokines; Dermatitis, Contact; Dinoprostone; Gene Expression; Hypersensitivity, Delayed; Interleukin-1; Interleukin-6; Macrophages; Male; Mice; Mice, Inbred CBA; Oxazolone; Picryl Chloride; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

Neuropeptides in primary afferent neurons have been found to be engaged in the immediate type of hypersensitivity. However, their role in the delayed form of hypersensitivity is not yet established. The hypothesis that substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) are involved in delayed hypersensitivity was tested in oxazolone-induced, murine ear allergic contact dermatitis. Concentrations of immunoreactive SP, NKA, and CGRP were measured in extracts of the eczema ears (n = 26), whereas extracts of the opposite ears were used as controls. The SP, NKA, and CGRP contents in the treated ears were on the average 28% (p = 0.001), 32% (p = 0.004), and 15% (p = 0.016), respectively, lower than in the control ears. Lower peptide concentrations in the eczema ears indicate increased release of the peptides because the peptides are rapidly metabolized locally when released and only replenished by axonal transport from the cell bodies. Our results indicate that peptides released from primary afferent neurons play a role in the delayed type of hypersensitivity reactions.

Topics: Animals; Calcitonin Gene-Related Peptide; Dermatitis, Contact; Ear; Male; Mice; Mice, Inbred C57BL; Neurokinin A; Oxazoles; Oxazolone; Substance P; Tachykinins

The requirements for the induction of antigenic competition in murine contact sensitivity have been examined. Experiments with a variety of skin-sensitizing chemicals revealed a correlation between immunogenicity and the ability to inhibit subsequent responses to an unrelated contact allergen, oxazolone. Previous studies have suggested that, in contact sensitivity at least, antigenic competition is the consequence of a reduced lymphocyte proliferative response to the second antigen. We investigated whether the regulatory events which impair proliferation following exposure to the second antigen are induced as the result of a strong proliferative response to the first (competitor) antigen. It was found, however, that significant inhibition of the primary proliferative response to picryl chloride, by pretreatment of mice with either picryl sulphonic acid or 2,4-dinitrochlorobenzene, failed to prevent picryl chloride inducing antigenic competition for oxazolone. Our studies suggest that following topical exposure to potent skin allergens events other than proliferation in draining lymph nodes induce active immunoregulatory processes, one consequence of which is the appearance of antigenic competition.

Topics: Animals; Antigens; Dermatitis, Contact; Dinitrochlorobenzene; Immune Tolerance; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazolone; Picryl Chloride; Trinitrobenzenesulfonic Acid

The frequency and antigen-bearing characteristics of dendritic cells (DC) within draining lymph nodes have been examined during antigenic competition in contact sensitivity. Pre-exposure of mice to oxazolone on the flank resulted in a marked depression of subsequent fluorescein isothiocyanate (FITC)-induced lymph node cell (LNC) proliferation. Antigenic competition was associated with an increased frequency of DC in the draining lymph nodes, but also with a reduced amount of antigen per cell. Thus, at 24 hr the draining nodes of FITC-challenged mice previously exposed to oxazolone exhibited an increased number of DC compared with control animals. Flow cytometric analysis revealed, however, that the percentage of antigen-bearing DC was reduced and that the median amount of antigen borne by DC was lower. Since exposure to oxazolone caused a significant increase in the frequency of DC in distant nodes, the changes observed in antigenic competition may, at least in part, be attributable to systemic effects on DC migration following application of the first antigen. These data indicate that the reduced primary proliferative response which characterizes antigenic competition in contact sensitivity is associated with, and may result from, induced changes in dendritic cell behaviour.

Topics: Animals; Antigens; Cell Division; Cell Movement; Dendritic Cells; Dermatitis, Contact; Fluorescein-5-isothiocyanate; Fluoresceins; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone; Thiocyanates

It was shown previously that experimental autoimmune diseases could be prevented or treated specifically by administering suitably attenuated autoimmune T lymphocytes to animals, a process termed T cell vaccination (Cohen, I. R., Sci. American 1988. 256: 52). We now report that T cell vaccination is an effective way of inducing tolerance to contact sensitivity to simple chemical haptens. Vaccines were prepared from populations of lymph node cells from specifically sensitized mice by activating the T cells with the T cell mitogen concanavalin A and then treating the T cell blasts with glutaraldehyde. The vaccinated mice showed decreased delayed sensitivity responses to the specific sensitizing antigen and developed significant delayed sensitivity responses to the T cells of the same specificity as those used for vaccination. Thus, T cell vaccination against contact sensitivity reactions appears to function similarly to T cell vaccination against autoimmune disease.

Topics: Animals; Dermatitis, Contact; Dinitrofluorobenzene; Female; Hypersensitivity, Delayed; Immune Tolerance; Immunoglobulin Idiotypes; Immunotherapy, Adoptive; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazolone; T-Lymphocytes; Vaccination

Biologically active interleukin-1 (IL-1) has been detected in supernatants of draining lymph node cells isolated from contact-sensitized mice. Induction of IL-1 was dependent upon the concentration of sensitizer applied and occurred within 2 hr of exposure. The IL-1 activity could not be attributed to other interleukins and was neutralized by a specific antiserum. Reduced concentrations of IL-1 were produced by cells isolated from the draining nodes of mice that had been pre-exposed to the sensitizer. Since IL-1 has the potential to influence several aspects of lymphocyte activation, the production of significant concentrations of biologically active IL-1 by draining lymph node cells indicates that it is likely to play an important role in the afferent phase of contact sensitization.

Topics: Animals; Cell Division; Cells, Cultured; Dermatitis, Contact; Ear, External; Interleukin-1; Kinetics; Lymph Nodes; Mice; Mice, Inbred BALB C; Oxazolone

Various models of delayed hypersensitivity (DH) were used in mice: contact hypersensitivity reactions to picryl chloride and oxazolone and reactions to methylated bovine serum albumin (MBSA) and sheep red blood cells (SRBC). Drugs of different classes were tested in these models by systemic treatment around the challenge period: non-steroidal anti-inflammatory drugs (cyclooxygenase inhibitors, and inhibitors of both cyclooxygenase and lipoxygenase); glucocorticoids and immunosuppressants (cyclosporin A. CsA; cyclophosphamide, Cy; methotrexate, Mtx; azathioprine, Aza). These compounds were also studied and compared for their effects on the 3-h and 24-h phase of the carrageenin mouse-paw edema (in which inflammation is maximal after 24 h). Non-steroidal anti-inflammatory drugs (including double inhibitors of cyclooxygenase and lipoxygenase) had little or no effect on DH models, except indometacin. Glucocorticoids inhibited all immune reactions except that to MBSA. Of the immunosuppressants, CsA reduced all the DH reactions while Aza mainly reduced the reaction to SRBC; Cy and Mtx were mainly active on SBRC and MBSA inflammations. On another hand CsA, Cy and Mtx were inactive on the 3-h phase but decreased the 24-h phase of carrageenin edema. At doses active on the DH models and on carrageenin inflammation, Cy induced a lasting blood leukopenia, but CsA and Mtx did not. This combination of tests in the mouse seems to be of interest to demonstrate any action on DH and any anti-inflammatory effect and to suggest whether these activities are related to a possible leukopenic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Dermatitis, Contact; Edema; Glucocorticoids; Hypersensitivity, Delayed; Immunosuppressive Agents; Inflammation; Kinetics; Leukocyte Count; Male; Mice; Oxazolone; Picryl Chloride; Serum Albumin, Bovine; Sheep

Numerous studies of the histology of allergic contact dermatitis reactions to potent allergens in guinea pigs and humans have indicated that there is significant tissue infiltration with basophilic leukocytes. In this study we determined whether this histologic finding could be of value in distinguishing weak sensitization reactions from primary irritation, thereby aiding in the predictive identification of weak or moderate contact allergens. Guinea pigs were sensitized by the Buehler test method. Skin reactions were graded 24, 48, and 72 h post-challenge with duplicate patch sites biopsied at the 24- or 72-h grading timepoints. The biopsies were fixed, embedded in glycol methacrylate, thin sectioned, and Giemsa stained. The number of basophils per 400 leukocytes were counted along the upper dermis just below the dermal/epidermal junction. Challenge patch sites from animals sensitized to a relatively low dose of the strong contact allergen, oxazolone, were compared with patch sites from animals challenged only with a strong irritant, sodium lauryl sulfate (SLS). Compared to normal skin (7.5 +/- 1.0 basophils/400 leukocytes +/- SEM) only the oxazolone patch sites showed significant basophil infiltration (36.8 +/- 6.5), despite the fact that the skin reactions to the low oxazolone challenge dose were relatively weak. SLS patch sites showed no basophil infiltration above normal skin levels (4.8 +/- 0.9). Subsequent blinded studies compared weak/moderate presumptive sensitization reactions (as defined by accepted visual skin grading criteria) to various chemicals (citronellal, vanillin, cinnamic aldehyde, and ethylenediamine) to primary irritation reactions to the same chemicals. In each case, low-challenge-dose sensitization sites on previously treated (induced) animals showed mean basophil infiltration (range, 11.9-69.2 basophils/400 leukocytes) significantly greater than higher-dose irritant reactions (range, 1.6-13.3). The range for normal skin was 0.2-10.2 and the range for strong patch reactions to higher concentrations of oxazolone was 59.8-209.3. These data strongly indicate that light-microscopic quantitation of the CBH response can be used to distinguish relatively weak to moderate contact sensitization reactions from primary irritation reactions to the same chemicals.

Topics: Animals; Basophils; Dermatitis, Contact; Dose-Response Relationship, Drug; Ethylenediamines; Female; Guinea Pigs; Leukocyte Count; Male; Oxazolone; Skin Tests

Epicutaneous exposure of mice to the contact sensitizing chemicals 4-ethoxymethylene-2-phenyl-oxazol-5-one (oxazolone) and 2,4,6-trinitrochlorobenzene (picryl chloride) causes an inhibition of proliferative responses induced following subsequent topical challenge. The effects on lymphocyte proliferation comprise both transient antigen non-specific and more persistent hapten-specific mechanisms. Pretreatment of mice with one chemical 5 days prior to sensitization with a second, at which time antigen non-specific influences on proliferative responses are manifest, results in depression of contact sensitization as measured by changes in ear thickness following challenge. If, however, the period between pretreatment and sensitization is extended the inhibition of contact sensitization disappears in parallel with a decline in the antigen non-specific depression of lymph node cell proliferation. These data reveal that there exist two homeostatic mechanisms which control proliferation in response to challenge with at least some antigens, and that the extent of lymphocyte proliferation directly influences the degree of contact sensitization achieved. Moreover these results demonstrate that, in some instances at least, competition between antigens may be a function of immunoregulatory influences on lymphocyte proliferation.

Topics: Animals; Binding, Competitive; Dermatitis, Contact; Epitopes; Homeostasis; Kinetics; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazoles; Oxazolone; Picryl Chloride

The evaluation of contact reactions in previously sensitized mice is assessed conventionally by measurement of increases in ear thickness following challenge. In an attempt to develop a serological method for the investigation of contact sensitization in mice, we have examined whether analysis of changes in the concentration of acute-phase proteins in response to challenge provides a reliable alternative means of evaluating elicitation reactions. Measurement of either the relative serum haptoglobin concentration, using radial immunodiffusion, or the absolute concentration of serum amyloid A, by an enzyme-linked immunosorbent assay, has been found to correlate well with induced increases in ear thickness following challenge. Changes in the concentration of acute-phase proteins proved to be of sufficient sensitivity to reflect the specificity of contact sensitization and its inhibition by antigenic competition.

Topics: Acute-Phase Proteins; Animals; Dermatitis, Contact; Haptoglobins; Mice; Mice, Inbred BALB C; Oxazolone; Serum Amyloid A Protein; Time Factors

Using the model of allergic contact dermatitis in mice, we have demonstrated that local administration of recombinant (r) murine gamma interferon (gamma-rIFN) to the sensitization site substantially enhances the acquisition of delayed-type hypersensitivity. Single doses of gamma-rIFN from 500 to 2,000 U immunopotentiate successfully, with a suggestion of a better response at the higher doses. The immunoadjuvant effect is lost when the injections of gamma-rIFN are at a site distant from the sensitization site. Kinetic studies imply that gamma-rIFN is most effective when given at the time of sensitization, but significant immunopotentiation can be seen when gamma-rIFN is given several days before or after sensitization.

Topics: Adjuvants, Immunologic; Animals; Dermatitis, Contact; Dinitrofluorobenzene; Female; Immunization; Injections, Intradermal; Interferon-gamma; Mice; Mice, Inbred BALB C; Oxazolone; Recombinant Proteins

The immunostimulatory properties of dendritic cells (DC) which rapidly accumulate within the draining lymph nodes of mice topically exposed to contact allergens have been assessed as a function of their ability to initiate proliferative responses by sensitized lymph node cells (LNC) in vitro. A proportion of such DC bear antigen which is in a highly immunogenic form. Thus, at low stimulator:responder ratios, DC-enriched populations prepared from the draining nodes of mice exposed to the skin-sensitizing fluorochrome fluorescein isothiocyanate (FITC) induced significant proliferative responses by FITC-primed responder lymphocytes. Under the same experimental conditions, unfractionated or DC-enriched LNC isolated from naive mice and coated with at least equivalent amounts of FITC in vitro were without effect. The influence of DC populations on lymphocyte proliferation was largely, but not wholly, antigen-specific in nature. Dendritic cell fractions isolated 18 h following topical exposure to high concentrations of FITC or oxazolone induced a marked proliferative response by lymphocytes sensitized to the homologous chemical and a significantly less vigorous response by lymphocytes primed to unrelated haptens. In contrast DC isolated from the nodes of mice sensitized with 2,4-dinitrochlorobenzene (DNCB) caused proliferation only of DNCB-primed lymphocytes. The relative potential of DC populations prepared from FITC- and oxazolone-primed mice to induce antigen-specific and non-specific stimulation was dependent upon both the concentration of chemical used for sensitization and the period of exposure.

Topics: Animals; Antigen-Presenting Cells; Dendritic Cells; Dermatitis, Contact; Fluorescein-5-isothiocyanate; Fluoresceins; Haptens; Histocompatibility Antigens Class II; Lymph Nodes; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Oxazolone; T-Lymphocytes; Thiocyanates; Time Factors

A murine local lymph node assay for the identification of contact allergens has been developed. Contact sensitizing activity is measured as a function of lymphocyte proliferation in the draining lymph node following repeated application of the test agent to the dorsum of the ear. In original studies, lymphocyte proliferative activity was measured in vitro. In an attempt to remove the requirement for tissue culture, and thereby enhance the utility of the local lymph node assay as a predictive screening method, a new protocol has been evaluated in which proliferation in draining nodes is measured following i.v. injection of 3H-thymidine.

Topics: Allergens; Animals; Dermatitis, Contact; Dinitrochlorobenzene; Dose-Response Relationship, Immunologic; Evaluation Studies as Topic; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred CBA; Oxazolone; Picryl Chloride; T-Lymphocytes

Treatment of mice with rifampin (Ri, 100-200 mg/kg) affected the course of contact sensitivity (CS) reactions to oxazolone. The effects which were seen as either partial inhibition or enhancement of the response, under one set of conditions, could be abrogated or even reversed if conditions of either induction, elicitation and time of measuring reactions were altered. In addition, amount of Ri used for treatment and time of treatment in relation to the induction of CS reactions also influenced the effects observed.

Topics: Animals; Dermatitis, Contact; Dose-Response Relationship, Drug; Male; Mice; Mice, Inbred BALB C; Oxazolone; Rifampin; Time Factors

Offspring of mice treated with cyclophosphamide (Cy; 1, 2.5 or 5 mg/kg) during pregnancy (6-18 days of gestation) and tested for immunocompetence from 5 to 10 weeks of age were found to have defective reticuloendothelial clearance. The main effects were: a) increased elimination half time (T 1/2) of 51Cr-labeled SRBC from circulation, b) decreased liver uptake of 51Cr and c) impaired ability of the spleen, mostly affecting the female pups, to compensate for decreased liver uptake. The highest dose group suffered the most pronounced effects. This group was also found to have increased IgG immunoglobulin levels at 7 weeks of age. IgG antibody production in response to specific antigenic stimulation and delayed hypersensitivity reactions to oxazolone did not appear to be affected by Cy treatment.

Topics: Aging; Animals; Body Weight; Chromium Radioisotopes; Cyclophosphamide; Dermatitis, Contact; Female; Immunocompetence; Immunoglobulin G; Immunoglobulin M; Mice; Mononuclear Phagocyte System; Ovalbumin; Oxazolone; Pregnancy; Prenatal Exposure Delayed Effects; Reticulocytes; Sheep

Current methods for the predictive and diagnostic assessment of contact sensitization rely on the visual scoring of skin reactions. Predictive animal tests, generally using guinea pigs, require a relatively large number of animals to produce a sufficient database for interpreting skin reaction scores. In vitro assays have the potential of being more quantitative than skin testing and, if so, would require fewer animals. However, although in vitro assays are commonly used to study the cellular immune response to strong contact sensitizers, there has been little effort to validate them for predictive assessment purposes. We have optimized an in vitro lymphocyte blastogenesis assay for detecting the response of mouse lymphocytes to strong contact sensitizers with the eventual objective of applying this assay to moderate and weak sensitizers as well. Lymph node lymphocytes from mice sensitized to the strong contact allergens, dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), or trinitrochlorobenzene (TNCB), responded [greater than or equal to 12,000 counts per minute (CPM) above background] when cultured with water soluble chemical analogues, di- or trinitrobenzene sulfonic acid (DNBS or TNBS). However, the strong sensitizer, oxazolone (OXAZ), has no water soluble analogue and lymphocytes from mice sensitized to OXAZ responded poorly in vitro (less than 2000 CPM) to an ethanol-solubilized OXAZ preparation in spite of very strong in vivo sensitization (ear swelling assay). To increase the assay sensitivity, for OXAZ, we modified the antigen presentation conditions by using 1) solubilized antigen-modified adherent spleen cells, 2) dendritic cells from the draining lymph nodes of antigen painted mice, and 3) antigen-modified Langerhans cell-enriched cultured epidermal cells (EC). These approaches increased OXAZ-directed responses to greater than 7000, greater than 20,000, and greater than 100,000 CPM, respectively, under culture conditions optimized for cell density, responder: stimulator cell ratio, culture duration, and responder cell type. Our results represent a first attempt to directly modify cultured epidermal cells with OXAZ and use these cells to stimulate OXAZ-directed blastogenesis in microtiter plate cultures. This optimized assay is now under evaluation for predictive assessment of contact sensitizers relevant to occupational and consumer exposures.

Topics: Animals; Antigens; Cells, Cultured; Dermatitis, Contact; Dinitrochlorobenzene; Dinitrofluorobenzene; Dose-Response Relationship, Immunologic; Epidermal Cells; Epidermis; Female; Haptens; Langerhans Cells; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Nylons; Oxazolone; Picryl Chloride; Spleen; Time Factors

The effects of local hyperthermia treatment on contact sensitivity (CS) and on the number of Langerhans cells (LCs) were studied in mice. CS was significantly suppressed when mice were sensitized in the hyperthermia treated skin I, 2 or 4 days after treatment (43 degrees C for 45 min). This suppressive effect was not observed 7 or 14 days after the treatment. CS was also suppressed when mice were sensitized in non-treated skin I day after the treatment. The density of LCs detected as ATPase-positive cells also decreased significantly 1, 2, 4 and 7 days after the treatment. There appeared to be a positive correlation between the number of LCs and the extent of CS when mice were sensitized at hyperthermia treated skin. It was observed that this suppressive effect on CS was dose- and temperature-dependent. It could be transferred by spleen cells from the hyperthermia treated and DNFB-sensitized donors, and was antigen specific when spleen cells were transferred before sensitization of the recipient mice. This indicated it was, in part, associated with the induction of suppressor cells. These findings suggest that local hyperthermia treatment reduces the number of LCs with subsequent suppression of the induction phase of delayed-type hypersensitivity by the generation of antigen-specific suppressor cells.

Topics: Animals; Cell Count; Dermatitis, Contact; Dinitrofluorobenzene; Female; Hot Temperature; Immunization, Passive; Langerhans Cells; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Oxazolone; Spleen; Temperature

Draining lymph node cells isolated from mice 24 h following topical exposure to a variety of contact-sensitizing chemicals, including the dinitrobenzene derivatives, 2,4-dinitrochlorobenzene and 2,4-dinitrothiocyanobenzene, contained increased numbers of dendritic cells (DCs). The increase in frequency of DCs was time-dependent and preceded significant changes in either lymph node cellularity or lymph node cell proliferative activity. The degree of DC accumulation was also influenced by the chemical used and the concentration employed for sensitization. In the context of contact allergy, the biological relevance of this phenomenon to the induction of hapten-specific responses is indicated by the fact that relatively small numbers of DC--enriched fractions of lymph node cells (comprising approximately 70% DCs), but not unfractionated or DC--depleted populations, transferred sensitization to naive animals. Moreover, using the skin-sensitizing fluorochrome, fluorescein isothiocyanate, it was observed that 24 h following exposure the majority of lymph node cells bearing high concentrations of antigen were within the DC-rich fraction.

Topics: Animals; Cell Movement; Dendritic Cells; Dermatitis, Contact; Dose-Response Relationship, Immunologic; Flow Cytometry; Immunization, Passive; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Oxazolone; Time Factors

Skin painting of guinea pigs with either 4-ethoxymethylene-2-phenyloxazol-5-one or 2,4-dinitrofluorobenzene induced not only a primary proliferative response in the draining lymph node but also the systemic suppression of subsequent proliferative responses to topically applied hapten. The inhibition of lymphocyte proliferation, as assessed by the incorporation of [3H]-thymidine and the presence of large pyroninophilic cells in the paracortex, was hapten-specific and long-lasting. This study demonstrates that, in common with the mouse, the sensitization of guinea pigs results in the induction of a hapten-specific suppressor mechanism, which serves to control the proliferative response following reexposure to hapten. However, the antigen-nonspecific suppression of proliferation observed in the mouse following exposure to some potent contact sensitizers was not, under the conditions employed, detectable in the guinea pig.

Topics: Allergens; Animals; Dermatitis, Contact; Dinitrofluorobenzene; Female; Guinea Pigs; Haptens; Lymphocyte Activation; Oxazolone; T-Lymphocytes

Immunopotentiation by cytostatic drugs continuously released from osmotic minipumps, was investigated in a guinea-pig contact-sensitivity model. These pumps are designed to release their content within a period of 7 days. Vepeside (VP-16) and 5-fluorouracil were released into oxazolone-stimulated lymph nodes by subcutaneous implantation of pumps containing either of these drugs. The pumps were implanted at the intended sensitization site, 2 days before sensitization. Strong potentiation of T-cell-mediated immunity, evaluated by delayed-type hypersensitivity measurements, was observed with both drugs tested. Daily injections with VP-16 also caused an enhancement of the immune response. However, daily injections with 5-fluorouracil, a drug assumed to be cell-cycle-specific in its action, failed to potentiate delayed hypersensitivity to oxazolone. Intralesional administration of cytostatic drugs has been put forward as an effective treatment modality in various types of cancer. Therapeutic effects may depend on both local tumor cytotoxic and immunopotentiating activities. Our present results suggest that osmotic minipumps can be applied to broaden the applicability and effectiveness of local chemotherapy.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cyclophosphamide; Dermatitis, Contact; Dose-Response Relationship, Immunologic; Drug Administration Schedule; Etoposide; Female; Fluorouracil; Guinea Pigs; Immunity, Cellular; Infusion Pumps; Oxazoles; Oxazolone

Vitiligo is a human disorder which destroys pigment cells in the skin, ears, eyes, and meningeal tissues and has often been associated with a variety of autoimmune disorders. The C57BL/Ler-vit/vit mouse is a mutant strain that exhibits a loss of epidermal pigment cells and a selective cell-mediated immune deficiency to epicutaneous-administered allergens. This observation is consistent with that observed in humans with vitiligo, who also exhibit loss of contact hypersensitivity (CHS), that appears to be associated with loss of pigment cells from the epidermis. Other cellular immune parameters such as delayed type hypersensitivity and antibody generation to both particulate and soluble Ag are normal or even hyperimmune in the vit/vit mice compared with congenic C57BL/6 controls. Cyclophosphamide treatment could reconstitute CHS responsiveness of the vit/vit mice to the allergen dinitrofluorobenzene. Further, this loss of CHS responsiveness to dinitrofluorobenzene could be restored with skin transplants from normal pigmented C57BL/6 mice to vit/vit mice. Normal C57BL/6 mice bearing white skin grafts from vit/vit mice did not contact sensitize. We suggest that this vit/vit mouse strain may serve as an excellent system to investigate various aspects of other contact hypersensitivity reactions as well as vitiligo.

Topics: Animals; Antibody Formation; Antigens, Surface; Dermatitis, Contact; Dinitrofluorobenzene; Disease Models, Animal; Ficoll; Histocompatibility Antigens Class II; Hypersensitivity, Delayed; Immunization, Passive; Langerhans Cells; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Oxazolone; Phenotype; Picryl Chloride; Skin Transplantation; Thy-1 Antigens; Vitiligo

In the present study, immunopharmacological effects of clonidine-TTS on allergic contact dermatitis (ACD) to non-related, established contact sensitizers were investigated in guinea pigs. First, to evaluate the hypotensive effect of clonidine-TTS in guinea pigs, intra-arterial blood pressure was recorded. After 4 days of treatment with one (or two) TTS per animal, a reduction of arterial blood pressure from 71 +/- 1 to 51 +/- 2 mm Hg was observed. We subsequently assessed the effects of clonidine-TTS on contact hypersensitivity reactions to 2,4-dinitrochlorobenzene (DNCB) and 4-ethoxymethylene-2-phenyl-oxazolone (Ox). This study indicates that clonidine-TTS suppressed the elicitation of contact hypersensitivity reactions. The observed immunosuppressive effect of clonidine may account for the relatively weak hypersensitivity reactions to this drug in experimental animal studies. Further studies are needed to determine whether such findings are of relevance to the clinical use of clonidine in patient populations.

Topics: Administration, Cutaneous; Animals; Blood Pressure; Clonidine; Dermatitis, Contact; Dinitrochlorobenzene; Drug Hypersensitivity; Female; Guinea Pigs; Oxazolone

The purpose of this investigation was to determine the relative roles of the humoral and cell-mediated immune responses in the resolution of chlamydial genital infection of mice and resistance to reinfection. To this end, female BALB/c mice were rendered B cell deficient by treatment with heterologous anti-immunoglobulin M (IgM) serum from birth. Controls were similarly treated with either normal serum or phosphate-buffered saline. Before inclusion in each experiment, anti-IgM-treated mice were screened for the absence of IgM in serum and for the presence of cell-mediated immune responses. In addition, spleen cells from anti-IgM-treated mice responded to concanavalin A and phytohemagglutinin but not to lipopolysaccharide. By these criteria, mice were designated B cell deficient. B-cell-deficient mice and controls were inoculated intravaginally with a suspension of mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. All B-cell-deficient mice resolved the infection. Additionally, no significant difference was seen in the course of the infection in B-cell-deficient mice when compared with controls. In contrast to control mice, B-cell-deficient mice displayed no detectable antibody responses to MoPn in serum or in genital secretions. However, both B-cell-deficient mice and controls developed delayed-type hypersensitivity and T-cell proliferative responses to MoPn. When challenged 53 days after primary infection, no significant difference was seen in the resistance of B-cell-deficient mice to reinfection when compared with that of the controls. These data indicate that cell-mediated immune mechanisms play an important role in the resolution of and resistance to chlamydial genital infection in this model.

Topics: Animals; Antibodies, Bacterial; B-Lymphocytes; Chlamydia Infections; Dermatitis, Contact; Female; Hypersensitivity, Delayed; Immunity, Cellular; Lymphocyte Activation; Mice; Oxazolone; Spleen; Vaginitis

The effects of mast cell activation/degranulation on the elicitation of contact sensitivity (CS) to oxazolone and dinitrofluorobenzene were investigated. Mice were actively sensitized to oxazolone by epicutaneous painting followed by ear challenge. Passive sensitization to DNFB was induced by intradermal injections of dinitrophenol (DNP)-specific cloned T cells in the ears. Mast cells in the challenged ears were activated in various time periods by inducing a passive cutaneous anaphylaxis reaction where passive sensitization with monoclonal IgE anti-DNP antibodies was followed by iv injection of DNP-BSA. This combination of immediate and delayed-type hypersensitivity reactions resulted in a significant increase of ear swelling without any noticeable effect on cellular infiltration when the contact response was evaluated a short time (3-4 hr) after mast cell activation. The very same results were obtained in naive (unsensitized) mice, indicating that this reaction was nonspecific. However, when the CS reaction was evaluated at its peak, i.e., 24 hr post challenge, mast cell activation that had been induced 0.5-11 hr after ear challenge did not have any significant effect on both swelling and cellular infiltration when the latter was evaluated by a radiometric assay. We conclude that in these systems mast cell activation/degranulation makes little or no contribution to the modulation of T-cell activity.

Topics: Animals; Dermatitis, Contact; Dinitrofluorobenzene; Drug Hypersensitivity; Exocytosis; Female; Histamine Release; Immunoglobulin E; Mast Cells; Mice; Mice, Inbred BALB C; Oxazolone; Passive Cutaneous Anaphylaxis; T-Lymphocytes

The influence of topical exposure to two sensitizing chemical on draining lymph node cell proliferative responses in BALB/c mice has been examined. Conventional contact sensitization with 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone) has been shown to induce a rapid and systemic suppression of subsequent proliferative responses to topically applied chemical which can be adoptively transferred to recipient mice with immune lymph node cells. In contrast to some previous reports in which such suppression was found to be largely antigen-specific in nature, we report that, at least initially, the inhibition of lymphocyte proliferation induced by skin sensitization is hapten-non-specific. The relevance of this phenomenon to the regulation of contact sensitization is discussed.

Topics: Animals; Dermatitis, Contact; Haptens; Hyperplasia; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Oxazoles; Oxazolone; Picryl Chloride; T-Lymphocytes, Regulatory

Topical application of trifluoroperazine (TFP), a calmodulin inhibitor, on the skin of CBA mice previously immunized with 2-phenyl-4-ethoximethylene-oxazolone (oxazolone) blocks the expression phase of contact sensitivity. More detailed analysis of the TFP-mediated inhibition of contact sensitivity shows that TFP significantly suppresses the passive transfer of contact sensitivity when added in vitro to oxazolone-immune cells at a molar concentration of 10(-4) -10(-5). Furthermore, the DNA synthesis of draining lymph node cells from immune mice challenged with oxazolone was suppressed when cultured in the presence of TFP. The exposure of spleen and lymph node cells from immune animals to recombinant IL-2 fails to modify the TFP-mediated inhibition of passive transfer and cell proliferation. The TFP topical application opens the possibility to use this compound in the treatment of delayed hypersensitivity-caused skin disorders.

Topics: Administration, Topical; Animals; Dermatitis, Contact; In Vitro Techniques; Interleukin-2; Lymph Nodes; Mice; Mice, Inbred CBA; Oxazolone; Spleen; Trifluoperazine

In previous studies, the alpha 2-adrenoceptor agonist clonidine has been shown to suppress the wheal and flare reaction in guinea pigs sensitized to ovalbumin. This phenomenon has been further studied with special reference to effects on the dermal inflammatory cell infiltrate and mast cells. Clonidine lessens the degranulation of mast cells seen in control untreated immediate hypersensitivity reactions. Less neutrophils and eosinophils arrive to the treated reactions. Basophils and mononuclear cells (chiefly lymphocytes) which characterize the late phase of the wheal and flare reaction were not influenced by clonidine. Clonidine had a possible minimal effect on allergic contact (delayed hypersensitivity) reactions. The toxic contact reaction to croton oil (nonspecific cutaneous inflammation) was not affected.

Topics: Animals; Cell Movement; Clonidine; Croton Oil; Dermatitis, Contact; Drug Hypersensitivity; Female; Granulocytes; Guinea Pigs; Histamine; Hypersensitivity, Delayed; Hypersensitivity, Immediate; Inflammation; Mast Cells; Ovalbumin; Oxazolone; Skin; Skin Tests

Frequent skin exposure of guinea pigs to the contact sensitizing agents dinitrochlorobenzene or 4-ethoxymethylene-2-phenyloxazolone induced both systemic hyposensitization and local unresponsiveness within 8 weeks. Both phenomena were hapten-specific. Decreased systemic reactivity in repeatedly painted guinea pigs is probably not due to receptor blockade or the development of hapten-specific antibodies, but rather to transient sequestration of hapten-specific effector cells within lymph nodes draining the site of hapten exposure. After discontinuation of allergen exposure, effector cells return into the circulation, as indicated by a reversal of systemic hyporesponsiveness within 5 weeks. The persistence of a cellular infiltrate at the site of repeated application and the hapten-specific unresponsiveness at this site suggest that suppressor cells play a role in local unresponsiveness. Upon discontinuation of allergen exposure, local unresponsiveness rapidly dissolves (within one week). Since the circulation is still depleted of effector cells, residual hyporesponsiveness may persist for longer periods.

Topics: Administration, Topical; Animals; Antibodies; Ascitic Fluid; Dermatitis, Contact; Desensitization, Immunologic; Dinitrochlorobenzene; Guinea Pigs; Immunization, Passive; Lymph Nodes; Lymphocyte Transfusion; Lymphocytes; Oxazoles; Oxazolone; Skin Tests

Recognition that delayed-type hypersensitivity (DTH) reactions, such as contact sensitivity (CS) in mice, are initiated by Ly-1+ T cell-derived, antigen-specific factors has led to identification of a new kind of suppressor T cell that regulates this initiation phase of CS. Regulation by these suppressor T cells is T cell isotype-like in that initiation of DTH of various antigenic specificities is suppressed, whereas, Ly-1+ T cells mediating the antigen/major histocompatibility complex-restricted, classic delayed phase of CS responses are not affected, nor are other T cell activities. This study shows that these isotype-specific suppressor T cells probably act by release of soluble, isotype-specific, suppressor factors. These isotype-specific T cell factors bind to and can be eluted from columns linked with antigen-specific Ly-1+ T cell factors that initiate CS, and are of different antigen specificities. These T cell regulating, anti-isotypic suppressor factors are derived from Lyt-2+ I-J- T cells and suppress CS-initiating T cells, but do not affect the delayed-acting T cells of CS. This is in contrast with antigen-specific T cell suppressor factors that affect the late-acting and not the early-acting T cells of CS. It is suggested that the antigen-binding, CS-initiating, T cell factors, and their regulatory, anti-isotypic T cell factors are, respectively, T cell analogues of immunoglobulin(Ig)E antibody, and IgE-binding factors, that regulate IgE antibody production by IgE+ B cells.

Topics: Animals; Antigens, Heterophile; Antigens, Ly; Dermatitis, Contact; Epitopes; Immunity, Cellular; Immunization, Passive; Immunoglobulin Allotypes; Male; Mice; Mice, Inbred CBA; Molecular Weight; Oxazolone; Picryl Chloride; Sheep; Suppressor Factors, Immunologic; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Time Factors

We investigated the clotting associated with delayed hypersensitivity (DH) responses in the mouse by sensitizing the animals to the contactant oxazolone (Ox), and then administering 125I-guinea pig fibrinogen i.v. 10 to 30 min before antigen challenge 5 days later. Early (4 to 8 hr) contact sensitivity (CS) responses in immunized mice were barely detectable by three conventional measures of CS, but the total 125I-cpm in ears challenged with hapten was 3.6 to 4.5 X that in control ears challenged with vehicle alone; moreover, the amount of urea-insoluble cpm (cross-linked 125I-fibrin-associated cpm) in the reactions to Ox was 6.5-fold to 8.2-fold that present in the control reactions. In 24 hr reactions that were near peak intensity by measurements of ear swelling, ear weight ratios, and ratios of 125I-5-iodo-2-deoxyuridine-labeled leukocyte infiltration, the cpm in antigen-challenged ears exceeded that in control ears by 13-fold to 53-fold. In addition, antigen-challenged ears contained 27 to 300 X the urea-insoluble cpm present in control ears. 125I-Fibrin deposition was not a specific characteristic of CS reactions, because a small amount of urea-insoluble reactivity was also detected in some reactions to Ox in native mice. Nevertheless, the assay was exquisitely sensitive and readily detected quantitative differences between the immunologically specific and nonspecific reactions at very early intervals after challenge or with suboptimal doses of antigen. Furthermore, it was more sensitive than conventional tests of CS in detecting the reactions elicited in mice that had been passively sensitized to Ox by adoptive transfer of immune lymph node cells. Finally, we showed that the assay gave similar results when we tested CS reactions elicited in mast cell deficient WBB6F1-W/Wv and littermate normal (+/+) mice, demonstrating yet another similarity in the phenotype of DH reactions elicited in the presence or absence of mast cells.

Topics: Animals; Blood Coagulation; Dermatitis, Contact; Female; Fibrin; Fibrinogen; Hypersensitivity, Delayed; Immunization, Passive; Mast Cells; Mice; Oxazolone; T-Lymphocytes

Electrokinetic behaviour of LNC and their subpopulations from CBA mice contact-sensitized with dinitrofluorobenzene and oxazolone were studied. The sensitized LNC showed a significant reduction of mean EPM on the days of maximum DH response. Histogram analysis revealed that LNC of unsensitized as well as contact-sensitized mice were heterogeneous populations. While the LNC of unsensitized mice resolved into two subpopulations, sensitized LNC resolved into three distinct subpopulations. The emergence of a new populations with mean EPM intermediate between those of low and high mobility lymphocytes was a consequence of contact sensitization. Enriched subpopulations were also obtained by nylon adherence-dependent and surface marker-dependent procedures. histograms of these subpopulations revealed that in the mice sensitized to DNFB and oxazolone both T and B cells were electrokinetically altered and were heterogeneous in the distribution of their EPM. These findings are similar to the earlier observations on EPM of LNC in allograft-sensitized mice.

Topics: Animals; B-Lymphocytes; Cell Movement; Dermatitis, Contact; Dinitrofluorobenzene; Electrophoresis; Lymph Nodes; Lymphocytes; Mice; Mice, Inbred CBA; Oxazolone; Spleen; T-Lymphocytes

Contact sensitization induces two different kinds of T cells (both Ly 1) that act in sequence to produce upon challenge with antigen a classical 24-hour local skin swelling reaction. One of these cells produces an antigen-specific factor. It has been suggested that it sensitizes mast cells, similar to IgE antibody, and causes them to release vasoactive amines in the presence of antigen. This results in an early (2-hour) swelling reaction. Increased vascular permeability facilitates the entry of the second, lymphokine-producing Ly 1 cell into the site of reaction to elicit the classical 24-hour delayed-type hypersensitivity reaction. In alloxan diabetic mice, contact sensitivity reactions are reduced significantly, and our experiments show that insulin deficiency affects only the activity of the late acting, lymphokine-producing cell and leaves the factor-producing cell responsible for the early swelling reaction unaffected. Our experiments demonstrate that insulin deficiency has different effects on distinct subpopulations of T lymphocytes.

Topics: Animals; Antigens, Ly; Dermatitis, Contact; Diabetes Mellitus, Experimental; Edema; Glucose; Immunity, Cellular; Immunization, Passive; Mice; Mice, Inbred CBA; Oxazolone; Picryl Chloride; T-Lymphocytes; Time Factors

Streptolysin S, a hemolytic toxin produced by strains of Streptococcus pyogenes, was examined for its effect on cellular immune reaction in mice. The toxin given intraperitoneally for six consecutive days did not influence intensiveness of delayed hypersensitivity to oxazolone which has been used as a model of cellular immune reaction. Streptolysin S injected subcutaneously, closely to lymph nodes directly involved in immune response, markedly suppressed delayed hypersensitivity. Significant inhibition of lymphocyte proliferation by streptolysin S was observed both in vivo as well as in vitro experiments.

Topics: Animals; Bacterial Proteins; Dermatitis, Contact; Immunity, Cellular; Injections, Intraperitoneal; Injections, Subcutaneous; Lymphocyte Activation; Male; Mice; Oxazolone; Streptococcus pyogenes; Streptolysins

Oxazolone-induced allergic contact dermatitis in mice has been used as an in vivo model for identifying potential anti-inflammatory and immune-modulating drugs. Studies were conducted with this model to detail the histokinetics of the inflammatory response and to examine the adequacy of three assays for measuring the intensity of the inflammatory response when compared to histologic findings. CD-1 mice were sensitized with oxazolone on the flank and challenged on the dorsum of the ear pinna. The inflammatory response was quantitated by measuring the change in pinna weight and thickness and presence of 125I-2-deoxyuridine (125IUDR)-labeled cells. Cellular infiltrate was mixed with general predominance of mononuclear cells over neutrophils. Correlation with the histologic data suggests that both the ear weight and thickness assays are suitable primary screening assays, since both provided a sensitive, accurate and objective measure of the inflammatory response and its suppression by dexamethasone. The 125IUDR assay worked well in untreated mice but was unsatisfactory in mice treated topically or systemically with dexamethasone.

Topics: Administration, Topical; Animals; Dermatitis, Contact; Dexamethasone; Injections, Subcutaneous; Male; Mice; Oxazolone; Time Factors

Single intraperitoneal injections of cyclophosphamide were administered 6 days before testing guinea pigs sensitized to oxazolone in order to study the effects on inflammatory cell populations in blood and dermis. Skin tests were assessed macroscopically (erythema and oedema) and microscopically (counting of the dermal inflammatory cell infiltrate). At the highest dose (300 mg/kg) the allergic contact reaction was augmented with increases in erythema and oedema and the mononuclear dermal infiltrate. At the lowest dose (75 mg/kg), redness and oedema and all components of the dermal inflammatory cell infiltrate decreased. Total and differential white blood counts up to 20 days after administration of cyclophosphamide showed that a dose-dependent leukopenia maximal around 6 days occurred. During the leukopenia the differential count showed a lymphocytosis with a marked granulocyte depletion. The augmentation of the contact allergic reaction produced at the highest dose of cyclophosphamide occurs despite a marked peripheral blood leukopenia. Cyclophosphamide's effects at the lower dose would appear to be of a non-specific anti-inflammatory nature.

Topics: Animals; Blood Cells; Capillaries; Cyclophosphamide; Dermatitis, Contact; Dose-Response Relationship, Drug; Female; Guinea Pigs; Leukocyte Count; Oxazolone; Skin

The mechanisms of non-specific unresponsiveness to contact sensitivity (CS) induced with intravenous (i.v.) administration of oxazolone (Ox)-conjugated syngeneic spleen cells was investigated. Non-specific suppressor cells were found in spleen cells of mice which had been injected i.v. with Ox-conjugated cells 7 days before. These non-specific suppressor cells blocked the efferent stage of CS profoundly, i.e. inhibited the activity of effector cells for picryl chloride (PCl). Since these suppressor cells were plastic-adherent and resistant to treatment with anti-Thy 1.2 antibody and complement, they were considered to be macrophage-like. These suppressor cells released non-specific suppressor factor(s) (NSF) during culture for 1 hr without any antigenic triggering. Effector cells for PCl which were treated with NSF for 1 hr at 4 degrees lost their ability to transfer CS. NSF was easily absorbed by normal spleen cells. Furthermore, these NSF-treated spleen cells acquired the ability to inhibit the passive transfer of CS non-specifically. We also discussed the pathway for the induction of these macrophage-like suppressor cells.

Topics: Animals; Cells, Cultured; Dermatitis, Contact; Female; Immunization, Passive; Lymphokines; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oxazolone; Spleen; Suppressor Factors, Immunologic; T-Lymphocytes, Regulatory; Time Factors

Staphylococcus aureus strain Cowan I, a strong polyclonal B-cell activator (PBA), inhibited contact sensitivity to oxazolone in mice when administered 24 hr before sensitization. This suppression was mediated by idiotype-positive (Id+) B lymphocytes, which arose very early during the sensitization process and induced anti-Id B cells. These cells were found at Day 3 of the sensitization process and exerted their effect by activating antigen-specific suppressor T lymphocytes, which affected the efferent phase of the immune response. S. aureus strain Wood 46, which lacks of the ability to act as a PBA, was unable to inhibit contact sensitivity. These results indicate that PBA may play an important role in the regulation of cell-mediated immune reactions.

Topics: Animals; B-Lymphocytes; Dermatitis, Contact; Female; Immune Tolerance; Lymphocyte Activation; Male; Mice; Oxazolone; Staphylococcus aureus; T-Lymphocytes, Regulatory

Primary contact sensitivity (CS) to Ox and non-specific unresponsiveness to other unrelated delayed-type hypersensitivity (DTH) containing CS were induced simultaneously by i.v. administration of oxazolone(Ox)-conjugated spleen cells. The subpopulation of spleen cells which serves as carriers for the induction of primary CS consisted of plastic-adherent, Thy 1-negative macrophage-like cells, and the carriers for the induction of non-specific unresponsiveness were non-adherent, Thy 1-positive cells. Further, allogeneic spleen cells were not capable of acting as carriers for the induction of primary CS, though H-2 restriction was not observed in the induction of non-specific unresponsiveness by Ox-conjugated cells. Thus, these two phenomena were shown to be induced by distinct pathways or distinct mechanisms. Adult thymectomy or treatment with cyclophosphamide (CY) prevents the induction of non-specific unresponsiveness. Moreover, an interval of 7 days was required for almost complete suppression of CS between treatment with Ox-conjugated spleen cells and sensitization with picryl chloride (PCl). These results suggest that relatively short-lived, CY-sensitive T cells participate in the induction of the non-specific unresponsiveness induced with Ox-conjugated cells.

Topics: Animals; Antilymphocyte Serum; Ascitic Fluid; Cell Adhesion; Cyclophosphamide; Dermatitis, Contact; Female; Hypersensitivity, Delayed; Male; Mice; Mice, Inbred Strains; Oxazolone; Picryl Chloride; Spleen; T-Lymphocytes; Thymectomy

An eluate prepared by brief incubation of spleen cells from 2,4,6-trinitrophenyl (TNP) lysyl-Ficoll immunized mice with TNP-epsilon-amino-n-caproic acid causes a specific inhibition of the induction of contact sensitivity by 2,4,6-trinitrochlorobenzene skin painting. The active factor in the eluate binds to an anti-mouse immunoglobulin (Ig) immunoadsorbent column but not to a TNP immunoadsorbent column and is therefore Ig but not anti-TNP antibody. The active factor does bind to an immunoadsorbent prepared from anti-TNP antibody, suggesting that the factor has anti-idiotype specificity. Evidence based upon hapten-reversible inhibition of plaque formation and an enzyme-linked immunosorbent assay (ELISA) indicates that the eluates contain auto-anti-idiotype antibody specific for anti-TNP antibody. It is suggested that auto-anti-idiotype antibody spontaneously produced during the immune response to a T-independent antigen can specifically downregulate contact sensitization to the same epitope.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibody Formation; Autoantibodies; Dermatitis, Contact; Immune Sera; Immunoglobulin Idiotypes; Immunosorbent Techniques; Male; Mice; Mice, Inbred AKR; Mice, Inbred BALB C; Oxazolone; Receptors, Antigen, T-Cell; Spleen; Trinitrobenzenes

The antigen-presenting capability of macrophage modified with a single hapten (dinitrofluorobenzene) is partially or totally abolished by an additional haptenation with a second related (picryl chloride) or unrelated (oxazolone) hapten. This effect, 'antigenic competition', is only partially mediated by suppressor cells. There also seems to be an inhibition of the association of the hapten with particular Ia antigens. The prerequisite for antigenic competition is that both hapten responses are controlled by the same immune response gene. Hapten responses which are controlled by different genes, e.g., dinitrofluorobenzene, picryl chloride, and oxazolone on the one hand and benzilidene acetone on the other, do not compete. The pattern of competition thus varies with the strain of guinea pig.

Topics: Animals; Antigen-Presenting Cells; Binding, Competitive; Butanones; Cyclophosphamide; Dermatitis, Contact; Dinitrochlorobenzene; Dinitrofluorobenzene; Female; Freund's Adjuvant; Genes, MHC Class II; Guinea Pigs; Macrophages; Male; Oxazolone; Picryl Chloride; Trinitrobenzenesulfonic Acid

The effects upon the contact allergic reaction in the guinea pig of the 3 cytostatic agents most commonly used for their immunosuppressant properties, cyclophosphamide, methotrexate and azothioprine, have been investigated in an experimental model which allows comparison of the qualitative and quantitative aspects of the dermal inflammatory infiltrate and the macroscopic response. The 3 agents were given in single, relatively high doses and had effects on the dermal inflammatory infiltrate which varied in nature, degree and time course. Changes often showed a cyclical development with time, with "rebound" often following initial changes. Although cyclophosphamide had the most marked effect, all agents affected the mononuclear, basophil and eosinophil dermal infiltrates at some point in the experiment. Cyclophosphamide was the only agent found to affect the total white cell count of peripheral blood, but the leukopenia did not affect recruitment of mononuclear cells to the dermis. In the period immediately after administration of cyclophosphamide and methotrexate, the macroscopic score increased. Whilst the mechanism of this effect is unclear, it may be a manifestation of a basophil, perhaps IgE mediated reaction. Further manipulation of dose and dose interval may result in administration schedules relevant to the treatment of cell-mediated hypersensitivity in clinical practice.

Topics: Animals; Azathioprine; Basophils; Cyclophosphamide; Dermatitis, Contact; Female; Guinea Pigs; Immunity, Cellular; Immunoglobulin E; Immunosuppressive Agents; Leukocyte Count; Methotrexate; Oxazolone; Skin Tests; Time Factors

A series of substituted 5,6-diaryl-2,3-dihydroimidazo[2,1-b]thiazoles were synthesized and evaluated in the rat adjuvant-induced arthritis and mouse oxazolone-induced contact sensitivity assays to determine the potential of these compounds for use as immunoregulatory antiinflammatory agents. This class of compounds was derived by combining salient structural features of the antiinflammatory agent flumizole and the immunoregulatory drug levamisole. Unlike the latter two, a number of compounds in the target series were found to possess the desired combination of activities. Exploration of structure-activity relationships in the adjuvant-induced arthritic rat assay revealed that optimal potency was exhibited by symmetrically substituted 5,6-diaryl compounds having one of the following alkyl heteroatom or halogen functions at the para position: methoxy, ethoxy, methylthio, N-ethyl-N-methylamino, fluoro, or chloro. Scrambling of these two substituent classes to yield the asymmetrically substituted 5,6-diaryl compounds resulted in potent activity only with the 5-alkyl heteroatom, 6-halo-substituted regioisomers. However in the oxazolone-induced contact sensitivity assay, no consistent relationship of variation in activity with structural change was apparent. The initial target compound 5,6-bis(4-methoxyphenyl)-2,3-dihydroimidazo[2,1-b]thiazole (1) was compared with its progenitors in additional models of inflammation and immunoregulation.

Topics: Animals; Arthritis; Arthritis, Experimental; Chemical Phenomena; Chemistry; Dermatitis, Contact; Hemagglutination Tests; Imidazoles; Levamisole; Male; Mice; Mice, Inbred C57BL; Oxazolone; Rats; Rats, Inbred Strains; Structure-Activity Relationship; Thiazoles

Topics: Animals; Carrageenan; Dermatitis, Contact; Macrophages; Mice; Mice, Inbred AKR; Oxazoles; Oxazolone

Peptides derived from fibrinogen, known from earlier studies to inhibit the stimulation of lymphocytes in vitro and to suppress the humoral immune response in vivo, were investigated for their effect on cell-mediated immune reactivity in mice. An unfractioned mixture of peptides with molecular weights under 3,500 injected intraperitoneally at repeated intervals suppressed the contact hypersensitivity to oxazolone but did not influence the skin inflammatory reaction to croton oil. Local injections of peptides had a stronger effect on contact hypersensitivity. Four 200 micrograms local injections of peptides prior to sensitization abolished the increase in lymph node weight and the uptake of 125I-iododeoxyuridine in the draining lymph node after sensitization. Three previously isolated peptides with vasoactive effects inhibited Con A-stimulated incorporation of 3H-thymidine into spleen cells. The first, a pentapeptide (Ala-Arg-Pro-Ala-Lys), and the second, an undecapeptide (Ser-Glu-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys) both with an enhancing effect on microvascular permeability, were more potent than the third, a pentapeptide with slight vasoconstrictive properties (Thr-Ser-Glu-Val-Lys). Cell viability was not altered, as measured by trypan blue exclusion and the release of 86Rb. Accumulating evidence indicates that peptides derived from fibrin may be of importance as modulators of cellular immunoreactivity in a number of clinical conditions.

Topics: Animals; Capillary Permeability; Cells, Cultured; Concanavalin A; Dermatitis, Contact; Fibrinogen; Humans; Hypersensitivity, Delayed; Immunity, Cellular; Immunosuppressive Agents; Lymphocytes; Mice; Molecular Weight; Oxazolone; Peptides; Spleen; Thymidine

In the T suppressor circuit which affects contact sensitivity, the T acceptor cell (Tacc) armed with T suppressor factor (TsF) and then triggered by antigen and major histocompatibility complex products (I-J) releases non-specific inhibitor (nsINH). These non-specific inhibitor(s) affect both the efferent and afferent stage of the contact sensitivity reaction and were originally detected by the inhibition of the passive transfer of contact sensitivity. The nsINH also blocks the induction of contact sensitivity when given intravenously at the time of immunization but has no effect when given at the time of challenge. Similarly, it blocks proliferation in the regional lymph nodes induced by contact sensitizer in a dose-dependent fashion; it acts when given at the time of immunization but not 1 day later. This effect is antigen non-specific and H-2 unrestricted. The nsINH bears I-J determinants as shown by affinity chromatography on monoclonal antibody. The nsINH comes from the Tacc and is not a breakdown product of the TsF. This is shown by the fact that, when the Tacc and TsF have I-J of different genotypes, the genotype of the nsINH corresponds to that of the Tacc. Parallel measurements of inhibition of lymphoproliferation and of passive transfer show that the nsINH has a molecular weight of 50-60 Kd and a pI around 6.8 and suggest that similar or identical molecules block both the afferent and efferent stage of the contact sensitivity reaction.

Topics: Animals; Dermatitis, Contact; DNA; Epitopes; Lymphocyte Activation; Lymphokines; Mice; Mice, Inbred CBA; Molecular Weight; Oxazolone; T-Lymphocytes

Purified protein derivative from Mycobacterium tuberculosis inhibits contact sensitivity to oxazolone in mice when given intravenously 24 to 72 h before the antigen. Transfer experiments showed that various types of suppressor cells occurred in the lymph nodes draining the site of sensitization: (i) anti-oxazolone idiotype + B lymphocytes, found at day 3 after sensitization, transferred suppression to syngeneic recipients at the moment of their sensitization; (ii) anti-idiotype B lymphocytes, found at day 3 after sensitization, transferred suppression to syngeneic recipients when sensitization of these animals had been performed 3 days before cell transfer; (iii) T lymphocytes, found only at day 6 after sensitization, inhibited the passive transfer of contact sensitivity, indicating that they were effective on the efferent phase of the immune response. These results indicate that purified protein derivative from M. tuberculosis interferes with contact sensitivity by activating a complex and multiple immunoregulatory circuit.

Topics: Animals; B-Lymphocytes; Dermatitis, Contact; Haptens; Immune Tolerance; Immunization, Passive; Immunoglobulin Idiotypes; Mice; Mycobacterium tuberculosis; Oxazoles; Oxazolone; T-Lymphocytes, Regulatory; Tuberculin

Classical 24- to -48 hr delayed-type hypersensitivity (DTH) skin reactions are preceded by an early skin swelling reaction that peaks 2 hr after challenge. The ability to elicit this early component of DTH is T cell dependent and is also dependent on tissue mast cells and release of serotonin, mainly from these mast cells. The current study presents pharmacologic and kinetic evidence that the late response depends on the occurrence of the early response. A variety of pharmacological agents known to deplete, prevent release of, or block the activity of serotonin, when given just before skin challenge, blocked both the early and late components of DTH, but had no effect when given (even repeatedly) after the occurrence of the early component. Thus, the serotonin-dependence of the 24-hr component of DTH represents a dependence on the early component in which serotonin release is required. A temporal dependence of the late component of DTH on the early component was also demonstrated. The early and late phases occur at different times in recipients of sensitized T cells, depending on the interval between transfer and challenge, but there is a fixed 10- to 12-hr gap. Delayed onset of the late component occurs in recipients challenged immediately after transfer and appears to be due to a delay in the onset of the early component. This delay can be abolished by adoptive cell transfer into mice that are able to elicit a normal early component because of prior transfer of T cells that are able to mediate just an early component. On the basis of these findings, we conclude that DTH consists of a cascade of events. T cells mediating the early aspect of DTH release antigen-specific factors that, upon encountering antigen activate local serotonin-containing cells, such as mast cells, to release serotonin, which opens gaps between adjacent endothelial cells. Through these interendothelial gaps a second T cell population enters the extravascular space and interacts with local antigen to induce the late response by releasing the chemoattractant lymphokines that are classically associated with DTH and that cause recruitment of bone marrow-derived circulating leukocytes to infiltrate the reaction site. The ability of the second T cell population to mediate the late component of DTH is independent of further release of serotonin.

Topics: Animals; Dermatitis, Contact; Hypersensitivity, Delayed; Immunization, Passive; Kinetics; Male; Mast Cells; Mice; Mice, Inbred CBA; Oxazolone; Picryl Chloride; Serotonin; T-Lymphocytes

There is considerable confusion over whether the antigen-specific T suppressor factors (TsF) described by different authors are indeed equivalent. This paper investigates whether monoclonal TsF3, obtained from hybridomas derived from mice injected subcutaneously with NP derived spleen cells, is functionally equivalent to the conventional T suppressor factor, produced by mice injected intravenously with chemically reactive, water soluble haptene (picrylsulphonic acid and oxazolone thioglycolic acid). Comparison of monoclonal anti-NP TsF3 with conventional anti-picryl and anti-oxazolone T suppressor factor showed that both armed the non-specific T acceptor cell (Tacc) which was sensitive to cyclophosphamide and adult thymectomy. Moreover, non-specific inhibitor (nsINH) of the transfer of contact sensitivity was released when antigen, together with major histocompatibility complex products (MHC), reacted with conventional or monoclonal TsF on the surface of the non-specific T acceptor cell. The interaction of monoclonal TsF3 with antigen, which led to the release of NsINH, required the presence of MHC and was I-J restricted. However, there was no Igh-1 restriction. The equivalence of conventional anti-picryl and anti-oxazolone TsF has been demonstrated by arming the Tacc with a mixture of these two suppressor factors, and then triggering the release of nsINH with the mixed haptene 'picryl-oxazolone-lysine' which crosslinks separate molecules of TsF. A similar equivalence of conventional anti-oxazolone TsF and monoclonal anti-NP TsF3 was demonstrated using the mixed hapten 'NP-oxazolone-lysine' to trigger the release of nsINH. It was concluded that monoclonal TsF3 and conventional TsF were equivalent, and that both had an indirect mode of action through the non-specific T acceptor cell which led to the production of non-specific inhibitor.

Topics: Animals; Cyclophosphamide; Dermatitis, Contact; Haptens; Hybridomas; Immunization, Passive; Lymphokines; Major Histocompatibility Complex; Mice; Mice, Inbred Strains; Nitrophenols; Oxazolone; Phenylacetates; Picryl Chloride; Suppressor Factors, Immunologic; T-Lymphocytes; Thymectomy

Picryl chloride applied to the ears of Swiss mice induced a clearcut primary irritation inflammation (maximal after 3 to 6 hr) and after contact sensitization performed 7 days before a delayed hypersensitivity reaction. Oxazolone produced only a weak primary irritation reaction. After contact sensitization in the same conditions as above, oxazolone induced an immune response that was already substantial 3 to 6 hr after the challenge and generally reached a maximum after 24 hr. We tried to alter these four types of inflammation (the primary irritation and delayed hypersensitivity to picryl chloride, and the 6-hr and 24-hr phases of hypersensitivity to oxazolone) by various types of compounds administered cutaneously or sytemically. Mepyramine, methysergide, cimetidine, disodium cromoglycate, phenylbutazone, and acetylsalicylic acid reduced to varying degrees after cutaneous application the primary irritation and the delayed hypersensitivity inflammation induced by picryl chloride. Methysergide was the only one of these drugs that on topical application clearly reduced the immune response to oxazolone (decrease in the 6-hr phase). After systemic administration, these same drugs had no effect on the four types of reaction. Both the corticosteroids tested (hydrocortisone acetate and desonide) reduced all the inflammations to various degrees and were always more active (particularly desonide) when applied topically than when administered systemically. On the other hand indomethacin, which inhibited all types of inflammation, was more active when administered systemically. Study of the kinetics and trials of pharmacological modulation of the various reactions induced by picryl chloride and oxazolone in Swiss mice provided evidence of differences in behavior between the two agents.

Topics: Adrenal Cortex Hormones; Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Hypersensitivity, Delayed; Inflammation; Irritants; Kinetics; Male; Mice; Oxazoles; Oxazolone; Picryl Chloride

Various methods have been used to enhance the ability of laboratory animals to develop contact sensitivity to so-called weak sensitizers. The present studies have examined whether diets supplemented with vitamin A acetate (VAA) could enhance induction and elicitation of contact sensitivity to oxazolone in the mouse. Application of 0.3% oxazolone to VAA-fed mice resulted in increased DNA synthesis in the draining lymph nodes and increased ear thickness after challenge, in comparison with mice on a stock (standard) diet. Moreover, VAA-fed (but not control) mice were sensitized to 0.03% oxazolone as shown by DNA synthesis, increased arrival of syngeneic 51Cr-labelled lymphocytes in the draining lymph nodes, and ear swelling after challenge. These findings demonstrate that vitamin A enhances delayed hypersensitivity responses, and point to a role for vitamin A in developing an animal model capable of detecting weak contact sensitizers after topical application.

Topics: Animal Feed; Animals; Cell Movement; Dermatitis, Contact; Diterpenes; DNA; Hypersensitivity, Delayed; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Oxazolone; Retinyl Esters; Skin; Stimulation, Chemical; Vitamin A

Lymph node cells collected 4 days after painting the skin with picryl chloride activate the first components of the classical pathway of complement cascade, as shown by consumption of C4 of rabbit complement with total sparing of C5 and factor B activity. In contrast, lymph node cells collected 1 or 6 days after sensitization fail to do so. The ability of "4-day" cells to activate complement is inhibited by treating the cells with specific low-molecular-weight hapten, which is known to dissociate the immune complex present on the cell surface. When mouse serum was used as source of complement, a different behavior in complement activation between CBA/J and B10.D2-New/SnJ serum was observed: "4-day" cells failed to consume CBA/J serum whereas a normal complement activation was detected when B10.D2-New/SnJ serum was used. Using these two sera which differ in the level of C4, an inverse relationship between the ability of "4-day" cells to activate complement and their capacity to induce contact sensitivity when injected into the footpad of normal recipients was reported. Experiments performed using sera from C5 genetically deficient mice demonstrate that only the early complement components are involved, suggesting that membrane immune complexes are solubilized as a result of complement activation; on the other hand, membrane bound activated complement components could alter the immunizing potential of "4-day" cells.

Topics: Animals; Antigen-Antibody Complex; Complement Activation; Complement Pathway, Classical; Dermatitis, Contact; Immunity, Cellular; Lymph Nodes; Lymphocytes; Male; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Oxazolone; Picryl Chloride

Killed Staphylococcus aureus strain Cowan I cells inhibit contact sensitivity to oxazolone in mice, when given intravenously 24-72 h before sensitization. With transfer experiments it was found that the cells responsible for the suppression are antigen-specific, nylon-adherent, resistant to antitheta serum + C, and sensitive to anti-mouse Ig serum + C. These suppressor B cells bear anti-oxazolone immunoglobulins and appear to exert their suppressive activity by preventing the contact sensitizer from reaching the specific reactive T cells.

Topics: Animals; B-Lymphocytes; Dermatitis, Contact; Female; Immune Tolerance; Immunity, Cellular; Immunization, Passive; Lymph Nodes; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Oxazolone; Staphylococcal Infections; Staphylococcal Vaccines

Treatment of strain 2 guinea pigs with ultraviolet b (uvb) (280-320 nm) radiation or methoxsalen, followed by ultraviolet a (uva) (320-400 nm) radiation, decreased the contact hypersensitivity (CHS) reaction to sensitizing agents applied subsequently to unirradiated sites. The decreased reactivity could be transferred to syngeneic animals and appeared to be caused by antigen-specific suppressor T lymphocytes. Ultraviolet b irradiation of sensitized animals did not affect elicitation of CHS in unirradiated skin.

Topics: Animals; Dermatitis, Contact; Dinitrofluorobenzene; Female; Guinea Pigs; Immune Tolerance; Methoxsalen; Oxazolone; Ultraviolet Rays

The mechanisms of the anti-inflammatory effects of corticosteroids are uncertain but could be explained by an influence on infiltrating leukocytes. Our method for the qualitative and quantitative investigation of the dermal cellular infiltrate makes it possible to study the effects of topically applied corticosteroid preparations and vehicles on the infiltrating leukocytes of normal skin, allergic and toxic reactions in guinea pig skin. Ointment and cream vehicles as well as corticosteroid cream and ointment preparations often caused erythema and increased mononuclear infiltrate after only short periods of application (24-72 h). The strongest steroid ointment gave the most marked macroscopic response and propylene glycol preparations the most marked cellular response. In both toxic and allergic reactions, application of steroid preparations after the provocation gave no beneficial result either macroscopically or microscopically. Macroscopic scores were worsened by cream and ointment preparations. Although steroid solutions had no beneficial effect, they caused no detrimental effect. The guinea pig seems to be extremely sensitive to irritants and has not proved to be a suitable model for this approach to the study of the efficacy of topically applied steroids.

Topics: Administration, Topical; Adrenal Cortex Hormones; Animals; Croton Oil; Dermatitis, Contact; Dinitrochlorobenzene; Guinea Pigs; Ointments; Oxazolone; Pharmaceutic Aids; Skin; Time Factors

It is known that the skin is the natural route for induction of allergic contact dermatitis (ACD). Because the lung is another potential portal of entry for sensitizing chemicals, studies were performed to evaluate immune responses to haptens deposited in the lung. As a result, a new method for inducing ACD was developed. Intratracheal (IT) inoculation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) led to maximal ear swelling 24 hr after challenge on the ear with 2,4,6-trinitrochlorobenzene (TNCB) in carrier. This response was specific for immunizing hapten. Furthermore, it was equally possible to induce ACD by intratracheal inoculation of trinitrophenyl- (TNP) modified bronchoalveolar cells (BAC) or haptenated spleen cells. Adoptive transfer studies demonstrated that the hypersensitivity that resulted from IT inoculation of TNBS or TNP BAC could be transferred with cells. With a monoclonal anti-hamster Ia reagent, it was demonstrated that, like Langerhans cells, 80 to 90% of cells in the bronchial lavage fluid expressed Ia determinants on their membrane. These cells were morphologically indistinguishable from macrophages. Because an individual is capable of being sensitized when hapten is introduced by the pulmonary or epicutaneous routes, the possibility is raised that the alveolar macrophages in the lung possess a similar capability for antigen presentation of hapten in the induction of ACD as does the Langerhans cell.

Topics: Animals; Ascitic Fluid; Cricetinae; Dermatitis, Contact; Female; Haptens; Histocompatibility Antigens Class II; Immunization, Passive; Intubation, Intratracheal; Mesocricetus; Mice; Mice, Inbred BALB C; Oxazolone; Pulmonary Alveoli; Trinitrobenzenes; Trinitrobenzenesulfonic Acid

Using an experimental contact sensitivity model in guinea-pigs, evidence is presented that hapten (DNCB or oxazolone) specific T lymphocytes may persist for several months in previous sites of inflammation. Immunological memory, revealed by accelerated contact skin reactions upon retesting with the hapten, was limited to the original contact skin reaction sites. This 'local skin memory' to DNCB or oxazolone could be induced in both specific and non-specific skin inflammatory reactions, provided the animals had been sensitized to the hapten not longer than 2 weeks before. In animals which had been sensitized more than 1 month earlier, local skin memory could be induced if the animals received a booster application of hapten shortly (0-2 days) before primary skin testing. From these results we conclude that recently activated T cells may enter inflammatory sites non-specifically, producing specific local immunological memory. This memory may last several months. Accumulation of hapten specific T cells at inflammatory sites may be important in retest reactivity, in flare-up reactivity and in chronic inflammation.

Topics: Animals; Dermatitis, Contact; Dinitrochlorobenzene; Female; Guinea Pigs; Haptens; Immunization; Immunologic Memory; Oxazolone; Skin; Skin Tests; T-Lymphocytes; Time Factors

Topics: Animals; Complement System Proteins; Dermatitis, Contact; Haptens; Immunization, Passive; Immunoglobulin M; Immunosuppression Therapy; Lymph Nodes; Mice; Mice, Inbred CBA; Oxazoles; Oxazolone; Picryl Chloride; Receptors, Antigen, B-Cell; Time Factors

Four colonies of rats were tested for their ability to produce adjuvant-induced arthritis, oxazolone contact hypersensitivity and the dextran anaphylactoid reaction. Tuck Wistar and Hooded Lister rats, both of which respond to dextran, showed disseminated inflammatory lesions after adjuvant and exhibited oxazolone sensitisation, regardless of the age of the animal. Spontaneously hypertensive rats, which at all ages respond to dextran, also responded to adjuvant and oxazolone but only when they were young; as they grew older, this response to these two agents diminished. Wistar NELP rats, which at all ages do not respond to dextran, responded to oxazolone; sensitivity to adjuvant, however, was maximal only in young animals. The link between the ability of rats to resist the dextran anaphylactoid reaction and their failure to respond to adjuvant with disseminated inflammatory lesions has not been confirmed.

Topics: Anaphylaxis; Animals; Arthritis, Experimental; Dermatitis, Contact; Dextrans; Female; Hypersensitivity, Delayed; Male; Oxazolone; Rats; Rats, Inbred Strains

The subcutaneous administration of 0.01-10 mg/kg of the antiallergic agent disodium cromoglycate on day 0 30 min prior to sensitization of C57Bl/6 male mice with 5% 2-phenyl-4-ethoxy-methylene oxazolone proved to cause a significant stimulation of the low-grade volume response as measured plethysmographically in 24 h after challenge. The investigation of the mechanism favors the conclusion that histamine release is involved in the action of disodium cromoglycate as judged by the ability of the antihistaminics chlorpheniramine and metiamide to inhibit the disodium cromoglycate action and by the inhibitory effect of polymyxin B induced depletion of histamine.

Topics: Animals; Chlorpheniramine; Cromolyn Sodium; Dermatitis, Contact; Histamine; Imidazoles; Male; Mice; Mice, Inbred C57BL; Oxazoles; Oxazolone; Polymyxin B; Stimulation, Chemical

R-830, a di-tert-butylphenol, has been shown to be anti-inflammatory in a number of animal models. These include conventional systems such as carrageenan-induced edema and adjuvant arthritis of the rat and ultraviolet-induced erythema in the guinea pig in which the acidic nonsteroidal anti-inflammatory drugs (e.g., indomethacin) are effective. The anti-inflammatory activity of R-830 has also been demonstrated in other models (e.g., graft vs. host reaction and reversed passive cutaneous Arthus reaction in the rat, contact sensitivity in the mouse) in which the acidic nonsteroidal drugs are not effective. In vitro, R-830 inhibits guinea pig lung lipoxygenase and bovine seminal vesicle cyclo-oxygenase. The antioxidant properties of R-830 were demonstrated in two in vitro systems. We speculate that the antioxidant activity of this molecule might be related to its unusual profile of pharmacological activity.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Arthritis, Experimental; Butylated Hydroxytoluene; Dermatitis, Contact; Edema; Graft vs Host Reaction; Granuloma; Lipoxygenase Inhibitors; Male; Oxazolone; Passive Cutaneous Anaphylaxis; Prostaglandins; Rats; Rats, Inbred Strains

Levamisole, but not D-penicillamine, inhibited the oxazolone delayed hypersensitivity in rats. It was more effective when given locally to the ear (the tissue being challenged with oxazolone) or when injected intraperitoneally 24 h previously. Rats bred for resistance to the dextran anaphylactoid reaction exhibit contact sensitivity to oxazolone even better than rats which respond to clinical dextran.

Topics: Anaphylaxis; Animals; Arthritis, Rheumatoid; Dermatitis, Contact; Female; Levamisole; Oxazoles; Oxazolone; Penicillamine; Rats; Rats, Inbred Strains

The effect of selected compounds with known immunoregulatory activity was examined in a 45-h sensitization period oxazolone contact-sensitivity reaction. Oxazolone sensitivity was induced by applying 0.1 ml of 5% oxazolone in absolute ethanol to the shaved abdomen of C57Bl/6 mice on day 0. Challenge with oxazolone followed 45 h later and was accomplished by painting a 5% solution of oxazolone in absolute ethanol on the left hindpaw. The response at 24 h was determined plethysmographically. Histamine (0.062-1.0 mg/kg, subcutaneously, twice a day), concanavalin A (0.31-5.0 mg/kg intravenously), penicillamine (6.25-25 mg/kg, subcutaneously), chloroquine (6.25-25 mg/kg, subcutaneously), and thymosin fraction 5 (0.125-1.25 mg/kg subcutaneously) all stimulated the oxazolone reaction when administered on day 0. These data suggest that the low-grade oxazolone response may be a useful assay to detect immunostimulatory activity.

Topics: Adjuvants, Immunologic; Animals; Chloroquine; Dermatitis, Contact; Dose-Response Relationship, Drug; Histamine; Male; Mice; Mice, Inbred C57BL; Oxazolone; Penicillamine; Thymosin

Prevalent techniques for the assessment of cell-mediated immunity (CMI) in vitro are time consuming and may lack specificity. Deriving from the earlier observation of reduction in mean electrophoretic mobility (EPM) of lymphocytes following the development of humoral immune response, the possibility of using this electrokinetic method for the assessment of contact sensitivity in vitro was tested. CBA mice epicutaneously sensitized to dinitrofluorobenzene and 2-phenyl 4-ethoxy oxazolone showed a marked and reproducible reduction in the mean EPM of their lymph node cells (LNC). The specificity of this alteration in surface charge density was established by the enhancement in the EPM of contact sensitized LNC upon subsequent incubation in vitro with the specific antigen only. The profiles of time kinetics of changes in the EPM of LNC before and after incubation with antigen were virtually parallel to those of in vivo delayed hypersensitivity (DH) reaction as measured by the ear-swelling method. The coefficients of correlation between the reduction in EPM and in vivo DH response for DNFB and oxazolone were 0.89 and 0.86 respectively. EPM measurements may thus provide a sensitive, rapid, and quantitative parameter for the assessment of CMI in vitro.

Topics: Animals; Dermatitis, Contact; Dinitrofluorobenzene; Electrophoresis; Female; Hypersensitivity, Delayed; Immunity, Cellular; Kinetics; Lymphocytes; Male; Mice; Mice, Inbred CBA; Nitrobenzenes; Oxazoles; Oxazolone

The activation of B lymphocytes, which regulates contact sensitivity to oxazolone, was studied in mice injected with bacterial lipopolysaccharide (LPS) 24 h before sensitization. These regulatory cells bear Ig receptors, are sensitive to mitomycin C treatment in vitro, and have precursors sensitive to cyclophosphamide. Their early induction needed the mitogenic signal of LPS, in that LPS deprived of lipid A did not induce B-cell-mediated suppression, and required little or no T-cell help, as they were produced in B mice. Possible mechanisms by which LPS-induced regulatory cells act and their relevance to other suppressor systems are discussed.

Topics: Alkalies; Animals; B-Lymphocytes; Cyclophosphamide; Dermatitis, Contact; Female; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Oxazoles; Oxazolone; Spleen; T-Lymphocytes, Regulatory

The suppression of the contact sensitivity of oxazolone in murine malaria is shown to be mediated by non-specific T suppressor cells, but to a different extent in infection caused by two different species of parasite. Depletion of T suppressor cells in vivo and/or anti-Thy 1.2 treatment in vitro indicated that in mice infected with P. berghei the suppressor effect was largely mediated by T cells. By contrast, in mice infected with a lethal strain of P. yoelii it was only partly due to T cells; B suppressor cells and/or macrophages may also be involved. However, depletion of T suppressor cells in vivo had no effect on the course of the parasitaemia or on the survival time. Therefore, we postulate that this kind of non-specific immunosuppression cannot be regarded as a major cause of lethality.

Topics: Animals; B-Lymphocytes; Dermatitis, Contact; Female; Immune Tolerance; Lymphocyte Depletion; Malaria; Mice; Mice, Inbred Strains; Oxazolone; Plasmodium berghei; T-Lymphocytes; T-Lymphocytes, Regulatory

The intravenous injection of 2,4,6-trinitrophenyl (TNP)-labeled peritoneal exudate cells (TNP-PEC) into CBA mice fails to produce a state of hypersensitivity; rather, it renders recipient mice incapable of mounting a contact hypersensitivity response when they are subsequently immunized with a reactive form of the specific hapten. However, if precultured neonatal spleen cells are injected along with the cells that induce tolerance (TNP-PEC), not only is the development of tolerance inhibited but sensitization to TNP develops. The neonatal spleen cell responsible for turning the tolerogenic signal into an immunogenic one is I-J+ and adheres to the Vicia villosa lectin. Thus, it expresses markers that distinguish contrasuppressor effector cells from helper cells (D. R. Green et al., Eur. J. Immunol. 1981. 11:973), indicating that activated contrasuppressor cells can act as potent, helpful regulatory cells in vivo.

Topics: Animals; Antigens, Ly; Cells, Cultured; Dermatitis, Contact; Immune Tolerance; Immunity, Cellular; Male; Mice; Mice, Inbred CBA; Oxazolone; T-Lymphocytes, Regulatory; Trinitrobenzenes

Topics: Animals; Antibody Formation; B-Lymphocytes; Cricetinae; Dermatitis, Contact; Dinitrofluorobenzene; Immune Tolerance; Immunity, Cellular; Immunization, Passive; Langerhans Cells; Lymphocyte Activation; Mesocricetus; Oxazolone; Rabbits; T-Lymphocytes

The paramyxovirus of Newcastle disease (NDV) impairs the contact sensitivity to oxazolone in CBA/J mice: in vitro treatment with the infectious virus inhibits the passive transfer of contact sensitivity. Experiments performed with three different virus preparations demonstrate that only the infectious virus is able to inhibit the hypersensitivity reaction while treatment of virus by heat or ultraviolet irradiation wipes out its inhibitory activity. These results suggest that I-NDV depresses the response to oxazolone by its action on T immunoblasts which mediate the response to contactant.

Topics: Animals; Antigens; Cell Movement; Dermatitis, Contact; Female; Hypersensitivity, Delayed; Immunization, Passive; Lymphocyte Activation; Lymphocytes; Male; Mice; Mice, Inbred CBA; Newcastle Disease; Oxazoles; Oxazolone; T-Lymphocytes

Several phases of plasma extravasation have been identified during the course of a contact sensitivity challenge reaction in mice. An early reaction at 1 h is seen which may be mast cell mediated. This is followed by the main phase of extravasation at 3-4 h. This phase is not seen in mice sensitized for less than 5 days and is transferable passively by serum. Between 8 and 15 h there is a prolonged period of moderately intense extravasation which represents the delayed hypersensitivity component of the reaction. Beyond 15 h there is virtually no further extravasation.

Topics: Animals; Antibody Formation; Dermatitis, Contact; Ear; Erythrocytes; Humans; Immunization, Passive; Iodine Radioisotopes; Male; Mice; Mice, Inbred CBA; Organ Size; Oxazolone; Serum Albumin; Sheep; Time Factors

Unresponsiveness was produced in mice by three feeds of high doses of oxazolone or picryl chloride. The mice were then sensitized and contact sensitivity assessed by ear swelling. Adult thymectomy prevented the unresponsiveness caused by feeding. In some but not all experiments adult thymectomy also increased the response to a single skin paint. This effect of thymectomy was apparent at 1 week and reached a plateau at 2-3 weeks. The effect of adult thymectomy, on unresponsiveness caused by feeding and its ability to increase the contact sensitivity skin reaction, was reversed by treatment with crude thymosin fraction V.

Topics: Animals; Dermatitis, Contact; Dose-Response Relationship, Drug; Immune Tolerance; Mice; Oxazolone; Picryl Chloride; Spleen; Thymectomy; Thymosin; Trinitrobenzenesulfonic Acid

The specificity of the contact sensitivity induced by various chemically related oxazolones was studied. Mice were painted with 1 of them (immunogen). They were divided into several groups and challenged (ear painting on day 5) with various concentrations of either the immunogen or a related oxazolone. The degree of the contact sensitivity was estimated by ear weight or by incorporation of a DNA precursor. Higher concentrations of a challenge compound elicited a stronger DTH reaction than lower concentrations. The efficiency of each compound was defined by the concentration that caused a 30% increase of the ear weight or 150% increase of incorporation. Both represented ca. one-third of the maximal response (1/3 max), and both methods gave similar results. Two types of specificity patterns were observed. One is based on limited data, and was exhibited by the pair furyl Ox and propenyl Ox. Furyl Ox was a more efficient challenge compound than propenyl Ox when mice were primed with furyl Ox. The reverse was true when mice were primed with propenyl Ox. The other pattern was exhibited by the pair propenyl Ox and phenyl Ox. When mice had been primed with phenyl Ox, the 1/3 max response required either 3 mM phenyl Ox or 17 mM propenyl Ox (6-fold difference). The unexpected finding was that phenyl Ox was also a (2.5-fold) more efficient challenge compound for mice that had been primed with propenyl Ox (a heteroclitic contact sensitivity).

Topics: Animals; Antibody Formation; Antibody Specificity; Cyclophosphamide; Dermatitis, Contact; Female; Hypersensitivity, Delayed; Male; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Oxazoles; Oxazolone; Piperazines; Time Factors

Guinea pigs were sensitized systemically with the contact sensitizer oxazolone and challenged by topical application of the chemical to the eye and the skin. Inflammatory reactions of the conjunctiva, cornea and skin were uniformly elicited, but these were most intense clinically following intradermal sensitization with mycobacterial adjuvants. Mononuclear, neutrophilic and eosinophilic leukocytes infiltrated the ocular tissues and skin 24 hours after challenge. Basophils, which were prominent in the cutaneous reactions, were virtually absent from ocular tissues.

Topics: Adjuvants, Immunologic; Administration, Topical; Animals; Conjunctivitis; Dermatitis, Contact; Female; Guinea Pigs; Injections, Intradermal; Keratitis; Leukocytes; Male; Oxazolone

Topics: Adenosine Triphosphatases; Animals; Dermatitis, Contact; Dinitrofluorobenzene; Epidermis; Epitopes; Immune Tolerance; Langerhans Cells; Mice; Mice, Inbred C57BL; Nitrobenzenes; Oxazolone; Skin; Time Factors; Ultraviolet Rays

Allergic contact dermatitis to strong, low molecular weight contact allergens can regularly be induced in the hamster. By its clinical course, histopathology and susceptibility to intensification with complete Freund's adjuvant, this hypersensitivity appears congruent with the allergic contact dermatitis observed in other experimental animals and the allergic contact dermatitis seen in humans. Further, in the hamster, we find that pretreatment with cyclophosphamide intensifies the acquisition of allergic contact dermatitis to dinitrochlorobenzene and to oxazolone; the target of cyclophosphamide immunopotentiation has been shown in the mouse and guinea pig to be a regulator suppressor cell. In addition, we have induced in the hamster specific immune tolerance to dinitrochlorobenzene with dinitrobenzene sulfonate; in the mouse and guinea pig it has been demonstrated that the induction of specific immune tolerance to contact allergens by parenteral hapten involves the elaboration of specific suppressor cells. These findings, then, imply the existence of regulatory suppressor cells for T-cell phenomena in the hamster. This contrasts with reports that suppressor cell function in hamsters, as against other rodents, is defective as it relates to the regulation of, for instance, allogeneic reactions, antibody formation and tolerance to contact allergens.

Topics: Animals; Cricetinae; Cyclophosphamide; Dermatitis, Contact; Dinitrochlorobenzene; Female; Freund's Adjuvant; Immune Tolerance; Mesocricetus; Oxazolone; T-Lymphocytes, Regulatory

The distribution of fluorescent cells in the draining lymph nodes of mice painted with the contact sensitizing agent fluorescein isothiocyanate (FITC) was investigated using a fluorescence-activated cell sorter. Up to 30% of the cells were fluorescent after 18 h and this decreased thereafter becoming undetectable after 4-5 days. Most of the fluorescent cells were morphologically lymphocytes, theta - ve and adherent to nylon wool. Immunogenicity of these cells was tested by injecting them into the footpads of normal mice and measuring contact sensitivity after 6 days. This was restricted to large cells which represented less than 5% of the white cell population and nearly all of which became fluorescent after skin painting. The large fluorescent cells were a mixture of monocytes and lymphocytes. Most of the lymphocytes had surface immunoglobulin. The immunogenicity was reduced by nylon filtration but was not affected by silica and anti-theta. These results showed that the immunogenicity is not associated with T cells. In contrast, similar immunogenic activity in the draining lymph nodes of mice painted with oxazolone is associated with T cells. The results therefore showed that different sensitizers form immunogenic complexes with different cell populations, perhaps in this case becuase of the different water solubilities of FITC and oxazolone. They also suggested that this may cause important differences in antigen presentation, for example in their association with different MHC products.

Topics: Animals; Antibody-Producing Cells; Cell Separation; Dermatitis, Contact; Fluoresceins; Fluorescent Antibody Technique; Lymph Nodes; Lymphocytes; Male; Mice; Mice, Inbred CBA; Mice, Nude; Monocytes; Oxazolone; Receptors, Antigen, B-Cell

Cyclophosphamide was used to assess the role of suppressor cells in the contact sensitivity reaction. A single painting with 300 microgram and 30 microgram oxazolone produced poor contact sensitivity reactions (ear swelling). Cyclophosphamide (200 mg/kg) 2 days before painting increased the response to the lower doses but had less effect on the response to 3 mg oxazolone. A single feed with 10 mg oxazolone caused strong contact sensitivity while lower doses (10-1000 microgram) caused poor responses. Cyclophosphamide increased the response to the lower doses but not to the highest dose of oxazolone. These results suggested that the poor response to painting and feeding lower doses of oxazolone was due to a suppressor system which was sensitive to cyclophosphamide. A different result was obtained when contact sensitivity was measured by arrival of radioactively labelled cells. Cyclophosphamide had the greatest effect on cell arrival when high doses were fed. This indicates that ear swelling and cell arrival measure separate aspects of the contact sensitivity response. The lower doses of oxazolone, which caused little contact sensitivity, reduced the response to a standard immunizing dose. This low dose unresponsiveness occurred after either painting or feeding (Chase-Sulzberger phenomenon). It did not occur in mice treated with cyclophosphamide before the first exposure to oxazolone. This suggested that the low dose unresponsiveness was due to suppressor cells. The response to oxazolone was also assessed by DNA synthesis in the regional lymph nodes. A small dose of oxazolone (30 microgram) caused a peak of DNA synthesis on day four while a high dose (3 mg) caused a peak on day three. Pretreatment with cyclophosphamide depressed the response to 30 microgram although it increased contact sensitivity. The secondary response was smaller than the primary on days 3, 4 and 5 after immunization but larger on day two. The depression but not the increase was prevented by cyclophosphamide and was probably due to a suppressor system.

Topics: Animals; Antibody Formation; Antigens; Cyclophosphamide; Dermatitis, Contact; DNA; Dose-Response Relationship, Immunologic; Immunologic Memory; Lymph Nodes; Mice; Mice, Inbred CBA; Oxazoles; Oxazolone; T-Lymphocytes; Time Factors

Donor mice were painted on the skin of the abdomen with the contact sensitizing agent, oxazolone. One day later 2-5 x 10(6) cells from the regional lymph nodes were injected into the footpads of recipient mice. Contact sensitivity was detected 6 days later by challenging the ears of the recipients and measuring the increase of thickness at 24 h. Good contact sensitivity was obtained when CBA cells were injected into CBA mice and BALB/c cells injected into BALB/c mice; the injection of BALB/c (H-2d) cells into CBA (H-2k) mice and vice versa failed to give rise to contact sensitivity. Hybrid F1 cells gave intermediate responses. The contact sensitivity caused by the injection of small numbers of lymph node cells into the footpad is interpreted as a mode of active immunization and the present results show that this only occurs when there is genetic matching at the major histocompatibility complex between the donor and the recipient mouse.

Topics: Animals; Dermatitis, Contact; Foot; Lymph Nodes; Major Histocompatibility Complex; Mice; Oxazolone; Transplantation, Homologous; Transplantation, Isogeneic

Contact sensitivity responses to dinitrofluorobenzene (DNFB) or oxazolone were enhanced by amphotericin B (AmB) administration. This adjuvant effect of AmB was documented in mice by ear thickness measurements, ear histology, and the 5-iodo-2'-deoxyuridine-125I ear assay. The optimum immunopotentiating effect of AmB required its simultaneous administration at the time of skin sensitization. AmB-induced adjuvant effects were also observed in adoptive transfer experiments in which syngeneic recipients of lymph node cells from animals sensitized with DNFB plus AmB gave stronger contact sensitivity responses than recipients of cells from mice sensitized with DNFB alone. AmB also interfered with tolerance induction by i.v. dinitrobenzene sulfonic acid, suggesting that its adjuvant effects involve inhibition of suppressor cells or their precursors.

Topics: Adjuvants, Immunologic; Amphotericin B; Animals; Dermatitis, Contact; Dinitrofluorobenzene; Ear; Female; Idoxuridine; Immune Tolerance; Immunization, Passive; Mice; Mice, Inbred AKR; Oxazolone

Contact hypersensitivity to dinitrochlorobenzene (DNCB), picryl chloride (PCl) or oxazolone was induced in mice by skin painting with these agents, and was measured by skin testing in vivo using the ear swelling method. Prior administration of dinitrobenzene sulfonate (DNBS) or picryl sulfonic acid prevented sensitization to DNCB and PCl, respectively. Both this tolerization and the original sensitization were specific for the hapten used. Cyclophosphamide given before sensitization enhanced skin reactions, but when given before tolerization it interfered with establishment of tolerance. Cells from both sensitized and tolerized mice were shown to be reactive with the corresponding haptens in vitro in the leukocyte adherence inhibition (LAI) reaction. LAI specificity was similar to that found for cutaneous reactivity. The reaction of DNCB-sensitized cells with DNBS led to the production of a soluble mediator which induced LAI in normal cells. The demonstration of potentially reactive cells in mice judged to be tolerant by skin testing indicates the concomitant existence of suppressor factors.

Topics: Animals; Cell Adhesion; Cyclophosphamide; Dermatitis, Contact; Dinitrochlorobenzene; Drug Hypersensitivity; Female; Hypersensitivity, Delayed; Immune Tolerance; Immunity, Cellular; Leukocytes; Mice; Oxazolone; Picryl Chloride; Skin Tests

Topics: Animals; Antigens; Dermatitis, Contact; DNA; Female; Germ-Free Life; Lymph Nodes; Male; Mice; Oxazolone; Picryl Chloride; Time Factors

alpha-Globulins prepared from different biological sources (human and bovine sera, mouse ascitic fluid, human ovarian cyst fluid) were separated by ion exchange chromatography on DEAE-cellulose into fractions A, B and C. Whereas fractions A and B had no immunosuppressive activity, fraction C injected into mice shortly before antigen administration, but not after, significantly inhibited the contact sensitivity to oxazolone. Single high doses were preferential over divided doses given on several occasions. In transfer experiments when cells of animals sensitized to oxazolone were injected into recipients treated previously with fraction C, the response was also inhibited. Fraction C, when administered into F1 hybrid mice before injection of parental splenocytes, prevented the development of GVH reaction. The authors point out that the critical concentration of alpha-globulins at the time of antigen administration is necessary to make T-lymphocytes hyporesponsive, and when once triggered by antigen they become refractory to alpha-globulins action.

Topics: Alpha-Globulins; Animals; Cattle; Dermatitis, Contact; Graft vs Host Reaction; Humans; Immunity, Cellular; Immunization, Passive; Mice; Oxazolone; Spleen

The skin of CBA mice was painted with the contact sensitizing agent 4-ethoxymethylene-2-phenyloxazolone (oxazolone). One day later the regional lymph node cells were injected into the footpads of normal recipients. The recipients were tested 6 days later for contact sensitivity by challenging the ears with oxazolone and measuring the increase of ear thickness at 24 h. T cells and macrophages in the regional lymph nodes each independently gave rise to contact sensitivity in the recipient following injection into the footpad. This activity of T cells and macrophages was found in lymph nodes taken 1, 3 and 4 days after painting the donors. The role of T cells in the injected population was shown by purifying T cells by nylon-wool filtration and rosetting with sheep red cells coated with antibody and complement (EAC rosetting) and by destroyed T cells with anti-0 serum and complement. The activity of purified T cells resisted 2000 rad in vitro. The activity of cells from T-deprived (B) mice showed that a second cell type was important in the footpad transfer. This cell behaved like a macrophage, and not like a B cell, on EAC rosetting in the presence or absence of divalent cations and on treatment with silica and carrageenan--agents which damage macrophages. Our working hypothesis is that the footpad transfer may be caused independently by macrophages or T cells with oxazolone (probably linked to major histocompatibility complex antigens) on their surface and that these cells act by collaborating with T cells in the recipient which give rise to the effector cells for contact sensitivity.

Topics: Animals; Cell Separation; Dermatitis, Contact; Immunity, Cellular; Lymph Nodes; Macrophages; Mice; Oxazolone; T-Lymphocytes; Time Factors; Transplantation, Homologous

Topics: Animals; Antibody Formation; B-Lymphocytes; Dermatitis, Contact; DNA; Female; Immunization, Passive; Lymph Nodes; Lymphoid Tissue; Male; Mice; Mice, Inbred CBA; Oxazoles; Oxazolone; Picryl Chloride; Rosette Formation; T-Lymphocytes; Time Factors

Infection with the avirulent piroplasm Babesia microti in mice is accompanied by a marked depression in the ability of the mice to mount an immune response to sheep red blood cells. The period of immunodepression begins 3 days after peak parasitaemia and is maximal 4 days later. Thereafter, there is a slow return to normal immune responsiveness, correlated with the gradual disappearance of the parasites from the blood. Both IgM and IgG responses are depressed. Cell-mediated responses as determined by contact sensitivity to oxazolone and allograft survival are apparently unaffected. Phagocytic activity was measured by carbon clearance tests is increased, and is correlated with the parasitaemia.

Topics: Animals; Antibody Formation; Babesiosis; Dermatitis, Contact; Immunoglobulin G; Immunoglobulin M; Immunosuppression Therapy; Mice; Oxazolone; Phagocytosis; Skin Transplantation; Transplantation, Homologous

The immunostimulatory activity of levamisole [1-(-),2,3,5,6-tetrahydro-6-phenylimidazo(2,1-beta)-thiazole] was investigated using oxazolone-induced contact sensitivity in C57Bl mice. Oxazolone sensitivity was induced by applying 0.1 ml of 3 or 5% oxazolone in ethanol to the shaved abdomen (day 0). Challenge with oxazolone followed on day 2 (45 h) or day 3, and was accomplished by painting a 1--5% solution of oxazolone in ethanol to the left hind paw. The response at 24 h was determined plethysmographically. Levamisole (50 mg/kg, p.o., days 0--3 or day 0 only) failed to stimulate consistently the oxazolone response in a 3-day (minimal) sensitization period regimen. Use of a subliminal (45 h) sensitization, by contrast, revealed a consistent immunostimulatory effect of levamisole (12.5--100 mg/kg, p.o., day 0 only). It is speculated that the observed difference in levamisole effectiveness is attributable to (1) the ability of levamisole to stimulate both effector and suppressor mechanisms, and (2) the apparent absence of significant suppressor influence at 2-day postsensitization, leaving only the effector mechanism to be stimulated by levamisole.

Topics: Animals; Dermatitis, Contact; Immunity, Cellular; Levamisole; Male; Mice; Mice, Inbred C57BL; Oxazolone

The topical effects of N0164 (a phenyl phosphonate derivative which is a partially selective antagonist of prostagladin E2), indomethacin and triamcinolone acetonide have been shown to reduce the erythema and ear weight gain from inflammation induced by experimental contact allergic eczema. Oxazolone sensitized Swiss Webster mice were used, ear erythema and ear weights being used as a measure of the anti-inflammatory response to the drugs. N0164 was also shown to have systemic anti-inflammatory activity after intraperitoneal injection.

Topics: Administration, Topical; Animals; Anti-Inflammatory Agents; Dermatitis, Contact; Erythema; Indomethacin; Inflammation; Injections, Intraperitoneal; Mice; Organophosphonates; Oxazolone; Prostaglandin Antagonists; Triamcinolone Acetonide

Delayed-type hypersensitivity (DTH) to 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) was found to be under multigenic control in inbred, H-2 congenic and intra-H-2 recombinant strains of mice. A high response was associated with haplotypes H-2d,a,k and low response with haplotype H-2b. DTH to oxazolone was high or intermediate in different F1 hybrids of high and low responder mice. In F2 and backcross generations a higher response was associated with the "dd", than with "bb" phenotype, while intermediate response was found in the heterozygote "db" mice. A study of H-2 recombinant strains suggests that a gene controlling the DTH response maps in the I-B subregion of the H-2 complex. The response was significantly modified by gene(s) which are not linked to the H-2 complex and have not been mapped. Since congenitally athymic nude (nu/nu) mice did not respond to oxazolone, this contact sensitivity is belived to be a T cell-dependent immune response.

Topics: Administration, Topical; Alleles; Animals; Dermatitis, Contact; Genes; Genetic Linkage; Histocompatibility; Immunity, Cellular; Mice; Mice, Inbred Strains; Oxazoles; Oxazolone; Phenotype; Species Specificity

Chickens can easily be induced to develop delayed-type skin reactions to oxazolone when animals are sensitized 7 days before the challenge. The reaction is quantitated by assessing the increase in wattle thickness: maximum reactions occur 24 h after challenge. The reaction is inhibited by neonatal thymectomy or bursectomy; these findings therefore suggest also an important B-derived component in delayed hypersensitivity to oxazolone.

Topics: Animals; Bursa of Fabricius; Chickens; Dermatitis, Contact; Drug Hypersensitivity; Hypersensitivity, Delayed; Male; Oxazolone; Thymus Gland

Treatment with cyclophosphamide (Cy) can modulate the acquisition of allergic contact dermatitis in the mouse. We compared the effect of a single dose of Cy given at different times before or after allergen. Cy one or more days prior to allergen intensified, and Cy several days after allergen inhibited the acquisition of sensitivity. Mice whose immunological response to an allergen had been suppressed by Cy were specifically immunologically tolerant to that allergen, but not to an unrelated allergen. This tolerance probably represents a combination of clone deletion and inhibition; almost certainly it does not depend on the generation of enhancing ('blocking') antibody.

Topics: Allergens; Aniline Compounds; Animals; Cyclophosphamide; Dermatitis, Contact; Epitopes; Immune Tolerance; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Nitroso Compounds; Oxazolone

Inbred strain 2 and random-bred guinea pigs injected oxazolone in incomplete or complete Freund's adjuvant showed contact reactions within an hour after topical application when tested 3 weeks post-sensititization. The application when tested 3 weeks post-sensitization. The time for appearance of skin reactions at 5 days post-sensitization was 8 to 24 hr, more along classic delayed-type hypersensitivity lines. Immune serum harvested several weeks after sensititazation was effective for passive transfer of early skin reactivity, while serum at 5 to 10 days was not. Lymph node cells taken 5 days after sensitization of donors could transfer only the late skin reactivity to histocompatible recipients; lymph nodes taken at 15 days did not contain cells capable of such transfers. The heterogeneity of mechanisms involved in the production of contact dermatitis suggested by these results provides new approaches to studies in this area of allergy research.

Topics: Animals; Cyclophosphamide; Dermatitis, Contact; Guinea Pigs; Immunity, Active; Immunization, Passive; Oxazoles; Oxazolone; Skin Tests; Time Factors

Pseudomonas aeruginosa infection depresses contact sensitivity to 2-phenyl-4-ethoximethylene-oxazolone (oxazolone), and enhances the antibody response to sheep erythrocytes (SRBC) in the mouse. Anti-oxazolone antibody titres were found not to be significantly different in infected and uninfected animals; thus, the major circulating classes of antibodies do not seem to be responsibile for the observed depression of skin reactivity. Low dose (20 mg/Kg) cyclophosphamide (CY) pretreatment induced a further potentiation of antibody response to SRBC, and prevented depression of contact sensitivity in infected mice. On the other hand, when infected animals were pretreated with high doses (200 mg/Kg) of CY, antibody production was completely suppressed, whereas contact sensitivity was unaffected. Since CY treatment is known to selectively inhibit B lymphocytes, and since it can abrogate the infection-induced depression of reactivity to oxazolone, it is suggested that suppressor cells, which may have B-cell characteristics, are stimulated during P. aeruginosa infection in the mouse.

Topics: Animals; Antibody Formation; Cyclophosphamide; Dermatitis, Contact; Erythrocytes; Female; Immunity, Cellular; Immunization; Immunosuppression Therapy; Male; Mice; Mice, Inbred C57BL; Oxazoles; Oxazolone; Pseudomonas Infections; Skin; Time Factors

Suppressor cells were demonstrated in the spleen of guinea-pigs made specifically unresponsive to dinitrofluorobenzene (DNFB) with dinitrobenzene sulphonic acid (DNBSO3). Transfusion of these cells at the same time as sensitization with DNFB, produced a significant reduction in the immunoblasts proliferating in the draining lymph node 4 days later. Transfusion on the day of skin testing produced no greater suppression of skin reactivity than cells taken from animals made hypo-reactive to DNFB by contact with dinitrothiocyanate benzene (DNTB). It is concluded that there are at least two sites that suppressor cells can act. In the case of total unresponsiveness induced by DNBSO3, action is both central and in the periphery. In the case of hyporeactivity induced by DNTB, in which there is no defect in proliferation of T cells in response to antigen, the action of these cells is confined to the periphery. results of spleen weight studies suggest that suppressor cells homing in the spleen respond by proliferation to epicutaneously applied DNFB.

Topics: Animals; Antigens; Cell Division; Dermatitis, Contact; Dinitrofluorobenzene; Female; Guinea Pigs; Lymph Nodes; Male; Nitrobenzenes; Organ Size; Oxazolone; Skin Tests; Spleen; T-Lymphocytes

Allergic contact dermatitis to oxazolone was induced in three chickens rendered B-cell deficient by combined chemical bursectomy with testosterone and cyclophosphamide.

Topics: Animals; B-Lymphocytes; Bursa of Fabricius; Chickens; Cyclophosphamide; Dermatitis, Contact; Immunity, Cellular; Male; Oxazoles; Oxazolone; Testosterone

We have produced allergic contact dermatitis (ACD) to low molecular weight allergens in the chicken. Sensitization was induced by the application of a high concentration of allergen to a site on the comb previously treated with dimethyl sulfoxide. Challenge, which was made on the wattle with a nonirritating concentration of allergen, resulted in a dermatitis that was grossly and histologically similar to ACD in mammals. Chickens sensitized to oxazolone reacted to that allergen and not to picryl chloride, and conversely. This model provides a unique system wherein to study the T-cell and B-cell contributions to the generation, maintainance and expression of the ACD response.

Topics: Allergens; Animals; Chickens; Dermatitis, Contact; Dinitrofluorobenzene; Male; Molecular Weight; Oxazolone; Picryl Chloride

The arrival of cells from lymph nodes immunized with the contact sensitizing agents oxazolone and picryl chloride at ears challenged with these antigens was studied inmice using the technique of labelling with -51Cr. An apparent specificity of arrival was seen because the immune cells transfered contact sensitivity passively, giving rise to an inflammatory response in the ear, to which a subpopulation of cells (T blasts) was non-specifically attracted. It was also shown that there are at least two distinct populations of cells with the ability to move to inflammatory sites: the first, found in immunized lymph nodes, moves to contact sensitivity reactions in both actively and passively sensitized mice; the second, found in bone marrow and oil-induced peritoneal exudates, moves to contact sensitivity reactions in actively sensitized mice, whereas in passively sensitized mice, the arrival of these cells at contact sensitivity reaction is poor. It is suggested that the ability of T blasts to move to sites of inflammation my be useful as an assay technique for contact sensitivity reactions.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Movement; Chromium Radioisotopes; Dermatitis, Contact; Ear, External; Immunity, Active; Immunity, Cellular; Immunity, Maternally-Acquired; Immunization, Passive; Lymph Nodes; Lymphocyte Activation; Male; Mice; Mice, Inbred CBA; Oxazolone; Picrates; T-Lymphocytes

DNA--guinea-pig erythrocyte rosette-forming cells (RFC), as found after contact sensitization of guinea-pigs with DNCB, were shown to be highly sensitive to pretreatment with cyclophosphamide or X-irradiation, which suggests that these cells are B cells. The finding was confirmed by rosette-blocking experiments using an anti-immunoglobulin serum. It has already been shown that they are not directly related to antibody production. Possibly they represent precursors of antibody-producing cells. An increase in rosette formation, using non-antigenically related erythrocytes, was shown to parallel development of cell-mediated immunity (CMI) in the lymph nodes. These non-specifically induced RFC were also found to be B cells. Data were obtained which make it improbable that either proliferation or influx of B cells during development of CMI plays a major role in this increase. An alternative mechanism is suggested, while a possible role for these non-specifically induced RC is proposed.

Topics: Animals; Antibody Formation; Antibody-Producing Cells; Antilymphocyte Serum; B-Lymphocytes; Cyclophosphamide; Dermatitis, Contact; Dinitrochlorobenzene; Erythrocytes; Female; Guinea Pigs; Hemolytic Plaque Technique; Immune Adherence Reaction; Immunity, Cellular; Immunosuppression Therapy; Lymph Nodes; Nitrobenzenes; Ovalbumin; Oxazolone; Polysaccharides, Bacterial; Spleen; T-Lymphocytes

Contact sensitivity to 2-phenyl-4-ethoxymethilene oxazolone, as a probe for cell-mediated immunity, was investigated in susceptible BALB/c and resistant C57BL/6 mice after infection with Friend leukemia complex (FLC) or with Rowson-Parr virus (RPV). In BALB/c mice, FLC depressed contact sensitivity when given before primary sensitization but had no effect on established contact sensitivity nor on the response elicited by a booster application of the sensitizer. These findings, together with the failure to alter reactivity to an aspecific inflammatory stimulus, indicate that FLC impairs the afferent limb of the response. In the same strain of mice RPV infection did not significantly depress contact sensitivity, as judged by the extent of the reaction 24 h after challenge, but slightly inhibited the early antibody-mediated phase of this reaction. In C57BL/6 mice neither viral preparation affected contact sensitivity.

Topics: Allergens; Animals; Dermatitis, Contact; Ear; Female; Friend murine leukemia virus; Immunization; Immunization, Secondary; Immunosuppression Therapy; Leukemia, Experimental; Mice; Mice, Inbred BALB C; Oxazoles; Oxazolone

Alloxan-diabetic mice of Swiss, CBA and DBA/2 strains show a significant depression of contact sensitivity to oxazolone, as compared with normoglycaemic control animals, which is accompanied by the involution of the thymus and spleen. Insulin treatment partially restores the contact sensitivity in diabetic animals and also increases the weight of lymphatic organs. In contrast, the non-specific inflammatory response to oxazolone is not impaired in insulin-deficient mice. Further experiments have shown that neither sensitized lymphocytes of control animals given to diabetic mice, nor sensitized lymphocytes of diabetic mice injected into normoglycaemic recipients, were able to transfer passively any significant contact sensitivity. It is suggested that in alloxan-diabetic mice the function of T lymphocytes is affected.

Topics: Animals; Antigens; Blood Glucose; Dermatitis, Contact; Diabetes Mellitus, Experimental; Ear; Female; Immunization, Passive; Insulin; Male; Mice; Mice, Inbred CBA; Mice, Inbred DBA; Organ Size; Oxazoles; Oxazolone; Spleen; Thymus Gland

Topics: Animals; Antigens; Cyclophosphamide; Cytarabine; Dermatitis, Contact; Female; Fluorouracil; Immunization; Male; Mice; Mice, Inbred A; Mice, Inbred C3H; Oxazoles; Oxazolone; Plethysmography; Time Factors