calcimycin and Inflammation

Various cationic polyelectrolytes (poly-alpha-amino acids and histones), lectins, the chemotactic peptide, f-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were investigated regarding their capacity to induce luminol-dependent chemiluminescence (LDCL) and superoxide production by human blood leukocytes. Although when tested individually, poly-L-arginine (PARG), phytohemagglutinin (PHA), concanavalin A (Con A), or fMLP induced only a low to moderate LDCL response, very intense synergistic CL reactions were obtained by mixtures of PARG + PHA, PARG + Con A, PARG + PHA + fMLP, Ca2 + ionophore + PARG + PHA + fMLP, and PARG + PMA. The sequence of addition of the various agents to WBC in the presence of luminol absolutely determined the intensity of the LDCL signals obtained, the highest reactions being achieved when the WBC were preincubated for 2-3 min with A23187 followed by the sequential addition of fMLP, PARG, and PHA. These "multiple hits" induced CL reactions which were many times higher than those obtained by each factor alone. On the other hand, neither poly-L-lysine, poly-L-ornithine, poly-L-histidine, nor poly-L-asparagine, when employed at equimolar concentrations, cooperated efficiently with PHA and fMLP to trigger synergistic LDCL responses in leukocytes. Concomitantly with the induction of LDCL, certain ligand mixtures also triggered the production of superoxide. The LDCL which was induced by the "cocktail" of agents was markedly inhibited by sodium azide (93% inhibition), but to a lesser extent by catalase (10% inhibition) or by superoxide dismutase (20%-60% inhibition). On the other hand, scavengers of singlet oxygen and OH (sodium benzoate, histidine) did not affect the synergistic LDCL responses induced by these multiple ligands. Cytochalasin B also markedly inhibited the LDCL responses induced either by soluble stimuli or by streptococci preopsonized either with histone or with polyanethole sulfonate. The LDCL responses which were induced by mixtures of PARG and concanavalin A were also strongly inhibited by mannose, alpha-methyl mannoside, and poly-L-glutamic acid. The data suggest that the LDCL responses induced by the soluble ligands involved a myeloperoxidase-catalyzed reaction. The possible employment of "cocktails" of ligands to enhance the bactericidal effects of PMNs, macrophages, and natural killer cells on microbial cells and mammalian targets is discussed.

Topics: Antimetabolites; Calcimycin; Carbohydrates; Cytochalasin B; Drug Synergism; Electrolytes; Histones; Humans; Inflammation; Lectins; Leukocytes; Ligands; Luminescent Measurements; Luminol; Lysophosphatidylcholines; N-Formylmethionine Leucyl-Phenylalanine; Peptides; Polymyxin B; Superoxides; Tetradecanoylphorbol Acetate

Current knowledge of the mechanism of inflammatory mediator release from mast cells is reviewed with particular reference to the role of cyclic nucleotides and calcium and their interrelationship with one another as defined by studies in highly purified rat peritoneal mast cells. Data are presented indicating an important role for intracellular cAMP and calcium in the mediation or modulation of release, as well as evidence for a close relationship between these two regulatory systems. Releasing agents which clearly act at the level of the plasma membrane (concanavalin A and anti-IgE antibody) are shown to differ from releasing agents that may not (48/80 and the ionophore A23187) in regard to the early cellular cAMP response, dependency of the release reaction on phosphatidyl serine, and kinetics of release. Pharmacologic modulators of release are discussed; these include: PGE1 and theophylline, which raise cAMP and inhibit release; and diazoxide, adenine, and carbachol which lower cAMP and potentiate release. Adenosine was also found to enhance release with marked effects at concentrations in the low nanomolar range.

Topics: Animals; Antibodies; Calcimycin; Calcium; Catecholamines; Cell Membrane; Concanavalin A; Cyclic AMP; Cyclic GMP; Histamine Release; Humans; Immunoglobulin E; Inflammation; Kinetics; Mast Cells; p-Methoxy-N-methylphenethylamine; Prostaglandins E; Rats; Theophylline

IV magnesium (Mg2+) has been proposed as an emergent treatment for acute asthma exacerbations. Recent studies have focused on the effects of Mg2+ on bronchial smooth muscle, yet asthma is primarily an inflammatory disease.. To assess the effects of Mg2+ on the neutrophil respiratory burst of adult patients with asthma.. A prospective, blind study of volunteer adult asthmatic patients was performed. The patients' polymorphonuclear neutrophils (PMNs) were isolated, purified, and placed into phosphate-buffered saline with the following test conditions: concentrations of magnesium chloride (MgCl2) added: 0 mmol MgCl2, 1 mmol MgCl2 (low), and 10 mmol MgCl2 (high) both with and without the calcium (Ca2+) ionophore A23187 (0.1 mmol). PMNs were activated using N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 mumol), and the production of superoxide (O2-) was measured by the spectrophotometric reduction of cytochrome c.. Mg2+ reduced activated PMN O2- production compared with that for no Mg2+ (1.0 +/- 0.1 nmol O2-/5 x 10(5) PMN/min) in both low (-0.52* +/- 0.3 nmol O2-/5 x 10(5) PMN/min) and high (-0.76* +/- 0.3 nmol O2-/5 x 10(5) PMN/min; *p < 0.05) concentrations. The addition of A23187 increased O2- production in both the high (0.53* +/- 0.02 nmol O2-/5 x 10(5) PMN/min) and the low (1.5* +/- 0.6 nmol O2-/5 x 10(5) x 10(5) PMN/min) Mg2+ groups, with no change in the control group (1.2 +/- 0.2 nmol O2-/10(5) PMN/min).. In clinically relevant concentrations, Mg2+ attenuates the neutrophil respiratory burst in adult asthmatic patients. Mg2+ appears to affect PMNs by interfering with extracellular Ca2+ influx. Mg2+ may have a beneficial anti-inflammatory effect in asthmatic individuals.

Topics: Acute Disease; Adult; Asthma; Calcimycin; Calcium Channels; Double-Blind Method; Drug Therapy, Combination; Female; Humans; Inflammation; Ionophores; Magnesium Chloride; Male; Middle Aged; Prospective Studies; Respiratory Burst

Platelets play a modulatory role in inflammatory response by secreting a vast array of granules and disintegrating into membrane-bound microparticles upon activation. The interplay between eosinophils and platelets is postulated to be implicated in the pathology of allergic airway inflammation. In this study, we investigated whether activated platelets can induce eosinophil extracellular trap (EET) formation, a cellular process by which activated eosinophils release net-like DNA fibers.. Platelets were stimulated with the calcium ionophore, A23187, and the platelet agonists, thrombin and adenosine diphosphate (ADP). Platelet cultures were fractionated into conditioned medium (CM) and pellet, which were then overlaid on eosinophils to examine EET formation.. The CM and pellet from A23187-activated platelets stimulated eosinophils to generate EET, whereas those from thrombin- or ADP-activated platelets failed to induce such generation. The EET-inducing activity of the A23187-activated platelet culture was linearly proportional to the number of activated platelets. Interestingly, while EET formation induced by the direct stimulation of eosinophils with A23187 was NADPH oxidase (NOX)-dependent, EET formation induced by A23187-activated platelets was NOX-independent and significantly inhibited by necroptosis pathway inhibitors.. Activated platelets and their products may induce EET formation, thereby potentiating their role in eosinophilic airway inflammation.

Topics: Adenosine Diphosphate; Blood Platelets; Calcimycin; Calcium; Calcium Ionophores; Extracellular Traps; Humans; Inflammation; Thrombin

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with

Topics: Calcimycin; Chromogranin A; DNA, Mitochondrial; Extracellular Vesicles; Humans; Inflammation; Interleukin-6; Macrophages; Membrane Proteins; Neutrophils; Nucleotidyltransferases; Staphylococcus aureus

Oleanolic acid (OA) is an active compound found in a variety of medicinal herbs and plants. Though OA has been widely attributed with a variety of biological activities, studies focused on its anti-allergic inflammation properties are insufficient.. Given the rapid increase in allergic diseases and the lack of fundamental treatment options, this study aimed to find a safe and effective therapy for allergic disorders.. We evaluated the inhibitory effect of OA on allergic inflammatory response and the possible mechanisms underlying the effect using phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell (HMC)-1, and a mouse model of compound 48/80-induced anaphylactic shock.. OA suppressed pro-inflammatory cytokine expressions in PMACI-induced HMC-1 cells by inhibiting activation of the Akt, p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT) 1 signaling pathways. Moreover, OA showed a protective effect against compound 48/80-induced anaphylactic shock through inhibition of histamine release and immunoglobulin E level via regulation of NF-κB and STAT1 activation.. The results showed that OA suppressed mast cell-mediated allergic response by transcriptional regulation. We suggest that OA has potential effect against allergic inflammatory disorders, including anaphylaxis, and might be a useful therapeutic agent for allergic disease.

Topics: Anaphylaxis; Animals; Anti-Allergic Agents; Calcimycin; Cell Line; Cytokines; Histamine Release; Humans; Inflammation; Inflammation Mediators; Male; Mast Cells; Mice, Inbred ICR; NF-kappa B; Oleanolic Acid; p-Methoxy-N-methylphenethylamine; p38 Mitogen-Activated Protein Kinases; Phorbol Esters; Proto-Oncogene Proteins c-akt; STAT1 Transcription Factor

Aging has pronounced effects on mammalian tissues and cells, but the impacts of aging on salivary gland function are relatively unknown. This study aims to evaluate the effects of aging on submandibular gland (SMG) and parotid gland (PG) functions in the male senescence-accelerated mouse. In vivo analysis at the systemic level revealed that salivary secretion induced by pilocarpine, a muscarinic agonist, from the SMG was significantly decreased in aged mice, whereas salivary secretion from the PG was not affected. To evaluate organ-level function, the SMG was perfused with the muscarinic agonists carbachol and calcium ionophore A23187 ex vivo to induce salivary secretion, and decreased saliva production was also observed in the aged SMG. Histological analysis revealed the presence of CD4-positive lymphocytes infiltrating the aged SMG. Furthermore, real-time PCR revealed that the aged SMG exhibited accelerated cell aging, increased levels of the inflammatory cytokine interleukin-6, and decreased mRNA levels of the water channel protein aquaporin-5 (AQP5). In summary, these results demonstrate that SMG function in aged mice was diminished, and that cell senescence, chronic inflammation, and the decreased gene expression of AQP5 are the likely causes of hyposalivation in the SMG of aged mice.

Topics: Animals; Aquaporin 5; Calcimycin; Calcium Ionophores; Carbachol; CD4-Positive T-Lymphocytes; Cellular Senescence; Cholinergic Agonists; Down-Regulation; Inflammation; Interleukin-6; Male; Mice; Parotid Gland; Submandibular Gland; Treatment Outcome; Xerostomia

micro-RNAs (miRNAs) are non-coding RNAs which play important role in human diseases. Dysregulated miRNAs have been identified in asthma patient while their precise roles in asthma are not well elucidated. We compared the expression level of total 11 miRNAs between PMA/A23187-treated and control HMC-1 mast cells. We determined the effect of miR-20a on inflammation by overexpressing miR-20a mimic or its antagonist. We further predicted histone deacetylase 4 (HDAC4) as potential target of miR-20a and explored the effects of miR-20a on HDAC4 expression and histone modification. miR-20a was down-regulated in PMA/A23187-treated HMC-1 cells. miR-20a inhibited expression of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and Interferon gamma (IFN-γ) while promoted Interleukin 10 (IL-10) production. miR-20a targeted HDAC4 and suppressed its expression, which contributed to epigenetically regulation of IL-10 expression by miR-20a. hsa-miR-20a-5p attenuates allergic inflammation in HMC-1 cells by targeting HDAC4.

Topics: Base Sequence; Calcimycin; Cell Line; Cytokines; Down-Regulation; Epigenesis, Genetic; Histone Deacetylases; Humans; Hypersensitivity; Inflammation; Inflammation Mediators; MicroRNAs; Ovalbumin; Repressor Proteins; Tetradecanoylphorbol Acetate

Inflammation‑associated damage may occur in any tissue following infection, exposure to toxins, following ischemia, and in allergic and auto‑immune reactions. Inflammation may also result from mast cell degranulation induced by the intracellular calcium concentration. The inflammatory process may be inhibited by compounds that affect mast cells. Bisdemethoxycurcumin [1,7‑bis(4‑hydroxyphenyl) hepta‑1,6‑diene‑3,5‑dione, BDCM] is the active component of turmeric. It has anticancer, antioxidant and antibacterial properties. To investigate the molecular mechanism associated with the anti‑inflammatory activity of BDCM, human mast cell line 1 (HMC‑1) cells were treated with phorbol‑12‑myristate‑13‑acetate (PMA) and calcium ionophore A23187 (A23187) to induce the inflammatory process. Various HMC‑1 cells were pretreated with BDCM prior to stimulation of inflammation. BDCM inhibited the inflammation‑triggered production of cytokines including interleukin (IL)‑6, IL‑8, and tumor necrosis factor (TNF)‑α. BDCM inhibition extended to the gene level. In activated HMC‑1 cells, phosphorylation levels of extracellular signal‑regulated kinase, c‑jun N‑terminal kinase and p38 mitogen‑activated protein kinase were decreased by treatment with BDCM. BDCM also inhibited nuclear factor‑(NF)‑κB activation and IκB degradation. In conclusion, BDCM suppresses the expression of TNF‑α, IL‑8, and IL‑6 by inhibiting the NF‑κB and mitogen activated protein kinase signaling pathways.

Topics: Calcimycin; Cell Line; Curcumin; Diarylheptanoids; Gene Expression Regulation; Humans; Inflammation; Mast Cells; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor RelA

Allergic inflammation is the foundation of allergic rhinitis and asthma. Although microRNAs are implicated in the pathogenesis of various diseases, information regarding the functional role of microRNAs in allergic diseases is limited. Herein, we reported that microRNA-302e (miR-302e) serves as an important regulator of allergic inflammation in human mast cell line, HMC-1 cells. Our results showed that miR-302e is the dominant member of miR-302 family expressed in HMC-1 cells. Moreover, the expression of miR-302e was significantly decreased in response to phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 or ovalbumin (OVA) stimulation. Overexpression of miR-302e blocked PMA/A23187 or OVA induced the increase in inflammatory cytokines levels, such as IL-1β, IL-6, tumor necrosis factor (TNF)-α and thymic stromal lymphopoietin, while miR-302 inhibition further promoted the release of these cytokines. Mechanistically, we found that miR-302e is a novel miRNA that targets RelA, a gene known to be involved in regulating inflammation, through binding to the 3'-UTR of RelA mRNA. Ectopic miR-302e remarkably suppressed the luciferase activity and expression of RelA, whereas down-regulation of miR-302e increased RelA luciferase activity and expression. Pharmacological inhibition of NF-κB reversed the augmented effect of miR-302e down-regulation on inflammatory cytokines level. Taken together, the present study demonstrates miR-302e limits allergic inflammation through inhibition of NF-κB activation, suggesting miR-302e may play an anti-inflammatory role in allergic diseases and function as a novel therapeutic target for the treatment of these diseases.

Topics: Calcimycin; Cell Line; Gene Expression Regulation; Humans; Inflammation; Mast Cells; MicroRNAs; NF-kappa B; Ovalbumin; Rhinitis, Allergic; Tetradecanoylphorbol Acetate; Transcription Factor RelA

Cynanchum atratum Bunge (Apocynaceae) is a folk medicine to treat skin inflammatory diseases. However, the effects of C. atratum on atopic dermatitis have not been elucidated. In this study, we evaluate the effects of aqueous extract of C. atratum (CA) and its molecular mechanism on atopic dermatitis (AD). 1 and 100mg/mL CA were topically applied to 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions for 11 days. The number of scratching behavior was evaluated for 20min. AD-like symptoms including elevated serum IgE, skin hyperplasia and mast cell infiltration were investigated. The expressions of pro-inflammatory cytokines and mediators were analyzed in AD-like skin legions. In addition, pro-inflammatory cytokine production was confirmed in human mast cells (HMC)-1 stimulated with PMA plus A23187 (PMACI). Topical application of CA attenuated total serum IgE level and scratching behavior. Skin hyperplasia including epidermis and dermis was ameliorated in CA-treated skin legions. The number of infiltrated mast cells was significantly decreased by CA treatment. In addition, CA reduced pro-inflammatory cytokines, such as IL-6, IL-1β and TNF-α and Th2 cytokine, IL-4, in both of AD-like skin lesions and PMACI-sensitized HMC-1 cells. Furthermore, CA decreased the expressions of NF-κB, phospho-IκBα and MAP kinase. These results suggest the inhibitory effects of CA on the development of AD by regulating pro-inflammatory cytokines and mediators. CA could be an effective substance for the treatment of AD.

Topics: Animals; Calcimycin; Cytokines; Dermatitis, Atopic; Dermis; Dinitrochlorobenzene; Epidermis; Female; Hyperplasia; Immunoglobulin E; Inflammation; Interleukin-6; Mast Cells; Mice; Mice, Inbred BALB C; NF-kappa B; NF-KappaB Inhibitor alpha; Plant Extracts; Tumor Necrosis Factor-alpha; Vincetoxicum

Basophils (BAs) are the least common granulocytes of all leukocytes, but they play an important role in orchestrating of chronic allergic inflammation. The Notch signaling pathway is a highly conserved pathway that influences cell lineage decisions and differentiation during various stages of development. However, the relationship between Notch signaling and BA remains to be elucidate. Here, we report that several Notch signaling molecules were found to be expressed in BAs. γ-secretase inhibitor (GSI) treatment increase BAs apoptosis, and suppress BAs proliferation. Furthermore, GSI reduced BAs in the S phase, with a concomitant accumulation in G1 and G2 phases. In addition, GSI also significantly down-regulated mRNA levels of cytokines IL-4, IL-6 and IL-13 induced by A23187, and this effect was dependent on MAPK pathway. Finally, IL-6 inhibition was specifically associated with ERK and IL-13 with JNK. Therefore, Notch signaling regulates BA biological function, at least partially via the modulation of MAPK.

Topics: Animals; Basophils; Calcimycin; Cell Cycle; Cells, Cultured; Chronic Disease; Cytokines; Extracellular Signal-Regulated MAP Kinases; Hypersensitivity; Inflammation; Inflammation Mediators; Mice; Oligopeptides; Receptors, Notch; Signal Transduction

Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE.  ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

Topics: Aspirin; Azepines; Caco-2 Cells; Calcimycin; Humans; Inflammation; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; omega-N-Methylarginine; Pancreatic Elastase; Respiratory Burst; Triazoles

Bacterial endotoxin lipopolysaccharide (LPS) activates inflammatory pathways, induces cytokine expression in the endothelium, augments reactive oxygen species (ROS) production in the vascular wall, and induces endothelial dysfunction. The aim of the present study was to analyze the effects of peroxisome proliferator-activated receptor (PPAR)β/δ activation on LPS-induced inflammation, oxidative stress and endothelial dysfunction and to determine whether uncoupling protein-2 (UCP2) plays a role in these effects. In vivo, the PPARβ/δ agonist GW0742 treatment prevented the LPS-induced reduction in aortic relaxation, the increase in vascular ROS production, the upregulation of NOX1, NOX2, p47(phox), and p22(phox) mRNA levels, and the endoplasmic reticulum (ER) stress markers in mice. We show that in mouse aortic endothelial cells (MAECs), GW0742 prevented the decreased A23187-stimulated nitric oxide (NO) production, and the increased intracellular ROS levels caused by exposure to LPS in vitro. The PPARβ/δ antagonist GSK0660 abolished all these in vivo and in vitro protective effects induced by GW0742. This agonist also restored the reduced expression of UCP2 and mitofusin-2 induced by LPS. The effects of GW0742 on NO and ROS production in MAEC exposed to LPS were abolished by the UCP2 inhibitor genipin or by siRNA targeting UCP-2. Genipin also suppressed the expressional changes on NADPH oxidase and ER stress markers induced by GW0742. In conclusion, PPARβ/δ activation restored the LPS-induced endothelial dysfunction by upregulation of UCP2, with the subsequent alleviation of ER stress and NADPH oxidase activity, thus reducing intracellular ROS production and increasing NO bioavailability.

Topics: Animals; Aorta; Calcimycin; Cytochrome b Group; Endoplasmic Reticulum Stress; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation; Inflammation; Lipopolysaccharides; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidases; Nitric Oxide; PPAR gamma; PPAR-beta; Primary Cell Culture; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Sulfones; Thiazoles; Thiophenes; Tissue Culture Techniques; Uncoupling Protein 2

Salidroside [2-(4-hydroxyphenyl)ethyl β-D-gluco-pyranoside (SAS)] has been identified as the most potent ingredient of the plant Rhodiola rosea L. Previous studies have demonstrated that it possesses a number of pharmacological properties, including anti-aging, anti-fatigue, antioxidant, anticancer and anti-inflammatory properties. In this study, to ascertain the molecular mechanisms responsible for the anti-inflammatory activity of SAS, we used phorbol-12-myristate-13-acetate (PMA) plus A23187 to induce inflammation in human mast cell line-1 (HMC-1). The HMC-1 cells were treated with SAS prior to being stimulated with PMA plus A23187. Pro-inflammatory cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis was used to examine the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). SAS inhibited the mRNA expression and production of interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF). In cells stimulated with PMA plus A23187, SAS suppressed the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and c-jun N-terminal kinase 1/2 (JNK1/2), but not that of p38 MAPK. SAS suppressed the expression of NF-κB in the nucleus. On the whole, our results suggest that SAS exerts an anti-inflammatory effect by inhibiting the production of pro-inflammatory cytokines through the blocking of the NF-κB and MAPK signaling pathways.

Topics: Anti-Inflammatory Agents; Calcimycin; Cell Line, Tumor; Cytokines; Gene Expression Regulation; Glucosides; Humans; Inflammation; Inflammation Mediators; Mitogen-Activated Protein Kinases; NF-kappa B; Phenols; Phorbol Esters; Signal Transduction

Damnacanthal, an anthraquinone obtained from the noni fruit (Morinda citrifolia L.), has been described to possess anti-cancer and anti-inflammatory properties. Since mast cells are key players in various inflammatory conditions as well as in cancer, we considered the possibility that the biological actions of damnacanthal, at least partly, could be due to effects on mast cells. Many of the biological activities of mast cells are mediated by IgE receptor cross-linking, which results in degranulation with release of preformed granule mediators, as well as de novo synthesis and release of additional compounds. Here we show that damnacanthal has profound inhibitory activity on mast cell activation through this pathway. The release of the granule compounds beta-hexosaminidase and tryptase release was completely abrogated by damnacanthal at doses that were non-toxic to mast cells. In addition, damnacanthal inhibited activation-dependent pro-inflammatory gene induction, as well as cytokine/chemokine release in response to mast cell stimulation. The mechanism underlying damnacanthal inhibition was linked to impaired phosphorylation of Syk and Akt. Furthermore, damnacanthal inhibited mast cell activation in response to calcium ionophore A23187. Altogether, the data presented here demonstrate that damnacanthal inhibits mast cell activation induced by different stimuli and open a new window for the use of this compound as a mast cell stabilizer.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents; beta-N-Acetylhexosaminidases; Bone Marrow Cells; Calcimycin; Cell Degranulation; Cytokines; Inflammation; Intracellular Signaling Peptides and Proteins; Mast Cells; Mice; Mice, Inbred C57BL; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Receptors, IgE; Syk Kinase; Tryptases

The aim of the present study was to investigate the biological activity of 20 essential oils (EOs) derived from herbal plants and citrus fruits. The in vitro anti-allergic and anti-inflammatory activities of these oils were investigated, and the EO which was found to have the strongest activity of the 20 EOs examined, was investigated further to identify its components and bioactive compounds. The in vitro anti-allergic activity was determined by measuring the release of β-hexosaminidase from rat basophilic leukemia (RBL-2H3) cells treated with the calcium ionophore, A23187. The in vitro anti-inflammatory activity was determined by measuring the production of tumor necrosis factor-α (TNF-α) in RAW264.7 murine macrophages treated with lipopolysaccharide. Among the EOs examined, lemongrass [Cymbopogon citratus (DC.) Stapf] elicited the strongest anti-allergic and anti-inflammatory effects. A principal component of this EO is citral (3,7-dimethyl-2,6-octadien-1-al) (74.5%), a mixture of the stereoisomers, geranial (trans-citral, 40.16%) and neral (cis-citral, 34.24%), as determined by chromatography-mass spectrometry analysis. The activities of citral and geranial are similar to those of lemongrass EO. These compounds elicited significant in vivo anti-allergic and anti-inflammatory effects, suppressing an immunoglobulin E (IgE)-induced passive cutaneous anaphylactic reaction in mice and a 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, respectively. Our data demonstrate that lemongrass EO and its constituents, citral and geranial, may be a therapeutic candidate for allergic and inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Calcimycin; Cell Line, Tumor; Citrus; Cymbopogon; Immunoglobulin E; Inflammation; Mice; Oils, Volatile; Tumor Necrosis Factor-alpha

Zinc (Zn) is an essential trace metal for eukaryotes. The roles of Zn in the numerous physiological functions have been elucidated. Bamboo salt contains Zn that was shown to have anti-inflammatory effect and other health benefits. Nanoparticles of various types have found application in the biology, medicine, and physics. Here we synthesized tetrapod-like, zinc oxide nanoparticles (ZO-NP; diameter 200 nm, source of Zn) using a radio frequency thermal plasma system and investigated its effects on mast cell-mediated allergic inflammatory reactions. ZO-NP was found to inhibit the productions and mRNA expressions of inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α on the phorbol 12-myristate 13-acetate plus A23187 (PMACI)-stimulated human mast cell line, HMC-1 cells. In these stimulated cells, caspase-1 and nuclear factor-κB activations were abolished by ZO-NP, and the expressions of receptor interacting protein2 (RIP2) and IκB kinaseβ (IKKβ) induced by PAMCI were reduced. On the other hand, ZO-NP alone increased the expressions of RIP2 and IKKβ in normal condition. ZO-NP inhibited the phosphorylation of extracellular signal-regulated protein kinase in the PMACI-stimulated HMC-1 cells. Furthermore, ZO-NP significantly inhibited passive cutaneous anaphylaxis activated by anti-dinitrophenyl IgE. These findings indicate that ZO-NP effectively ameliorates mast cell-mediated allergic inflammatory reaction, and suggest that ZO-NP be considered a potential therapeutic for the treatment of mast cell-mediated allergic diseases.

Topics: Animals; Anti-Inflammatory Agents; Calcimycin; Caspase 1; Cell Line; Cytokines; Enzyme Activation; Gene Expression Regulation; Humans; Hypersensitivity; I-kappa B Kinase; Inflammation; Male; Mast Cells; Mice; Mitogen-Activated Protein Kinases; Nanoparticles; NF-kappa B; Passive Cutaneous Anaphylaxis; Phosphorylation; Proteolysis; Receptor-Interacting Protein Serine-Threonine Kinase 2; RNA, Messenger; Tetradecanoylphorbol Acetate; Zinc Oxide

Mast cells are central players in immediate-type hypersensitvity and inflammatory responses. In the present study, the effects of britanin on the passive cutaneous anaphylaxis (PCA) reaction in mice and on the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced production of pro-inflammatory cytokines in human mast cell line (HMC-1) were evaluated. The oral administration of britanin (10-20 mg/kg) decreased the mast cell-mediated PCA reaction in IgE-sensitized mice. In the activity and mechanism of britanin in vitro assay, britanin suppressed the gene expression and secretion of pro-inflammatory cytokines in a dose-dependent manner in HMC-1. In addition, britanin attenuated PMACI-induced activation of NF-κB as indicated by the inhibition of the degradation of IκBα, nuclear translocation of NF-κB, NF-κB/DNA binding activity assay, and blocked the phosphorylation of p38 MAP kinase, in a dose-dependent manner. We conclude that britanin may have potential as a treatment for allergic-inflammatory diseases.

Topics: Administration, Ophthalmic; Animals; Calcimycin; Calcium Ionophores; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Humans; Hypersensitivity, Immediate; Inflammation; Inflammation Mediators; Inula; Lactones; Male; Mast Cells; Mice, Inbred ICR; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Passive Cutaneous Anaphylaxis; Phosphorylation; Phytotherapy; Sesquiterpenes; Tetradecanoylphorbol Acetate

[6]-Shogaol is a major bioactive component of Zingiber officinale. Although [6]-shogaol has a number of pharmacological activities including antipyretic, analgesic, antitussive and anti-inflammatory effects, the specific mechanisms of its anti-allergic effects have not been studied. In this study, we present the effects of [6]-shogaol on mast cell-mediated allergic reactions in vivo and in vitro. Sprague-Dawley rats received intradermal injections of anti-DNP IgE was injected into dorsal skin sites. After 48 h, [6]-shogaol was administered orally 1 h prior to challenge with DNP-HSA in saline containing 4% Evans blue through the dorsal vein of the penis. In addition, rat peritoneal mast cells (RPMCs) were cultured and purified to investigate histamine release. In vitro, we evaluated the regulatory effects of [6]-shogaol on the level of inflammatory mediators in phorbol 12-myristate 13-acetate plus calcium ionomycin A23187-stimulated human mast cells (HMC-1). [6]-Shogaol reduced the passive cutaneous anaphylaxis reaction compared to the control group, and histamine release decreased significantly following the treatment of RPMCs with [6]-shogaol. In HMC-1 cells, [6]-shogaol inhibited the production of TNF-α, IL-6 and IL-8, as well as the activation of nuclear factor-κB (NF-κB) and phosphorylation of JNK in compound 48/80-induced HMC-1 cells. [6]-shogaol inhibited mast cell-mediated allergic reactions by inhibiting the release of histamine and the production of proinflammatory cytokines with the involvement of regulation of NF-κB and phosphorylation of JNK.

Topics: Animals; Calcimycin; Calcium Ionophores; Carcinogens; Catechols; Cell Line; Cytokines; Histamine Release; Humans; Inflammation; Male; MAP Kinase Kinase 4; Mast Cells; Mutagens; NF-kappa B; Phosphorylation; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate

In this study, the anti-inflammatory and anti-allergic effects of the chloroform-soluble extract of Agaricus blazei in mouse bone marrow-derived mast cells (BMMCs) were investigated. The chloroform-soluble extract inhibited IL-6 production in PMA plus A23187-stimulated BMMCs, and down-regulated the phosphorylation of Akt. In addition, this extract demonstrated inhibition of the degranulation of β-hexosaminidase and the production of IL-6, prostaglandin D(2) and leukotriene C(4) in PMA plus A23187-induced BMMCs. In conclusion, the chloroform-soluble extract of Agaricus blazei exerted anti-inflammatory and anti-allergic activities mediated by influencing IL-6, prostaglandin D(2), leukotriene C(4), and the phosphorylation of Akt.

Topics: Agaricus; Animals; Anti-Allergic Agents; Anti-Inflammatory Agents; beta-N-Acetylhexosaminidases; Biological Products; Bone Marrow Cells; Calcimycin; Cell Degranulation; Down-Regulation; Female; Inflammation; Interleukin-6; Leukotriene C4; Mast Cells; Mice; Mice, Inbred BALB C; Phosphorylation; Phytotherapy; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Tetradecanoylphorbol Acetate

Insamhodo-tang (IHT) has traditionally been used in Korea to treat a variety of diseases, including chronic cough, tuberculosis, and chronic bronchitis. However, the anti-allergic and anti-inflammatory effects of IHT and its molecular mechanisms have yet to be clearly elucidated. In this study, we attempted to evaluate the effects of IHT on mast cell-mediated allergy inflammation in vitro and in vivo.. We investigated to ascertain the pharmacological effects of IHT on both compound 48/80-induced and 2,4-dinitrofluorobenzene (DNFB)-induced allergic reactions under in vivo conditions. Additionally, to find a possible explanation for the anti-inflammatory mechanisms of IHT, we evaluated the regulatory effects of IHT on the level of inflammatory mediators in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cells (HMC-1).. The finding of this study demonstrated that IHT reduced compound 48/80-induced systemic anaphylactic shock, DNFB-induced dermatitis, and ear swelling responses in mice. Additionally, IHT inhibited the production of interleukin (IL)-6, IL-8, and TNF-α, as well as the activation of nuclear factor-κB and caspase-1 in PMACI-stimulated HMC-1.. Collectively, the findings of this study provide us with a novel insight into the pharmacological actions of IHT as a potential molecule for use in the treatment of allergic inflammation diseases.

Topics: Anaphylaxis; Animals; Anti-Inflammatory Agents; Calcimycin; Dermatitis; Dinitrofluorobenzene; Edema; Humans; Hypersensitivity; Inflammation; Inflammation Mediators; Ionophores; Male; Mast Cells; Medicine, Korean Traditional; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; p-Methoxy-N-methylphenethylamine; Phytotherapy; Plant Extracts

Chelidonic acid (CA) is a γ-pyrone which is contained in the rhizome of Chelidonium majus L. It has multiple pharmacological effects including those of a mild analgesic, an antimicrobial, an oncostatic and a central nervous system sedative, but the anti-inflammatory effect of CA and its molecular mechanisms are poorly understood. In this study, we investigated the regulatory mechanism of CA in mast cell-mediated inflammatory response by phorbol 12-myristate 13-acetate and calcium ionophore A23187. The results indicate that CA inhibits the production of interleukin-6 (IL-6) and the expression of IL-6 mRNA through the regulation of nuclear factor-κB. In addition, CA suppresses the activation and expression of caspase-1. These results provide new insights into the pharmacological actions of CA as a potential molecule for use in therapy in mast cell-mediated inflammatory diseases.

Topics: Calcimycin; Calcium Ionophores; Carcinogens; Caspase 1; Caspase Inhibitors; Cell Line; Chelidonium; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Inflammation; Interleukin-6; Mast Cells; NF-kappa B; Pyrans; Rhizome; Tetradecanoylphorbol Acetate

Gomisin N is a bioactive compound and a prominent anti-allergic agent found in the fruits of tree Schizandra chinensis. However, its effects on the bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanism remain unknown. In this study, the biological effects of gomisin were evaluated while focusing on its effects on the allergic mediator in PMA + A23187-stimulated BMMCs. The anti-allergic effect of gomisin has shown that inhibited PMA + A23187-induced interleukin-6 (IL-6) production. An investigation was also conducted to determine its effects on the production of several allergic mediators including prostaglandin D(2) (PGD(2)), leukotriene C(4) (LTC(4)), β-hexosaminidase (β-Hex), and cyclooxygenase-2 (COX-2) protein. The results revealed that gomisin inhibited the PMA + A23187-induced production of IL-6, PGD(2), LTC(4), β-Hex, and COX-2 protein. Taken together, these findings indicate that gomisin N has the potential for use in the treatment of allergy.

Topics: Animals; Anti-Allergic Agents; Bone Marrow Cells; Calcimycin; Calcium Ionophores; Carcinogens; Cells, Cultured; Cyclooctanes; Gene Expression Regulation; Hypersensitivity; Inflammation; Inflammation Mediators; Interleukin-6; Lignans; Male; Mast Cells; Mice; Mice, Inbred BALB C; Polycyclic Compounds; Tetradecanoylphorbol Acetate

Thymic stromal lymphopoietin (TSLP) plays a pivotal role in allergic diseases such as atopic dermatitis, asthma, and chronic obstructive pulmonary disease. Although there are many reports regarding function and regulatory mechanism of TSLP in dendritic cells and/or T cells, the regulatory mechanism of TSLP in mast cells has not been fully elucidated. Here, we describe how TSLP is expressed and produced by inflammatory stimulus in mast cells. TSLP mRNA was expressed by phorbol myristate acetate (PMA) plus A23187 stimulation in HMC-1 cells and reached its peak 5h after PMA plus A23187 stimulation. The expression of TSLP mRNA was inhibited by nuclear factor (NF)-κB inhibitor. In addition, NF-κB luciferase activity was inhibited by caspase-1 inhibitor, indicating that caspase-1 is an upstream of NF-κB in mast cells. Furthermore, caspase-1 inhibitor decreased the expression of TSLP mRNA induced by PMA plus A23187. Finally, TSLP production was inhibited by both caspase-1 inhibitor and NF-κB inhibitor. These results provide proof of principle that TSLP can be expressed and produced through caspase-1 and NF-κB in mast cells and open new perspectives to pharmacologically manipulate the expression and production of TSLP by molecules acting on the caspase-1 and NF-κB pathway.

Topics: Calcimycin; Caspase 1; Cell Line; Cytokines; Gene Expression Regulation; Humans; Inflammation; Ionophores; Mast Cells; NF-kappa B; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymic Stromal Lymphopoietin; Time Factors

Topics: Actins; Calcimycin; Cytokines; Extracellular Matrix; Fibroblasts; Fibrosis; Humans; Inflammation; Mast Cells; Muscle, Smooth; Plasminogen Activator Inhibitor 2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Urokinase-Type Plasminogen Activator

Fritillaria ussuriensis (FU, derived from the bulbs of various species of the genus Fritillaria, including Fritillaria thunbergii Miq.) is used in herbal medicine to treat conditions such as eczema, skin burns, and frostbite. In this study, we investigated the mechanism of the anti-allergy effect of FU. FU extract (80 mg/kg), orally administered to Sprague-Dawley (SD) rats, significantly inhibited the passive cutaneous anaphylaxis (PCA) reaction. It inhibited the compound 48/80-induced release of histamine from rat peritoneal mast cells in a concentration-dependent manner. Significant inhibitory effects of the FU extract on IL-6, IL-8, and TNF-α (1, 10, and 100 µg/mL) were observed in HMC-1 cells. Treatment with FU attenuated PMA plus A23187-induced phosphorylation of all three MAPKs, especially at concentrations of 10 and 100 µg/mL. Further, it (80 mg/kg) led to significant inhibition of mast-cell accumulation in ear tissue at the chronic phase. These results indicate that it inhibits allergic reactions.

Topics: Administration, Oral; Animals; Anti-Allergic Agents; Calcimycin; Fritillaria; Histamine Release; Hypersensitivity; Inflammation; Interleukin-6; Interleukin-8; Male; Mast Cells; Mitogen-Activated Protein Kinase Kinases; p-Methoxy-N-methylphenethylamine; Passive Cutaneous Anaphylaxis; Phosphorylation; Phytotherapy; Plant Extracts; Plant Roots; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Aristolochia tagala Cham. (syn: Aristolochia acuminata Lam.) (Aristolochiaceae), known as Nallayishwari in Telugu, has been of interest to researchers because of its traditional uses for treating rheumatic pains and fever.. The anti-inflammatory activity of the petroleum ether, ethyl acetate, and ethanol extracts of A. tagala roots were investigated for the first time.. In vivo and in vitro anti-inflammatory effects were investigated employing the carrageenan-induced hind paw edema in rats and the macrophage cell line RAW264.7 stimulated with proinflammatory stimuli (lipopolysaccharide interferon γ or the calcium ionophore A23187) to determine PGE(2) or LTB(4) release, respectively.. All the extracts exhibited anti-inflammatory effects which were found to be significant (p < 0.001) at 200 and 400 mg/kg, p.o, in rats tested and the ethyl acetate extract inhibited the induction of PGE(2) with IC(50) = 39.1 mg mL(-1) and LTB(4) with IC(50) = 29.5 mg mL(-1).. These findings demonstrate that the A. tagala roots have excellent anti-inflammatory activity and validate the traditional indications of this plant in its origin country.

Topics: Acetates; Alkanes; Animals; Anti-Inflammatory Agents; Aristolochia; Calcimycin; Calcium Ionophores; Carrageenan; Cell Line; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Ethanol; Female; Inflammation; Interferon-gamma; Leukotriene B4; Macrophages; Male; Mice; Plant Extracts; Plant Roots; Plants, Medicinal; Rats; Rats, Wistar; Time Factors

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Activation of mast cells in rheumatoid synovial tissue has often been associated with tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 production and disease pathogenesis by adjacent cell types. Butea monosperma (BM) is a well known medicinal plant in India and the tropics. The aim of this study was to examine whether a standardized extract of BM flower (BME) could inhibit inflammatory reactions in human mast cells (HMC) using activated HMC-1 cells as a model. Four previously characterized polyphenols--butrin, isobutrin, isocoreopsin, and butein--were isolated from BME by preparative thin layer chromatography, and their purity and molecular weights were determined by liquid chromatography/mass spectrometry analysis. Our results showed that butrin, isobutrin, and butein significantly reduced the phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced inflammatory gene expression and production of TNF-alpha, IL-6, and IL-8 in HMC-1 cells by inhibiting the activation of NF-kappaB. In addition, isobutrin was most potent in suppressing the NF-kappaB p65 activation by inhibiting IkappaBalpha degradation, whereas butrin and butein were relatively less effective. In vitro kinase activity assay revealed that isobutrin was a potent inhibitor of IkappaB kinase complex activity. This is the first report identifying the molecular basis of the reported anti-inflammatory effects of BME and its constituents butrin, isobutrin, and butein. The novel pharmacological actions of these polyphenolic compounds indicate potential therapeutic value for the treatment of inflammatory and other diseases in which activated mast cells play a role.

Topics: Butea; Calcimycin; Chalcones; Chromatography, Thin Layer; Enzyme-Linked Immunosorbent Assay; Flavonoids; Gas Chromatography-Mass Spectrometry; Gene Expression; Humans; I-kappa B Kinase; Inflammation; Interleukin-6; Interleukin-8; Mast Cells; NF-kappa B; Plant Extracts; Plants, Medicinal; Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Motherwort (MW), a Korean folk medicine, has been applied to treat inflammatory disease. However, its effect on inflammatory cytokine release from mast cells is not well known. We investigated the anti- inflammatory effect of MW on the secretion of inflammatory cytokine such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and IL-8 in human mast cell line (HMC-1). MW was treated in vitro before activation of HMC-1 cells with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187. MW had no cytotoxic effects on HMC-1 cell viability. MW (1 mg/ml) inhibited PMA plus A23187-stimulated gene expression and production of TNF-alpha, IL-6, and IL-8. Stimulation with PMA plus A23187 induced NF-kappaB activation in HMC-1 cells, which was inhibited by MW (1 mg/ml). MW inhibited secretion of TNF-alpha, IL-6, and IL-8 possibly by inhibiting NF-kappaB activation. These results indicate that MW may be helpful in regulating inflammatory diseases.

Topics: Anti-Inflammatory Agents; Calcimycin; Humans; Inflammation; Interleukin-6; Interleukin-8; Leonurus; Mast Cells; Medicine, Traditional; NF-kappa B; Phytotherapy; Plant Extracts; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional Chinese medicine with a long history of anti-inflammatory use, but its pharmacological effects have not been thoroughly investigated. This study aimed to evaluate the anti-inflammatory activity of HLJDT in vivo and in vitro.. The carrageenan rat air pouch model was used to investigate the anti-inflammatory action of HLJDT after oral administration. Moreover, we exploited a modified method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique to assay the effects of HLJDT on arachidonic acid metabolites.. Our data demonstrate that oral administration of HLJDT significantly inhibited the inflammatory responses in carrageenan-injected rat air pouches, and also significantly reduced the production of nitric oxide (NO) and leukotriene B(4) (LTB(4)) in vivo, without any influence on biosynthesis of cyclooxygenase (COX)-derived eicosanoids. Similar behaviour of HLJDT was also observed by using calcium ionophore A23187-stimulated peritoneal macrophages, where HLJDT markedly inhibited eicosanoids derived from different lipoxygenases. The NO production and the mRNA expression of inducible nitric oxide synthase (iNOS) and chemotactic factors (CCL3, CCL4, CCL5 and CXCL2) were also inhibited by HLJDT in RAW 264.7 macrophages stimulated by lipopolysaccharide.. Our data revealed, for the first time, that HLJDT could inhibit biosynthesis of eicosanoids derived from different lipoxygenases. Also, HLJDT may exert its anti-inflammatory effects by its suppression on eicosanoid generation, NO production and gene transcription of chemotactic factors, which supports its effectiveness in the treatment of inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Calcimycin; Carrageenan; Cell Line; Chemotactic Factors; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Eicosanoids; Inflammation; Leukotriene B4; Lipopolysaccharides; Lipoxygenase; Macrophages; Metabolic Networks and Pathways; Models, Animal; Nitric Oxide; Nitric Oxide Synthase Type II; Phytotherapy; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Receptors, Eicosanoid; RNA, Messenger; Tandem Mass Spectrometry

The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.

Topics: Air; Animals; Arachidonate 5-Lipoxygenase; Biological Assay; Calcimycin; Carrageenan; Cell Line, Tumor; Chromatography, Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Activators; Enzyme-Linked Immunosorbent Assay; Humans; Hydroxyurea; Imidazoles; Immunoglobulin E; Inflammation; Ionophores; Leukotrienes; Lipoxygenase Inhibitors; Male; Mast Cells; Oxidation-Reduction; Rats; Rats, Inbred Lew; Reproducibility of Results; Sulfides; Tandem Mass Spectrometry

Human bronchial epithelial cells synthesize cyclooxygenase and 15-lipoxygenase products, but the 5-lipoxygenase (5-LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro-inflammatory mediators is thought to be restricted to leucocytes.. We hypothesized that human bronchial epithelial cells (HBECs) express a complete and active 5-LO pathway for the synthesis of LTB4 and LTC4, either constitutively or after stimulation.. Flow cytometry, RT-PCR, Western blotting, enzyme immunoassays and reverse-phase high-performance liquid chromatography were used to investigate constitutive and stimulated expression of 5-LO pathway enzymes and the synthesis of LTs B4 and C4 in primary HBECs and in the 16-HBE 14o- cell line.. Constitutive mRNA and protein expression for 5-LO, 5-LO-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase were demonstrated in primary HBECs and in the 16-HBE 14o- cell line. In 16-HBE 14o- cells, treatment with calcium ionophore A23187, bradykinin or LPS up-regulated the expression of these enzymes. The up-regulation of 5-LO was blocked by the anti-inflammatory glucocorticoid dexamethasone. Human bronchial epithelial cells were shown to generate bioactive LTs, with primary HBECs generating 11-fold more LTC4 and five-fold more LTB4 than 16-HBE 14o- cells. LT production was enhanced by ionophore treatment and blocked by the FLAP inhibitor MK-886.. Expression of an active and inducible 5-LO pathway in HBEC suggests that damaged or inflamed bronchial epithelium may synthesize LTs that contribute directly to bronchoconstriction and leucocytosis in airway inflammation.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Bradykinin; Bronchi; Bronchoconstriction; Bronchoconstrictor Agents; Calcimycin; Carrier Proteins; Cell Line; Epithelial Cells; Gene Expression Regulation, Enzymologic; Glutathione Transferase; Humans; Inflammation; Inflammation Mediators; Ionophores; Leukotriene B4; Leukotriene C4; Lipopolysaccharides; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Up-Regulation; Vasodilator Agents

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 +/- 377 seconds) and arterioles (858 +/- 289 seconds) than in WT vessels (559 +/- 241 seconds, P < .001; 443 +/- 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively.

Topics: ADAMTS13 Protein; Animals; Blood Platelets; Calcimycin; Endothelium, Vascular; Humans; Inflammation; Ionophores; Metalloendopeptidases; Mice; Mice, Knockout; Platelet Adhesiveness; Splanchnic Circulation; Thrombosis; Thrombospondin 1; von Willebrand Factor

The epidemiologic association between pulmonary exposure to ambient particulate matter (PM) and cardiovascular dysfunction is well known, but the systemic mechanisms that drive this effect remain unclear. We have previously shown that acute pulmonary exposure to PM impairs or abolishes endothelium-dependent arteriolar dilation in the rat spinotrapezius muscle. The purpose of this study was to further characterize the effect of pulmonary PM exposure on systemic microvascular function and to identify local inflammatory events that may contribute to these effects. Rats were intratracheally instilled with residual oil fly ash (ROFA) or titanium dioxide at 0.1 or 0.25 mg/rat 24 hr before measurement of pulmonary and systemic microvascular responses. In vivo microscopy of the spinotrapezius muscle was used to study systemic arteriolar responses to intraluminal infusion of the Ca2+ ionophore A23187 or iontophoretic abluminal application of the adrenergic agonist phenylephrine (PHE). Leukocyte rolling and adhesion were quantified in venules paired with the studied arterioles. Histologic techniques were used to assess pulmonary inflammation, characterize the adherence of leukocytes to systemic venules, verify the presence of myeloperoxidase (MPO) in the systemic microvascular wall, and quantify systemic microvascular oxidative stress. In the lungs of rats exposed to ROFA or TiO2, changes in some bronchoalveolar lavage markers of inflammation were noted, but an indication of cellular damage was not found. In rats exposed to 0.1 mg ROFA, focal alveolitis was evident, particularly at sites of particle deposition. Exposure to either ROFA or TiO2 caused a dose-dependent impairment of endothelium-dependent arteriolar dilation. However, exposure to these particles did not affect microvascular constriction in response to PHE. ROFA and TiO2 exposure significantly increased leukocyte rolling and adhesion in paired venules, and these cells were positively identified as polymorphonuclear leukocytes (PMNLs). In ROFA- and TiO2-exposed rats, MPO was found in PMNLs adhering to the systemic microvascular wall. Evidence suggests that some of this MPO had been deposited in the microvascular wall. There was also evidence for oxidative stress in the microvascular wall. These results indicate that after PM exposure, the impairment of endothelium-dependent dilation in the systemic microcirculation coincides with PMNL adhesion, MPO deposition, and local oxidative stress. Collectively, th

Topics: Air Pollutants; Albumins; Animals; Arterioles; Bronchoalveolar Lavage Fluid; Calcimycin; Carbon; Coal Ash; Inflammation; L-Lactate Dehydrogenase; Lung; Macrophages, Alveolar; Male; Neutrophils; Particulate Matter; Peroxidase; Phenylephrine; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Titanium; Vasoconstriction; Vasodilation

To investigate whether FK506 (tacrolimus) can inhibit Fas- or A23187-induced interleukin (IL)-8 expression and cell death in A549 human alveolar epithelial cells, plus Fas-mediated acute lung injury in vivo.. Assays for IL-8, cell death, and caspase-3 activity were performed. A549 cells were treated with 25 micromol A23187 or 0.2 microg/ml agonistic anti-Fas antibody plus 5 ng/ml interferon-gamma (IFN-gamma). Tacrolimus was treated at 0.1-10 ng/ml. For in vivo experiment, agonistic anti-Fas antibody (Jo2) at 2.5 microg/g was intratracheally instilled into C57BL/6 mice. Neutrophils and protein contents in bronchoalveolar lavage (BAL) fluid were measured within 24 h of instillation. Mice were orally treated with 32 mg/kg of tacrolimus 24 h and 1 h prior to instillation.. Both Fas and A23187 caused significant IL-8 expression and cell death in A549 cells. Tacrolimus inhibited A23187-induced IL-8 expression alone while it protected all Fas-mediated responses. Mice instilled intratracheally with Jo2 at 2.5 microg/g had significant increases in neutrophils, protein contents in BAL fluid and in expression of chemoattractants for neutrophils. These increases were reversed by tacrolimus.. Tacrolimus serves as a therapeutic option for improving lung injury through inhibition of Fas-mediated inflammation.

Topics: Animals; Antibodies, Monoclonal; Calcimycin; Caspase 3; Cell Death; Cell Line; Dose-Response Relationship, Drug; fas Receptor; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Humans; Immunosuppressive Agents; Inflammation; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Alveoli; Respiratory Mucosa; RNA, Messenger; Tacrolimus

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

Topics: Adult; Amides; Calcimycin; Cathepsin G; Cathepsins; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Humans; Inflammation; Ionomycin; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplasm Metastasis; Neutrophils; Oligopeptides; Pancreatic Elastase; Phenylalanine; Protease Inhibitors; Proteins; RNA, Messenger; Serine Endopeptidases; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiophenes; Tissue Inhibitor of Metalloproteinase-2; Umbilical Veins

Virgin olive oil (VOO) compared with fish oil (FO) and evening primrose oil (PO) on the ability of stimulated leukocytes to produce inflammatory mediators was investigated in rats. Weaned Wistar rats were fed a basal diet (BD) (2% by weight of corn oil) or diets containing 15% by weight of VOO, PO, or FO. After 8 weeks, glycogen-elicited peritoneal polymorphonuclear leukocytes, mainly neutrophils, were isolated. The calcium-ionophore stimulated neutrophils (2.5 x 10(6) cells/mL) obtained from rats fed the different oils produced a higher release of lysosomal enzymes (beta-glucuronidase, lysozyme, and myeloperoxidase [MPO]) compared with those fed BD. The production of reactive oxygen species (ROS) in response to the stimulant, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), by neutrophils from the VOO group (15.44 nmol of O(2)(-) and 6.56 nmol of H(2)O(2)) was similar to the BD group (12.01 nmol O(2)(-) and 8.49 nmol H(2)O(2)) and significantly lower than the PO (20.90 nmol O(2)(-) and 10.84 nmol H(2)O(2)) and FO (20.93 nmol O(2)(-) and 12.79 nmol H(2)O(2)) groups. The cyclooxygenase-derived eicosanoid production was reduced by the lipid enrichment of the diets. Whereas the generation of prostaglandin E(2) (PGE(2)) was significantly decreased in VOO (5.40 ng/mL), PO (4.95 ng/mL), and FO (1.44 ng/mL) groups compared with BD (8.19 ng/mL), thromboxane B(2) (TXB(2)) reduction was especially significant in neutrophils from the FO diet group (14.67 ng/mL compared with 26.69 ng/mL from BD). These experimental data suggest that FO and PO, as well as VOO, could be considered a valuable strategy in preventing the generation of some inflammatory mediators.

Topics: Animals; Arachidonic Acid; Calcimycin; Dietary Fats, Unsaturated; Dinoprostone; Eicosanoids; Fatty Acids; Fatty Acids, Essential; Fish Oils; gamma-Linolenic Acid; Glucuronidase; Glycogen; Hydrogen Peroxide; Inflammation; Linoleic Acid; Linoleic Acids; Lysosomes; Male; Muramidase; Neutrophils; Oenothera biennis; Oleic Acid; Olive Oil; Peritoneum; Peroxidase; Plant Oils; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxides; Tetradecanoylphorbol Acetate; Thromboxane B2

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 microg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Amino Acid Motifs; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blood Platelets; Calcimycin; Calcium Signaling; Carrier Proteins; Collagen; Cyclooxygenase 1; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Humans; Inflammation; Isoenzymes; Leukotrienes; Membrane Proteins; p38 Mitogen-Activated Protein Kinases; Peptides; Phosphorylation; Platelet Activation; Platelet Endothelial Cell Adhesion Molecule-1; Platelet Membrane Glycoproteins; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Quinolines; Receptors, IgG; Thrombin

Prostaglandins (PGs) and thromboxane (TX) are important mediators of inflammation. Recent studies revealed that PG and TX synthesis is controlled by the regulation of PG- and TX-synthesizing enzymes. In this study, we examined the TX synthesis and the expression of TX-synthesizing enzymes in activated peripheral polymorphonuclear leukocytes (PMNs) obtained from children with bacterial infection. Blood samples were obtained from controls and patients with bacterial infection. A23187-stimulated production of TXB(2), a stable metabolite of TXA(2) in PMNs, was measured by a specific radioimmunoassay. The mRNA expression of cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase (COX)-1, COX-2, and TXA(2) synthase was determined by RT-PCR. The synthesis of TXB(2) in PMNs was significantly increased in the patients [925.0 (550.0-1100.0) pg/10(6) cells], compared with the controls [550.0 (450.0-775.0) pg/10(6) cells]. The mRNA expression for cPLA(2) and COX-2 in PMNs was also enhanced in the patients. The results indicate that TX production in PMNs is significantly increased through possible transcriptional mechanisms of cPLA(2) and COX-2 during bacterial infection in children. The upregulation of TXA(2) synthesis may contribute to the process of acute inflammatory reaction caused by bacterial infection.

Topics: Adolescent; Anti-Bacterial Agents; Bacterial Infections; Calcimycin; Case-Control Studies; Child; Child, Preschool; Cyclooxygenase 1; Cyclooxygenase 2; Cytosol; Female; Humans; Infant; Inflammation; Isoenzymes; Male; Membrane Proteins; Neutrophils; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Thromboxane A2; Thromboxane B2; Up-Regulation

Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A2, the initial rate-limiting step. In this study, ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1beta (IL-1beta), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, ceramide kinase, was activated in response to IL-1beta and A23187. RNA interference targeted to ceramide kinase specifically down-regulated ceramide kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1beta and A23187. Down-regulation of ceramide kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that ceramide kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A2 activation. This study identifies previously unknown roles for ceramide kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.

Topics: Animals; Arachidonic Acid; Calcimycin; Calcium; Cell Line; Ceramides; Cytokines; Cytosol; Dinoprostone; Dose-Response Relationship, Drug; Down-Regulation; Eicosanoids; Enzyme Activation; Humans; Inflammation; Interleukin-1; Ionophores; Lipid Metabolism; Phospholipases A; Phospholipases A2; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Thromboxane B2; Time Factors; Transfection; Tumor Cells, Cultured

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

Topics: Administration, Cutaneous; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Calcimycin; Cell Movement; Chemotaxis, Leukocyte; Croton Oil; Disease Models, Animal; Diterpenes; Female; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; Hypersensitivity, Delayed; Iloprost; Inflammation; Leukotriene B4; Lipoxins; Mice; Phthalic Anhydrides; Skin; Terpenes

Four sesquiterpenes isolated from Jasonia glutinosa D.C. (Asteraceae), namely lucinone, glutinone, 5-epi-kutdtriol and kutdtriol, have been evaluated for their in vitro anti-inflammatory activity in cellular systems generating cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) metabolites. None of the compounds assayed had a significant effect on leukotriene C4 (LTC4)-release from calcium ionophore-stimulated mouse peritoneal cells. However, the release of prostaglandin E2 (PGE2) by mouse peritoneal cells stimulated with calcium ionophore was inhibited by these compounds, although with less potency than the reference drug indomethacin (IC50=0.24 microM). The IC50 values of the active compounds were: lucinone 42.69 microM, glutinone 3.61 microM, 5-epi-kutdtriol 1.28 microM and kutdtriol 39 microM. Of the tested compounds, only glutinone (IC50=24 microM) showed a significant effect on thromboxane B2 (TXB2)-release induced by calcium ionophore in human platelets, although with less potency than the reference drug ibuprofen (IC50=1.27 microM).

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Asteraceae; Blood Platelets; Calcimycin; Cell Survival; Cyclooxygenase 1; Cyclooxygenase Inhibitors; Dinoprostone; Humans; In Vitro Techniques; Inflammation; Isoenzymes; Leukotriene C4; Lipoxygenase Inhibitors; Membrane Proteins; Mice; Peritoneal Cavity; Plant Leaves; Plants, Medicinal; Prostaglandin-Endoperoxide Synthases; Sesquiterpenes; Thromboxane B2

Infection by Helicobacter pylori causes an acute inflammatory response followed by a chronic infection of the human gastric mucosa. A neutrophil-activating protein (HP-NAP) has been identified in H.pylori, and its role in infection and immune response is currently under investigation. Here, we show that HP-NAP induces beta-hexosaminidase release and interleukin-6 production in peritoneal mast cells, two actions which are completely inhibited by pertussis toxin. We also show that in polarized epithelial cell monolayers HP-NAP translocates from the apical to the basolateral domain, where mast cells are located. These findings characterize HP-NAP as an inflammatory factor of H.pylori that is effective from the beginning of the inflammatory cascade.

Topics: Adenocarcinoma; Animals; Bacterial Proteins; beta-N-Acetylhexosaminidases; Calcimycin; Calcium; Cell Polarity; Chemotactic Factors; Colonic Neoplasms; Cytoplasmic Granules; Epithelial Cells; Exocytosis; Helicobacter pylori; Histamine Release; Inflammation; Interleukin-8; Ionophores; Male; Mast Cells; Peritoneal Cavity; Pertussis Toxin; Protein Transport; Rats; Rats, Wistar; Tumor Cells, Cultured; Virulence Factors, Bordetella

From the galls of Pistacia terebinthus we obtained an extract that proved to be effective against chronic and acute inflammation. Now we report on the isolation and identification of three triterpenes: two tirucallane-type lanostanoids and one oleanane, which we have identified as masticadienonic acid (1), masticadienolic acid (2), and morolic acid (3), respectively. All of them showed effectiveness on the mouse ear inflammation induced by repeated applications of 12-O-tetradecanoylphorbol 13-acetate and on the phospholipase A2-induced foot paw edema. The pharmacological activity of the compounds was ratified by a histological study of the ear samples. In addition, they inhibited leukotriene B4 production in rat polymorphonuclear leukocytes stimulated with calcium ionophore A 23187.

Topics: Animals; Anti-Inflammatory Agents; Calcimycin; Cyproheptadine; Dexamethasone; Dose-Response Relationship, Drug; Hindlimb; Inflammation; Ionophores; Leukotriene B4; Magnetic Resonance Spectroscopy; Mice; Neutrophils; Phospholipases A; Phospholipases A2; Pistacia; Plant Extracts; Tetradecanoylphorbol Acetate; Triterpenes

Elevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail.. Overnight stimulation of HUVEC with either LPS or TNFalpha, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 microM camptothecin, documented by 60-70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (approximately 80,000 MP/103 cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3-1 microm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.. Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.

Topics: Annexin A5; Antigens, CD; Apoptosis; Calcimycin; Camptothecin; CD59 Antigens; Cell Extracts; Cell Membrane; Cells, Cultured; Endoglin; Endothelium, Vascular; Humans; Inflammation; Ionophores; Protein Binding; Receptors, Cell Surface; Umbilical Veins; Vascular Cell Adhesion Molecule-1

Chemical examination of the MeOH extract of the root of Taraxacum officinale, which exhibited inhibitory activity on the formation of leukotriene B4 from activated human neutrophils, has resulted in the isolation of 14-O-beta-D-glucosyl-11,13-dihydro-taraxinic acid (1) and 14-O-beta-D-glucosyl-taraxinic acid (2). The absolute stereostructure of 1 has been established by X-ray chrystallographic examination.

Topics: Anti-HIV Agents; Asteraceae; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Crystallography, X-Ray; Dose-Response Relationship, Drug; Glucosides; HeLa Cells; HIV-1; Humans; Inflammation; Japan; Leukotriene B4; Molecular Conformation; Molecular Structure; Neutrophils; Nuclear Magnetic Resonance, Biomolecular; Plant Extracts; Plant Roots; Plants, Medicinal; Sesquiterpenes; Stereoisomerism; Structure-Activity Relationship

We examined the in vivo actions of LY293111 sodium (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]pro poxy]phenoxy] benzoic acid sodium salt). Guinea pigs were used to evaluate the effect of this agent on (1) acute airway obstruction produced by intravenous leukotriene B4, (2) pulmonary granulocyte infiltration and delayed onset airway obstruction resulting from a 4-h leukotriene B4 inhalation and (3) lung inflammation after aerosol challenge with the divalent cationic ionophore A23187 (6S-[6alpha(2S*,3S*),8beta(R*),9beta,11alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)e thyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazole carboxylic acid). Airway obstruction was quantitated using pulmonary gas trapping measurements and lung inflammation was evaluated by bronchoalveolar lavage (BAL) and histology. LY293111 sodium produced a dose-related inhibition of acute leukotriene B4-induced airway obstruction when administered i.v. (ED50=14 microg/kg) or p.o. (ED50=0.4 mg/kg). In contrast, LY293111 sodium did not inhibit the pulmonary gas trapping caused by aerosols of histamine, leukotriene D4, or the thromboxane mimetic U46619 (15 [(S)-hydroxy11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid]). Oral LY293111 sodium inhibited leukotriene B4-induced bronchoalveolar lavage granulocyte infiltration and delayed onset airway obstruction at doses as low as 0.3 mg/kg. In A23187-challenged animals, pulmonary inflammation was markedly inhibited at 1 h, but not 2 h and 4 h post-exposure. We conclude that LY293 11 sodium is a selective leukotriene B4 receptor antagonist with potent pulmonary anti-inflammatory activity.

Topics: Airway Obstruction; Animals; Benzoates; Benzopyrans; Bronchoalveolar Lavage Fluid; Calcimycin; Chemotaxis, Leukocyte; Dinoprostone; Granulocytes; Guinea Pigs; Inflammation; Leukotriene Antagonists; Leukotriene B4; Lung; Male; Receptors, Leukotriene B4; Thromboxane B2

Eosinophil accumulation has been associated with the pathogenesis of numerous allergic inflammatory disorders. Despite the great interest in this response, many aspects of eosinophil accumulation remain unknown. This is particularly true with respect to tissue-specific mechanisms that may regulate the accumulation of eosinophils in different organs. This study addressed this issue by investigating and comparing the roles of alpha(4)-integrins and vascular cell adhesion molecule 1 (VCAM-1) adhesion pathways in interleukin 4 (IL-4)-induced eosinophil accumulation in 2 different rat models of inflammation, namely pleural and cutaneous inflammation. Similar to our previous findings in studies in rat skin, locally administered IL-4 induced a time- and dose-dependent accumulation of eosinophils in rat pleural cavities, a response that was associated with generation of the chemokine eotaxin. The IL-4-induced eosinophil accumulation in skin and pleural cavities was totally inhibited by an antirat alpha(4)-integrins monoclonal antibody (mAb) (TA-2). In contrast, whereas an antirat VCAM-1 mAb (5F10) totally blocked the response in skin, IL-4-induced eosinophil accumulation in rat pleural cavities was not affected by VCAM-1 blockade. A radiolabeled mAb technique demonstrated that endothelial-cell VCAM-1 expression was induced in response to IL-4 in both skin and pleural membrane. The results indicate that although endothelial-cell VCAM-1 is present in skin and pleura, a functional role for it in IL-4-induced eosinophil accumulation was evident only in skin. These findings suggest the existence of tissue-specific adhesive mechanisms in regulating leukocyte migration in vivo and demonstrate a dissociation between VCAM-1 expression and eosinophil accumulation.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Calcimycin; Cell Adhesion Molecules; Cell Movement; Chemokine CCL11; Chemokines, CC; Cytokines; Dose-Response Relationship, Drug; Endothelium; Eosinophils; Inflammation; Integrin alpha4; Interleukin-4; Ligands; Male; Models, Animal; Pleura; Rats; Rats, Sprague-Dawley; Skin; Time Factors; Vascular Cell Adhesion Molecule-1

Prostaglandin (PG) release in cells expressing constitutive cyclooxygenase-1 is known to be regulated by liberation of arachidonic acid by phospholipase A2 followed by metabolism by cyclooxygenase. However, the relative contribution of phospholipase A2 to the release of PGs in cells expressing cyclooxygenase-2 is not clear. We addressed this question by using radioimmunoassay to measure PGE2 release by human cells (A549) induced to express cyclooxygenase-2 (measured by Western blot analysis) by interleukin-1beta. Cells were either unstimulated or stimulated with agents known to activate phospholipase A2 (bradykinin, Des-Arg10-kallidin, or the calcium ionophore A23187) or treated with exogenous arachidonic acid. When cells were treated to express cyclooxygenase-2, the levels of PGE2 released over 15 min were undetectable; however, in the same cells stimulated with bradykinin, A23187, or arachidonic acid, large amounts of prostanoid were produced. Using selective inhibitors/antagonists, we found that the effects of bradykinin were mediated by B2 receptor activation and that prostanoid release was due to cyclooxygenase-2, and not cyclooxygenase-1, activity. In addition, we show that the release of PGE2 stimulated by either bradykinin, A23187, or arachidonic acid was inhibited by the phospholipase A2 inhibitor arachidonate trifluoromethyl ketone. Hence, we have demonstrated that PGE2 is released by two components: induction of cyclooxygenase-2 and supply of substrate, probably via activation of phospholipase A2. This is illustrated in A549 cells by a clear synergy between the cytokine interleukin-1beta and the kinin bradykinin.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Arachidonic Acid; Bradykinin; Calcimycin; Cell Line; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Humans; Inflammation; Interleukin-1; Isoenzymes; Kallidin; Membrane Proteins; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Receptors, Bradykinin

The contribution of platelets to arachidonic acid transcellular metabolism may represent an important pathway of leukotriene (LT) production. The aim of this study was to investigate the role of platelets on LT production in an acute inflammatory model in the rabbit. Preliminary experiments showed that rabbit whole blood (5 ml) stimulated in vitro with the calcium ionophore A23187 produced LTB4 (52.7+/-13.9 ng) and the mixed 5,12-DiHETE (7.25+/-0.75 ng). In A23187-stimulated thrombocytopenic blood, LTB4 was significantly reduced to 19.5+/-8.6 ng and 5,12-DiHETE was undetectable. Peptido-LTs were undetectable in both conditions. In experiments using washed cells, addition of thrombin-activated platelets to fMLP-activated PMN resulted in the appearance of 5,12-DiHETE and in more than twofold increase of LTB4 synthesis. When 3H-arachidonic acid-labelled platelets were mixed with unlabelled PMN and challenged with fMLP and thrombin, radioactive LTB4 and 5,12-DiHETE were produced, indicating that platelet-derived arachidonic acid was utilized by PMN 5-lipoxygenase. Intravenous infusion of fMLP (2.5 nmol/kg/min) in the rabbit induced marked granulocytopenia, thrombocytopenia and increased TxB2 plasma concentrations within 3 min. Electron microscopy of lungs showed morphologically activated and aggregated platelets occluding the capillary lumen. Activation and recruitment of circulating cells was accompanied by the production of LTB4 (peak levels at 1 min: 30.0+/-9.5 ng/ml) and LTE4 (peak levels at 10 minutes: 77.8+/-11.6 ng/ml). The areas under the blood concentration-time curve (AUC, ng min/ml) corresponded to 812+/-182 and 3692+/-658 for LTB4 and LTE4, respectively. In immunologically thrombocytopenic rabbits, the AUC for LTB4 (86.0+/-23.0) and LTE4 (1165+/-542) were both significantly different from controls while in rabbits treated with an anti-leukocyte antiserum, both LTB4 and LTE4 were similar to controls. This experimental model provides in vivo evidence that platelets, involved in an acute inflammatory event contribute to the transcellular production of LTs.

Topics: Animals; Arachidonic Acid; Blood Platelets; Calcimycin; Inflammation; Ionophores; Leukotriene B4; Leukotrienes; Male; Platelet Activation; Rabbits

Recently, the immediate early gene h-sgk was cloned as a hypertonicity-induced gene from human hepatoma cells. The aim of this study was to localize h-sgk messenger RNA (mRNA) expression in normal and inflamed intestinal mucosa and to identify potential transcriptional regulators.. h-sgk mRNA in small intestinal mucosa from healthy persons and patients with Crohn's disease was determined by in situ hybridization. Transcriptional regulation was studied by Northern blot analysis of total RNA isolated from cultured human Intestine 407, U937, and HepG2 cells.. In normal ileum, h-sgk mRNA was selectively localized to the apical villus enterocytes, whereas no staining was detected in crypt cells. In Crohn's disease, enterocytes of the crypts expressed h-sgk and abundant h-sgk positive inflammatory cells appeared in the lamina propria. Combined h-sgk in situ hybridization and immunohistochemical analysis of CD68 antigen expression identified a part of these cells as macrophages. In addition to spatial correlation of transforming growth factor (TGF)-beta1 protein and h-sgk mRNA expression, h-sgk transcription in human Intestine 407 and HepG2 cells as well as in U937 monocytes/macrophages was strongly induced by TGF-beta1 in vitro.. h-sgk expression in normal and inflamed intestinal mucosa may be regulated by TGF-beta1 and may contribute to the pleiotropic actions of TGF-beta1 in mucosal cell populations.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blotting, Northern; Calcimycin; Cells, Cultured; Crohn Disease; Cycloheximide; Gene Expression Regulation; Humans; Ileum; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Inflammation; Interleukin-1; Intestinal Mucosa; Ionophores; Nuclear Proteins; Phorbol Esters; Protein Serine-Threonine Kinases; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; U937 Cells

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.

Topics: Air; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Arachidonic Acids; Caffeic Acids; Calcimycin; Carcinoma, Squamous Cell; Carrageenan; Cell Membrane; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Activation; Enzyme Induction; Epithelial Cells; Genetic Vectors; Humans; Indomethacin; Inflammation; Ionophores; Isoenzymes; Male; Membrane Lipids; Membrane Proteins; Mouth Mucosa; Nucleopolyhedroviruses; Phenylethyl Alcohol; Phospholipids; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Lew; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

The effect of a thrombin receptor agonist peptide (TRAP-6) on the release of nitric oxide (NO) and platelet activating factor (PAF) from resting and calcium-ionophore (A23187)-activated rat peritoneal mast cells (RPMC) was studied using a platelet aggregation bioassay. RPMC spontaneously released NO, which inhibited TRAP-6-, ADP-, and PAF-stimulated platelet aggregation. This effect of NO was abolished by the addition of an NO binding agent, oxyhemoglobin (oxyHb), to the platelet suspension. The RPMC-induced suppression of platelet aggregation was completely inhibited by the NO-synthase inhibitor L-NAME. TRAP-6 and its high affinity analog haTRAP stimulated the rapid release of NO from RPMC. The effect of TRAP-6 was inhibited by pretreatment of the RPMC with L-NAME or with the inhibitor of the constitutive NO-synthase isoform (cNOS) calmidazolium. TRAP-6 inhibited PAF release from A23187-activated RPMC via an NO-dependent mechanism. Platelet aggregation induced by PAF release from activated RPMC was also confirmed in experiments using the PAF receptor antagonist ginkgolide B. Thus, TRAP-6 is a rapidly acting modulator of mast cell reactivity; it stimulates NO release and inhibits PAF secretion.

Topics: Animals; Calcimycin; Inflammation; Mast Cells; Nitric Oxide; Peptide Fragments; Peritoneum; Platelet Activating Factor; Rats; Receptors, Thrombin; Thrombin

Bleomycin and asparaginase are widely used antineoplastic agents which may induce allergic or inflammatory side-effects. Mast cells are implicated as effector cells in allergic and inflammatory responses. The aim of this study was to establish whether bleomycin or asparaginase modulate leukotriene production in vitro and in vivo. Leukotriene C4 (LTC4) production by murine bone marrow-derived mast cells (BMMC) was determined by radioimmunoassay (RIA). Leukotriene production in patients was assessed by determining leukotriene E4 and N-acetyl-leukotriene E4 in urine by means of combined HPLC and RIA. Bleomycin induced an up to 2.1-fold increase in LTC4 production both in unstimulated and in calcium ionophore-stimulated mast cells. In 3 of 7 patients treated with bleomycin, a greater than 2-fold increase in endogenous leukotriene production was observed. This effect was associated with febrile responses and was most pronounced in a patient who developed an Adult Respiratory Distress Syndrome (ARDS). Asparaginase increased leukotriene production up to 10-fold in stimulated but not in unstimulated BMMC. In a patient who developed an anaphylactic reaction after treatment with asparaginase, a pronounced increase in urinary leukotriene concentration was observed. In contrast to bleomycin or asparaginase, a number of other cytostatic agents did not significantly change leukotriene production by BMMC. Our data indicate that some of the inflammatory and allergic side-effects of bleomycin and asparaginase could be mediated by leukotrienes, a possible source of which may be mast cells.

Topics: Adult; Anaphylaxis; Animals; Antineoplastic Agents; Asparaginase; Bleomycin; Calcimycin; Drug Hypersensitivity; Humans; In Vitro Techniques; Inflammation; Ionophores; Leukotriene C4; Leukotriene E4; Leukotrienes; Lymphoma, Non-Hodgkin; Mast Cells; Mice; Mice, Inbred BALB C; Respiratory Distress Syndrome

Muscle fibers are the target of T cell-mediated cytotoxic reactions in polymyositis and inclusion body myositis, while the success of myoblast transplantation depends on the absence of an immune rejection against the myofibers. In order to study the behaviour of muscle cells in an inflammatory milieu, we investigated the production of IL-6 and its modulation, including the second messenger pathways controlling it, in in vitro highly purified human myoblast cultures. We found that IL-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) stimulated myoblast IL-6 secretion in a dose- and time-dependent manner, whereas forskolin and cholera toxin did not. HA1004 at 10 microM did not significantly affect the IL-1beta- and TNF-alpha-induced IL-6 secretion, suggesting that cAMP and protein kinase A are not sufficient to stimulate this process. To investigate the role of protein kinase C (PKC) in this signal transduction, we employed the inhibitor calphostin C, and the activators phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A23187. Calphostin C blocked IL-6 secretion, PMA had a small stimulatory effect and A23187 had no effect; moreover, PKC down-regulation by PMA did not inhibit IL-1beta stimulation, while it reduced TNF-alpha stimulation. These data indicate that different PKC isoforms may be involved in TNF-alpha and IL-1beta signal transduction. Such a difference can distinguish the action of two traditionally 'overlapping' inflammatory cytokines. Our data suggest that muscle cells, like myoblasts, satellite cells and in vivo regenerating myofibers, may discriminate between different stimuli and produce IL-6 when activated in response to muscle injury.

Topics: Adenylyl Cyclases; Calcimycin; Cells, Cultured; Humans; Inflammation; Interleukin-1; Interleukin-6; Muscles; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Surfactin inhibits platelet and spleen cytosolic 100 kDa phospholipase A2 (PLA2). In contrast, this same compound enhances rat platelet group II PLA2 activity by approximately 2-fold and slightly increases group I PLA2 activity from porcine pancreas and Naja naja venom in vitro. Surfactin does not affect a Ca2+ -independent PLA2 partially purified from bovine brain. Thus, this compound inhibits selectively the cytosolic form of PLA2. Based on in vitro studies utilizing preincubation of surfactin with the enzyme, dialysis, and increased concentrations of substrates, the inhibitory effect of surfactin appears to be due to a direct interaction with the enzyme. Linear regression analysis of the linear portion of a concentration-response curve reveals an IC50 of 8.5 microM. To further determine the inhibitory pattern, a Dixon plot was constructed to show that the inhibition by surfactin is competitive, but not uncompetitive, with an inhibition constant of Ki = 4.7 microM in 50 mM Tris-HCl buffer, pH 8.0, at 37 degrees. Surfactin blocked non-stimulated and calcium ionophore A23187-stimulated release of arachidonic acid from monkey kidney CV-1 cells, which contain a cytosolic 100 kDa PLA2 as the major activity, as shown in an anionic exchange DEAE-5PW high performance liquid chromatography profile and western blotting analysis. Surfactin ameliorated inflammation induced by several chemicals. That is, it exhibited in vivo anti-inflammatory activity in several tested inflammatory reactions including 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, carrageenan-induced rat paw edema, and acetic acid-induced mouse writhing. These results demonstrate that surfactin is a selective inhibitor for cytosolic PLA2 and a putative anti-inflammatory agent through the inhibitory effect produced by direct interaction with cytosolic PLA2, and that inhibition of cytosolic PLA2 activity may suppress inflammatory responses.

Topics: Acetic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Bacterial Proteins; Blood Platelets; Blotting, Western; Calcimycin; Cytosol; Edema; Enzyme Inhibitors; Haplorhini; Inflammation; Isoenzymes; Kidney; Lipopeptides; Male; Mice; Pain; Peptides, Cyclic; Phospholipases A; Phospholipases A2; Rats

RANTES (regulated on activation, normal T expressed and secreted) has been shown to possess chemotactic activity for eosinophils. Eosinophils have been considered to play a key role in the allergic inflammation through the release of inflammatory molecules such as radical oxygen products. Thus, in this study, we examined the effect of RANTES on radical oxygen products from eosinophils.. Eosinophils were isolated from heparinized venous blood of patients with bronchial asthma by the modified CD16-negative depletion method. Radical oxygen products were examined in terms of lucigenin-dependent chemiluminescence. To a mixture of 50 microl of eosinophils (2x10(6)/ml) and 50 microl of lucigenin (5x10(-4)M), 50 microl of calcium ionophore A23187 (final concentration 10(-5)M) was added, and radical oxygen products were determined for 600 s.. RANTES treatment resulted in the enhancement of peak value (0.64+/-0.23 RLU) and integrated value (119.08+/-20.52 RLU) as compared to untreated cells (0.15+/-0.03 RLU, 29.48+/-8.92 RLU, respectively). CONCLUSIONS We could conclude that RANTES might play an important role in the pathogenesis of allergic inflammation through involvement in selective eosinophil infiltration and eosinophil activation by augmentation of eosinophil oxidative metabolism.

Topics: Acridines; Asthma; Calcimycin; Chemokine CCL5; Eosinophils; Free Radicals; Humans; In Vitro Techniques; Inflammation; Inflammation Mediators; Ionophores; Luminescent Measurements; Reactive Oxygen Species

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC. The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC "memory" of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

Topics: Calcimycin; Cells, Cultured; Endothelium, Vascular; Golgi Apparatus; Histamine; Humans; Inflammation; Interleukin-1; Interleukin-8; Ionophores; Microscopy, Fluorescence; Microscopy, Immunoelectron; Organelles; Tetradecanoylphorbol Acetate; Umbilical Veins

Through its pro-inflammatory effects on leukocytes, endothelial cells, and keratinocytes, the lipid mediator platelet-activating factor (PAF) has been implicated in cutaneous inflammation. Although the 1-alkyl PAF species has been considered historically the most abundant and important ligand for the PAF receptor (PAF-R), other putative ligands for this receptor have been described including 1-acyl analogs of sn-2 acetyl glycerophosphocholines. Previous bioassays have demonstrated a PAF-like activity in lesions of the autoimmune blistering disease bullous pemphigoid. To assess the actual sn-2 acetyl glycerophosphocholine species that result in this PAF agonistic activity, we measured PAF and related sn-2 acetyl GPCs in fresh blister fluid samples from bullous pemphigoid and noninflammatory (suction-induced) bullae by mass spectrometry. We report the presence of 1-hexadecyl as well as the 1-acyl PAF analog 1-palmitoyl-2-acetyl glycerophosphocholine (PAPC) in inflammatory blister fluid samples. Because PAPC is the most abundant sn-2 acetyl glycerophosphocholine species found in all samples examined, the pharmacological effects of this species with respect to the PAF-R were determined using a model system created by transduction of a PAF-R-negative epidermoid cell line with the PAF-R. Radioligand binding and intracellular calcium mobilization studies indicated that PAPC is approximately 100x less potent than PAF. Though a weak agonist, PAPC could induce PAF biosynthesis and PAF-R desensitization. Finally, intradermal injections of PAF and PAPC into the ventral ears of rats demonstrated that PAPC was 100x less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous diseases.

Topics: Animals; Binding, Competitive; Blister; Blotting, Northern; Calcimycin; Calcium; Dose-Response Relationship, Drug; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycerylphosphorylcholine; Humans; Inflammation; KB Cells; Pemphigoid, Bullous; Phosphatidylcholines; Phospholipid Ethers; Platelet Activating Factor; Rats; Rats, Wistar; Retroviridae; Time Factors; Transduction, Genetic

Rat peritoneal mast cells are stimulated to generate superoxide anion (O2) by the addition of compound 48/80 and A23187. Recently, we demonstrated by immunohistochemical and Western blot analysis that the mast cells contained the p47phox protein, which was one of cytosolic component of the NADPH oxidase system. In the present study, it was demonstrated that the mast cells contained the p47phox mRNA, much similar to that of mouse leukocyte. The permeabilized mast cells were stimulated to generate O2- by the addition of Ca2+, phospholipase A2 (PLA2) and arachidonic acid. Our data suggest the following:(1) cytosolic PLA2 may be activated by the elevation of [Ca2+]i; (2) the conjugation of membrane component with cytosolic component may be stimulated by the released arachidonic acid. The mast cell granules contained superoxide dismutase (SOD)-like enzyme, which degradated O2-, generated in xanthine-xanthinoxidase system. SOD-like enzyme was released from the granules by the treatment with Ca2+ and trapped by the treatment with heparin. In conclusion, our studies suggest that the disorder of the degradation system of O2- may contribute to the development of allergic inflammation.

Topics: Animals; Ascitic Fluid; Calcimycin; Hypersensitivity; Inflammation; Male; Mast Cells; NADPH Oxidases; p-Methoxy-N-methylphenethylamine; Phosphoproteins; Rats; Rats, Wistar; Superoxide Dismutase; Superoxides

We have examined the synthesis of leukotriene C4 from bovine aortic and pulmonary artery endothelium. Under basal conditions, neither aortic nor pulmonary artery endothelium revealed significant amounts of hydroxy fatty acids. Following incubation with ionophore A23187, several peaks including one which co-migrated with authentic LTC4 could be demonstrated from both aortic and pulmonary endothelium. LTC4 production was maximal after 30 min incubation, was inhibitable by the lipoxygenase inhibitor nordihydroguairetic acid, and was synthesized by bovine endothelium from tritiated arachidonic acid substrate. The putative LTC4 from endothelium was shown to be identical to authentic LTC4 by chromatography and scanning UV spectroscopy. Endothelial-derived LTC4 increased the adherence of bovine aortic endothelium for neutrophils in a concentration dependent pattern similar to authentic LTC4. These data suggest that vascular endothelium may influence leukocyte-endothelial interactions through synthesis of biologically active arachidonic acid metabolites such as LTC4.

Topics: Animals; Arachidonic Acid; Calcimycin; Cattle; Cell Adhesion; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelium, Vascular; Humans; Inflammation; Ionophores; Leukotriene C4; Neutrophils; Spectrophotometry

Neutrophils (PMNs) from patients with thermal injury are dysfunctional for the CD11b/CD18-dependent functions of diapedesis, chemotaxis, and phagocytosis. The expression of CD11b/CD18 on normal PMNs is increased after an inflammatory stimulus. We proposed that CD11b/CD18 expression on burn patient PMNs would respond abnormally to inflammatory stimuli. PMNs were obtained from nonseptic burn patients during the second week after thermal injury. PMNs from burn patients incubated with lipopolysaccharide (LPS), N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, or A23187 did not increase the expression of CD11b/CD18 to the same degree exhibited by normal PMNs. This inability to increase CD11b/CD18 was not due to differences in CD14 receptor expression, LPS binding, or factors present in the serum of burn patients. The upregulation of CD35 also was decreased on burn patient PMNs. Western blot analysis revealed decreased quantities of CD11b protein in burn patient PMNs compared with normal control PMNs. The deficiency in CD11b/CD18 expression after inflammatory stimuli may explain some of the abnormalities observed in burn PMN function.

Topics: Burns; Calcimycin; CD18 Antigens; Humans; Inflammation; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophage-1 Antigen; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Antigen; Receptors, Complement 3b; Stimulation, Chemical; Up-Regulation

To define the isoform of phospholipases A2 active in inflammation we evaluated the effects of low-molecular-weight inhibitors of secretory and cytosolic phospholipases A2. We found that inhibitors of cytosolic phospholipase A2 had therapeutic efficacy in an in vivo model of chronic inflammation (rat adjuvant arthritis), whereas inhibitors of secretory phospholipase A2 had no beneficial effect. In vitro, inhibitors of cytosolic phospholipase A2 diminished surface expression of Mac-1 (CD11b/CD18) beta2-integrin on calcium ionophore-stimulated human blood granulocytes and suppressed synthesis of interleukin-1beta in lipopolysaccharide-stimulated human blood monocytes and U937 cells by reducing mRNA levels. Lipid mediators promote Mac-1 exocytosis and transcription of interleukin-1beta, which further enhances cytosolic phospholipase A2 activity and expression. Thus, superinduction of cytosolic phospholipase A2 may establish a positive feedback loop, converting acute inflammation into chronic inflammation. Consequently, inhibitors of cytosolic phospholipase A2 may prevent inflammation in vivo by interfering with cellular activation and infiltration. We conclude that cytosolic phospholipase A2 but not secretory phospholipase A2 is the predominant enzyme in inflammatory signalling.

Topics: Animals; Arthritis, Experimental; Calcimycin; Cell Line; Chronic Disease; Cytokines; Cytosol; Depression, Chemical; Disease Models, Animal; Enzyme Inhibitors; Humans; Inflammation; Integrins; Male; Molecular Weight; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Lew

Topics: Animals; Animals, Newborn; Arachidonic Acid; Bradykinin; Calcimycin; Carcinogens; Cells, Cultured; Cyclooxygenase 1; Cyclooxygenase 2; Inflammation; Isoenzymes; Kinetics; Membrane Proteins; Mice; Mice, Inbred Strains; Phospholipases A; Prostaglandin-Endoperoxide Synthases; Protein Biosynthesis; Proteins; Skin; Tetradecanoylphorbol Acetate

Eosinophils are thought to be one of the pathophysiologically pivotal cells in atopic-type inflammation. In the present experiments, the in vitro responsiveness to stimuli of eosinophils, which had infiltrated into the airway following intravenous administration of Sephadex G-200 (Sephadex), was mainly studied in non-sensitized and [antigen + Al(OH)3]-sensitized guinea pigs. In sensitized, Sephadex-treated guinea pigs, a large number of eosinophils were found in the bronchoalveolar lavage fluid, whereas a much smaller number of cells were recovered in either non-sensitized or sensitized, Sephadex-untreated animals and a smaller number were recovered in non-sensitized Sephadex-treated animals. The eosinophils from non-sensitized Sephadex-treated guinea pigs released superoxide anion (.O2-) and thromboxane (TX) B2 in response to platelet-activating factor (PAF), leukotriene B4 and Ca ionophore A23187. Either spontaneous or PAF-induced .O2- generation from eosinophils of sensitized, Sephadex-treated guinea pigs was significantly greater than that from non-sensitized animals, while TXB2 release stimulated by any of the above stimuli was not further enhanced by sensitization. These results indicate that active sensitization can change some eosinophil functions and that the functionally altered cells could play a pathophysiological role in atopic inflammation.

Topics: Aluminum Hydroxide; Animals; Antigens; Bronchoalveolar Lavage Fluid; Calcimycin; Dextrans; Eosinophils; Gels; Guinea Pigs; Indicators and Reagents; Inflammation; Injections, Intraperitoneal; Injections, Intravenous; Ionophores; Leukocyte Count; Leukotriene B4; Male; Passive Cutaneous Anaphylaxis; Platelet Activating Factor; Respiratory System; Superoxides; Thromboxane B2

To investigate whether modulation of the fatty acid profile can be achieved by the short-term infusion of a fish oil emulsion which may attenuate the pulmonary response to inflammatory stimulation. Changes of fatty acid pattern in-lung tissue and perfusate were analyzed and correlated with physiologic data after a 3-hr infusion of fish oil in comparison with a soybean oil preparation.. Prospective, randomized, controlled trial.. Experimental laboratory in a university teaching hospital.. Forty standard breed rabbits of either gender.. Isolated lungs from anesthetized rabbits were ventilated and recirculation-perfused (200 mL/min) with 200 mL of cell-free buffer solution to which either 2 mL of saline (control, n = 6), 2 mL of a 10% soybean oil preparation (n = 6), or 2 mL of a 10% fish oil emulsion (n = 6) were added. Samples of perfusate and lung tissue were collected for analysis of fatty acid composition. Tissue and perfusate fatty acid composition were analyzed by capillary gas chromatography. To study metabolic alterations in states of inflammatory stimulation, lungs of each group were stimulated with small doses of the calcium ionophore, A23187 (10(-8) M), during the 180-min lipid perfusion period and again after washing out the lipids by exchanging the perfusion fluid. Pulmonary arterial pressure and lung weight gain were monitored, and eicosanoids were analyzed in the perfusate.. Free eicosapentaenoic acids increased several-fold in lung tissue and perfusate during a 3-hr infusion with fish oil. The intravenously administered n-3 fatty acids were rapidly hydrolyzed, as indicated by the appearance of substantial quantities of eicosapentaenoic acid in the perfusate free fatty acid fraction. This increase of perfusion levels of eicosapentaenoic acid was paralleled by an attenuated pressure increase and edema formation due to calcium ionophore challenge and an altered eicosanoid spectrum determined in the perfusate compared with soybean oil-treated lungs.. Short-term n-3 lipid application (fish oil emulsion) exerts anti-inflammatory effects on lung vasculature, which may be due to the metabolism of eicosapentaenoic acid resulting in the generation of less potent inflammatory eicosanoids.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Eicosapentaenoic Acid; Fatty Acids, Omega-3; Female; Fish Oils; Inflammation; Ionophores; Leukotriene C4; Lung; Male; Rabbits; SRS-A

Four coumarins were isolated from the EtOAc extract of the flower-tops of Santolina oblongifolia Boiss. (Compositae). They were identified as 7-methoxycoumarin (herniarin) (1), 6,7-dihydroxycoumarin (aesculetin) (2), 6-methoxy-7-glucosidylcoumarin (scopolin) (3), and 6-hydroxy-7-methoxycoumarin (scopoletin) (4). This is the first report of the isolation of aesculetin and scopolin from the genus Santolina. The isolated coumarins showed marked activity as inhibitors of eicosanoid-release from ionophore-stimulated mouse peritoneal macrophages.

Topics: Animals; Anti-Inflammatory Agents; Calcimycin; Carrageenan; Chromatography, Thin Layer; Coumarins; Dinoprostone; Inflammation; Ionophores; Leukotrienes; Macrophages, Peritoneal; Magnetic Resonance Spectroscopy; Mice; Plant Extracts; Plants, Medicinal; Rats; Rats, Wistar; Spain; Spectrophotometry, Ultraviolet

The amyloidogenic peptides, amyloid-beta (A beta) and human amylin, are the major constituents of amyloid deposits found in patients with the chronic degenerative disorders Alzheimer's disease (AD) and type 2 diabetes, respectively. Recent studies have shown that a variety of inflammatory proteins such as cytokines are associated with the amyloid deposits of AD brain tissues. Therefore, in the present study, we sought to determine whether A beta and/or human amylin could modulate the various inflammatory activities of eosinophils. We observed that human amylin but not A beta peptides inhibited the in vitro interleukin-5 (IL-5)-mediated survival of cord blood-derived eosinophils (CBEs) in a concentration-dependent manner. By contrast, rat amylin, a nonamyloidogenic peptide that is highly homologous to human amylin, failed to affect the IL-5-mediated survival of CBEs. Similar inhibitory effects of human amylin were observed for peripheral blood eosinophils. Human amylin also enhanced the release of the cytokine granulocyte-macrophage colony-stimulating factor by CBEs that were stimulated with the calcium ionophore A23187 but was incapable of directly stimulating CBEs to release cytokines. In addition, the A23187-induced release of the inflammatory lipid mediator leukotriene C4 by CBEs was augmented by human amylin. These results suggest that the amyloidogenic peptide human amylin is capable of amplifying the various inflammatory activities of eosinophils.

Topics: Amyloid; Amyloid beta-Protein Precursor; Animals; Calcimycin; Cell Survival; Cells, Cultured; Eosinophils; Fetal Blood; Humans; Inflammation; Inflammation Mediators; Interleukin-5; Ionophores; Islet Amyloid Polypeptide; Leukotriene C4; Peptide Fragments; Rats

The marine metabolites pacifenol, stypotriol triacetate and epitaondiol were tested for their effects on a number of inflammatory responses. Epitaondiol exhibited a potent topical anti-inflammatory activity related to inhibition of leukocyte accumulation. The other compounds showed a lower potency, similar to that of indomethacin. None of the compounds affected superoxide generation by human neutrophils but pacifenol effectively inhibited the degranulation response. This compound and epitaondiol decreased the release of eicosanoids with a higher potency on the cyclo-oxygenase pathway. Only epitaondiol inhibited human recombinant synovial phospholipase A2 activity in a concentration-dependent manner.

Topics: Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Blood Platelets; Calcimycin; Cytochrome c Group; Ear, External; Edema; Humans; Inflammation; Leukotriene B4; Mice; Neutrophils; Oxidation-Reduction; Phospholipases A; Phospholipases A2; Sesquiterpenes; Steroids; Stimulation, Chemical; Superoxides; Terpenes; Tetradecanoylphorbol Acetate; Thromboxane B2

alpha-naphthylisothiocyanate (ANIT) administration to rats results in periportal hepatic inflammation and injury. Glutathione (GSH) appears to be necessary for the liver injury to occur. The leukotrienes (LTs) are metabolites of arachidonic acid and potent mediators of inflammation that have been implicated in certain liver injury models. Inasmuch as GSH is a cofactor for the synthesis of cysteinyl-LTs and since inflammation is a prominent component of ANIT injury, we hypothesized that LTs are involved in producing the hepatic insult that results from ANIT administration. To test this hypothesis, rats were treated with one of several inhibitors of LT biosynthesis, A63162, Zileuton or MK-886. Each of these agents prevented the formation of LTB4 in Ca++ ionophore-stimulated whole blood from rats treated with the inhibitors. A63162 attenuated the hepatic parenchymal injury caused by ANIT and resulted in a modest decrease in ANIT-induced cholestasis. In contrast, neither Zileuton nor MK-886 attenuated liver injury. AT-125 (Acivicin) inhibits gamma-glutamyl transferase (GGT), the enzyme that catalyzes the formation of LTD4 from LTC4. AT-125 pretreatment did not prevent ANIT-induced hepatic parenchymal insult. It did, however, ameliorate the cholestasis caused by ANIT. In conclusion, the partial protection afforded by A63162 and AT-125 likely results from effects unrelated to the formation of LTs, since Zileuton and MK-886 inhibited LT synthesis without affording protection. The lack of protection by Zileuton and MK-886 in the face of LT synthesis inhibition suggests that LTs are not necessary for the expression of injury after ANIT administration.

Topics: 1-Naphthylisothiocyanate; Acetamides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Glutathione; Hydroxyurea; Indoles; Inflammation; Leukotriene Antagonists; Leukotrienes; Lipoxygenase Inhibitors; Liver; Liver Diseases; Male; Phenyl Ethers; Rats; Rats, Sprague-Dawley

Topics: Animals; Animals, Newborn; Arachidonic Acid; Bronchoalveolar Lavage Fluid; Calcimycin; Cells, Cultured; Culture Media, Conditioned; Epithelium; Inflammation; Macrophages, Alveolar; Rats; Tetradecanoylphorbol Acetate; Trachea

Exocytosis from Weibel-Palade bodies, the secretory granules of vascular endothelial cells, causes the rapid release of von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, and cell surface expression of P-selectin, a membrane protein involved in neutrophil binding. Thus, exocytosis may represent a link between hemostasis and inflammation. We investigated the effect of reactive oxygen intermediates (ROIs) on vWF secretion. Incubation of cultured endothelial cells with xanthine oxidase (XO), which generates superoxide anions (O2-), induces a potent, rapid secretory response. However, vWF release was not observed in response to H2O2. Extracellular, subendothelial vWF deposits typically seen after exocytosis from Weibel-Palade bodies were observed after exposure to XO. XO caused a rapid, sustained increase in intracellular free calcium concentration ([Ca2+]i). vWF secretion was markedly inhibited by BAPTA-AM, a cell-permeant calcium chelator. Removal of extracellular calcium did not inhibit vWF release, although the sustained phase of the [Ca2+]i increase was suppressed. These results suggest that XO-induced vWF release is mediated by the initial increase in [Ca2+]i which is caused by calcium mobilization from intracellular stores rather than by calcium influx. Exocytosis from Weibel-Palade bodies may contribute to the pathogenic effect of ROIs in atherosclerosis and inflammation.

Topics: Calcimycin; Calcium; Cells, Cultured; Chelating Agents; Cytoplasmic Granules; Egtazic Acid; Endothelium, Vascular; Exocytosis; Hemostasis; Humans; Hydrogen Peroxide; Inflammation; Reactive Oxygen Species; Secretory Rate; Signal Transduction; Umbilical Veins; von Willebrand Factor; Xanthine Oxidase

A new water-soluble, orally absorbable de-N-acetyl-lysoganglioside (WILD20), breakdown product of the monosialoganglioside GM1, was found to influence some parameters of neutrophil response to inflammation stimuli. Superoxide anion production appears inhibited, along with neutrophil killing properties. A block of both pathways of arachidonic acid cascade and PAF was also found, as well as neutrophil ICAM-1-mediated adhesion to endothelial cells. Of particular interest was the significant reduction of neutrophils observed at the site of inflammation, whichever agonist was used. The effects on neutrophil physiology found in normal or in pathological conditions, are in favour of a WILD20-related inhibitory effect on neutrophil contribution to inflammation.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Calcimycin; Cell Adhesion; Chemotaxis, Leukocyte; Eicosanoids; Erythrocytes; Gangliosides; Hemolysis; Humans; In Vitro Techniques; Inflammation; Leukocyte Count; Mice; Neutrophils; Phagocytosis; Platelet Activating Factor; Platelet Aggregation Inhibitors; Superoxides

In order to determine whether hydroxyapatite modulates the response of polymorphonuclear leukocyte (PMN) to oxidative stimuli, human PMNs were incubated with a non-activating concentration (1 or 10 micrograms/ml) of hydroxyapatite prior to stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 or 1 microM), phorbol 12-myristate 13-acetate (PMA; 100 pg/ml), sodium fluoride (50 microM), zymosan (1 microgram/ml), or the calcium ionophore A23187 (0.1 microM). Chemiluminescence was measured with an automatic microcomputer-controlled luminescence analyzer at 37 degrees C. Hydroxyapatite alone did not stimulate chemiluminescence at concentrations below 10 micrograms/ml. Levels 300-400% higher than 'stimulus only' controls without preincubation with hydroxyapatite have been recorded. This synergism between hydroxyapatite and subsequent stimuli reveals a new activity of hydroxyapatite and suggests that particulate material may prepare PMNs for an exaggerated inflammatory response to other phlogistic mediators. This is the first report demonstrating PMNs primed with particulate material.

Topics: Calcimycin; Dose-Response Relationship, Drug; Durapatite; Humans; Inflammation; Luminescent Measurements; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Sodium Fluoride; Tetradecanoylphorbol Acetate; Time Factors; Zymosan

The antiinflammatory activity of rolipram, a selective inhibitor of the cyclic AMP-specific phosphodiesterase (PDE IV), was studied. Rolipram did not inhibit 5-lipoxygenase activity but did inhibit human monocyte production of leukotriene B4 (LTB4, IC50 3.5 microM). Likewise, murine mast cell release of leukotriene C4 and histamine was inhibited. In vivo, rolipram inhibited arachidonic acid-induced inflammation in the mouse, while the low Km-cyclic-GMP PDE inhibitor, zaprinast, did not inhibit. Rolipram had a modest effect on LTB4 production in the mouse, but markedly reduced LTB4-induced PMN infiltration. Beta-adrenergic receptor activation of adenylate cyclase was important for rolipram antiinflammatory activity since beta blockade abrogated arachidonic acid-induced inflammation. Thus, the antiinflammatory profile of rolipram is novel and may result from inhibition of PMN function and perhaps vasoactive amine release and leukotriene biosynthesis. These actions may be dependent upon endogenous beta-adrenergic activity and are likely mediated through inhibition of PDE IV.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Calcimycin; Cells, Cultured; Colforsin; Cyclic Nucleotide Phosphodiesterases, Type 4; Ear, External; Eicosanoids; Histamine Release; Humans; Imidazoles; Inflammation; Leukotriene B4; Male; Mast Cells; Mice; Mice, Inbred BALB C; Monocytes; Nadolol; Naproxen; Neutrophils; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrazoles; Pyrrolidinones; Receptors, Adrenergic, beta; Rolipram; SRS-A; Thiazoles

The inhibitory effects of azelastine hydrochloride on PAF-induced and fMLP-induced Ca2+ influx, actin polymerization and calcium ionophore A23187-induced and aggregated IgG-induced release of eosinophil cationic protein (ECP) of an eosinophilic leukaemia cell line, EoL-1, were examined. EoL-1 cells cultured with 0.2 mM dibutyryladenosine-cyclic monophosphate for 48 hours showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) and actin polymerization when stimulated by PAF and fMLP. Azelastine hydrochloride inhibited PAF-induced and fMLP-induced Ca2+ influx ([Ca2+]i) in a dose-dependent manner with an IC50 of 1 x 10(-8) M and 1 x 10(-7) M, respectively. It also inhibited PAF-induced and fMLP-induced actin polymerization in a dose-dependent manner up to 40% and 30%, respectively. EoL-1 cells were differentiated to contain ECP in their eosinophilic granules when cultured for 9 days with supernatants of a human adult T cell leukaemia cell line, HIL-3 (HIL-3 sup). Calcium ionophore A23187 and aggregated IgG induced the secretion of ECP by EoL-1 cells. Azelastine hydrochloride inhibited the secretion of ECP in a dose-dependent manner. These inhibitory effects were seen even at therapeutic concentrations of 10(-8) M to 10(-9) M. These results indicate that the therapeutic effects of azelastine hydrochloride as an anti-allergic agent may include inhibition of the accumulation of eosinophils into the locus of allergic inflammation and of the release of cytotoxic granules from eosinophils.

Topics: Actins; Blood Proteins; Bucladesine; Calcimycin; Calcium-Transporting ATPases; Cell Differentiation; Culture Media; Dose-Response Relationship, Drug; Eosinophil Granule Proteins; Eosinophils; Humans; Hypersensitivity; Immunoglobulin G; Inflammation; Leukemia, Eosinophilic, Acute; Leukemia, T-Cell; Lipoxygenase Inhibitors; N-Formylmethionine Leucyl-Phenylalanine; Phthalazines; Platelet Activating Factor; Polymers; Ribonucleases; Tumor Cells, Cultured

Staphylococcus aureus alpha-toxin is a pore-forming exotoxin that probably represents a significant virulence factor in staphylococcal infections. Previous studies demonstrated that this agent stimulated arachidonate metabolism with subsequent formation of prostacyclin in endothelial cells and leukotriene B4 in granulocytes. We now examined the effect of alpha-toxin on the synthesis of platelet-activating factor (PAF) in cultured porcine pulmonary artery endothelial cells. PAF was labeled by bioincorporation of tritiated acetate and separated/quantitated using thin-layer chromatography, straight-phase-HPLC, or post-HPLC-bioassay. Alpha-toxin induced synthesis of small amounts of PAF in a time- and dose-dependent manner. The maximal amount of PAF elicited by alpha-toxin was approximately 7% of that observed after application of the ionophore A23187. Staphylococcal alpha-toxin but not the ionophore induced a rapid fall in cellular ATP-content. On kinetic grounds, the decrease in ATP-levels did not explain the differences in stimulated PAF-synthesis. Low amounts of PAF induced by the action of alpha-toxin on endothelial cells may contribute to the development of inflammatory lesions in infectious disease.

Topics: Adenosine Triphosphate; Animals; Bacterial Toxins; Calcimycin; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Hemolysin Proteins; Inflammation; Platelet Activating Factor; Pulmonary Artery; Staphylococcus aureus; Swine

Topics: Calcimycin; Dinoprostone; HEPES; Humans; Inflammation; Leukotriene B4; Luminescent Measurements; Neutrophils; Psoriasis; Reference Values; Skin Diseases; Taurine

Intraperitoneal and intra-articular (knee joint) injection of zymosan in the rat caused two phases of increased vascular permeability, a rapid increase (0.25-0.5 h) and a secondary increase (2-3 h) which was temporally associated with the onset of leukocyte infiltration. Intraperitoneal injection of zymosan led to a single peak of eicosanoid production (LTB4, C4, D4, E4 and 6-oxo-PGF1 alpha) which was maximal at 0.125-0.25 h. Intra-articular injection led to an initial peak of LTB4 production (maximal at 0.25 h) and a secondary peak of LTB4 and PGE2 production (maximal at 3 h). Oral administration of the 5-lipoxygenase (5-LO) inhibitors phenidone, BW A4C (N-hydroxy-N-[3-(3-phenoxyphenyl)-2-propenyl] acetamide), A63162 (N-hydroxy-N-[1-(4-(phenylmethoxy) phenyl)ethyl] acetamide and ICI 207 968 (2-[3-pyridylmethyl]-indazolinone inhibited LTB4 production in A23187 stimulation blood ex vivo. The glucocorticosteroid dexamethasone had no effect in this model. The initial phase of increased vascular permeability in the peritoneal cavity and LTB4 production was dose dependently inhibited by the 5-LO inhibitors phenidone, BW A4C, A63162, and ICI 207 968 but not by dexamethasone or colchicine. The initial phase of increased permeability in the joint was unaffected by phenidone, BW A4C, dexamethasone or colchicine. However the latter two drugs inhibited the later phase of increased permeability and leukocyte infiltration in the joint and peritoneal cavity. These results demonstrate that zymosan induces eicosanoid production in vivo but the relative importance of these mediators varies depending on the inflammatory site.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arthritis; Benzeneacetamides; Calcimycin; Colchicine; Dexamethasone; Dinoprostone; Disease Models, Animal; Hydroxamic Acids; Inflammation; Kinetics; Knee Joint; Leukocytes; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Male; Peritonitis; Pyrazoles; Rats; Zymosan

The effects of dietary alpha-linolenate (18:3, n-3) and linoleate (18:2, n-6) on platelet-activating factor (PAF) production were examined. Rats were fed an alpha-linolenic acid-rich (perilla oil) diet or a linoleic acid-rich (safflower oil) diet for 6 wk, and polymorphonuclear leukocytes (PMN) were elicited by peritoneal injection of casein. The overall phospholipid content and composition as well as the subclass distribution of choline and ethanolamine glycerophospholipids in PMN were not altered by these diets. However, with the perilla oil diet their content of a putative precursor of PAF, 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine was approximately 50% of that with safflower oil diet. On exposure to various concentrations of FMLP, PAF formation by PMN in the perilla oil group was less than 50% of that by PMN in the safflower oil group. A larger difference in PAF productions by PMN in the two dietary groups was observed on their stimulation with calcium ionophore A23187. These results demonstrate that PAF production is modulated in some as yet unknown way by changing the alpha-linolenate/linoleate balance of the diet.

Topics: alpha-Linolenic Acid; Animals; Calcimycin; Dietary Fats, Unsaturated; Inflammation; Leukotriene B4; Linolenic Acids; Male; Neutrophils; Phosphatidylcholines; Phosphatidylethanolamines; Platelet Activating Factor; Radioimmunoassay; Rats; Rats, Inbred Strains

The beta 2-integrin CD11a/CD18 binds to the intercellular adhesion molecules (ICAM)-1 (CD54) and ICAM-2. ICAM-1 has a wide distribution, and its expression is up-regulated by various cytokines. In contrast, ICAM-2 has a more restricted distribution, and is mainly expressed on endothelial cells. In the present study we show that it is not induced by inflammatory cytokines or other treatments on any of several cells studied. Moreover, antibodies to the intercellular adhesion ligands were not able to block all CD11a/CD18-dependent adhesion, indicating the presence of additional CD11a/CD18 ligands.

Topics: Antigens, CD; Blotting, Western; Calcimycin; Cell Adhesion; Cell Adhesion Molecules; Cytokines; Endothelium, Vascular; Granulocyte-Macrophage Colony-Stimulating Factor; Hemin; Humans; In Vitro Techniques; Inflammation; Intercellular Adhesion Molecule-1; Leukocytes; Phorbol 12,13-Dibutyrate

RU 41740 (Biostim), a biological response modifier of bacterial origin obtained from the 01.K2 strain of K. pneumoniae, was studied with regard to its effect to modulate the chemiluminescence response, phagocytosis and leukotriene formation from polymorphonuclear leukocytes (PMNs), the histamine release from basophils and the aggregation of human platelets. For histamine release human basophils were analyzed with either the calcium ionophore A23187 or hemolysin producing bacteria. RU 41740 reduced the E. coli K12 pANN5211 induced histamine release whereas the calcium ionophore A23187 induced histamine release was not affected. The studies with regard to adherence and phagocytosis showed a significant increase in phagocytosis for S. aureus in the presence of RU 41740. With regard to the chemiluminescence response of human granulocytes, donor cells were divided into cells with a high and a low response. The chemiluminescence response of high responder cells towards a potent stimulus, e.g. the calcium ionophore A23187, was decreased by RU 41740 whereas weak stimuli (e.g. E. coli K12) were not affected. In contrast to these results the chemiluminescence response of low responder cells to a weak stimulus e.g. E. coli K12 was increased by RU 41740 and the potent stimuli were not affected. RU 41740 also modulates the eicosanoid synthesis of human polymorphonuclear granulocytes. For the calcium ionophore A23187 induced leukotriene production an increase of LTB4 and a decrease of LTC4 by RU 41740 was observed. Furthermore, preincubation of platelets with various concentrations of RU 41740 for 5 min shows an inhibition of platelet aggregation, when an additional stimulation with the calcium ionophore A23187 was carried out.

Topics: Adjuvants, Immunologic; Bacterial Proteins; Basophils; Blood Platelets; Calcimycin; Escherichia coli; Granulocytes; Hemolysin Proteins; Histamine Release; Humans; Immune Adherence Reaction; In Vitro Techniques; Inflammation; Klebsiella pneumoniae; Leukotrienes; Luminescent Measurements; Phagocytosis; Platelet Aggregation; Platelet Aggregation Inhibitors

Topics: Animals; Calcimycin; Cell Differentiation; Dexamethasone; Endotoxins; Fibroblasts; Humans; Inflammation; Interleukins; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; Lipopolysaccharides; Macrophages; Mice; Neoplasm Proteins; Prostaglandin-Endoperoxide Synthases; Tumor Cells, Cultured

Irradiation of whole blood with 137Cs gamma rays intensifies the oxidative burst. Oxidant production was used as an indicator of inflammatory cell reactions and was measured by luminol-amplified chemiluminescence after treatment with inflammatory activators including bacteria, the neutrophil taxin formyl-Met-Leu-Phe, the Ca2+ ionophore A23187, the detergent saponin, and the tumor promoter phorbol ester. The irradiation response is dose-dependent up to about 100 microGy, is detectable within minutes, persists at least 1 h, and is transmitted intercellularly by a soluble mediator. The response is completely inhibited by Ca2+ sequestration in the presence of A23187 or by adenosine, indicating its Ca2+ dependency, and by the phospholipase A2 blocker p-bromphenacyl bromide. However, inhibition by the cyclooxygenase blocker aspirin is sporadic or absent. Blood taken after diagnostic examination of lungs with X rays also exhibited intensified chemiluminescence. These reactions implicate a role for specific amplifying mediator pathways, especially metabolites of the arachidonic acid cascade, in the response: "damage and repair" to cells or DNA plays little or no role. Our results provide evidence for a new mechanism of radiation action with possible consequences for the homeostasis of reactions involving inflammation and second messengers in human health and early development.

Topics: Arachidonic Acid; Blood; Blood Cells; Calcimycin; Dose-Response Relationship, Radiation; Escherichia coli; Gamma Rays; Humans; Inflammation; Luminescent Measurements; Luminol; N-Formylmethionine Leucyl-Phenylalanine; Phospholipases A; Phospholipases A2; Radiation Dosage; Respiratory Burst; Saponins; Tetradecanoylphorbol Acetate

Platelet activating factors (PAFs) are a family of ether lipids with properties that suggest a major role in inflammation. We have previously implicated PAFs in ocular inflammation based on the inhibition of several rabbit models of iritis with a specific PAF receptor antagonist. We have tested ocular tissues for the ability to synthesize PAF. Iris, ciliary body, cornea, and/or retina were carefully dissected from New Zealand white rabbits, and tissue from four eyes was pooled. Tissues were stimulated with calcium ionophore (10 mumol/L), and supernatants were extracted with chloroform-methanol. Platelet-aggregating activity was found in the chloroform phase in 2 of 9, 1 of 8, 0 of 9, and 3 of 9 studies involving iris, retina, ciliary body, or cornea, respectively. Twenty-four hours after the intravitreal injection of 125 ng of endotoxin, aggregating activity was consistently detectable from supernatants of stimulated iris and ciliary body, occasionally present from stimulated retina but not detectable from cornea. The shape of the aggregation curve resembled that produced by 0.5 to 2.0 ng of authentic PAF. Moreover, the aggregation could be completely inhibited by a PAF receptor antagonist and the aggregating activity chromatographed identically on high-performance liquid chromatography to a PAF standard. These studies indicate that PAF-like activity could be detected from several ocular tissues subsequent to inflammation. Iris, ciliary body, retina, vascular endothelium, and/or leukocytes could each contribute to the presence of this inflammatory mediator.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Ciliary Body; Cornea; Endotoxins; Eye; Female; Inflammation; Iris; Male; Platelet Activating Factor; Platelet Aggregation; Rabbits; Retina

The early cellular infiltrate at inflammatory sites consists predominantly of neutrophils and cells of the monocyte/macrophage lineage. The mechanism by which circulating, unsensitized lymphocytes accumulate at sites of inflammation is unknown. The pattern of accumulation of 111indium-labeled circulating thymocytes in response to local injections of the ionophore A23187 was studied and compared with the pattern of (125)iodinated albumin accumulation as a measure of vascular permeability. The kinetics of thymocyte accumulation differed from those of vascular permeability. Sublethal total-body irradiation (750 rads) markedly decreased thymocyte accumulation but had little effect on vascular permeability. Irradiation of the local site alone had no effect. T lymphocyte, T lymphoblast, and platelet accumulation generally followed the same pattern as thymocytes. Intravenous injection of neutrophils, but not platelets, partially restored lymphocyte accumulation in vivo in irradiated mice via a pathway involving the circulating neutrophil, and seemed to be independent of changes in vascular permeability.

Topics: Animals; Blood Platelets; Calcimycin; Capillary Permeability; Female; Indium Radioisotopes; Inflammation; Kinetics; Mice; Mice, Inbred BALB C; Neutrophils; T-Lymphocytes; Time Factors; Whole-Body Irradiation

Neutrophil (PMNL) infiltration is a prominent feature of human psoriasis. Psoriatic skin lesions contain abnormally high amounts of leukotriene B4 (LTB4), itself a potent PMNL chemoattractant both in vivo and in vitro. SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid), an orally active LTB4 receptor antagonist, was tested topically in models of skin inflammation induced by 200 nmol of the calcium ionophore A23187 or 200 micrograms phorbol-12-myristate-13-acetate (PMA) applied topically to the guinea pig ear as assessed by ear weight, levels of the PMNL marker enzyme myeloperoxidase (MPO), and histological examination (PMA model) at 4 and 18 h respectively. When coapplied topically with A23187 or PMA, SC-41930 significantly inhibited epidermal inflammation with ED50 values of 0.6 and 4 mg, respectively. SC-41930 treatment also was associated with lowered dermal LTB4 levels in both models. The PMA-induced skin inflammation model also was assessed histologically and revealed acanthosis, edema, PMNL infiltration, and rete ridge prominence as long as 96 h after a single application that was completely inhibited by SC-41930 topical coapplication. Furthermore, oral treatment (40 mg/kg) significantly reduced edema and inflammatory cell infiltration in both models. These models possess many of the characteristics of human psoriasis, and agents such as SC-41930 that demonstrate activity in these models may well have therapeutic utility in the treatment of human psoriasis.

Topics: Administration, Cutaneous; Animals; Benzopyrans; Calcimycin; Disease Models, Animal; Guinea Pigs; Inflammation; Male; Psoriasis; Receptors, Immunologic; Receptors, Leukotriene B4; Skin; Tetradecanoylphorbol Acetate

Thrombin is a serine protease that plays an essential role in blood coagulation and also induces various responses in endothelial cells. The actions of thrombin on the conversion of fibrinogen to fibrin are inhibited by peptides based on the amino acid sequence of hirudin, a natural anticoagulant from leeches. We show in these studies that the peptides Hir45-64 and sulfated Hir53-64 block the effects of thrombin on endothelial cells. These peptides inhibited, in a concentration-dependent manner, the synthesis of prostaglandin I2 and platelet-activating factor, and the acquisition of an adhesive surface for leukocytes that occur in response to thrombin. These actions of the peptides occurred even though the catalytic site of thrombin was not blocked.

Topics: Calcimycin; Cells, Cultured; Endothelium, Vascular; Epoprostenol; Hirudins; Humans; Inflammation; Kinetics; Peptide Fragments; Platelet Activating Factor; Thrombin

The inflammatory peptide bradykinin stimulated a rapid and transient increase in cytoplasmic [Ca2+] in primary pig chondrocytes, as measured by the fluorescent indicator dye Fura-2. This increase occurred in the absence of extracellular Ca2+, indicating a mobilization from intracellular stores. The elevation in intracellular [Ca2+] was mediated by authentic bradykinin receptors, since it was blocked by the specific bradykinin antagonist [beta-(2-thienyl)-L-Ala5,8,D-Phe7]bradykinin. Activation of chondrocytes by bradykinin induced a concentration-dependent [ED50 (dose for half-maximal response) approximately 40 nM] accumulation of inositol monophosphate in the presence of LiCl and a concentration-dependent increase in production of prostaglandin E2. The generation of the secondary mediator prostaglandin E2 was a biologically relevant output response induced by bradykinin, but chondrocyte responses, such as the rate of entry into DNA synthesis, the rate and pattern of new protein synthesis and the rate of synthesis and resorption of cartilage proteoglycan, were unaltered by bradykinin treatment. Chondrocytes were also shown to be activated by two pharmacological mediators of cytosolic [Ca2+] elevation, i.e. the ionophore A23187 and thapsigargin, which both produced alterations in protein synthesis which were mimicked by bradykinin. Thus Ca2+-sensitive pathways exist which are not functionally responsive to a Ca2+-mobilizing and inositol phosphate-generating hormone, potentially indicating other routes of regulation. These results call attention to bradykinin and related peptides as another class of inflammatory mediators which may regulate physiological and pathological chondrocyte metabolism.

Topics: Animals; Binding Sites; Bradykinin; Calcimycin; Calcium; Cartilage, Articular; Dinoprostone; DNA; Glycosaminoglycans; Inflammation; Inositol Phosphates; Plant Extracts; Protein Biosynthesis; Swine; Thapsigargin

Pre-treatment of the endothelial cells lining the guinea pig inferior vena cava with 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) (10 microM) in vitro significantly reduced the shape changes resulting from subsequent exposure to platelet activating factor (PAF) (0.1 microM), calcium ionophore A23187 (10 microM), histamine (300 microM), bradykinin (2 microM), prostaglandin E2 (PGE2) (30 microM), or leukotrienes (LT) C4, D4 or E4 (1 microM). Since TMB-8 is an intracellular calcium antagonist, this provides evidence to support the suggestion that these inflammatory agents increase the concentration of intracellular calcium which brings about a contraction of the actin-myosin complex resulting in endothelial cell shape changes, and the formation of interendothelial cell gaps.

Topics: Animals; Bradykinin; Calcimycin; Calcium Channel Blockers; Dinoprostone; Endothelium, Vascular; Gallic Acid; Guinea Pigs; Histamine; In Vitro Techniques; Inflammation; Leukotrienes; Platelet Activating Factor; Vena Cava, Inferior

Topics: Adolescent; Adult; Asthma; Calcimycin; Calcium; Humans; Inflammation; Influenza A virus; Influenza, Human; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Respiratory Syncytial Viruses; Respirovirus Infections; Superoxides

In polymorphonuclear neutrophils, phospholipase A2 activity is the rate-limiting step for platelet-activating factor (PAF) formation and for the biosynthesis of arachidonic acid derivatives, leukotrienes and prostaglandins. Glucocorticosteroids inhibit phospholipase A2 activity by inducing in target cells the synthesis and release of phospholipase A2 inhibitory proteins named 'lipocortins'. Here, we report that rat pleural inflammatory polymorphonuclear neutrophils, treated with dexamethasone, decrease their production of eicosanoids and PAF. Evidence is presented which may implicate lipocortin 's' in these inhibitions since (i) phospholipase A2 inhibitory proteins are found in the supernatant of dexamethasone-treated cells, (ii) this supernatant inhibits the formation of lipid mediators in untreated cells, inhibition being reversed either by incubating the supernatant with a monoclonal antibody against rat lipocortin or by boiling it and (iii) a 36 kDa lipocortin from mice lungs mimics the effects of dexamethasone when added exogenously on untreated cells. Our results favour the hypothesis that the newly formed lipocortin 's' could be responsible for the antiphospholipase A2 activity of glucocorticosteroids.

Topics: Annexins; Calcimycin; Dexamethasone; Dinoprostone; Glycoproteins; Humans; Inflammation; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor

To assess the relative contribution of different leucocyte subpopulations to LTB4 production, peripheral blood leucocytes from human donors were separated into polymorphonuclear neutrophils (PMN) and lymphocytes/monocytes (L/M) and were then stimulated in vitro with the Ca-ionophore A 23187 for different times. The supernatants were analysed for their contents of leukotriene B4 (LTB4) and its omega-metabolites by HPLC-analysis and column fractions were also examined for their chemotactic activities towards eosinophils in vitro. PMN supernatants contained greater quantities of LTB4, 20-OH-LTB4, 20-COOH-LTB4, and chemotactic activities than did L/M supernatants. On the other hand, the time dependent decrease of LTB4 and chemotactic activity and the increase of omega-metabolites were higher in PMN than in L/M. These results would correlate with the greater role of PMN in acute and that of monocytes in chronic inflammation.

Topics: Calcimycin; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Humans; In Vitro Techniques; Inflammation; Leukotriene B4; Lymphocytes; Monocytes; Neutrophils; Time Factors

Leukotrienes are potent mediators of allergy and inflammation. Polymorphonuclear granulocytes which participate in acute and chronic disease processes can be activated by immunological and nonimmunological stimuli to generate leukotrienes. It appears that on activation of the cells 5-lipoxygenase is released into the supernatant and thus may exert a potent role with regard to interdependent cellular interactions. The release of leukotriene-metabolizing enzymes (e.g., gamma-glutamyl-transpeptidase, dipeptidase) is stimulus-dependent. Polymorphonuclear granulocytes alter their release profile once the cells have been prestimulated. Thus, a precise knowledge on the leukotriene-inducing and -metabolizing enzymes is of utmost importance for future immunopharmacological interventions. It appears that the enzyme activity reflects the state of cellular activation.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Calcimycin; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Dipeptidases; Enzyme Activation; Exotoxins; gamma-Glutamyltransferase; Humans; Inflammation; Leukotriene B4; Mixed Function Oxygenases; Neutrophils; SRS-A

Rat leukocytes from inflammatory peritoneal exudates respond in vitro to a variety of chemotactic and phagocytic stimuli by releasing both elastase and cathepsin G neutral proteinase enzyme activities. PAF, FMLP, and PMA stimulated a rapid, cytochalasin B-dependent, dose-related release of both enzymes; however, leukotriene B4 was inactive. It was not possible to measure the activity of zymosan-activated serum on these cells as rat serum contains high levels of proteinase inhibitors. The calcium ionophore A23187 stimulated a dose-related, time-dependent, cytochalasin B-independent enzyme release. Concanavalin A stimulated a weak, nondose-related release of enzyme activity. Zymosan and serum-coated zymosan stimulated enzyme secretion which was markedly inhibited by the presence of cytochalasin B. These data indicate that release of azurophillic granule neutral proteinases from rat inflammatory leukocytes can be detected and quantitated in vitro. This model could provide a test system for monitoring the pattern and specificity of enzyme release from azurophil granules. The ability of a variety of stimuli to induce proteolytic enzyme release from inflammatory neutrophils may be of considerable relevance to chronic inflammatory diseases.

Topics: Animals; Calcimycin; Cathepsin G; Cathepsins; Cytoplasmic Granules; Dose-Response Relationship, Drug; Inflammation; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Peritoneal Cavity; Platelet Activating Factor; Rats; Rats, Inbred Strains; Serine Endopeptidases

Stimulation of rat astrocytes in vitro by calcium ionophore A23187 and/or lipopolysaccharide results in the generation of a cytotoxic factor that is functionally similar to the previously described macrophage-derived cytotoxic factor, tumor necrosis factor. Like the macrophage product, the astrocyte cytotoxic factor kills murine L 929 cell targets. In addition, it kills rat oligodendrocytes, the myelin-producing cells of the central nervous system. Human recombinant tumor necrosis factor also has cytotoxic activity directed against rat oligodendrocytes.

Topics: Animals; Astrocytes; Calcimycin; Cytotoxins; Demyelinating Diseases; Inflammation; Lipopolysaccharides; Neuroglia; Oligodendroglia; Rabbits; Rats; Recombinant Proteins; Tumor Necrosis Factor-alpha

The subcutaneous sponge implant model of acute inflammation in the rat has been evaluated as a suitable test system for evaluating the potential anti-inflammatory efficacy of 5-lipoxygenase inhibitors. The inflammatory parameters measured were exudate volume and leukocyte recruitment. Specific radioimmunoassays were used to measure (1) 5-lipoxygenase (LPO) and cyclo-oxygenase (CO) activity in exudate leukocytes stimulated ex vivo with A23187, and (2) the LTB4 and PGE2 content of inflammatory exudate. The NSAIDs flurbiprofen and indomethacin inhibited cell recruitment, exudate volume and CO activity with ED50S of approximately 1 mg per kg p.o. but failed to inhibit LPO activity at 10 mg per kg p.o. Nafazatrom (Bayer 6575), quercetin and NDGA, which inhibit LPO activity in vitro, were inactive against all parameters when dosed at 100 mg per kg p.o. The "mixed inhibitors" BW755C and phenidone were approximately equipotent inhibitors of LPO activity but BW755C was 10 times more potent than phenidone against CO activity. BW755C was also greater than 10 times more potent at inhibiting cell recruitment and exudate volume than phenidone suggesting that the anti-inflammatory efficacy of the mixed inhibitors reflect their potency against CO rather than LPO activity. Time course studies demonstrated that the inhibitor effects of BW755C and phenidone on leukocyte recruitment reflected a reduction in the PGE2 but not the LTB4 content of the inflammatory exudate. Polyester sponges soaked in high concentrations of LTB4 caused only a modest (2-fold) increase in leukocyte recruitment whilst physiological levels were inactive. The results taken together suggest that CO products make a major contribution to leukocyte recruitment in this model whilst the LPO product LTB4 has little role. This model therefore is of little value for evaluating the anti-inflammatory efficacy of 5-lipoxygenase inhibitors. Moreover, the rat would appear to be unsuitable for evaluating the role of LTB4 in acute inflammation.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Acute Disease; Animals; Blood Proteins; Calcimycin; Chemotaxis, Leukocyte; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Flurbiprofen; Indomethacin; Inflammation; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Male; Prostaglandins E; Pyrazoles; Pyrazolones; Rats; Rats, Inbred Strains; Skin

Arachidonic acid was converted to a series of hydroxyeicosatetraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored 'on line' by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode (M - H-:m/z 319) for each of the isomeric HETEs. The (M + H+ - H2O) ion at m/z 303 is the major species in the positive ion spectra of HETEs. Mass spectra were obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.

Topics: Arachidonic Acid; Arachidonic Acids; Ascitic Fluid; Calcimycin; Chromatography, High Pressure Liquid; Cyclosporins; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Inflammation

The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell-bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes.

Topics: Animals; Arachidonic Acids; Calcimycin; Humans; Inflammation; Leukotriene A4; Leukotriene B4; Lipoxygenase; Mast Cells; Mice; Neutrophils; Phospholipases; SRS-A

Rat neutrophils isolated from 4-h reverse passive Arthus reaction (RPAR) pleural exudates actively metabolize arachidonic acid via cyclooxygenase and lipoxygenase. Utilizing this system, the effect of oral doses of nonsteroidal antiinflammatory drugs on the ability of these cells to produce HHT, 5-HETE, and LTB from exogenously added arachidonic acid has been investigated. In vitro and ex vivo, indomethacin and timegadine inhibit cyclooxygenase activity in rat pleural neutrophils. In vitro, timegadine is a lipoxygenase as well as a cyclooxygenase inhibitor. This dual inhibition is confirmed by the observation that ex vivo timegadine inhibits the production of lipoxygenase as well as cyclooxygenase metabolites. While indomethacin, a cyclooxygenase inhibitor, primarily inhibits edema formation, the inhibition of both pathways of arachidonic acid metabolism by timegadine is reflected in the drug's ability to reduce cellular influx as well as edema formation in the RPAR pleural cavity inflammatory reaction.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Arthus Reaction; Calcimycin; Edema; Guanidines; Indomethacin; Inflammation; Leukocyte Count; Male; Neutrophils; Rats; Rats, Inbred Lew; Rats, Inbred Strains

We have studied Microciona prolifera cells as a model for inflammation and secretion. Dissociated in Ca-, Mg-free seawater with 2.5 mM EDTA, the cells aggregate when exposed to Ca (greater than 5 mM) and Ca ionophores. Extracellular Ca is not required over the course of aggregation; brief pulses of Ca suffice. Aggregation was induced by A23187 in excess EDTA after cells were prepared by pulse Ca. It appeared that Ca ionophore stimulated the secretion of Microciona aggregation factor (MAF) to a locus or in a form inaccessible to external EDTA. Pulse-induced aggregation depended on MAF because it was inhibited by MAF fragments, which are ligands for MAF-binding sites. Sponge cells were preloaded with three fluorescent dyes that monitor aspects of stimulus-secretion coupling: 1) 3,3'-dipropylthiadicarbocyanine iodide (dis-C3-(5)), a carbocyanine dye presumed to report changes in membrane potential; 2) 9-aminoacridine (9AA), which presumably reports secretion from acid vesicles; and 3) chlortetracycline (CTC), presumed to report mobilization of membrane-associated Ca. Exposure of cells either to constant Ca or to pulse Ca stimuli caused prompt decreases in the fluorescence of cells with diS-C3-(5) and increases in fluorescence of cells with 9AA. In contrast, although constant Ca provoked decreases in fluorescence of cells with CTC, a pulse Ca was without effect. Moreover, inhibitors of stimulus-response coupling (e.g., aspirin, sodium salicylate, 5 mM; diclofenac, 100 microM) inhibited sponge aggregation induced by either constant or pulse stimuli. In contrast, like the endogenous mediator of inflammation, leukotriene B4, trienoic alkyl catechols (urushiol) from poison ivy provoked aggregation. These studies suggest the utility of this marine model for analysis of stimulus-response coupling in cells of higher species that also respond to secretagogues in the absence of external Ca.

Topics: Aminacrine; Animals; Anti-Inflammatory Agents; Benzothiazoles; Calcimycin; Calcium; Carbocyanines; Catechols; Cell Adhesion Molecules; Cell Aggregation; Chlortetracycline; Fluorescence; Fluorescent Dyes; Inflammation; Porifera; Potassium; Proteins

Leukocyte numbers and Leukotriene B4- (LTB4-) and LTC4-immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containing 1% uric acid, carrageenan or zymosan were implanted for 5h and compared to saline sponges. Increases in leukocyte numbers and LTB4-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB4 was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltration was found to coincide with the maximal levels of LTB4-immunoreactivity. The LTC4-immunoreactivity was low and following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB4-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionophore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with indomethacin; 3 and 10 mg/kg p.o.). Impregnation of sponges with 3 X 10(-6)M LTB4 but not 3 X 10(-5) and 3 X 10(-7)M LTB4 induced a significant leukocyte migration. It was concluded that LTB4 can induce leukocyte migration into sponge exudates in the rat but that measurements of LTB4 in such exudates can not be correlated with the degree of leukocyte infiltration.

Topics: Animals; Antibody Specificity; Calcimycin; Carrageenan; Chromatography, High Pressure Liquid; Exudates and Transudates; Indomethacin; Inflammation; Leukocyte Count; Leukotriene B4; Neutrophils; Rats; SRS-A; Zymosan

Leukotriene B4 (LTB4) has been detected by radioimmunoassay in inflammatory exudates obtained following the implantation of saline- or carrageenan-soaked polyester sponges in rats. The immunoreactive material was confirmed as LTB4 after extraction and purification by high pressure liquid chromatography. The peak concentration (6.9 +/- 0.5 ng/ml) was detected 6 hr after implantation of sponges soaked in 0.5% carrageenan; thereafter the level declined and was undetectable after 16-24 hr. The concentration of LTB4 during the early phase of the inflammatory response (4-8 hr) is sufficient to induce leukocyte aggregation, chemotaxis and degranulation of polymorphonuclear leukocytes (PMN) in vitro. Therefore, LTB4 may mediate, at least in part, the influx of PMN and contribute to other events which characterise the inflammatory response. The level of thromboxane B2 (TXB2) in the inflammatory exudate followed a similar time-course to that of LTB4 although the maximum concentration was higher (15-30 ng/ml). However, prostaglandin E2 (PGE2) exhibited a different time-course; the maximum level (20-30 ng/ml) was also reached 6-8 hr after implantation but remained elevated at 24 hr. The PMN count in the sponges and the concentrations of both LTB4 and TXB2, but not PGE2, were significantly reduced by prior treatment of the animals with colchicine. This suggests that PMN are the major source of LTB4 and TXB2 in the inflammatory exudate whereas PGE2 is produced in significant amounts by other tissues.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Colchicine; Dinoprostone; Inflammation; Leukocyte Count; Leukotriene B4; Male; Prostaglandins E; Rats; Rats, Inbred Strains; Thromboxane B2

Synthetic 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) and 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF) have previously been shown to induce chemotaxis and chemokinesis of human neutrophils. We present here data showing that these agents are inactive by themselves, but that they enhance neutrophil secretion once it has been initiated by a calcium ionophore or by zymosan. Two substances, the lipid eosinophil chemotactic factor (ECF) and the lysosomal enzyme beta-glucuronidase, are used as markers for neutrophil release. PAF augments secretion of both substances in a dose-dependent fashion, with lyso-PAF being less potent. The kinetics of enhancement are very rapid (less than 2 min) and are not reversible by washing of the cells. A pyrazoline derivative that inhibits arachidonate cyclo-oxygenation and lipoxygenation, reduces the enhancing effect of PAF and lyso-PAF. PAF, and less so lyso-PAF, are thus potentially important modulators of neutrophil secretion during inflammatory processes.

Topics: Calcimycin; Chemotactic Factors; Chemotactic Factors, Eosinophil; Chemotaxis, Leukocyte; Complement System Proteins; Drug Synergism; Eosinophils; Glucuronidase; Humans; Inflammation; Neutrophils; Platelet Activating Factor; Zymosan

Topics: Animals; Blood Bactericidal Activity; Calcimycin; Complement C3b; Cytotoxicity, Immunologic; Dinoprostone; Humans; Inflammation; Macrophages; Phagocytosis; Prostaglandins E

When applied to mouse skin in vivo, both the strong tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 nmol) and the divalent cation ionophore A 23187 (200 nmol) caused the same responses, i.e., skin inflammation and prostaglandin E2-mediated epidermal hyperplasia. In both cases, these events were accompanied by certain biochemical reactions in the epidermis such as an increase in the biosynthesis of and sensitivity to prostaglandin E2, increase in ornithine decarboxylase and phosphodiesterase activities, and refractoriness of cyclic adenosine 3':5'-monophosphate production to beta-adrenergic stimulation. In contrast to A 23187, TPA did not induce degranulation of mast cells; whereas, in contrast with TPA, A 23187 did not show tumor-promoting activity. These results indicate that the observed biological effects of TPA are no indication of tumor-promoting ability and that, on the other hand, the mitogenic effects of A 23187 are possibly not due to its properties as a calcium ionophore.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Catecholamines; Cyclic AMP; Enzyme Induction; Epidermal Cells; Epidermis; Inflammation; Mast Cells; Mice; Mitosis; Phorbols; Prostaglandins E; Stimulation, Chemical; Tetradecanoylphorbol Acetate

Human, monkey and rat alveolar macrophages (AM) release PAF-acether in a dose-dependent fashion in the presence of 1 to 5 microgram/ml ionophore A 23187 (2.5 pmol of PAF-acether from 2.5 x 10(5) cells) but not in the presence of zymosan. Arachidonic acid (AA) metabolites released from AM from these species were studied. Thromboxane A2 TxA2) - detected by its action on rabbit arteries - was released from human, monkey and rat AM upon addition of 0.5 mM AA. This release was inhibited by aspirin and indomethacin. Lipoxygenase and cyclooxygenase AA metabolites from rat AM were identified using high efficiency glass capillary column gas chromatography coupled to mass spectrometry. The cyclooxygenase metabolites PGF2 alpha, E2 and D2 and TxB2 were identified. The lipoxygenase-dependent AA metabolites were explored using aspirin-pretreated AM. Only 12 HETE was found. These data indicate that AM secrete several substances with bronchoconstrictive activity: PGF2 alpha, D2, TxA2 and PAF-acether. Therefore an active role of AM in human and experimental bronchoconstriction must be considered.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Humans; In Vitro Techniques; Inflammation; Lysophosphatidylcholines; Macrophages; Papio; Platelet Activating Factor; Pulmonary Alveoli; Rats

Topics: Animals; Arachidonic Acids; Calcimycin; Chemotactic Factors; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Guinea Pigs; Inflammation; Leukocyte Count; Leukotriene B4; Neutrophils; Rats

Arachidonic acid is metabolised either by cyclooxygenases to produce prostaglandins and thromboxanes or by lipoxygenases to produce mono-, di- and trihydroxyeicosatetraenoic acids (HETEs). Polymorphonuclear leukocytes (PMNs) release HETEs, including mono- and dihydroxy fatty acids, when exposed to stimuli such as the calcium ionophore A23187 (refs 1, 2). The mono-HETEs are assumed to be of particular importance with respect to effects on leukocyte function because they have been shown to possess both chemotactic and chemokinetic activities towards PMNs and eosinophils. However, we have now shown that the chemokinetic and aggregating activities released from rat and human PMNs exposed to ionophore A23187 (ref. 5) are not due to the release of mono-HETEs but to that of 5, 12-di-HETE (leukotriene B). This compound is active over the concentration range 10 pg ml-1 to 5 ng ml-1.

Topics: Animals; Arachidonic Acids; Calcimycin; Cell Aggregation; Chemotaxis, Leukocyte; Humans; Hydroxy Acids; Inflammation; Leukotriene B4; Lipoxygenase; Neutrophils; Rats

Topics: Calcimycin; Chemotaxis; Eosinophils; Humans; Inflammation; Neutrophils; Phagocytosis