leukotriene-b4 and Granuloma

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Substance P (SP) is an important neuropeptide involved in neurogenic inflammation and most of its pathophysiological functions are mediated through binding to the neurokinin-1 receptor. SP exerts various proinflammatory actions on immune-cells, including macrophages. Several compounds such as cytokines have the capacity to activate and stimulate macrophages to produce arachidonic acid oxygenation and lipoxygenation products. Leukotriene B4 (LTB4) is one of the most important mediators of leukocyte activation in acute and chronic inflammatory reactions. LTB4 stimulates chemotaxis, lysosomal enzyme release, and cell aggregation. In this report, we studied the effect of SP on rat adherent granuloma macrophages (RAGMs). The chronic granuloma in rat was induced by dorsal injections of a potassium permanganate (KMnO4) saturated crystal solution (200 microl of a 1:40 dilution). After 7 days, all rats developed a subcutaneous granuloma in the injection site from which infiltrated macrophages were extracted, isolated, and cultured in vitro. We tested the hypothesis that SP stimulates the production of LTB4 in RAGMs and increases lipoxygenase expression. Here we show that the cell-free supernatant of RAGMs stimulated with SP (10 microM), resulted in statistically significant increases of LTB4 Preincubation of RAGMs with NDGA (nordihydroguaiaretic acid (10 microM), completely abolished the production of LTB(4) in the supernatants and lipoxygenase expression on RAGMs challenged with SP, or the cation ionophore A23187 (positive control). Similar effects were obtained when the cells were pretreated with dexamethasone (10 microM). Our results suggest that SP is able to stimulate the release of LTB4 and lipoxygenase expression in macrophages from chronic inflammatory granuloma and provide further evidence for a neuroinflammatory pathway.

Topics: Animals; Arachidonate 5-Lipoxygenase; Calcimycin; Dexamethasone; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Granuloma; Ionophores; Leukotriene B4; Lipoxygenase Inhibitors; Macrophages; Male; Masoprocol; Microscopy, Electron, Transmission; Potassium Permanganate; Radioimmunoassay; Rats; Rats, Wistar; Substance P; Time Factors; Up-Regulation

The activation of monocytes/macrophages by several stimuli is an initial event in the inflammatory response. To ascertain the importance of LTB(4) and 5-lypoxigenase in the inflammatory site, we isolated and stimulated rat adherent granuloma macrophages (RAGMs) with calcium ionophore in the presence or absence of regulated on activation, normal T expressed and secreted (RANTES) [CCL5] at different concentrations. We tested the hypothesis that RANTES may influence the production of LTB(4) stimulated by calcium ionophore A23187 (2.5 microM/ml) in rat adherent granuloma macrophages derived from granuloma induced by potassium permanganate diluted 1:40 saturated solution. To test this hypothesis, we measured LTB(4) production, in rat granuloma macrophages stimulated with A23187 (2.5 microM) alone and in combination with RANTES at different concentrations. In these studies, the cell-free supernatant of stimulated RAGMs with the ionophore A23187, resulted in a drastic increase of LTB(4). However, when the cells were treated with the combination RANTES plus A23187 the stimulatory effect was more pronounced than A23187 alone. LTB(4) production was quantitated. The calcium ionophore A23187 directly induced LTB(4) in macrophages, this production was markedly enhanced when the cells were pretreated with RANTES. However, the addition of RANTES in the absence of calcium ionophore A23187 did not directly induce LTB(4) release, nor was lypoxigenase expression augmented. Preincubation of RAGMs with NDGA (nordihydroguiaretic acid) (10(-5)M) completely abolished the production of LTB4 on RAGMSs challenged with A23187 in combination with RANTES or A23187 alone in the supernatants. Similar effects were obtained when the cells were pretreated with dexamethasone. These data suggest, for the first time, that RANTES may stimulate the release of LTB(4), only when it is associated to other stimuli and for this reason we conclude that RANTES modulates inflammatory diseases, and may require other stimuli to be effective in amplifying its spectrum of action(s).

Topics: Animals; Arachidonate 5-Lipoxygenase; Calcimycin; Chemokine CCL5; Dose-Response Relationship, Drug; Drug Synergism; Granuloma; Leukotriene B4; Macrophages; Male; Masoprocol; Potassium Permanganate; Rats; Rats, Wistar; RNA, Messenger

A novel series of heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic acids was studied as leukotriene biosynthesis inhibitors. A hypothesis of structure-activity optimization by insertion of an oxime moiety was investigated using REV-5901 as a starting point. A systematic structure-activity optimization showed that the spatial arrangement and stereochemistry of the oxime insertion unit proved to be important for inhibitory activity. The promising lead, S-(E)-11, inhibited LTB(4) biosynthesis in the intact human neutrophil with IC(50) of 8 nM and had superior oral activity in vivo, in a rat pleurisy model (ED(50) = 0.14 mg/kg) and rat anaphylaxis model (ED(50) = 0.13 mg/kg). In a model of lung inflammation, S-(E)-11 blocked LTE(4) biosynthesis (ED(50) of 0.1 mg/kg) and eosinophil influx (ED(50) of 0.2 mg/kg). S-(E)-11 (A-93178) was selected for further preclinical evaluation.

Topics: Acrylic Resins; Anaphylaxis; Animals; Anti-Allergic Agents; Anti-Inflammatory Agents, Non-Steroidal; Ascitic Fluid; Granuloma; Humans; In Vitro Techniques; Leukotriene B4; Male; Mice; Neutrophils; Pleuropneumonia; Pneumonia; Quinolines; Rats; Stereoisomerism; Structure-Activity Relationship

The changing levels of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) were compared over a period of 1 h to 15 days in two kinds of inflammatory responses induced in the rat air pouch by chemically similar polymers of peptidoglycan-polysaccharide (PG-PS) isolated from cell walls of group A streptococci. The smaller polymers (100 s, average mol. wt 5.3 x 10(6)) induced a more severe acute response over the first 24 h, whereas, from 3 to 15 days the larger polymers (10 p, average mol. wt 5 x 10(8)) induced greater chronic inflammation, as measured by pouch fluid volume, number of infiltrating cells and weight of granulation tissue. The most prominent difference between the two kinds of responses in the pattern of PGE2 and LTB4 were: (a) A shift between 1 to 6 h to much higher production of PGE2 in the exudate induced by large polymers. During this time the level of LTB4 also increased, but continued to be higher in the exudate induced by small polymers. (b) The sustained production of relatively high levels of LTB4 and PGE2 in the large polymer exudate at 15 days. Changes such as these indicate that this model is useful for analyzing mediators which regulate the evolution of acute into chronic inflammation.

Topics: Acute Disease; Animals; Cell Wall; Chronic Disease; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Female; Granulation Tissue; Granuloma; Inflammation; Leukotriene B4; Molecular Weight; Peptidoglycan; Rats; Rats, Inbred Lew; Streptococcus pyogenes

Interleukin-1 (IL-1) is a polypeptide which mediates several systemic changes associated with infection, inflammation and injury, such as fever, neutrophilia, increased acute phase protein synthesis, and arachidonic acid metabolites. Recently, a natural inhibitor of IL-1 has been cloned, called IL-1 receptor antagonist (IL-1ra), which prevents Escherichia coli-induced shock in rabbits and blocks PGE2 induced by IL-1. In this report we study the effect of human recombinant (hr) IL-1ra on chronic inflammation induced by dorsal injections of 200 microliters of a 1:40 saturated crystal solution of potassium permanganate (KMnO4) in mice. After 7 days, all mice developed a subcutaneous granulomatous tissue indicative of a chronic inflammatory response, at the site of injection. KMnO4-treated mice, injected intraperitoneally twice with hrIL-1ra, 20 micrograms/dose (the first at the same time of induction of the granuloma and the second 24 hr later), show significant decreases in size and weight of the granuloma when compared to mice not treated with hrIL-1ra (controls); the inhibitory effect was approximately 32-46 and 25-51%, respectively. In addition, in all mice treated with hrIL-1ra, there was a strong inhibition of PGE2 and LTB4 on assay of freshly minced granuloma tissue. Moreover, when hrIL-1 beta (1.0 ng/ml) or LPS (100 ng/ml) were added overnight to the minced granuloma tissue cultures, these compounds enhanced the production of LTB4 and PGE2 from the untreated mice, whereas in IL-1ra-treated mice they failed. In the histological studies of the granuloma, animals treated with hrIL-1ra show a lesser degree of mononuclear cell (MC) accumulation. The inhibitory effect of hrIL-1ra on PGE2 production was also confirmed on peritoneal macrophages from untreated mice, stimulated overnight with hrIL-1 beta or LPS in vitro. The resulting inhibition was dose-dependent. In these studies we show, for the first time, the anti-inflammatory effect of hrIL-1ra on chronic inflammation as assessed by the inhibition of granuloma formation, PGE2, LTB4, and white cell accumulation in inflamed tissue.

Topics: Animals; Calcium; Dinoprostone; Granuloma; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Leukotriene B4; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Potassium Permanganate; Receptors, Interleukin-1; Recombinant Proteins; Sialoglycoproteins

Interleukin-1 (IL-1) is a monokine that exerts multiple biological activity, including immunity and inflammation. Moreover, IL-1 is involved in Ca2+ release causing hypercalcemia and bone resorption. Recently, a 22 kDa natural inhibitor to IL-1 called interleukin-1 receptor antagonist (IL-1ra) has been described in human fluids, which specifically binds IL-1 alpha or IL-1 beta receptors. In this study, we found that experimental granuloma induced by subcutaneous injections (0.2 ml) of potassium permanganate (KMnO4) 1:40 saturated crystal solution, after 7 days was strongly inhibited in size, weight and calcium content (measured as dry ash weight by incineration of granuloma tissue) compared with untreated controls, in mice treated intraperitoneally with IL-1ra (20 micrograms/bolus) given twice; the first at the same time of the induction of the granuloma and the second 24 hours later. In addition, leukotriene B4 and prostaglandin E2 were also inhibited in fresh granuloma of mice treated with IL-1ra. Taken together, these findings conclude for the first time, that the accumulation of calcium in chronic inflammatory states is strongly inhibited by IL-1ra, which decreases tissue calcergy and can potentially be useful for the treatment of calcium-related inflammatory diseases and malignancy-associated hypercalcemia.

Topics: Animals; Calcium; Data Interpretation, Statistical; Dinoprostone; Granuloma; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Leukotriene B4; Male; Mice; Potassium Permanganate; Radioimmunoassay; Recombinant Proteins; Sialoglycoproteins

Release of arachidonic acid metabolites (eicosanoids) by alveolar macrophages may be important in regulating pulmonary inflammatory reactions. The purpose of this study was to characterize eicosanoids released by rat alveolar macrophages during the evolution of experimentally induced pulmonary inflammation. Immunization with subcutaneous bacillus Calmette-Guerin (BCG) followed 2 wk later by intravenous BCG challenge resulted in mild granulomatous pulmonary inflammation for up to 30 days. At serial intervals, alveolar macrophages were lavaged from the BCG-treated rats as well as from control normal rats. Lavaged macrophages were cultured in vitro, and culture supernatants were assayed by radioimmunoassay for release of prostaglandin E2 (PGE2), Leukotriene B4 (LTB4), and thromboxane B2 (TXB2). Cells were cultured alone, or with added LPS or calcium ionophore A23187 to stimulate eicosanoid release. During BCG-induced inflammation, spontaneous release of PGE2 and LTB4 was unchanged, while spontaneous release of TXB2 was depressed acutely and then returned to control levels. The capacity of alveolar macrophages to release specific eicosanoids in response to an in vitro stimulus was dramatically altered during the course of BCG-induced inflammation. Stimulated release of PGE2 was transiently increased during acute lung injury, but stimulated release of LTB4 was significantly decreased at all stages of inflammation. Stimulated release of TXB2 was unchanged. These results indicate that during the course of granulomatous pulmonary inflammation there are dynamic changes in the profile of eicosanoids released by alveolar macrophages, both spontaneously and in response to in vitro stimulation. This alteration in the release of eicosanoids by alveolar macrophages may be an important factor in the resolution of pulmonary inflammation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cells, Cultured; Culture Media; Dinoprostone; Eicosanoids; Granuloma; Inflammation; Leukotriene B4; Lung Diseases; Macrophages; Male; Mycobacterium bovis; Pulmonary Alveoli; Radioimmunoassay; Rats; Rats, Inbred F344; Thromboxane B2

The injection of polyacrylamide gel (Bio Gel P-4, 200-400 mesh) into the subcutaneous area of mice, induced an inflammatory reaction which was characterized by the migration of leukocytes (mainly neutrophils) from the blood vessels towards the polyacrylamide gel. A rapid protein accumulation was observed during the migration of cells towards the inflamed site. Neutrophils released some pro-inflammatory lipids (prostaglandins, leukotriene B4) and lysozyme, these products were assayed in the granuloma exudate at various times of the granuloma formation. In our experimental inflammatory model, the results suggest that neutrophils that are attracted by the polyacrylamide gel produce eicosanoids and lysozyme, which could act synergistically to potentiate cell migration and protein accumulation in the inflamed site.

Topics: Acrylic Resins; Animals; Arachidonic Acid; Arachidonic Acids; Cell Count; Granuloma; Leukotriene B4; Male; Mice; Muramidase; Prostaglandins; Proteins