calcimycin and Granuloma

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