Formation and metabolism of leukotriene C₄ in macrophages exposed to bacterial lipopolysaccharide

Abstract

Lipopolysaccharide (10 pg/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 k 1.3 ng/106 cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was < 1.6 ng/106 cells. Leukotriene C4 was characterized by reversed-phase highperformance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C,. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5 -60 pg/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C, was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins Ez and Fza by radioimmunoassay demonstrated that leukotriene C, and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.Lipopolysaccharides (endotoxins) represent characteristic surface components of gram-negative bacteria. They are believed to contribute to the pathogenesis and manifestations of gram-negative infection (for review see [l, 21). The mechanisms underlying the toxic events of lipopolysaccharide in vivo, however, are not clearly understood.It has previously been demonstrated that treatment of mouse peritoneal macrophages [3 -51, alveolar macrophages [6] and rat Kupffer cells [7] with lipopolysaccharide resulted in the release of several cyclooxygenase products. These metabolites of arachidonic acid are believed to mediate some of the biological effects induced by lipopolysaccharide [8,9] released leukotrienes were not dependent on the lipopolysaccharide concentration, this cell type may nevertheless represent a cellular source of leukotrienes in septic shock. In the present study the question was addressed as to whether macrophages, which are well known target cells of lipopolysaccharide, are capable of producing lipoxygenase products in response to lipopolysaccharide treatment. It will be shown that under specified conditions, lipopolysaccharide activates mouse peritoneal macrophages (MPM) to secrete LTC, and that these cells exhibit the capacity to metabolize released LTC,. Some of the present data have been presented in a preliminary form [21,22]. MATERIALS AND METHODS MaterialsIscove's modified Dulbecco's medium (Iscove's medium) was purchased from Gibco Limited (Paisley, Scotland). Prostaglandins Ez, Fza and L-glutathione (reduced) were obtained from Serva (Heidelberg, FRG). Synthetic LTC, was kindly donated by Dr J. Rokach (Merk Frosst, Canada). 5,6,8,9,11,12,14,15-[3H]Arachidonic acid (specific activity 62.2 Ci/mmol), tritium-labeled leukotrienes (LTC,, LTD,, LTE,, LTB,), prostaglandins (PGEz, PGFza) and a LTC4 RIA kit were purchased from New England Nuclear (Dreieich, FRG). L-(+)-Ascorbic acid was from Merck (Dar...

Leukotrienes as mediators in frog virus 3-induced hepatitis in rats

“…We demonstrate that Kupffer cells can indeed produce and metabolize proinflammatory leukotrienes upon stimulation (Figure 3). Compared to the leukotriene production by other murine macrophages (37)(38)(39), the Kupffer cell capacity is low but sufficient to account for a major part of the in uiuogenerated leukotrienes detected after FV3 administration ( Figure 1). Preliminary data from our laboratories indicate that cultured endothelial cells from rat liver sinusoids generate leukotrienes only in minimal amounts as compared to Kupffer cells.…”

Stimulation of fibroblast chemotaxis by human recombinant tumor necrosis factor alpha (TNF-alpha) and a synthetic TNF-alpha 31-68 peptide.

“…(c) Most (up to 80%) of PDGF secreted by macrophages is bound to acid labile proteins (mostly a2-macroglobulin) and, like TGF-(31, require acidification for expression of biologic and receptor binding activity (30). (d) Macrophages do not synthesize LTB4 in response to LPS stimulation (31).…”

Effect of bacterial endotoxin on lipoxygenase and cyclo-oxygenase metabolites by rat neutrophils and correlation with cellular functional parameters

“…Studies by Luderitz et al [28] for instance, have shown that endotoxin stimulates LTC4 synthesis in murine macrophages. The discrepancy between these observations and lack of stimulation of endotoxin in rat peritoneal macrophages reported by Rogers [31] and murine macrophages reported by Humes et al [25] is likely attributable to differences in species or strains of mice.…”