Mouse peritoneal macrophages release leukotriene C in response to a phagocytic stimulus.

Abstract

Mouse peritoneal macrophages that had ingested zymosan particles released a polar metabolite of arachidonic acid possessing slow-reacting substance activity in the guinea pig ileum assay. The metabolite was purified by solvent extraction, Sephadex G-25 column chromatography, and highpressure reverse-phase liquid chromatography. The purified metabolite absorbed light at 280 nm and contained a free amino group. When macroha ges were preincubated overnight with [3Hjarachidonic acid, [ mSIcysteine, or ['4Cloutamic acid, each radiolabel was incorporated into the compound. Direct amino acid analysis revealed glycine, glutamic acid, and cysteine at molar ratios of 0.97:1.00:0.82. The above data were consistent with the structure of leukotriene C, an adduct of arachidonic acid and glutathione. Quantification of the leukotriene C based on incorporation of [3Hlarachidonic acid or amino acid analysis indicated that 6 X 107 macrophages (3.6 mg of cell protein) released 7.5 nmol after a maximal phagocytic stimulus. The purified leukotriene C had a slow reacting substance activity of 11,500 units/nmol (1 unit has the activity of 5 ng of histamine in a guinea pig ileum contraction assay).Recent studies in this and other laboratories have shown that macrophages contain large amounts of esterified arachidonic acid (20:4) in their phospholipids (1,2) and that up to 50% of this 20:4 is released in the form of oxygenated metabolites in response to a phagocytic stimulus (1). These metabolites have been identified as prostaglandins and hydroxyicosatetraenoic acids (1). More detailed recovery experiments, however, revealed that significant quantities of a more polar species were being lost in previous fractionation procedures. The (Taconic Farms, Germantown, NY) weighing 25-30 g as described (5). Peritoneal cells (6 X 106) in alpha minimal essential medium (a-MEM, GIBCO) containing 10% fetal calf serum were added to 35-mm-diameter plastic culture dishes. After 2 hr at 37°C in 95% air/5% C02, cultures were washed three times in a-MEM to remove nonadherent cells and incubated overnight (16 hr) in fresh medium plus 10% fetal calf serum containing radiolabeled precursors as described below.Preparation of Unopsonized Zymosan. Zymosan was purchased from ICN, and stock solutions in a-MEM were prepared as described (6).Synthesis of Radiolabeled LTC by Macrophage Cultures. RPMI medium deficient in cysteine or glutamic acid and glutathione was prepared using an RPMI Select-Amine Kit (GIBCO) with replacement of RPMI vitamins with minimal essential medium vitamins. To obtain LTC labeled in the cysteine moiety, 1 ml of cysteine-deficient RPMI plus 10% fetal calf serum containing [a5S]cysteine (specific activity 284 mCi/mmol, Amersham/Searle; 1 Ci = 3.7 X 1010 becquerels) at 14.6 ,uCi/ml was added to each culture dish prior to the 16-hr incubation period. Labeling with [14C]glutamic acid was similarly performed, using glutamic acid-deficient RPMI and L-[1,2,3,4,5-14C]glutamic acid (specific activity 267 mCi/mmol, New England Nucle...

Dynamics of leukotriene C production by macrophages.

“…The medium was extracted and chromatographed on silicic acid. The methanol eluate (fraction 2) was dried under reduced pressure, redissolved in HPLC starting buffer, and subjected to chromatography on a column of Lichrosorb C-8 as described previously (9). Elution profiles for aH (---) and relative fluorescence after reaction with fluorescamine (--) are shown.…”

“…[3HI-LTC standard was obtained from macrophages prelabeled with [3H]-20:4 and challenged with zymosan as described previously (9). Purified standard was dissolved in a-MEM (64,000 dpm/ml) and two samples (1 ml each) were extracted and chromatographed on silicic acid.…”

“…The macrophage offered obvious advantages for the detailed study of the release of LTC because purified cell populations were readily available, and because methods for the labeling of maerophage phospholipid with [SH]-20:4 had already been developed for the study of PG production (15). Furthermore, a simple method for the extraction of LTC from macrophage culture medium had been devised, allowing the recovery of the compound under very mild conditions (9).…”

“…ct-MEM, Minimum essential alpha medium; 20:4, arachidonic acid; [3H]-20:4, [5,6,8,9,11,12,14,15-aH]20:4; FCS, fetal calf serum; HPLC, reverse-phase high pressure liquid chromatography; LTC, leukotriene C; LTD, leukotriene D; PD, calcium-and magnesium-free phosphatebuffered saline; PG, prostaglandin; PGE, prostaglandin E; SRS, slow reacting substances; SRS-A, SRS of anaphylaxis.…”

Augmentation of reverse arthus reaction by mast cells in mice.

“…A-63 162 had no effect on mast cell degranulation. (29)(30)(31); and these cells can be stimulated by immune complexes (30,32). We interpret the data of the present study to mean that the strategic location of mast cells near blood vessels makes these cells critical for the production of leukotrienes which mediate certain early inflammatory effects.…”