During the course of our studies on the lipid composition of macrophage membranes, it was noted that 25% of the total fatty acid content of this cell was composed of arachidonie acid (20:4)) Similar values have been reported for both rabbit (1) and human mononuclear phagocytes (2), whereas the fatty acid complement of most other cell types rarely exceeds a few percent. This unusual enrichment with esterified 20:4 has focused attention on the ability of mononuclear phagocytes to produce prostaglandins (PG) from endogenous stores. PG, depending upon their structure, are thought to play a variety of important roles in the initiation and control of the inflammatory process (3).It is now well established that mononuclear phagocytes produce PG in response to a variety of stimuli that perturb their surface membranes. These include phagocytizable particulates such as zymosan (4) and immune complexes (5) or soluble agents such as phorbol myristate acetate and lipopolysaccharide (6, 7). Bonney et al. (4) have reported that the major synthetic product of mouse peritoneal cells is PGE with smaller amounts of 6-oxo-PGFl~ Unfortunately, there is little information concerning the regulation and control of the oxygenation products of 20:4. For this reason we have established a group of defined conditions in which we have examined the relationship between phagocytosis and PG synthesis, the localization and fate of 20:4 in the phospholipid pool of resident peritoneal macrophages, and the proportions of other 20:4 products released upon a zymosan challenge. Materials and MethodsCell Cultures. Primary cultures of peritoneal macrophages were established from resident cells of female NCS mice that weighed 95-30 g, as previously described (8). For measurements of PG synthesis, 6 × 106 peritoneal cells were added in minimum essential medium alpha medium (a-MEM) that contained 10% fetal calf serum (FCS) to 35-ram-diameter plastic culture dishes For determinations of phagocytosls and cell vlabihties, 7 × 105 peritoneal cells were added to 12-mm-diameter glass coverslips placed in similar dishes After 2 h at 37°C in 5% CO2, the cultures were washed three times in a-MEM to remove nonadherent cells and incubated overnight (16 h) in fresh a-MEM plus 10% FCS * Supported by National Institutes of Health grant AI-07012 and American Heart Society grant 79-1009 1Abbrevzatzons used m this paper a-MEM, minimum essential medmm alpha medmm, FCS fetal calf serum, HETE, hydroxy-elcosatetraenolc acid(s); wMEM, minimum essential medmm alpha medium, PG, prostaglandin(s), RIA, radioimmunoassay, 16 0, palmiuc acid, 18.0, steanc acid, 18-1, oleic acid, 18 2, hnolelc acid; 18'3, hnolemc acid, 20.4, arachldomc acid, [aH]20 4, [5, 6, 8, 9, 11, 12, 14, 15-aH]
Levels of zymosan-induced arachidonic acid (20:4) metabolism by peritoneal macrophages elicited with inflammatory agents and resident macrophages were similar. Thyioglycollate (THIO)-elicited macrophages represented the exception; however, the diminished metabolism by these cells was reproduced by exposing resident cells to 5 mg/ml THIO broth in vitro. In contrast, reduced prostaglandin synthesis by macrophages from mice variously treated with the immunologic agents, Corynebacterium parvum or Bacille Calmette Guérin (BCG), closely correlated with enhanced antitoxoplasma activity, one measure of macrophage activation. This relationship, although not causative, suggested that the capacity for 20:4 metabolism is a function of the macrophage activation state. Modulation of macrophage 20:4 metabolism in vivo apparently required factors in addition to lymphocyte-derived products. Treatment of resident macrophages in vitro with BCG lymphokine was without effect on 20:4 release or prostaglandin synthesis. Activated macrophages from animals inoculated i.p. with C. parvum exhibited reduced 20:4 release and also failed to metabolize 70% of the 20:4 released in response to a zymosan stimulus. Consequently, the quantities of 20:4 metabolites formed were significantly less than expected from 20:4 release. These activated macrophages displayed greatly reduced synthesis of prostacylcin and leukotriene C compared with other 20:4 metabolites. It appeared that factors that regulate macrophage 20:4 metabolism influence the level of the inducible phospholipase and synthetic enzymes for specific 20:4 oxygenated products.
A method for the radiochemical assay of LTC production by mouse peritoneal macrophages in vitro is presented. The method involves labeling macrophages in culture with [5,6,8,9,11,12,14,15-3H]20:4 followed by stimulation of arachidonic acid (20:4) release under the experimental conditions desired. Radiolabeled leukotriene C (LTC) is recovered from the culture medium by extraction and silicic acid chromatography in 40% yield with full retention of biological activity. Because this LTC is radiochemically pure, the quantity of LTC release may be estimated from the amount of radioactivity in the sample. Use of the radioassay to study parameters affecting LTC synthesis by macrophages indicated that the time course of LTC synthesis and its relationship to the dose of a phagocytic stimulus (zymosan) were very similar to those of prostaglandin (PG) release. LTC release was also similar to that of PG in that lower levels of both metabolites were produced by Corynebacterium parvum-elicited macrophages than by resident cells. Finally, LTC release was stimulated in response to a challenge with antigen-antibody complexes, but lower maximal levels were attained than those with zymosan. The data presented here are consistent with the hypothesis that challenge of macrophages with a phagocytic stimulus leads to the release of 20:4 by an inducible phospholipase. Cyclooxygenase and lipoxygenase then compete for the released 20:4, leading to the production of PG, hydroxyeicosatetraenoic acids, and LTC.
Prior studies have shown that mouse peritoneal macrophages contain unusually high levels of arachidonic acid (20:4)1 in their phospholipids. Under resting conditions of in vitro cultivation, 20:4 is retained in cell phospholipids and insignificant amounts are metabolized via lipoxygenase and cyclo-oxygenase pathways (1, 2). However, when stimulated by the appropriate membrane-perturbing agents of both soluble and particulate nature (1-3), up to 50% of the 20:4 is released in the form of oxygenated metabolites. Release occurs under conditions in which tritiated 20:4 has been previously incorporated into phospholipids (1) and leads to the formation of radiolabeled prostaglandins, hydroxyeicosatetraenoic acids (HETEs), and leukotriene C, a slowreacting substance (1-2, 4, 5).In this paper we report the unique response of resting macrophages to exogenously supplied 20:4 and its rapid metabolism in the absence of phagocytic or pharmacologic stimuli. We show that within 5 rain approximately one-third of the radiolabeled 20: 4 supplied in serum-free medium is incorporated into cell phospholipids and approximately two-thirds is converted to a spectrum of oxygenated products--primarily prostacyclin and HETEs. Qualitatively different results are obtained when Corynebacterium parvum-elicited macrophages are employed. Materials and MethodsMacrophage Cultures. Primary cultures of peritoneal macrophages were established from resident cells of female and male Swiss Webster (Taconic Farms, Germantown, N. Y.) or CD-1 (The Trudeau Institute, Saranae Lake, N. Y.) mice, as previously described (1). Approximately 6 × 10 e peritoneal cells suspended in 1 ml of minimum essential a medium (a-MEM, Grand Island Biological Co., Grand Island, N. Y.) containing 10% fetal calf serum (FCS) were added to 35-ram Diam plastic culture dishes. After 2 h at 37°C in 5% CO2/95% air, cultures were washed three times in calcium and magnesium-free phosphate buffered saline (PD) to remove nonadherent cells and incubated overnight (16 h) in fresh a-MEM plus 10% FCS.C. parvum-elicited Macrophages. C. parvum-elicited peritoneal macrophages were obtained from
Murine peritoneal macrophages cultured in minimal essential medium (ct-MEM; 118 mM Na +, 5 mM K+) 1 released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K ÷ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K ÷ on 20:4 release was fully reversed on returning cells to medium containing Na + (118 mM). Preingestion of zymosan particles by macrophages maintained in high K + medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na ÷. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K ÷ medium, suggesting no direct effect of high K + on the phospholipase. Macrophages stimulated with zymosan in a-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K + medium was recovered as 6-Keto-PGFlct, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K ÷, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in a-MEM. These findings and the similarity of products (including LTC) produced by A23187 stimulated cells in a-MEM and high K + medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K ÷. We conclude that high concentrations of extracellular K ÷ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.Stimulation of macrophages by appropriate membrane-perturbing agents of both soluble and particulate nature results in the induction ofphospholipase activity and the quantitative oxygenation of the released arachidonic acid (20:4) via the cyclooxygenase and lipoxygenase pathways (1-3). The 20:4 cascade can proceed maximally under conditions which prevent particle interiorization indicating that the triggering event is due to the interaction of the particle-bound ligands with plasma membrane receptors (4). Knowledge concerning the Abbreviations used in this paper. 20:4, arachidonic acid; a-MEM, alpha-minimal essential medium; PBS, calcium-and magnesium-free phosphate-buffered saline; HETEs, hydroxyeicosatetraenoic acids; HHT, 12-Hydroxyheptadecanoic acid; LTC, leukotriene C; 6-Keto-PGF~, 6-Keto prostaglandin Flu; PGE2, prostaglandin E2; HPLC, reverse-phase high performance liquid chromatography; TXB2, Thromboxane B2; PGF2,, prostaglandin F2~.next step in the cascade, i.e., the signal between the receptorligand complex and the phospholipase is scant.In this study we report that high con...
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