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]

Mouse peritoneal macrophages incubated in serumless medium containing a 19:0 or trans-18:1 fatty acid complexed to bovine serum albu'min incorporate the exogenous fatty acid supplement into cellular phospholipids. Within 8 hr, 25% of the total phospholipid fatty acids are derived from the supplement, with cell viability remaining >95%. The incorporation of either of these supplements increases the saturated/ unsaturated fatty acid ratio in the phospholipids 2-fold over that of cells cultured in serum and effects striking changes in endocytic activities. The levels of both fluid-phase pinocytosis and receptor-mediated phagocytosis are decreased at all temperatures examined between 150 and 37°. The increased degree of saturation of cell phospholipids correlates with decreased endocytic rates for both processes and with increased activation energies (Eact) for phagocytosis. The Eact values for phagocytosis, which range from 54 to 90 kcal/mol, depend on the supplementation conditions used. Although the levels of pinocytosis are depressed, the Ea. values for pinocytosis (17-25 kcal/mol)

Murine macrophages synthesize specific cyclo-oxygenase and lipoxygenase metabolites of arachidonic acid (20:4) 1 after stimulation with zymosan (1-3) or particles coated with immune complexes of immunoglobulin G or E (4, 5). In man, less is known about the spectrum of oxygenated 20:4 compounds produced by mononuclear phagocytes. A number of studies have examined the cyclooxygeanse products of the human monocyte (6-1 1), the blood-borne precursor of the tissue macrophage. In addition, the synthesis of the lipoxygenase metabolite leukotriene B4 by the human alveolar macrophage has been reported (12).A particular problem in the study of 20:4 metabolism by the human monocyte has been platelet contamination. We now report a means of removing platelets adherent to the surfaces of freshly isolated monocytes. As a result, quantitative and qualitative comparisons of the biosynthetic pathways of platelets and monocytes were possible. Thromboxane was the predominant metabolite of monocytes incubated with particulate stimuli whereas exposure to the calcium ionophore A23187 yielded chiefly lipoxygenase products.

Recent studies in our laboratory (1) have shown that resident, mouse peritoneal macrophages respond to particulate immune complexes of immunolgobulin E (IgE) (IgE-IC) a with the release of arachidonic acid (20:4). We have now defined the optimum in vitro conditions for IgE-IC-mediated 20:4 metabolism and have examined the nature of the products synthesized. In this report, we demonstrate that macrophages challenged with particle-bound IgE-IC release large quantities of prostacyclin, prostaglandin (PG)E2, and the slow-reacting substance, leukotriene C [5(S)-hydroxy-6(R)-glutathionyl-7,9,11,14-eicosatetraenoic acid] (LTC). These observations suggest that the macrophage might be a source of slow-reacting substance and other 20:4 metabolites generated during IgE-mediated allergic reactions. Materials and MethodsPreparation of Fibronectin-coated surfaces. Fibronectin-coated plastic tissue culture dishes for the isolation of peritoneal macrophages were prepared by the method of Bevilacqua et al. (2). Briefly, 35-mm dishes containing 1 ml of a 30 mg/ml sterile solution of gelatin (type II; Sigma Chemical Co., St. Louis, MO) in water were incubated at 37°C for 2 h. The gelatin solution was then removed, and the plates were allowed to dry for at least 2 h at 37°C. To coat the dishes with fibronectin, 1 ml of human, heparinized, platelet-free plasma was added to each dish. After incubation at room temperature for 1 h, the plates were washed three times with calcium-and magnesium-free phosphate-buffered saline (Pi/NaC1).

Recent work from this laboratory (1, 2) has demonstrated that mouse resident peritoneal macrophages release the slow-reacting substance (SRS), 1 leukotriene C [5(S)-hydroxy-6(R)-glutathionyl-7,9,11,14-eicosatetraenoic acid; LTC] in response to a phagocytic stimulus. No data are as yet available concerning leukotriene synthesis by other mouse macrophage populations. It is of particular interest to know whether pulmonary macrophages are also able to produce large quantities of leukotrienes because of the sensitivity of the peripheral airways of the lung to SRS (3,4). In this report, we present the results of studies of zymosan-induced leukotriene synthesis by mouse pulmonary alveolar macrophages, obtained by bronchoalveolar lavage and by pulmonary interstitial macrophages, isolated by enzymatic digestion of lung tissue. Materials and MethodsPulmonary Macrophage Cultures. Pulmonary alveolar macrophages (PAM) and pulmonary tissue macrophages (PTM) were obtained from male and female ICR mice weighing 25-30 g (Trudeau Institute Inc., Saranac Lake, NY) by a modification of the method of Bluss~ Van Oud Alblas and Van Furth (5). Each mouse was anesthetized with an intraperitoneal injection of 5 mg of sodium pentobarbital (Abbott Laboratories, North Chicago, IL). The chest cavity was opened, and the aorta and inferior vena cava were cut. Blood was removed from the lungs by perfusion with calcium-and magnesium-free phosphate-buffered saline containing 1 mM EDTA, which was injected into the right ventricle of the heart until the lung tissue was uniformly white. The trachea was then cannulated, and the lungs were washed five times with 1 ml of buffered saline containing EDTA at 37°C. These washes, which contained PAM, were combined and placed on ice. For culture, the cells were washed once with buffered saline and resuspended at a cell density of 1 × 106/ml in minimum essential alpha medium (e~-MEM; Gibco Laboratories, Grand Island Biological Co., Grand Island, NY) containing 10% fetal calf serum. The cell suspensions were then added to 60-mm Diam plastic tissue culture dishes (3 ml), 15-mm culture wells (0.3 ml), or 12-mm glass cover slips (0.1 ml). After 2 h of incubation at 37°C in 95% air/5% CO2, the cultures were washed vigorously to remove nonadherent cells and overlaid with fresh a-MEM containing 10% serum. Cultures contained 2.5 zt: 0.1 × 105 adherent cells/60-mm dish.