When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [l-14C- Glucocorticoids exhibit a variety of biological effects, such as enzyme induction and anti-inflammatory actions (1, 2). The steroids act as a consequence of their binding to cytoplasmic receptors, followed by the translocation of the ligand-receptor complex into the nucleus, which, in turn, affects the transcription of various RNA species (3). The anti-inflammatory action of glucocorticoids is expressed in their ability to inhibit both chemotaxis and release of lysosomal enzymes in phagocytic cells (4-6). Recently, it has been demonstrated that chemotactic peptides enhance the release of arachidonic acid, a product of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4), in rabbit neutrophils (7). Steroids possibly exert their anti-inflammatory action by preventing the release of arachidonic acid from phospholipids and its conversion to prostaglandins (8). These observations prompted a search for a steroid-induced inhibitor of phospholipase A2 in neutrophils. Here, we report the existence of a protein in rabbit neutrophils that inhibits phospholipase A2 and whose synthesis is induced by glucocorticoids.METHODS AND MATERIALS Assay of Phospholipase A2 Activities in Situ and in Vitro. Rabbit peritoneal neutrophils were obtained as described (7) and treated with various glucocorticoids in RPMI 1640 medium (GIBCO) for 16 hr at 37°C under a humid atmosphere of 95% 02/5% CO2. Phospholipase A2 activity was measured by the release of ["4C]arachidonic acid from the cellular lipids, mainly phospholipids, with a slight modification of the method described (7). Bovine serum albumin (1%) was included in Gey's balanced salt solution buffered with 10 mM Hepes, pH 7.4 (modified Gey's buffer). The cells (8-11 X 106 cells per ml) were preincubated in a total volume of 5 ml with 1.25 ,uCi of [1-'4C]arachidonic acid (55.5 mCi/mmol) at 370C for 1 hr (1 Ci = 3.7 X 1010 becquerels). The cells were washed twice with 5 ml of modified Gey's buffer and resuspended in 5 ml of the same buffer. For arachidonic acid release, the cells were stimulated with 10 nM fMet-Leu-Phe for 10 min at 370C (7). 16 hr. After three washings with 0.84% NaCl solution buffered with 10 mM sodium phosphate buffer, pH 7.4, the cells (8 X 106 cells) were lysed in 3 ml of distilled water. After centrifugation, at 27,000 X g for 50 min, the precipitates were solubilized with 0.5 ml of 2% Nonidet P40. Samples (0.5 ml) of 2533The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

When rabbit peritoneal leukocytes were treated with chemoattractants such as fMet-Leu-Phe, an apparent decrease of [3Hlmethyl incorporation into the lipid fraction from L4methyl-3Hlmethionine was observed. This decrease was a result of increased degradation of methylated phospholipids, not of decreased synthesis. Chemotactic peptides did not affect the metabolism of the phospholipids in which Imethyl-'4Cjcholine was incorporated. The disappearance of the [3Hlmethyl group was associated with the release of (1-14Cjarachidonic acid from phospholipids prelabeled with these compounds. These findings suggested the activation by chemoattractants of phospholipase A2, an enzyme that removes an unsaturated fatty acid from phospholipids. The order of potency of chemoattractants for the stimulated degradation of phospholipids was in good agreement with that for chemotaxis. Mepacrine (quinacrine) and hydrocortisone inhibited and a phorbol ester enhanced both chemotaxis and phospholipase A2 activity. These results, taken together, suggest close association of the metabolism of methylated phospholipids with chemotaxis in rabbit peritoneal leukocytes.Leukocytes respond to various chemoattractants by interaction with specific receptors on the cell surface (1). The biochemical mechanism by which stimulation of chemotactic receptors leads to directed movement of leukocytes is still poorly understood. Recently, our laboratory showed that the activation of protein carboxy-O-methylase in leukocytes is one of the early events in chemotaxis (2). We have also found that phospholipid methylation alters biomembrane structure and functions (3-5). Because the chemotactically responding cells show a marked polarization and have alterations in properties of membranes, we examined the effect of chemoattractants on phospholipid methylation in leukocyte membranes. Here, we report that chemotactic peptides enhance the degradation of phosphatidylcholine synthesized by the transmethylation but not by the choline pathway(s).METHODS AND MATERIALS Cell Preparations. Rabbit leukocytes were obtained by lavage of the peritoneal cavity of rabbits injected with 150 ml of 0.1% glycogen as described (6). When necessary, the peritoneal exudates were exposed to hypotonic saline (0.2%) in the cold for 30 sec to lyse contaminating erythrocytes and then diluted with an equal volume of 1.6% saline. After collection by centrifugation at 600 X g for 10 min, the cells were suspended inGey's balanced salt solution containing 0.1% bovine serum albumin and 0.01 M Hepes buffer at pH 7.4 (modified Gey's solution), at a concentration of 8-11 X 106 cells per ml. A typical cell preparation contained 90% neutrophils and 10% lymphocytes and macrophages. Chemotaxis was measured in modified Boyden chambers as described (7).Assay of Phospholipid Methylation. The phospholipid methylation was assayed by using intact leukocytes and measuring [3H]methyl group from L-[methyl-3H]methionine incorporated into the phospholipid fraction. The cells were preincubated in a total volum...

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