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.

Many types of cells methylate phospholipids using two methyltransferase enzymes that are asymmetrically distributed in membranes. As the phospholipids are successively methylated, they are translocated from the inside to the outside of the membrane. When catecholamine neurotransmitters, lectins, immunoglobulins or chemotaxic peptides bind to the cell surface, they stimulate the methyltransferase enzymes and reduce membrane viscosity. The methylation of phospholipids is coupled to Ca2+ influx and the release of arachidonic acid, lysophosphatidylcholine, and prostaglandins. These closely associated biochemical changes facilitate the transmission of many signals through membranes, resulting in the generation of adenosine 3',5'-monophophate in many cell types, release of histamine in mast cells and basophils, mitogenesis in lymphocytes, and chemotaxis in neutrophils.

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...

Inflammation is a common feature in the pathogenesis of cigarette smoke-associated diseases. The recruitment of inflammatory cells into the lung following cigarette smoke exposure presents a risk of tissue damage through the release of toxic mediators, including proteolytic enzymes and reactive oxygen species. This review represents a toxicological approach to investigation of cigarette smoke-induced lung injury, with a focus on laboratory studies and an emphasis on inflammatory mechanisms. The studies discussed in this review analyze the role of inflammation and inflammatory mediators in the development of injury. In cases where information relating to cigarette smoke is limited, examples are taken from other models of lung injury applicable to cigarette smoke. The primary aim of the review is to summarize published work so as to permit (1) an evaluation of chronic lung injury and inflammatory responses in animal models, (2) a discussion of inflammatory mediators in the development of chronic injury, and (3) identification of immunological mechanisms of injury. These studies discuss the currently understood roles of cytokines, cell adhesion molecules, and oxidative stress in inflammatory reactions and lung injury. A role for lipocortin 1 (annexin 1), a naturally occurring defense factor against inflammation, is discussed because of the possibility that impaired synthesis and degradation of lipocortin 1 will influence immune responses in animals exposed to cigarette smoke either by augmenting T helper cell Th1 response or by shifting Th1 to Th2 response. While Th1 augmentation will increase the risk for development of emphysema, Th1 to Th2 shift will favor development of asthma.

Normal rat mast cells were stimulated by antibodies against IgE receptors (anti-RBL) or by anti-IgE, and[3H]methyl group incorporation into phospholipids, 45Ca uptake, and histamine release were examined. Anti-RBL (8). Previous studies have shown that the anti-receptor antibodies in anti-RBL are responsible for histamine release and 4Ca uptake (6, 7). The F(ab')2 fragments of anti-RBL were obtained by pepsin digestion and these fragments were split into Fab' monomer by reduction and alkylation (6). Because of the low protein concentration of purified anti-RBL (1.6 mg/ml), an 8-fold excess of normal rabbit IgG was added to the antibody preparation before digestion.Monoclonal rat IgE (IR 162) and the IgG fraction of a goat antiserum specific for rat IgE (anti-IgE) were those described (7). The concentration of anti-IgE antibody in the goat antiserum was 3.25 mg/ml.Purification of Mast Cells. Mast cells were obtained from peritoneal cells of Sprague-Dawley rats (Holtzman, Madison, WI) by the method of Bach and colleagues (9) with slight modifications (10). Purity of mast cell preparations was in the range 89-92%. Viability of the cells was >97% as assessed by trypan blue exclusion.Measurement of 45Ca Influx. The method described by Foreman et al. (11) was followed. Purified mast cells were suspended in Tyrode's solution (pH 7.0) containing 1 mM CaCl2, 0.5 mM MgCl2, 5 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (Hepes), 5 mM 2-(N-morpholino)-ethanesulfonic acid (Sigma), and 0.5 g of gelatin per liter.Phosphatidylserine (50,ug/ml; Supelco, Bellefonte, PA) was dispersed in the solution by sonication. One hundred microliters of Versilube F50 silicone oil (General Electric, Waterford, NY) was placed in the bottom of Microfuge tubes; 40 ,ul of Tyrode's solution containing 45Ca (3 ,Ci/ml; 1 Ci = 3.7 X 1010 becquerels; Amersham) and 10 Ml of an appropriate concentration of either anti-RBL or anti-IgE was layered on top of the silicone oil. The Microfuge tubes were warmed at 370C, and then 50 ,ul of a mast cell suspension containing 1-2 X 105 cells was Abbreviations: RBL cells, rat basophilic leukemia cells; Con A, concanavalin A.

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Two methyltransferases involved in the methylation of phosphatidylethanolamine to form phosphatidytcholine were demonstrated in a microsomal fraction of bovine adrenal medulla. The first methyltransferase catalyzes the methylation of phoshatidylethanol'amine to form phosphatidyl-N-monomethylethanolamine. This enzyme has an optimum pH of 6.5, a low Km for S-adenosyl-L-methionine (1.4 ;M), and an absolute requirement for Mg2+. The second methyltransferase catalyzes the two successive methylations of phosphatidyl-N-monomethylethanolamine to phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In contrast to the first methyltransferase, it has an optimum pH of 10 and a high Km for S-adenosyl-L-methionine (0.1 mM) and does not require MgS+.Several investigations have shown that enzymatic methylations can occur on the amino group of phospholipids to form phosphatidyicholine (1-4). The enzyme(s) catalyzing this sequence of methylation were shown to reside in the microsomes of rat liver and Neurospora. A preparation of rat liver microsomes has been described that catalyzed the stepwise methylation of phosphatidyl-N-monomethylethanolamine to phosphatidylcholine but not of phosphatidylethanolamine (1; 4). The enzyme catalyzing the first methylation step has been suggested to be rate-limiting (1), but its properties have not yet been described. Recently, our laboratory reported on the ability of the enzyme, protein carboxymethylase, to transfer a methyl group from S-adenosyl-L-methionine to carboxy groups of membrane proteins of chromaffin granules in the adrenal medulla (5-7). In studies to examine the effects of cations on this enzyme activity with various membrane fractions, it was observed that methylation of lipids also occurred which depended upon the presence of Mg2+. Since the methylation of phospholipids has not been shown to require Mg2+ (1-4), this led us to search for and characterize the Mg2+ dependent enzyme that methylates lipids. This communication presents evidence that this Mg2+-dependent enzyme is involved in the conversion of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine and that a second methyltransferase converts the latter compound to phosphatidylcholine. METHODS AND MATERIALSAssay of Phosphatide Methyltransferases. The methylation of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine was assayed by measuring incorporation of the methyl group from S-adenosyl-L-[methyl-3H]methionine into phospholipids. The assay medium, in a 6-ml stoppered polyethylene tube, contained 4 ,uM S-adenosyl-L-[methyl-3H]-The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "'advertisement"9 in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1718 methionine (2 ,uCi), 10 mM MgCl2, 0.1 mM sodium EDTA, 50 mM sodium acetate buffer (pH 6.5), and tissue extract (0.1 mg of protein) in a total volume of 50 1l. The reaction was started by the addition of radioactive S-adenosyl-L...

The challenge of previously sensitized guinea pigs with aerosolized ovalbumin resulted in impairment of the beta-adrenoceptor-mediated relaxation as measured by the in vitro isometric assay of tracheas preconstricted with endothelin-1 or carbamylcholine. Numbers and affinities of beta-adrenoceptors in lung membranes of these animals were not altered under these conditions, although the antigen challenge caused an inflammatory response, as evident from the accumulation of inflammatory cells in the bronchoalveolar lavage fluids. In order to investigate the pathophysiologic role of inflammation in hyperreactive airways, isolated guinea pig tracheas were cultured with proinflammatory cytokines such as human recombinant tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or interleukin-2 (IL-2). None of these cytokines affected the contractile response of tracheas to carbamylcholine. After preconstriction with carbamylcholine, the TNF-alpha- and IL-1 beta-pretreated tissues produced a significant reduction in the maximal relaxation induced by isoproterenol, whereas the IL-2 pretreatment had no effect. The reduction of the isoproterenol-mediated relaxation by the IL-1 beta treatment was time and dose dependent. Our present observations suggest that in vitro incubation of naive tracheas with proinflammatory cytokines is able to reproduce apparent beta-adrenoceptor impairment as seen in the airways of antigen-challenged guinea pigs of asthma model.