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Glucocorticoids induce the synthesis of a family of phospholipase inhibitory proteins, lipocortins. This family of lipocortins includes inhibitory proteins on phospholipase A2, phospholipase C and phosphatidylinositol phospholipase C. Hence, glucocorticoids reduce the formation of prostaglandins and leukotrienes by inhibiting cellular phospholipases, enzymes that degrade membrane phospholipids to release arachidonic acid, a precursor. The induction by glucocorticoids requires 1 h for the synthesis of mRNA and 5 h for the synthesis of proteins in various tissues and cells. However, glucocorticoids often exert their suppressive effects before the induction of lipocortins. This is now attributed to the nonenzymic formation of the adducts between glucocorticoids and lipocortins. These adducts are easily inserted into the membranes and more resistant to digestion of proteases, thus being more biologically potent with respect to suppression of the release of arachidonic acid, a precursor of prostaglandins and leukotrienes.

The treatment of murine thymocytes with anti-lipomodulin antibody during Con A stimulation causes selective loss of suppressor activity, but not of helper activity on PFC assay, when co-cultured with T cell-depleted spleen cells. Interaction of the antibody with responder cells in thymocyte culture were necessary in the early stage rather than in the later stage of lymphocyte activation by Con A, which suggests that anti-lipomodulin antibody acts in the stage of suppressor T cells generation. When thymocytes were cultured with purified lipomodulin for 48 hr, suppressor activity was induced. Lipomodulin as detected by radioimmunoassay was found to be released from T cells with the phenotype of I-J+, Lyt-1-, Lyt-2+. The immunoprecipitates from the media of Con A-stimulated thymocyte with anti-I-Kk antibody and anti-lipomodulin antibody were analyzed on SDS-gel electrophoresis. I-J products had m.w. 36,000 and 24,000, whereas lipomodulin had m.w. 36,000, 24,000, and 15,000. Because anti-I-Jk antibody could precipitate 125I-labeled lipomodulin purified from rabbit neutrophils, these results suggest that lipomodulin is a product of I-J genes that induces suppressor T cells.

The actions of glucocorticoids require the synthesis of new proteins in many, if not all, cases. One such protein that mimics the actions of glucocorticoids has been isolated and characterized as lipocortin. This protein can mimic the anti-inflammatory activity of glucocorticoids by inhibiting phospholipase A2. The effect of glucocorticoids on various stages of brain and spinal cord injury may be partly, although not totally, explained by the activity of lipocortin.

Lipocortin was found to be secreted by human endometrium incubated for 1-2 days under organ culture conditions. Levels of lipocortin in the culture medium, measured by RIA, were increased by dexamethasone (10(-8)-10(-6) M) and decreased by progesterone (10(-8)-10(-6) M). Both steroids, however, decreased prostaglandin F2 alpha (PGF2 alpha) output, also estimated by RIA. These results suggest that lipocortin does not mediate the inhibition of PGF2 alpha production evoked by progesterone. Dexamethasone may inhibit PGF2 alpha production by mechanisms involved in the inhibition by progesterone in addition to those mediated by an elevation in the levels of lipocortin, a phospholipase inhibitor.

Lipocortin is a phospholipase inhibitory protein whose synthesis is induced in various cells by glucocorticoids. At least three species with molecular weights of 40,000, 30,000, and 15,000 are presently known. This protein mimics the anti-inflammatory action of glucocorticoids in vitro as well as in vivo. The synthesis of the protein appears to be associated with the MHC genes.

When U 937 cells, a human histiocytic lymphoma cell line, were cultured with purified lipomodulin for 3 days, morphological and functional differentiation was induced as detected by microscopical examination of Giemsa stained smears, expression of mature monocyte antigen, and antibody dependent cellular cytotoxicity tests. Essentially similar differentiation was observed by the treatment with dexamethasone for 6 days and this differentiation by dexamethasone was blocked by monoclonal anti-lipomodulin antibody. Furthermore, the synthesis of immunoprecipitable lipomodulin in these cells was induced by dexamethasone treatment. These results, taken together, suggest that the induction of lipomodulin synthesis might be the primary event in dexamethasone-induced cellular differentiation of U 937 cells.

When murine thymocytes were stimulated by mitogens such as concanavalin A, the Ca2+ ionophore A23187, or 4 beta-phorbol 12-myristate 13-acetate, there was a marked increase of 32P incorporation into immunoprecipitable lipomodulin, a phospholipase inhibitory protein. These compounds enhanced 45Ca2+ influx into thymocytes, which, in turn, increased protein phosphorylation, probably by Ca2+- and phospholipid-dependent protein kinase (protein kinase C). Cyclic 8-bromo-AMP, an inhibitor of lymphocyte mitogenesis, blocked the mitogen-stimulated phosphorylation of lipomodulin, although it stimulated the protein phosphorylation via cyclic AMP-dependent kinase (protein kinase A). On electrophoresis, the hydrolysates of 32P-labeled lipomodulin showed a single radioactive spot, which comigrated with authentic phosphotyrosine. The partially purified middle-sized tumor antigen was able to phosphorylate lipomodulin after being phosphorylated by protein kinase C but not by the catalytic subunit of protein kinase A. Our findings suggest that the activity of a tyrosine-specific kinase, which phosphorylates lipomodulin in vivo as well as in vitro, is stimulated by protein kinase C and inhibited by protein kinase A.

Splenic T lymphocytes of rats treated with complete Freund's adjuvant (CFA) spontaneously release a lymphokine that inhibits the glycosylation of IgE-binding factors during their biosynthesis and provides the factors with biologic activity: selective suppression of the IgE response. The lymphokine, which is called glycosylation-inhibiting factor, also prevents IgE-induced increases in Fc epsilon R+ cells. The glycosylation inhibiting factor is formed by stimulation of BCG-primed spleen cells with PPD and participates in the selective formation of IgE-suppressive factors. The lymphokine is derived from OX 8+ T cells in both the CFA and BCG systems. The glycosylation-inhibiting factor is a 16,000-dalton peptide, as estimated by gel filtration, and specifically binds to monoclonal antibody against lipomodulin, a phospholipase inhibitory protein. Furthermore, "lipomodulin" was detected by radioimmunoassay in the 16,000-dalton fraction that contained glycosylation inhibiting factor. The fraction did not inhibit phospholipase A2 but after alkaline phosphatase treatment, the fraction did inhibit phospholipase A2. Furthermore, purified lipomodulin obtained from glucocorticoid-treated rabbit neutrophils had the same biologic activities as glycosylation inhibiting factors; i.e., it inhibited both protein glycosylation of IgE-binding factors and IgE-induced expression of Fc epsilon R. The results collectively indicate that glycosylation-inhibiting factor is a fragment of phosphorylated lipomodulin.

Lipomodulin, a protein with a MW of 40,000, is induced in many cells by glucocorticoids. This protein can mimic a variety of biological activities of glucocorticoids such as anti-inflammation, immunosuppression, and promotion of cellular differentiation. Since anti-lipomodulin antibody can block these effects of both glucocorticoids and lipomodulin, it is quite possible that lipomodulin is a mediator of glucocorticoids.

A highly purified preparation of lipomodulin, a phospholipase-inhibitory protein from rabbit neutrophils treated with glucocorticoids, inhibited NK and antibody-dependent cellular cytotoxicity (ADCC) activities of human peripheral blood lymphocytes in a dose-dependent manner. The presence of lipomodulin during the early period of the cytotoxicity assay was necessary to obtain maximal inhibition. The inhibition of NK or ADCC activity by lipomodulin was greater when effector cells were treated with lipomodulin than when target cells were incubated with lipomodulin. As lipomodulin did not block binding of effector cells to target cells, our results suggest that lipomodulin inhibits the cytolytic phase of NK and ADCC activities after binding to target cells, and imply that phospholipase(s) may be involved in NK and ADCC activities.

Lipomodulin, purified to near homogeneity from rabbit peritoneal neutrophils, was phosphorylated by cyclic AMP-dependent protein kinase from bovine heart with concomitant loss of its ability to inhibit phospholipase A2 from porcine pancreas. Phosphorylation of lipomodulin was confirmed by the incorporation of 32P from [gamma-32P]ATP. To demonstrate that lipomodulin undergoes phosphorylation in vivo, rabbit peritoneal neutrophils were incubated with 32P and lipomoculin was isolated by immunoprecipitation with serum from a patient with systemic lupus erythematosus which has anti-lipomodulin antibody. Analysis of 32P-labeled immunoprecipitates by sodium dodecyl sulfate electrophoresis revealed a single peak of radioactivity that comigrated with [35S]methionine-labeled lipomodulin. The administration of a chemoattractant, N-formyl-methionyl-leucyl-phenylalanine to intact rabbit neutrophils, resulted in a marked increase in arachidonate release from the cells and an increase in 32P incorporation into lipomodulin. A close correlation was found between the extent of phosphorylation of lipomodulin and the rate of arachidonate release. Phosphorylation of lipomodulin in neutrophils gradually returned to the control level with corresponding cessation of arachidonate release. In contrast to the in vitro system, phosphorylation of lipomodulin and release of arachidonic acid from peptide-stimulated neutrophils required Ca2+ entry into the cells. These results suggest that the phosphorylation-dephosphorylation of lipomodulin, phospholipase inhibitory protein, is an important mechanism for chemotactic receptor-mediated regulation of arachidonic acid release in rabbit neutrophils.

The activity of phospholipase inhibitory protein, lipomodulin, partially purified from rabbit neutrophils, was markedly decreased after treatment with sera from patients with rheumatic diseases such as systemic lupus erythematosus, rheumatoid arthritis, and dermatomyositis. The decrease of the protein's inhibitory activity on phospholipase A2 paralleled the amount of [35S]methionine-labeled lipomodulin precipitated by the sera. Absorption of patients' sera with anti-human IgM (mu chain) or protein A-agarose, but not with anti-human IgG (gamma chain), decreased their ability to decrease the activity of lipomodulin on phospholipase A2 or to precipitate the radioactive lipomodulin. The IgM fraction of patients' sera could precipitate [35S]methionine-labeled lipomodulin (40,000 daltons) which comigrated with highly purified lipomodulin on gel electrophoresis with sodium dodecyl sulfate. All of these observations suggest that the sera of many patients with rheumatic diseases contain autoantibody against lipomodulin. A monoclonal antibody against lipomodulin was also obtained. Stimulating human fibroblasts with bradykinin in the presence of monoclonal antilipomodulin antibody markedly enhanced arachidonic acid release due to the activation of phospholipase(s) in the intact cells, and this stimulatory effect was blocked by adding purified lipomodulin. These findings suggest that lipomodulin regulates the activity of phospholipase(s) on the cell surface and that autoantibodies against lipomodulin may play a role in certain symptoms of rheumatic diseases, especially by the formation of prostaglandins and other metabolites of arachidonic acid.

The phospholipase-dependent liberation of arachidonic acid (AA) from membrane phospholipids has been proposed as the rate-limiting step in the synthesis of bioactive AA metabolites, which play an important role in the expression of inflammatory and immune reactions. We have examined the effects of steroids in vitro on the release of AA by rat alveolar macrophages exposed to zymosan. Fluocinolone (1 microM) significantly inhibited the zymosan-induced release of radiolabeled AA from phosphatidylcholine as well as the production of radiolabeled prostaglandin E2. (PGE2). Dose-response curves gave the following rank order of potency: fluocinolone greater than dexamethasone greater than hydrocortisone. The maximal degree of inhibition of radiolabeled AA release observed was approximately 70%. Inhibition was not observed after 3 h of glucocorticoid pretreatment, but maximal inhibition was achieved after 10 h of pretreatment. Pretreatment with gonadal sex hormones (1 microM) did not inhibit AA release. Concurrent incubation of macrophages with hydrocortisone and excess concentrations of the partial glucocorticoid agonist, progesterone, blunted the degree of inhibition observed with hydrocortisone alone. These data are consistent with a receptor-mediated process. The time course suggests a response dependent on new protein synthesis, and the increased concentration of the phospholipase-inhibitory protein, lipomodulin, in steroid-treated cultures is putative evidence of new protein synthesis.

Chemotaxis of rabbit peritoneal leucocytes stimulated by fMet-Leu-Phe, a synthetic chemoattractant, was inhibited by Glu-Glu-Glu-Glu-Tyr-Pro-Met-Glu (MT peptide) and Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Glu-Gly (Src peptide). Both peptides did not inhibit the binding of [3H] formyl-NLe-Leu-Phe, a chemoattractant, to neutrophils, suggesting that the peptides inhibit the events distal to the chemotactic receptors. These peptides blocked the release of arachidonic acid from phospholipids in neutrophils stimulated with chemoattractants, whereas they had no effect on phospholipase A2 activity itself. The peptides markedly reduced the phosphorylation of lipomodulin, a phospholipase inhibitory protein, in either intact cells or isolated plasma membranes. Lipomodulin immunoprecipitated by monoclonal anti-lipomodulin antibody had phosphorylserine and phosphoryltyrosine as analyzed upon electrophoresis. The MT peptide which does not contain threonine or serine was phosphorylated by isolated plasma membranes. These results, taken together, suggest that a tyrosine phosphorylating kinase is involved in biochemical events of chemotactic receptors, and that lipomodulin is a substrate for this kinase.

The effect of glucocorticoids on respiratory glycoconjugate (RGC) secretion was studied in a cat tracheal organ culture system. Dexamethasone (10(-5) to 10(-9) M) added to culture medium for 24 h caused a dose-related reversible inhibition of RCG of as much as 40% with a peak effect at 24 to 60 h after initiation of dexamethasone treatment. A monoclonal antilipocortin antibody added to the cultures blocked the inhibitory effect of dexamethasone on RGC secretion and accelerated the reversal of the dexamethasone effect after discontinuation of dexamethasone treatment. A control antibody without antilipocortin activity had no effect on RGC secretion or dexamethasone-induced inhibition of RGC secretion. Measurement of the concentration of lipocortin in airways revealed a 220% increase after treatment with dexamethasone for 24 h. We conclude that dexamethasone inhibits RGC secretion through the induction of lipocortin synthesis.