Glucocorticoids inhibit superoxide (O°) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2 generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2 generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2 generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2 generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.
1 The injection of a suspension of a polyacrylamide gel (bio gel) into the dorsal subcutaneous area of mice induced an inflammatory reaction and the migration of neutrophils towards the inflamed site.2 The intravenous administration of anti-inflammatory drugs (dexamethasone, indomethacin and lysine-acetylsalicylate) to polyacrylamide gel-treated mice inhibited the accumulation of neutrophils in the inflamed site. 3 A similar administration of a 36 K mouse lipocortin, induced a strong dose-dependent inhibition of neutrophil accumulation in the inflamed site. 4 Dexamethasone and lipocortin inhibited the production of eicosanoids, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the inflamed site of polyacrylamide gel-treated mice. 5 Lipocortin impaired both phospholipase A2 (PLA2) activity and chemotaxis of isolated inflammatory neutrophils. 6 The present studies show an in vivo anti-inflammatory effect of lipocortin similar to that of glucocorticosteroids. In agreement with recent data on the extracellular effects of various lipocortins, these results might implicate lipocortin(s) in the anti-inflammatory effects of glucocorticosteroids.
A 32-kDa protein was isolated from human monocytes after calcium precipitation and chromatography. The protein activity was assessed by the inhibition of soluble phospholipase A 2 (PLA2). This in vitro inhibitory effect on phospholipases A 2 was found only with negatively charged phospholipids. The protein was also able to inhibit cellular PLA~ in mouse thymocytes. The biochemical properties and amino acid composition strongly suggest that the protein shares similarities with endonexin. Using a neutralizing monoclonal antibody against rat lipocortin, we found a cross-reactivity with the 32-kDa protein. According to the biochemical and immunological properties, we propose to relate this PLA 2 inhibitory protein from human monocytes to lipocortin.
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