Inhibition by glucocorticoids of tumor necrosis factor-mediated cytotoxicityEvidence against lipocortin involvement (original) (raw)
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Inhibition by glucocorticoids of tumor necrosis factor‐mediated cytotoxicity
FEBS Letters, 1990
The role of the phospholipase inhibitor proteins, lipocortin‐I and ‐II, in tumor necrosis factor (TNF)‐mediated cytotoxicity against L929 fibrosarcoma cells was investigated. We previously reported that TNF‐mediated cytotoxicity was inhibited by dexamethasone (DEX), suggesting an involvement of lipocortins [1]. Now we show that, despite inhibition by DEX of TNF‐induced arachidonic acid release, DEX has no effect on the synthesis of these lipocortins. Moreover, TNF itself has no effect on the synthesis and phosphorylation of lipocortin‐I and ‐II. Also there was no difference in expression levels of lipocortin‐I and ‐II between TNF‐sensitive and ‐resistant cells. These data strongly suggest that the protective effect of DEX and other glucocorticoids is not mediated by lipocortins.
British Journal of Pharmacology, 1989
1 Prostanoid synthesis was induced in bone marrow-derived macrophages by addition of exogenous arachidonic acid to the cell cultures. When the cells were preincubated with dexamethasone (10'and 10 6m) overnight, prostaglandin synthesis was inhibited by 66.5 + 2.8% and 56.7 + 2.9% (mean + s.d.; n = 3) respectively. 2 Endogenous membrane bound phospholipase A2 was measured with labelled phospholipids used as substrates. The enzyme activity with phosphatidylcholine and phosphatidylethanolamine as substrates was inhibited by 27.0 + 8.3% and 23.3 + 11.1% (n = 4) respectively, in dexamethasonetreated macrophages compared to control cells. Neither the distribution of radiolabelled arachidonic acid among the different phospholipid species nor the release of arachidonic acid from prelabelled cells were significantly impaired by pretreatment of the macrophages with dexamethasone (1 gM).
Immunology
We have investigated the modulating effect of steroids on the in vitro production of tumour necrosis factor (TNF) by lipopolysaccharide (LPS)-stimulated human monocytes. Dexamethasone, at concentrations ranging from 10-8 to 10-6 M, and cortisol, at concentrations 10-7 and 10-6 M, suppressed the TNF production in a dose-dependent manner. The highest concentrations of dexamethasone or cortisol reduced the TNF production to 21 + 2% and 48 + 8% of the control value, respectively. The effect of dexamethasone was time dependent, and an incubation time of 48 hr was required to reduce the TNF production to 21 % of control. The effect of dexamethasone decreased when the incubation time became shorter, and the mean TNF production ranged from 49% to 72% of control when dexamethasone was added later than 8 hr before LPS addition, at the time of LPS addition, or within 1 hr after LPS addition. The magnitude of the TNF-suppressing effect of dexamethasone varied greatly from donor to donor. Only the glucocorticoids, and not the sex steroids or the mineralocorticoids, significantly reduced the TNF production.
Journal of Endocrinological Investigation, 1993
Adaptive responses to the environment depend on the induction of the "stress response" in less differentiated organisms and cultured cells and the activation of the hypothalamic-pituitary-adrenal axis in animals and humans. This indicates that adrenal steroids and stress proteins play an important role in regulating cell survival in response to noxious stimuli. In an in vitro model, we analyzed the effects of either dexamethasone (DEX) treatment or environmental changes which can elicit a stress response, on the survival of cultured L-929 mouse fibroblasts exposed to the cytotoxic cytokine tumor necrosis factor alpha (TNF-a). DEX treatment produced a significant reduction in the apoptotic death of L-929 cells produced by TNF-a. Abrogation of the protective effect of DEX by actinomycin D and cy-1This study was supported by grants from AIRC and PF ACRO to C.R., and Regione Umbria and MURST to I.N. .
A purified lipocortin shares the anti-inflammatory effect of glucocorticosteroids in vivo in mice
British Journal of Pharmacology, 1989
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.
European Journal of Biochemistry, 1991
L929, a murine fibrosarcoma cell line highly sensitive to the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF), was used as a target cell in our studies. We Biochem. Biophys. Res. Commun. 149, 735 -7431, as well as others, have previously provided evidence that a phospholipase (PL), most probably a PL-A,-type enzyme, is likely to be involved in TNF-mediated cell killing. We now further document this conclusion and provide suggestive evidence that the enzyme activity specifically involved in TNF cytotoxicity differs from activities associated with the eventual cell death process itself or with non-toxic serum treatment. We also show that the 5,8,11,14-icosatetraenoic acid (arachidonic acid, A4Ach) released by PL, and possibly metabolized, is unlikely to be a key mediator of the TNF-mediated cytotoxicity. These conclusions are based on the following experimental findings.
Role of lipocortin-1 in the anti-hyperalgesic actions of dexamethasone
British Journal of Pharmacology, 1997
1 The eect of dexamethasone, lipocorton-1 2 ± 26 and an antiserum to lipocortin-1 2 ± 26 (LCPS1) upon the hyperalgesic activities in rats of carrageenin, bradykinin, tumour necrosis factor a (TNFa), interleukin-1 2 , interleukin-6 (IL-6), interleukin-8 (IL-8), prostaglandin Eb (PGE 2 ) and dopamine were investigated in a model of mechanical hyperalgesia. 2 Hyperalgesic responses to intraplantar (i.pl.) injections of carrageenin (100 mg), bradykinin (500 ng), TNFa (2.5 pg), IL-1b (0.5 pg), and IL-6 (1.0 ng), but not responses to IL-8 (0.1 ng), PGE 2 (100 ng) and dopamine (10 mg), were inhibited by pretreatment with dexamethasone (0.5 mg kg 71 , subcutaneously, s.c., or 0.04 ± 5.0 mg/paw). 3 Inhibition of hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng) and IL-1b (0.5 pg) by dexamethasone (0.5 mg kg 71 , s.c.) was reversed by LCPS1 (0.5 ml kg 71 , injected s.c., 24 h and 1 h before hyperalgesic substances) and hyperalgesic responses to injections (i.pl.) of bradykinin (500 ng), TNFa (2.5 pg) and IL-1b (0.5 pg), but not responses to PGE 2 (100 ng), were inhibited by pretreatment with lipocortin-1 2 ± 26 (100 mg/paw). Also, lipocortin-1 2 ± 26 (30 and 100 mg ml 71 and dexamethasone (10 mg ml 71 ) inhibited TNFa release by cells of the J774 (murine macrophage-like) cell-line stimulated with LPS (3 mg ml 71 ), and LCPS1 partially reversed the inhibition by dexamethasone. These data are consistent with an important role for endogenous lipocortin-1 2 ± 26 in mediating the anti-hyperalgesic eect of dexamethasone, with inhibiton of TNFa production by lipocortin-1 2 ± 26 contributing, in part, to this role. 4 Although arachidonic acid by itself was not hyperalgesic, the hyperalgesic response to IL-1b (0.25 pg, i.pl.) was potentiated by arachidonic acid (50 mg) and the potentiated response was inhibited by dexamethasone (50 mg, i.pl.) and lipocortin-1 2 ± 26 (100 mg, i.pl.). Also, lipocortin-1 2 ± 26 (30 and 100 mg ml 71 ) inhibited/abolished PGE 2 release by J774 cells stimulated with LPS (3 mg ml 71 ). These data suggest that, in in¯ammatory hyperalgesia, inhibition of the induction of cyclo-oxygenase 2 (COX-2), rather than phospholipase A 2 , by dexamethasone and lipocortin-1 2 ± 26 accounts for the antihyperalgesic eects of these agents. 5 The above data support the notion that induction of lipocortin by dexamethasone plays a major role in the inhibition by dexamethasone of in¯ammatory hyperalgesia evoked by carrageenin, bradykinin and the cytokines TNFa, IL-1b and IL-6, and provides additional evidence that the biological activity of lipocortin resides within the peptide lipocortin-1 2 ± 26 . Further, the data suggest that inhibition of lipocortin-1 2 ± 26 of eicosanoid production by COX-2 also contributes to the anti-hyperalgesic eect of lipocortin-1.
Inhibition of O2- generation by dexamethasone is mimicked by lipocortin I in alveolar macrophages
Journal of Clinical Investigation, 1989
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.