Effect Of Lipocortin Research Articles

Glucocorticoid inhibition of adjuvant arthritis synovial macrophage nitric oxide production: role of lipocortin 1

Nitric oxide (NO) is a mediator of inflammatory injury which is inhibited by glucocorticoids and is implicated in rheumatoid (RA) and adjuvant arthritis (AA). The glucocorticoid-induced anti-inflammatory molecule lipocortin 1 is expressed in RA synovium, but the effects of lipocortin 1 on synovial inflammation have been little studied. We investigated the effects of glucocorticoids and lipocortin 1 on inducible NO synthase (iNOS) and glucocorticoids on the induction of lipocortin 1 in AA synovial macrophages. NO production was measured by Griess assay in supernatants of day 14 AA rat synovial explants and of synovial macrophages purified from enzyme-digested synovium and treated with lipopolysaccharide (LPS) 1 microg/ml, dexamethasone (DEX) 10(-7) M, and anti-lipocortin 1 MoAb. iNOS and lipocortin 1 expression were detected by flow cytometry using specific MoAb. Cell surface lipocortin was determined by Western blot. NO was produced by all AA synovial explants and NO was released by cultured synovial macrophages (14.5 +/- 2.1 micromol/24 h). iNOS was detected in synovial macrophages (ED-1+) by permeabilization flow cytometry. LPS increased synovial macrophage NO release (P < 0.0001) and iNOS expression (P = 0.04). DEX inhibited constitutive (P = 0.002) and LPS-induced (P < 0.001) NO release and iNOS expression (P = 0.03). DEX inhibition of synovial macrophage NO was associated with induction of cell surface and intracellular lipocortin 1. Anti-lipocortin 1 MoAb treatment reduced the inhibition of NO release by DEX (P = 0.002), but had no effect on iNOS expression. These findings demonstrate a role for lipocortin I in the inhibition by glucocorticoids of AA synovial macrophage iNOS activity.

Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: effect of lipocortin 1.

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5-6 cells per 100-micrometers of vessel length) and emigration (maximal at 4 hr: 8-10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2-26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (>/=50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2-26 (13 mg/kg) or recombinant human LC1 (0.7-2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 +/- 0.75 min and 2.36 +/- 0.31 min, respectively (n = 20-25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.

The effect of lipocortin 1 on neutrophil deformability

Lipocortn 1 (Lc1) is an anti-inflammatory protein, which, given systemically, inhibits polymorphonuclear neutrophil (PMN) emigration from the circulation to sites of inflammation; delivery of Lc1 to the inflamed site is ineffective. We have examined the effect of Lc1 on changes in PMN deformability, and observed a consistent improvement in the deformability of unstimulated PMN; N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated cell deformability was unaltered. A Lc1-induced increase in cell deformability may reduce PMN sequestration so contributing to the anti-migratory effects of systemic Lc1 previously demonstrated in vivo.

Effects of lipocortin 1 and dexamethasone on the secretion of corticotrophin-releasing factors in the rat: in vitro and in vivo studies.

Lipocortin 1 (LC1: also called annexin 1) was first described as a putative second messenger protein for the anti-inflammatory steroids in peripheral tissues. In the present study, in vitro and in vivo methods were used to examine its potential role within the hypothalamus as a mediator of the regulatory actions of the glucocorticoids on the hypothalamo-pituitary-adrenocortical axis of the rat. In the in vitro studies, the effects of human recombinant LC1 (hu-r-LC1) on the concomitant release of the two major corticotrophin-releasing factors (CRF-41 and arginine vasopressin, AVP) from isolated hypothalami removed from chronically adrenalectomized rats were compared with those of dexamethasone in the presence and absence of appropriate secretagogues, namely phospholipase A2 (PLA2), interleukin-6 (IL-6) and a non-specific depolarizing agent, K+ (56 mM). The spontaneous release of CRF-41 in vitro was unaffected by either hu-r-LC1 (5 to 100 ng/ml) or dexamethasone (1 microM). Both compounds however reduced the release of the neuropeptide evoked by IL-6 (5 ng/ml) but failed to modify the secretory responses to PLA2 (25 U/ml) or K+ (56 mM). Dexamethasone (1 microM) had no effect on the basal release of AVP but effectively blocked the secretion of the peptide induced by either IL-6 (10 ng/ml) or PLA2 (25 U/ml). In complete contrast, hu-r-LC1 (5 to 100 ng/ml) stimulated the release of AVP and potentiated the secretory responses to IL-6 (10 ng/ml) and PLA2 (25 U/ml) but not to K+ (56 mM). The hypothalamic responses to PLA2 stimulation (25 U/ml) were associated with significant (P < 0.01) increases in prostaglandin E2 release which, in some instances, were potentiated by hu-r-LC1 (5 to 20 ng/ml). In vivo, administration of histamine (0.6 mg/100 g body wt, ip) produced significant (P < 0.01) increases in the serum corticosterone concentration and in the hypothalamic LC1 content. Neither hu-r-LC1 (0.6 to 1.2 micrograms) nor a polyclonal anti-LC1 antibody (3 microliters, diluted 1:200), injected intracerebroventricularly (icv), influenced either the resting serum corticosterone concentration or the hypersecretion of the steroid evoked by histamine stress. A lower dose of the recombinant protein (0.3 micrograms icv) also failed to alter basal corticosterone release but, in contrast to the higher doses, potentiated the pituitary-adrenocortical responses to histamine. The results suggest that LC1 may contribute to some aspects of peptide release in the hypothalamus but that its actions are not necessarily related to those of the glucocorticoids.