Local amplification of glucocorticoids by 11 beta-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic leukocytes.

Glucocorticoids promote macrophage phagocytosis of leukocytes undergoing apoptosis. Prereceptor metabolism of glucocorticoids by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) modulates cellular steroid action. 11beta-HSD type 1 amplifies intracellular levels of active glucocorticoids in mice by reactivating corticosterone from inert 11-dehydrocorticosterone in cells expressing the enzyme. In this study we describe the rapid (within 3 h) induction of 11beta-HSD activity in cells elicited in the peritoneum by a single thioglycolate injection in mice. Levels remained high in peritoneal cells until resolution. In vitro experiments on mouse macrophages demonstrated that treatment with inert 11-dehydrocorticosterone for 24 h increased phagocytosis of apoptotic neutrophils to the same extent as corticosterone. This effect was dependent upon 11beta-HSD1, as 11beta-HSD1 mRNA, but not 11beta-HSD2 mRNA, was expressed in these cells; 11-dehydrocorticosterone was ineffective in promoting phagocytosis by Hsd11b1(-/-) macrophages, and carbenoxolone, an 11beta-HSD inhibitor, prevented the increase in phagocytosis elicited in wild-type macrophages by 11-dehydrocorticosterone. Importantly, as experimental peritonitis progressed, clearance of apoptotic neutrophils was delayed in Hsd11b1(-/-) mice. These data point to an early role for 11beta-HSD1 in promoting the rapid clearance of apoptotic cells during the resolution of inflammation and indicate a novel target for therapy.

Differential modulatory effects of annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages.

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-alpha) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-alpha production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1.