PROINFLAMMATORY AGENTS, IL-8 AND IL-10, UP-REGULATE INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION AND NITRIC-OXIDE PRODUCTION IN AVIAN OSTEOCLAST-LIKE CELLS

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a s ensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in r esponse to proinflammatory agents, many of which also regulate local b one metabolism. NO is a potent inhibitor of osteoclast bone resorption , whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated INOS and produce NO as a potential autocrine signal followi ng inflammatory stimulation was investigated in well-characterized avi an marrow-derived osteoclast-like cells. NO production (reflected by m edium nitrite levels) was markedly elevated in these cells by the proi nflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1 alpha, TNF alpha, and IFN gamma. Inhibitors of NOS activity ( aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGF beta) re duced LPS-stimulated nitrite production. LPS also increased the NOS-as sociated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human INOS primers, yielded a novel 900 bp cDNA with high seq uence homology (76%) to human, rat, and mouse iNOS genes. In probing o steoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induc tion of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred pr ior to the rise of medium nitrite in time course studies and was dimin ished by dexamethasone. Moreover, osteoclast-like cells demonstrated a n upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regul atory mechanism's not previously described. It is concluded that osteo clast-like cells express a novel INOS that is upregulated by inflammat ory mediators, leading to NO production. Therefore, NO may serve as bo th a paracrine and autocrine signal for modulating osteoclast bone res orption. (C) 1996 Wiley-Liss, Inc.