RESUMEN
OBJECTIVE: To determine whether rabbit synovial fibroblasts can synthesize nitric oxide (NO) and, if so, how production is regulated by cytokines. METHODS: Primary cultures of synovial fibroblasts (type B synoviocytes) were established from synovia excised from the knee joints of New Zealand white rabbits. Synthesis of NO was measured as nitrite accumulation in conditioned media in the presence or absence of various cytokines and other activators. RESULTS: Resting cultures of synoviocytes normally produced little or no NO. However, production of this free radical was induced by interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha) or the phagocytosis of latex beads; in some cultures, the synthesis of NO occurred spontaneously. In each case, NO synthesis began approximately 9 h after the addition of cytokines, suggesting the involvement of an inducible form of NO synthase. Antagonists of this phenomenon included interferon gamma (IFN-gamma), which weakly inhibited NO production, and transforming growth factor beta (TGF-beta), a very strong inhibitor. Platelet derived growth factor (PDGF) inhibited NO synthesis by cells stimulated with IL-1, but not by cells stimulated with TNF-alpha. Synovial autocrine factors (CAF) modestly induced NO synthesis, but inhibited synthesis by IL-1; TGF-beta was identified as an inhibitory component of CAF. Phorbol myristate acetate (PMA) had only a small inductive effect, and inhibited induction by IL-1. However, the protein kinase inhibitor staurosporin was a strong inducer. Modulators of cyclic nucleotides, in contrast, had relatively modest effects on NO synthesis. Inhibition of NO biosynthesis by NG-monomethyl-L-arginine (NMA) had no effect upon the increase in the production of prostaglandin E2 (PGE2), matrix metalloproteinases (MMP) or lactate by synoviocytes responding to IL-1. The rabbit synoviocyte cell line, HIG-82, did not synthesize detectable NO under any of the culture conditions tested. CONCLUSION: Synoviocytes are a potential source of NO in arthritic joints.
Asunto(s)
Citocinas/fisiología , Fibroblastos/metabolismo , Óxido Nítrico/biosíntesis , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Alcaloides/farmacología , Animales , Línea Celular , Células Cultivadas , Citocinas/farmacología , Dinoprostona/metabolismo , Fibroblastos/citología , Radicales Libres , Interferón gamma/farmacología , Interleucina-1/farmacología , Metaloendopeptidasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Conejos , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND AND METHODS: Nitric oxide synthesis occurs both in vitro and in vivo in response to inflammatory stimuli and can have profound effects on the local cellular environment. Hepatocytes, Kupffer cells, and endothelial cells produce nitric oxide in vitro, but the in vivo role of this reactive mediator in the liver is unknown. We assessed the role of nitric oxide synthesis during endotoxemia in mice by inhibiting its synthesis with NG-monomethyl-L-arginine after lipopolysaccharide injection and by determining the effects of this inhibition on hepatic damage. RESULTS: Injection of lipopolysaccharide in mice increased plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, and caused mild hepatic damage as measured by increased circulating hepatocellular enzyme levels. NG-monomethyl-L-arginine decreased plasma nitrite and nitrate values, but increased the lipopolysaccharide-induced hepatic injury. NG-monomethyl-L-arginine caused no hepatic damage when given without lipopolysaccharide. The extent of hepatic damage with NG-monomethyl-L-arginine was proportional to the dose of lipopolysaccharide used and could be reduced with concurrent administration of L-arginine but not D-arginine. CONCLUSIONS: Nitric oxide synthesis provides a protective function against lipopolysaccharide-induced liver injury that increases in importance as the degree of endotoxemia increases. The production of nitric oxide is, therefore, an important part of the liver's response to a systemic inflammatory stimulus.