Inhibition of neutrophil-mediated chondrocyte cytotoxicity by nitric oxide (NO) generated by articular chondrocytes.

一氧化氮(NO)是L精氨酸氧化裂解的活性产物。NO由内皮细胞、巨噬细胞和中性粒细胞产生，在类风湿滑液和血清中检测到较高浓度的亚硝酸盐，这是NO的稳定终产物。最近报道，NO抑制中性粒细胞超氧阴离子的产生，并通过这种方式与炎症过程中的组织降解有关。我们已经报道了关节软骨细胞被激活的中性粒细胞产生的过氧化氢损伤。在本研究中，我们研究了NO在中性粒细胞介导的软骨细胞毒性中的作用。用胶原酶消化法从牛关节软骨获得原代单层软骨细胞培养，并从健康人分离中性粒细胞。在中性粒细胞存在的情况下，通过预先标记的软骨细胞释放铬-51(^&lt；51&gt；Cr)来测量软骨细胞的细胞毒性。用Grisse反应测定NO浓度作为亚硝酸盐浓度。关节软骨细胞在白细胞介素1α(IL-1α)和肿瘤坏死因子-α(TNF-α)刺激24小时后合成NO，并呈剂量依赖性。在100U L精氨酸存在下，200U/mlIL-1α可显著(P&lt；0.01)产生2M亚硝酸当量的NO。中性粒细胞促进关节软骨细胞释放铬，在中性粒细胞-软骨细胞共培养中加入100mU的L-精氨酸可显著逆转这种细胞毒性。提示关节软骨细胞产生的NO对炎症软骨的氧化应激具有保护作用。

Nitric oxide (NO) was identified as an active product of the oxidative cleavage of L-arginine. NO is generated by endothelial cells, macrophages and neutrophils and the higher concentration of nitrite, which is the stable endproduct of NO,is detected in the rheumatoid synovial fluid and serum. It is recently reported that NO inhibits neutrophil superoxide anion production and, in this way, associated with tissue degradation in inflammatory process. We have reported that articular chondrocytes were damaged by hydrogen peroxide generated by activated neutrophils. We have studied, in this investigation, the effect of NO on neutrophil-mediated chondrocyte cytotoxicity. Primary monolayr culture of chondrocytes was obtained from bovine articular cartilage by collagenase digestion and neutrophils were separated from healthy human doners. Chondrocyte cytotoxicity was measured by chromium-51 (^<51>Cr) release from the prelabeled chondrocytes in the presence of neutrophils. The concentration of NO was measured as nitrite concentration by Grisse reaction. Articular chondrocytes synthesized NO by the stimulation of interleukin (IL-1alpha) and TNF-alpha for 24h in a dose-dependent fashion. In the presence of 100 muM of L-arginine, 200 U/ml of IL-1alpha yielded the significant generation (P<0.01) of NO (2muM nitrite equivalent) from chondrocytes. Neutrophils increased ^<51>Cr-release from articular chondrocytes, and this chondrocyte cytotoxicity was significantly (p<0.05) reversed by addition of 100 muM of L-arginine in the neutrophil-chondrocyte co-cultures In contrast, D-arginine failed to inhibit the neutrophil mediated chondrocyte cytotoxicity. These result suggested that NO generated from articular chondrocytes have a protective effect against the oxidative stress in inflammatory cartilage.