Amin A R, Di Cesare P E, Vyas P, Attur M, Tzeng E, Billiar T R, Stuchin S A, Abramson S B
Department of Rheumatology, Hospital for Joint Diseases Orthopaedic Institute, New York 10003, USA.
J Exp Med. 1995 Dec 1;182(6):2097-102. doi: 10.1084/jem.182.6.2097.
Classically, osteoarthritis (OA) has been considered a noninflammatory disease. However, the detection of selected inflammatory mediators in osteoarthritic fluid, in the absence of significant inflammatory cell infiltrate, is increasingly appreciated. We sought to identify the inflammatory component in human OA-affected cartilage that may be involved in cartilage damage/destruction. Using Western blot analysis and an antibody to the conserved region of nitric oxide synthase (NOS), we have observed up-regulation of NOS, one of the "key players" of inflammation, in chondrocytes of OA-affected patients. Remarkably, none of the cartilage samples examined from normal joints demonstrated detectable amounts of this NOS. Western blot analysis using the same alpha-NOS antibody indicated that this NOS from OA-affected cartilage (OA-NOS) was larger in size than (and distinct from) transfected human hepatocyte or murine inducible NOS (iNOS) (150 versus 133 kD) and similar in size to neuronal constitutive NOS (ncNOS). Antibodies specific for iNOS showed binding to murine and human iNOS but not to OA-NOS, endothelial constitutive NOS, or ncNOS. Antibodies specific for ncNOS bound to ncNOS and also to OA-NOS, but not to murine or human iNOS or endothelial constitutive NOS. Incubation of OA cartilage in serum-free medium resulted in spontaneous release, for up to 72 h, of substantial amounts of nitrite (up to approximately 80 microM/100 mg wet tissue), which could be inhibited by at least 80% with various inhibitors of iNOS, including inhibitors of protein synthesis and transcription factor NF-kappa B, but which (unlike murine macrophage iNOS) was not sensitive to hydrocortisone or TGF-beta. Exposure of OA-affected cartilage to interleukin 1 beta, tumor necrosis factor-alpha, and lipopolysaccharide resulted in approximately 20-50% augmentation of nitrite accumulation, which was also sensitive to cycloheximide and pyrrolidine dithiocarbamate. Hence, our data indicate that OA-NOS (based on immunoreactivity and molecular weight) is similar to ncNOS and that it releases nitric oxide, which may contribute to the inflammation and pathogenesis of cartilage destruction in OA.
传统上，骨关节炎（OA）一直被认为是一种非炎症性疾病。然而，在骨关节炎关节液中检测到某些炎症介质，且不存在明显的炎症细胞浸润，这一点越来越受到重视。我们试图确定人类OA病变软骨中可能参与软骨损伤/破坏的炎症成分。使用蛋白质印迹分析和针对一氧化氮合酶（NOS）保守区域的抗体，我们观察到在OA患者的软骨细胞中，炎症的“关键参与者”之一——NOS上调。值得注意的是，从正常关节检查的软骨样本中均未检测到这种NOS。使用相同的α-NOS抗体进行蛋白质印迹分析表明，来自OA病变软骨的这种NOS（OA-NOS）在大小上比转染的人肝细胞或小鼠诱导型NOS（iNOS）更大（且不同）（150 kDa对133 kDa），并且在大小上与神经元组成型NOS（ncNOS）相似。针对iNOS的特异性抗体显示与小鼠和人iNOS结合，但不与OA-NOS、内皮组成型NOS或ncNOS结合。针对ncNOS的特异性抗体与ncNOS结合，也与OA-NOS结合，但不与小鼠或人iNOS或内皮组成型NOS结合。将OA软骨在无血清培养基中孵育长达72小时，会自发释放大量亚硝酸盐（高达约80 μM/100 mg湿组织），各种iNOS抑制剂，包括蛋白质合成抑制剂和转录因子NF-κB抑制剂，可将其抑制至少80%，但（与小鼠巨噬细胞iNOS不同）它对氢化可的松或转化生长因子-β不敏感。将OA病变软骨暴露于白细胞介素1β、肿瘤坏死因子-α和脂多糖会导致亚硝酸盐积累增加约20 - 50%，这也对环己酰亚胺和吡咯烷二硫代氨基甲酸盐敏感。因此，我们的数据表明，OA-NOS（基于免疫反应性和分子量）与ncNOS相似，并且它释放一氧化氮，这可能导致OA中软骨破坏的炎症和发病机制。
