Jasin H E, Noyori K, Takagi T, Taurog J D
Division of Rheumatology and Clinical Immunology, University of Arkansas for Medical Sciences, Little Rock 72205.
Arthritis Rheum. 1993 May;36(5):651-9. doi: 10.1002/art.1780360512.
Previous studies suggested that it was possible to characterize the intact surface of articular cartilage by probing it with antibodies against matrix macromolecules. The present studies were undertaken to investigate type II collagen (CII) epitope availability on the intact surface of articular cartilage.
Normal bovine, rabbit, and human cartilage specimens were used to measure binding of anti-CII antibodies to the articular and cut surfaces of cartilage. Antisera were raised against the material obtained after brief extraction of the cartilage surface with 4M guanidine solution.
Anti-CII did not bind to the intact surface of rabbit articular cartilage when compared with control rat sera, but did bind significantly to the cut surface. With normal human articular cartilage, the cut surfaces bound approximately 4 times as much anti-CII antibody as the intact articular surfaces. These findings suggested that the CII epitopes are normally protected by the superficial cartilage layer. In experiments carried out to characterize this layer, binding of anti-CII antibodies was unchanged after exhaustive washing of bovine or rabbit cartilage with phosphate buffered saline or 1M NaCl solution, whereas it was significantly increased after brief exposure to 4M guanidine solution or after incubation with neutrophil elastase. No restoration of the protection of CII epitopes in guanidine-treated cartilage could be achieved by incubation with undiluted normal bovine synovial fluid; however, 8-day culture of elastase-treated cartilage explants resulted in partial restoration of protection of the CII epitopes. Rat and rabbit antisera raised against the cartilage surface material extracted with 4M guanidine contained antibodies that bound to the cartilage surface. By Western blotting, rat antibodies identified 50-65-kd protein bands present in the guanidine extract, but not present either in the material obtained from brief digestion of cartilage with neutrophil elastase or in synovial fluid. The rabbit antisera identified a broad 70-95-kd band. Exposure of elastase-treated cartilage to the guanidine-extracted material resulted in a partial decrease of anti-CII antibody binding.
These results suggest that CII on intact cartilage is protected from antibody binding, and that the protective material is at least partly proteinaceous in nature, is unlikely to be derived from synovial fluid, is noncovalently bound to the underlying intercellular matrix, and is synthesized by resident chondrocytes. Further characterization of the protective material may provide important information on the mechanisms of early cartilage damage in inflammatory arthritis.
先前的研究表明，通过用针对基质大分子的抗体探测关节软骨的完整表面来对其进行表征是可行的。本研究旨在调查关节软骨完整表面上II型胶原（CII）表位的可及性。
使用正常牛、兔和人的软骨标本测量抗CII抗体与软骨关节面和切割面的结合情况。用4M胍溶液对软骨表面进行短暂提取后获得的材料制备抗血清。
与对照大鼠血清相比，抗CII不与兔关节软骨的完整表面结合，但与切割面有显著结合。对于正常人关节软骨，切割面结合的抗CII抗体量约为完整关节面的4倍。这些发现表明CII表位通常受到表层软骨的保护。在表征该层的实验中，用磷酸盐缓冲盐水或1M氯化钠溶液彻底洗涤牛或兔软骨后，抗CII抗体的结合未发生变化，而在短暂暴露于4M胍溶液后或与中性粒细胞弹性蛋白酶孵育后，结合显著增加。用未稀释的正常牛滑液孵育胍处理的软骨，无法恢复对CII表位的保护；然而，弹性蛋白酶处理的软骨外植体培养8天导致对CII表位的保护部分恢复。用4M胍提取的软骨表面材料制备的大鼠和兔抗血清含有与软骨表面结合的抗体。通过蛋白质印迹法，大鼠抗体识别出胍提取物中存在但在中性粒细胞弹性蛋白酶短暂消化软骨获得的材料或滑液中不存在的50 - 65kd蛋白条带。兔抗血清识别出一条宽的70 - 95kd条带。将弹性蛋白酶处理的软骨暴露于胍提取物中导致抗CII抗体结合部分减少。
这些结果表明完整软骨上的CII免受抗体结合，保护物质至少部分是蛋白质性质的，不太可能源自滑液，与下方的细胞间基质非共价结合，并且由驻留软骨细胞合成。对保护物质的进一步表征可能为炎性关节炎中早期软骨损伤的机制提供重要信息。
