Tiku K, Thakker-Varia S, Ramachandrula A, Tiku M L
Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson, Medical School, New Brunswick 08903-0019.
Cell Immunol. 1992 Mar;140(1):1-20. doi: 10.1016/0008-8749(92)90172-l.
Previous work from our laboratory has shown that rabbit articular chondrocytes, like macrophages, produce reactive oxygen intermediates, express Ia antigen, and can mediate immunologic functions such as antigen presentation and induction of mixed and autologous lymphocyte reactions. We were interested in seeing if these cells could secrete interleukin-1 (IL-1) or express membrane form of IL-1 (mIL-1). Using the standard C3H/HeJ thymocyte assay, neither secreted IL-1 nor mIL-1 activity was detected in untreated or LPS-treated chondrocytes. However, the D10.G4.1 proliferation assay showed that chondrocytes, stimulated with LPS, secrete IL-1 and express the mIL-1 in a dose- and time-dependent manner. The IL-1 activity in LPS-stimulated chondrocyte supernatant and on fixed cells could be inhibited by anti-IL-1 antibodies. Sephadex G-75 chromatography of pooled, concentrated LPS culture supernatant resolved into two peaks of IL-1 activity at 13-17 and at 45-70 kDa, respectively. The bioactivity of chromatographic fractions were similar using both the thymocyte and D10.G4.1 bioassays. Western blot analysis of chondrocyte supernatant detects 17-kDa IL-1 beta; no processed 17-kDa IL-1 alpha was seen but IL-1 alpha-specific reactivity was observed at 64 kDa. Immunoblot analysis of chondrocyte lysates shows that cell-associated IL-1 is IL-1 alpha and is 37 kDa in size. PCR analysis shows the presence of mRNA for IL-1 beta and IL-1 alpha in LPS-treated cells; IL-1 beta mRNA was detected in untreated chondrocytes. The inability to detect IL-1 by the thymocyte assay is due to the presence of a chondrocyte inhibitor of IL-1 that can be demonstrated in cell sonicates, supernatants, and on paraformaldehyde-fixed chondrocytes. Chromatography of LPS-stimulated supernatant showed a peak of IL-1 inhibitory activity at 21-45 kDa. Chondrocytes which secrete IL-1 and express mIL-1 could play a critical role in maintaining chronic inflammation in rheumatoid arthritis. Therefore, the ability of chondrocytes to produce both IL-1 and an inhibitor to IL-1 is important in interpreting the mechanism of cartilage matrix maintenance and degradation.
我们实验室之前的研究表明，兔关节软骨细胞与巨噬细胞一样，能产生活性氧中间体，表达Ia抗原，并可介导免疫功能，如抗原呈递以及诱导混合淋巴细胞反应和自体淋巴细胞反应。我们想了解这些细胞是否能分泌白细胞介素-1（IL-1）或表达IL-1的膜形式（mIL-1）。使用标准的C3H/HeJ胸腺细胞检测法，在未处理或经脂多糖（LPS）处理的软骨细胞中均未检测到分泌的IL-1或mIL-1活性。然而，D10.G4.1增殖检测显示，经LPS刺激的软骨细胞以剂量和时间依赖性方式分泌IL-1并表达mIL-1。LPS刺激的软骨细胞上清液和固定细胞上的IL-1活性可被抗IL-1抗体抑制。对合并、浓缩的LPS培养上清液进行葡聚糖G-75层析，分别在13 - 17 kDa和45 - 70 kDa处解析出两个IL-1活性峰。使用胸腺细胞和D10.G4.1生物检测法，层析组分的生物活性相似。对软骨细胞上清液进行蛋白质印迹分析可检测到17 kDa的IL-1β；未观察到加工后的17 kDa的IL-1α，但在64 kDa处观察到IL-1α特异性反应性。对软骨细胞裂解物进行免疫印迹分析表明，与细胞相关的IL-1是IL-1α，大小为37 kDa。聚合酶链反应（PCR）分析显示，在经LPS处理的细胞中存在IL-1β和IL-1α的信使核糖核酸（mRNA）；在未处理的软骨细胞中检测到IL-1β mRNA。胸腺细胞检测法无法检测到IL-1是由于软骨细胞中存在IL-1抑制剂，这在细胞超声裂解物、上清液以及经多聚甲醛固定的软骨细胞中均可得到证实。对LPS刺激的上清液进行层析显示，在21 - 45 kDa处有一个IL-1抑制活性峰。分泌IL-1并表达mIL-1的软骨细胞可能在类风湿性关节炎慢性炎症的维持中起关键作用。因此，软骨细胞产生IL-1及其抑制剂的能力对于解释软骨基质维持和降解机制很重要。
