Henrotin Y E, Zheng S X, Labasse A H, Deby G P, Crielaard J M, Reginster J Y
Bone and Cartilage Metabolism Research Unit, University Hospital, CHUSart-Tilman, 4000 Liège, Belgium.
Osteoarthritis Cartilage. 2000 Nov;8(6):474-82. doi: 10.1053/joca.1999.0323.
Interferon gamma (IFN gamma) is found to be elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis, suggesting its implication in joint disease pathogenesis. In this study, we investigated the effects of IFN gamma on the production of cytokines (IL-6, IL-8, IL-10), prostaglandin E(2)(PGE(2)), proteoglycans (PG), nitric oxide (NO), interleukin-1 receptor antagonist (IL-1ra) and stromelysin by non-stimulated and IL-1 beta-treated human chondrocytes. The role played by NO in the responses of chondrocytes to IFN gamma was also examined by incubation of chondrocytes with N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase.
Enzymatically isolated human chondrocytes were cultured for 48 h in the absence or presence of IL-1 beta, IFN gamma or N(G)-monomethyl-L-arginine (L-NMMA) added solely or in combination. The productions of IL-6, IL-8, IL-10, IL-1ra and stromelysin were measured by enzyme amplified sensitivity immunoassays (EASIA). PG and PGE(2)were quantified by specific radioimmunoassays (RIA). Nitrite concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction.
As expected, IL-1 beta highly stimulated NO, IL-6, IL-8, IL-10, IL-1ra, PGE(2)and stromelysin synthesis, but dramatically decreased PG production. NO, IL-6, IL-1ra and PGE(2)production by non-stimulated chondrocytes was dose-dependently increased by IFN gamma while PG production was inhibited. In the absence of IL-1 beta, IL-10 was undetectable in the culture supernatants. At the doses of 10 and 100 U/ml, IFN gamma markedly inhibited the constitutive and IL-1 beta-stimulated IL-8, IL-10 and stromelysin productions. Interestingly, IFN gamma synergized with IL-1 beta to increase NO, IL-6, IL-1ra and to depress PG production. As previously reported, the inhibition of NO synthesis by the competitive inhibitor L-NMMA led to enhancement of IL-6, IL-8 and PGE(2)production by IL-1 beta treated chondrocytes, but did not significantly modify IL-10, PG and MMP-3 productions. Inhibition of NO synthase significantly inhibited the stimulating effect of IFN gamma on IL-6 and IL-1ra but did not affect the inhibitory effect of IFN gamma on IL-8, PG or stromelysin production.
These findings suggest that IFN gamma and IL-1 synergistically stimulate the production of IL-6, IL-1ra, NO and PGE(2)and inhibit PG synthesis. By contrast, IL-1 beta and IFN gamma have opposite effects on IL-8, IL-10 and stromelysin productions. These effects are not reversed by L-NMMA, suggesting that NO is not the principal mediator involved in responses of chondrocytes to IFN gamma.
研究发现，类风湿性关节炎和骨关节炎患者滑液中的γ干扰素（IFNγ）水平升高，提示其与关节疾病发病机制有关。在本研究中，我们调查了IFNγ对未受刺激和经白细胞介素-1β（IL-1β）处理的人软骨细胞产生细胞因子（IL-6、IL-8、IL-10）、前列腺素E2（PGE2）、蛋白聚糖（PG）、一氧化氮（NO）、白细胞介素-1受体拮抗剂（IL-1ra）和基质溶解素的影响。通过用NO合酶的竞争性抑制剂N（G）-单甲基-L-精氨酸（L-NMMA）孵育软骨细胞，还研究了NO在软骨细胞对IFNγ反应中所起的作用。
将酶解分离的人软骨细胞在单独或联合添加IL-1β、IFNγ或N（G）-单甲基-L-精氨酸（L-NMMA）的情况下培养48小时。通过酶放大敏感性免疫测定法（EASIA）测量IL-6、IL-8、IL-10、IL-1ra和基质溶解素的产生。通过特异性放射免疫测定法（RIA）对PG和PGE2进行定量。基于Griess反应的分光光度法测定培养上清液中的亚硝酸盐浓度。
正如预期的那样，IL-1β高度刺激NO、IL-6、IL-8、IL-10、IL-1ra、PGE2和基质溶解素的合成，但显著降低PG的产生。IFNγ剂量依赖性地增加未受刺激软骨细胞产生的NO、IL-6、IL-1ra和PGE2，同时抑制PG的产生。在没有IL-1β的情况下，培养上清液中未检测到IL-10。在10和100 U/ml的剂量下，IFNγ显著抑制组成性和IL-1β刺激的IL-8、IL-10和基质溶解素的产生。有趣的是，IFNγ与IL-1β协同作用以增加NO、IL-6、IL-1ra并降低PG的产生。如先前报道的那样，竞争性抑制剂L-NMMA抑制NO合成导致IL-1β处理的软骨细胞产生的IL-6、IL-8和PGE2增加，但对IL-10、PG和基质金属蛋白酶-3的产生没有显著影响。抑制NO合酶显著抑制IFNγ对IL-6和IL-1ra的刺激作用，但不影响IFNγ对IL-8、PG或基质溶解素产生的抑制作用。
这些发现表明，IFNγ和IL-1协同刺激IL-6、IL-1ra、NO和PGE2的产生并抑制PG合成。相比之下，IL-1β和IFNγ对IL-8、IL-10和基质溶解素的产生具有相反的作用。L-NMMA不会逆转这些作用，表明NO不是软骨细胞对IFNγ反应中涉及的主要介质。
