Rodenburg R J, Ganga A, van Lent P L, van de Putte L B, van Venrooij W J
University of Nijmegen, The Netherlands.
Arthritis Rheum. 2000 Sep;43(9):1941-50. doi: 10.1002/1529-0131(200009)43:9<1941::AID-ANR4>3.0.CO;2-O.
Sulfasalazine (SSZ) is a commonly used drug in the treatment of inflammatory diseases such as rheumatoid arthritis and Crohn's disease. In both diseases, the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) plays a prominent role. In these studies, we investigated the mechanism by which SSZ inhibits TNFalpha expression in macrophages and macrophage-like cell lines.
Monocyte-derived macrophages and several macrophage-like cell lines were exposed to SSZ in vitro, and the effect on TNFalpha expression was monitored by reverse transcriptase-polymerase chain reaction and Western blot analysis. In addition, the effects of SSZ in vivo were examined by intraperitoneally injecting mice with SSZ, after which peritoneal cells were harvested and examined using various staining methods.
Preincubation of macrophages with SSZ, but not with methotrexate, inhibited lipopolysaccharide (LPS)-induced TNFalpha expression. Inhibition of TNFalpha expression by SSZ coincided with the induction of apoptosis, as judged by the appearance of morphologic changes typical of apoptosis, such as nuclear condensation and fragmentation. Induction of apoptosis by SSZ was confirmed by TUNEL analysis and by the detection of cleaved U1-70K, a substrate of caspase 3. Intraperitoneal injections of SSZ in mice resulted in the induction of apoptosis of peritoneal cells within a few hours. SSZ-induced cleavage of the U1-70K protein was inhibited by Zn2+ and by specific inhibitors of caspases 3 and 8, but not caspases 1 and 9. Interestingly, the reduced expression of LPS-induced TNFalpha in the presence of SSZ was restored by inhibition of caspase 8.
Inhibition of TNFalpha expression in macrophages by SSZ is due to the induction of apoptosis and involves the activation of caspase 8.
柳氮磺胺吡啶（SSZ）是治疗类风湿性关节炎和克罗恩病等炎症性疾病的常用药物。在这两种疾病中，促炎细胞因子肿瘤坏死因子α（TNFα）发挥着重要作用。在这些研究中，我们探究了SSZ抑制巨噬细胞和巨噬细胞样细胞系中TNFα表达的机制。
将单核细胞衍生的巨噬细胞和几种巨噬细胞样细胞系在体外暴露于SSZ，通过逆转录聚合酶链反应和蛋白质免疫印迹分析监测对TNFα表达的影响。此外，通过向小鼠腹腔注射SSZ来检测SSZ在体内的作用，之后收集腹腔细胞并使用各种染色方法进行检测。
用SSZ而非甲氨蝶呤预孵育巨噬细胞可抑制脂多糖（LPS）诱导的TNFα表达。SSZ对TNFα表达的抑制与细胞凋亡的诱导同时发生，这可通过凋亡典型形态学变化的出现来判断，如核浓缩和碎片化。通过TUNEL分析以及检测半胱天冬酶3的底物裂解的U1-70K证实了SSZ诱导的细胞凋亡。向小鼠腹腔注射SSZ会在数小时内导致腹腔细胞凋亡。Zn2+以及半胱天冬酶3和8的特异性抑制剂可抑制SSZ诱导的U1-70K蛋白裂解，但半胱天冬酶1和9的抑制剂则无此作用。有趣的是，通过抑制半胱天冬酶8可恢复在SSZ存在下LPS诱导的TNFα表达降低。
SSZ对巨噬细胞中TNFα表达的抑制是由于细胞凋亡的诱导，且涉及半胱天冬酶8的激活。
