Objective: 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.
Methods: 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.
Results: 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.
Conclusion: Inhibition of TNFalpha expression in macrophages by SSZ is due to the induction of apoptosis and involves the activation of caspase 8.