Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of caspase pathway

P>Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti-inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC). Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2-12 h with or without 10, 30 or 100 mu mol/L SSZ and 25 mu g/mL IC. In some experiments, cells were co-incubated with 20 mu mol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 mu mol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grunwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. At 100 mu mol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 mu mol/L of the caspase 8 or 9 inhibitors Z-IETD-fmk or Z-LEHD-fmk, respectively, demonstrated that the SSZ-induced pro-apoptotic effect was mediated by a caspase 8- but not caspase 9-dependent pathway. The caspase 3 activity assay showed that treatment with 100 mu mol/L SSZ increased caspase 3 activation. In conlusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil-mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8-dependent pathway.