Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of caspase pathway.
1. 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). 2. 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 micromol/L SSZ and 25 microg/mL IC. In some experiments, cells were co-incubated with 20 micromol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 micromol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grünwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. 3. At 100 micromol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 micromol/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 micromol/L SSZ increased caspase 3 activation. 4. In conclusion, 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.
Bertolotto
M
M
Clinic of Internal Medicine I, Department of Internal Medicine, University of Genoa, Medical School, Genoa, Italy.
Dallegri
F
F
Dapino
P
P
Quercioli
A
A
Pende
A
A
Ottonello
L
L
Montecucco
Fabrizio
F
D000936:Antigen-Antibody Complex / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D012460:Sulfasalazine / Q000494:pharmacology*
D000936:Antigen-Antibody Complex; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D012460:Sulfasalazine; D020169:Caspases