CYSTATINS UP-REGULATE NITRIC-OXIDE RELEASE FROM INTERFERON-GAMMA-ACTIVATED MOUSE PERITONEAL-MACROPHAGES

Up-regulation of nitric oxide (NO) production by activated murine macr ophages was observed during infection by Trypanosoma cruzi, the etiolo gical agent of Chagas' disease. Cell infection by T. cruzi depends at least in part on cruzipain, a membrane-associated papain-related prote inase which is sensitive to inhibition by synthetic inhibitors of cyst eine proteinases. Using the natural cysteine proteinase inhibitor chic ken cystatin, a representative member of cystatin family 2, to investi gate the effect of cruzipain on macrophage infection and NO release, w e found that the inhibitor alone up-regulated NO release from interfer on-gamma-activated macrophages. A 12-fold increase in NO production wa s observed in the presence of 1 mu M chicken cystatin. This overproduc tion was concentration-dependent and could be detected at concentratio ns as low as 10 nM and remained in the presence of polymyxin B. Repres entative members of the other cystatin families, i.e. stefin B (family 1), T-kininogen, and its inhibitory domains (family 3), were also abl e to enhance NO production from interferon-gamma-activated macrophages . Neither E64, an irreversible inhibitor of cysteine proteinases, nor inhibitors of aspartyl and serine proteinases (aprotinin, pepstatin, a nd soybean trypsin inhibitor) enhanced NO production. Upon complexatio n with saturating amounts of reduced-alkylated papain, cystatins still remained active in increasing NO production, suggesting that the cyst atin inhibitory site was not involved in the mechanism. The results de monstrate that members of all 3 cystatin families share another common property unrelated to their function of cysteine proteinase inhibitor s, i.e. upregulation of NO production, which biological significance r emains to be elucidated.