Up-regulation of nitric oxide (NO) production by activated murine macrophages was observed during infection by Trypanosoma cruzi, the etiological agent of Chagas' disease. Cell infection by T. cruzi depends at least in part on cruzipain, a membrane-associated papain-related proteinase which is sensitive to inhibition by synthetic inhibitors of cysteine proteinases. Using the natural cysteine proteinase inhibitor chicken cystatin, a representative member of cystatin family 2, to investigate the effect of cruzipain on macrophage infection and NO release, we found that the inhibitor alone up-regulated NO release from interferon-␥-activated macrophages. A 12-fold increase in NO production was observed in the presence of 1 M chicken cystatin. This overproduction was concentration-dependent and could be detected at concentrations as low as 10 nM and remained in the presence of polymyxin B. Representative members of the other cystatin families, i.e. stefin B (family 1), T-kininogen, and its inhibitory domains (family 3), were also able to enhance NO production from interferon-␥-activated macrophages. Neither E64, an irreversible inhibitor of cysteine proteinases, nor inhibitors of aspartyl and serine proteinases (aprotinin, pepstatin, and soybean trypsin inhibitor) enhanced NO production. Upon complexation with saturating amounts of reduced-alkylated papain, cystatins still remained active in increasing NO production, suggesting that the cystatin inhibitory site was not involved in the mechanism.The results demonstrate that members of all 3 cystatin families share another common property unrelated to their function of cysteine proteinase inhibitors, i.e. upregulation of NO production, which biological significance remains to be elucidated.
Nitric oxide (NO)1 is a multipotent physiologic molecule detected in the immune, neuronal, and vascular systems and in many other tissues (1). NO is synthesized by a wide variety of cell types from L-arginine by NO synthases (NOS), three distinct isoforms of which have now been identified (2, 3). Two isoforms are constitutive and Ca 2ϩ -calmodulin dependent: the first is membrane-bound, and was initially discovered in endothelial cells; the second is soluble and was first identified in neurons. The third isoform is an inducible Ca 2ϩ -independent NOS first discovered in murine macrophages and induced by appropriate stimulation with cytokines (4 -7).Among other cells, macrophages allow the intracellular multiplication of Trypanosoma cruzi, the etiological agent of Chagas' disease, a major public health problem in South and Central America (8). A close relationship has been demonstrated between NO production in activated murine macrophages and T. cruzi infection (9). The parasite modulates the NO production by cytokine and LPS-activated macrophages (10, 11). Processes involved in macrophage infection with T. cruzi are extensively studied (12). The membrane cysteine proteinase cruzipain of T. cruzi, which corresponds to the major antigen gp 57/51 (13-17), has been shown to be involved i...