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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...

The impact of a pathogen-induced inflammatory response on dendritic cells (DCs) and on their expression of galectin-3 (Gal-3) was studied on splenic DCs (sDCs) from Trypanosoma cruzi-infected mice. We determined the lectin expression and also presentation of ligands using the labeled galectin as probe. By reverse transcriptase polymerase chain reaction, western blot analysis, quantitative glycocytochemistry, and computer-assisted quantitative microscopy, we demonstrate that, in sDCs from infected mice, expression of Gal-3 and Gal-3-specific ligands were markedly up-regulated and adhesiveness was increased with Gal-3-coated substratum. Gal-3 expression was also enhanced in T. cruzi-infected D2SC-1 cells. To assess influence on migration, we had to work exclusively with D2SC-1 cells because sDCs rapidly lost their capacity to adhere to substratum. Migration of infected- and TCM-treated D2SC-1 cells were reduced when substratum was coated with Gal-3. Expression of Gal-3 by D2SC-1 was reduced when they were incubated with anti-Gal-3 antisense oligonucleotide without effect on cell invasion by the parasite. By using seven neoglycoconjugates, we probed the cellular capacity to specifically bind carbohydrate ligands. Similar to Gal-3, an up-regulation was noted with respect to sites specific for Man and alpha-GalNAc, respectively, revealing that infection-dependent changes are not confined to Gal-3-dependent parameters. Considered together, these data document for the first time that a parasitic infection can modulate both in vivo and in vitro the expression of Gal-3 and of ligands for this lectin in DCs with functional consequences on their capacities of adhesion and migration. These results suggest a new immunomodulatory property of T. cruzi.

SummarySerum levels and liver expression of CCL2 are increased in patients with alcoholic hepatitis (AH). In an experimental model of alcoholic liver disease (ALD), CCL2 was implicated in proinflammatory cytokines activation and hepatic lipid metabolism, but its role in human disease is currently unknown. In a large cohort of ALD patients, we analysed plasma levels and liver expression of CCL2 and their association with liver disease severity and histological lesions. We also studied the relationship between -2518 A > G CCL2 and CCR2 190 A/G polymorphisms and severity of ALD. We show that CCL2 plasma levels are increased in ALD patients compared with healthy subjects. AH patients had significantly higher plasma levels and hepatic expression of CCL2 than patients without AH. Plasma levels and hepatic expression of CCL2 were associated with disease severity. CCL2 liver expression was correlated with neutrophil infiltrate and interleukin (IL)-8 expression, but not with steatosis. Moreover, there were more G-allele carriers of -2518 A > G CCL2 polymorphism in severe AH patients than in other ALD patients. Our results demonstrate that CCL2 is increased in ALD, particularly in severe forms, and suggest a role for CCL2 in the pathogenesis of ALD via neutrophil recruitment.

Trypanosoma cruzi, the etiological agent of Chagas' disease, may persist for many years in its mammalian host. This suggests escape from the immune response and particularly a suboptimal CD8 + T cell response, since these cells are involved in infection control. In this report, we show that T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I molecules at the surface of human dendritic cells (DC). To further investigate the functional consequences of this inhibition, a trypomastigote surface antigen-derived peptide (TSA-1 514±522 peptide) was selected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzispeci®c human CD8 + T cell line in vitro. We observed that DC infected with T. cruzi or treated with T. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the speci®c CD8 + T cell line as shown in an IFN-g ELISPOT assay. Interestingly, T. cruzi or TCM also reduced the antigen presentation capacity of DC to CD8 + T cell lines speci®c for the in¯uenza virus M 58±66 or HIV RT 476±484 epitopes. This dysfunction appears to be linked essentially to reduced MHC class I molecule expression since the stimulation of the RT 476±484 peptide-speci®c CD8 + T cell line was shown to depend mainly on the MHC class I±TCR interaction and not on the co-stimulatory signals which, however, were also inhibited by T. cruzi. This impairment of DC function may represent a novel mechanism reducing in vivo the host's ability to combat ef®ciently T. cruzi infection.

Cystatins are natural tight‐binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon‐γ‐activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077–28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon‐γ and then stimulated with interferon‐γ plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon‐γ. Interferon‐γ‐activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon‐γ plus chicken cystatin produced increased amounts of both tumor necrosis factor α and interleukin 10. The addition of recombinant murine tumor necrosis factor α alone or in combination with recombinant murine interleukin‐10 mimicked the effect of chicken cystatin. The addition of neutralizing anti‐(tumor necrosis factor α) antibodies reduced sharply NO production by chicken cystatin/interferon‐γ‐activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor α and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon‐γ‐activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.