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Cystatins up-regulate nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages
L Verdot1, G Lalmanach, V Vercruysse
1Laboratoire d'Enzymologie et de Chimie des Protéines, Université François Rabelais, F37032 Tours, France. bvray@med.ulb.ac.be
Abstract:
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-gamma-activated macrophages. A 12-fold increase in NO production was observed in the presence of 1 microM 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-gamma-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. up-regulation of NO production, which biological significance remains to be elucidated.
Insights
Chicken cystatin and other cystatins up-regulate nitric oxide (NO) production in activated macrophages, independent of their cysteine proteinase inhibitory activity. This finding reveals a novel function for cystatins in modulating immune responses during Chagas
Area of Science:
- Immunology
- Parasitology
- Biochemistry
Background:
- Trypanosoma cruzi infection, the cause of Chagas' disease, leads to increased nitric oxide (NO) production by activated macrophages.
- Cruzipain, a proteinase essential for T. cruzi infection, is inhibited by cysteine proteinase inhibitors.
- Chicken cystatin, a cysteine proteinase inhibitor, was used to investigate its effect on macrophage NO release.
Purpose of the Study:
- To investigate the effect of chicken cystatin on nitric oxide (NO) production by interferon-gamma-activated macrophages.
- To determine if other cystatin families share this NO-modulating property.
- To explore the mechanism by which cystatins influence NO production.
Main Methods:
- Measurement of NO production in interferon-gamma-activated murine macrophages treated with chicken cystatin.
- Dose-response studies with chicken cystatin and other cystatin family members (stefin B, T-kininogen).
- Testing the effect of E64, aprotinin, pepstatin, and soybean trypsin inhibitor on NO production.
- Assessing cystatin activity after complexation with papain.
Main Results:
- Chicken cystatin alone significantly up-regulated NO production in a concentration-dependent manner.
- A 12-fold increase in NO was observed with 1 microM chicken cystatin, detectable at 10 nM.
- Members of all three cystatin families (stefin B, chicken cystatin, T-kininogen) enhanced NO production.
- Inhibitors of other proteases and E64 did not enhance NO production.
- Cystatins retained NO-upregulating activity even after complexation with papain, suggesting the inhibitory site is not involved.
Conclusions:
- Members of all three cystatin families possess a novel property of up-regulating NO production.
- This NO-modulating function is independent of their cysteine proteinase inhibitory activity.
- The biological significance of this cystatin-mediated NO up-regulation requires further investigation.