Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. Because these cysteine proteases exist in all living organisms and because they are involved in various biological and pathological processes, the control of these protease functions by cystatins is of cardinal importance. Cystatins are found in mammals but cystatin-like molecules are also present in mammals and parasites. In the immune system, cystatins modulate cathepsin activities and antigen presentation. They also induce tumor necrosis factor alpha and interleukin 10 synthesis, and they stimulate nitric oxide production by interferon gamma-activated murine macrophages. In turn, nitric oxide has inhibitory activity on cysteine proteases, especially those from parasitic protozoa. Cystatins isolated from parasitic nematodes also have immunomodulatory activities that are distinguishable from those induced by lipopolysacharide-like molecules from endosymbiotic bacteria. On the whole, cystatins and cystatin-like molecules belong to a new category of immunomodulatory molecules. Doubtless increasing data will improve our knowledge of this property, leading to practical applications in immunotherapy.
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...
Interaction between CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ligand CD40L, a 39-kDa glycoprotein, is essential for the development of humoral and cellular immune responses 1,2 . Selective blockade or activation of this pathway provides the ground for the development of new treatments against immunologically based diseases 3,4 and malignancies 5,6 . Like other members of the TNF superfamily, CD40L monomers self-assemble around a threefold symmetry axis to form noncovalent homotrimers that can each bind three receptor molecules 7,8 . Here, we report on the structure-based design of small synthetic molecules with C 3 symmetry that can mimic CD40L homotrimers. These molecules interact with CD40, compete with the binding of CD40L to CD40, and reproduce, to a certain extent, the functional properties of the much larger homotrimeric soluble CD40L. Architectures based on rigid C 3 -symmetric cores may thus represent a general approach to mimicking homotrimers of the TNF superfamily.CD40L is expressed mainly on activated T cells, whereas its cognate receptor, CD40, is constitutively expressed on dendritic cells (DC), macrophages and B cells. The engagement of CD40 by its ligand contributes to regulation of B cell proliferation, immunoglobulin production, immunoglobulin class switching, germinal center formation and development of B cell memory 1 . Moreover, CD40-CD40L interaction has an essential role in cellular immune response in which CD40 ligation activates DCs, 'licensing' them to present antigen to cytotoxic T cells by increasing MHC and costimulatory molecule expression and by producing high levels of IL-12, a T cell-stimulating cytokine [9][10][11] . Antibodies against CD40 with agonist activity have been used to increase immune response in infectious diseases 12,13 and in cancer immunotherapy 5,6 . All of these results underscore the important therapeutic applications that could emerge from the development of small-molecule CD40 agonists.Although ligand-induced dimerization is a general mechanism for activating receptors of cytokines and growth factors 14 , signaling through receptors of the TNFR superfamily strongly relies on the formation of stoichiometrically defined C 3 -symmetric complexes 7 . The structures of several TNF family members in complex with their cognate receptors show that each ligand homotrimer interacts with three monomeric receptor chains 7,15,16 . The geometry of the resulting 3:3 hexameric complex is favorable to the formation of an internal 3:3 signaling complex between the intracellular tail of the receptor and transduction proteins, ultimately activating downstream effector pathways 7 .Despite the difficulty in identifying small molecules that can disrupt protein-protein interactions, synthetic agonists of homodimeric cytokine receptors have been reported 17,18 . The ability of these molecules to dimerize cell-surface receptors is a major determinant of their effector functions 19 . In the present study, we have developed CD40L mimetics by integr...
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.
Cystatins of two filarial nematodes were studied with regard to their capacity to up-regulate the production of nitric oxide (NO) in vitro, and the effects were analysed. Recombinant cystatin of the human pathogenic filaria Onchocerca volvulus and of the rodent filaria Acanthocheilonema viteae significantly enhanced the NO production of interferon (IFN)-gamma-activated macrophages of BALB/c and C3H/HeJ mice. Truncated cystatins lacking the N-terminal protease inhibitory active site, and showing marginal protease inhibitory activity, up-regulated the NO production to the same extent as the full-length proteins, indicating that the effect on the NO production is independent of cysteine protease inhibition. NO did not contribute to the suppression of proliferative T cell responses exerted by filarial cystatins, as shown in other studies, since NO synthase inhibitors did not restore proliferative responses. The up-regulation of NO production induced by filarial cystatins was partly dependent on the production of interleukin-10 and tumour necrosis factor-alpha, since depletion of both cytokines by antibodies led to a diminution of the enhanced NO production by 22-48%. Our data suggest that filarial cystatins are potent triggers of the production of NO, a mediator which was shown to have a role as an effector molecule against filarial worms in vitro and in vivo.
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