Bernard Vray

Immunomodulatory properties of cystatins

Abstract. 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 α and interleukin 10 synthesis, and they stimulate nitric oxide production by interferon γ-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.

Cystatins of filarial nematodes up-regulate the nitric oxide production of interferon-γ-activated murine macrophages

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)‐γ‐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‐α, 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.

Antiinflammatory properties of mycophenolate mofetil in murine endotoxemia: inhibition of TNF-α and upregulation of IL-10 release

Mycophenolate mofetil (MMF) is a new immunosuppressive agent currently used in organ transplantation and under evaluation in immune-mediated inflammatory disorders such as rheumatoid arthritis. Although MMF was shown to inhibit purine nucleotide synthesis in lymphocytes, it is still unclear whether it might also exert direct antiinflammatory actions in vivo. To address this question, we evaluated the effects of MMF administration on the responses of mice to a single challenge with bacterial lipopolysaccharide (LPS). We observed that MMF treatment inhibits the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) upon LPS injection whereas it promotes IL-10 production. In parallel, MMF was found to protect mice from LPS-induced lethality. Inhibition of TNF-alpha release was also observed in IL-10-deficient mice indicating that it does not exclusively depend on the upregulation of IL-10 endogenous synthesis. In view of the differential effects of MMF on the LPS-induced production of TNF-alpha and NO on one hand and that of IL-10 on the other hand, we conclude that beside its immunosuppressive action at the lymphocyte level, MMF is also endowed with antiinflammatory properties.

Trypanosoma cruzi upregulates nitric oxide release by IFN-γ-preactivated macrophages, limiting cell infection independently of the respiratory burst

The relationship between nitric oxide (N = O) produced by mouse peritoneal macrophages (MPM) and Trypanosoma cruzi infection is still poorly understood. The conditions of MPM activation by gamma‐interferon (IFN‐γ) to trigger a N = O‐dependent trypanocidal activity, as well as the effect of parasite infection or of reactive oxygen species (ROS) inhibitors on the N = O release were studied. T. cruzi infection occurring after a previous 24 h MPM activation induced an enhancement of nitrite levels (the stable degradation product of N = O) in cell supernatants; both the percentage of infected MPM and the number of amastigotes per infected cell were decreased in comparison to infected but non‐activated MPM. Addition of superoxide dismutase or catalase to non‐infected but activated MPM increased the nitrite levels; these were not detectable when L‐arginine inhibitors were added together with ROS inhibitors. The latter had no effect on infection nor on nitrite levels when infection occurred after pre‐activation, and induced only a weak nitrite release when infection took place before MPM activation. Altogether, these results support the involvement of N = O in the inhibition of T. cruzi infection by IFN‐γ‐preactivated macrophages, together with the upregulation of N = O release by T. cruzi infection independently of the respiratory burst.

Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the β1-adrenergic and M2-muscarinic receptors and regulate calcium channels in isolated cardiomyocytes

Sera from T. cruzi infected mice were tested in an enzyme immunoassay on peptides corresponding to the second extracellular loops of the β−, the β2-adrenergic receptor and the M2 muscarinic receptor. All sera of mice (4/4) in the acute phase recognized the β1-adrenergic receptor and the M2 muscarinic receptor peptides but not the β2-adrenergic receptor peptide. The same peptides were recognized during the chronic phase in half of the mice (6/12). The immunoglobulin fractions of the mice were tested for their activity on L-type Ca−+ channels of isolated guinea-pig cardiomyocytes using the whole-cell patch clamp technique. The immunoglobulin fractions of acute phase mice were able to activate the Ca−+ channels by stimulation of the β-adrenergic receptors, as assessed by inhibition with propranolol. Those of the chronic phase mice reduced the Ca++ current by stimulation of the muscarinic receptors, as assessed by inhibition with atropine.These results confirm the existence of functional epitopes on the second extracellular loops of both receptors. They suggest that, as in humans, the parasite is able to elicit functional autoantibodies against these epitopes. They give evidence that these autoantibodies mediate their physiological effects by modulating the cAMP activated Ca+− channels.

IL‐10 up‐regulates nitric oxide (NO) synthesis by lipopolysaccharide (LPS)‐activated macrophages: improved control of Trypanosoma cruzi infection

We examined the effects of IL‐10 on tumour necrosis factor‐alpha (TNF‐α) and NO production by LPS‐activated macrophages and on the ability of these cells to control Trypanosoma cruzi infection. We first observed that the addition of rIL‐10 to macrophages of the J774 cell line decreased their synthesis of TNF‐α but increased their release of NO in a dose‐dependent manner. In parallel, treatment of J774 cells with rIL‐10 resulted in a better control of T. cruzi infection involving up‐regulation of NO synthesis, as it was not observed in presence of N‐nitro‐ L‐arginine methyl ester ( L‐NAME), a competitive inhibitor of NO synthase. The enhancing effect of rIL‐10 on NO production was not observed on peritoneal macrophages from wild‐type C57Bl/6 mice, but well on macrophages from IL‐10 knock‐out mice. The control of NO production by endogenous IL‐10 was confirmed by the demonstration that neutralization of IL‐10 secreted by LPS‐activated macrophages from wild‐type mice inhibited their production of NO and, in parallel, their ability to control T. cruzi infection. Taken together, these data demonstrate that both exogenous and endogenous IL‐10 up‐regulate the production of NO by LPS‐activated macrophages and improve thereby their ability to clear T. cruzi infection.

Alteration of Migration and Maturation of Dendritic Cells and T-Cell Depletion in the Course of Experimental Trypanosoma cruzi Infection

Trypanosoma cruzi, the etiologic agent of Chagas disease, induces infection that affects most immunocompetent cells. However, its effect on dendritic cells (DC) is still unknown in vivo. In this report, we show, by immunohistochemical staining, that T. cruzi infection triggers a huge increase in the number of CD11c+ DC in the spleen of infected mice at Days 14 and 21 post-inoculation (pi). In mice reaching the chronic phase (starting on Day 35 pi), the number of splenic DC (sDC) returned progressively to normal (ending on Day 98 pi). In the spleens of noninfected mice, most of the CD8α+CD11c+ and CD8α−CD11c+ DC were found in the red pulp and the marginal and T-cell zones. However, starting on Day 14 pi, a progressive decline of CD8α+CD11c+ was observed. In addition, sDC expressed low levels of the costimulatory molecule B7.2 at Days 14 and 21 pi, suggesting that they remained immature in the course of the infection. As expected, in lipopolysaccharide-treated and noninfected mice, the expression of B7.2 molecules was sharply up-regulated on sDC that migrated toward the T-cell zone. In contrast, upon lipopolysaccharide stimulation, sDC from T. cruzi-infected mice did not migrate toward the T-cell zone nor did they undergo maturation. Finally, white pulp was severely depleted in both CD4+ and CD8+ T cells at the peak of infection. Taken together, these results indicate that profound alterations of migration and maturation of sDC and depletion/redistribution of T cells occur during the acute phase of T. cruzi infection and could be part of another strategy to escape immune surveillance and to persist in the host.

Effects of alpha - and beta -adrenergic stimulation on hepatosplanchnic perfusion and oxygen extraction in endotoxic shock.

ObjectiveTo examine the effects of adrenergic stimulation on hepatosplanchnic perfusion, oxygen extraction, and tumor necrosis factor-&agr; production during endotoxic shock. DesignIn vivo, prospective, randomized, controlled, repeated-measures, experimental study. SettingExperimental physiology laboratory in a university teaching hospital. SubjectsTwenty-one anesthetized and mechanically ventilated dogs. InterventionsAn intrapericardial catheter was positioned. Catheters for blood sampling were inserted into the right femoral artery, hepatic vein, portal vein, and pulmonary artery. Ultrasonic flow probes were placed around the portal vein, the hepatic artery, the mesenteric artery, the left renal artery, and the left femoral artery. Animals received 2 mg/kg of Escherichia coli endotoxin, followed by fluid resuscitation. Seven dogs received intravenous isoproterenol (0.1 &mgr;g/kg·min-1), seven received phenylephrine (1 &mgr;g/kg·min-1), and seven served as controls. Thirty minutes later, cardiac tamponade was introduced to study organ perfusion and tissue oxygen extraction capabilities. Main ResultsThe isoproterenol group had a higher cardiac index and stroke index and lower systemic vascular resistance than the other groups. The phenylephrine group had a higher arterial pressure but a lower cardiac index than the isoproterenol group. The isoproterenol group had a higher hepatic artery blood flow than the other groups and a higher portal and mesenteric flow than the control group. Liver and gut mucosal blood flow was greater in the isoproterenol than in the phenylephrine group. The isoproterenol group had a lower global critical oxygen delivery than the other groups (8.8 ± 1.3 vs. 13.1 ± 2.0 (control) and 11.8 ± 3.3 mL/kg·min-1 (phenylephrine); both p < .05) and a higher liver critical oxygen extraction ratio than the control group. Isoproterenol tended to attenuate, but phenylephrine significantly increased, blood tumor necrosis factor levels. ConclusionsDuring endotoxic shock, &bgr;-stimulation can improve hepatosplanchnic perfusion and enhance tissue oxygen extraction capabilities, whereas &agr;-stimulation does not. In addition, &agr;-adrenergic stimulation can increase tumor necrosis factor levels.

A new quantitative fluorimetric assay for phagocytosis of bacteria

We describe a new quantitative fluorimetric assay for phagocytosis of bacteria. A suspension of fluorescein‐labelled bacteria (Micrococcus lysodeikticus) is mixed and incubated with phagocytes. After fixation with paraformaldehyde, the excess non‐phagocytosed and adsorbed bacteria are lysed with a solution of lysozyme in phosphate‐buffered saline. After washing of the phagocytes, the fluorescence of those that have Ingested the labelled bacteria is. measured with a spectro fluorimeter. We report results obtained with different types of phagocytes which show that this method allows sensitivity, saturation and kinetic studies and the calculation of a phagocytic index.

Relationship between granulocyte macrophage-colony stimulating factor, tumour necrosis factor-α and Trypanosoma cruzi infection of murine macrophages

Gamma interferon (IFN‐γ)‐activated macrophages control Trypanosoma cruzi infection via nitric oxide (NO), recently recognized as a major effector molecule. Granulocyte macrophage‐colony stimulating factor (GM‐CSF) is a multipotent cytokine secreted by macrophages and many other cells. It induces the production of tumour necrosis factor alpha (TNF‐α), another cytokine also secreted by macrophages and involved in the control of T. cruzi infection. However, no data are available on the relationship between GM‐CSF, TNF‐a and NO produced by macrophages activated by IFN‐γ and infected with T. cruzi. To highlight this relationship, mouse peritoneal macrophages (MPM) and two c‐myc retrovirus‐induced macrophage cell lines (9.1.1 and BMM8), respectively characterized by a constitutive and an inducible production of GM‐CSF, were activated with I FN–γ and/or GM‐CSF and infected with T. cruzi. Our results indicate that T. cruzi upregulates GM‐CSF release from MPM and from the two macrophage cell lines, activated (or not) by IFN‐γ. A high autocrine production of GM‐CSF or an exogenous supply of GM‐CSF is correlated with an enhanced release of TNF‐α and NO, inducing an improved control of T. cruzi infection by IFN‐γ‐activated MPM.