Immunomodulatory properties of cystatins.

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.

Metabolic effects of arginine addition to the enteral feeding of critically ill patients.

BACKGROUND: Some studies have suggested that the addition of arginine to enteral feeding solutions may improve outcome in critically ill patients, but the mechanism is incompletely explained. In particular, the availability and utilization of arginine administered enterally is not well defined. METHODS: This prospective, randomized, double-blind, placebo-controlled study performed in a Department of Medicosurgical Intensive Care included 51 patients likely requiring long-term enteral feeding. Thirty-seven patients (57 +/- 7 years, SAPS II 33 +/- 6) completed the 7-day study, of whom 20 received the formula enriched with free arginine (6.3 g/L) and 17 received an isocaloric and isonitrogenous control solution. Arginine absorption was assessed from plasma arginine concentrations in serial samples. Three pathways of arginine utilization were explored: (1) the production of nitric oxide, assessed by the plasma concentration of nitrite/nitrate (NOx) and citrulline, and 24-hour urinary excretion of NOx; (2) the protein turnover, estimated by the phenylalanine concentrations; and (3) the activity of arginase, reflected by the ornithine concentration. RESULTS: The plasma concentrations of arginine and ornithine increased in the group fed with the enriched formula (from 55 +/- 9 micromol/L to 102 +/- 9 micromol/L and from 57 +/- 7 to 135 +/- 11 micromol/L, respectively, p < .05), but not with the control formula. There was no difference between groups in either NO production or phenylalanine concentration. CONCLUSIONS: Supplemental arginine in enteral feeding is readily absorbed, and mainly metabolized into ornithine, presumably by the arginase enzyme.

Effects of nucleoside transport inhibition on hepatosplanchnic perfusion, oxygen extraction capabilities, and TNF release during acute endotoxic shock.

We explored the effects of the nucleoside transport inhibitor draflazine on regional blood flow, O2 extraction capabilities, and tumor necrosis factor (TNF) release in acute endotoxic shock. Fourteen anesthetized and mechanically ventilated dogs received 2 mg/kg of Escherichia coli endotoxin and were divided into two groups. Seven dogs received 0.1 mg/kg of draflazine 30 min before endotoxin, and 7 dogs served as a control group. Draflazine decreased arterial pressure without influencing cardiac index. Mesenteric and portal blood flow and ileum mucosal perfusion increased, but renal blood flow dramatically decreased. After endotoxemia, the draflazine-treated dogs had a lesser fall in cardiac index, filling pressures, and left ventricular stroke work index, and a lesser increase in pulmonary vascular resistance. After fluid resuscitation, they had a consistently lower renal blood flow and ileum mucosal perfusion, but a higher mixed venous and hepatic oxygen saturation and arterial pH than the control group. When cardiac index was reduced by tamponade to study the O2 extraction capabilities, renal blood flow and ileum mucosal perfusion remained lower in the draflazine group. Draflazine did not influence whole-body O2 extraction capabilities, but it delayed the occurrence of liver O2 supply dependency as indicated by a significantly lower liver DO2crit (27.7 +/- 3.9 vs. 43.3 +/- 10.8 mL/min) and a higher O2ERcrit (62.7 +/- 9.5 vs. 42.5 +/- 7.1%) than controls (both P< 0.05). On the other hand, draflazine increased intestinal DO2crit (42.4 +/- 15.4 vs. 27.7 +/- 6.5 mL/min, P < 0.05) compared to the control group. TNF levels remained higher in the draflazine group than in the control group, particularly 3 and 4 h after endotoxin administration. We conclude that nucleoside transport inhibition with draflazine does not alter global and hepatosplanchnic hemodynamics but may decrease gut mucosal perfusion and renal blood flow. However, this intervention can improve liver O2 extraction capabilities in acute endotoxic shock.

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

OBJECTIVE: To examine the effects of adrenergic stimulation on hepatosplanchnic perfusion, oxygen extraction, and tumor necrosis factor-alpha production during endotoxic shock. DESIGN: In vivo, prospective, randomized, controlled, repeated-measures, experimental study. SETTING: Experimental physiology laboratory in a university teaching hospital. SUBJECTS: Twenty-one anesthetized and mechanically ventilated dogs. INTERVENTIONS: An 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 microg/kg x min(-1)), seven received phenylephrine (1 microg/kg x min(-1)), and seven served as controls. Thirty minutes later, cardiac tamponade was introduced to study organ perfusion and tissue oxygen extraction capabilities. MAIN RESULTS: The 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 x 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. CONCLUSIONS: During endotoxic shock, beta-stimulation can improve hepatosplanchnic perfusion and enhance tissue oxygen extraction capabilities, whereas alpha-stimulation does not. In addition, alpha-adrenergic stimulation can increase tumor necrosis factor levels.

Time course of inducible nitric oxide synthase activity following endotoxin administration in dogs.

An increased production of nitric oxide (NO) via the inducible isoform of NO synthase (iNOS) has been incriminated in the pathogenesis of septic shock. Since the time course of iNOS activity is not known during endotoxic shock in dogs, we measured iNOS activity, estimated by the rate of conversion of (14)C-arginine to (14)C-citrulline in the absence of calcium, in the heart, lung, liver, kidney, and gut at 1, 2, 3, 4, and 6 h after a bolus of Escherichia coli endotoxin (2 mg/kg, iv), in the dog. This model, including generous fluid administration, is associated with typical features of human septic shock, including low systemic vascular resistance, altered myocardial function and limited oxygen extraction capability. An increase in iNOS activity was observed at 4 h in the liver (0.24 vs 0.04 mU/mg/min) and at 6 h in the heart (0.26 vs 0.09 mU/mg/min). These findings may contribute to a better delineation of the involvement of NO in endotoxic shock, and to the evaluation of the therapeutic effects of NO inhibitors.

Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production.

Indomethacin (0.14-.5 mM concentration) inhibits nitric oxide production in murine peritoneal macrophages. This was evidenced by measuring both nitrite production or 14C-L-citrulline formation. The inhibition was caused by the diminution of de novo inducible nitric oxide synthase production as demonstrated by Western blotting experiment. The effect of indomethacin after 4 h treatment was irreversible. NO synthase and arginase activities and the uptake of arginine were not directly affected by the drug. Indomethacin also decreased uridine incorporation in macrophages. The effect of indomethacin on the induction of other enzymes (i.e. arginase) was weaker.

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity.

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

Biochemical and genetic evidence for phospholipase C activity in Mycobacterium ulcerans.

This study reports the existence of phospholipase C and D enzymatic activities in Mycobacterium ulcerans cultures as determined by use of thin-layer chromatography to detect diglycerides in hydrolysates of radiolabeled phosphatidylcholine. M. ulcerans DNA sequences homologous to the genes encoding phospholipase C in Mycobacterium tuberculosis and Pseudomonas aeruginosa were identified by sequence analysis and DNA-DNA hybridization. Whether or not the phospholipase C and D enzymes of M. ulcerans plays a role in the pathogenesis of the disease needs further investigation.

Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis.

Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-gamma-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-gamma and then stimulated with interferon-gamma plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-gamma. Interferon-gamma-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-gamma plus chicken cystatin produced increased amounts of both tumor necrosis factor alpha and interleukin 10. The addition of recombinant murine tumor necrosis factor alpha alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor alpha) antibodies reduced sharply NO production by chicken cystatin/interferon-gamma-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor alpha and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-gamma-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.

Antiinflammatory properties of mycophenolate mofetil in murine endotoxemia: inhibition of TNF-alpha 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 infects human dendritic cells and prevents their maturation: inhibition of cytokines, HLA-DR, and costimulatory molecules.

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas' disease. Despite the many immune system disorders recognized in this infection and the crucial role played by dendritic cells (DC) in acquired immune responses, it was not known whether these cells could be infected by T. cruzi trypomastigotes and the consequences of such an infection on their immune functions. We now provide evidence that human monocyte-derived DC can be infected by T. cruzi and can support its intracellular multiplication. Interestingly, this infection has functional consequences on immature DC and on their maturation induced by lipopolysaccharide (LPS). First, after T. cruzi infection, the basal synthesis of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) was impaired. Furthermore, the process of maturation of DC induced by LPS was drastically affected by T. cruzi infection. Indeed, secretion of cytokines such as IL-12, TNF-alpha, and IL-6, which are released normally at high levels by LPS-activated DC, as well as the up-regulation of HLA-DR and CD40 molecules, was significantly reduced after this infection. The same effects could be induced by T. cruzi-conditioned medium, indicating that at least these inhibitory effects were mediated by soluble factors released by T. cruzi. Taken together, these results provide new insights into a novel efficient mechanism, directly involving the alteration of DC function, which might be used by T. cruzi to escape the host immune responses in Chagas' disease and thus might favor persistent infection.

CD40 ligation prevents Trypanosoma cruzi infection through interleukin-12 upregulation.

Because of the critical role of the CD40-CD40 ligand (CD40L) pathway in the induction and effector phases of immune responses, we investigated the effects of CD40 ligation on the control of Trypanosoma cruzi infection. First, we observed that supernatants of murine spleen cells stimulated by CD40L-transfected 3T3 fibroblasts (3T3-CD40L transfectants) prevent the infection of mouse peritoneal macrophages (MPM) by T. cruzi. This phenomenon depends on de novo production of nitric oxide (NO) as it is prevented by the addition of N-nitro-L-arginine methyl ester, a NO synthase inhibitor. NO production requires interleukin (IL)-12-mediated gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) synthesis as demonstrated by inhibition experiments using neutralizing anti-IL-12, anti-IFN-gamma, and anti-TNF-alpha monoclonal antibodies (MAb). We found that an activating anti-CD40 MAb also directly stimulates IFN-gamma-activated MPM to produce NO and thereby to control T. cruzi infection. To determine the in vivo relevance of these in vitro findings, mice were injected with 3T3-CD40L transfectants or 3T3 control fibroblasts at the time of T. cruzi inoculation. We observed that in vivo CD40 ligation dramatically reduced both parasitemia and the mortality rate of T. cruzi-infected mice. A reduced parasitemia was still observed when the injection of 3T3-CD40L transfectants was delayed 8 days postinfection. It was abolished by injection of anti-IL-12 MAb. Taken together, these data establish that CD40 ligation facilitates the control of T. cruzi infection through a cascade involving IL-12, IFN-gamma, and NO.

The metabolic fate of long-term inhaled nitric oxide.

PURPOSE: The fate of inhaled nitric oxide (NO) has not been precisely defined in critically ill patients. This study aimed at defining the effects of long-term NO inhalation on circulating NO byproduct levels. MATERIAL AND METHODS: During NO therapy, plasma and urine from 13 critically ill patients were sampled daily for determination of the stable byproducts of NO (nitrite [NO2-] and nitrate [NO3-]. Routine monitoring data included inhaled NO concentration, hemodynamic parameters, arterial blood gases, creatinine clearance, and C-reactive protein. RESULTS: For the first 24 hours of NO inhalation (6.3+/-1.1 ppm), NO3- plasma concentration increased (from 13.3+/-5.4 to 52.3+/-17.6 micromol/L), but NO2- plasma concentration was not affected. The NO3- plasma concentration was correlated with the C-reactive protein level, the inhaled NO concentration. Renal excretion of NO metabolites was unaltered by NO inhalation. The NO3 concentrations returned to baseline when NO therapy was discontinued. CONCLUSION: Long-term NO inhalation was associated with a consistent increase in the NO3- plasma concentration. NO byproducts may be implicated in the systemic effects associated with this treatment.

Action of chloroquine on nitric oxide production and parasite killing by macrophages.

Chloroquine is known to inhibit several functions of macrophages, but its effect on the nitric oxide (NO)-dependent parasite killing capacity of macrophages has not been documented. NO synthesis by interferon-gamma-induced mouse and casein-elicited rat macrophages was significantly and irreversibly inhibited by chloroquine. The activity of the inducible NO synthase was not directly altered, but previous incubation of macrophages with chloroquine decreased it. Chloroquine did not alter arginase activity or arginine uptake. NADPH diaphorase activity, an indicator of NO synthase was impaired. Western blotting showed that inducible NO synthase synthesis was blocked by chloroquine. The blocking of NO formation by chloroquine resulted in increased infection of mouse peritoneal macrophages by Trypanosoma cruzi (T. cruzi). This suggests that chloroquine decreases NO formation by macrophages by inhibiting the induction of NO synthase. The findings are further evidence that NO is involved in the anti-parasitic response of macrophages.

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-alpha) 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-alpha 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.

Effects of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha on Trypanosoma cruzi trypomastigotes.

We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limits Trypanosoma cruzi infections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135-141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-alpha) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-alpha might have a direct parasitocidal effect on T. cruzi trypomastigotes, independently of NO release. To address this question, T. cruzi trypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-alpha (rmTNF-alpha), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-alpha caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-alpha was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-alpha-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-alpha was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-alpha. Collectively, these results suggest that cytokines such as GM-CSF and TNF-alpha may directly control the level of T. cruzi trypomastigotes at least in vitro and so could determine the outcome of infection in vivo.

Methotrexate inhibits LPS-induced tumor necrosis factor production in vivo.

We investigated the effects of methotrexate (MTX) on the production of tumor necrosis factor (TNF), nitric oxide (NO) and interleukin-10 (IL-10) in vivo. Administration of low doses of MTX (450 mug/kg once a week for 4 weeks) in Balb/c mice resulted in a significant inhibition of the systemic release of TNF and NO upon LPS challenge, whereas the production of IL-10 remained unchanged. These anti-inflammatory effects of MTX were exerted at the macrophage level since peritoneal macrophages from mice, injected for 4 weeks with MTX produced lower levels of TNF than cells from control mice upon LPS stimulation ex vivo. In parallel, we found that MTX-treated mice were less susceptible to a lethal challenge with LPS and D-galactosamine. MTX did not inhibit TNF release after injection of Concanavalin A (ConA) or anti-CD3 monoclonal antibody (mAb), indicating that MTX does not inhibit TNF production at the T cell level. We conclude that repeated administration of a low dose of MTX in mice is associated with a decreased production of TNF by macrophages upon LPS activation.

Cystatins up-regulate nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages.

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.

Antibodies from Trypanosoma cruzi infected mice recognize the second extracellular loop of the beta 1-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 beta 1-, the beta 2-adrenergic receptor and the M2 muscarinic receptor. All sera of mice (4/4) in the acute phase recognized the beta 1-adrenergic receptor and the M2 muscarinic receptor peptides but not the beta 2-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 beta-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.

Granulocyte-macrophage colony-stimulating factor: involvement in control of Trypanosoma cruzi infection in mice.

Several cytokines play crucial roles in Trypanosoma cruzi infection in mice, but the involvement of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) is poorly documented. This report shows that T. cruzi infection of mice triggered an early and sharp increase in plasma GM-CSF during the ascending phase of parasitemia. The plasma GM-CSF concentration remained stable at the peak of parasitemia and subsequently increased in those mice that survived to the acute phase. GM-CSF level increased again sharply, while parasitemia was rapidly decreasing. Finally, GM-CSF was undetectable, soon after the disappearance of circulating parasites. Injection of T. cruzi-infected mice with neutralizing anti-GM-CSF monoclonal antibodies induced the early appearance of parasitemia and aggravated cumulative mortality. In contrast, recombinant mouse GM-CSF (rmGM-CSF) caused sharp decreases in both parasitemia and cumulative mortality in T. cruzi-infected mice. Peritoneal macrophages from rmGM-CSF-treated and infected or uninfected mice were less infected ex vivo than those from control mice. Taken together these data demonstrate the protective action of endogenous GM-CSF in T. cruzi infection. Neutralization of endogenous GM-CSF aggravates infection, while exogenous rmGM-CSF decreases both parasitemia and host mortality.