Apoptotic CD8 T-lymphocytes disable macrophage-mediated immunity to Trypanosoma cruzi infection.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi. CD8 T-lymphocytes help to control infection, but apoptosis of CD8 T cells disrupts immunity and efferocytosis can enhance parasite infection within macrophages. Here, we investigate how apoptosis of activated CD8 T cells affects M1 and M2 macrophage phenotypes. First, we found that CD8 T-lymphocytes and inflammatory monocytes/macrophages infiltrate peritoneum during acute T. cruzi infection. We show that treatment with anti-Fas ligand (FasL) prevents lymphocyte apoptosis, upregulates type-1 responses to parasite antigens, and reduces infection in macrophages cocultured with activated CD8 T cells. Anti-FasL skews mixed M1/M2 macrophage profiles into polarized M1 phenotype, both in vitro and following injection in infected mice. Moreover, inhibition of T-cell apoptosis induces a broad reprogramming of cytokine responses and improves macrophage-mediated immunity to T. cruzi. The results indicate that disposal of apoptotic CD8 T cells increases M2-macrophage differentiation and contributes to parasite persistence.

Evasion of immune responses by Trypanosoma cruzi, the etiological agent of Chagas disease

Infection with the protozoan parasite Trypanosoma cruzi leads to Chagas disease, which affects millions of people in Latin America. Infection with T. cruzi cannot be eliminated by the immune system. A better understanding of immune evasion mechanisms is required in order to develop more effective vaccines. During the acute phase, parasites replicate extensively and release immunomodulatory molecules that delay parasite-specific responses mediated by T cells. This immune evasion allows the parasite to spread in the host. In the chronic phase, parasite evasion relies on its replication strategy of hijacking the TGF-β signaling pathway involved in inflammation and tissue regeneration. In this article, the mechanisms of immune evasion described for T. cruzi are reviewed.

Neutrophils, apoptosis and phagocytic clearance: an innate sequence of cellular responses regulating intramacrophagic parasite infections.

In complex organisms, apoptosis is a constitutive cell death process that is involved in physiological regulation of cell numbers and that can also be induced in the course of inflammatory and immune responses. Neutrophils are among the first cells recruited during inflammation. Neutrophils constitutively die by apoptosis at inflamed sites, and are ingested by macrophages. Recent studies investigated how phagocytic clearance of senescent neutrophils influences the survival of intracellular protozoan parasites that have been phagocytosed by, or have invaded phagocytes. The results indicate that neutrophil clearance plays an unexpected role in regulation of intramacrophagic protozoan parasite infection.

The central role of Fas-ligand cell signaling in inflammatory lung diseases.

Following inflammation and injury in the lung, loss of epithelial cell precursors could determine the balance between tissue regeneration and fibrosis. This review discusses evidence that proapoptotic Fas-Fas ligand (FasL) signaling plays a central role in pulmonary inflammation, injury and fibrosis. FasL signaling induces inflammatory apoptosis in epithelial cells and alveolar macrophages, with concomitant IL-1 beta and chemokine release, leading to neutrophil infiltration. FasL signaling plays a critical role in models of acute lung injury, idiopathic pulmonary fibrosis and silicosis; blockade of Fas-FasL interactions either prevents or attenuates pulmonary inflammation and fibrosis. Serologic and immunohistochemical studies in patients support a major pathogenic role of Fas and FasL molecules in inflammatory lung diseases. Identification of the pathogenic role of FasL could facilitate the discovery of more effective treatments for currently untreatable inflammatory lung diseases.

The dual role of CTLA-4 in Leishmania infection.

The role of CTLA-4 in inducing the production of transforming growth factor beta (TGF-beta) from T cells during a Leishmania infection has only recently been recognized. However, CTLA-4 and TGF-beta affect T helper cells differently, depending on the maturation. This review discusses the data obtained from different experimental models and demonstrates that CTLA-4 is a target molecule for vaccination and therapy against leishmaniasis.

Fas ligand triggers pulmonary silicosis.

We investigated the role of Fas ligand in murine silicosis. Wild-type mice instilled with silica developed severe pulmonary inflammation, with local production of tumor necrosis factor (TNF)-alpha, and interstitial neutrophil and macrophage infiltration in the lungs. Strikingly, Fas ligand-deficient generalized lymphoproliferative disease mutant (gld) mice did not develop silicosis. The gld mice had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-alpha production, nor pulmonary inflammation. Bone marrow chimeras and local adoptive transfer demonstrated that wild-type, but not Fas ligand-deficient lung macrophages recruit neutrophils and initiate silicosis. Silica induced Fas ligand expression in lung macrophages in vitro and in vivo, and promoted Fas ligand-dependent macrophage apoptosis. Administration of neutralizing anti-Fas ligand antibody in vivo blocked induction of silicosis. Thus, Fas ligand plays a central role in induction of pulmonary silicosis.

Early in vitro priming of distinct T(h) cell subsets determines polarized growth of visceralizing Leishmania in macrophages.

An in vitro priming system of murine naive splenocytes was established to investigate early immune responses to Leishmania chagasi, the agent of visceral leishmaniasis in the New World. Priming of splenocytes from resistant C3H and CBA or susceptible BALB and B10 mice with L. chagasi resulted in blast transformation and in proliferating parasite-specific CD4(+) T cells secreting a differential complement of cytokines (IFN-gamma and low IL-10 levels for resistant T cells; IFN-gamma, IL-4 and high IL-10 levels for susceptible T cells). After priming, intracellular parasite load was much higher in susceptible than in resistant-type splenocyte cultures. On the other hand, infection of purified splenic macrophages from either resistant or susceptible mice with live L. chagasi promastigotes, resulted in comparable parasite loads. Moreover, when early CD4(+) T cell priming in splenocyte cultures was disrupted with anti-CD4 mAb, polarized parasite growth was abolished, becoming comparable in resistant and susceptible cultures. Neutralizing IL-4 activity during splenocyte priming did not affect the final parasite load in susceptible cultures. However, neutralizing IL-10 activity markedly decreased parasite load in susceptible, but not in resistant splenic macrophages. These results suggest that IL-10 plays an important role in L. chagasi infection in susceptible hosts. The results also indicate that innate control of growth of a visceralizing Leishmania in splenic macrophages results from the ability to activate different CD4(+) T cell subsets.

TGF-beta mediates CTLA-4 suppression of cellular immunity in murine kalaazar.

Recent studies indicate important roles for CTLA-4 engagement in T cells, and for TGF-beta production in the immunopathogenesis of murine kalaazar or visceral leishmaniasis, but a functional link between these two pathways in helping intracellular parasite growth is unknown. Here we report that Ag or anti-CD3 activation of splenic CD4+ T cells from visceral leishmaniasis leads to intense CTLA-4-mediated TGF-beta1 production, as assessed either by CTLA-4 blockade or by direct CTLA-4 cross-linkage. Production of TGF-beta1 accounted for the reciprocal regulation of IFN-gamma production by CTLA-4 engagement. Following CD4+ T cell activation, intracellular growth of Leishmania chagasi in cocultured splenic macrophages required both CTLA-4 function and TGF-beta1 secretion. Cross-linkage of CTLA-4 markedly increased L. chagasi replication in cocultures of infected macrophages and activated CD4+ T cells, and parasite growth could be completely blocked with neutralizing anti-TGF-beta1 Ab. Exogenous addition of rTGF-beta1 restored parasite growth in cultures protected from parasitism by CTLA-4 blockade. These results indicate that the negative costimulatory receptor CTLA-4 is critically involved in TGF-beta production and in intracellular parasite replication seen in murine kalaazar.

Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages.

After apoptosis, phagocytes prevent inflammation and tissue damage by the uptake and removal of dead cells. In addition, apoptotic cells evoke an anti-inflammatory response through macrophages. We have previously shown that there is intense lymphocyte apoptosis in an experimental model of Chagas' disease, a debilitating cardiac illness caused by the protozoan Trypanosoma cruzi. Here we show that the interaction of apoptotic, but not necrotic T lymphocytes with macrophages infected with T. cruzi fuels parasite growth in a manner dependent on prostaglandins, transforming growth factor-beta (TGF-beta) and polyamine biosynthesis. We show that the vitronectin receptor is critical, in both apoptotic-cell cytoadherence and the induction of prostaglandin E2/TGF-beta release and ornithine decarboxylase activity in macrophages. A single injection of apoptotic cells in infected mice increases parasitaemia, whereas treatment with cyclooxygenase inhibitors almost completely ablates it in vivo. These results suggest that continual lymphocyte apoptosis and phagocytosis of apoptotic cells by macrophages have a role in parasite persistence in the host, and that cyclooxygenase inhibitors have potential therapeutic application in the control of parasite replication and spread in Chagas' disease.

Increased susceptibility of Fas ligand-deficient gld mice to Trypanosoma cruzi infection due to a Th2-biased host immune response.

Infection of BALB/c mice with Trypanosoma cruzi resulted in up-regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation-induced CD4+ T cell death (AICD), and in Fas: FasL-mediated cytotoxicity. When CD4+ T cells from infected mice were co-cultured with T. cruzi-infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi-infected FasL-deficient BALB gld/gld mice had no detectable AICD in vitro and their activation with anti-TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld/gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild-type mice. Secretion of Th2 cytokines IL-10 and IL-4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti-IL-4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild-type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas: FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2-type immune response to the parasite.

Unresponsive CD4+ T lymphocytes from Leishmania chagasi-infected mice increase cytokine production and mediate parasite killing after blockade of B7-1/CTLA-4 molecular pathway.

Infection of BALB/c mice with Leishmania chagasi results in progressive increase of parasite burden in spleen, in spite of extensive T cell activation in situ. Explanted splenic CD4+ T cells showed decreased proliferation to anti-CD3, compared with controls, and no response to L. chagasi recombinant antigen Lcr1. Blockade of the negative costimulatory receptor CTLA-4 restored responses to anti-CD3 and induced vigorous responses to Lcr1. Blockade of B7-1, but not B7-2, also enhanced T cell responsiveness. CTLA-4 blockade completely restored activation-induced interleukin-2 secretion and increased interferon-gamma production. The effect, however, was not restricted to Th1 responses, since CTLA-4 blockade also enhanced antigen-induced interleukin-4 secretion. CTLA-4 blockade induced almost complete elimination of parasite burden in splenocyte cultures activated with anti-CD3 or Lcr1. These results indicate that CTLA-4 engagement by B7-1 plays an important role in maintaining unresponsiveness in CD4+ T cells in this model of chronic visceral leishmaniasis.

Proapoptotic activity of a Trypanosoma cruzi ceramide-containing glycolipid turned on in host macrophages by IFN-gamma.

The effects of glycoinositolphospholipid (GIPL), from the pathogenic protozoan Trypanosoma cruzi, and its isolated glycan and lipid (dihydroceramide) components, were investigated in J774 cells and primary macrophages. Isolated GIPL ceramide, but not intact GIPL or its glycan, induced intense fluid phase endocytosis when added exogenously. In the presence of the cytokine IFN-gamma, GIPL ceramide induced marked apoptosis in J774 cells and macrophages, independent of nitric oxide secretion. When cells were preincubated with the GIPL-derived glycan chain, addition of intact GIPL induced macrophage apoptosis in the presence of IFN-gamma. Synthetic C2-dihydroceramide also induced apoptosis in the presence of IFN-gamma. Induction of apoptosis in T. cruzi-infected macrophages by GIPL ceramide plus IFN-gamma led to increased parasite release compared with IFN-gamma treatment alone. Viable parasites released comprised both infective trypomastigote and spheromastigote forms. These results identify a novel pathway by which T. cruzi glycosylphosphatidylinositol family molecules affect host macrophages, with implications for the infectious process.

Activation-induced T cell death exacerbates Trypanosoma cruzi replication in macrophages cocultured with CD4+ T lymphocytes from infected hosts.

Activation-induced cell death (AICD) of CD4+ T lymphocytes was described in infection with Trypanosoma cruzi, but a role for AICD in modulating parasite spread in host cells has not been investigated. In this study, replication of T. cruzi in vitro in murine macrophage (Mphi) monolayers was investigated. Long term (5 to 13 day) replication of infective (trypomastigote) T. cruzi forms was blocked by supernatants from activated (anti-TCR) CD4+ T cells of infected mice or by rIFN-gamma. However, when CD4+ T cells from infected mice were cocultured with Mphi and activated by anti-TCR, marked exacerbation of trypomastigote growth in Mphi ensued. The deleterious effect required contact between T cells and infected Mphi. Both anti-Fas and TCR activation killed a proportion of CD4+ T cells. Ly-6 activation did not induce AICD and did not exacerbate parasite growth. However, Fas-mediated killing of T cells before Ly-6 activation led to exacerbated parasite growth. Although a minor population, Fas-susceptible cells were the major source of IFN-gamma production by activated T cells. Addition of a neutralizing anti-Fas ligand antibody blocked 50 to 60% of CD4+ T cell AICD and reduced trypomastigote growth in T/Mphi cocultures stimulated by anti-TCR. The results demonstrate that in CD4+ T cells from infected mice, the onset of AICD selectively ablates IFN-gamma production and up-regulates parasite replication in Mphi in vitro. These findings suggest a deleterious role for AICD in T. cruzi infection.

Apoptosis as a cause of T-cell unresponsiveness in experimental Chagas' disease

A murine model of Chagas'disease induced by metacyclic forms of T. cruzi was used to evaluate T-cell function during infection. T-cell unresponsiveness to TcR; CD3 stimulation in vitro and lymphocyte activation in vivo occurred simultaneously. These paradoxical findings ar discussed in the light of recent evidence that mature activated T cells become susceptible to TcR-mediated apoptosis. Activation-induced death in T cells from T. cruzi-infected mice has recently been demonstrated in this model. Evidence that TcR-induced death of activated T cells could be a cause for T-cell unresponsiveness in vitro and in vivo, as well as the possible molecular mechanisms involved, are discussed.

T-lymphocytes in experimental Leishmania amazonensis infection: comparison between immunized and naive BALB/c mice.

Highly susceptible naive BALB/c mice or mice that had previously been immunized i.v. with solubilized homologous antigen (partially resistant) were infected with Leishmania amazonensis. Histologically, the main differences between the two groups were lymphocytic infiltration and macrophage activation. Assays of T-cell function at 3 and 10 weeks after infection revealed that purified T-cells did not proliferate following treatment with leishmania antigen. A mitogenic anti-CD3 monoclonal antibody (mAb) failed to activate T-cells after 3 weeks of infection as judged by proliferation and IL-2 secretion assays. After 10 weeks of infection, anti-CD3 mAb fully activated T-cells to proliferation and IL-2 secretion. On the other hand, T-cells released IL-3 in response to leishmania antigen, anti-CD3 mAb and anti-Thy1 mAb at 3 and 10 weeks post-infection. Surprisingly, a mitogenic anti-Thy 1 mAb (G7) fully activated T-cells even at 3 weeks of infection as judged by proliferative and IL-2 secretion assays. No significant differences were found in the proliferative or interleukin secretory responses of T-cells from animals that had been infected in either the presence or the absence of prior immunization. Since the Thy1 triggering pathway has different accessory cell and cytokine requirements than does the CD3: TCR lymphocyte activation pathway, it is possible that immunization was more effective in changing the cellular interactions of the T-lymphocyte than in altering its intrinsic capabilities.

Comparative analysis of splenic cell proliferation induced by interleukin 3 and by syngeneic accessory cells (syngeneic mixed leukocyte reaction): evidence that autoreactive T-cell functioning instructs hematopoietic phenomena.

Murine syngeneic mixed leukocyte reaction (SMLR) was studied under totally autologous culture conditions using syngeneic normal mouse serum in the culture. SMLR was detected in splenic, but not in lymph node, nonadherent responding cell populations (NWNAC). In the absence of stimulator, accessory cells (AC), IL3-containing fluids also induced splenic, but not lymph node, NWNAC growth. SMLR-derived supernatants contained IL3, but not IL2, activity, and production of this IL3 activity could be prevented by adding anti-CD4 mAbs to SMLR cultures. Precursor frequencies of both SMLR and IL3 splenic responses were very low and similar, and there was a synergism between IL3 and AC in induction of NWNAC growth. Growth of responding NWNAC was further enhanced by T-cell depletion with anti-Thy1 mAb and complement. Lack of T-cell proliferation in the SMLR was confirmed by BUdR and light protection experiments. Autoradiographs indicated that the same cell type grew in both SMLR and IL3-induced NWNAC cultures. Besides blast cells, cells with the appearance of immature monocytes with 3H-labeled nuclei were found in both kinds of culture. No labeled lymphocytes could be found. Both SMLR and IL3-induced NWNAC cultures contained expanded numbers of M-CSF-responsive monocyte precursors. On the other hand, SMLR- but not IL3-induced cultures contained expanded numbers of IL3-responsive, immature precursors capable of giving rise to large colonies of monocytic-like cells. Although IL2 could not be detected in SMLR supernatants, both cell growth and IL3 production could be blocked with anti-IL2 receptor and anti-IL2 mAbs. Exogenous IL2, on the other hand, enhanced both cell growth and IL3 production in the SMLR. These results indicate that, under totally autologous conditions, CD4+ autoreactive T-cells do not proliferate in the SMLR, but rather instruct the growth of splenic hematopoietic precursors capable of differentiating along the monocytic lineage. Autoreactive T-cell activation in the SMLR seems to involve minimal IL2 production, which is critically necessary for triggering IL3 production in a markedly amplified manner. These results suggest a link between normal regulation of hematopoiesis and MHC-restricted, autoreactive T-cell activation.

Independent and cooperative bheravior of THY1 and CD3:TcR T cell signalling pathways

Normal resting spleen T lymphocytes form mice were stimulated in vitro by monoclonal antibodies (mAbs) against either Thy1 or CD3:TcR surface protein molecules. Although both mAbs were mitogenic, anti-Thy1 activation generated 5 times more IL2 secretiom than anti-CD3 activation under similar conditions. Priduction of IL-like activity was comparable for both Thy1 and CD3-mediated activation. In addition, non-mitogenic doses of anti-CD3 and anti-Thy1 (0.16microng/ml and 0.0125% ascites, repectively) mAbs induced T cell activation when provided together. These reults indicate that Thy1 signalling cooperates with the CD3:TcR pathway to activate T cells. However, the pathway is also regulated independently since IL2 production is larger when stimulated by anti-Thy1 than anti-CD3mAbs

Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3',5'-cyclic adenosine monophosphate accumulation.

The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyryl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T-cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)