Apoptosis 2000; 5: 221–224 ° C 2000 Kluwer Academic Publishers
Experimental Chagas disease: Phagocytosis of apoptotic lymphocytes deactivates macrophages and fuels parasite growth M. F. Lopes and G. A. DosReis Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21944-970, Brazil
Keywords: apoptosis; macrophages; Chagas disease; Trypanosoma cruzi ; T lymphocytes; vitronectin receptor; transforming growth factor-beta; prostaglandins.
(Received 6 March 2000; accepted 14 March 2000)
American trypanosomiasis, or Chagas disease, is a chronic parasitic infection that affects more than 16 million people in Latin America.1 It is caused by Trypanosoma cruzi, a protozoan parasite that replicates inside host macrophages and other cell types. Today, Chagas disease represents the fourth biggest economic loss due to an infectious disease in Latin America, surpassed only by upper respiratory infections, gastrointestinal infections, and AIDS.2 The intriguing hypothesis that the misterious illness of Charles Darwin could be Chagas’ disease has been discussed, although it has never been proved.3 In past years, a great deal of research effort has been made to clarify the immunopathogenesis of T. cruzi infection, specially using the mouse as a disease model.4 Chagas disease is one of the best examples of a linkage between infection by a pathogen and late autoimmune attack, targeting the heart, skeletal muscle, and the neurons of the myoenteric plexuses.5 In spite of clear genetic differences in degree of host susceptibility, infection with T. cruzi is always for life, in both mice and men. Using PCR amplification of parasite DNA, it was shown that chronically infected mice had 5–10 parasites/ml in the bloodstream, while a chronically infected human harbored 4 parasites/ml.6 Recently,
The authors’ cited work was supported by the following brazilian agencies: PADCT/CNPq/World Bank, PRONEX/ MCT, CNPq, CAPES and FAPERJ. Correspondence to: G. A. DosReis, Instituto de Biof´ısica, Univer´ sidade Federal do Rio de Janeiro, Centro de Cieˆ ncias da Saude, Bloco G, Ilha do Funda˜ o, Rio de Janeiro, RJ 21944-970, Brasil. Tel: (+55)-21-590-9522 (ext. 297); Fax: (+55)-21-280-8193; E-mail: [email protected]
several studies identified host lymphocyte apoptosis in the course of T. cruzi infection, and suggested an important link between apoptosis, immunosuppression and parasite escape.
Apoptosis of cells of the immune system in Chagas disease Like other protozoan infections, acute infection of mice with T. cruzi leads to massive polyclonal lymphocyte activation in vivo and to depressed cellular immune responses in vitro.7 It is well known that lymphocyte expansion is followed by apoptosis of terminally differentiated effector cells,8 which helps to control the extent and duration of immune responses. Experimental Chagas disease was the first parasitic disease where spontaneous and activationinduced lymphocyte apoptosis were described.9 In acute infection of mice with T. cruzi, helper CD4+ , but not cytotoxic CD8+ T cells undergo activation-induced cell death (AICD) in vitro upon stimulation of the T-cell antigen receptor complex (TCR;CD3).9 On the other hand, CD8+ T cells undergo more spontaneous cell death in culture.9 Splenic T cells freshly isolated from T. cruzi-infected mice already contain apoptotic cells, indicating that programmed cell death (PCD) is an ongoing process in vivo.9 Recently, increased apoptosis of B lymphocytes has also been described in the acute phase of T. cruzi infection.10 PCD of responding T lymphocytes causes reduced cellular responses in vitro, a marker of the acute phase of Chagas disease.11 Infection-activated CD4+ T cells are eliminated through the Fas/FasL pathway. Acute infection leads to Fas hyperexpression and to increased susceptibility to Fasmediated death in T cells,12 and to increased Fas expression in B cells.10 Infection also upregulates FasL mRNA message and expression; anti-FasL antibody blocks AICD and increases otherwise depressed mitogenic CD4+ T cell responses.12 Furthermore, AICD is absent in CD4+ T cells from T. cruzi-infected FasL-deficient gld mice.12
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M. F. Lopes and G. A. DosReis
AICD mediated by Fas/FasL interaction preferentially targets Th1 effector T cells.13 This immunoregulatory loop could be important in the pathogenesis of Chagas disease, since Th1 T cells have been associated with increased resistance against T. cruzi.14 There is indirect evidence that Th1 T cells are preferentially targeted by apoptosis in T. cruzi infection. Production of both IFN-γ and nitric oxide (NO), which correlates with functional Th1 activity, cooperate to suppress in vitro CD4+ T cell responses in T. cruzi infection.15 Moreover, IFN-γ upregulates Fas/FasL expression and cooperates with NO production for induction of lymphocyte apoptosis in vivo during infection.16 These results are consistent with the hypothesis that PCD of fully functional parasite-specific Th1 T cells could play a pathogenic role by allowing parasite escape and spread.17 Surprisingly, FasL-deficient gld mice are more susceptible to T. cruzi infection than wild-type mice, due to enhanced Th2-type immune responses mediated by IL4 and IL-10 secretion.12 The reason for a Th2 bias in gld mice is unknown. The abnormal CD4/CD8 doublenegative T cells that accumulate in the disease,18 could play a role. It is also possible that Fas-mediated death of T. cruzi-infected host cells is required for adequate control of the parasite load in tissues, an issue that remains to be investigated. Besides immunoregulatory apoptosis, protozoan parasites such as T. cruzi, elaborate proapototic molecules that either kill leucocytes or promote parasite spread. The enzyme Trans-sialidase from T. cruzi is released into the extracellular medium, and induces lymphocyte apoptosis in thymus, spleen and lymph nodes when injected in mice.19 A ceramide-containing glycoinositolphospholipid from T. cruzi induces macrophage apoptosis in the presence of either IFN-γ or GM-CSF.20 Induction of macrophage apoptosis with the ceramide moiety of this glycolipid promotes the release of viable infective forms of T. cruzi,20 suggesting a virulence mechanism. The role of macrophage apoptosis in T. cruzi infection has not been investigated. However, a recent study suggests that resistance to T. cruzi infection in mice correlates with macrophage expression of heat-shock protein 65 (HSP65), and with HSP65-mediated protection from macrophage apoptosis in vitro.21 Finally, apoptosis has been described in vivo in acute myocarditis following T. cruzi infection in dogs.22 Apoptosis affected leucocytes and myocytes in the cardiac inflammatory infiltrates, and even parasites with signs of apoptosis were described.22 On the other hand, apoptosis was found only in the infiltrating leucocytes, but not in myocardiocytes from chronic Chagas disease patients.23 However, it should be noted that in humans, the earlier stages of progressive myocarditis cannot be investigated by similar in situ nick end labelling techniques.
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Phagocytosis of apoptotic lymphocytes fuels parasite growth in macrophages Lymphocyte apoptosis has been postulated as a mechanism for parasite escape in Chagas disease.17 Evidence for this possibility came from studies employing cocultures of T. cruzi-infected macrophages and CD4+ T cells from infected mice.24 Induction of T-cell activation with antiTCR or anti-CD3 antibodies produced an intense exacerbation of T. cruzi growth inside macrophages, and led to parasite accumulation in the extracellular medium.24 Parasite replication could be modulated by the Fas/FasL pathway, and required lymphocyte apoptosis.24 However, parasite replication also required physical contact between macrophages and lymphocytes. Moreover, cocultures of macrophages and activated T cells contained macrophages that have ingested apoptotic lymphocytes.17 These findings suggest a functional link between phagocytosis of apoptotic lymphocytes and increased parasite replication within macrophages. Previous studies demonstrated that TGF-β production increases susceptibility to T. cruzi infection.25 In addition, an intact TGF-β signaling pathway is required for effective T. cruzi replication inside epithelial cells.26 Interestingly, it turned out that TGF-β appears to be the link. In contrast to necrosis, rapid and efficient engulfment of apoptotic cells by macrophages, followed by intracellular digestion, is a safe and nonphlogistic pathway for disposal of the potentially dangerous molecules that result from cellular injury.27 Macrophage uptake of apoptotic cells is critical for many physiological processes, such as embryogenesis, tissue repair and cell turnover.28 Molecular alterations in the cell membrane of apoptotic cells are targeted by phagocytes, and have been termed apoptotic cellassociated molecular patterns, or ACAMPs.28 ACAMPs are recognized by a set of pattern recognition receptors expressed by phagocytes, that includes the Vitronectin Receptor (VNR) integrin αV β3 , CD14 and the scavenger receptor CD36.27 The extracellular matrix protein Thrombospondin binds to VNR and to CD36, and plays a critical role in phagocytosis, by opsonizing apoptotic bodies for phagocyte ingestion.27,28 Uptake of apoptotic cells is not neutral, but rather transduces deactivating signals to macrophages, leading to the secretion of antiinflammatory mediators PGE2 , platelet-activating factor (PAF) and TGF-β.29 Moreover, crosslinkage of CD36 with either antibodies or apoptotic cells, inhibits secretion of proinflammatory cytokines IL-12, IL-1β and TNF-α, but promotes secretion of the antiinflammatory cytokine IL-10.30 Apoptotic lymphocytes drive the growth of T. cruzi in murine macrophages through surface receptors for ACAMPs. Apoptotic, but not necrotic or live T cells,
Apoptotic cells increase parasite growth
increase T. cruzi growth by several fold in in vitro- or in vivo-infected peritoneal macrophages.31 Increased parasite replication was preceded by a cascade of interconnected biochemical events that included PGE2 and TGF-β secretion, followed by induction of Ornithine Decarboxylase (ODC) activity in macrophages.31 In the study of Freirede-Lima et al.,31 a monovalent Fab fragment of anti-VNR antibody blocked, while intact anti-VNR mimicked all the effects of apoptotic cells, including PGE2 and TGF-β secretion, ODC induction, and increased parasite growth.31 TGF-β induced ODC activity in macrophages, resulting in synthesis of the polyamine putrescine.31 Cyclooxygenase (COX) inhibitors aspirin and indomethacin, neutralizing anti-TGF-β antibodies, and an irreversible ODC inhibitor (α-methylornithine), all blocked replication of T. cruzi driven by apoptotic cells.31 These results indicate that ACAMP recognition by phagocytes initiates a cascade of events starting close to VNR at the cell surface, and ending up with putrescine accumulation. Most likely, this is an ancient biochemical pathway, involved in tissue repair and remodeling.28 By inducing lymphocyte apoptosis, the pathogenic parasite appears to exploit this pathway to fuel its own growth inside macrophages. Injection of apoptotic, but not necrotic T cells, increases parasitemia in infected mice,31 indicating that apoptosis helps parasite growth in vivo. Interestingly, injection of COX inhibitors aspirin or indomethacin into T. cruzi-infected mice reduced parasitemia by 90%.31 Finally, a general antiinflammatory effect was also found. Phagocytosis of apoptotic cells blocked NO production by macrophages activated with LPS plus IFN-γ , an effect that could be reversed by neutralizing TGF-β.31 Taken together, these results help to establish a critical role for lymphocyte apoptosis, both in parasite spread and in suppression of macrophage activation and cellular immune responses in the infected host. It is noteworthy that endogenous production of prostaglandins by macrophages has long been associated with immunosuppression in experimental Chagas disease,32 and that TGF-β suppresses T cell activation33 and blocks Th1 T-cell differentiation under certain conditions.34
Perspectives for future directions T. cruzi is an emerging opportunistic pathogen, causing fatal infections in immunocompromised patients.35 Immunosuppressive treatments, such as cyclosphosphamide and gamma irradiation, exacerbate acute T. cruzi infection, and reactivate chronic infection in mice, leading to high parasitemia and death.36 The possibility that, besides HIV infection, these agents act by inducing apoptosisdependent parasite growth should be investigated. Identification of a new pathway for T. cruzi growth in the host opens the possibility for better pharmacological control
of the infection. However, the use of cyclooxygenase inhibitors in T. cruzi infection has led to conflicting results,37 probably related to the dosage and timing of drug administration. Identification of additional molecular components and mediators of this signal-transducing pathway would expand the possibilities for drug development. Finally, the findings we have discussed suggest the possibility of a link between host cell apoptosis and intracellular replication of other relevant pathogens. Infections that target macrophages and induce marked lymphocyte apoptosis, such as Toxoplasmosis and AIDS, are natural candidates to be investigated.
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