Felipe Kierszenbaum

Views on the autoimmunity hypothesis for Chagas disease pathogenesis

Initially, the notion that the pathogenesis of Chagas disease has an autoimmune component was based on the finding that sera from Trypanosoma cruzi-infected patients or laboratory animals contain antibodies that recognize both parasite and host tissue antigens. Subsequent work suggested that T lymphocytes from chagasic patients and animals also displayed such cross-reactivity. However, the autoimmunity hypothesis has remained controversial because of experimental pitfalls, incomplete or inadequate controls, difficulties in reproducing some key results, and a lack of persuasive evidence that the cross-reactive antibodies or lymphocytes can truly effect the multifaceted pathological features of Chagas disease. Whether the immunologic autoreactivities described to date cause chagasic pathology or result from it is another unresolved question. Discussed herein are the most recent contributions to this topic and the reservations they have raised.

Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi

The role of mannose residues on the membranes of Trypanosoma cruzi and its host cells in their association (surface binding plus internalization of the parasite) leading to infection was studied. Used in this work were the bloodstream (trypomastigote), intracellular (amastigote) and insect-transmissible (metacyclic trypomastigote) forms of the parasite; mouse macrophages and rat heart myoblasts were used as the host cells. Removal of mannose residues from the surface of all forms of the parasite by treatment with alpha-mannosidase produced a marked increase in their respective abilities to associate with either host cell. The increase was more pronounced with the bloodstream and insect-derived trypomastigotes (which can penetrate cell membranes) than with the amastigotes (which can not do so). By contrast, mannosidase treatment of the macrophages and the myoblasts caused a significant decrease in the ability of these cells to associate with either bloodstream or insect-derived trypomastigote forms. The capacity of mannosidase-treated macrophages to take up the non-invasive amastigotes was also reduced. These results, as a whole, suggest that mannose residues on the surface of the parasite modulate their binding to macrophages and myoblasts and that mannose residues on the surface of these host cells play a role in cell association with the parasite.

Modulation of macrophage interaction with Trypanosomacruzi by phospholipase A2-sensitive components of the parasite membrane

The presence of phospholipase A2 (PLA2) significantly increased the association between Trypanosoma cruzi and macrophages. This effect reflected alterations to the parasite membrane since it was reproduced only when the parasite but not the macrophage was pretreated with PLA2. That PLA2 activity was responsible for the noted enhancement was indicated by the ability of the specific substrate phosphatidylcholine to block it. The presence of the PLA2 inhibitors quinacrine, 4-bromophenacyl bromide or phentermine markedly inhibited parasite-macrophage association. Quinacrine also inhibited association of the parasite with a non-phagocytic host cell. These results suggested a role for endogenous PLA2 in the initial stages of cell infection by T. cruzi.

Host-cell invasion by Trypanosomacruzi: Role of cell surface galactose residues

Alpha-galactosidase treatment of blood, insect and intracellular forms of T. cruzi enhanced their ability to associate with mouse peritoneal macrophages or rat heart myoblasts as evidenced by significant increases in both the percentage of infected cells and the number of parasites per cell. The magnitude of the enhancement was greater with invasive (blood and insect) than with noninvasive (intracellular) forms of the parasite. The enzyme effect was reversible, attaining total recovery in 2.5 hr. By contrast, when either host cell was pretreated with the enzyme, the extent of cell-parasite association was significantly reduced. These results indicate that galactose residues on T. cruzi and host cells modulate their association in opposite ways.

Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi.

Trypanosoma cruzi was found to release 14CO2 from radiolabeled arginine, and this effect was inhibited by either DL-alpha-difluoromethylarginine or monofluoromethylagmatine, both specific inhibitors of arginine decarboxylase (ADC). Furthermore, agmatine, which can be derived metabolically only by ADC-mediated arginine decarboxylation, was produced when T. cruzi was incubated with radiolabeled arginine, and agmatine production was inhibited in the presence of DL-alpha-difluoromethylarginine. These results constitute direct biochemical evidence for the presence in T. cruzi of ADC, an enzyme that does not occur in mammalian cells.

Inhibitory effects of the Trypanosoma cruzi membrane glycoprotein AGC10 on the expression of IL-2 receptor chains and secretion of cytokines by subpopulations of activated human T lymphocytes.

The Trypanosoma cruzi membrane glycoprotein AGC10 has been shown to alter some human macrophage functions (De Diego, J. L. et al., J. Immunol. 1997. 159: 4983 – 4989). We show here that, in the presence of AGC10, [3 H] thymidine incorporation by normal human lymphocytes stimulated with anti‐CD3 or phytohemagglutinin (PHA) is severely curtailed. This effect was found to involve down‐regulation of the expression of both CD25 (IL‐2Rα) and CD122 (IL‐2Rβ) on the lymphocyte membrane and a marked decrease in the level of up‐regulation of the expression of surface CD132 (IL‐2Rγ or γc ). These alterations occurred in fairly large proportions of CD4+ and CD8+ lymphocytes. AGC10 also inhibited proliferation and expression of IL‐2 receptor chains by activated T lymphocytes virtually depleted of monocytes/macrophages, indicating that these effects do not necessarily require prior modification of monocyte/macrophage function by AGC10. Human lymphocytes stimulated with anti‐CD3 or PHA also displayed a markedly decreased capacity to secrete IL‐2 and IFN‐γ, suggesting that AGC10 affected at least Th1 cell functions. Cell viability in cultures containing or lacking AGC10 was comparable over a 72‐h period, and neither CD25 expression by, nor the viability of, PHA‐stimulated Jurkat cells was altered by AGC10, ruling out that the effects of AGC10 are due to cell killing. These results highlight down‐regulatory effects on activated T lymphocytes exerted by a membrane molecule from a parasite causing a disease whose acute phase is accompanied by immunosuppression.

Mechanisms of pathogenesis in Chagas disease

Chagas disease, caused by the obligate unicellular parasite Trypanosoma cruzi, presents itself in a diverse collection of clinical manifestations, ranging from severe, fatal heart and digestive tract pathologies to unapparent or minor alterations that do not compromise survival. Over the years, a number of mechanisms have been proposed to explain the pathogenesis of chagasic tissue lesions, all of which have faced some criticism or been received with skepticism. This article excludes the autoimmunity hypothesis for Chagas disease because it has been extensively reviewed elsewhere, and summarizes the various alternative hypotheses that have been advanced over the years. For each of these hypotheses, an outline of its main tenets and key findings that support them is presented. This is followed by the results and comments that have challenged them and the caveats that stand on their way to wider acceptance. It is hoped that this writing will draw attention to our shortcomings in understanding the pathogenesis of Chagas disease, which, unfortunately, continues to figure among the most serious health problems of the American continent.

INHIBITION OF MACROPHAGE-TRYPANOSOMA CRUZI INTERACTION BY CONCANAVALIN A AND DIFFERENTIAL BINDING OF BLOODSTREAM AND CULTURE FORMS TO THE MACROPHAGE SURFACE

The initial interaction between the surfaces of mouse peritoneal macrophages and Trypanosoma cruzi was examined using bloodstream (trypomastigote) and culture (epimastigote) forms of a predominantly reticulotropic strain of the parasite. Pretreatment with Con A resulted in a marked inhibition of macrophage binding of both forms of the parasite. Con A inhibition of epimastigote binding persisted for at least 4 hr after exposure to Con A whereas the trypomastigote-binding ability of macrophages showed a significant spontaneous recovery (57-79%) after 1 hr whether or not the parasites were present in the cell cultures during that time. Binding of Con A to the macrophage was required for inhibition of parasite attachment since incubation of Con A-treated cells with alpha-methyl mannoside prevented the inhibitory phenomenon when either epimastigotes or trypomastigotes were used. This monosaccharide had an inhibitory effect of its own which was not as marked as that produced by Con A and affected epimastigote but not trypomastigote binding to the phagocytic cells, thus representing an additional difference in the modes of interaction of these forms of the parasite with the macrophage surface. Furthermore, inhibition of either trypomastigote or epimastigote binding to macrophages was not caused by succinyl-Con A (which consists of two monomeric Con A subunits whereas Con A has four) unless the succinyl-Con A-treated macrophages were further incubated with anti-Con A antibodies. This observation suggests the importance of either molecular size or crosslinking of Con A subunits with consequent membrane rearrangement in causing the inhibitory phenomenon. The antibody preparation had no effect on macrophage binding of T. cruzi when tested by itself. These results highlight distinct characteristics of the binding of two forms of T. cruzi differing in their infective capacity to the surface of a host cell.

CHARACTERIZATION OF THE ANTIVIRAL ACTIVITY PRODUCED DURING TRYPANOSOMA CRUZI INFECTION AND PROTECTIVE EFFECTS OF EXOGENOUS INTERFERON AGAINST EXPERIMENTAL CHAGAS' DISEASE

Mice infected with Trypanosoma cruzi have been shown to develop an antiviral activity in their sera as early as 24 hr postinfection. This activity was characterized in this work as alpha/beta interferon by the following criteria: resistance to pH 2 treatment, sensitivity to incubation at 56 C, or to trypsin treatment, or to actinomycin D treatment, and inactivation by antibody specific for alpha/beta interferon produced in L cells. Mice receiving exogenous alpha/beta interferon in seven daily doses starting on the day of infection with T. cruzi displayed an enhanced resistance evidenced by significantly decreased parasitemias with respect to those of infected animals receiving a mock preparation lacking interferon activity. Direct incubation of T. cruzi with interferon had no consequence on the motility, infectivity or virulence of the parasite. These results suggest a possible role for interferon in inducing enhancement of host resistance against T. cruzi infection.

The effect of swainsonine on the association of Trypanosoma cruzi with host cells

The role of glycoprotein processing in Trypanosoma cruzi association with mammalian host cells (i.e., surface binding and internalization) was studied by using swainsonine - an inhibitor of glycoprotein processing. The presence of swainsonine in co-cultures of blood forms of T. cruzi with either mouse peritoneal macrophages or rat heart myoblasts markedly reduced parasite-host cell association as evidenced by significant decreases in both the percentage of cells associating with the organisms and the number of parasites per cell. This inhibition appeared to result from effects on both the parasite and the host cells since pretreatment of either one reduced its association with the untreated counterpart. The inhibitory effect of swainsonine on the host cells was demonstrable with trypomastigote forms derived from either infected mouse blood or the feces of infected insect vectors, and also with amastigotes. Conversely, treatment of the parasites with swainsonine inhibited their association with the host cells. Studies with blood trypomastigotes revealed that the swainsonine effect was reversible within 3 h. Two observations suggested that swainsonine had induced the parasite to produce defective oligosaccharides rich in mannose. First, swainsonine-treated organisms displayed a greater capacity to bind concanavalin than medium-treated organisms. Second, presence of alpha-methyl mannoside reduced such increased concanavalin A binding. Taken together, the present results suggest important roles for glycoprotein processing and oligosaccharide structures on the surface of both T. cruzi and its host cells in the initial stages of host cell infection.