Down-regulation of human B lymphocyte activities by a Trypanosoma cruzi membrane glycoprotein.

The effects of purified AGC10, a Trypanosoma cruzi membrane glycoprotein, on normal human B lymphocytes were studied in this work. In the presence of AGC10, [3H]-thymidine uptake by human peripheral blood mononuclear cells stimulated with the B cell-specific mitogen SACI (killed Staphylococcus aureus Cowan I) was markedly decreased. This alteration was accompanied by others such as decreased expression of the CD122 and CD132 chains of the IL-2R complex. These inhibitory effects appeared to be somewhat selective, as expression of CD25, another IL-2R chain, was not affected by AGC10 and no significant modification occurred in the expression of the B-cell-specific marker CD19 or CD21. In contrast, AGC10 did reduce the levels of expression of CD86 and CD80, molecules known to play critical roles in B cell interactions with T lymphocytes. Fairly large subpopulations of, but not all, B lymphocytes had their expression of CD122(+), CD132(+), CD86(+) and CD80(+) reduced to undetectable levels in the presence of AGC10. However, the SACI-activated B cells that remained capable of expressing these molecules in the presence of AGC10 did so at normal levels. This was denoted by comparable mean fluorescence intensity values representing the expression of CD122, CD132, CD86 or CD80 molecules on the surface of SACI-stimulated CD19(+) cells cultured without or with AGC10. These results indicated that AGC10, derived from an organism that causes immunosuppression in infected hosts, down-regulates B cell activities and suggested that the relevant mechanism could involve the molecular alterations described above.

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, [3H]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-2R alpha) and CD122 (IL-2R beta) on the lymphocyte membrane and a marked decrease in the level of up-regulation of the expression of surface CD132 (IL-2R gamma or gamma(c)). 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-gamma, 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.

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs.

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

Chagas' disease and the autoimmunity hypothesis.

The notion that the pathology of Chagas' disease has an autoimmune component was initially based on the finding of circulating antibodies binding heart tissue antigens in patients and mice chronically infected with Trypanosoma cruzi. Later, T lymphocytes reactive with heart or nerve tissue antigens were found in chagasic mice and patients, extending the concept to include cell-mediated immunity. However, there is disagreement about whether the observed immunologic autoreactivities are triggered by T. cruzi epitopes and then affect host tissue antigens by virtue of molecular mimicry or are elicited by host antigens exposed to lymphocytes after tissue damage caused by the parasite. There is also disagreement about the relevance of immunologic autoreactivities to the pathogenesis of Chagas' disease because of the lack of reproducibility of some key reports supporting the autoimmunity hypothesis, conflicting data from independent laboratories, conclusions invalidated by advances in our understanding of the immunologic mechanisms underlying cell lysis, and, last but not least, a lack of direct, incontrovertible evidence that cross-reacting antibodies or autoreactive cells mediate the typical pathologic changes associated with human Chagas' disease. The data and views backing and questioning the autoimmunity hypothesis for Chagas' disease are summarized in this review.

The Trypanosoma cruzi immunosuppressive factor (TIF) targets a lymphocyte activation event subsequent to increased intracellular calcium ion concentration and translocation of protein kinase C but previous to cyclin D2 and cdk4 mRNA accumulation.

Many immunosuppressive effects of Trypanosoma cruzi can be reproduced in vitro by a preparation consisting of molecules spontaneously released by this protozoan (termed trypanosomal immunosuppressive factor (TIF)). In this work, we attempted to establish whether TIF-induced inhibition of lymphoproliferation results from preventing lymphocyte activation or impairing a post-activation process. Although [3H]thymidine uptake and expression of CD25 by normal human T lymphocytes stimulated with a phorbol ester were markedly reduced by T. cruzi or TIF, translocation of cytosolic protein kinase C (PKC) to the cell membrane was not affected. Lymphoproliferation induced by ionomycin was also inhibited by T. cruzi or TIF but the typical elevation of intracellular calcium ions [Ca2+]i caused by this calcium ionophore was not altered. The increase in [Ca2+]i induced with anti-CD3 antibody was also unaffected by TIF. TIF did not preclude lymphocytes stimulated with phytohemagglutinin from accumulating normal mRNA levels of NFAT1 (also known as NFATp) and NFATc. NFAT1 and NFATc are components of the NFAT complex that controls transcription of genes coding for several cytokines and whose translocation to the nucleus is dependent upon PKC activation and increased [Ca2+]i. In contrast, the mRNA levels of cyclin D2 and cdk4, which form a holoenzyme complex known to regulate cell progression through the G1 phase, were markedly reduced by TIF. These results indicated that TIF did not inhibit lymphocyte activation leading to early secondary signaling but curtailed a mechanism controlling cell progression through G1 and necessary for reaching S phase.

Trypanosoma cruzi downregulates the production of interleukin-2, interferon-gamma, interleukin-4 and interleukin-5 by activated human lymphocytes.

The intracellular levels of interleukin (IL)-2, IL-4, IL-5 and interferon-gamma (IFN-gamma) produced by mitogen-stimulated normal human blood lymphocytes were found to be markedly reduced in cultures containing purified Trypanosoma cruzi trypomastigotes. This was evidenced by marked decreases in the proportion of lymphocytes whose cytoplasm was stained with fluorochrome-conjugated monoclonal antibodies specific for the selected cytokines and a somewhat small but systematically demonstrable reduction in the mean fluorescence intensity of the stained lymphocytes relative to values obtained with parasite-free cultures. The percentage of activated CD3+ cells containing both IL-2, IL-4, i.e., THO-like cells, was also much smaller in the cultures containing trypomastigotes, indicating that T. cruzi can concomitantly inhibit the production of more than 1 cytokine by individual activated T lymphocytes. These results evidence the ability of T. cruzi to down-regulate the production of several important cytokines and raise the possibility of a direct role for this parasite in altering cytokine production during infection.

Mechanisms of Trypanosoma cruzi-induced down-regulation of lymphocyte function. Inhibition of transcription and expression of IL-2 receptor gamma (p64IL-2R) and beta (p70IL-2R) chain molecules in activated normal human lymphocytes.

Acute infection with Trypanosoma cruzi, the agent of Chagas' disease, is accompanied by multiple manifestations of immunosuppression, and this parasite has been shown to inhibit T and B lymphocyte functions in vitro through a mechanism involving impaired membrane expression of at least the p55IL-2R (IL-2R alpha) and p70IL-2R (IL-2R beta) components of the multimeric high affinity IL-2R complex. We document in this paper that addition of T. cruzi or a parasite-conditioned medium (trypanosomal immunosuppressive factor (TIF) to cultures of PHA-stimulated normal human blood lymphocytes markedly decreases the cytoplasmic levels of p64IL-2R and p70IL-2R molecules, i.e., the two IL-2R chains that bind IL-2 and participate in signal transduction. These effects highlighted the ability of T. cruzi to curtail biosynthesis of these IL-2R chains and weakened the possibility that decreased membrane IL-2R expression results exclusively from altered chain transportation to the lymphocyte surface. Additional support for impaired lymphocyte production of IL-2R components was derived from northern blot analyses demonstrating TIF-induced decreases in p64IL-2R and p70IL-2R mRNA levels. These reductions are unlikely to be due to conditions affecting mRNA stability, since the rates of decay of both mRNA species were nearly identical in the presence and in the absence of TIF. In addition, nuclear run-on studies revealed that transcription of the p64IL-2R, p70IL-2R, and p55IL-2R genes, but not that of the CD69 gene, was below normal levels in PHA-stimulated lymphocytes incubated with TIF. These results suggest that altered gene transcription is involved in the mechanism by which T. cruzi down-regulates the expression of all the known components of the high affinity IL-2R.

Trypanosoma cruzi immunosuppressive factor decreases the interleukin-2 mRNA level in cultured normal activated human lymphocytes.

We show here that the marked inhibition of interleukin-2 production seen in hosts acutely infected with Trypanosoma cruzi can be reproduced in vitro by adding a T. cruzi suspension filtrate to cultures of activated human peripheral blood mononuclear cells. This effect was not due to an accelerated decay of interleukin-2 mRNA and appeared to be selective since the levels of other mRNA molecules relevant to lymphocyte activation were not noticeably decreased. The use of normal lymphoid cells in this in vitro system makes it possible to examine the mechanisms used by T. cruzi to induce abnormalities in lymphocyte functions that also occur in infected hosts.

Regulatory effect of the level of free Ca2+ of the host cell on the capacity of Trypanosoma cruzi to invade and multiply intracellularly.

We studied whether modification of the free intracellular Ca2+ level of a mammalian host cell would affect its susceptibility to infection by Trypanosoma cruzi or its capacity to support trypomastigote-->amastigote transformation and amastigote replication. Pretreatment of rat heart myoblasts (RHM) with BAPTA.AM or Quin-2.AM, intracellular Ca2+ chelators, decreased the susceptibility of these cells to infection by untreated trypomastigotes. This was evidenced by a significant drop in both the percentage of infected RHM and the average number of organisms per 100 host cells relative to control values. Similar RHM treatment with the Ca2+ ionophore ionomycin had the opposite effects. The rate of trypomastigote-->amastigote transformation measured in RHM that had been treated with BAPTA.AM, Quin-2.AM, or ionomycin before and after, but not during co-culture with trypomastigotes was not significantly altered. The rate of intracellular amastigote multiplication measured in RHM exposed to the intracellular Ca2+ chelators only after virtually all of the internalized trypomastigotes had transformed into amastigotes was significantly decreased by incubation with BAPTA.AM or Quin-2.AM but was increased by ionomycin. None of the drug treatments affected RHM viability to any significant extent. These results suggest that T. cruzi relies on host cell Ca(2+)-dependent events, utilizes host cell free Ca2+ during invasion, or both, and highlight a requirement for an adequate free Ca2+ level for effective intracellular T. cruzi multiplication but not for trypomastigote-->amastigote transformation.

Differential effects of Trypanosoma cruzi on the transcription of the p55IL-2R, c-fos, c-myc and CD69 genes in activated human lymphocytes.

Mitogen-activated lymphocytes co-cultured with either purified Trypanosoma cruzi trypomastigotes or the filtrate of trypomastigote suspensions in culture medium manifest a significant decrease in their capacities to express p55 interleukin-2 receptor molecules (p55IL-2R) on their membrane and proliferate. In this study we found that the cytoplasmic levels of p55IL-2R are also markedly reduced under these conditions. This inhibition appeared to result from altered gene transcription since the levels of p55IL-2R mRNA in phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) dropped substantially in the presence of parasite suspension filtrate. The rates of decay for p55IL-2R mRNA determined in cultures lacking and containing the parasite filtrate after addition of actinomycin D to inhibit further RNA synthesis were comparable. These results indicated that decreased p55IL-2R mRNA was not due to decreased stability of this mRNA under our conditions and pointed to a transcriptional or pre-transcriptional modification as the likely mechanism by which T. cruzi affects activated lymphocytes. The parasite filtrate did not appear to affect transcription of c-fos or c-myc (known to occur in the very early stages of lymphocyte activation) or that of CD69 (which is concomitant with p55IL-2R transcription). Thus, decreased p55IL-2R gene transcription appears to be a somewhat selective effect of a T. cruzi-derived molecule(s) rather than the consequence of an overall shutdown of gene transcription.

Trypanosoma cruzi-induced decrease in the level of interferon-gamma receptor expression by resting and activated human blood lymphocytes.

A substantial proportion of human peripheral blood mononuclear cells (PBMC) manifested a decreased capacity to express membrane interferon-gamma receptors (IFN-gamma R) when co-cultured with Trypanosoma cruzi. Among the lymphocytes, B cells accounted for the bulk of this effect, evidenced by a marked drop in the proportion of CD19+ or CD20+ cells expressing IFN-gamma R. Decreased IFN-gamma R expression by B lymphocytes was seen as early as 3 h after co-culture with T. cruzi and persisted for at least 24 h. The parasite had no detectable effect on CD19, CD20 or DR antigen expression by B lymphocytes. Neither the proportion of B cells expressing these markers nor the membrane density of these molecules varied significantly in the presence of T. cruzi. In PBMC cultures stimulated with Staphlyococcus aureus Cowan I (SACI), T. cruzi decreased the percentages of both IFN-gamma R+ and IFN-R+bright (cells expressing above-normal levels of surface IFN-gamma R) B lymphocytes. Cell-free filtrates of T. cruzi suspensions reproduced the suppressive effects of living parasites on IFN-gamma R expression by B cells. When T. cruzi present, the intracellular levels of IFN-gamma R molecules in resting or SACI-activated B lymphocytes, represented by fluorescence intensity, were well below control values, suggesting that decreased surface expression resulted from suppressed IFN-gamma R synthesis. Among T (CD3+) cells, 10.8% to 39.6% (7 donors) expressed surface IFN-gamma R and did so at a very low level. These percentages were also reduced by T. cruzi.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Trypanosoma cruzi infectivity by treatments that affect calcium ion levels.

The possible role of the intracellular Ca2+ level in the regulation of Trypanosoma cruzi infectivity was explored by measuring the capacity of trypomastigote forms of this organism to invade mammalian host cells after treatments which decrease or elevate cytoplasmic Ca2+. Parasites loaded with either bis-(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid (BAPTA) or 2-([2-bis(carboxymethyl)-amino-5-methylphenoxy]methyl)-6-nethoxy-8 - bis(carboxymethyl)aminoquinoline (Quin-2) to chelate Ca2+ displayed significantly decreased infectivity. This effect was denoted by reductions in both the proportion of rat heart myoblasts invaded by the parasite in vitro and the number of trypanosomes penetrating these host cells, the extents of which were BAPTA or Quin-2 concentration dependent. Consistent with these observations, inhibitory effects were also recorded when the parasite was pretreated with the calmodulin-binding phenothiazines trifluoperazine and chlorpromazine or with felodipine, a chemically different type of calmodulin antagonist, for as little as 5 min. In contrast, pretreatment with the Ca2+ ionophore ionomycin, which elevated Ca2+ levels in T. cruzi, significantly enhanced the infective capacity of the parasite. These results point to the existence of a Ca(2+)-dependent mechanism that regulates the invasive capacity of T. cruzi.

Inhibition of Trypanosoma cruzi-specific immune responses by a protein produced by T. cruzi in the course of Chagas' disease.

Immunosuppression is readily demonstrable in the acute phase of Trypanosoma cruzi infection but subsides during the chronic phase. In vitro, living T. cruzi induces important alterations in mitogen-activated human T and B lymphocytes and inhibits their capacity to proliferate. These effects are reproduced by a protein spontaneously released by this parasite, termed trypanosomal immunosuppressive factor (TIF). In this study we asked whether TIF would also inhibit a T. cruzi-specific immune response and if it is produced in a mammalian host during infection. A significant reduction in the level of [3H]thymidine incorporation by spleen cells from chronically infected mice stimulated with a T. cruzi antigen preparation ensued when TIF was added to the cultures. Production of TIF in T. cruzi-infected individuals was denoted by the ability of serum IgG from either chronically infected patients or mice to abolish, in a concentration-dependent manner, the capacity of TIF to suppress interleukin-2 receptor expression by phytohaemagglutinin-stimulated human lymphocytes. This neutralizing activity was absent in the IgG fractions prepared from sera of healthy volunteers, noninfected mice or mice killed at different times during acute T. cruzi infection. Circulating anti-TIF antibodies represent indirect evidence of TIF production in vivo which, together with TIF-mediated inhibition of T. cruzi-specific lymphoproliferation, raise the possibility that TIF controls anti-parasite immune responses in vivo. The presence of TIF-neutralizing antibodies during chronic but not acute T. cruzi infection may be one of the reasons why immunosuppression is confined to the acute stage.

Does interleukin-2 restore lymphocyte responses suppressed by Trypanosoma cruzi?

There has been disagreement about the ability of exogenous interleukin-2 (IL-2) to restore responsiveness to lymphocytes from either Trypanosoma cruzi-infected animals or normal individuals co-cultured with this parasite. The discrepancy has been attributed to the use of different strains of mice or T. cruzi isolates, or to the use of lymphoid cells from different organs. As T. cruzi inhibits the expression of IL-2 receptors by activated lymphocytes in vitro, we were able to test whether restoration of responsiveness by exogenous IL-2 might depend on the level of suppression present in the system. Human or mouse lymphocytes stimulated with phytohaemagglutinin (PHA) exhibited gradual decreases in IL-2 receptor expression, [3H]thymidine incorporation and IL-2 secretion as the concentration of T. cruzi in the culture increased. Exogenous IL-2 afforded a degree of restoration of both IL-2 receptor expression and [3H]thymidine uptake which was substantial at the lower, but very small--if any--at the higher, parasite concentrations tested. Trypanosoma cruzi could not have competed with the lymphocytes for IL-2 because it did not bind significant amounts of this cytokine. These results suggested that the controversy about the corrective effects of IL-2 may be more apparent than real, reflecting variations in the extent of immunosuppression present in different model systems of T. cruzi-associated immunosuppression.

Mechanisms of development of immunosuppression during Trypanosoma infections.

Acute infection with Trypanosoma cruzi or its African relatives, including T. brucei rhodesiense, T. b. gambiense, T. b. brucei and T. congolense, is frequently accompanied by manifestations of immunological dysfunction. Initially investigators catalogued the ensuing immunologic alterations and identified a number of modifications in lymphoid or accessory cell properties. More recently, the emphasis has switched towards the molecular underpinnings of immunosuppression in these infections. In this article, Marcelo Sztein and Felipe Kierszenboum focus on recent progress made in the quest to delineate the mechanisms behind altered lymphocyte functions in tryponosomal infections, point out particular and common features of immunosuppression induced by T. cruzi and African trypanosomes, and outline possible directions for future research.

Inhibition of host cell invasion and intracellular replication of Trypanosoma cruzi by N,N'-bis(benzyl)-substituted polyamine analogs.

We studied the effects of two N,N'-bis(benzyl)-substituted polyamine analogs on the capacities of Trypanosoma cruzi to invade and multiply within a mammalian host cell. At concentrations as low as 1 microM, these compounds reduced significantly the infectivity of the parasite for rat heart myoblasts in a time-dependent manner. Pretreatment of virulent T. cruzi trypomastigotes, but not myoblast pretreatment, reduced the level of infectivity. The inhibitory effects started to subside 3 h after removal of the drugs and were no longer detectable after 4 h. A significant decrease in the rate of intracellular amastigote multiplication was also seen when the drugs were added to myoblast cultures which had been previously infected with untreated T. cruzi. These results show that N,N'-bis(benzyl)-substituted polyamine analogs meet the two most important criteria for potential chemotherapeutic agents against T. cruzi infection, namely, inhibition of both host cell invasion and intracellular replication by this parasite.

N,N'-thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypanosoma cruzi.

We studied the effects of two N,N'-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N1-acetylspermine and N1-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [14C]spermine to [14C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued.(ABSTRACT TRUNCATED AT 250 WORDS)