Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages

Kierszenbaum F., Lima M. F. and Wirth J. J. 1985. Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages. International Journal for Parasitology15: 409–413. The contribution of phagocytic function to host defense against infection with metacyclic forms of Trypanosoma cruzi isolated from insect vectors was investigated in mice passively transferred with anti-T. cruzi serum. The protective effect resulting from the passive transfers was significantly reduced by administration of either silica or cobra venom factor (CVF). A more pronounced curtailment of the protective effect was seen when both silica and CVF were administered to the mice. This effect was greater than that calculated by adding the effects produced by silica and CVF alone. In in vitro experiments, presence of anti-T. cruzi antibodies enhanced the capacity of mouse macrophages to take up the metacyclic organisms and increased the proportion of macrophages associating with the parasites. Increased macrophage-parasite association was also seen when either the flagellates or the macrophages were preincubated with the antiserum. Antibody-treated metacyclic forms of T. cruzi were more rapidly cleared by untreated macrophages than parasites pretreated with normal mouse serum. These results support a role for macrophages in host defense against the form of T. cruzi responsible for natural infections and emphasize the role played by anti-T. cruzi antibodies. The combined effect of the silica and CVF treatments suggests that C activity may contribute to the protective action of antibodies through its opsonic properties, though a concomitant role for C-dependent immune lysis cannot be ruled out. These results highlight the protective role of antibodymediated mechanisms against infection with the form of T. cruzi responsible for natural infections.     

Role of polymorphonuclear cells in Chagas' disease. I. Uptake and mechanisms of destruction of intracellular (amastigote) forms of Trypanosoma cruzi by human neutrophils

The ability of human polymorphonuclear cells (PMN) to take up and destroy intracellular forms of Trypanosoma cruzi (AMA) was investigated as a part of our efforts to elucidate the mechanisms of clearing of these parasites from infected tissues. PMN were found to take up AMA and destroyed parasites were seen after 30 min of cell-parasite interaction. Under our experimental conditions, the rate of uptake of AMA by PMN was maximal during the first 30 min of interaction. AMA were found to be located and destroyed inside the phagolysosomal vacuoles of PMN. The parasite was never found outside these vacuoles despite electron microscopic examination of numerous preparations derived from several experiments. Intracellular destruction of AMA by PMN was visible by electron microscopy and could be monitored by measuring the release of 3H-labeled substances by PMN that had ingested radiolabeled AMA. PMN incubated after removal of unbound parasites destroyed over 90% of the ingested organisms within 3 hr and close to 99% after 12 hr. In cellfree systems, 44% of the AMA were destroyed in the presence of 10(-4) M H2O2 and all of the parasites died at 10(-3) M. Addition of lactoperoxidase and iodide resulted in 100% killing at 10(-5) M H2O2. These mechanisms appeared to be involved in the lysis of AMA by PMN since both H2O2 and peroxidase activity were demonstrated to be present in PMN vacuoles containing the parasite. Addition of NaN3, KCN (inhibitors of myeloperoxidase activity) or catalase (to decompose H2O2) caused a marked reduction in the extent of AMA killing by PMN. Xanthine oxidase was toxic for the AMA in the presence of acetaldehyde. This microbicidal activity was inhibited by catalase but not by heat-inactivated catalase or by reagents that scavenge the intermediate products of reduction of molecular oxygen, O - X 2, X OH, and 1O2. These results suggest that PMN have the potential of clearing AMA liberated in infected chagasic tissues and that parasite killing within the phagolysosomal vacuoles is mediated by myeloperoxidase activity and H2O2.     

Cross-linking of human FcgammaRIIIb induces the production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils

We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcgammaRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab')(2) to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab')(2) exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of beta(2) integrins, particularly CD11b, which is the alpha-chain of complement receptor type 3, but also CD18, which is its beta-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoantibodies.     

Crystal structure of the tryparedoxin peroxidase from the human parasite Trypanosoma cruzi

Tryparedoxin peroxidase from Trypanosoma cruzi (TcTXNPx) belongs to the family of typical 2-Cys peroxiredoxins. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. In T. cruzi, as in all trypanosomatids, this enzyme is the final electron acceptor of a unique system for detoxifying hydroperoxides, constituting a relevant target for drug design. We have determined the crystal structure of TcTXPNx in the reduced active state. The structure comprises 10 subunits in the asymmetric unit, associated to form a decamer of toroidal shape obeying 52 (D5) point group symmetry. We have analyzed the structure of TcTXNPx by comparing it with other structures of typical 2-Cys peroxiredoxins in both redox states, and have identified key residues in the structural rearrangement taking place in the enzymatic cycle. This is the first report of the structure of an active peroxiredoxin that has peroxidase and peroxynitrite reductase activity, and it is noteworthy that it is from a human parasite. This knowledge is of interest for further understanding peroxide metabolism in these parasites, and in the design of new trypanosomatidal drugs against Chagas disease.     

Effect of granulocyte-macrophage colony-stimulating factor on chemiluminescence of human neutrophils

We investigated the capacity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance the function of neutrophils. Neutrophil function was measured in terms of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced luminol-dependent chemiluminescence (LDCL). LDCL of fMLP-stimulated neutrophils was enhanced up to 4.5 fold following preincubation with rhGM-CSF. This enhancement depended on the length of preincubation, reaching an optimal level at 120 min. The dose-response relationship for fMLP-induced LDCL of neutrophils preincubated with rhGM-CSF revealed that half-maximum enhancement was achieved at an approximately 20-fold higher concentration than that of colony-forming units in culture-derived colony formation. These results suggest that differences in dose dependency may be explained by differences in the distribution of receptor(s) for GM-CSF. This may also enable GM-CSF to affect the hematopoietic system, which contains cells at various levels of differentiation, thus mediating the host-defense mechanism.     

Down-modulation of receptors for granulocyte colony-stimulating factor on human neutrophils by granulocyte-activating agents

Purified human blood neutrophils were able to bind radioiodinated murine granulocyte-colony-stimulating factor (G-CSF) in a specific manner. This factor has previously been shown to stimulate functional activities of human and murine neutrophilic granulocytes and to be functionally analogous to human-derived CSF beta. The binding of 125I G-CSF to human neutrophils was competed for equally by unlabeled G-CSF and CSF beta but not by other CSF's. Saturation analysis indicated that human neutrophils displayed about 700-1,500 receptors for G-CSF/CSF beta per cell. Three other agents (N-formyl-methionine-leucine phenylalanine, bacterial lipopolysaccharide, and human CSF alpha) known to activate neutrophils did not compete directly for G-CSF binding sites but, in preincubation experiments at 37 degrees C, were able to down-modulate the expression of G-CSF receptors on human neutrophils in a dose- and time-dependent manner. This effect was specific since the same agents have been shown elsewhere to up-regulate the expression of other granulocyte surface antigens and other agents were much less effective at down-modulating G-CSF receptors. Since the granulocyte-activating agents increase the sensitivity of human neutrophils to G-CSF/CSF beta and mimic some of the actions of G-CSF on neutrophils, it is suggested that G-CSF receptor down-modulation might be a mechanism whereby these agents activate G-CSF receptors and thereby exert some of their effects.     

Granulocyte-macrophage colony-stimulating factor is a stimulant of platelet-activating factor and superoxide anion generation by human neutrophils.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.     

Stimulation and priming of human neutrophils by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor: qualitative and quantitative differences.

Granulocyte colony-stimulating factor(G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) increased neutrophil C3bi-receptor expression and adherence and rapidly (less than 10 min) primed neutrophils to enhanced O2- release and membrane depolarization stimulated by chemotactic peptide. Direct triggering of O2- release in suspended neutrophils was also provoked by GM-CSF but not by G-CSF. GM-CSF-induced O2- release was inhibited by cyclic AMP agonists and cytochalasin B. The biological activity was greater in non-glycosylated GM-CSF than in glycosylated GM-CSF, whereas it was identical in glycosylated and non-glycosylated G-CSFs. Direct stimulation and priming by GM-CSF were consistently greater than those by G-CSF and the combined addition of the optimal concentrations of G-CSF and GM-CSF resulted in the effects of GM-CSF alone. These findings indicate that the effects of G-CSF and GM-CSF on neutrophil functions are qualitatively and quantitatively different from each other.     

Biochemical characterization of a low-affinity arginine permease from the parasite Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, uses arginine for several metabolic processes, including energy reserves management. In the present work, a novel low-affinity arginine transport system has been studied. Maximum velocity (97 pmol min(-1) per 10(7) cells), and an estimate for the apparent Km value (350 microM) of this arginine transporter, were 6-fold and 80-fold higher respectively, when compared with the previously described high-affinity arginine transport system. This transport activity seems to be H+ -mediated, presents a broad specificity by other amino acids such as methionine, and is regulated along the parasite growth curve and life cycle.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) primes the respiratory burst and stimulates protein biosynthesis in human neutrophils

Pre-treatment of human neutrophils with rGM-CSF resulted in a 3-fold increase in the rate of fMet-Leu-Phe stimulated reactive oxidant generation, as assessed by luminol- and lucigenin-chemiluminescence and O2- secretion. When blood-stream neutrophils were incubated in RPMI 1640 medium supplemented with [35S]methionine, both fMet-Leu-Phe (0.1 microM) and gamma-interferon (100 U/ml) stimulated a 3-4-fold increased incorporation of label into TCA-precipitable material. Similarly, rGM-CSF (50 U/ml) also stimulated protein biosynthesis in bloodstream neutrophils, and newly labelled polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Two classes of polypeptides were visualised on these gels: the relative rate of labelling of one class changed very little upon rGM-CSF treatment whereas the relative rate of labelling of a second group increased 3-12-fold.     

Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion

During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca(2+) concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca(2+)-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.     

Sialylated ligands on pathogenic Trypanosoma cruzi interact with Siglec-E (sialic acid-binding Ig-like lectin-E)

Trypanosoma cruzi causes a suppression of the immune system leading to persistence in host cells. The trans -sialidase expressed by T. cruzi is a major virulence factor and transfers sialic acid from host glycoconjugates to mucin-like molecules on the parasite. Here we demonstrate that these sialylated structures play a role in the immunosuppression. We used two T. cruzi strains, whose TS activity correlated with their pathogenicity. The Tulahuen strain, characterized by a high TS activity efficiently infected mice, whereas the Tehuantepec strain showing a reduced TS activity could not establish a patent parasitemia. In vitro analysis revealed that these two strains invaded phagocytic and non-phagocytic host cells at a comparable rate, but they exhibited different potentials to modulate dendritic cell function. In contrast to Tehuantepec, the Tulahuen strain suppressed the production of the proinflammatory cytokine IL-12 and subsequent T-cell activation. This inhibitory effect was absent upon desialylation of the parasite. Therefore, we analysed whether sialylated structures of T. cruzi interact with the inhibitory sialic acid-binding protein Siglec-E on DC. Indeed, Siglec-E interacted with the pathogenic Tulahuen strain, but showed a diminished binding to the Tehuantepec strain. Ligation of Siglec-E on DC using antibodies confirmed this inhibitory effect on DC function.     

Characterization of receptor for granulocyte colony-stimulating factor on human circulating neutrophils

We have made a mutein of human G-CSF with more stable, and potent biological activity. Using 125 I-labeled mutein human G-CSF, high affinity binding sites were identified on human circulating neutrophils. Receptor number per cell was 560 with a Kd of 250 pM. The human G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000.     

The short non-coding transcriptome of the protozoan parasite Trypanosoma cruzi

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.     

Recombinant tumor necrosis factor enhances macrophage destruction of Trypanosoma cruzi in the presence of bacterial endotoxin

We examined in this work whether rTNF inhibits the capacity of Trypanosoma cruzi to multiply within murine macrophages or enhances the ability of the phagocytic host cells to destroy internalized parasites. We found that rTNF would not alter the fate of the trypanosomes within macrophages over a 48-h incubation period unless the latter cells were also treated with 1 ng/ml bacterial endotoxin (LPS). Treatment of macrophages with rTNF plus LPS, but not separate treatment with either rTNF or LPS, resulted in a significant decrease in the number of organisms per 100 macrophages with respect to values obtained with mock-treated macrophages. In addition, there was a significant reduction in the proportion of infected macrophages over the 48-h incubation period, indicating parasite clearance by the host cells. The combined effects of rTNF and LPS were seen when macrophages from CBA/J were used but not with LPS-insensitive macrophages from C3H/HeJ mice. Increased trypanosome killing by CBA/J macrophages treated with rTNF plus LPS was not seen when catalase was present in the culture medium, indicating a role for hydrogen peroxide in the cytotoxic effect. These results show that rTNF can affect the fate of T. cruzi within macrophages if LPS is present and point to destruction of internalized organisms rather than inhibition of parasite multiplication as the most likely explanation.     

Trypanosoma brucei: host parasite interaction in parasite destruction by salicylhydroxamic acid and glycerol in mice

Mouse blood and serum contain a synergistic factor which affects both the speed and completeness of destruction of Trypanosoma brucei in the presence of salicylhydroxamic acid (SHAM) and glycerol. The action of this factor is dose dependent producing complete killing in infected whole blood with 2 mM SHAM and 12 mM glycerol but not in a mixture of 20% infected whole blood and 80% buffer containing the same final concentration of SHAM and glycerol. This factor may account for the discrepancy in reports showing that SHAM-glycerol does not kill 100% of the exposed parasites in vitro yet is able to cure infected animals. The factor is not due to an acquired immune response, complement action, nor lipoproteins. Should the level of the factor be able to be increased, this could greatly increase the effectiveness of SHAM-glycerol.     

Trypanosoma cruzi: effect of parasite subpopulation on murine pregnancy outcome

C3H/HeN female mice infected with distinct Trypanosoma cruzi subpopulations (RA strain [pantropic/reticulotropic] and K98 clone of the CA-I strain [myotropic]) show differences both in inflammatory compromise of the genital tract and in the outcome of pregnancy. The group of mice infected with the K98 clone show lymphomononuclear infiltrates in pelvian fat and in uterus interstitium, coexisting with the presence of T. cruzi DNA, and show moderate oophoritis, perioophoritis, and vasculitis. However, neither parasite DNA nor inflammatory foci were detected in the uterus, and only mild oophoritis was observed among RA-infected mice at mating time. Independently from the parasite subpopulation, females developed estrous 30 days postinoculation (PI), and at the same time, parasite counts were similar for K98 and for RA-infected mice. However, fertility was significantly diminished in K98-infected females. On day 14 of gestation, fetal resorptions increased in this group and cannot be attributed to hormonal disbalance because similar serum progesterone levels were found in all groups. At this time (44 days PI), parasitemia was higher in K98- than in RA-infected mice. However, resorptions were not triggered by massive infection because polymerase chain reaction failed to prove parasite DNA in resorbing fetuses. In contrast with K98 females, RA-infected mice delivered T. cruzi-infected newborns.     

In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi

We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exerting its anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.