Interferon- or interleukin-10 production is induced by related Trypanosoma cruzi antigens.

The purpose of this study was to evaluate the effects of a crude Trypanosoma cruzi antigen (TCA) and its partially purified subfractions TCF1, TCF2 on peripheral blood mononuclear cells (PBMC) of normal donors and chagasic patients. TCFI and TCF2 stimulated cells from normal donors and chagasic patients in association with a significant production of interleukin (IL)-10. Only PBMC from chagasic patients multiplied after incubation with TCA and released mainly interferon-y but also IL-10. Neither the production of IL-2 and IL-4 nor CD4/CD8 ratios were changed after culture with antigens. These data suggest that some antigens active during the acute phase of T. cruzi infection would stimulate the production of cytokines that promote progression of infection, and the immune system can produce a desired cytokine(s) once the appropriate antigenic stimulus is used.

Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology.

Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC) and cloned. These T cell clones (TCC) were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%). On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%), bradycardia with megacolon (75%) and bradycardia (75%). Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

Impairment of monocytic function during Trypanosoma cruzi infection.

During acute infection, Trypanosoma cruzi, the etiologic agent of Chagas' disease, causes immunosuppression by mechanisms that are not fully delineated. Since mononuclear phagocytes are major target cells in trypanosomiasis, we investigated monocytic function during acute T. cruzi infection. A series of human monocyte and macrophage hybridomas, which represent clonal expansions of subpopulations of human macrophages and possess many normal monocytic functions, were successfully infected with T. cruzi. Clones 63 and 53, chosen for stability in long-term culture, were studied extensively after infection with T. cruzi. Following infection of clone 63, the trypomastigote did not transform into the amastigote multiplicative form, suggesting that clone 63 did not support the entire T. cruzi life cycle. The typical life cycle was completed in clone 53, and thus, clone 53 was used in subsequent studies. Following infection, clone 53 lost expression of class II antigens compared with uninfected cells (DR of 2.2% versus 29.3% and mean channel fluorescence intensity [mean channel] of 4.1 versus 30.5, DQ of 2.3% versus 15.6% and mean channel of 5.4 versus 11.4, and DP of 6.3% versus 27.2% and mean channel of 10.3 versus 33.4). The expression of Class I antigens (87.9% versus 82.8%; mean channel, 20 versus 120) and the adhesion molecules LFA-1 (72.9% versus 28.7%; mean channel, 50.7 versus 23.7) and LFA-3 (10.8% versus 0.7%; mean channel, 20.7 versus 15.1) was increased in infected cells compared with that in uninfected cells. Production of interleukin-1 alpha was decreased and interleukin-6 production was increased in infected clone 53 compared with those in the uninfected cells, while production of tumor necrosis factor alpha was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosomal immunosuppressive factor: a secretion product(s) of Trypanosoma cruzi that inhibits proliferation and IL-2 receptor expression by activated human peripheral blood mononuclear cells.

Coculture of blood forms of Trypanosoma cruzi with human PMBC suppresses the expression of several molecules involved in lymphocyte activation, including receptors for IL-2. Our work was initially undertaken to establish whether this effect required physical parasite-PBMC contact or was mediated by a T. cruzi secretion product. Using culture inserts with cell-impermeable membranes, we were able to demonstrate significant suppression of PHA-induced lymphoproliferation whether the trypanosomes were placed in the same compartment as, or separated from, the PBMC. Similar effects were observed by using supernatants from T. cruzi suspensions. These supernatants, which we refer to as trypanosomal immunosuppressive factor, also inhibited IL-2R expression in response to PHA stimulation. The suppressive effect of the secretion product(s) of T. cruzi was reversible, as evidenced by significant recovery of the proliferative capacity of PBMC after removal of the parasite-containing inserts. Moreover, the extent of the suppression produced by trypanosomal immunosuppressive factor subsided as culture time increased. Treatment of trypanosomal immunosuppressive factor with proteases abrogated its suppressive activity, suggesting that the relevant principle(s) was of protein nature. From ultrafiltration experiments, the molecular mass of the suppressive molecule(s) was estimated to be between 30,000 and 100,000 Da. These results demonstrate for the first time the capacity of T. cruzi to spontaneously secrete a factor that suppresses human lymphocyte responses in vitro. This factor, which may play a role in the down-regulation of host immune function observed in acute chagasic patients, might be a useful tool in exploring the mechanisms that regulate the expression of IL-2R and other surface molecules playing key roles in lymphocyte activation.

Trypanosoma cruzi inhibits the expression of CD3, CD4, CD8, and IL-2R by mitogen-activated helper and cytotoxic human lymphocytes.

We studied the effects of Trypanosoma cruzi secretion products on the capacity of helper (Th) and cytotoxic (Tc) cells to express IL-2R, CD3, CD4, and CD8 in response to PHA or anti-CD3 stimulation. To this end, we used a culture system in which blood forms of the parasite were cocultured with human PBMC. Two-color flow cytometry studies revealed that, under these conditions, there was a significant decrease in the percentage of both Th and Tc cells expressing IL-2R (inhibition range = 30 to 65%). These effects were already demonstrable 18 h after mitogenic activation, and maximal reductions occurred after 48-72 h. T cruzi-induced suppression of IL-2R expression was accompanied by a marked decrease in surface CD3, CD4, and CD8. The surface densities of these markers on the CD3+, CD4+, and CD8+ cells was markedly inhibited by the parasite. This effect was sometimes accompanied by a significant drop in the percentage of positive cells. Diminished expression of all of these surface molecules was observed on activated, but not on resting cells, i.e., no effects occurred in the absence of mitogen. The suppressed expression of CD3, CD4, and CD8 was also seen at 18 h, and occurred concomitantly with a marked decrease in cell proliferation. The inhibited expression of IL-2R, CD3, CD4, and CD8 molecules by Th and Tc might underlie the immunosuppression that occurs during the acute phase of Chagas disease (caused by T. cruzi). Our recent observation that the parasite suppresses responses of human lymphocytes separated by a cell-impermeable membrane, raises hopes that parasite product(s) may be useful reagents for studying the regulation of the expression of IL-2R, CD3, CD4, and CD8, which are known to play key roles during lymphocyte activation.

Trypanosoma cruzi reduces the number of high-affinity IL-2 receptors on activated human lymphocytes by suppressing the expression of the p55 and p70 receptor components.

We previously established that Trypanosoma cruzi, the causative agent of Chagas' disease, has the ability to suppress expression of the p55 component of the IL-2R by activated human PBMC. We explored in this work whether the parasite alters the expression of high affinity IL-2R, responsible for the internalization of IL-2 and signal transduction. Radiobinding measurements revealed that the trypanosome indeed inhibited the expression of high affinity IL-2R. Thus, a considerably smaller number of 125I-IL-2 molecules was necessary to saturate the IL-2R on PHA-stimulated PBMC cocultured with T. cruzi than those of control PBMC that had not been exposed to the organisms. Scatchard analysis of equilibrium binding data showed that, in the presence of T. cruzi, the number of high affinity IL-2R per cell was reduced by approximately 80%. The Kd for IL-2 binding to the fewer IL-2R expressed on PBMC exposed to T. cruzi was not significantly different from that of IL-2R on nonsuppressed PBMC. Independent measurements made after cross-linking 125I-IL-2 to its specific receptors with disuccinimidylsuberate showed that both the p55 and p70 components of the IL-2R were markedly suppressed and to comparable extents. These results demonstrate for the first time that T. cruzi suppresses the expression of high affinity IL-2R by human cells, including the p70 chain of the heterodimeric IL-2R. It is noteworthy that the in vitro model system we used in this work to study the mechanisms whereby T. cruzi may induce the immunosuppression that accompanies acute Chagas' disease also lends itself to the exploration of the regulatory mechanisms governing the expression of IL-2R by human PBMC.

Evaluation of a competitive antibody enzyme immunoassay for specific diagnosis of Chagas' disease.

A competitive enzyme immunoassay based on the use of a monoclonal antibody (MAb) specific for "component 5" of Trypanosoma cruzi was evaluated. The antigenicity and immunogenicity of this component has been observed in natural and experimental infections. The studies were conducted in an area of Bolivia where mixed infections with Leishmania braziliensis are frequent and present a problem in the accurate diagnosis of T. cruzi infections. The specificity and sensitivity of this assay as compared to the indirect immunofluorescence and ELISA tests were demonstrated. The present test has proved to be more specific than the immunofluorescence and ELISA tests.

Tolerance in rats by transplacental transfer of Dipetalonema viteae microfilariae: recognition of putative tolerogen(s) by antibodies that inhibit antigen-specific lymphocyte proliferation.

We have previously reported (Nature 1982. 299:361) that the transplacental transfer of Dipetalonema viteae microfilariae (mf) can induce an antigen-specific tolerance in rats. Rats thus tolerized have serum factor(s) which block(s) antigen-specific lymphocyte proliferation. The results of experiments involving fractionation of antisera from tolerant animals indicate that the inhibitory activity for antigen-specific blastogenesis resides in IgG antibodies. Absorption of IgG (eluted from protein A) with specific filarial antigens reduced the inhibition from 58% to 9% whereas a similar immunosorption of IgG size fraction (obtained by applying to AcA 34 Ultrogel) resulted in a decrease from 72% to 35%. This suggests that IgG size fraction might include factor(s) derived from mf and was partially blocking the blastogenic response. Since the tolerant animals harbor only mf, we have used radiolabeled mf surface antigens for immunoprecipitation by antisera from tolerant animals. Antibodies from tolerant animals have a different specificity for filarial antigens compared to those from immunocompetent and mf-resistant rats.

Platelet mediated killing of larvae from different filarial species in the presence of Dipetalonema viteae stimulated IgE antibodies.

The platelets from normal rats interact with microfilariae of Dipetalonema viteae in vitro in the presence of antibodies leading to the killing of the parasite. The antibody involved in this reaction is identified as IgE because the absorption of immune rat serum on anti-rat IgE column or the pretreatment of platelets with anti-Fc epsilon receptor resulted in a significant reduction in the percentage of killing of microfilariae. This antibody, which mediates platelet activity towards microfilariae, appears early in the secondary infection and persists for a short period of time. This short-lasting IgE antibody is not apparently present in the form of large complexes since the supernatant but not the pellet after ultracentrifugation was able to mediate killing of microfilariae by platelets. IgE-dependent platelet-mediated parasite killing is neither stage- nor species-specific because the microfilariae (LI) of Brugia malayi or of Loa loa and infective larvae (L3) of D. viteae or of B. malayi were killed when they were incubated with the serum obtained from rats at day 8 after secondary infection with adult D. viteae worms. The results of the present study suggest that platelets can actively participate in the immunological killing of filarial larvae.

Resistance against Brugia malayi microfilariae induced by a monoclonal antibody which promotes killing by macrophages and recognizes surface antigen(s).

Several monoclonal antibodies were produced following the immunization of mice with infective larvae of Brugia malayi. One of these gives a positive fluorescence reaction on the surface of B. malayi microfilariae and this particular monoclonal antibody (IgM isotype) was able to mediate mouse peritoneal macrophage adherence to, and killing of, B. malayi microfilariae in vitro. Adherence and killing were enhanced by fresh normal mouse serum, suggesting a role for complement. When the same monoclonal antibody was passively transferred to mice harbouring microfilariae in their circulation, a complete clearance of microfilariae was observed in 70% of the animals. This monoclonal antibody was able to recognize antigenic determinants (of 110,000 MW) present on the surface of B. malayi microfilariae by radioimmunoprecipitation.

The use of monoclonal antibodies in studies of filarial parasite antigens.

The strength of the hybridoma technology in providing probes for the study of parasite immunology is obvious. The use of monoclonal antibodies should greatly expedite the task of the identification and the isolation of antigens which are relevant to host protection, of immunodiagnosis and of immunopathology. We have produced a series of monoclonal antibodies against Brugia malayi which causes lymphatic filariasis in man. One of these monoclonal antibodies (IgM isotype) can detect circulating antigens in the sera of individuals and of laboratory animals infected with B. malayi by radioimmunoprecipitation-PEG assay. This monoclonal antibody can bind to the relevant epitope of the circulating antigen or to the antigenic determinants of the immune complexes that remained exposed in the complex. A few monoclonal antibodies raised against B. malayi were selected, one of which gave positive fluorescence reaction on the surface of microfilariae. This particular monoclonal antibody showed such biological activities as conferring resistance to circulating microfilariae and inducing cell-mediated killing of microfilariae in vitro. The same monoclonal antibody was able to identify antigenic determinants (of mol. wt, 110 Kd) on the surface of B. malayi microfilariae which may be involved in effector mechanisms related to the development of transmission inhibiting immunity in lymphatic filariasis.