Opsonization of Toxoplasma gondii tachyzoites with nonspecific immunoglobulins promotes their phagocytosis by macrophages and inhibits their proliferation in nonphagocytic cells in tissue culture.

We have recently shown that Toxoplasma gondii tachyzoites grown in in vitro culture can bind unspecific immunoglobulin (Ig) through their Fc moiety. We show now that Fc receptors are also present on T. gondii within the host animal, and that intraperitoneal parasites in immunocompetent mice are saturated with unspecific Ig. We have also investigated the effect of the parasite's Fc receptor on the interaction of tachyzoites with mammalian cells, using the Vero cell line as a model for nonphagocytic host cells and murine peritoneal macrophages in primary culture as a model for phagocytic cells. Coating of tachyzoites with parasite-unrelated Ig did not enhance their invasive capacity in either target cell type, but slightly decreased the parasite proliferation. Moreover, phagocytosis by macrophages was increased by approximately 50% when parasites were coated with unspecific Ig. These results indicate that the Fc receptor on T. gondii affects the balance between invasion and phagocytosis in a way that is detrimental to the parasites.

Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response.

The combination of BCG with killed Leishmania promastigotes, demonstrated to be efficient in the cure of patients suffering American cutaneous leishmaniasis and in the induction of a long-term immune response in healthy vaccinated volunteers, was tested in BALB/c mice infected with Trypanosoma cruzi, in comparison to BCG or Leishmania alone, and a vehicle (PBS) control. BCG-Leishmania vaccination, applied intra-peritoneally 10 and 3 days before T. cruzi trypomastigote inoculation, prolonged the survival, and reduced blood parasitaemia of infected animals. Proliferation studies indicated that splenocytes of mice vaccinated with BCG-Leishmania and harvested in the acute phase of T. cruzi infection displayed stimulation indices higher than cells from PBS-treated mice when stimulated with PHA mitogen, PPD, Leishmania or T. cruzi antigens. Injections of a monoclonal antibody able to neutralise IFN-gamma into BCG-Leishmania vaccinated mice increased parasitaemia to levels similar to those of control animals (treated with PBS) and reversed the beneficial effect of vaccination on the proliferative response to T. cruzi antigen. These results show that vaccination of mice with BCG plus killed Leishmania promastigotes delayed acute T. cruzi infection, stimulated a T-cell response to T. cruzi antigen and promoted IFN-gamma production.

Identification and characterization of a Fc receptor activity on the Toxoplasma gondii tachyzoite.

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.

Maternal Trypanosoma cruzi-specific antibodies and worsening of acute infection in mouse offspring.

The role of antibodies in the previously demonstrated harmful effect of Trypanosoma cruzi-infected mothers on progeny infection was studied by injecting either serum from chronically infected animals or purified T. cruzi-specific antibodies into uninfected mice during gestation and lactation periods. It was verified that injected antibodies were transferred to offspring. Pregnant or lactating animals exhibited lower circulating antibody levels than nonpregnant or pregnant but nonlactating mice, respectively, suggesting that such antibody transfer occurred in both fetuses and suckling offspring. When infected two months after birth, offspring of mice treated with chronic serum or purified antibodies displayed significantly higher parasitemia than offspring from mothers receiving control serum or immunoglobulins unrelated to T. cruzi. These results indicate that soluble factors contained in sera of infected mice, and particularly antibodies, when transferred from mothers to their young, are able to worsen T. cruzi acquired infection in the offspring.

Trypanosoma cruzi infection in mice induces a polyisotypic hypergammaglobulinaemia and parasite-specific response involving high IgG2a concentrations and highly avid IgG1 antibodies.

Trypanosoma cruzi infection in BALB/c mice induced a reversible polyisotypic hypergammaglobulinaemia, with particularly high levels of IgG2a, IgM and IgE. Hypergammaglobulinaemia started during the acute phase of infection and persisted during chronic disease until 11-13 weeks post-infection (w.p.i.), when immunoglobulin levels, with the exception of IgE, returned near normal values. Parasite-specific antibodies counted for 14 to 23% of gammaglobulinaemia, in acute and chronic infection respectively. The titres of IgM antibodies rose from two w.p.i. IgA, IgE and IgG subclass antibodies built up gradually over the time of parasite clearance (i.e., between three and six w.p.i.). All antibody isotypes, including IgM reached significant and stable titres throughout chronic infection. IgG2a, IgG1 and IgM antibodies had constantly higher titres than the other antibody isotypes. The dominance of IgG2a antibodies was due to their high plasma concentrations, around 70% of all antibodies available in the chronic infection. IgG1 had the highest functional avidity, whereas its concentration corresponded to only 10% of the whole antibody fraction. These results indicate that T. cruzi infection in mice induces a polyisotypic humoral immune response, dominated by some antibody isotypes, with major differences in concentrations and functional avidities. This could be of crucial importance in determining the outcome of infection.

An Fc gamma RII-, Fc gamma RIII-specific monoclonal antibody (2.4G2) decreases acute Trypanosoma cruzi infection in mice.

In order to study the role of Fc gamma Rs in Trypanosoma cruzi infection in mice, the 2.4G2 monoclonal antibody (MAb), specific to the extracellular domains of Fc gamma RII and Fc gamma RIII, was injected intraperitoneally into mice. Flow cytometry studies of uninfected mice showed that 2.4G2 MAb bound to peritoneal and lymph node cells, respectively, on days 2 and 6 after injection. Repeating 2.4G2 injections every 3 to 4 days decreased the availability of Fc gamma Rs on peritoneal, lymph node, and spleen cells. Injections of 2.4G2 MAb into T. cruzi-infected mice, at days -1, 3, 7, 11, 16, 20, and 24 relative to infection, reduced mortality in comparison with that in infected animals injected with an unrelated MAb (50 versus 93.3% mortality; P < 0.01). Parasitemia in 2.4G2-treated mice was significantly (three times) lower than in control animals on days 21 and 24 postinfection (P < 0.05), before parasite-specific antibodies were detectable at significant levels. Immunoglobulin and T. cruzi-specific antibody levels were similar in all groups of mice. These results indicate that repeated injections of 2.4G2 MAb administered to acutely infected mice reduce the in vivo infection level, suggesting that Fc gamma Rs play a role in the early host invasion by T. cruzi parasites.

Trypanosoma cruzi infection in mice enhances the membrane expression of low-affinity Fc receptors for IgG and the release of their soluble forms.

The membrane expression of low-affinity Fc receptors for IgG (Fc gamma RII/III) on cells and the number of Fc gamma RII/III(+) cells were studied by flow cytometry, using the 2.4G2 MoAb, in mice infected by Trypanosoma cruzi. Cells from spleen, mesenteric lymph nodes and peritoneum were collected on days 10, 20, 30 and 40 post infection (p.i.). The in vivo serum level of soluble Fc gamma RII/III, as well as its in vitro release by cells from infected mice were studied. Parasitaemia and IgG1, IgG2a and IgG2b T. cruzi-specific antibody titres were also recorded. Both the expression of Fc gamma R on cell membrane and the absolute number of Fc gamma R(+) cells increased in spleen and in mesenteric lymph nodes, but not in peritoneum. The modifications in spleen occurred in the early and late parasitaemic phase of infection, i.e., before and after detection of T. cruzi-specific antibodies (from day 10 to 40 p.i.). In mesenteric lymph nodes, the variations were observed only in the early acute infection, when antibodies were not yet detectable at significant levels (on days 10 and 20 p.i.). Higher levels of soluble Fc gamma R were detected in sera and in culture supernatants of spleen and lymph node cells from day 20 to 40 p.i. These results show that T. cruzi infection in mice upregulates the expression and the release of Fc gamma RII/III, in the acute phase of infection, before as well as after the rise of antibody response.