The effect of C3 depletion on the clearance of Trypanosoma cruzi induced by IgG antibodies

Passive transfer of homologous immune serum or IgG anti-Trypanosoma cruzi antibodies to normal mice containing circulating T. cruzi bloodstream trypomastigotes (Btrys) induces a very fast clearance of the parasites. Previous work from this laboratory has shown that F(ab')2 fragments obtained from IgG anti-T. cruzi antibodies retain the ability to induce lysis of the Btrys but are unable to induce clearance of the parasites. This suggests that the clearance is dependent on the Fc region. The removal of the Btrys may then be effected by attachment of the antibodies to the parasites and removal of the opsonized parasites by the mononuclear phagocytic system. Attachment of the opsonized parasites to macrophages may be effected either through the Fc receptor that binds specifically to the heavy chain of IgG or through C3b fragments after cleavage of C3 by C3-convertases. In order to find out the possible role of C3b in this phenomenon the clearance of Btrys was determined in mice depleted of C3 by previous treatment with cobra venom factor. The results of these experiments showed that depletion of C3 completely abolished the immune clearance induced by anti-T. cruzi antibodies. It is suggested that C3 is required for the clearance of Btrys from circulation.     

Role of the antibody Fc in the immune clearance of Trypanosoma cruzi

Passive transfer of immune serum obtained from mice chronically infected with Trypanosoma cruzi to mice containing circulating bloodstream trypomastigotes induces a very fast clearance of the parasites. Comparison of trypomastigotes clearance in normocomplementemic and C5-deficient mice showed no difference. IgG fraction obtained from immune serum was very efficient at inducing complement-mediated lysis and immune clearance of bloodstream trypomastigotes, whereas its Fc-missing F (ab') 2 fragments, although able to induce lysis, were unable to induce clearance. It is suggested that the immune clearance of bloodstream trypomastigotes is dependent on the antibody Fc region and that complement-mediated lysis is not a prerequisite for elimination of the parasites from circulation.     

Isotype of antibodies responsible for immune lysis in Trypanosoma cruzi infected mice

Serum obtained from mice infected with Trypanosoma cruzi have antibodies able to induce immune lysis in presence of complement. Gel filtration in Sephadex G-200 and affinity chromatography with rabbit anti-mouse IgG and protein A-Sepharose showed that the antibodies responsible for the immune serum lytic activity are exclusively located in Ig of IgG isotype, including IgG1, IgG2a, IgG2b and probably IgG3. It is suggested that antibodies responsible for protection against T. cruzi infection and antibodies responsible for immune lysis are the same.     

Immune complex-mediated glomerulopathy in experimental Chagas' disease.

To investigate the development of glomerulopathy during the chronic phase of experimental Chagas' disease, C3H-Hej mice were infected with Trypanosoma cruzi trypomastigotes. Deposits of IgG, IgM, and C3 in renal mesangium were observed by immunofluorescence (IF) to increase in size as a function of time after infection (4-6 months). T. cruzi antigens were codeposited in glomeruli with Ig and C3. Electron-dense deposits were visualized in mesangial and paramesangial areas by electron microscopy. Anti-T. cruzi and rheumatoid factor (RF) antibodies (of IgG isotypes) were detected both in serum and in renal eluates. In serum, the titers of both antibodies progressively decreased as a function of time after infection. In renal eluates, titers of anti-T. cruzi antibodies appeared to be stable during the three time periods after infection. By contrast, titers of RF antibodies in renal eluates were shown to increase progressively during these same time periods, paralleling the increase in size of mesangial Ig deposits observed by IF. Several T. cruzi proteins were immunoprecipitated from radiolabeled renal eluates by a control anti-T. cruzi antibody. In addition, antibodies from renal eluates specifically precipitated a 85-kDa protein from radiolabeled T. cruzi lysates, whereas serum antibodies precipitated a broad pattern of T. cruzi proteins. These results demonstrate that mice experimentally infected with T. cruzi can develop a mesangial glomerulopathy during the chronic phase of the disease, which appears to be mediated through immune complexes containing parasite antigens associated with secondary deposition of RF.     

Biological activity of chronic phase antibodies eluted from sensitized Trypanosoma cruzi trypomastigotes

Chronic infection with Trypanosoma cruzi induces high levels of antibodies that have lytic and clearance activities on bloodstream trypomastigote forms. These two activities were tested with antibodies eluted from parasites sensitized with serum obtained from mice in the chronic phase of infection. Parasites submitted to treatment for antibody elution were also tested. Our results show that antibodies eluted from the parasites are very efficient to induce lysis but unable to induce clearance. In addition, we observed that after being submitted to treatment for antibody elution the parasites still presented a slower but significant clearance and a high lytic activity. These results allow us to suggest that clearance inducing antibodies are mostly high affinity antibodies that could not be eluted from the parasites in our experimental conditions.     

IFN-gamma, but not nitric oxide or specific IgG, is essential for the in vivo control of low-virulence Sylvio X10/4 Trypanosoma cruzi parasites

Highly virulent strains of Trypanosoma cruzi are frequently used as murine models of Chagas' disease. However, these strains do not fully represent the spectrum of parasites involved in the human infection. In this paper, we analysed parasitaemia, mortality, tissue pathology and parasite-specific IgG serum levels in immune-deficient mice infected with Sylvio X10/4 parasites, a T. cruzi derived from a chagasic patient that yields very low parasitaemias and in C3H/HePAS mice induces a chronic cardiopathy resembling the human disease. IFN-gamma was identified as a crucial element for parasite control as its absence determined a drastic increase in parasitaemia, tissue parasitism, leukocyte infiltrates at the heart and striated muscles and mortality. The lack of IFN-gamma or IL-12p40, a molecule shared by IL-12 and IL-23, also resulted in spinal cord lesions and a progressive paralysis syndrome. Whereas IgG2a was the main Ig isotype in infected C57BL/6 mice, IL-12p40-KO mice produced IgG2a and IgG1 and IFN-gamma-KO mice produced only IgG1. The IFN-gamma-protective effect was not essentially mediated by nitric oxide (NO), inasmuch as infected iNOS-KO mice showed no parasitaemia and low tissue damage. Mice deficient in CD4(+) or CD8(+) T cells showed an intermediate phenotype with increased mortality and tissue pathology but no parasitaemia. Interestingly, CD28-KO mice were unable to produce anti-T. cruzi IgG antibodies but presented moderate tissue pathology and managed to control the infection. Thus, differently from infections with high virulence parasites, neither IgG, NO nor CD28-mediated signalling are essential for the non-sterile control of Sylvio X10/4 parasites.     

Restricted IgG isotype profiles in T. cruzi infected mice and Chagas'' disease patients.

IgG2 was the predominant specific antibody isotype in mice chronically infected with Y strain Trypanosoma cruzi; IgG1 and IgG3 antibodies were absent or present only at very low levels. Isotype analyses of the acute phase of infection confirmed no early production of IgG1 or IgG3 and no failure in the switch from IgM to IgG. In vivo passive transfer studies of immune serum fractions showed protection to be associated only with the IgG2 isotype. A characteristic specific anti-T. cruzi IgG isotype profile (IgG1, IgG3, greater than IgG2, IgG4) was detected in a majority (39 of 50) of sera from Chagas' disease patients.     

Pregnancy and humoral immune response in mice chronically infected by Trypanosoma cruzi.

The effect of pregnancy on the humoral immune response induced by Trypanosoma cruzi was studied in groups of chronically infected and pregnant mice (IP) or chronically infected and nonpregnant mice (INP) of strain BALB/c. Groups of noninfected and nonpregnant mice (NINP) or noninfected and pregnant mice (NIP) served as controls. The pregnant mice were killed on day 17 of pregnancy. Anti-T. cruzi immunoglobulin G (IgG) and IgM antibodies, detected by immunofluorescence or enzyme-linked immunosorbent assay or both, underwent a pregnancy-associated decrease of 20 to 40%, whereas complement-mediated lytic antibodies were unaffected by pregnancy. Immunoblotting analysis indicated identical specificities of the anti-T. cruzi antibodies in IP and INP groups. The levels of all the immunoglobulin isotypes (particularly IgG2a and IgG3), circulating immune complexes, rheumatoid-like factor, and anti-DNA antibodies were considerably increased during chronic infection (NINP versus INP), which could be related to the high degree of polyclonal B-cell activation occurring in T. cruzi infection. However, pregnancy significantly decreased (by 20 to 60%) such parameters. IgG levels were particularly affected (by 40 to 60%), and the decreases could be ordered as follows: IgG3 greater than IgG2a greater than IgG1 greater than IgG2b for IP versus INP. Comparisons between the noninfected groups indicated differences only in IgG levels. These results indicate the following. (i) The specific humoral anti-T. cruzi immune response is weakly affected by pregnancy, which is not sufficient to modify the course of the mother's infection. (ii) Pregnancy does not modify the expression of the anti-T. cruzi antibody repertory. (iii) Pregnancy reduces the polyclonal B-cell activation, particularly the levels of the IgG isotypes undergoing the greatest activation.     

Clearance and tissue uptake of immune complexes in complement-depleted and control mice

The clearance kinetics, specific hepatic uptake and specific splenic uptake of immune complexes were examined in control mice and in mice treated with large doses of purified cobra venom factor (CoF) to deplete serum C3. At least 90% depletion of C3 was achieved as tested by double diffusion with antiserum specific to antigenic determinants on C3. A saturating dose of preformed immune complexes, consisting of HSA and rabbit antibodies to HSA, was used in these experiments. No differences in clearance kinetics and organ uptake of the immune complexes containing IgG as antibodies were observed between the two groups of mice. Within the limits of the experimental system no evidence was obtained for the participation of serum C3 and C3b receptors on Kupffer cells in the hepatic uptake of circulating immune complexes. The apparent discrepancies on the role of C3 and C3b receptors between these experiments and the in vitro studies on the uptake of immune complexes by macrophages is most likely related to the differences in the lattice of immune complexes employed by investigators.     

Parasite-derived trans-sialidase binds to heart tissue in Trypanosoma cruzi-infected animals

Trypanosoma cruzi is an obligate intracellular protozoan parasite that actively penetrates into non-phagocytic mammalian cells. To accomplish this, the parasite relies on the binding of cell surface ligands. It is reported herein that the T. cruzi trans-sialidase (TS), which is exposed on the parasite surface, binds to mouse heart cells, and should therefore be further studied as a possible cell penetration-related ligand. In addition, as has been proposed elsewhere, the binding of T. cruzi to tissues may turn them into targets for parasite-specific immune reactions. Washed heart sections from T. cruzi-infected mice were subjected to immunoenzymatic staining with antisera against whole T. cruzi and with polyclonal or monoclonal antibodies against TS. The anti-TS antibodies stained both parasites and uninfected heart cells in the vicinity of T. cruzi nest remains/trypomastigotes. On the other hand, an anti-T. cruzi serum, which did not recognize TS, only stained the parasites. In addition, normal heart sections from uninfected nude mice were shown to react with both enzymatically active and inactive recombinant TS molecules, probably through their amino-terminal region, since a recombinant TS lacking this region failed to bind.     

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.     

Determination of the specificity of seric IgA produced in response to antigens of Leishmania (Leishmania) mexicana in murine leishmaniasis

In experimental leishmaniasis, the role of antibodies is not entirely clear, as some authors consider that these proteins are not involved in protection against infection. However, histopathological studies in human and experimental leishmaniasis lesions, show plasma cell infiltrates positive for IgA and secretion of IgM, IgG and IgA could mediate the formation of immune complexes with parasite antigens or self components, favoring necrosis leading to the elimination of the parasite. In this study, we determined if the serum IgA in the murine model has specific reactivity against antigens of Leishmania (Leishmania) mexicana of diagnostic utility. To do this, we used mice either susceptible or resistant to cutaneous leishmaniasis, and demonstrated by indirect ELISA that serum IgA is elevated in susceptible mice compared with that produced by resistant mice. Although other studies in murine models show that the serum IgG from mice infected with L. (L) mexicana present cross reactivity with unrelated parasite antigens derived from Trypanosoma cruzi, the analysis of the specificity of IgA by antigens of L. (L) mexicana and T. cruzi, by Western Blot, showed that the IgA serum of mice infected with T. cruzi reacts too with antigens of L. (L) mexicana. These findings suggest that IgA may be useful for the clinical management and prognosis of the disease.     

Endotoxin-induced auto-immunity in mice. I. Time and dose dependence of production and serum levels of antibodies against bromelain-treated mouse erythrocytes and circulating immune complexes

Injection of mice with endotoxin results in the formation of auto-antibodies and the appearance of soluble immune complexes in the blood. In this study the relationship between the production and serum levels of autohaemolysins and circulating immune complex titres was investigated. Immune complexes were detected by a solid-phase C1q-binding assay and found to contain antibodies of the IgM, but not of the IgG class. Individual mice showed marked differences as to their splenic plaque-forming cells, serum autohaemolysin, and circulating immune complex responses, both in kinetic studies and dose-response experiments. The dissociation between production and serum levels of auto-antibodies was ascribed to extrasplenic synthesis or a disproportionate production per plasma cell. The independent behaviour of the circulating immune complex response could, at least partially, be attributed to differential complement-dependent clearance from the circulation. The implications of our findings for the laboratory diagnosis of auto-immunity at the blood level are being discussed.     

The adjuvant effect of jacalin on the mouse humoral immune response to trinitrophenyl and Trypanosoma cruzi.

We have evaluated the adjuvant action of jacalin, a lectin obtained from seeds of Artocarpus integrifolia, on humoral immune response against the trinitrophenyl (TNP) hapten when conjugated to it and to Trypanosoma cruzi. The protective effect of parasite-specific antibodies generated in mice immunized with epimastigote forms of T. cruzi plus jacalin was also evaluated by determining the parasitemia levels of animals after infection with 1000 trypomastigote forms. Immunization of mice with trinitrophenylated jacalin (TNP-JAC) in saline resulted in an antibody response to the TNP hapten that was eight and 16 times higher than that found in mice immunized with TNP-human gamma globulin (TNP-HGG) or TNP-bovine serum albumin (TNP-BSA), respectively. In addition, immunization with either a lysate or viable epimastigote forms of T. cruzi in the presence of jacalin resulted in a marked increase in the levels of anti-T. cruzi antibodies. The protective action of antibodies against acute infection by T. cruzi was evident when mice were immunized with 1.0 x 10(5) epimastigotes plus jacalin. These animals had a significantly lower parasitemia than those immunized with epimastigotes alone. In contrast, mice immunized with 1.0 x 10(6) epimastigotes developed very low levels of parasitemia, regardless of the presence of jacalin. These data suggest that jacalin is a potent adjuvant in the humoral response to TNP and T. cruzi, and that the protective action of the T. cruzi-specific antibodies depends on the number of parasites used in the immunization protocol.     

A comparative study of anti-Trypanosoma cruzi serum obtained in acute and chronic phase of infection in mice

The IgG antibody content, specificity, lytic activity, clearance capacity and protective ability of mouse anti-Trypanosoma cruzi serum was determined during the course of infection. The IgG antibody content increased during the course of infection, reaching its highest level in the serum collected in the chronic phase of the infection. The T. cruzi antigens recognized by antibodies using the protein transfer technique also increased with time of infection. Antibodies present in day 22 post-infection (p.i.) serum were already able to recognize all the antigens detected by antibodies present in serum from the chronic phase. The lytic and clearance ability were not detected on day 7 p.i., but appeared on day 14 p.i. and reached their highest level on day 45 p.i. The protective ability was present in serum of the chronic phase, but was absent from the acute serum. The IgG antibody content of the acute serum was four times less than that of the chronic serum. When the IgG antibody concentration of the acute serum was equalized to that of the chronic serum, the acute serum was as able to protect the infected animals as the chronic serum. It is suggested that the disagreement between the protective ability of anti-T. cruzi antisera collected in the acute or in the chronic phase of the infection is due to a quantitative rather than a qualitative difference.     

Effects of complement depletion in experimental chagas disease: immune lysis of virulent blood forms of Trypanosoma cruzi.

In mice infected with virulent blood (trypomastigote) forms of Trypanosoma cruzi, complement depletion with cobra venom factor caused a marked exacerbation of the disease evidenced by significantly increased parasitemia levels and early mortality as compared with those of untreated infected animals. The effect was greater in mice receiving cobra venom factor on day 7 postinfection, i.e., at the time when the parasites had had time to localize and multiply in the tissues and appeared in the circulation in appreciable numbers. The possibility that complement participates in host defense against T. cruzi infection through a mechanism involving immune lysis was explored in vitro. T. cruzi trypomastigotes were found to undergo immune lysis in sera of patients with chronic Chagas' disease, in sera of immunized mice, and in solutions containing both immune mouse gamma globulin and a source of active complement. This phenomenon failed to take place either in the absence of complement or after complement inactivation by heat or utilizing complement inactivators. The lytic capacity of heated sera was restored by the addition of active complement to the system. During the immune lysis of T. cruzi blood forms, complement was activated in human sera via both the classical and the alternate pathways. In mouse sera, activation followed at least the alternate pathway.     

Anatomical route of invasion and protective mucosal immunity in Trypanosoma cruzi conjunctival infection

Trypanosoma cruzi is a protozoan parasite that can initiate mucosal infection after conjunctival exposure. The anatomical route of T. cruzi invasion and spread after conjunctival parasite contamination remains poorly characterized. In the present work we have identified the sites of initial invasion and replication after contaminative conjunctival challenges with T. cruzi metacyclic trypomastigotes using a combination of immunohistochemical and real-time PCR confirmatory techniques in 56 mice between 3 and 14 days after challenge. Our results demonstrate that the predominant route of infection involves drainage of parasites through the nasolacrimal duct into the nasal cavity. Initial parasite invasion occurs within the ductal and respiratory epithelia. After successive waves of intracellular replication and cell-to-cell spread, parasites drain via local lymphatic channels to lymph nodes and then disseminate through the blood to distant tissues. This model of conjunctival challenge was used to identify immune responses associated with protection against mucosal infection. Preceding mucosal infection induces mucosal immunity, resulting in at least 50-fold reductions in recoverable tissue parasite DNA in immune mice compared to controls 10 days after conjunctival challenge (P < 0.05). Antigen-specific gamma interferon production by T cells was increased at least 100-fold in cells harvested from immune mice (P < 0.05). Mucosal secretions containing T. cruzi-specific secretory immunoglobulin A harvested from immune mice were shown to protect against mucosal parasite infection (P < 0.05), demonstrating that mucosal antibodies can play a role in T. cruzi immunity. This model provides an important tool for detailed studies of mucosal immunity necessary for the development of mucosal vaccines.     

Protected Trypanosoma cruzi infection in rats born to mothers receiving interferon-gamma during gestation is associated with a decreased intramacrophage parasite growth and preferential synthesis of specific IgG2b antibodies

We demonstrated that administration of interferon gamma (IFN-gamma) to pregnant rats conferred partial resistance in their offspring to further challenge with Trypanosoma cruzi. Because of the effects of IFN-gamma on macrophage activation and immunoglobulin isotype selection, offspring were now studied to ascertain whether this intervention modifies the in vitro replication of T. cruzi and nitric oxide (NO) production by peritoneal macrophages (PE), together with the anti-T. cruzi IgG isotypes. To evaluate the possibility of a detrimental effect of IFN-gamma, serum levels of anti-sulphatide autoantibodies were also investigated. Offspring were born to mothers undergoing one of the following procedures during gestation: treatment with recombinant rat IFN-gamma, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating; infection with 10(6) trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gamma treatment as given to the former group; the same protocol except that physiological saline was injected instead of IFN-gamma; injection of physiological saline only. Offspring were challenged at weaning with a similar dose of T. cruzi, to constitute four groups of infected young, plus an additional group of age-matched uninfected rats born to control mothers. PE were harvested at day 7 postinfection (pi), exposed to parasites and further investigated for the replication of T. cruzi and NO production, whereas ELISA studies for measuring serum anti-T. cruzi IgG subclasses and anti-sulphatide autoantibodies were performed at day 30 pi. The number of intracellular parasites in PE was markedly decreased in young born to IFN-gamma-treated mothers, this not being accompanied by higher nitrite levels in culture supernatants. Offspring delivered by IFN-gamma-treated mothers showed no higher serum concentrations of anti-sulphatide autoantibodies, but exhibited a preferential synthesis of anti-T. cruzi IgG2b antibodies. This rat isotype is known to fix complement and constitutes the rat counterpart of IgG2a mouse immunoglobulins whose synthesis is favoured by IFN-gamma.     

A mouse monoclonal antibody that reacts specifically with immune-complexed human IgG

After human IgG binds to antigen, it attains biological functions that are not properties of monomeric, uncomplexed IgG, including the ability to activate complement and to bind to cellular receptors. Associated with antigen binding, we have recently demonstrated that IgG itself has neoantigenic epitopes. Antibodies to these neoantigens on immune-complexed IgG may represent a significant proportion of circulating anti-human IgG in rabbits immunized with immune complexes. In contrast, mice immunized in an identical fashion have very little circulating anti-neoantigen antibody. This is true whether the mice are genetically easy to tolerize to monomeric human IgG (DBA/2 and C57Bl/6) or difficult to tolerize (BALB/c). Fusions were made between the NS-1 myeloma cell line and spleen cells from mice of each strain, which had been made tolerant to monomeric human IgG and then immunized with immune complexes containing IgG. Like the serum antibody, antibodies made by these fusions showed little specificity for immune complexes since 99% of the hybridoma antibodies that recognized IgG in immune complexes also bound to uncomplexed IgG. Only 1 hybridoma produced antibody that preferentially recognized human IgG in immune complexes. This antibody, called CE9, is an IgM that binds to IgG in plate-bound immune complexes with 100-1000-fold greater avidity than it does to plate-bound uncomplexed IgG. Because CE9 will not bind to immune complexes made with F(ab')2 antibody, the epitope it recognizes requires the Fc fragment of IgG. The minimal binding of CE9 to uncomplexed IgG is easily inhibited by soluble aggregates of IgG, but binding to immune complexes is not inhibited by aggregated IgG. CE9 does recognize fluid-phase immune complexes as well as solid-phase immune complexes. We conclude that, while mice produce much less anti-immune complex antibody than rabbits, anti-neoantigen is still a component of their response to immunization with immune complexes. Using hybridoma techniques to amplify these anti-neoantigen antibodies, we have shown that they resemble rheumatoid factors in their isotype and binding properties.     

Relationship between complement activation and renal deposition of immune complexes made with IgG2a monoclonal antibodies

Previous studies identified a single murine monoclonal IgG2a anti-dinitrophenyl antibody that, when combined with the antigen, formed immune complexes (IC) that were preferentially deposited in glomeruli. The present study examined the clearance and organ localization in Balb/c mice of expanded panels of radiolabeled IC containing murine monoclonal antibodies. The results identified a second IgG2a antibody that formed IC with a predilection for renal deposition. IC made with the two IgG2a antibodies that were preferentially deposited in the kidney were the least efficient binders of human C1q or homologous murine C3b and C4b within the IgG2a panel. These observations suggest a new model of IC-mediated renal disease initiation in which relatively weak complement activation leads to inefficient IC clearance by complement receptor-bearing circulating cells and consequent IC deposition in tissues susceptible to IC-mediated injury.