RESUMO
Although a complete cellular and humoral immune response is elicited in Chagas' disease, recent data suggest that other natural elements of innate immunity may also contribute to the initial host primary defense. alpha-Macroglobulins are a family of plasma proteinase inhibitors that are acute-phase reactants in Trypanosoma cruzi-infected mice and humans. Mice contain a tetrameric alpha-2-macroglobulin (MAM) and a monomeric murinoglobulin (MUG). Heterogeneity in their reactions was observed in murine T. cruzi-infected plasma A2M levels despite an overall increase. In addition, up-regulation of the A2M receptor (A2MR/LRP) was observed in peritoneal macrophages during T. cruzi infection. Here, we show that during T. cruzi infection (Y strain), the MAM and MUG hepatic mRNA levels and the corresponding plasma protein levels were up-regulated in C3H and C57BL/6 (B6) mice, but with different kinetics. On the contrary, A2MR/LRP mRNA levels increased in acutely infected C3H mice, but decreased in B6 mice, in both liver and heart. Immunocytochemistry of infected B6 heart cryosections confirmed a less intense endothelium labeling by the fluoresceinated ligand for A2MR/LRP. On the other hand, infected B6 spleen cells displayed higher F-A2M-FITC binding and MAC1 expression, confirming higher A2MR/LRP expression in macrophages. In uninfected mice, as well as after T. cruzi infection, higher A2M plasma levels were measured in C3H mice than in B6 mice. The lower tissue T. cruzi parasitism found in C3H-infected mice could reflect an inhibitory effect of A2M on parasite invasion. Our present data further contribute to clarifying aspects of the role of A2MR/LRP in a model of acute Chagas' disease in different mouse strains.
Assuntos
Doença de Chagas/metabolismo , Receptores Imunológicos/biossíntese , alfa-Macroglobulinas/biossíntese , Doença Aguda , Animais , Doença de Chagas/genética , Doença de Chagas/parasitologia , Expressão Gênica , Coração/parasitologia , Fígado/química , Fígado/metabolismo , Fígado/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/patologia , Tamanho do Órgão , Parasitemia/genética , Parasitemia/metabolismo , Parasitemia/parasitologia , RNA Mensageiro/análise , Receptores Imunológicos/genética , Soroglobulinas/biossíntese , Soroglobulinas/genética , Baço/química , Baço/metabolismo , Baço/patologia , Trypanosoma cruzi/fisiologia , Regulação para Cima , alfa-Macroglobulinas/genéticaRESUMO
Mannosyl binding sites were detected "in vitro" on cardiomyocytes (CM) surface using horseradish peroxidase (HRP) as the ligand. Binding assays revealed a specific recognition system, which was time- and concentration-dependent. The binding required physiological pH and was inhibited by EDTA and trypsin treatments. HRP binding was reduced by pre-incubations with low concentrations of D-mannose. Ultrastructural analysis of the endocytic process was followed using HRP coupled to colloidal gold particles (HRP-Au). The tracer was found within caveolae characterizing early steps of the receptor-mediated endocytosis. The addition of 10 mM D-mannose to the interaction medium blocked Trypanosoma cruzi uptake by CM. The labeling of CM with a subsaturating concentration of HRP-Au before their infection showed, by ultrastructural studies, that its association with trypomastigote forms occurred frequently near to HRP-gold particles that could also be seen to comprise the parasitophorous vacuole. After infection of CM with T. cruzi, a considerable reduction on HRP binding was noticed. Binding was almost completely restored by treating the infected cultures with the trypanocidal drug Nifurtimox. Our "in vitro" findings suggest that cardiomyocyte's mannose receptors localized at the sarcolemma mediates T. cruzi recognition and can be down-modulated by parasite infection.
Assuntos
Lectinas Tipo C , Lectinas de Ligação a Manose , Miocárdio/metabolismo , Receptores de Superfície Celular/metabolismo , Trypanosoma cruzi/metabolismo , Tripanossomíase/metabolismo , Animais , Regulação para Baixo , Endocitose , Ensaio de Imunoadsorção Enzimática , Galactose/metabolismo , Coração/embriologia , Peroxidase do Rábano Silvestre/metabolismo , Peroxidase do Rábano Silvestre/ultraestrutura , Concentração de Íons de Hidrogênio , Manose/metabolismo , Receptor de Manose , Camundongos , Microscopia Eletrônica , Tripanossomíase/parasitologiaRESUMO
The surface charge of heart muscle cells (HMC) and Trypanosoma cruzi trypomastigotes was estimated during their interaction by means of zeta potential (ZP). Metacyclic and bloodstream trypomastigote, but not amastigote forms, are able to decrease the surface charge of HMC as well as other nonphagocytic cells. However, no alteration could be detected on T. cruzi-infected macrophage cell line. Trypomastigote forms collected from the supernatant after 20 h of contact with HMC also have their ZP value decreased. The analysis of the surface components of both the parasite and HMC involved in such interaction was also carried out. Assays concerning the kinetics of the cell-parasite interaction demonstrated the influence of parasite surface anionogenicity during its interaction with HMC. The binding of bloodstream forms to HMC was enhanced after their incubation with cationized ferritin (CF), whereas phospholipase C and neuraminidase treatments improved and trypsin treatment inhibited parasite uptake in HMC. Conversely, the incubation of HMC with phospholipase C impaired, and with trypsin enhanced, the interiorization of the parasites. These results suggest that trypomastigote forms of T. cruzi may process the surface of HMC and its own surface either by removing molecules or by exposing ligands for their internalization.
Assuntos
Doença de Chagas/fisiopatologia , Coração/parasitologia , Trypanosoma cruzi/patogenicidade , Animais , Ânions , Células Cultivadas , Camundongos , Miocárdio/patologia , Neuraminidase/farmacologia , Inibidores de Proteases/farmacologia , Propriedades de SuperfícieRESUMO
We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels--hitherto observed in all experiments especially in those using HMC--CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continuous fibroblastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background.