RESUMEN
Trypanosoma cruzi, a protozoan parasite and the causative agent of Chagas' disease, induces a state of lymphocyte hyporesponsiveness to both mitogenic and antigenic stimuli in mice during the acute phase of infection. Addition of spleen cells from T. cruzi-infected mice (SCinf) to microcultures of spleen cells from noninfected mice (SCn) suppresses the responsiveness of such cultures to antigenic challenge and to mitogenic stimulation. We analyzed the regulatory cell populations in SCinf by limiting dilution-partition analysis and found a complex regulatory circuit in T. cruzi-infected mice consisting of two suppressive macrophage subsets and an enhancing T-cell population. This T-cell population was able to abrogate or escape the suppressive ability of one suppressor macrophage subset, yet was suppressed by the other macrophage subset. To further study the cellular interactions of this regulatory circuit and analyze the suppressive abilities of the two suppressor macrophage subsets, we examined the effect of adding either primed T helper cells of known specificity or interleukin-2 to the limiting dilution-partition analysis microcultures. The results of these experiments suggest that one suppressor macrophage subset, which is abundant and, therefore, detected with low doses of SCinf, is able to suppress both mitogen- and primary antigen-specific responses but is unable to inhibit cells once they are already activated or primed. The other macrophage subset, which is presumably a less abundant or less active population (since high doses of SCinf are required to detect it), is able to suppress the response of activated or primed T cells by the inhibition of interleukin-2 production.
Asunto(s)
Enfermedad de Chagas/inmunología , Interleucina-2/inmunología , Macrófagos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Femenino , Técnica de Placa Hemolítica , Técnicas In Vitro , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fitohemaglutininas , Bazo/citologíaRESUMEN
Mice infected with the protozoan parasite, Trypanosoma cruzi, are known to be immunosuppressed in responsiveness to heterologous antigens and parasite-specific antigens. This suppression is mediated by suppressor macrophages and is exemplified by deficient T cell activity and abnormal cytokine production. Neither the mechanism by which suppressor macrophages effect suppression nor the characteristics of these suppressor macrophages is known. In the present study, we analyzed the regulatory cell populations in splenocytes of infected mice (SCinf) and their interactions by limiting dilution-partition analysis, an approach which allows the functional separation of multiple regulatory cell subpopulations within cell mixtures. Our results demonstrate the presence of a complex immunoregulatory circuit in SCinf affecting the generation of anti-sheep erythrocyte antibody responses in vitro. Titration of SCinf (but not peritoneal exudate or lymph node cells) into Mishell-Dutton microcultures of normal spleen cells generated complex dose-response curves with two zones of suppressed responses following the addition of either low or high doses of SCinf to the cultures. Addition of intermediate doses of SCinf to the microcultures restored responsiveness. Both the low- and high-dose zones of suppression were shown to be mediated by macrophages, whereas T cells were responsible for the restored responsiveness at intermediate doses of SCinf. Examination of the development of this complex regulatory pattern during the course of the acute phase of infection indicated the sequential development of one suppressor macrophage population, followed by the development of the beneficial T cell population, and finally the expression of the second suppressive macrophage population.
Asunto(s)
Enfermedad de Chagas/inmunología , Macrófagos/inmunología , Bazo/inmunología , Animales , Células Productoras de Anticuerpos/fisiología , Técnicas In Vitro , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Linfocitos T Colaboradores-Inductores/fisiología , Linfocitos T Reguladores/fisiologíaRESUMEN
Limiting dilution analysis has been a valuable approach for both determining the frequency of cell subpopulations elicited during immune responses, as well as for the analysis of immunoregulatory circuits. We describe a simple, visually scored spot test for evaluating the response of Mishell-Dutton microcultures used in limiting dilution analysis. This spot test is based on a microELISA using immunoreagents conjugated to the enzyme, urease, as an alternative to the hemolytic spot test. The assay as performed in Terasaki trays requires minute quantities (less than 10 microliters) of culture supernatant, yet the ELISA yields a distinct color difference between tray wells containing culture supernatants derived from responding (purple) and nonresponding (yellow) microcultures. Although designed to be scored rapidly by visual inspection, the assay can be quantified by manual alignment of the Terasaki tray wells on commercially available ELISA plate readers with an accuracy and reproducibility comparable to assays performed in 96-well ELISA plates. Determination of anti-sheep RBC responses in limiting dilution Mishell-Dutton microcultures with both the hemolytic spot test and the urease-microELISA spot test showed a very close correlation between the results of the two assays. However, the urease-microELISA should be amenable for use with antigens not readily conjugated to an indicator RBC, and should be useful in those situations where determination of the antibody subclass(es) produced by responding microcultures is desired.
Asunto(s)
Anticuerpos/análisis , Ensayo de Inmunoadsorción Enzimática , Ureasa , Animales , Femenino , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BLRESUMEN
We isolated and sequenced the gene for the chlamydial heat shock protein 60 (HSP-60) from a Chlamydia trachomatis genomic library by molecular genetic methods. The DNA sequence derived revealed an operon-like gene structure with two open reading frames encoding an 11,122- and a 57,956-Da protein. The translated amino acid sequence of the larger open reading frame showed a high degree of homology with known sequences for HSP-60 from several bacterial species as well as with plant and human sequences. By using the determined nucleotide sequence, fragments of the gene were cloned into the plasmid vector pGEX for expression as fusion proteins consisting of glutathione S-transferase and peptide portions of the chlamydial HSP-60. HSP-60 antigenic identity was confirmed by an immunoblot with anti-HSP-60 rabbit serum. Sera from patients that exhibited both high antichlamydial titers and reactivity to chlamydial HSP-60 showed reactivity on immunoblots to two fusion proteins that represented portions of the carboxyl-terminal half of the molecule, whereas fusion proteins defining the amino-terminal half were nonreactive. No reactivity with the fusion proteins was seen with sera from patients that had been previously screened as nonreactive to native chlamydial HSP-60 but which had high antichlamydial titers. Sera from noninfected control subjects also exhibited no reactivity. Definition of recognized HSP-60 epitopes may provide a predictive screen for those patients with C. trachomatis infections who may develop damaging sequelae, as well as providing tools for the study of immunopathogenic mechanisms of Chlamydia-induced disease.
Asunto(s)
Chlamydia trachomatis/genética , Clonación Molecular , Proteínas de Choque Térmico/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/química , Glutatión Transferasa/análisis , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/fisiología , Datos de Secuencia MolecularRESUMEN
Two cysteine-rich proteins of Chlamydia trachomatis are essential structural components of the unique outer membrane of the infectious elementary body. These 58,000 (outer membrane protein 2; OMP2) and 15,000 (OMP3) proteins also differ structurally and chemically between biovariants that differ in invasive capability. We have identified the gene for OMP3 and sequenced both trachoma and lymphogranuloma venereum (LGV) omp3 genes. We have previously sequenced omp2 from the LGV biovar and now describe the omp2 sequence for a trachoma biovariant. Amino acid sequence differences between biovariants were few but, significantly, these changes have altered the charge of both OMP2 and OMP3 such that the net charge of each protein differs between biovariants. These compensatory charge alterations have implications for the outer membrane organization of these proteins. In addition, examination of the OMP3 sequence suggests that OMP3 may be a lipoprotein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Chlamydia trachomatis/genética , Variación Genética , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Secuencia de Bases , Chlamydia trachomatis/metabolismo , Cisteína , Genes Bacterianos , Immunoblotting , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
These studies showed that increased natural killer (NK) cell activity develops during experimental American Trypanosomiasis in mice. Increased cytotoxicity against YAC target cells by peritoneal cavity cells (PEC) was detected as early as 1 day into infection in both C3H/He and C57BL/6 mice. Peak activity occurred 2 days into infection, with significant but variable increases detected in the spleen cells (SC) from these mice. The response subsided in PEC and to a lesser extent in SC by 4 days post-infection. Similar responses were not detected in infected C57BL/6 bgJ/bgJ (beige mutant) mice until the fourth day of infection and peaked at significantly lower levels than the peak on day 2 in beige hybrid mice ([C57BL/6 +/+ X C57BL/6 bgJ/bgJ]F1). The effector cell from infected mice was sensitive to pretreatment with anti-NK 1.2 serum plus complement (C), partially sensitive to anti-Thy 1.2 + C, and insensitive to polyvalent anti-mouse immunoglobulin + C. The cytotoxic activity induced in cells from mice infected for 2 days was recovered in subpopulations nonadherent to plastic or Sephadex G-10. Thus, the anti-YAC effector cell found very early in mice infected with T. cruzi possessed many of the characteristics of natural killer (NK) cells found in normal mice. Other experiments demonstrated that injection of heat-killed preparations of blood-form trypomastigotes (BFT) induced increases in NK activity. The presence of augmented NK activity against YAC tumor cells in both resistant and susceptible strains of mice, together with the presentation of the resistance phenotype in beige mutant mice, which had a lower NK response to infection, is not indicative of a direct role for host protection during infection with T. cruzi by NK cells.