RESUMEN
Regulatory B cells are now recognized as an essential component of the immune system. The function of regulatory B cells is dependent on IL-10. The cell-surface phenotype of murine IL-10-producing regulatory B cells is reported to be CD1d(hi)CD5(+) B cell or CD21(hi)CD23(hi) T2 marginal zone precursor B cells. B cells play several critical roles in the pathogenesis of systemic lupus erythematosus (SLE). It is now apparent that regulatory B cells are important for disease suppression in SLE. Regulatory B cells inhibit disease onset of NZB/W F1 mice, a spontaneous SLE mouse model. Furthermore, the potential therapeutic effect of regulatory B cells in NZB/W F1 mice is highlighted by the adoptive transfer of splenic CD1d(hi)CD5(+) B cells. Regulatory B cells also suppress the disease manifestation in MRL-Fas(lpr) mice, a SLE mouse model. Thus, regulatory B cells have protective role and therapeutic effects in mouse modes of SLE. Herein, the methods for evaluating SLE mouse model, B-cell depletion, and regulatory B-cell analysis are provided. These methods should facilitate the study of regulatory B cells in SLE.
Asunto(s)
Linfocitos B Reguladores/inmunología , Linfocitos B Reguladores/patología , Modelos Animales de Enfermedad , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones Endogámicos NZB/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antinucleares/análisis , Humanos , Riñón/inmunología , Riñón/patología , Enfermedades Renales/complicaciones , Enfermedades Renales/diagnóstico , Enfermedades Renales/inmunología , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/diagnóstico , Proteinuria/complicaciones , Proteinuria/diagnósticoRESUMEN
H.P. Acthar Gel® (Acthar) is a highly purified repository gel preparation of adrenocorticotropic hormone (ACTH1-39), a melanocortin peptide that can bind and activate specific receptors expressed on a range of systemic lupus erythematosus (SLE)-relevant target cells and tissues. This study was performed to evaluate the effects of Acthar in a mouse model of SLE, using an F1 hybrid of the New Zealand Black and New Zealand White strains (NZB/W F1). Twenty-eight week old NZB/W F1 mice with established autoimmune disease were treated with Acthar, Placebo Gel (Placebo), or prednisolone and monitored for 19 weeks. Outcomes assessed included disease severity (severe proteinuria, ≥ 20% body weight loss, or prostration), measurement of serial serum autoantibody titers, terminal spleen immunophenotyping, and evaluation of renal histopathology. Acthar treatment was linked with evidence of altered B cell differentiation and development, manifested by a significant reduction in splenic B cell follicular and germinal center cells, and decreased levels of circulating total and anti-double-stranded DNA (IgM, IgG, and IgG2a) autoantibodies as compared with Placebo. Additionally, Acthar treatment resulted in a significant decrease of proteinuria, reduced renal lymphocyte infiltration, and attenuation of glomerular immune complex deposition. These data suggest that Acthar diminished pathogenic autoimmune responses in the spleen, peripheral blood, and kidney of NZB/W F1 mice. This is the first preclinical evidence demonstrating Acthar's potential immunomodulatory activity and efficacy in a murine model of systemic lupus erythematosus.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Modelos Animales de Enfermedad , Hormonas/farmacología , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos NZB/inmunología , Animales , Femenino , Geles , RatonesRESUMEN
The lupus-prone New Zealand Black (NZB) strain uniquely develops a genetically imposed severe spontaneous autoimmune hemolytic anemia (AIHA) that is very similar to the corresponding human disease. Previous studies have mapped anti-erythrocyte Ab (AEA)-promoting NZB loci to several chromosomal locations, including chromosome 4; however, none of these have been analyzed with interval congenics. In this study, we used NZB.NZW-Lbw2 congenic (designated Lbw2 congenic) mice containing an introgressed fragment of New Zealand White (NZW) on chromosome 4 encompassing Lbw2, a locus previously linked to survival, glomerulonephritis, and splenomegaly, to investigate its role in AIHA. Lbw2 congenic mice exhibited marked reductions in AEAs and splenomegaly but not in anti-nuclear Abs. Furthermore, Lbw2 congenics had greater numbers of marginal zone B cells and reduced expansion of peritoneal cells, particularly the B-1a cell subset at early ages, but no reduction in B cell response to LPS. Analysis of a panel of subinterval congenic mice showed that the full effect of Lbw2 on AEA production was dependent on three subloci, with splenomegaly mapping to two of the subloci and expansions of peritoneal cell populations, including B-1a cells to one. These results directly demonstrated the presence of AEA-specific promoting genes on NZB chromosome 4, documented a marked influence of background genes on autoimmune phenotypes related to Lbw2, and further refined the locations of the underlying genetic variants. Delineation of the Lbw2 genes should yield new insights into both the pathogenesis of AIHA and the nature of epistatic interactions of lupus-modifying genetic variants.
Asunto(s)
Anemia Hemolítica Autoinmune/genética , Anemia Hemolítica Congénita/genética , Lupus Eritematoso Sistémico/genética , Ratones Endogámicos NZB/genética , Anemia Hemolítica Autoinmune/inmunología , Anemia Hemolítica Autoinmune/patología , Anemia Hemolítica Congénita/inmunología , Anemia Hemolítica Congénita/patología , Animales , Líquido Ascítico/inmunología , Líquido Ascítico/patología , Autoanticuerpos/biosíntesis , Autoanticuerpos/genética , Subgrupos de Linfocitos B/inmunología , Mapeo Cromosómico , Prueba de Coombs , Cruzamientos Genéticos , Eritrocitos/inmunología , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Ratones , Ratones Congénicos , Ratones Endogámicos NZB/inmunología , Ratones Endogámicos , Bazo/inmunología , Bazo/patología , Esplenomegalia/etiologíaRESUMEN
Adjuvants, commonly used in vaccines, may be responsible for inducing autoimmunity and autoimmune diseases, both in humans and mice. The so-called 'ASIA' (Autoimmune/inflammatory Syndrome Induced by Adjuvants) syndrome has been recently described, which is caused by the exposure to a component reproducing the effect of adjuvants. The aim of our study was to evaluate the effect of injection of complete Freund's adjuvant (CFA) in NZB/NZWF1 mice, a lupus-prone murine model. We injected 10 NZB/NZWF1 mice with CFA/PBS and 10 with PBS, three times, 3 weeks apart, and followed-up until natural death. CFA-injected mice developed both anti-double-stranded DNA and proteinuria earlier and at higher levels than the control group. Proteinuria-free survival rate and survival rate were significantly lower in CFA-treated mice than in the control mice (p = 0.002 and p = 0.001, respectively). Histological analyses showed a more severe glomerulonephritis in CFA-injected mice compared with the control mice. In addition, lymphoid hyperplasia in spleen and lungs, myocarditis, and vasculitis were observed in the former, but not in the latter group. In conclusion, the injection of CFA in NZB/NZWF1 mice accelerated autoimmune manifestations resembling 'ASIA' syndrome in humans.
Asunto(s)
Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Adyuvante de Freund/efectos adversos , Adyuvante de Freund/inmunología , Ratones Endogámicos NZB/inmunología , Animales , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/fisiopatología , ADN/inmunología , Femenino , Adyuvante de Freund/administración & dosificación , Humanos , Ratones , Ratones Endogámicos BALB C , Tasa de Supervivencia , SíndromeRESUMEN
The New Zealand Black (NZB) Lbw2 locus (lupus NZB x New Zealand White (NZW) 2 locus) was previously linked to mortality and glomerulonephritis, but not to IgG autoantibodies, suggesting that it played a role in a later disease stage. To define its contribution, (NZB x NZW)F1 hybrids (BWF1) containing two, one, or no copies of this locus were generated. Lack of the NZB Lbw2 indeed reduced mortality and glomerulonephritis, but not serum levels of total and anti-DNA IgG Abs. There were, however, significant reductions in the B cell response to LPS, total and anti-DNA IgM and IgG Ab-forming cells, IgM Ab levels, and glomerular Ig deposits. Furthermore, although serum IgG autoantibody levels correlated poorly with kidney IgG deposits, the number of spontaneous IgG Ab-forming cells had a significant correlation. Genome-wide mapping of IgM anti-chromatin levels identified only Lbw2, and analysis of subinterval congenics tentatively reduced Lbw2 to approximately 5 Mb. Because no known genes associated with B cell activation and lupus are in this interval, Lbw2 probably represents a novel B cell activation gene. These findings establish the importance of Lbw2 in the BWF1 hybrid and indicate that Lbw2, by enhancing B cell hyperactivity, promotes the early polyclonal activation of B cells and subsequent production of autoantibodies.
Asunto(s)
Autoinmunidad/genética , Ratones Endogámicos NZB/genética , Ratones Endogámicos NZB/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/genética , Linfocitos B/inmunología , Secuencia de Bases , Mapeo Cromosómico , Femenino , Dosificación de Gen , Ligamiento Genético , Hibridación Genética , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/genética , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Lipopolisacáridos/toxicidad , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/genética , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Activación de Linfocitos , Masculino , Ratones , Ratones Congénicos , Repeticiones de Microsatélite , Fenotipo , Bazo/inmunología , Bazo/patologíaRESUMEN
F(1) hybrids among New Zealand Black (NZB), New Zealand White (NZW), and BXSB lupus-prone strains develop accelerated autoimmunity in both sexes regardless of the specific combination. To identify BXSB susceptibility loci in the absence of the Y chromosome accelerator of autoimmunity (Yaa) and to study the genetics of this complementation, genome-wide quantitative trait locus (QTL) mapping was performed on female (BXSB x NZW)F(2) mice. Six QTL were identified on chromosomes 1, 4, 5, 6, 7, and 17. Survival mapped to chromosomes 5 and 17, anti-chromatin Ab to chromosomes 4 and 17, glomerulonephritis to chromosomes 6 and 17, and splenomegaly to chromosomes 1, 7, and 17. QTL on chromosomes 4 and 6 were new and designated as Lxw1 and -2, respectively. Two non-MHC QTL (chromosomes 1 and 4) were inherited from the BXSB and the rest were NZW-derived, including two similar to previously defined loci. Only two of 11 previously defined non-MHC BXSB QTL using male (Yaa(+)) crosses were implicated, suggesting that some male-defined BXSB QTL may require coexpression of the Yaa. Findings from this and other studies indicate that BXSB and NZB backgrounds contribute completely different sets of genes to complement NZW mice. Identification of susceptibility genes and complementing genes in several lupus-prone strain combinations will be important for defining the epistatic effects and background influences on the heterogeneous genetic factors responsible for lupus induction.
Asunto(s)
Cruzamientos Genéticos , Prueba de Complementación Genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos NZB/genética , Ratones Endogámicos NZB/inmunología , Sitios de Carácter Cuantitativo/inmunología , Animales , Arteritis/genética , Arteritis/inmunología , Autoanticuerpos/biosíntesis , Cromatina/inmunología , Mapeo Cromosómico , Femenino , Prueba de Complementación Genética/métodos , Ligamiento Genético/inmunología , Predisposición Genética a la Enfermedad , Genoma , Lupus Eritematoso Sistémico/mortalidad , Ratones , Infarto del Miocardio/genética , Infarto del Miocardio/inmunología , Sitios de Carácter Cuantitativo/genética , Índice de Severidad de la Enfermedad , Esplenomegalia/genética , Esplenomegalia/inmunología , Análisis de SupervivenciaRESUMEN
New Zealand Black (NZB) mice are a well-known animal model of human autoimmune disease. Although the mechanism for development of autoimmunity is unclear, NZB mice are well known for severe thymic microarchitecture abnormalities. It is thought that thymic dendritic cells (DC) may play a role in thymic education and contribute to the autoimmune process. To address this issue and, in particular, that qualitative and/or quantitative differences exist in thymic DC, we took advantage of a novel restriction analysis system that allow definition of differences in the expression of tyrosine kinases using highly enriched populations of thymic DC from NZB compared to BALB/c and C57BL/6 mice. The method chosen, restriction analysis of gene expression, allowed the determination of protein tyrosine kinase transcription profiles. We report herein that NZB mice have a significant upregulation of C-met compared to the control strains. The abnormality of the C-met transcription was confined to thymic DC. We believe that its abnormal expression reflects the resistance of thymic cells to apoptosis, which will ultimately lead to defects and/or abnormal signaling by the interaction of thymic DC and thymocytes. Further studies involving such interactions are under way.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ratones Endogámicos NZB/inmunología , Ratones Endogámicos NZB/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Autoinmunidad , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NZB/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-met/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la Especie , Timo/citología , Timo/inmunología , Timo/metabolismo , Regulación hacia Arriba , Proteína bcl-XRESUMEN
Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B-C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre-Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre-Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor component Ig alpha (mb-1). Furthermore, levels of expression of the Rug2, lambda5 and Ig beta (B29) genes are also reduced in Pre-Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre-Pro B cell population occurs at the most primitive stage of B cell differentiation.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Ratones Endogámicos NZB/inmunología , Animales , Apoptosis , Subgrupos de Linfocitos B/metabolismo , Bromodesoxiuridina/metabolismo , Ciclo Celular , Diferenciación Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Lupus Eritematoso Sistémico/etiología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos NZB/genética , Ratones Endogámicos NZB/crecimiento & desarrollo , Especificidad de la EspecieRESUMEN
Intrinsic defects in the B lymphoid lineage are involved in predisposition for systemic lupus erythematosus in (NZB x NZW)F(1) (NZB/W) mice. In addition, a contribution of CD4(+) T cells has been shown to be crucial for the development of fatal glomerulonephritis. To further dissect the role of B and T cells in lupus immunopathology we used Ig mu-heavy chain (muHC) transgenic (Tg) NZB/W mice that we recently established to study mechanisms of B cell tolerance. The Tg NZB/W mice have a very restricted B cell repertoire and only a very minor population of B cells having endogenously rearranged muHC Ig loci are able to undergo isotype switch. Here we analyzed the influence of the restricted B cell repertoire on the development of IgG anti-DNA antibodies and glomerulonephritis as well as the hyperactivation of T(h) cells. IgG anti-DNA antibodies developed delayed but consistently in the Tg NZB/W mice, suggesting that a strong selective mechanism for the development of these autoantibodies is operative. Despite significant autoantibody titers in Tg NZB/W mice, very little immune deposits in the glomeruli and no evidence for renal inflammation were found. The Tg mice have a significantly prolonged survival time and most of the Tg mice lived much longer than 1 year. Interestingly, the generalized T cell activation that normally correlates and coincides with the progression of the disease in NZB/W mice is strongly reduced in older Tg animals. The absence of IgG3 anti-DNA antibodies and the strong reduction of IgG2a anti-DNA antibodies in the Tg mice suggests that particularly the activation of T(h)1 cells is inhibited. This result shows that a significant restriction in the B cell repertoire prevents hyperactivation of T(h) cells and supports the model that T cell hyperactivation in NZB/W mice is secondary to specific interactions with a subpopulation of presumably autoreactive B lymphocytes.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Lupus Eritematoso Sistémico/prevención & control , Ratones Endogámicos NZB/genética , Ratones Endogámicos NZB/inmunología , Transgenes/inmunología , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/sangre , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Movimiento Celular/genética , Movimiento Celular/inmunología , Cruzamientos Genéticos , ADN/inmunología , Femenino , Genes de Inmunoglobulinas/fisiología , Glomerulonefritis/genética , Glomerulonefritis/inmunología , Glomerulonefritis/prevención & control , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Cadenas Pesadas de Inmunoglobulina/fisiología , Cadenas mu de Inmunoglobulina/fisiología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/mortalidad , Lupus Eritematoso Sistémico/patología , Masculino , Ratones , Ratones Transgénicos , Tasa de Supervivencia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patologíaRESUMEN
Defects in T cell development have been suggested to be a factor in the development of systemic autoimmunity in NZB mice. However, the suggestion of a primary T cell defect has often been by extrapolation, and few direct observations of T cell precursors in NZB mice have been performed. Moreover, the capacity of NZB bone marrow T cell precursors to colonize the thymus and the ability of the NZB thymic microenvironment to support T lymphopoiesis have not been analyzed. To address this important issue, we employed the fetal thymic organ culture system to examine NZB T cell development. Our data demonstrated that NZB bone marrow cells were less efficient at colonizing fetal thymic lobes than those of control BALB/c or C57BL/6 mice. In addition, NZB bone marrow cells did not differentiate into mature T cells as efficiently as bone marrow cells from BALB/c or C57BL/6 mice. Further analysis revealed that this defect resulted from an intrinsic deficiency in the NZB Lin-Sca-1+c-kit+ bone marrow stem cell pool to differentiate into T cells in fetal thymic organ culture. Taken together, the data document heretofore unappreciated deficiencies in T cell development that may contribute to the development of the autoimmune phenotype in NZB mice.
Asunto(s)
Linfopenia/genética , Linfopenia/inmunología , Ratones Endogámicos NZB/genética , Ratones Endogámicos NZB/inmunología , Linfocitos T/inmunología , Timo/embriología , Timo/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Prueba de Histocompatibilidad , Leucopoyesis/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Células Madre/inmunología , Células Madre/patología , Linfocitos T/patología , Timo/patologíaRESUMEN
NZB mice spontaneously develop an autoimmune disease characterized by autoimmune hemolytic anemia, thymic atrophy, lymphoid hyperplasia, and hypergammaglobulinemia. The aim of this study was to examine the hypothesis that cryoglobulins may have an immunoregulatory effect on the autoimmune process. The effect of human monoclonal IgM cryoglobulin preparations (including Cryo13, Cryo14, and Cryo16) isolated from the serum of patients with Waldenström's macroglobulinemia on the autoimmune disease of NZB mice was therefore studied. The effect of cryoglobulin preparations was evaluated on several disease parameters, i.e., survival, severity of anemia, and serum IgM and IgG levels (hypergammaglobulinemia). We found that immunization of NZB mice with Cryo13 at 3 months of age delayed the course of the disease, whereas Cryo14 and Cryo16 were ineffective. Furthermore, the effect of Cryo13 was long lasting. On the other hand, Cryo13 was able to react with 8 of 32 mouse monoclonal natural IgM autoantibodies. In contrast, Cryo14 was able to bind only 2 and Cryo16 none of these mouse monoclonal IgM antibodies. These results indicate that, in this model of autoimmune pathology, the beneficial effect of Cryo13 is mediated by its idiotypic interaction with the murine natural autoantibody network.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Crioglobulinas/uso terapéutico , Inmunoglobulina M/uso terapéutico , Nefritis Lúpica/prevención & control , Ratones Endogámicos NZB/inmunología , Anemia Hemolítica Autoinmune/inmunología , Anemia Hemolítica Autoinmune/prevención & control , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Crioglobulinas/química , Crioglobulinas/metabolismo , Femenino , Humanos , Hipergammaglobulinemia/inmunología , Hipergammaglobulinemia/prevención & control , Técnicas para Inmunoenzimas , Idiotipos de Inmunoglobulinas/metabolismo , Inmunoglobulina M/química , Inmunoglobulina M/metabolismo , Inyecciones Intraperitoneales , Nefritis Lúpica/inmunología , Nefritis Lúpica/mortalidad , Ratones , Ratones Endogámicos C57BL , Análisis de SupervivenciaRESUMEN
Polyclonal B cell activation is a hallmark of autoimmune disease in NZB and (NZB x NZW)F(1) (NZB/W) mice. However, the mechanism by which this activated cell subset facilitates disease development is unknown. We recently showed that resting B cells from these mice demonstrate enhanced expression of costimulatory molecules in response to CD40 crosslinking (Jongstra-Bilen et al., J. Immunol. 159,5810-5820, 1997). This led us to question whether activated B cells expressed costimulatory molecules in vivo. Using flow cytometry we found that NZB and NZB/W mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1. These B cells isolate within the low-density activated population and possess the phenotypic characteristics of marginal zone B cells. The levels of B7.1 on the activated B cell population are similar to those induced by CD40 stimulation raising the possibility that activated B cells in NZB and NZB/W mice provide costimulatory signals to self-reactive T cells leading to loss of tolerance.
Asunto(s)
Linfocitos B/inmunología , Antígeno B7-1/metabolismo , Ratones Endogámicos NZB/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos de Superficie/genética , Enfermedades Autoinmunes/metabolismo , Linfocitos B/química , Antígenos CD40/biosíntesis , Ligando de CD40 , Centrifugación por Gradiente de Densidad , Femenino , Ligandos , Activación de Linfocitos/inmunología , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Fenotipo , Povidona , Dióxido de Silicio , Regulación hacia ArribaRESUMEN
There are distinct microenvironmental abnormalities of thymic architecture in several murine models of SLE defined using immunohistochemistry and a panel of mAb dissected at thymic epithelial markers. To address the issue of the relationship between the thymic microenvironment and autoimmunity, we studied backcross (NZB x NZW) F1 x NZW mice in which 50% of offspring develop nephritis associated with proteinuria and anti-DNA antibodies. We reasoned that if thymic abnormalities are associated with development of disease, the correlation of abnormalities with lupus-like disease in individual backcross mice will form the foundation for identification of the mechanisms involved. In parallel, we directed a genetic linkage analysis, using markers previously shown to be linked to nephritis and IgG autoantibody production, to determine if such loci were similarly associated with microenvironmental changes. Our data demonstrate that all (NZB x NZW) F1 x NZW backcross mice with disease have microenvironmental defects. Although the microenvironmental defects are not sufficient for development of autoimmune disease, the severity of thymic abnormalities correlates with titers of IgG autoantibodies to DNA and with proteinuria. Consistent with past studies of (NZB x NZW) F1 x NZW mice, genetic markers on proximal chromosome 17 (near MHC) and distal chromosome 4 showed trends for linkage with nephritis. Although the markers chosen only covered about 10-15% of the genome, the results demonstrated trends for linkage with thymic medullary abnormalities for loci on distal chromosome 4 and distal chromosome 1. We believe it will be important to define the biochemical nature of the molecules recognized by these mAbs to understand the relationships between thymic architecture and immunopathology.
Asunto(s)
ADN/inmunología , Nefritis Lúpica/inmunología , Ratones Endogámicos NZB/inmunología , Proteinuria/inmunología , Timo/inmunología , Animales , Autoinmunidad , Cruzamientos Genéticos , ADN de Cadena Simple/inmunología , Predisposición Genética a la Enfermedad , Inmunoglobulina G/sangre , Isotipos de Inmunoglobulinas/sangre , Nefritis Lúpica/complicaciones , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos NZB/genética , Polimorfismo Genético , Proteinuria/complicaciones , Proteinuria/patología , Timo/patologíaRESUMEN
NZB mice spontaneously develop an autoimmune disease characterized by production of anti-RBC, -lymphocyte, and -ssDNA Abs. Evidence suggests that the NZB mouse strain has all of the immunologic defects required to produce lupus nephritis but lacks an MHC locus that allows pathogenic anti-dsDNA Ab production. The capacity to produce diverse autoantibodies in these mice raises the possibility that they possess a generalized defect in self-tolerance. To determine whether this defect is found within the T cell subset, we backcrossed a transgene encoding bovine insulin (BI) onto the NZB background. In nonautoimmune BALB/c mice, the BI transgene induces a profound but incomplete state of T cell tolerance mediated predominantly by clonal anergy. Comparison of tolerance in NZB and BALB/c BI-transgenic mice clearly demonstrated that NZB T cells were at least as tolerant to BI as BALB/c T cells. NZB BI-transgenic mice did not spontaneously produce anti-BI Abs, and following antigenic challenge, BI-specific Ab production was comparably reduced in both BI-transgenic NZB and BALB/c mice. Further, in vitro BI-specific T cell proliferation and cytokine secretion were appropriately decreased for primed lymph node and splenic T cells derived from NZB BI-transgenic relative to their nontransgenic counterparts. These data indicate that a generalized T cell tolerance defect does not underlie the autoimmune disease in NZB mice. Instead, we propose that the T cell-dependent production of pathogenic IgG autoantibodies in these mice arises from abnormal activation of T cells in the setting of normal but incomplete tolerance.
Asunto(s)
Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/inmunología , Autoinmunidad , Anergia Clonal , Inmunoglobulina M/biosíntesis , Lupus Eritematoso Sistémico/inmunología , Cooperación Linfocítica , Ratones Endogámicos NZB/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Especificidad de Anticuerpos , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/genética , Bovinos , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Femenino , Inmunidad Celular , Insulina/genética , Insulina/inmunología , Lupus Eritematoso Sistémico/genética , Activación de Linfocitos , Linfocinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB/genética , Ratones Transgénicos , TransgenesRESUMEN
To elucidate the mechanism of Ag drive in the anti-DNA response, the Ab response to bacterial DNA has been analyzed in normal and autoimmune mice. Preautoimmune NZB/W mice immunized with Escherichia coli dsDNA produce Abs that resemble spontaneous autoantibodies and bind mammalian dsDNA. In contrast, normal mice, when immunized similarly, produce Abs that bind only bacterial dsDNA. To characterize further the responsiveness of NZB/W mice to bacterial DNA, we determined the molecular properties of mAbs from preautoimmune NZB/W mice immunized with E. coli DNA. Of nine Abs studied, all were IgM and all bound mammalian ssDNA, while four had appreciable reactivity with mammalian dsDNA. The induced anti-dsDNA resembled spontaneous anti-DNA from autoimmune mice in V gene utilization and V(H) CDR3 arginine content. These Abs lacked evidence of somatic mutation, however, indicating that affinity maturation via somatic mutation is not essential for dsDNA reactivity. The findings suggest that preautoimmune NZB/W mice have immunoregulatory defects that allow activation of mammalian dsDNA reactive B cells by bacterial DNA.
Asunto(s)
Anticuerpos Antinucleares/genética , ADN Bacteriano/inmunología , Ratones Endogámicos NZB/inmunología , Secuencia de Aminoácidos , Animales , Arginina/química , Autoinmunidad/inmunología , Linfocitos B/inmunología , Secuencia de Bases , Escherichia coli/inmunología , Femenino , Genes de Inmunoglobulinas , Ratones , Datos de Secuencia Molecular , MutaciónRESUMEN
OBJECTIVE: Short courses of intermittent pulse cyclophosphamide (CY) have mitigated ovarian toxicity but have also led to incomplete or unsustained remissions of active systemic lupus erythematosus (SLE) in many patients, prompting an evaluation of the immunologic effects of this regimen in the NZB/W female mouse (B/W) model of human SLE. METHODS: Phenotypic and functional characteristics of spleen lymphocytes from B/W mice treated with short courses of intraperitoneal (i.p.) CY were compared to those from untreated control B/W mice. RESULTS: After a single dose (250 mg/kg) of i.p. CY, spleen lymphocyte subpopulations fell abruptly but recovered within 4 weeks. Four monthly doses of i.p. CY (starting at 5 months of age) led to a sustained reduction in spleen lymphocyte subpopulations and a parallel decrease in the number of spleen cells spontaneously secreting immunoglobulin and anti-DNA antibody to about 30% of the number seen in untreated control B/W mice. Lipopolysaccharide induced secretion of total IgG and IgG anti-DNA by cultured spleen cells was not diminished one month after the 4 month course of i.p. CY. CONCLUSION: The 4 month course of i.p. CY produced a marked reduction in the number of activated B cells producing autoantibody, but did not achieve sustained immunomodulation judged by the unaltered proportion of spleen B cells spontaneously secreting immunoglobulin and anti-DNA, as well as the response to polyclonal activators of B cells. These results suggest a continued susceptibility to flares of SLE activity after brief courses of intensive immunosuppressive therapy.
Asunto(s)
Ciclofosfamida/administración & dosificación , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos NZB/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antinucleares/inmunología , Formación de Anticuerpos , Modelos Animales de Enfermedad , Esquema de Medicación , Femenino , Inyecciones Intraperitoneales , Subgrupos Linfocitarios/efectos de los fármacos , Ratones , Bazo/citologíaRESUMEN
To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Interfase/inmunología , Nefritis Lúpica/inmunología , Activación de Linfocitos , Ratones Endogámicos NZB/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Subgrupos de Linfocitos B/metabolismo , Antígenos CD40/inmunología , Cruzamientos Genéticos , Citocinas/farmacología , Susceptibilidad a Enfermedades , Femenino , Inmunoglobulina M/biosíntesis , Inmunofenotipificación , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Nefritis Lúpica/genética , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Cooperación Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Endogámicos NZB/genética , Bazo/citologíaRESUMEN
The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105-2H and IgG2a MoAb 34-3C) immunoprecipitated a 105-kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34-2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34-2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105-2H and 34-3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecipitation and immunoblotting, the IgM antibodies 103-7E and 106-10E reacted poorly with intact erythrocytes by flow cytometry, but the IgG1 antibody 31-9D bound well. ELISAs demonstrated that all four IgM MoAbs are polyreactive, since they bound to histones from a panel of nuclear antigens, and additionally 103-7E reacted with phosphatidyl choline. It is concluded that band 3 is an important autoantigen in NZB AIHA. However, since 3/5 haemolytic MoAbs failed to participate this antigen, either these antibodies represent minor components of the total autoantibody response, or responses to diverse possibly non-protein surface antigens also contribute to the pathogenesis of the disease.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Eritrocitos/inmunología , Ratones Endogámicos NZB/inmunología , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/análisis , Especificidad de Anticuerpos , Citometría de Flujo , Immunoblotting , Ratones , Fosfolípidos/inmunología , Pruebas de PrecipitinaRESUMEN
Helper T cells are required for development of the autoantibody responses to native mouse erythrocytes (MRBC) that spontaneously develop in NZB mice. However, the stimulus for these Th is not known. Therefore, we compared the abilities of splenic T cells from actively autoimmune old NZB mice and preautoimmune, young NZB mice with those of T cells from nonautoimmune strains of mice to respond to autologous erythrocytes. We found that autologous RBC ghosts, washed free of haemoglobin, induced low, but statistically significant, proliferative responses in T cells from old NZB mice but not in T cells from young NZB or from normal young and old BALB/c mice. In addition, autologous RBC lysates induced proliferative responses detectable by 3H-thymidine uptake in T cells from nonautoimmune as well as autoimmune mice. CD4+ T cells accounted for most of the observed RBC lysate-induced proliferation, with virtually no response made by CD8+ T cells or B cells. T cells from actively autoimmune NZB mice were not more active in their responses to RBC lysates than T cells from normal strains of mice in terms of their level of proliferation, kinetics, or dose response. Haemoglobin was the major stimulus in the autologous RBC lysates and a similar stimulation was seen with lysates and haemoglobins from horse, human, and mouse sources. Haptoglobin, a haemoglobin-binding serum protein, inhibited T cell responses to haemoglobin and haemoglobin-containing RBC lysates but did not have the same effect on these responses in T cells from either young or old NZB mice. Therefore, either or both of the RBC stimuli from autologous RBC might account for the helper T cell activity in autoimmune NZB mice. T cells in normal mice do not respond either to RBC lysates in the presence of haptoglobin or to RBC ghosts.