RESUMO
We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4(+) T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4(+) T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8(+) T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8(+) cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8(+) T cells show more limited migration from the graft draining lymph node into other tissues.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sobrevivência de Enxerto/imunologia , Antígeno H-Y/imunologia , Fragmentos de Peptídeos/imunologia , Tolerância ao Transplante , Administração Intranasal , Transferência Adotiva , Animais , Movimento Celular , Citocinas/genética , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Expressão Gênica , Antígeno H-Y/administração & dosagem , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Reação em Cadeia da Polimerase , Transplante de Pele/imunologiaRESUMO
Interleukin 21 (IL-21) is a pleiotropic cytokine produced by CD4 T cells that affects the differentiation and function of T, B, and NK cells by binding to a receptor consisting of the common cytokine receptor gamma chain and the IL-21 receptor (IL-21R). IL-21, a product associated with IL-17-producing CD4 T cells (T(H)17) and follicular CD4 T helper cells (T(FH)), has been implicated in autoimmune disorders including the severe systemic lupus erythematosus (SLE)-like disease characteristic of BXSB-Yaa mice. To determine whether IL-21 plays a significant role in this disease, we compared IL-21R-deficient and -competent BXSB-Yaa mice for multiple parameters of SLE. The deficient mice showed none of the abnormalities characteristic of SLE in IL-21R-competent Yaa mice, including hypergammaglobulinemia, autoantibody production, reduced frequencies of marginal zone B cells and monocytosis, renal disease, and premature morbidity. IL-21 production associated with this autoimmune disease was not a product of T(H)17 cells and was not limited to conventional CXCR5(+) T(FH) but instead was produced broadly by ICOS(+) CD4(+) splenic T cells. IL-21 arising from an abnormal population of CD4 T cells is thus central to the development of this lethal disease, and, more generally, could play an important role in human SLE and related autoimmune disorders.
Assuntos
Lúpus Eritematoso Sistêmico/etiologia , Receptores de Interleucina-21/metabolismo , Transdução de Sinais , Animais , Formação de Anticorpos , Sequência de Bases , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interleucinas/genética , Nefropatias/prevenção & controle , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Subpopulações de Linfócitos , Camundongos , Receptores de Interleucina-21/genéticaRESUMO
The MHC class I family-like Fc receptor, FcRn, is normally responsible for extending the life span of serum IgG Ab's, but whether this molecule contributes to autoimmune pathogenesis remains speculative. To determine directly whether this function contributes to humoral autoimmune disease, we examined whether a deficiency in the FcRn heavy chain influences autoimmune arthritis in the K/BxN mouse model. FcRn deficiency conferred either partial or complete protection in the arthritogenic serum transfer and the more aggressive genetically determined K/BxN autoimmune arthritis models. The protective effects of an FcRn deficiency could be overridden with excessive amounts of pathogenic IgG Ab's. The therapeutic saturation of FcRn by high-dose intravenous IgG (IVIg) also ameliorated arthritis, directly implicating FcRn blockade as a significant mechanism of IVIg's anti-inflammatory action. The results suggest that FcRn is a potential therapeutic target that links the initiation and effector phases of humoral autoimmune disease.
Assuntos
Doenças Autoimunes/etiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Anticorpos/sangue , Anticorpos/metabolismo , Artrite Reumatoide/etiologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Extremidades/patologia , Feminino , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/sangue , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Injeções Intraperitoneais , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Transgênicos , Receptores Fc/deficiência , Receptores Fc/genética , Soro/metabolismo , TransgenesRESUMO
PURPOSE: The present study was conducted to investigate healing of alkali-burned corneas in MRL/MpJ (MRL) mice. METHODS: Gross, clinical, and histologic criteria were used to compare healing of alkali-burned corneas in MRL and control C57BL/6J (B6) mice. Effects of neutrophil depletion of B6 mice and allogeneic reconstitution of B6 mice with MRL bone marrow on wound healing were evaluated. Gene expression patterns in normal and wounded corneas were surveyed with array-based quantitative real-time RT-PCR (AQPCR). RESULTS: MRL mice showed accelerated reepithelialization and decreased corneal opacity compared with B6 mice after alkali wounding. Marked inflammatory cell infiltration and fibrosis were evident in the corneas and anterior chambers of B6 mice. MRL mice showed less severe lesions, except for stromal edema. Rapid reepithelialization and reduced keratitis/iritis were also observed in neutrophil-depleted B6 mice, but not in B6 mice reconstituted with MRL bone marrow. AQPCR showed transcriptional changes of fewer genes associated with inflammation and corneal tissue homeostasis in alkali-burned corneas from MRL mice. Increased expression of an anti-inflammatory gene, Socs1, and a gene associated with healing, Mmp1a, were evident in MRL corneas. CONCLUSIONS: Alkali-burned corneas heal faster and more completely in MRL mice than in B6 mice, by means of rapid reepithelialization, reduced inflammation, and reduced fibrosis. Reduced inflammation, including decreased neutrophil infiltrates and the lack of a robust proinflammatory gene expression signature correlates with the rapid healing. However, the rapid-healing phenotype is not intrinsic to MRL hematopoietic progenitor cells.
Assuntos
Queimaduras Químicas/metabolismo , Doenças da Córnea/metabolismo , Queimaduras Oculares/induzido quimicamente , Cicatrização/fisiologia , Animais , Transplante de Medula Óssea , Queimaduras Químicas/imunologia , Queimaduras Químicas/patologia , Queimaduras Químicas/cirurgia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colagenases/genética , Colagenases/metabolismo , Doenças da Córnea/imunologia , Doenças da Córnea/patologia , Doenças da Córnea/cirurgia , Modelos Animais de Doenças , Epitélio Corneano/fisiologia , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Masculino , Metaloproteinase 1 da Matriz , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Neutrófilos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hidróxido de Sódio , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
BACKGROUND: Recent developments in sequence databases provide the opportunity to relate the expression pattern of genes to their genomic position, thus creating a transcriptome map. Quantitative trait loci (QTL) are phenotypically-defined chromosomal regions that contribute to allelically variant biological traits, and by overlaying QTL on the transcriptome, the search for candidate genes becomes extremely focused. RESULTS: We used our novel data mining tool, ExQuest, to select genes within known diabesity QTL showing enriched expression in primary diabesity affected tissues. We then quantified transcripts in adipose, pancreas, and liver tissue from Tally Ho mice, a multigenic model for Type II diabetes (T2D), and from diabesity-resistant C57BL/6J controls. Analysis of the resulting quantitative PCR data using the Global Pattern Recognition analytical algorithm identified a number of genes whose expression is altered, and thus are novel candidates for diabesity QTL and/or pathways associated with diabesity. CONCLUSION: Transcription-based data mining of genes in QTL-limited intervals followed by efficient quantitative PCR methods is an effective strategy for identifying genes that may contribute to complex pathophysiological processes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Regulação da Expressão Gênica , Locos de Características Quantitativas , Algoritmos , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Distribuição TecidualRESUMO
BACKGROUND: The cytokine-deficiency-induced colitis susceptibility (Cdcs)1 locus is a major modifier of murine inflammatory bowel disease (IBD) and was originally identified in experimental crosses of interleukin-10-deficient (Il10(-/-)) mice. Congenic mice, in which this locus was reciprocally transferred between IBD-susceptible C3H/HeJBir-Il10(-/-) and resistant C57BL/6J-Il10(-/-) mice, revealed that this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NF-kappaB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval. METHODS: In total, 15 reciprocal congenic strains were generated from Il10(-/-) mice of either C3H/HeJBir or C57BL/6J genetic backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis, and a high-throughput real-time reverse-transcription polymerase chain reaction (RT-PCR) approach using bone marrow-derived macrophages. RESULTS: Within the originally identified Cdcs1-interval, 3 independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains. CONCLUSIONS: Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions.
Assuntos
Biomarcadores Tumorais/genética , Colite/genética , Predisposição Genética para Doença , Interleucina-10/fisiologia , Animais , Biomarcadores Tumorais/metabolismo , Western Blotting , Colite/patologia , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aside from Myc-activating translocations characteristic of plasmacytomas (PCT), little is known about genetic factors and signaling pathways responsible for the development of spontaneous B-cell lineage lymphomas of mice. Here, we characterized the transcriptional profiles of PCT, centroblastic diffuse large B-cell lymphomas (CBL), and high-grade splenic marginal zone B-cell lymphoma (MZL++) using high-throughput quantitative reverse transcription-PCR. Expression profiles of CBL and MZL++ were strikingly similar and quite unlike that of PCT. Among the genes expressed at significantly higher levels by PCT were a number involved in NOTCH signaling, a finding supported by gene set enrichment analyses of microarray data. To investigate the importance of this pathway, NOTCH signaling was blocked in PCT cell lines by treatment with a gamma-secretase inhibitor (GSI) or transduction of a dominant-negative mutant of MAML1. These treatments resulted in reduced expression of NOTCH transcriptional targets in association with impaired proliferation and increased apoptosis. GSI treatment of transformed plasma cells in a primary PCT also induced apoptosis. These results integrate NOTCH activation with oncogenic signaling pathways downstream of translocated Myc in the pathogenesis of mouse PCT, two signaling pathways also implicated in development of human multiple myeloma and T-cell lymphoblastic lymphoma.
Assuntos
Perfilação da Expressão Gênica , Linfoma de Células B/patologia , Plasmocitoma/patologia , Receptores Notch/fisiologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Proliferação de Células , Sobrevivência Celular , Linfoma de Células B/etiologia , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Plasmócitos/metabolismo , Plasmocitoma/etiologia , Plasmocitoma/metabolismo , Reação em Cadeia da Polimerase , Receptores Notch/antagonistas & inibidores , Transdução de SinaisRESUMO
NKT cell activation by alpha-galactosylceramide (alpha-GalCer) inhibits autoimmune diabetes in NOD mice, in part by inducing recruitment to pancreatic lymph nodes (PLNs) of mature dendritic cells (DCs) with disease-protective effects. However, how activated NKT cells promote DC maturation, and what downstream effect this has on diabetogenic T cells was unknown. Activated NKT cells were found to produce a soluble factor(s) inducing DC maturation. Initially, there was a preferential accumulation of mature DCs in the PLNs of alpha-GalCer-treated NOD mice, followed by a substantial increase in T cells. Adoptive transfer of a diabetogenic CD8 T cell population (AI4) induced a high rate of disease (75%) in PBS-treated NOD recipients, but not in those pretreated with alpha-GalCer (8%). Significantly, more AI4 T cells accumulated in PLNs of alpha-GalCer than PBS-treated recipients, while no differences were found in mesenteric lymph nodes from each group. Compared with those in mesenteric lymph nodes, AI4 T cells entering PLNs underwent greater levels of apoptosis, and the survivors became functionally anergic. NKT cell activation enhanced this process. Hence, activated NKT cells elicit diabetes protection in NOD mice by producing a soluble factor(s) that induces DC maturation and accumulation in PLNs, where they subsequently recruit and tolerize pathogenic T cells.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Tolerância Imunológica , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Ativação Linfocitária , Pâncreas/imunologia , Subpopulações de Linfócitos T/imunologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Animais , Subpopulações de Linfócitos B/citologia , Linfócitos T CD8-Positivos/citologia , Agregação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Células Clonais , Células Dendríticas/citologia , Diabetes Mellitus Tipo 1/imunologia , Feminino , Galactosilceramidas/administração & dosagem , Galactosilceramidas/farmacologia , Galactosilceramidas/uso terapêutico , Tolerância Imunológica/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Linfonodos/citologia , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Pâncreas/citologia , Solubilidade , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Description of the molecular phenotypes of pathobiological processes in vivo is a pressing need in genomic biology. We have implemented a high-throughput real-time PCR strategy to establish quantitative expression profiles of a customized set of target genes. It enables rapid, reproducible data acquisition from limited quantities of RNA, permitting serial sampling of mouse blood during disease progression. We developed an easy to use statistical algorithm--Global Pattern Recognition--to readily identify genes whose expression has changed significantly from healthy baseline profiles. This approach provides unique molecular signatures for rheumatoid arthritis, systemic lupus erythematosus, and graft versus host disease, and can also be applied to defining the molecular phenotype of a variety of other normal and pathological processes.
Assuntos
Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Animais , Artrite Reumatoide/genética , Biologia Computacional/estatística & dados numéricos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Predisposição Genética para Doença/genética , Doença Enxerto-Hospedeiro/genética , Imunoensaio/métodos , Imunoensaio/estatística & dados numéricos , Lúpus Eritematoso Sistêmico/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Genéticos , Modelos Imunológicos , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Fenótipo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
IL-21 is a type I cytokine whose receptor is expressed on T, B, and NK cells. Within the B cell lineage, IL-21 regulates IgG1 production and cooperates with IL-4 for the production of multiple Ab classes in vivo. Using IL-21-transgenic mice and hydrodynamics-based gene delivery of IL-21 plasmid DNA into wild-type mice as well as in vitro studies, we demonstrate that although IL-21 induces death of resting B cells, it promotes differentiation of B cells into postswitch and plasma cells. Thus, IL-21 differentially influences B cell fate depending on the signaling context, explaining how IL-21 can be proapoptotic for B cells in vitro yet critical for Ag-specific Ig production in vivo. Moreover, we demonstrate that IL-21 unexpectedly induces expression of both Blimp-1 and Bcl-6, indicating mechanisms as to how IL-21 can serve as a complex regulator of B cell maturation and terminal differentiation. Finally, BXSB-Yaa mice, which develop a systemic lupus erythematosus-like disease, have greatly elevated IL-21, suggesting a role for IL-21 in the development of autoimmune disease.
Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Proteínas de Ligação a DNA/biossíntese , Interleucinas/fisiologia , Plasmócitos/citologia , Plasmócitos/imunologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras/biossíntese , Fatores de Transcrição/biossíntese , Animais , Apoptose/genética , Apoptose/imunologia , Subpopulações de Linfócitos B/metabolismo , Divisão Celular/genética , Divisão Celular/imunologia , Separação Celular , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Humanos , Switching de Imunoglobulina/genética , Interleucinas/biossíntese , Interleucinas/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Contagem de Linfócitos , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Fator de Transcrição PAX5 , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteoglicanas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-6 , Baço/anatomia & histologia , Baço/citologia , Baço/imunologia , Sindecanas , Fatores de Transcrição/antagonistas & inibidores , Regulação para Cima/imunologiaRESUMO
Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4b presented on Kb class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to C-->T nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4b while the anti-B6 response directed against H4a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.
Assuntos
Glicoproteínas de Membrana/genética , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA Complementar , Epitopos , Isoantígenos/genética , Camundongos , Dados de Sequência MolecularRESUMO
Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.