RESUMO
Stepwise degradation of the invariant chain (Ii) is required for the binding of antigenic peptides to MHC class II molecules. Cathepsin (Cat) L in the murine thymus and Cat S in peripheral APCs have both been implicated in the last step of Ii degradation that gives rise to the class II-associated invariant chain peptides (CLIP). Cat V has been recently described as highly homologous to Cat L and exclusively expressed in human thymus and testis, but with no mouse orthologue. We report that Cat V is the dominant cysteine protease in cortical human thymic epithelial cells, while Cat L and Cat S seem to be restricted to dendritic and macrophage-like cells. Active Cat V in thymic lysosomal preparations was demonstrated by active-site labeling. Recombinant Cat V was capable of converting Ii into CLIP efficiently, suggesting that Cat V is the protease that controls the generation of alphabeta-CLIP complexes in the human thymus, in analogy to Cat L in mouse. Comparison of Cat V expression between thymi from patients with myasthenia gravis and healthy controls revealed a significantly higher expression level in the pathological samples, suggesting a potential involvement of this protease in the immunopathogenesis of myasthenia gravis, an autoimmune disease almost invariably associated with thymic pathology.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Catepsinas/fisiologia , Cisteína Endopeptidases/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Miastenia Gravis/metabolismo , Timo/metabolismo , Adolescente , Animais , Antígenos de Diferenciação de Linfócitos B/química , Western Blotting , Catepsina L , Catepsinas/metabolismo , Criança , Pré-Escolar , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Antígenos de Histocompatibilidade Classe II/química , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Lasers , Camundongos , Peptídeos/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição TecidualRESUMO
The nicotinic acetylcholine receptor (nAChR) is the autoantigen in seropositive myasthenia gravis (MG) that is a T cell-dependent B cell-mediated autoimmune disorder. We tested the immunogenicity and myasthenogenicity of the extracellular and first transmembrane domain of the epsilon-chain(1-221) of the nAChR in inbred and MHC congenic rat strains. Immunodominant T and B cell determinants did not induce experimental autoimmune myasthenia gravis (EAMG), although immunization resulted in strong Th1 and B cell responses, which could be mapped with overlapping peptides of the nAChR epsilon-subunit in eight different rat strains. Our data underscores the concept that immunodominant autoantigen-specific T and B cell responses can lack pathogenicity in autoimmune disease and might be of relevance for the physiological integrity of the organism.
Assuntos
Linfócitos B/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Receptores Nicotínicos/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Animais Congênicos , Autoantígenos/imunologia , Modelos Animais de Doenças , Humanos , Masculino , Dados de Sequência Molecular , Miastenia Gravis Autoimune Experimental/imunologia , Peptídeos/imunologia , Ratos , Ratos Endogâmicos LewRESUMO
The immunological properties of muscle cells are of critical importance for both the pathogenesis of inflammatory muscle disorders as well as for understanding and controlling novel therapeutic strategies. Muscle cells can present antigens to both CD4 and CD8 cells. However, the cellular biochemistry of antigen processing and presentation by muscle cells is not clear. Cathepsins play a central role in the generation of antigenic peptide and control transport and maturation of MHC class II molecules. To further elucidate the molecular basis for the MHC class II-mediated antigen presentation by muscle cells, we here analyzed cultured human myoblasts and biopsies from inflammatory myopathies with respect to the expression and function of the constituents of the MHC class II antigen presentation machinery. We identified cathepsin S (CatS) as the dominant endocytic protease that is specifically upregulated under inflammatory conditions to significant mRNA levels, synchronously with HLA-DR, -DM and the class II invariant chain (Ii), both in muscle biopsies from affected individuals with inflammatory myopathies and in human myoblasts cultured in the presence of IFN-gamma. This led to translation of the mature CatS polypeptide that was enzymatically active in human myoblasts under inflammatory conditions. By contrast, expression of CatL and CatB was unaffected by IFN-gamma at both the expression and activity levels. CatS activity is required for efficient surface display of MHC class II in this cell type: functional inhibition of CatS using a CatS-selective inhibitor reduced the levels of surface class II alphabeta:peptide complexes on stimulated myoblasts by almost 50%. Surprisingly, and in contrast to B cells and dendritic cells, this was not due to inefficient processing of Ii in the absence of CatS, which was unaffected by the elimination of CatS activity. We therefore conclude that CatS is involved in the regulation of class II expression in human myoblasts independently from Ii processing.
Assuntos
Apresentação de Antígeno , Catepsinas/fisiologia , Antígenos HLA-D/biossíntese , Músculo Esquelético/enzimologia , Músculo Esquelético/imunologia , Miosite/imunologia , Regulação para Cima/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Biópsia , Catepsinas/antagonistas & inibidores , Catepsinas/biossíntese , Linhagem Celular Transformada , Membrana Celular/enzimologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Cultivadas , Criança , Pré-Escolar , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Lactente , Recém-Nascido , Interferon gama/farmacologia , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/enzimologia , Mioblastos/imunologia , Mioblastos/metabolismo , Mioblastos/patologia , Miosite/enzimologia , Miosite/patologiaRESUMO
Costimulatory signals play a key role in regulating T cell activation and are believed to have decisive influence in the inciting and perpetuating cellular effector mechanisms in autoimmune diseases such as multiple sclerosis (MS). Inducible costimulator protein (ICOS), a recently identified member of the CD28-family, presumably affects the differentiation of Th1/Th2 cells after primary activation and modulates the immune response of effector/memory T cells. This study examines the expression and functional role of ICOS costimulation in healthy donors and patients with MS. After nonspecific or antigen-specific stimulation, ICOS is preferentially expressed on CD4 Th2-T cells. ICOS-costimulation affects the production of Th1 and Th2 cytokines both in the absence and presence of B7/CD28 costimulation, thus suggesting that ICOS costimulation can modulate cytokine secretion also in a CD28-independent manner. Levels of constitutive and inducible ICOS expression on human T cell subsets from peripheral blood were quantified in healthy donors and patients with MS. Constitutive expression of ICOS on T cells varies between 0.1% and 42.3%. There were no significant differences between both groups in the baseline expression or inducibility of ICOS on CD4 or CD8 T cells. ICOS expression could be demonstrated on CSF T lymphocytes in patients with acute MS relapses but was not elevated compared with peripheral blood. In essence we show that ICOS is upregulated on human T cells after stimulation and can modulate both Th1 and Th2 cytokine production in the absence and presence of B7-costimulation. In MS patients we demonstrate the functionality of the ICOS costimulatory pathway. Potential implications of ICOSL/ICOS interactions for MS immunopathogenesis are discussed.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Glicoproteínas de Membrana/fisiologia , Esclerose Múltipla/imunologia , Subpopulações de Linfócitos T/imunologia , Adjuvantes Imunológicos/farmacologia , Adulto , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/líquido cefalorraquidiano , Antígenos de Diferenciação de Linfócitos T/genética , Antígeno B7-1/biossíntese , Antígeno B7-1/genética , Antígeno B7-2 , Linhagem Celular , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Acetato de Glatiramer , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis , Interferon beta/farmacologia , Interferon gama/biossíntese , Interleucina-4/biossíntese , Células L , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/metabolismo , Peptídeos/farmacologia , Receptores de Quimiocinas/biossíntese , Subpopulações de Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Transfecção , Regulação para Cima/imunologiaRESUMO
Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS with associated axonal loss. There is strong evidence for an autoimmune pathogenesis driven by myelin-specific T cells. Myelin oligodendrocyte glycoprotein (MOG) induces a type of experimental autoimmune encephalomyelitis in animals which is very MS-like since there are demyelinating CNS lesions and axonal loss. This underscores the potential role of MOG in MS pathogenesis. We performed a T cell reactivity pattern analysis of MS patients at the onset of relapse or progression of neurological deficits and controls that were stratified for the genetic risk factor HLA-DRB1*1501. For the first time, we show that there is an HLA-DR-restricted promiscuous dominant epitope for CD4(+) T cells within the transmembrane/intracellular part of MOG comprising aa 146-154 (FLCLQYRLR). Surprisingly, controls had broader T cell reactivity patterns toward MOG peptides compared with MS patients, and the transmembrane and intracellular parts of MOG were much more immunogenic compared with the extracellular part. Measurements of in vitro binding affinities revealed that HLA-DRB1*1501 molecules bound MOG 146-154 with intermediate and HLA-DRB1*0401 molecules with weak affinities. The binding of MOG 146-154 was comparable or better than myelin basic protein 85-99, which is the dominant myelin basic protein epitope in context with HLA-DRB1*1501 molecules in MS patients. This is the first study in which the data underscore the need to investigate the pathogenic or regulatory role of the transmembrane and intracellular part of MOG for MS in more detail.