RESUMEN
Most of our knowledge of the antigenic repertoire of autoreactive B lymphocytes in type 1 diabetes (T1D) comes from studies on the antigenic specificity of both circulating islet-reactive autoantibodies and peripheral B lymphocyte hybridomas generated from human blood or rodent spleen. In a recent study, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis, but with different degrees of susceptibility to T1D, to characterize the antigenic specificity of islet-infiltrating B lymphocytes during progression of the disease. We found that many hybridomas produced mAbs restricted to the peripheral nervous system (PNS), thus indicating an active B lymphocyte response against PNS elements in the pancreatic islet during disease development. The aim of this study was to identify the autoantigen recognized by these anti-PNS mAbs. Our results showed that peripherin is the autoantigen recognized by all anti-PNS mAbs, and, therefore, a relevant neuroendocrine autoantigen targeted by islet-infiltrating B lymphocytes. Moreover, we discovered that the immune dominant epitope of this B lymphocyte immune response is found at the C-terminal end of Per58 and Per61 isoforms. In conclusion, our study strongly suggests that peripherin is a major autoantigen targeted during T1D development and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during disease.
Asunto(s)
Autoantígenos/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Movimiento Celular/inmunología , Proteínas de Filamentos Intermediarios/inmunología , Proteínas de Filamentos Intermediarios/metabolismo , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Autoantígenos/inmunología , Subgrupos de Linfocitos B/patología , Línea Celular Tumoral , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/metabolismo , Femenino , Hibridomas , Insulinoma/inmunología , Insulinoma/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos NOD , Neuroblastoma/inmunología , Neuroblastoma/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Periferinas , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismoRESUMEN
There is increasing evidence that factors originally identified due to their neurotrophic activity also function within the immune system. This study focused on the related molecules glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) as well as their receptors. GDNF and NTN signaling is mediated by a two-component receptor: a signal-transducing component, RET, which is shared by both ligands, and a ligand-specific binding component, GFR alpha-1 (higher GDNF affinity) or GFR alpha-2 (higher NTN affinity). We report that human T cells, B cells, and monocytes produce NTN but not GDNF, as seen by RT-PCR and immunocytochemistry. RET was expressed by B cells, T cells, and monocytes. Exons 2-5 of RET encoding the cadherin-like domains 1-3 in the extracellular part and exons 16-19 encoding a section of the second tyrosine kinase domain were transcribed in CD4+ T cells, CD8+ T cells, B cells, and monocytes. Different splice variants encoding the C-terminal intracellular part (exons 19-21) of RET were detected. The ligand-binding receptors GFR alpha-1 and GFR alpha-2 were transcribed in all immune cell subsets. Quantitative PCR showed that GFR alpha-2 is by far the dominant ligand binding chain in T cells, B cells, and monocytes. Addition of GDNF or NTN to activated PBMCs reduced the amount of detectable TNF protein without altering its transcription. Together, this suggests that immune cells communicate with each other via NTN. Production of NTN by immune cells might also contribute to the neuroprotective immunity in the CNS observed in different model systems.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Familia de Multigenes , Células 3T3 , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular , Línea Celular Tumoral , Regulación de la Expresión Génica/inmunología , Células HL-60 , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/fisiología , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Ligandos , Activación de Linfocitos , Ratones , Monocitos/enzimología , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Células U937RESUMEN
Vascular malformations cause discomfort and pain in children and are often associated with skeletal hypertrophy. Their molecular basis is poorly understood. Ephrin ligands and Eph receptor tyrosine kinases are involved in embryonic vascular development. In mice, some ephrin/Eph family members show a complementary expression pattern in blood vessels, with ephrinB2 being expressed on arterial and EphB4 on venous endothelium. Targeted deletions of the genes reveal their essential roles for conduit vessel development in mice, suggesting similar functions during human vascular development and deregulation in vascular malformations. Here, we have defined the expression patterns of human ephrinB2, EphB4, and EphB2 in normal vessels of neonates (i.e. umbilici) and adults and compared them with those in congenital venous malformations. In adults, normal vessels of the skin, muscle, and legs express ephrinB2 and EphB2 on arterial endothelial cells (ECs), whereas EphB4 is found in arteries and veins. In the umbilicus, EphB2 is a specific marker of arterial ECs, whereas ephrinB2 is additionally expressed in venous ECs, suggesting an arterial function of the veins. In venous malformations, the expression of EphB4 is not altered, but both ephrinB2 and EphB2 are ectopically expressed in venous ECs. This may reflect a nonphysiologic arterialization of malformed veins. Our study shows that the arterial markers ephrin B2 and EphB2 are expressed in a subset of veins, and it remains to be studied whether this is cause or consequence of an altered vascular identity.
Asunto(s)
Malformaciones Arteriovenosas/metabolismo , Vasos Sanguíneos/metabolismo , Efrina-B2/metabolismo , Receptor EphB2/metabolismo , Receptor EphB4/metabolismo , Cordón Umbilical/irrigación sanguínea , Adulto , Animales , Malformaciones Arteriovenosas/patología , Biomarcadores , Vasos Sanguíneos/anomalías , Niño , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Ratones , Cordón Umbilical/anatomía & histologíaRESUMEN
Granzyme B (GzmB), a serine protease of cytotoxic T lymphocytes and natural killer (NK) cells, induces apoptosis by caspase activation after crossing the plasma membrane of target cells. The mechanism of this translocation during killer cell attack, however, is not understood. Killer cells release GzmB and the membrane-disturbing perforin at the contact site after target recognition. Receptor-mediated import of glycosylated GzmB and release from endosomes were suggested, but the role of the cation-independent mannose 6-phosphate receptor was recently refuted. Using recombinant nonglycosylated GzmB, we observed binding of GzmB to cellular membranes in a cell type-dependent manner. The basis and functional impact of surface binding were clarified. GzmB binding was correlated with the surface density of heparan sulfate chains, was eliminated on treatment of target cells with heparinase III or sodium chlorate, and was completely blocked by an excess of catalytically inactive GzmB or GzmK. Although heparan sulfate-bound GzmB was taken up rapidly into intracellular lysosomal compartments, neither of the treatments had an inhibitory influence on apoptosis induced by externally added streptolysin O and GzmB or by natural killer cells. We conclude that membrane receptors for GzmB on target cells are not crucial for killer cell-mediated apoptosis.
Asunto(s)
Apoptosis , Heparina/análogos & derivados , Células Asesinas Naturales/fisiología , Receptores de Superficie Celular/fisiología , Serina Endopeptidasas/metabolismo , Sitios de Unión , Línea Celular , Permeabilidad de la Membrana Celular , Granzimas , Heparina/análisis , Heparina/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Lisosomas/metabolismo , Proteoglicanos/análisis , Proteoglicanos/metabolismoRESUMEN
Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits.
Asunto(s)
Miastenia Gravis/genética , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/metabolismo , Timo/metabolismo , Transcripción Genética , Adolescente , Adulto , Anciano , Southern Blotting/métodos , Desmina/metabolismo , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Expresión Génica , Humanos , Microdisección/métodos , Persona de Mediana Edad , Músculos/metabolismo , Miastenia Gravis/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , ARN Mensajero/biosíntesis , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Timoma/genética , Timoma/metabolismo , Timo/citología , Neoplasias del Timo/genética , Neoplasias del Timo/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
An important feature of central nervous system (CNS) immune privilege is that antigens expressed in CNS are sequestered and not available for central tolerance induction. Tissue distribution and, more specifically, thymic expression of many of the CNS putative autoantigens have not yet been clearly established in humans. We have addressed this question for the putative multiple sclerosis(MS) autoantigens alphaB-crystallin, S100beta, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG)-alpha and MOG-beta isoforms, using quantitative RT-PCR on human thymus (total, cell fractions and microdissected specimen) and on a panel of peripheral tissues. alphaB-crystallin, S100beta and the DM20 isoform of PLP were clearly expressed in the thymus and also in selected peripheral tissues. In contrast, the expression of MOG out of the CNS was not observed. Within the human thymus, the level of CNS antigen expression was found higher in the stromal epithelial enriched cell fraction, and in microdissected samples of the medullary compartment. These results indicate that most of the antigens involved in MS are expressed in the thymus, suggesting a possible role in central tolerance. However, MOG and, to a lesser extent PLP, conform the classical concept of sequestered antigens, thus supporting the involvement of MOG in autoimmune demyelinating diseases.