RESUMEN
We have previously shown that B cells and Abs reactive with GAPDH and antitriosephosphate isomerase (TPI) are present in lesions and cerebrospinal fluid (CSF) in multiple sclerosis (MS). In the current study, we studied the effect of anti-GAPDH and anti-TPI CSF IgG on the glycolytic enzyme activity of GAPDH and TPI after exposure to intrathecal IgG from 10 patients with MS and 34 patients with other neurologic diseases. The degree of inhibition of GAPDH activity by CSF anti-GAPDH IgG in the seven MS samples tested varied from 13 to 98%, which seemed to correlate with the percentage of anti-GAPDH IgG in the CSF IgG (1-45%). Inhibition of GAPDH activity (18 and 23%) by CSF IgG was seen in two of the 34 patients with other neurologic diseases, corresponding to the low percentage of CSF anti-GAPDH IgG (1 and 8%). In addition, depletion of anti-GAPDH IgG from CSF IgG, using immobilized GAPDH, removed the inhibitory effect of the IgG on GAPDH. No inhibition of GAPDH activity was seen with CSF samples not containing anti-GAPDH IgG. No inhibition of TPI activity was seen with any purified CSF IgG sample. These findings demonstrate an increased percentage of anti-GAPDH Abs in the CSF of patients with MS that can inhibit GAPDH glycolytic enzyme activity and may contribute to neuroaxonal degeneration.
Asunto(s)
Autoanticuerpos/líquido cefalorraquídeo , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Inmunoglobulina G/líquido cefalorraquídeo , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/inmunología , Adulto , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/fisiología , Regulación hacia Abajo/inmunología , Activación Enzimática/inmunología , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/fisiología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Enfermedades Neurodegenerativas/líquido cefalorraquídeo , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/inmunología , Conejos , Triosa-Fosfato Isomerasa/inmunología , Triosa-Fosfato Isomerasa/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
Our previous results revealed that Igs in lesions and single chain variable fragment Abs (scFv-Abs) generated from clonal B cells in the cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) bind to axons in MS brains. To study the axonal Ags involved in MS, we identified the glycolytic enzymes, triosephosphate isomerase (TPI) and GAPDH, using Igs from the CSF and scFv-Abs generated from clonal B cells in the CSF and in lesions from MS patients. Elevated levels of CSF-Abs to TPI were observed in patients with MS (46%), clinically isolated syndrome (CIS) suggestive of MS (40%), other inflammatory neurological diseases (OIND; 29%), and other noninflammatory neurological diseases (ONIND; 31%). Levels of GAPDH-reactive Abs were elevated in MS patients (60%), in patients with CIS (10%), OIND (14%), and ONIND (8%). The coexistence of both autoantibodies was detected in 10 MS patients (29%), and 1 CIS patient (3%), but not in patients with OIND/ONIND. Two scFv-Abs generated from the CSF and from lesions of a MS brain showed immunoreactivity to TPI and GAPDH, respectively. The findings suggest that TPI and GAPDH may be candidate Ags for an autoimmune response to neurons and axons in MS.
Asunto(s)
Autoanticuerpos/líquido cefalorraquídeo , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Esclerosis Múltiple/inmunología , Triosa-Fosfato Isomerasa/inmunología , Adulto , Autoanticuerpos/análisis , Autoantígenos , Axones/inmunología , Linfocitos B , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Enfermedades del Sistema Nervioso/inmunología , Neuronas/inmunologíaRESUMEN
Immunoglobulin A (IgA), the predominant immunoglobulin class in mucosal secretions, has been found in the cerebrospinal fluid of patients with multiple sclerosis (MS). In this study we examined the infiltration of clonally expanded IgA plasma cells in lesions of MS brains. Sequences of complementarity-determining region 3 of IgA variable heavy chain (V(H)) genes demonstrated the clonal expansion of IgA-bearing plasma cells in MS lesions. Somatic mutations and ongoing intra-clonal mutations occurred in their V(H) genes. Immunohistochemical study demonstrated infiltration of dimer and polymer IgA1- and A2-positive plasma cells in perivascular spaces, in the parenchyma of MS lesions, and in the adjacent white matter. Double immunofluorescence staining showed binding of IgA antibody on axons and walls of microvessels in the areas of chronic active and inactive demyelination. Bielshowsky's silver impregnation revealed axonal damage in these areas. These findings suggest that IgA in the CNS are localized on axons in lesions and may contribute to axonal damage in MS.
Asunto(s)
Anticuerpos/farmacología , Axones/efectos de los fármacos , Inmunoglobulina A/metabolismo , Esclerosis Múltiple/inmunología , Células Plasmáticas/inmunología , Axones/fisiología , Linfocitos B/metabolismo , Northern Blotting/métodos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Análisis Mutacional de ADN/métodos , Femenino , Genes de Inmunoglobulinas/fisiología , Humanos , Inmunoglobulina A/genética , Región de Unión de la Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/metabolismo , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/fisiopatología , Proteína Básica de Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Cambios Post Mortem , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tinción con Nitrato de Plata/métodosRESUMEN
Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.
Asunto(s)
Axones/inmunología , Linfocitos B/inmunología , Líquido Cefalorraquídeo/citología , Esclerosis Múltiple/inmunología , Antígenos/análisis , Linfocitos B/patología , Encéfalo/inmunología , Encéfalo/patología , Proliferación Celular , Células Clonales/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina , Región Variable de Inmunoglobulina/inmunología , Inmunohistoquímica , Esclerosis Múltiple/etiología , Degeneración WallerianaRESUMEN
To investigate the hypothesis that pseudotumor cerebri (PTC) is associated with humoral immunity, we analyzed immunoglobulin heavy chain variable region (Ig-VH) genes of B cells in the cerebrospinal fluid (CSF) of 10 patients with PTC. Using RT-PCR and sequencing techniques, intrathecal B-cell Ig-VH genes were amplified in 6 of 10 PTC samples. Sequence analysis of complementarity-determining region 3 (CDR 3) and VH genes revealed a polyclonal intrathecal B-cell expansion in these patients. The nucleotide sequences showed that one-third of analyzed sequences had a high replacement to silent nucleotide substitution ratio, indicating an antigen-driven T-cell-dependent intrathecal B-cell proliferation. Moreover, other one-third had germline VH genes without or with a few nucleotide mutations, suggesting a T-cell-independent natural B-cell-mediated humoral immunity in the CNS of these patients. Our results suggest that both T-cell-dependent and T-cell-independent humoral immunity are present in the CSF of PTC.
Asunto(s)
Linfocitos B/inmunología , Líquido Cefalorraquídeo/inmunología , Seudotumor Cerebral/inmunología , Adolescente , Adulto , Formación de Anticuerpos , Linfocitos B/patología , Secuencia de Bases , Proliferación Celular , Líquido Cefalorraquídeo/citología , Femenino , Genes de Inmunoglobulinas/genética , Humanos , Activación de Linfocitos/inmunología , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Seudotumor Cerebral/líquido cefalorraquídeo , Linfocitos T/inmunologíaRESUMEN
The development of somatically mutated memory and plasma B cells is a consequence of T cell-dependent antigen-challenged humoral immunity. To investigate the role of B cell-mediated humoral immunity in the initiation and evolution of multiple sclerosis (MS), we analyzed Ig variable heavy chain genes of intrathecal B cells derived from patients with a first clinical manifestation suggestive of MS. Sequences of Ig variable regions showed that B cells in the cerebrospinal fluid from most of these patients were clonally expanded and carried somatic hypermutated variable heavy chain genes. The mutations showed a high replacement-to-silent ratio and were distributed in a way suggesting that these clonally expanded B cells had been positively selected through their antigen receptor. In comparison, intrathecal B-cell clonal expansion often precedes both oligoclonal IgG bands and multiple magnetic resonance imaging lesions. Clinical follow-up study showed that patients with clonally expanded intrathecal B cells had a high rate of conversion to clinically definite MS. The findings provide direct evidence of recruitment of germinal center differentiated B lymphocytes into the central nervous system during the initiation of MS. These results indicate B cell-mediated immune response in the cerebrospinal fluid is an early event of inflammatory reaction in the central nervous system of MS. This procedure also provides a more sensitive method to evaluate the association of humoral immunity in the evolution of MS.