RESUMO
Multiple sclerosis (MS) and neuromyelitis optica (NMO) are inflammatory demyelinating disorders of the central nervous system with evidence of antibody-mediated pathology. Using ex vivo organotypic mouse cerebellar slice cultures, we have demonstrated that recombinant antibodies (rAbs) cloned from cerebrospinal fluid plasmablasts of MS and NMO patients target myelin- and astrocyte-specific antigens to induce disease-specific oligodendrocyte loss and myelin degradation. In this study, we examined glial cell responses and myelin integrity during recovery from disease-specific antibody-mediated injury. Following exposure to MS rAb and human complement (HC) in cerebellar explants, myelinating oligodendrocytes repopulated the demyelinated tissue and formed new myelin sheaths along axons. Remyelination was accompanied by pronounced microglial activation. In contrast, following treatment with NMO rAb and HC, there was rapid regeneration of astrocytes and pre-myelinating oligodendrocytes but little formation of myelin sheaths on preserved axons. Deficient remyelination was associated with progressive axonal loss and the return of microglia to a resting state. Our results indicate that antibody-mediated demyelination in MS and NMO show distinct capacities for recovery associated with differential injury to adjacent axons and variable activation of microglia. Remyelination was rapid in MS rAb plus HC-induced demyelination. By contrast, oligodendrocyte maturation and remyelination failed following NMO rAb-mediated injury despite the rapid restoration of astrocytes and preservation of axons in early lesions.
Assuntos
Cerebelo , Imunoglobulina G/toxicidade , Esclerose Múltipla/imunologia , Neuroglia/efeitos dos fármacos , Neuromielite Óptica/imunologia , Remielinização/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Aquaporina 4/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa S-Transferase pi/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/sangue , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Neuroglia/fisiologia , Neuromielite Óptica/sangue , Técnicas de Cultura de Órgãos , Remielinização/fisiologia , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
Neuromyelitis optica-immunoglobulin G (NMO-IgG) binds to aquaporin-4 (AQP4) water channels in the central nervous system leading to immune-mediated injury. We have previously demonstrated that a high proportion of CSF plasma cells of NMO patients produce antibody to the extracellular domains of the AQP4 protein and that recombinant IgG (rAb) derived from these cells recapitulate pathogenic features of disease. We performed a comprehensive mutational analysis of the three extracellular loops of the M23 isoform of human AQP4 using both serial and single point mutations, and we evaluated the effects on binding of NMO AQP4-reactive rAbs by quantitative immunofluorescence. Whereas all NMO rAbs required conserved loop C ((137)TP(138) and Val(150)) and loop E ((230)HW(231)) amino acids for binding, two broad patterns of NMO-IgG recognition could be distinguished based on differential sensitivity to loop A amino acid changes. Pattern 1 NMO rAbs were insensitive to loop A mutations and could be further discriminated by differential sensitivity to amino acid changes in loop C ((148)TM(149) and His(151)) and loop E (Asn(226) and Glu(228)). Alternatively, pattern 2 NMO rAbs showed significantly reduced binding following amino acid changes in loop A ((63)EKP(65) and Asp(69)) and loop C (Val(141), His(151), and Leu(154)). Amino acid substitutions at (137)TP(138) altered loop C conformation and abolished the binding of all NMO rAbs and NMO-IgG, indicating the global importance of loop C conformation to the recognition of AQP4 by pathogenic NMO Abs. The generation of human NMO rAbs has allowed the first high resolution mapping of extracellular loop amino acids critical for NMO-IgG binding and identified regions of AQP4 extracellular structure that may represent prime targets for drug therapy.
Assuntos
Aquaporina 4/química , Autoanticorpos/química , Imunoglobulina G/química , Mutagênese , Neuromielite Óptica/imunologia , Alanina/química , Animais , Anticorpos Monoclonais/química , Células CHO , Separação Celular , Cricetinae , Cricetulus , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/química , Citometria de Fluxo , Glicina/química , Humanos , Mutação , Neuromielite Óptica/líquido cefalorraquidiano , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Studies of neuromyelitis optica (NMO), an autoimmune disease of the central nervous system (CNS), have demonstrated that autoantibodies against the water channel aquaporin-4 (AQP4) induce astrocyte damage through complement-dependent cytotoxicity (CDC). In developing experimental models of NMO using cells, tissues or animals from mice, co-administration of AQP4-IgG and normal human serum, which serves as the source of human complement (HC), is required. The sensitivity of mouse CNS cells to HC and CDC in these models is not known. METHODS: We used HC and recombinant monoclonal antibodies (rAbs) against AQP4 to investigate CDC on mouse neurons, astrocytes, differentiated oligodendrocytes (OLs), and oligodendrocyte progenitors (OPCs) in the context of purified monocultures, neuroglial mixed cultures, and organotypic cerebellar slices. RESULTS: We found that murine neurons, OLs, and OPCs were sensitive to HC in monocultures. In mixed murine neuroglial cultures, HC-mediated toxicity to neurons and OLs was reduced; however, astrocyte damage induced by an AQP-specific rAb #53 and HC increased neuronal and oligodendroglial loss. OPCs were resistant to HC toxicity in neuroglial mixed cultures. In mouse cerebellar slices, damage to neurons and OLs following rAb #53-mediated CDC was further reduced, but in contrast to neuroglial mixed cultures, astrocyte damage sensitized OPCs to complement damage. Finally, we established that some injury to neurons, OLs, and OPCs in cell and slice cultures resulted from the activation of HC by anti-tissue antibodies to mouse cells. CONCLUSIONS: Murine neurons and oligodendroglia demonstrate variable sensitivity to activated complement based on their differentiation and culture conditions. In organotypic cultures, the protection of neurons, OLs, and OPCs against CDC is eliminated by targeted astrocyte destruction. The activation of human complement proteins on mouse CNS cells necessitates caution when interpreting the results of mouse experimental models of NMO using HC.
Assuntos
Ativação do Complemento/fisiologia , Proteínas do Sistema Complemento/toxicidade , Modelos Animais de Doenças , Neuromielite Óptica/induzido quimicamente , Neuromielite Óptica/metabolismo , Animais , Aquaporina 4/toxicidade , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuromielite Óptica/imunologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Neuromyelitis optica (NMO) is a severe demyelinating disorder of the central nervous system (CNS) associated with the presence of an autoimmune antibody response (AQP4-IgG) against the water channel aquaporin-4 (AQP4). It remains unclear whether pathologic AQP4-IgG in the CNS is produced entirely by peripheral plasma cells or is generated in part by infiltrating B cells. To determine the overlap of AQP4-IgG idiotypes between the CNS and periphery, we compared the immunoglobulin G (IgG) transcriptome of cerebrospinal fluid (CSF) plasmablasts with the CSF and serum IgG proteomes in 7 AQP4-seropositive NMO patients following exacerbation. METHODS: CSF variable region Ig heavy- (VH) and light-chain (VL) transcriptome libraries were generated for each patient from CSF plasmablasts by single cell sorting, reverse transcriptase polymerase chain reaction (RT-PCR), and DNA sequencing. Recombinant antibodies were generated from clonally expanded, paired VH and VL sequences and tested for AQP4-reactivity by cell-binding assay. CSF and serum IgG fractions were searched for sequences that matched their respective CSF IgG transcriptome. Matching peptides within the same patient's CSF and serum IgG proteomes were also identified. RESULTS: In each NMO patient, we recovered CSF IgG VH and VL sequences that matched germline-mutated IgG protein sequences from the patient's CSF and serum IgG proteomes. Although a modest variation was observed between patients, the overlap between the transcriptome and proteome sequences was found primarily, but not exclusively, within the CSF. More than 50% of the CSF IgG transcriptome sequences were exclusively found in the CSF IgG proteome, whereas 28% were found in both the CSF and blood IgG proteome, and 18% were found exclusively in the blood proteome. A comparable distribution was noted when only AQP4-specific IgG clones were considered. Similarly, on average, only 50% of the CSF IgG proteome matched corresponding peptide sequences in the serum. CONCLUSIONS: During NMO exacerbations, a substantial fraction of the intrathecal Ig proteome is generated by an intrathecal B cell population composed of both novel and peripherally-derived clones. Intrathecal CSF B cell clones may contribute to NMO disease exacerbation and lesion formation and may be an important target for preventative therapies.
Assuntos
Aquaporina 4/imunologia , Linfócitos B/metabolismo , Sistema Nervoso Central/patologia , Imunoglobulina G/líquido cefalorraquidiano , Neuromielite Óptica/líquido cefalorraquidiano , Neuromielite Óptica/patologia , Sequência de Aminoácidos , Bases de Dados Factuais/estatística & dados numéricos , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Cadeias Pesadas de Imunoglobulinas/líquido cefalorraquidiano , Cadeias Leves de Imunoglobulina/líquido cefalorraquidiano , Espectrometria de Massas , Proteoma , TranscriptomaRESUMO
B cells are implicated in the etiology of multiple sclerosis (MS). Intrathecal IgG synthesis, cerebrospinal fluid (CSF) oligoclonal bands and lesional IgG deposition suggest a role for antibody-mediated pathology. We examined the binding of IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF expanded B cell clones to central nervous system (CNS) tissue. MS rAbs displaying CNS binding to mouse and human CNS tissue were further tested for their ability to induce complement-mediated tissue injury in ex vivo spinal cord explant cultures. The staining of CNS tissue, primary human astrocytes and human neurons revealed a measurable bias in MS rAb binding to antigens preferentially expressed on astrocytes and neurons. MS rAbs that recognize myelin-enriched antigens were rarely detected. Both myelin-specific and some astrocyte/neuronal-specific MS rAbs caused significant myelin loss and astrocyte activation when applied to spinal cord explant cultures in the presence of complement. Overall, the intrathecal B cell response in multiple sclerosis binds to both glial and neuronal targets and produces demyelination in spinal cord explant cultures implicating intrathecal IgG in MS pathogenesis.
Assuntos
Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Doenças Desmielinizantes/imunologia , Imunoglobulina G/imunologia , Esclerose Múltipla/imunologia , Plasmócitos/imunologia , Medula Espinal/imunologia , Adulto , Animais , Animais não Endogâmicos , Astrócitos/imunologia , Astrócitos/patologia , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/imunologia , Córtex Cerebral/patologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos Endogâmicos C57BL , Bainha de Mielina/imunologia , Bainha de Mielina/patologia , Neurônios/imunologia , Neurônios/patologia , Proteínas Recombinantes/imunologia , Medula Espinal/patologia , Técnicas de Cultura de TecidosRESUMO
Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacina contra Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas Virais/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/genética , Anticorpos Antivirais/isolamento & purificação , Vacina contra Herpes Zoster/administração & dosagem , Humanos , Imunoprecipitação , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Pessoa de Meia-Idade , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/antagonistas & inibidoresRESUMO
B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.
Assuntos
Esclerose Múltipla , Camundongos , Animais , Humanos , Esclerose Múltipla/patologia , Bainha de Mielina , Imunoglobulina G , Epitopos , ProteolipídeosRESUMO
A causal role for virus infection in the pathophysiology of multiple sclerosis (MS) has been suggested and widely debated since the landmark epidemiologic studies of Kurtzke revealed a strong environmental determinant to MS susceptibility. Despite multiple efforts, no virus has been unequivocally associated with lesion formation in the brain either by direct isolation or by indirect methods of detection. In many infectious diseases of the central nervous system, oligoclonal IgG bands are the product of a robust and specific humoral response against the causative agent; yet in MS, immunoreactivity to a primary target has been elusive. In the absence of any infectious agent fulfilling Koch's Postulates, new concepts that could plausibly explain the epidemiology of MS have been postulated. The initiation or activation of a nascent autoimmune response in genetically susceptible individuals following exposure to one or more common infectious agents is now a leading hypothesis to explain MS pathogenesis. Epstein-Barr Virus (EBV), a human herpes virus that infects B cells in ~95% of the human population and persists latently in the memory B cell pool throughout life, has received the most attention as a probable candidate; EBV has been implicated as both an environmental trigger and as a direct causative agent of CNS immunopathology. In this review, we will discuss the most salient features of EBV epidemiology, the immunological response to EBV in MS patients and whether EBV infection of the brain is a necessary prerequisite of MS pathology.
Assuntos
Encéfalo/virologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/patogenicidade , Esclerose Múltipla/virologia , Animais , Autoimunidade , Encéfalo/imunologia , Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/imunologia , Predisposição Genética para Doença , Herpesvirus Humano 4/imunologia , Humanos , Esclerose Múltipla/epidemiologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Fenótipo , Medição de Risco , Fatores de RiscoRESUMO
Simian varicella virus (SVV) open reading frame (ORF) 63, duplicated in the virus genome as ORF 70, is homologous to varicella zoster virus ORF 63/70. Transfection of bacterial artificial chromosome clones containing the wild-type SVV genome and mutants with stop codons in ORF 70, in both ORFs 63 and 70 and the repaired virus DNA sequences into Vero cells produced a cytopathic effect (CPE). The onset of CPE was much slower with the double-mutant transfectants (10 days vs. 3 days) and plaques were smaller. While SVV ORF 63 is not required for replication in culture, its expression leads to robust virus replication.
Assuntos
Varicela/genética , Varicela/virologia , Cromossomos Artificiais Bacterianos/genética , Herpesvirus Humano 3/genética , Fases de Leitura Aberta , Animais , Sequência de Bases , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/metabolismo , Efeito Citopatogênico Viral/genética , DNA Viral/genética , Genes Virais , Genoma Viral , Herpesvirus Humano 3/metabolismo , Dados de Sequência Molecular , Mutação , Transfecção , Células Vero , Replicação Viral/genéticaRESUMO
OBJECTIVE: The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis. METHODS: Fluorescence-activated cell sorting and single-cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE). RESULTS: Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition. INTERPRETATION: Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specific Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease.
Assuntos
Aquaporina 4/líquido cefalorraquidiano , Aquaporina 4/imunologia , Autoanticorpos/líquido cefalorraquidiano , Neuromielite Óptica/líquido cefalorraquidiano , Neuromielite Óptica/diagnóstico , Sequência de Aminoácidos , Animais , Aquaporina 4/genética , Biomarcadores/líquido cefalorraquidiano , Linhagem Celular , Células Cultivadas , Feminino , Feto , Humanos , Imunoglobulina G/líquido cefalorraquidiano , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Punção Espinal , Fatores de TempoRESUMO
Herpesvirions and varicella zoster virus (VZV) DNA were recently reported in all 15 cerebrospinal fluid (CSF) samples from patients with relapsing-remitting multiple sclerosis (MS) obtained within 1 week of exacerbation. Using identical electron microscopic and polymerase chain reaction techniques, including additional primer sets representing different regions of the VZV genome, we found no herpesvirions or VZV DNA in MS CSF or acute MS plaques. Although enzyme-linked immunosorbent assay analysis demonstrated a higher titer of VZV antibody in MS CSF than in inflammatory control samples, recombinant antibodies prepared from clonally expanded MS CSF plasma cells did not bind to VZV. VZV is not a disease-relevant antigen in MS.
Assuntos
Herpesvirus Humano 3/isolamento & purificação , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/líquido cefalorraquidiano , Antígenos Virais/imunologia , Criança , DNA Viral/líquido cefalorraquidiano , DNA Viral/imunologia , DNA Viral/ultraestrutura , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 3/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Transmissão/métodos , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/imunologia , Vírion/isolamento & purificação , Vírion/ultraestrutura , Adulto JovemRESUMO
OBJECTIVE: Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. METHODS: We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. RESULTS: Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. INTERPRETATION: The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/líquido cefalorraquidiano , Proliferação de Células , Imunoglobulina G/biossíntese , Imunoglobulina G/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/imunologia , Plasmócitos/imunologia , Plasmócitos/patologia , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Linhagem Celular , Células Clonais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Esclerose Múltipla/patologia , Plasmócitos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/líquido cefalorraquidianoRESUMO
Double negative (DN) (CD19+CD20lowCD27-IgD-) B cells are expanded in patients with autoimmune and infectious diseases; however their role in the humoral immune response remains unclear. Using systematic flow cytometric analyses of peripheral blood B cell subsets, we observed an inflated DN B cell population in patients with variety of active inflammatory conditions: myasthenia gravis, Guillain-Barré syndrome, neuromyelitis optica spectrum disorder, meningitis/encephalitis, and rheumatic disorders. Furthermore, we were able to induce DN B cells in healthy subjects following vaccination against influenza and tick borne encephalitis virus. Transcriptome analysis revealed a gene expression profile in DN B cells that clustered with naïve B cells, memory B cells, and plasmablasts. Immunoglobulin VH transcriptome sequencing and analysis of recombinant antibodies revealed clonal expansion of DN B cells that were targeted against the vaccine antigen. Our study suggests that DN B cells are expanded in multiple inflammatory neurologic diseases and represent an inducible B cell population that responds to antigenic stimulation, possibly through an extra-follicular maturation pathway.
Assuntos
Linfócitos B/imunologia , Proliferação de Células , Doenças Transmissíveis/imunologia , Imunogenicidade da Vacina , Inflamação/imunologia , Ativação Linfocitária , Vacinas Virais/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Antígenos CD19/metabolismo , Antígenos CD20/metabolismo , Linfócitos B/metabolismo , Estudos de Casos e Controles , Doenças Transmissíveis/sangue , Doenças Transmissíveis/genética , Doenças Transmissíveis/virologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Feminino , Humanos , Imunidade Humoral , Inflamação/sangue , Inflamação/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Transcriptoma , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Vacinação , Vacinas Virais/administração & dosagem , Adulto JovemRESUMO
Neuromyelitis optica (NMO) is an autoimmune CNS disorder mediated by pathogenic aquaporin-4 (AQP4) water channel autoantibodies (AQP4-IgG). Although AQP4-IgG-driven complement-dependent cytotoxicity (CDC) is critical for the formation of NMO lesions, the molecular mechanisms governing optimal classical pathway activation are unknown. We investigated the molecular determinants driving CDC in NMO using recombinant AQP4-specific autoantibodies (AQP4 rAbs) derived from affected patients. We identified a group of AQP4 rAbs targeting a distinct extracellular loop C epitope that demonstrated enhanced CDC on target cells. Targeted mutations of AQP4 rAb Fc domains that enhance or diminish C1q binding or antibody Fc-Fc interactions showed that optimal CDC was driven by the assembly of multimeric rAb platforms that increase multivalent C1q binding and facilitate C1q activation. A peptide that blocks antibody Fc-Fc interaction inhibited CDC induced by AQP4 rAbs and polyclonal NMO patient sera. Super-resolution microscopy revealed that AQP4 rAbs with enhanced CDC preferentially formed organized clusters on supramolecular AQP4 orthogonal arrays, linking epitope-dependent multimeric assembly with enhanced C1q binding and activation. The resulting model of AQP4-IgG CDC provides a framework for understanding classical complement activation in human autoantibody-mediated disorders and identifies a potential new therapeutic avenue for treating NMO.
Assuntos
Aquaporina 4/imunologia , Autoanticorpos/imunologia , Complemento C1q/imunologia , Neuromielite Óptica/imunologia , Animais , Astrócitos/imunologia , Células CHO , Ativação do Complemento , Proteínas do Sistema Complemento , Cricetinae , Cricetulus , Epitopos/imunologia , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Peptídeos/imunologia , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes/imunologiaRESUMO
Microglia are the principal resident immune cells in the central nervous system (CNS) and play important roles in CNS development, maintenance and repair. The survival and development of microglia depends on colony-stimulating factor 1 receptor (CSF1R), a member of the platelet-derived growth factor receptor (PDGFR) family of tyrosine kinases. Recently pharmacological CSF1R inhibition has been used to investigate the effects of microglial depletion in numerous animal models of CNS disease. However, the effects of CSF1R inhibitors on other cell types in the CNS remains incompletely characterized. In this report, we compared the effect of two commonly used CSF1R inhibitors, PLX5622 and PLX3397, on microglia and oligodendrocyte progenitor cell (OPC) numbers. In ex vivo cerebellar slices and adult mouse brain, both PLX compounds caused robust microglia loss; the kinetics of microglial depletion was more rapid with PLX5622. While high-doses of PLX5622 and PLX3397 reduced OPC number in primary cultures in vitro and ex vivo, low-doses of PLX5622 did not affect the number of OPCs or mature oligodendroglia in culture or in vivo. In adult mice, treatment with PLX5622 had no effect on OPC numbers for 7â¯days; however, a mild reduction was observed after 21â¯days in some CNS regions. In contrast, PLX3397 caused significant OPC loss after 7â¯days of treatment, despite only modest microglia depletion. Neither PLX compound had a remarkable effect on mature oligodendrocytes or myelin protein expression following long-term oral administration. Our results show that CSF1R inhibition with PLX5622 can selectively deplete microglia ex vivo and in vivo without affecting OPC number, demonstrating that microglia are not essential for OPC viability in ex vivo slice cultures or adult CNS tissues.
Assuntos
Aminopiridinas/farmacologia , Encéfalo/efeitos dos fármacos , Microglia/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Pirróis/farmacologia , Animais , Camundongos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidoresRESUMO
Using FACS and single cell reverse transcriptase polymerase chain reaction, we examined the cerebrospinal fluid (CSF) IgG VH repertoires from 10 subjects with a clinically isolated demyelinating syndrome (CIS). B and plasma cell repertoires from individual subjects showed similar VH family germline usage, nearly identical levels of post-germinal center somatic hypermutation, and significant overlap in their clonal populations. Repertoires from 7 of 10 CIS subjects demonstrated a biased usage of VH4 and/or VH2 family gene segments in their plasma or B cell repertoires. V-regionbias, however, was not observed in the corresponding peripheral blood CD19+ B cell repertoires from 2 CIS subjects or in normal healthy adults. Clinically, subjects with VH4 or VH2 CSF IgG repertoire bias rapidly progressed to definite MS, whereas individuals without repertoire bias did not develop MS after a minimum of 2 years of follow-up (p=0.01).
Assuntos
Doenças Desmielinizantes/líquido cefalorraquidiano , Doenças Desmielinizantes/imunologia , Imunoglobulina G/líquido cefalorraquidiano , Cadeias Pesadas de Imunoglobulinas/líquido cefalorraquidiano , Adulto , Linfócitos B/imunologia , Feminino , Citometria de Fluxo , Humanos , Região Variável de Imunoglobulina/líquido cefalorraquidiano , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Plasmócitos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Axônios/metabolismo , Biomarcadores/líquido cefalorraquidiano , Esclerose Múltipla Crônica Progressiva/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Neuroglia/metabolismo , Neuromielite Óptica/líquido cefalorraquidiano , Proteômica/métodos , Feminino , Humanos , MasculinoRESUMO
Fluorescence-activated cell sorting (FACS) analysis of B cell subtypes in 17 CSF samples from 15 patients with clinically-definite MS revealed that CD19+ B cells accounted for 2 to 11% (mean 5%) and CD138+ cells constituted 0 to 5% (mean 2%) of total CSF lymphocytes. Further stratification of CD138+ cells based on expression levels of CD19 showed that CD138+19+ plasma blasts constituted 89+/-2% (mean+/-SE) of the CD138+ cell population (P<0.00001), with more mature plasma cells (CD138+19-) constituting the remaining 11+/-2%. Sequence analysis of immunoglobulin variable regions in single CD138+19+ and CD138+19- cells sorted from MS CSF identified many of the same clonal populations in both populations, indicating a continuum of clonally related plasma cell subtypes of which CD138+19+ plasma blasts are most abundant.
Assuntos
Células Produtoras de Anticorpos/imunologia , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/imunologia , Adulto , Análise de Variância , Antígenos CD19/análise , Feminino , Citometria de Fluxo/métodos , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Sindecana-1/análiseRESUMO
Despite longstanding perceptions, robust innate and adaptive immune responses occur within the central nervous system (CNS) in response to infection and tissue damage. Although necessary to control infection, immune responses can lead to severe CNS pathology in the context of both viral infection and autoimmunity. Research into how the central nervous and immune systems communicate has accelerated over the past 20 years leading to a better understanding of pathways controlling immune activation and neuroinflammation that have guided the approval of new disease-modifying therapies to treat CNS immunopathology, particularly the inflammatory demyelinating disease multiple sclerosis. This article provides an introduction into the basic principles underlying immune responses within the CNS that developed from experimental animal models of both neurotropic virus infection and autoimmune T cell-mediated CNS demyelination.