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 PCR 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-specfic Ig is synthesized intrathecally at disease onset and directly
BACKGROUND Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. Many findings suggest that the disease has an autoimmune pathogenesis; the target of the immune response is not yet known. METHODS We screened serum IgG from persons with multiple sclerosis to identify antibodies that are capable of binding to brain tissue and observed specific binding of IgG to glial cells in a subgroup of patients. Using a proteomic approach focusing on membrane proteins, we identified the ATP-sensitive inward rectifying potassium channel KIR4.1 as the target of the IgG antibodies. We used a multifaceted validation strategy to confirm KIR4.1 as a target of the autoantibody response in multiple sclerosis and to show its potential pathogenicity in vivo. RESULTS Serum levels of antibodies to KIR4.1 were higher in persons with multiple sclerosis than in persons with other neurologic diseases and healthy donors (P<0.001 for both comparisons). We replicated this finding in two independent groups of persons with multiple sclerosis or other neurologic diseases (P<0.001 for both comparisons). Analysis of the combined data sets indicated the presence of serum antibodies to KIR4.1 in 186 of 397 persons with multiple sclerosis (46.9%), in 3 of 329 persons with other neurologic diseases (0.9%), and in none of the 59 healthy donors. These antibodies bound to the first extracellular loop of KIR4.1. Injection of KIR4.1 serum IgG into the cisternae magnae of mice led to a profound loss of KIR4.1 expression, altered expression of glial fibrillary acidic protein in astrocytes, and activation of the complement cascade at sites of KIR4.1 expression in the cerebellum. CONCLUSIONS KIR4.1 is a target of the autoantibody response in a subgroup of persons with multiple sclerosis. (Funded by the German Ministry for Education and Research and Deutsche Forschungsgemeinschaft.)
Overall, these findings suggest nMOG as a major target of the humoral immune response in a subgroup of children affected by inflammatory demyelinating diseases of the CNS. Children may provide valuable insight into the earliest immune mechanisms of CNS demyelination.
Background: Antibodies targeting membrane proteins play an important role in various autoimmune diseases of the nervous system. So far, assays allowing proper analysis of such autoantibodies are largely missing. A serum autoantibody to aquaporin 4 (AQP4) is associated with neuromyelitis optica (NMO). Although several assays are able to detect this autoantibody, they do not allow determination of the biological activity of anti-AQP4 antibodies.Objective: To develop a bioassay for quantification and characterization of human anti-AQP4 antibodies.Design, Setting, and Participants: We developed a novel bioassay for quantification and characterization of human anti-AQP4 antibodies based on high-level expression of native AQP4 (nAQP4) protein on the surface of human astroglioma cells. The test was validated in 2 independent cohorts of patients with NMO spectrum disease.Results: We detected anti-nAQP4-IgG with a sensitivity of 57.9% and specificity of 100% in patients with NMO spectrum diseases, suggesting that our bioassay is at least as sensitive and specific as the gold-standard NMO-IgG assay. The anti-AQP4 antibodies belonged predominantly to the IgG1 isotype and bound with high affinity to the extracellular domain of nAQP4. Our data suggest that the autoantibody exerts pathological properties because nAQP4-IgGpositive sera induced cell death of nAQP4-expressing cells by antibody-dependent cellular natural killer cell cytotoxic effect and complement activation. Furthermore, nAQP4-IgG titers strongly correlated with in vitro cytotoxic effect. Conclusions:In NMO, this assay may help to unravel the biological function of anti-nAQP4-IgG. Our findings demonstrate the potential of bioassays to characterize biologically relevant antibodies in human autoimmune diseases.
Antidepressants are known to impact on the immune system. In this study, we examined the immunomodulatory properties of venlafaxine, a selective serotonin/norepinephrine reuptake inhibitor (SNRI), in murine experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated CNS demyelinating disease model of multiple sclerosis. EAE was induced in SJL/J mice by adoptive transfer of myelin-specific T cells. Mice received different doses of venlafaxine before induction and after onset of disease. Sustained daily oral treatment with 6, 20 and 60 mg/kg significantly ameliorated the clinical symptoms of the disease compared to vehicle during both preventive and therapeutic intervention. Venlafaxine suppressed the generation of pro-inflammatory cytokines IL-12 p40, TNF-alpha and IFN-gamma in encephalitogenic T-cell clones, splenocytes and peritoneal macrophages in vitro. It also diminished mRNA expression of a number of inflammatory genes in the inflamed CNS tissue, among them CD3, CD8, Granzyme B, IL-12 p40, IFN-gamma, TNF-alpha and the chemokines Ccl2 and RANTES, whereas the expression of brain-derived neurotrophic factor was increased. These findings demonstrate the strong immunomodulatory property of the selective SNRI venlafaxine. Further studies are warranted to clarify whether venlafaxine may exert similar effects in humans.
BackgroundAntibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized.Methodology/Principal FindingsUsing overlapping 15-meric peptides of AQP4, we identified the immunogenic T cell epitopes of AQP4 that are restricted to murine major histocompatibility complex (MHC) I-Ab. The N-terminal region of AQP4 was highly immunogenic. More precisely, the intracellular epitope AQP422–36 was detected as major immunogenic determinant. AQP482–108 (located in the second transmembrane domain), AQP4139–153 (located in the second extracellular loop), AQP4211–225 (located in the fifth transmembrane domain), AQP4235–249 (located in the sixth transmembrane domain), as well as AQP4289–306 in the intracellular C-terminal region were also immunogenic epitopes. AQP422–36 and AQP4289–303 specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response.Conclusions and SignificanceAQP4 is able to induce an autoreactive T cell response. The identification of I-Ab restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4.
Antibody responses against gastrointestinal antigens are common in MS and AQP4-seropositive NMO/NMO-SD, especially in longitudinally extensive myelitis.
Serum antibodies to KIR4.1 are found in the majority of children with ADD but not in children with other diseases or in HCs. These findings suggest that KIR4.1 is an important target of autoantibodies in childhood ADD.
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