Neuromyelitis Optica: Deciphering a Complex Immune-Mediated Astrocytopathy

NEUROMYELITIS OPTICA: A NOVEL AUTOIMMUNE CENTRAL NERVOUS SYSTEM DISORDER In 1894, Eugene Devic (1) and Fernand Gault summarized multiple cases of acute, concurrent optic neuritis (ON) and transverse myelitis (TM), which they termed “neuromyélite optique aiguë.” Over the ensuing decades, the publication of multiple additional cases of neuromyelitis optica (NMO) documented that this relatively rare condition had both relapsing and monophasic presentations. Central nervous system (CNS) injury was usually dramatic: severe myelitis that was accompanied, preceded, or followed by severe vision loss progressing to complete blindness (2). Some early clinicopathologic studies argued for a distinct nosology, whereas others suggested classification with disseminated sclerosis (multiple sclerosis [MS]) or diffuse sclerosis (Schilder disease). During this time, it is likely that definitive categorization of NMO was hampered by both the overlapping clinical presentations and histopathology of demyelinating disorders. Although evidence for humoral-mediated disease pathology was observed in active NMO lesions (3), it was the identification of an autoantibody against the aquaporin-4 (AQP4) water channel in NMO patients that dramatically changed perspectives on the pathophysiology of disease and the relationship between NMO and other inflammatory CNS disorders (4,5). However, several fundamental questions needed to be addressed. First, are AQP4 autoantibodies (AQP4-IgG) causing pathology or are there additional NMO-specific immune targets? Second, how does an immune response against AQP4, the major astrocytic CNS water channel, result in myelin destruction? Third, why are the optic nerves and spinal cord predisposed to injury while AQP4-expressing tissues such as lung, stomach, kidney, and skeletal muscle spared? And finally, how can we use our understanding of disease mechanisms in NMO to preserve vision and neurologic function? In this review, we will assess progress in the field toward answering these important questions and examine how information from the bench is leading to the generation of novel therapeutic strategies at the bedside. AQP4-IgG: A SPECIFIC AND PATHOGENIC HUMORAL AUTOANTIBODY In 2004, Lennon et al (5) identified an autoantibody in NMO patient sera, termed NMO-IgG, that stained CNS microvessels, pia, subpia, and Virchow–Robin spaces in mouse midbrain and spinal cord by indirect immunofluorescence (IIF). Serum samples from NMO (5), MS, Japanese optic–spinal multiple sclerosis (O-S-MS), recurrent ON or TM, and various immune, vascular, nutritional, neoplastic, paraneoplastic, and idiopathic conditions were subsequently assayed for this immunoreactivity. NMO-IgG showed high sensitivity (73%; confidence interval [CI]: 60%–86%) and specificity (91%; CI: 79%–100%) for NMO patients. Interestingly, O-S-MS demonstrated similar sensitivity (58%; CI: 30%–86%) and specificity (100%; CI: 66%–100%), and 46% of cases of recurrent ON and TM cases were also positive for NMO-IgG. None of the classical MS or miscellaneous control samples were positive. The similar frequency of positive samples among NMO and Japanese O-S-MS patients also suggested that these 2 disorders were identical and that a significant fraction of individuals with recurrent ON and TM at high risk for NMO could be identified by IIF testing. The target of the serum autoantibody was rapidly identified as the AQP4 water channel (4), the unique CNS staining pattern on IIF resulting from localized expression of AQP4 on astrocyte foot processes on the abluminal face of CNS microvessels and pia mater. The identification of AQP4 as a specific immune target in NMO allowed the further development of specific quantitative and semiquantitative immunoassays for detection of AQP4-IgG in body fluids. Enzyme-linked immunosorbent assay (ELISA), fluorescence immunoprecipitation assay (FIPA) and radioimmunoprecipitation assay, and cell binding assays (CBA) with fluorescence microscopy or fluorescence-activated cell sorting (FACS) detection were subsequently developed and used to independently verify the sensitivity and specificity of AQP4-IgG for NMO (6,7). Although the number of studies and subjects varied considerably, they consistently demonstrated that AQP4-IgG was sensitive (range: 49%–77%) and specific (range: 96%–99%) for NMO (7). A multicenter comparison of IIF, ELISA, FIPA, CBA, and FACS assays demonstrated that CBA and FACS assays were most sensitive; IIF and FIPA assays lacked sensitivity (8). Therefore, multiple independent studies confirmed AQP4-IgG as a disease-specific biomarker of NMO and indicated that cell binding assays offer optimal sensitivity and specificity. Disease-specific autoantibodies, however, are not necessarily pathogenic. In NMO, the pathogenecity of AQP4-IgG was examined using multiple experimental strategies. Serum NMO-IgG, patient-derived AQP4-specific monoclonal recombinant antibodies (rAbs), or control human IgG were administered to rats with experimental autoimmune encephalomyelitis (EAE), and the CNS was examined for NMO-specific histopathology (9–11). NMO-IgG or patient-derived AQP4-specific rAb, but not control serum or control rAb, caused NMO-like pathology in the background of myelin-targeted EAE. CNS lesions demonstrated perivascular AQP4 and astrocyte loss, IgG and complement deposition, granulocytic and lymphocytic infiltrates, macrophage influx, and myelinolysis. NMO pathology was specific to NMO-IgG or AQP4-specific rAb. Using a distinct model of direct NMO-IgG and human complement (HC) intracerebral injection (ICI), Saadoun et al (12) were able to recapitulate the seminal histopathologic features of NMO lesions independent of a systemic immune response. Identical histopathology could be produced in the ICI model using AQP4-specific rAb and HC (13), indicating that intracerebral complement activation by AQP4-specific IgG is sufficient to induce the seminal features of NMO histopathology (Fig. 1).FIG. 1.: Inflammatory and noninflammatory mechanisms contributing to astrocyte injury in neuromyelitis optica. Inflammatory mechanisms include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), opsonization, and complement-induced degranulation. Potential noninflammatory mechanisms contributing to injury include aquaporin-4 (AQP4) and glutamate transporter (EAAT2) internalization and direct inhibition of AQP4-mediated water transport. AP, alternative pathway; C2aC4bC3b, C5 convertase; C4bC2a, C3 convertase; CP, classical pathway; MAC, membrane attack complex; NK, natural killer.Are There Other Target Antigens in Neuromyelitis Optica? Approximately 25% of patients meeting the 2006 Wingerchuk diagnostic criteria (14) for NMO are seronegative for AQP4-IgG (8). A small fraction of these seronegative patients have serum myelin oligodendrocyte glycoprotein autoantibodies (MOG-IgG) (15). AQP4-IgG and MOG-IgG are rarely observed in the same patient (16,17). Despite overlapping clinical presentations, multiple lines of data indicate that AQP4-IgG and MOG-IgG seropositive patients are distinct demyelinating conditions (18,19). Although both MOG-IgG and AQP4-IgG seropositive patients have relapsing clinical courses with significant disability (20), a lower female bias and higher predominance of monophasic disease are reported in multiple MOG-IgG–positive cohorts (18,19,21). In addition, radiographic features often distinguish MOG-IgG seropositive TM and ON from their AQP4-IgG counterparts: inflammation of the conus and cauda equina, perineuritic enhancement of the optic nerve, and shorter optic nerve lesion length (20,22). Moreover, functional recovery and steroid responsivity are more frequently reported for MOG-IgG seropositive ON (18,19,21,23,24). Importantly, the histopathology of brain lesions from MOG-IgG seropositive patients reveal Type 2 MS pathology (25), and MOG-IgG does not produce NMO lesion pathology in the ICI animal model (26,27). In recognition of the broadening clinical presentation of NMO, the International Panel for NMO Diagnosis recently established new criteria for the diagnosis of neuromyelitis optica spectrum disorder (NMOSD) (28). Novel clinical and radiologic criteria for seronegative NMOSD were formulated, and in a recent analysis of a large cohort of NMOSD patients, the fraction of AQP4-IgG seronegative patients fell from 15% to 10% (29). The remaining small fraction of AQP4-IgG seronegative NMOSD patients may be due to limited assay sensitivity (6–8), AQP4-IgG seroconversion (29), and clinically overlapping demyelinating conditions (15,19,30). The possibility of a pathogenic T-cell–restricted adaptive immune response against AQP4 is unlikely given the inability to reproduce complete AQP4-targeted pathology following the adoptive transfer of AQP4-specific T cells in experimental systems (31,32). Although additional autoantibodies are frequently observed in AQP4-IgG seropositive and seronegative NMOSD patients (33,34), candidate autoantigen targets, such as aquaporin-1, have failed to show reproducible disease association (35,36). NMO LESION PATHOLOGY: LINKING ASTROCYTE DESTRUCTION TO DEMYELINATION Myelinolysis Precedes Demyelination in NMO Lesions Understanding the link between AQP4-IgG–mediated astrocyte injury and downstream oligodendrocyte loss, myelin destruction, and neuronal injury may reveal critical stages for interventions to prevent or ameliorate CNS injury (Fig. 2). Early NMO lesions exhibit regions of intact myelin associated with either complete astrocyte loss or significantly reduced AQP4 and glial fibrillary acidic protein (GFAP) staining (37,38). The remaining astrocytes appear fragmented with surrounding GFAP-positive debris and perivascular GFAP-laden macrophages (38). Perivascular immunoglobulin and terminal complement complex (C5b-9; C9neo) deposition are evident, consistent with the primary humoral immune-mediated destruction of perivascular astrocytes (38,39). Myelin in these regions show numerous intracytoplasmic vacuoles or widening of the extracellular space between myelinated fibers, and immunohistochemical stains reveal early loss of myelin-associated glycoprotein (38,39). Oligodendrocytes are either reduced in number or absent, and many remaining oligodendrocytes demonstrate seminal features of apoptotic cell death including nuclear chromatin condensation (38), immunoreactivity for activated caspase-3 (38), and DNA fragmentation (39). Regions of demyelination in active NMO lesions are marked by the presence of lipid- and complement-laden macrophages (38,39). In contrast to still-myelinated areas, active demyelinating regions displayed more abundant infiltrating immune subsets, such as granulocytes, macrophages, eosinophils, lymphocytes, and plasma cells (38,40–42).FIG. 2.: Myelinolysis and progressive axonal swelling following astrocyte injury in neuromyelitis optica lesions. Astrocyte destruction mediated by AQP4-IgG results in early myelinolysis and progressive axonal swelling. The potential mechanisms driving these pathologies include inflammatory, metabolic, ionic, and excitotoxic mechanisms. Progressive axonal swellings identified in intravital microscopy may represent periaxonal swelling or myelin injury (splitting or focal bulging). Adapted from (27). Ca2+, calcium.Regions of asynchronous glial injury in human NMO lesions provide evidence that targeted destruction of CNS astrocytes leads to secondary oligodendrocyte loss and demyelination. Interestingly, spongiform demyelination is also evident in leukodystrophies associated with astrocyte dysfunction, such as Alexander disease and vanishing white matter disease (43). And, in a rat model of osmotic demyelination, astrocyte death occurs rapidly following correction of hyponatremia and outlines regions of future myelin loss. These observations suggest that the link between astrocyte damage and myelinolysis in NMO are independent of the mechanism of AQP4-IgG–mediated cytotoxicity. Possibilities include impaired potassium and cell volume regulation (44,45), excitotoxicity (46,47), altered astrocyte–oligodendroglial communication (48,49), release of inflammatory mediators (50), or deranged oligodendroglial metabolism (51) (Fig. 2). Experimental models of NMO lesion formation are uniquely positioned to decipher the complex pathophysiology linking astrocyte loss and myelinolysis. In EAE (9,13), passive transfer (52) and ICI (12,13,27) NMO models, oligodendrocyte apoptosis and myelin vacuolization are apparent in regions of isolated astrocyte loss before inflammatory cell infiltration. Oligodendroglial cells are relatively preserved 1 hour after ICI of an AQP4 rAb and HC despite significant astrocyte depletion; 3 hours after injection, oligodendrocyte loss is readily apparent but myelin sheaths are spared (13). Within 12 hours of ICI, there is significant myelin edema and vacuolation (12), and by 18 hours, there is significant myelin fragmentation and vesiculated membrane profiles at the innermost layers of the sheath (27). Mechanistically, pathophysiology appears linked to increased intracellular calcium. Ionomycin treatment of acute brain slices results in myelin vesiculation and fragmentation identical to NMO lesions, and calcium chelation lessens myelin fragmentation in the NMO ICI model (27). Recently, Herwerth et al (53) used intravital imaging to monitor the early phases of CNS lesion formation following the application of AQP4-IgG and HC to murine spinal cord in situ. Although oligodendrocytes remained relatively intact soon after the onset of astrocyte destruction, progressive axonal swellings were prominent (Fig. 2). Axonal swellings were not evident in the absence of the co-application of AQP-IgG and HC, and the number of axonal swellings correlated with AQP4-Ig titer and the degree of astrocyte loss. Previous research has shown that axonal swellings demonstrate complex ultrastructural changes in the subaxolemmal cytoskeleton that may result from a variety of extrinsic stimuli (54). Potential pathogenic mechanisms are similar to those linking astrocyte destruction and myelinolysis: excitotoxicity (55), altered ion homeostasis (45), increased oxidative stress (56), and reduced axonal metabolic support (57,58). Experimental manipulation of neuronal signaling and metabolism in NMO animal models should help to clarify the relationship of progressive axonal swelling, oligodendrocyte apoptosis, and myelinolysis in subacute NMO lesions. The results may identify novel therapeutic avenues for the treatment of acute attacks. Complement-Mediated Cytotoxicity: NMO Lesion Initiation and Propagation Although noninflammatory mechanisms are likely to drive oligodendrocyte injury after astrocyte loss, NMO animal models consistently indicate that AQP4-IgG alone is insufficient to initiate demyelination. NMO lesions in the ICI model require the addition of HC (13,59–61) (Fig. 1). In the absence of HC, in the presence of complement inhibitors, or CDC-deficient AQP4 rAb, there is no evidence of astrocyte or oligodendrocyte loss. Similarly, in the rat where there is a robust endogenous complement system, CNS lesion formation by peripherally administered AQP4-IgG is abrogated by the administration of cobra venom factor, an inhibitor of complement function (52). Similarly, NMO lesion formation is greatly reduced after inhibiting antibody-dependent cell-mediated cytotoxicity (ADCC) using mutated AQP4 rAb, FcγRIII receptor–deficient mice, or a Fcγ receptor blocking antibody (61). Interestingly, CNS lesions initiated by ADCC cause astrocyte destruction but limited myelin loss (62). Therefore, astrocyte destruction by AQP4-IgG requires antibody effector function (CDC or ADCC), but only CDC is sufficient to initiate secondary demyelination. CDC could be critical for demyelination in NMO lesions due to the combined production of anaphylatoxins (C3a, C4a, and C5a) and opsonins (C3b, C4b, and C1q) that recruit inflammatory cells (63), enhance polymorphonuclear-mediated ADCC (64), and facilitate phagocytosis (65) (Fig. 1). Anaphylatoxins are chemoattractants for polymorphonuclear cells (66). Indeed, experimental lesions initiated in the absence of complement show significantly reduced immune cell infiltration (62). After attracting granulocytes to the lesion site, C3a, C5a, and C1q promote further tissue injury by mediating eosinophil (67,68) and neutrophil degranulation (69) and free radical production (70). In NMO lesion models, inhibition of Fc receptor (FcR) binding or signaling significantly lessens tissue damage (62,68). Finally, opsonins (C3b and C4b) are important for phagocytic clearance of apoptotic cells (71,72). Macrophages are abundant in active demyelinating NMO lesions and constitute the major phagocytic population removing myelin debris (37,38,42). Opsonin production in NMO lesions may be critical for subsequent inflammatory demyelination by targeting apoptotic oligodendrocytes for phagocytosis (62). Potential Noninflammatory Mechanisms of CNS Injury In NMO, AQP4-IgG binding to CNS astrocytes has been reported to alter surface expression of AQP4 and diminish water channel function (Fig. 1). On the plasma membrane, AQP4 forms supramolecular assemblies, termed orthogonal arrays of particles (OAPs), that directly impact AQP4-IgG binding (7). In tissue culture cells, AQP4-IgG causes internalization of surface AQP4 (46,73–75). Hinson et al reported internalization of resulting in increased and Other however, have failed to reproduce these The of AQP4-IgG on surface AQP4 by primary murine astrocytes and glial have been In mouse models, systemic or intracerebral administration of AQP-IgG failed to cause internalization of astrocyte AQP4 or tissue injury However, in of large of AQP4-IgG 3 in of AQP4 in the spinal cord and Interestingly, similar histopathology has been in the of impaired NMO patients and in the of active NMO lesions The astrocyte glutamate transporter is with AQP4 in astrocyte and in In human NMO spinal cord lesions, there is reduced expression in regions that impaired glutamate and secondary excitotoxicity may to CNS tissue However, observations AQP4 internalization in cells and primary there are on changes in expression and glutamate after to AQP4-IgG In glial treatment with AQP-IgG in the absence of complement causes oligodendrocyte injury that is reduced with receptor expression is also reduced following of however, the of excitotoxicity to tissue injury in this model was not examined AQP4-IgG may also directly water channel In AQP4 water AQP4-IgG was observed to cell conditions In AQP4-IgG AQP4-specific rAb was to AQP4 water in or plasma The on water is likely due to an of experimental as astrocyte myelin edema is in or after administration of large of AQP4-IgG CENTRAL NERVOUS SYSTEM NEUROMYELITIS Although AQP4 is in multiple NMO pathology is limited to the serum in is the most of pathology muscle pathology with immune infiltrates, AQP4 loss, and complement deposition is evident injury in NMO is not by to AQP4 in Serum AQP4-IgG or AQP4-specific rAb readily to AQP4 in kidney, stomach, lung, and muscle in the and the and in the CNS NMO pathology and complement deposition are not observed in tissues in the absence or presence of inflammation indicating that CDC is not activated in these membrane complement and are with AQP4 in kidney, stomach, and skeletal whereas to be from astrocyte processes cells Indeed, injection of NMO-IgG and HC in spinal cord lesions and ON in passive transfer models AQP4 are for AQP4-IgG–mediated CDC Therefore, the and of AQP4 may to the observed of pathology in The expression of AQP4 and protein is higher in optic nerve and spinal cord tissues and optic nerve tissue the of large to small AQP4 supramolecular CNS injury may be further due to the of AQP4 supramolecular cord white matter astrocyte processes diffuse expression of AQP4 whereas AQP4 expression in matter astrocytes is microscopy of spinal cord large AQP4 in both perivascular and white matter but to perivascular matter The diffuse expression of AQP4 in spinal cord and optic nerve white matter may provide an for the of optic nerve and spinal cord lesions in patients NOVEL TO NEUROMYELITIS from NMO experimental models provide an for the development of novel therapeutic for the treatment of acute inflammatory and noninflammatory mechanisms may be various strategies. and that AQP4-IgG binding have been identified in and CNS these may offer a fundamental mechanism for CNS lesion an AQP4-specific blocking antibody of effector could be used to serum AQP4-IgG binding there is no definitive evidence for noninflammatory AQP4-IgG–mediated lesion pathology in experimental systems and there is no evidence that AQP4-IgG directly AQP4 water channel function CDC and ADCC are for complete lesion formation in the ICI animal model and ADCC to a critical in demyelination at the of lesions A inhibitor was recently shown to be in a small treatment of acute NMO TM however, of acute lesions in an animal model was In a monoclonal antibody demonstrated inhibition of AQP4-IgG–mediated CDC in and in of acute in NMOSD patients with complement inhibitors, however, may face unique therapeutic due to limited CNS abundant target and production of complement of eosinophil degranulation and neutrophil with and has shown in animal models, and clinical with an inhibitor and are may with ADCC the of expression administration of may offer for acute NMOSD to analysis of in optic nerves from NMOSD patients suggest that may to cell injury and loss optic nerve showed increased staining for receptor potential that may to axonal are directly by the a novel for axonal injury in ON In multiple avenues of in the have significantly our understanding of NMO The result is a for understanding and mechanisms that from a targeted antibody response against astrocytes to demyelination and neuronal the is with that target a of offer significant for acute by targeting that and CNS lesions. these from the bench to the the to identify and that CNS injury and preserve