Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) of unknown cause. Despite extensive pathological characterization of heterogeneous MS lesions, no consensus on the cellular and molecular mechanisms driving diverse lesion pathology has emerged [15, 24, 30]. The presence of persistent cerebrospinal fluid (CSF) oligoclonal immunoglobulin G (IgG) bands produced by intrathecal IgG synthesis in MS patients is one of the most striking biochemical hallmarks of disease [21]. Deposition of IgG and activated complement products are present in the most frequently seen Type II MS lesions [15], suggesting a possible role of intrathecal IgG in CNS tissue injury.

We have constructed recombinant monoclonal IgG1 antibodies (rAbs) from expanded CSF plasmablast clones isolated from MS patients [22] and demonstrated their differential patterns of binding to antigens expressed by astrocytes and neurons or to myelin-enriched antigens [3, 13]. In cDNA-transfected HEK cells or by protein immunoblotting of human brain lysate, myelin-specific rAbs failed to recognize myelin-enriched proteins, including myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) [22], and their specific targets remain elusive. Nevertheless, both myelin and neuron/astrocyte-targeted MS rAbs cause myelin loss when applied to mouse spinal cord explant cultures in the presence of human complement [3], indicating that, similar to autoantibodies against aquaporin-4 (AQP4-IgG) in neuromyelitis optica (NMO) [2, 4, 28], intrathecal IgGs in MS may contribute to lesion pathogenesis.

In this study, we further investigated the primary effect of myelin-specific MS rAbs on intact CNS tissue using organotypic mouse cerebellar slice cultures. Our results reveal that MS myelin-specific rAbs recognized surface antigens on oligodendrocyte processes and the outer layer of myelin ensheathing axons. In the presence of human complement, these rAbs initiated classical complement pathway activation leading to oligodendrocyte death and rapid demyelination. The extent and timing of glial and neuronal injury was distinct from damage driven by AQP4-IgG and reproduced some hallmark features of MS lesions, further distinguishing MS from NMO and supporting an active role for intrathecal MS IgG in CNS lesion formation.

Our studies demonstrate the presence of myelin-specific plasma blast clones in the CSF of some MS patients. Using structured illumination microscopy and confocal microscopy to assess live cell binding, rAb MS#30 and MS#49 label the surface of oligodendrocyte processes and myelinated axons adjacent to MOG and exterior to MAG. More importantly, these myelin-binding MS rAbs activate the complement pathway and induce robust oligodendrocyte loss and microglial activation, demonstrating their potential to contribute to demyelination in MS patients.

In organotypic cerebellar cultures, exposure to myelin-specific rAbs + HC resulted in rapid morphologic changes to oligodendrocytes and their processes accompanied by terminal complement deposition, microglial activation and mature oligodendrocyte cell loss, but with preservation of oligodendrocyte precursors (Figs. 3, 4 and 5). Demyelination induced by myelin-specific MS rAbs was distinct from AQP4 autoantibody-mediated demyelination. Whereas both cerebellar astrocytes and neurons remain intact at 48 h in slices when exposed to myelin-specific MS rAbs, AQP4 autoantibody initiated complement-dependent astrocyte damage, followed by oligodendrocyte cell death, loss of oligodendrocyte precursors [14] and extensive neuronal cell death (Fig. 4). We have previously reported that with ongoing astrocyte destruction induced by AQP4 autoantibody NMO#53 + HC, loss of oligodendrocytes increased from 30% at 12 h to 50% by 48 h. Also, approximately 50% of NG2+ Olig2+ oligodendrocyte precursors were lost in slices by 24 h after treatment with NMO #53 + HC [14]. The damage pattern in this model was thus consistent with in vivo murine models of NMO lesion formation that demonstrate initial astrocyte depletion followed by loss of oligodendrocytes and progenitors [32]. Rapid Iba1 upregulation in the presence of myelin-specific rAb plus HC is consistent with the role of microglia as early tissue-resident sensors of CNS injury [20]. Activated microglia were observed in focal areas around degenerating oligodendrocytes in MS#30 + HC-treated slices beginning at 8 h (Fig. 5), whereas in NMO#53 + HC treated slices, microglia activation began later becoming clearly prominent at the 48 h time point and dispersed evenly throughout the folia. Despite early complement-mediated killing of astrocytes by NMO#53, the differences in kinetics of microglia activation in the cerebellar slice cultures between MS#30 and NMO#53 indicates that microglia activation might be independent of complement activation. Instead, microglia may be activated by sensing debris of dead oligodendrocytes and/or damaged myelin, which leads to clearance of debris. Even with extended treatment, the morphology and survival of astrocytes and neurons remained unaffected in slices treated with MS#30 + HC, suggesting that early microglial activation may offer a protective or beneficial effect [27]. The distinct patterns of injury induced by the MS and NMO antibodies in the presence of complement indicate that the target of complement-dependent cytotoxicity, and not activation of the complement cascade itself, is important for delineating the spectrum of glial and neuronal injury (Fig. 8). The important supporting role of astrocytes to both oligodendrocytes and neurons [11] distinguishes damage by NMO antibody from that occurring by direct targeting of oligodendrocyte processes. Our results further distinguish these demyelinating disorders and their mechanisms of oligodendrocyte loss [1, 16, 23, 32, 33].

Extensive investigation has been performed to pathologically characterize and classify MS lesions. The staging system distinguishes active, chronic active, inactive and pre-active lesions [30]. In relapsing-remitting MS, the lesions are often active or chronic active, with axonal transections and massive infiltration and accumulation of inflammatory cells. In the progressive stage of disease, inactive lesions are more typically characterized by axonal loss, astrogliosis and minor infiltration of immune cells [26]. In the majority of active MS lesions, tissue histopathology demonstrates oligodendrocyte apoptosis, deposition of immunoglobulin and terminally activated complement [15] or T cell-mediated as separate and distinct cause of CNS demyelination, whereas others have reported oligodendrocyte apoptosis in the absence of inflammation as the earliest event in lesion development damage [25]. Importantly, our cerebellar slice model recapitulates some of the reported pathologic features of active MS lesions and provides strong evidence that antibodies produced by B cell populations expanded within the CNS compartment have the potential to drive complement-dependent oligodendrocyte cytotoxicity and contribute to lesion formation. Given the absence of a peripheral immune compartment in the cerebellar slice culture model, methodologic restrictions prevent the replication of some seminal features of inflammatory MS lesions, such as myelin phagocytosis, axonal loss and astrogliosis in this slice system. The development of an in vivo model of MS rAb antibody injury should allow investigators to further distinguish attributes of antibody-mediated from cell-mediated pathologies.

We have currently cloned myein-specific rAbs from 2 of 4 MS patients analyzed, but their antigenic target remains unknown. Localization of Ab binding to the outer surface of oligodendrocyte processes and myelin suggests a limited possibility of candidate antigens. MS#30 failed to bind to HEK cells expressing the myelin surface protein MOG [22], nor did it bind to the myelin glycolipids sulfatide and galC on lipid arrays [5]. We postulate that higher order multimolecular complexes may be driving antigen specificity. In addition to recognizing myelin-enriched antigens, antibodies cloned from other MS CSF plasmablasts bind to antigens expressed on astrocytes and neurons [3, 13]. It is possible that these antibodies may induce secondary demyelination as observed with AQP4-targeted rAbs [32, 33]. The impact of injured astrocytes and neurons on oligodendrocytes through glia-glia [12, 17] and axon-glia interactions [6, 7, 31] has been well documented. These antibodies may also cause primary degeneration or facilitate the removal of cellular debris.

The results of autologous hematopoietic stem cell transplantation (AHSCT) [19] and therapeutic B cell depletion [8] have called into question the role of antibody in MS lesion formation, despite numerous examples of antibody and complement-mediated demyelination in post-mortem MS autopsy specimens. In autopsy specimens from AHSCT-treated MS patients, extensive demyelination and axonal degeneration are observed in the presence of innate immune activation, but without notable B cell infiltration [19]. Following peripheral B cell depletion, significant reduction in inflammatory lesion formation is observed in the absence of changes in intrathecal IgG synthesis or oligoclonal bands [9, 10]. However, it must be noted that antibody deposition and complement activation were not directly evaluated in post-AHSCT lesions; and antibody-mediated complement cytotoxicity in MS patients may be suppressed following B cell depletion by limited serum complement extravasation into the CNS from the rapid and extensive reduction of blood-brain barrier breakdown.