Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination
© The Author(s). 2017
Received: 16 March 2017
Accepted: 16 March 2017
Published: 24 March 2017
Intrathecal immunoglobulin G (IgG) synthesis, cerebrospinal fluid (CSF) oligoclonal IgG bands and lesional IgG deposition are seminal features of multiple sclerosis (MS) disease pathology. Both the specific targets and pathogenic effects of MS antibodies remain poorly characterized. We produced IgG1 monoclonal recombinant antibodies (rAbs) from clonally-expanded plasmablasts recovered from MS patient CSF. Among these were a subset of myelin-specific MS rAbs. We examined their immunoreactivity to mouse organotypic cerebellar slices by live binding and evaluated tissue injury in the presence and absence of human complement. Demyelination, glial and neuronal viability, and complement pathway activation were assayed by immunofluorescence microscopy and compared to the effects of an aquaporin-4 water channel (AQP4)-specific rAb derived from a neuromyelitis optica (NMO) patient. MS myelin-specific rAbs bound to discrete surface domains on oligodendrocyte processes and myelinating axons. Myelin-specific MS rAbs initiated complement-dependent cytotoxicity to oligodendrocytes and induced rapid demyelination. Demyelination was accompanied by increased microglia activation; however, the morphology and survival of astrocytes, oligodendrocyte progenitors and neurons remained unaffected. In contrast, NMO AQP4-specific rAb initiated complement-dependent astrocyte damage, followed by sequential loss of oligodendrocytes, demyelination, microglia activation and neuronal death. Myelin-specific MS antibodies cause oligodendrocyte loss and demyelination in organotypic cerebellar slices, which are distinct from AQP4-targeted pathology, and display seminal features of active MS lesions. Myelin-specific antibodies may play an active role in MS lesion formation through complement-dependent mechanisms.
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 . Deposition of IgG and activated complement products are present in the most frequently seen Type II MS lesions , 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  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) , 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 , 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.
Materials and methods
The care and euthanasia of animals were in accordance with University of Colorado IUCAC policy for animal use, which is in agreement with the NIH Guide for the Care and Use of Laboratory Animals.
Myelin-specific MS monoclonal recombinant antibodies (rAbs) used in this study were constructed from expanded CSF plasma blast clones derived from a relapsing-progressive MS patient 6 months after disease onset (MS04-2, for rAb MS04-2#30  or from an isolated optic neuritis patient following their first clinical event who subsequently progressed to clinically-definite MS (ON07-7, for rAb MS07-7#49). NMO monoclonal recombinant anti-AQP4 antibody #53 (NMO#53) was cloned from seropositive NMO patient CSF plasma blast , and the isotype-control human antibody (Iso) was generated from a chronic meningitis patient CSF plasma blast clone . All rAbs were expressed as full-length bivalent human IgG1 antibodies containing a C-terminal Flag epitope using a dual vector transient transfection system and purified with protein A-sepharose (Sigma-Aldrich, St. Louis, MO) as previously described [2, 22]. All rAbs were used at 20 μg/ml in the slice cultures.
Cerebellar slice culture
Sagittal cerebellar slices (300 μm) were prepared from PLP-eGFP mice  at P10 and cultured on MilliCell 0.4 μm membrane inserts (Millipore, Billerica, MA) for 10–14 days in slice media (25% Hank’s balanced salt solution (HBSS), 25% heat-inactivated horse serum, 50% minimum essential media (MEM), 125 mM HEPES, 28 mM D-Glucose, 2 mM L-Glutamine, 10U/ml penicillin/streptomycin, all from Life Technologies, Carlsbad, CA) at 37 °C . Prior to treatment, slices were switched to a serum-free media (Neurobasal medium supplemented with B27, 2 mM L-glutamine, 10U/ml penicillin/streptomycin and 28 mM D-glucose).
Treatment of cerebellar slices
rAbs were applied at 20 μg/ml with or without 10% normal or C5-depleted human serum (Complement Technology, Tyler, TX). Media containing treatment reagents were applied both on top (50 μl) and below (250 μl) the membrane insert. For live binding assays, unfixed slices were incubated with 20 μg/ml rAbs for 4 h at 37 °C. Propidium iodide (PI) (Sigma) was used at 5 μg/ml in the culture medium to label dead cells in organotypic slice cultures. Normal and C5-depleted human serum (Complement Technology, Tyler, TX) were used at 10% (vol/vol) as the source of human complement (HC).
For immunostaining, adult C57bl/6 mice were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS), and the brain was subsequently removed, post-fixed overnight in 4% paraformaldehyde, and cryoprotected overnight in 20% sucrose at 4 °C. Mouse brain was embedded in optimal cutting temperature (OCT) freezing media, and 6–10 μm cryostat sections collected on Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Tissue sections were stored at −80 °C until immunostaining.
After treatment, cerebellar slices were rinsed twice with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 4 °C. For immunohistochemistry, slices were rinsed in PBS and permeabilized in 1.5 or 10% (myelin proteins) Triton X-100 in PBS for 20 min. Slices were rinsed, blocked with 5% normal donkey serum (NDS) in PBS with 0.3% Triton X-100 for 1 h, and incubated with primary antibodies overnight at room temperature. Following 3 washes in PBS, Alexa Fluor-labeled secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied (1:800) overnight at room temperature, washed 3 times in PBS and mounted in Fluoromount G (Southern Biotech, Birmingham, AL). The following primary antibodies were used: rabbit anti-GFAP (Sigma), rabbit anti-Calbindin (Millipore), mouse anti-MAG (Millipore), rabbit anti-MOG (Abcam, Cambridge, United Kingdom), mouse anti-MBP (Covance, Princeton, NJ), chicken anti- Neurofilament-H (Neuromics, Minneapolis, MN), goat anti-Iba1 (Abcam), mouse anti-C3d (a gift from Dr. Joshua Thurman, University of Colorado), rabbit anti-MAC complex (anti C5b-9, Abcam), guinea pig anti- NG2 and rabbit anti-Olig2 (are gifts from Dr. Charles Stiles, Harvard University).
Prior to immunostaining, PFA-fixed mouse cerebellum tissue sections were thawed for 10 min, re-hydrated in PBS for 10 min, and blocked in PBS containing 3% bovine serum albumin (BSA), 2% normal goat serum (NGS), and 0.3% Triton-X100 for 1 h. MS rAbs were applied at a final concentration of 20 μg/mL to mouse tissues for 16 h at 4 °C in PBS containing 3% BSA and 2% NGS. Tissue sections were then washed 3 times for 3 min with PBS. Alexa fluorescent secondary antibody (1:1000) against human IgG (Molecular probes, Life Science Technologies) was applied for 1 h at room temperature in PBS containing 3% BSA and 2% NGS. Tissue was then washed 5 times for 3 min in PBS and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) containing DAPI.
Fluorescence images were acquired by Zeiss fluorescence microscope with Axiovision software (Zeiss, Jena, Germany). Confocal images were acquired by Leica SP5 laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). Super-resolution structured illumination microscopy was performed using Nikon’s N-SIM (Nikon, Tokyo, Japan).
Quantification and statistical analysis
PLP-eGFP positive cell bodies in the cerebellar slices were imaged using a Zeiss fluorescence microscope with 20X objective. Images were quantified with ImageJ (National Institutes of Health open source). To quantify the extent of myelination, we calculated the percentage of total neurofilament-H staining that was co-labeled with MBP using a Matlab algorithm (MathWorks, Natick, MA). For each slice, 2–3 images were taken, quantified, and averaged. Slices from 3–4 independent experiments were analyzed. Statistical analyses were performed by unpaired Student’s t test for single comparisons or by two-way ANOVA for grouped comparisons using GraphPad Prism software. Data are expressed as means ± SD of independent experiments (n ≥ 3). Significance is reported for p < 0.05.
Myelin-specific MS rAb binds to oligodendrocyte processes and myelinated axons
Additional file 1: Video S1. 3D movie reconstructed by super-resolution structured illumination microscopy (SIM) imaging of live organotypic mouse cerebellar slices stained with MS#30 (red), then fixed and stained for MAG (blue) and NF-H (purple). MS#30 reactivity was on oligodendrocyte processes, including those contacting adjacent axons, and on myelinated MAG+ axons, outside of MAG layer. Scale bar: 2 μm. (MPG 90340 kb)
MS#30 causes loss of mature oligodendrocytes in the presence of human complement
MS#30-mediated demyelination is distinct from AQP4-targeted demyelination
Demyelination by MS#30 plus HC results in microglia activation, but not astrocyte or neuronal loss
Complement activation drives MS#30-mediated oligodendrocyte cytotoxicity
Myelin-binding MS rAbs are less frequent, but present in multiple MS patient CSF
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.
Extensive investigation has been performed to pathologically characterize and classify MS lesions. The staging system distinguishes active, chronic active, inactive and pre-active lesions . 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 . In the majority of active MS lesions, tissue histopathology demonstrates oligodendrocyte apoptosis, deposition of immunoglobulin and terminally activated complement  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 . 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 , nor did it bind to the myelin glycolipids sulfatide and galC on lipid arrays . 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)  and therapeutic B cell depletion  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 . 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.
In conclusion, we have identified a subset of early MS patients with expanded CSF plasma blast clones specific for antigenic epitopes present on the surface of oligodendrocyte processes and myelin. Our studies using an ex vivo slice model reveal that these myelin-specific antibodies drive complement-dependent oligodendrocyte loss and demyelination and could be contributors to type II MS lesions. Whether pathogenic myelin-specific Abs are rare or more common, both within MS CSF plasmablast repertoires and amongst a wider spectrum of MS patients are crucial questions that will require identification of the relevant myelin antigens and the development of assays to screen a large sampling of MS CSF and serum.
This work was supported by Collaborative Research Grant from National Multiple Sclerosis Society (W.B.M.), NEI EY022936 (J.L.B.), NINDS NS072141 (G.P.O.), NS25304 (W.B.M.) and the Guthy-Jackson Charitable Foundation (J.L.B.).
We are indebted to Hannah Schumann and Andre Navarro for the preparation of isotype control, NMO #53 rAbs and anti-C3d antibody. We thank Kristin Schaller for assistance with mouse breeding.
YL designed and coordinated the study, performed the experiments, analyzed the data, drafted the manuscript and revision. KG and DH performed the slice culture preparation, treatment, immuno-histochemistry and MATLAB analysis. AM produced and purified MS rAbs. WM, JB and GO designed the study, reviewed the data and the manuscript and revisions. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with University of Colorado IUCAC policy for animal use, which is in agreement with the NIH Guide for the Care and Use of Laboratory Animals.
This article does not contain any studies with human participants performed by any of the authors.
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