Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Although the cause of MS is still uncertain, many findings point toward an ongoing autoimmune response to myelin antigens. Because of its location on the outer surface of the myelin sheath and its pathogenicity in the experimental autoimmune encephalomyelitis model, myelin oligodendrocyte glycoprotein (MOG) is one of the potential disease-causing self antigens in MS. However, the role of MOG in the pathogenesis of MS has remained controversial. In this study we addressed the occurrence of autoantibodies to native MOG and its implication for demyelination and axonal loss in MS. We applied a high-sensitivity bioassay, which allowed detecting autoantibodies that bind to the extracellular part of native MOG. Antibodies, mostly IgG, were found in sera that bound with high affinity to strictly conformational epitopes of the extracellular domain of MOG. IgG but not IgM antibody titers to native MOG were significantly higher in MS patients compared with different control groups with the highest prevalence in primary progressive MS patients. Serum autoantibodies to native MOG induced death of MOG-expressing target cells in vitro. Serum from MS patients with high anti-MOG antibody titers stained white matter myelin in rat brain and enhanced demyelination and axonal damage when transferred to autoimmune encephalomyelitis animals. Overall these findings suggest a pathogenic antibody response to native MOG in a subgroup of MS patients.

Fig. 1.

Recombinant expression of native MOG to determine antibody binding. (a) Western blot analysis of MOG expression in cell lysates from LN18^MOG and LN18^Ctr cells in nonreducing condition. The 8–18C5 mAb stains two proteins corresponding to monomeric and dimeric MOG. (b) Immunocytochemistry of LN18^ctr cells [Left (×200)] and LN18^MOG [Center (×200) and Right (×600)] by 8–18C5 mAb. (c) Expression of MOG on LN18^MOG cells analyzed by flow cytometry at different concentrations of the 8–18C5 mAb. The antibody concentration is displayed on top of each curve in nanograms per milliliter. Duplicates are shown for each concentration. (d) Staining of LN18^Ctr (blue line) and LN18^MOG (red line) with sera of three MS patients. Serum antibody binding to MOG was detected by anti-human Ig (Left), IgM (Center), and IgG (Right) secondary antibodies and quantified by flow cytometry. (e) Competition assay for serum antibody binding to native MOG. Antibody-positive serum was applied at a 1:100 dilution to the LN18^MOG cells in the presence of different concentrations of 8–18C5 (filled circles) or an irrelevant IgG1 isotype antibody (open circles) (Left). Competition assay for antibody binding to recombinant MOG^1–125. Antibody-positive serum (Center) or 8–18C5 mAb (Right) was applied to the LN18^MOG cells in the presence of different concentrations of recombinant MOG^1–125 (filled circles) or two irrelevant recombinant proteins (open circles and filled triangles). Binding of antibodies was determined by secondary anti-human (Left and Center) or -mouse (Right) IgG antibodies and quantified by flow cytometry.

Fig. 2.

Increased IgG antibody titers to native MOG in MS patients. (a) Comparative analysis for serum IgG antibody titers to native MOG in MS (n = 47) and OIND (n = 47) patients. (b) Comparative analysis for IgG antibody titers to MOG in HD (n = 140) and a second group of MS patients stratified for disease course (54 patients with RR-MS, 80 patients with SP-MS, and 29 patients with PP-MS). Antibody binding to LN18^MOG and LN18^Ctr cells was determined in each patient by secondary anti-human IgG antibodies and quantified by flow cytometry. The MOG-specific antibody response was calculated by subtracting median fluorescence intensities obtained with LN18^Ctr from the one obtained with LN18^MOG cells. Titers were compared by the Kruskal-Wallis nonparametric analysis. The P values are shown for the comparison of different patient groups. The number of patients with titers exceeding the mean of OIND (a) and HD (b) by two standard deviations is shown.

Fig. 3.

Human antibodies to native MOG induce cell death of MOG-expressing target cells. LN18^MOG (gray bars) and LN18^Ctr (black bars) cells were incubated with anti-MOG antibody-positive and -negative MS sera. 8–18C5 mAb supplemented by serum from an antibody-negative patient was used as control. The cell numbers were determined after 20 h and normalized with the negative control sample (equals 100%). The experiment was performed in duplicate; mean and standard deviation are shown. The complement activity of all of the sera ranged between 50 and 55 CAE unit, without significant difference between anti-MOG antibody-positive and -negative sera. One representative experiment of three is shown.

Fig. 4.

Human antibodies to native MOG bind to intact myelin. Rat brain slices were stained with 8–18C5 mAb (a and d), anti-MOG antibody-positive and -negative sera. Staining was visualized by an anti-IgG antibody labeled with Alexa Fluor 488. Stainings of one representative anti-MOG antibody-positive serum of five (b and e) and one of four negative sera (c) are shown. (Magnification: a–c, ×100; d and e, ×200.)

Fig. 5.

Human antinative MOG antibodies induce demyelination and axonal damage in rat EAE. (a) EAE was induced in Lewis rats, and different sera or 8–18C5 mAb were injected intravenously. Demyelination was determined by LFB/PAS staining, and axonal damage was determined by APP staining on spinal cord sections. Representative perivascular areas are shown for animals treated with 8–18C5 mAb, anti-MOG antibody-positive or -negative sera. (b) Comparative analysis of demyelination, axonal damage, and perivascular infiltrates in EAE animals injected with 7-fold concentrated anti-MOG antibody negative [MOG−(7x), four animals], 7-fold concentrated anti-MOG antibody positive before [MOG+ (7x), four animals] and after IgG absorption [MOG+ (abs), two animals], and the control mAb of 1 mg of 8–18C5 mAb. ∗, P < 0.05 (t test). One representative experiment of three is shown. The mean EAE scores were 2 (8–18C5), 1 (MOG+), 1.25 (MOG−), and 0.75 [MOG+ (abs)]. Complement activity was 67 for MOG+ and 77 CAE units for MOG− serum.