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Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte cytoskeleton.

Dale RC, Tantsis EM, Merheb V, Kumaran RY, Sinmaz N, Pathmanandavel K, Ramanathan S, Booth DR, Wienholt LA, Prelog K, Clark DR, Guillemin GJ, Lim CK, Mathey EK, Brilot F - Neurol Neuroimmunol Neuroinflamm (2014)

Bottom Line: We used MO3.13 cells to examine immunoglobulin (Ig) G effects on oligodendrocyte cytoskeleton using 3D deconvolution imaging.MOG antibodies were found in 31/73 patients with DEM (42%) but in 0/24 controls.MOG antibody has functional effects on oligodendrocyte cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Group (R.C.D., E.M.T., V.M., R.-Y.A.K., N.S., K. Pathmanandavel, S.R., F.B.), Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, Sydney Medical School, University of Sydney, Westmead, Australia; Institute for Immunology and Allergy Research (D.R.B.), Westmead Millenium Institute for Medical Research, University of Sydney, Westmead, Australia; Clinical Immunology (L.A.W.), Royal Prince Alfred Hospital, Sydney Medical School Immunology & Infectious Diseases, University of Sydney, Camperdown, Australia; Department of Radiology (K. Prelog), the Children's Hospital at Westmead, Australia; Department of Paediatric Neurology (D.R.C.), Women's and Children's Hospital, North Adelaide, Australia; Neuroinflammation Group (G.J.G., C.K.L.), MND and Neurodegenerative Diseases Research Centre, Macquarie University, Australian School of Advanced Medicine, North Ryde, Australia; and Neuroinflammation Group (E.K.M.), Brain and Mind Research Institute, University of Sydney, Camperdown, Australia.

ABSTRACT

Objective: To examine the clinical features of pediatric CNS demyelination associated with positive myelin oligodendrocyte glycoprotein (MOG) antibodies and to examine the functional effects of MOG antibody on oligodendrocyte cytoskeleton.

Methods: We measured MOG antibody using a fluorescence-activated cell sorting live cell-based assay in acute sera of 73 children with CNS demyelination (DEM) (median age 8 years, range 1.3-15.3) followed for a median of 4 years. We used MO3.13 cells to examine immunoglobulin (Ig) G effects on oligodendrocyte cytoskeleton using 3D deconvolution imaging.

Results: MOG antibodies were found in 31/73 patients with DEM (42%) but in 0/24 controls. At first presentation, MOG antibody-positive patients were more likely to have bilateral than unilateral optic neuritis (ON) (9/10 vs 1/5, respectively, p = 0.03), less likely to have brainstem findings (2/31 vs 16/42, p = 0.005), more likely to have a raised erythrocyte sedimentation rate >20 mm/h (9/19 vs 3/21, p = 0.05), less likely to have intrathecal oligoclonal bands (0/16 vs 5/27, p = 0.18), and less likely to be homozygous or heterozygous for human leukocyte antigen DRB1*1501 (3/18 vs 7/22, p = 0.46). MOG antibody positivity varied according to clinical phenotype, with ON and relapsing ON most likely to be seropositive. Two relapsing MOG antibody-positive patients treated with mycophenolate mofetil remain in remission and have become MOG antibody seronegative. Oligodendrocytes incubated with purified IgG from MOG antibody-positive patients showed a striking loss of organization of the thin filaments and the microtubule cytoskeleton, as evidenced by F-actin and β-tubulin immunolabelings.

Conclusions: MOG antibody may define a separate demyelination syndrome, which has therapeutic implications. MOG antibody has functional effects on oligodendrocyte cytoskeleton.

No MeSH data available.


Related in: MedlinePlus

Human oligodendroglial MO3.13MOG+ cells express markers of oligodendrocytes and are immunolabeled with protein G- and human MOG-purified human IgG from MOG antibody–positive DEM patients(A) Immunocytochemistry on fixed permeabilized cells showed that MO3.13MOG+ cells expressed oligodendrocyte markers. (B) Protein G- and human MOG-immunopurified IgG from MOG antibody–positive serum immunolabeled live HEK293MOG+ cells but did not immunolabel HEK293Ctl cells compared to MOG antibody–negative serum. Binding to cells was determined by flow cytometry. Mean fluorescence intensity (MFI) values are shown in legends. (C) Protein G- and human MOG-purified IgG from MOG antibody–positive serum also immunolabeled fixed unpermeabilized MO3.13MOG+ cells compared to MOG antibody–negative serum. Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Bar: 10 μm. A2B5 = c-series ganglioside-specific antigen A2B5; Ab = antibody; CNPase = 2′, 3′-cyclic nucleotide 3′-phosphodiesterase; DEM = demyelinating diseases; GalC = galactocerebroside; HEK = human embryonic kidney; Ig = immunoglobulin; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; O4 = oligodendrocyte marker O4.
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Figure 3: Human oligodendroglial MO3.13MOG+ cells express markers of oligodendrocytes and are immunolabeled with protein G- and human MOG-purified human IgG from MOG antibody–positive DEM patients(A) Immunocytochemistry on fixed permeabilized cells showed that MO3.13MOG+ cells expressed oligodendrocyte markers. (B) Protein G- and human MOG-immunopurified IgG from MOG antibody–positive serum immunolabeled live HEK293MOG+ cells but did not immunolabel HEK293Ctl cells compared to MOG antibody–negative serum. Binding to cells was determined by flow cytometry. Mean fluorescence intensity (MFI) values are shown in legends. (C) Protein G- and human MOG-purified IgG from MOG antibody–positive serum also immunolabeled fixed unpermeabilized MO3.13MOG+ cells compared to MOG antibody–negative serum. Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Bar: 10 μm. A2B5 = c-series ganglioside-specific antigen A2B5; Ab = antibody; CNPase = 2′, 3′-cyclic nucleotide 3′-phosphodiesterase; DEM = demyelinating diseases; GalC = galactocerebroside; HEK = human embryonic kidney; Ig = immunoglobulin; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; O4 = oligodendrocyte marker O4.

Mentions: Human oligodendrocyte MO3.13MOG+ cells expressed MOG at their surface, whereas no MOG expression was observed on MO3.13Ctl cells (figure 3). MO3.13 cells also expressed oligodendrocyte markers, such as 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), galactocerebroside (GalC), oligodendrocyte marker O4, vimentin, and c-series ganglioside-specific antigen (A2B5) (figure 3A). Myelin basic protein (MBP), a specific marker of mature oligodendrocytes, was observed only in PMA-differentiated MO3.13MOG+ cells (figure 3A), suggesting that undifferentiated MO3.13 cells are immature oligodendrocytes. Using purified IgG and immunoaffinity-purified MOG IgG from MOG antibody–positive sera, we immunolabeled HEK293MOG+ and HEK293Ctl cells on live cells by FACS (figure 3B) and on fixed MO3.13MOG+ cells by immunocytochemistry (figure 3C) and showed a positive immunostaining compared to MOG antibody–negative sera, suggesting that protein G–purified IgG includes MOG-specific IgG. Due to small volumes of pediatric sera, we used protein G–purified IgG in pathogenic experiments. Next, we treated fixed and live MO3.13MOG+/Ctl cells with IgG from MOG antibody–positive and –negative patients with DEM and healthy controls (HCs). Then, all cells were immunolabeled for β-tubulin (marker of microtubule) or F-actin (marker of thin filaments). We quantified results and expressed them by the F-actin and β-tubulin relative enrichments in the cytoplasm and perinuclear region over the entire cell. All results were normalized using F-actin and β-tubulin in HC IgG-treated fixed cells. Organization of the microtubule and thin filament networks was similar in fixed cells incubated with HC (100% ± 10.1 and 100% ± 22.6, respectively, figure 4, A and C) or DEM IgGs (104.3% ± 11.3 and 85.8% ± 31.8, respectively, figure 4, A and C); there was clear visualization of “stress-fibers” and filopedia after F-actin immunolabeling (figure 4A, upper panels) and bright mesh-like staining spread out through the entire cell after β-tubulin immunolabeling (figure 4A, lower panels). Live cells treated by HC IgG displayed a small disorganization of F-actin thin filaments (91% ± 14.4, p < 0.0001, figure 4, B and C, upper images) and undisturbed microtubular β-tubulin network (107.4% ± 32.1, figure 4, B and C), similar to that observed in fixed DEM and HC IgG-treated cells (figure 4A, upper panels). Following treatment with DEM IgG on live MO3.13MOG+ cells, we observed a striking loss of organization in both F-actin (72.4% ± 11.7, p < 0.0001, figure 4, B and C) and β-tubulin networks (52.2% ± 13.1, p < 0.0001, figure 4, B and C) in otherwise healthy-looking undividing cells visualized by differential interference contrast imaging (figure 4B, dotted line, and data not shown) and analyzed by FACS, by which there was no difference in viability between live MO3.13MOG+ live cells treated with DEM IgG or HC IgG for 45 minutes (figure 4D, 87.2 ± 0.6 vs 87.2 ± 2.11) or 10 hours (data not shown). No loss of cytoskeleton organization was observed when live MO3.13Ctl cells were incubated with HC IgG and MOG antibody–positive DEM IgG, nor when live MO3.13MOG+ cells were incubated with MOG antibody–negative DEM IgG (figure e-1 and appendix e-3). In addition, successful immunoabsorption of MOG antibody–positive serum on HEK293MOG+ cells led to no change in cytoskeleton organization when immunoabsorbed serum was incubated on live MO3.13MOG+ cells (figure e-1 and appendix e-3). Overall, after incubation with MOG antibody–positive DEM IgG, both F-actin and β-tubulin immunolabelings appeared to be enriched within a perinuclear region in the center of the cells surrounding the nucleus.


Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte cytoskeleton.

Dale RC, Tantsis EM, Merheb V, Kumaran RY, Sinmaz N, Pathmanandavel K, Ramanathan S, Booth DR, Wienholt LA, Prelog K, Clark DR, Guillemin GJ, Lim CK, Mathey EK, Brilot F - Neurol Neuroimmunol Neuroinflamm (2014)

Human oligodendroglial MO3.13MOG+ cells express markers of oligodendrocytes and are immunolabeled with protein G- and human MOG-purified human IgG from MOG antibody–positive DEM patients(A) Immunocytochemistry on fixed permeabilized cells showed that MO3.13MOG+ cells expressed oligodendrocyte markers. (B) Protein G- and human MOG-immunopurified IgG from MOG antibody–positive serum immunolabeled live HEK293MOG+ cells but did not immunolabel HEK293Ctl cells compared to MOG antibody–negative serum. Binding to cells was determined by flow cytometry. Mean fluorescence intensity (MFI) values are shown in legends. (C) Protein G- and human MOG-purified IgG from MOG antibody–positive serum also immunolabeled fixed unpermeabilized MO3.13MOG+ cells compared to MOG antibody–negative serum. Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Bar: 10 μm. A2B5 = c-series ganglioside-specific antigen A2B5; Ab = antibody; CNPase = 2′, 3′-cyclic nucleotide 3′-phosphodiesterase; DEM = demyelinating diseases; GalC = galactocerebroside; HEK = human embryonic kidney; Ig = immunoglobulin; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; O4 = oligodendrocyte marker O4.
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Related In: Results  -  Collection

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Figure 3: Human oligodendroglial MO3.13MOG+ cells express markers of oligodendrocytes and are immunolabeled with protein G- and human MOG-purified human IgG from MOG antibody–positive DEM patients(A) Immunocytochemistry on fixed permeabilized cells showed that MO3.13MOG+ cells expressed oligodendrocyte markers. (B) Protein G- and human MOG-immunopurified IgG from MOG antibody–positive serum immunolabeled live HEK293MOG+ cells but did not immunolabel HEK293Ctl cells compared to MOG antibody–negative serum. Binding to cells was determined by flow cytometry. Mean fluorescence intensity (MFI) values are shown in legends. (C) Protein G- and human MOG-purified IgG from MOG antibody–positive serum also immunolabeled fixed unpermeabilized MO3.13MOG+ cells compared to MOG antibody–negative serum. Representative data are shown (volume projection of entire Z-stack acquired using 3D deconvolution microscopy). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Bar: 10 μm. A2B5 = c-series ganglioside-specific antigen A2B5; Ab = antibody; CNPase = 2′, 3′-cyclic nucleotide 3′-phosphodiesterase; DEM = demyelinating diseases; GalC = galactocerebroside; HEK = human embryonic kidney; Ig = immunoglobulin; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; O4 = oligodendrocyte marker O4.
Mentions: Human oligodendrocyte MO3.13MOG+ cells expressed MOG at their surface, whereas no MOG expression was observed on MO3.13Ctl cells (figure 3). MO3.13 cells also expressed oligodendrocyte markers, such as 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), galactocerebroside (GalC), oligodendrocyte marker O4, vimentin, and c-series ganglioside-specific antigen (A2B5) (figure 3A). Myelin basic protein (MBP), a specific marker of mature oligodendrocytes, was observed only in PMA-differentiated MO3.13MOG+ cells (figure 3A), suggesting that undifferentiated MO3.13 cells are immature oligodendrocytes. Using purified IgG and immunoaffinity-purified MOG IgG from MOG antibody–positive sera, we immunolabeled HEK293MOG+ and HEK293Ctl cells on live cells by FACS (figure 3B) and on fixed MO3.13MOG+ cells by immunocytochemistry (figure 3C) and showed a positive immunostaining compared to MOG antibody–negative sera, suggesting that protein G–purified IgG includes MOG-specific IgG. Due to small volumes of pediatric sera, we used protein G–purified IgG in pathogenic experiments. Next, we treated fixed and live MO3.13MOG+/Ctl cells with IgG from MOG antibody–positive and –negative patients with DEM and healthy controls (HCs). Then, all cells were immunolabeled for β-tubulin (marker of microtubule) or F-actin (marker of thin filaments). We quantified results and expressed them by the F-actin and β-tubulin relative enrichments in the cytoplasm and perinuclear region over the entire cell. All results were normalized using F-actin and β-tubulin in HC IgG-treated fixed cells. Organization of the microtubule and thin filament networks was similar in fixed cells incubated with HC (100% ± 10.1 and 100% ± 22.6, respectively, figure 4, A and C) or DEM IgGs (104.3% ± 11.3 and 85.8% ± 31.8, respectively, figure 4, A and C); there was clear visualization of “stress-fibers” and filopedia after F-actin immunolabeling (figure 4A, upper panels) and bright mesh-like staining spread out through the entire cell after β-tubulin immunolabeling (figure 4A, lower panels). Live cells treated by HC IgG displayed a small disorganization of F-actin thin filaments (91% ± 14.4, p < 0.0001, figure 4, B and C, upper images) and undisturbed microtubular β-tubulin network (107.4% ± 32.1, figure 4, B and C), similar to that observed in fixed DEM and HC IgG-treated cells (figure 4A, upper panels). Following treatment with DEM IgG on live MO3.13MOG+ cells, we observed a striking loss of organization in both F-actin (72.4% ± 11.7, p < 0.0001, figure 4, B and C) and β-tubulin networks (52.2% ± 13.1, p < 0.0001, figure 4, B and C) in otherwise healthy-looking undividing cells visualized by differential interference contrast imaging (figure 4B, dotted line, and data not shown) and analyzed by FACS, by which there was no difference in viability between live MO3.13MOG+ live cells treated with DEM IgG or HC IgG for 45 minutes (figure 4D, 87.2 ± 0.6 vs 87.2 ± 2.11) or 10 hours (data not shown). No loss of cytoskeleton organization was observed when live MO3.13Ctl cells were incubated with HC IgG and MOG antibody–positive DEM IgG, nor when live MO3.13MOG+ cells were incubated with MOG antibody–negative DEM IgG (figure e-1 and appendix e-3). In addition, successful immunoabsorption of MOG antibody–positive serum on HEK293MOG+ cells led to no change in cytoskeleton organization when immunoabsorbed serum was incubated on live MO3.13MOG+ cells (figure e-1 and appendix e-3). Overall, after incubation with MOG antibody–positive DEM IgG, both F-actin and β-tubulin immunolabelings appeared to be enriched within a perinuclear region in the center of the cells surrounding the nucleus.

Bottom Line: We used MO3.13 cells to examine immunoglobulin (Ig) G effects on oligodendrocyte cytoskeleton using 3D deconvolution imaging.MOG antibodies were found in 31/73 patients with DEM (42%) but in 0/24 controls.MOG antibody has functional effects on oligodendrocyte cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Group (R.C.D., E.M.T., V.M., R.-Y.A.K., N.S., K. Pathmanandavel, S.R., F.B.), Institute for Neuroscience and Muscle Research, The Kids Research Institute at the Children's Hospital at Westmead, Sydney Medical School, University of Sydney, Westmead, Australia; Institute for Immunology and Allergy Research (D.R.B.), Westmead Millenium Institute for Medical Research, University of Sydney, Westmead, Australia; Clinical Immunology (L.A.W.), Royal Prince Alfred Hospital, Sydney Medical School Immunology & Infectious Diseases, University of Sydney, Camperdown, Australia; Department of Radiology (K. Prelog), the Children's Hospital at Westmead, Australia; Department of Paediatric Neurology (D.R.C.), Women's and Children's Hospital, North Adelaide, Australia; Neuroinflammation Group (G.J.G., C.K.L.), MND and Neurodegenerative Diseases Research Centre, Macquarie University, Australian School of Advanced Medicine, North Ryde, Australia; and Neuroinflammation Group (E.K.M.), Brain and Mind Research Institute, University of Sydney, Camperdown, Australia.

ABSTRACT

Objective: To examine the clinical features of pediatric CNS demyelination associated with positive myelin oligodendrocyte glycoprotein (MOG) antibodies and to examine the functional effects of MOG antibody on oligodendrocyte cytoskeleton.

Methods: We measured MOG antibody using a fluorescence-activated cell sorting live cell-based assay in acute sera of 73 children with CNS demyelination (DEM) (median age 8 years, range 1.3-15.3) followed for a median of 4 years. We used MO3.13 cells to examine immunoglobulin (Ig) G effects on oligodendrocyte cytoskeleton using 3D deconvolution imaging.

Results: MOG antibodies were found in 31/73 patients with DEM (42%) but in 0/24 controls. At first presentation, MOG antibody-positive patients were more likely to have bilateral than unilateral optic neuritis (ON) (9/10 vs 1/5, respectively, p = 0.03), less likely to have brainstem findings (2/31 vs 16/42, p = 0.005), more likely to have a raised erythrocyte sedimentation rate >20 mm/h (9/19 vs 3/21, p = 0.05), less likely to have intrathecal oligoclonal bands (0/16 vs 5/27, p = 0.18), and less likely to be homozygous or heterozygous for human leukocyte antigen DRB1*1501 (3/18 vs 7/22, p = 0.46). MOG antibody positivity varied according to clinical phenotype, with ON and relapsing ON most likely to be seropositive. Two relapsing MOG antibody-positive patients treated with mycophenolate mofetil remain in remission and have become MOG antibody seronegative. Oligodendrocytes incubated with purified IgG from MOG antibody-positive patients showed a striking loss of organization of the thin filaments and the microtubule cytoskeleton, as evidenced by F-actin and β-tubulin immunolabelings.

Conclusions: MOG antibody may define a separate demyelination syndrome, which has therapeutic implications. MOG antibody has functional effects on oligodendrocyte cytoskeleton.

No MeSH data available.


Related in: MedlinePlus