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Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders.

Mader S, Gredler V, Schanda K, Rostasy K, Dujmovic I, Pfaller K, Lutterotti A, Jarius S, Di Pauli F, Kuenz B, Ehling R, Hegen H, Deisenhammer F, Aboul-Enein F, Storch MK, Koson P, Drulovic J, Kristoferitsch W, Berger T, Reindl M - J Neuroinflammation (2011)

Bottom Line: AQP4-IgG was found in patients with NMO (n = 43, 96%), HR-NMO (n = 32, 60%) and in one CIS patient (3%), but was absent in ADEM, MS and controls.High-titer MOG-IgG was found in patients with ADEM (n = 14, 42%), NMO (n = 3, 7%), HR-NMO (n = 7, 13%, 5 rON and 2 LETM), CIS (n = 2, 6%), MS (n = 2, 3%) and controls (n = 3, 3%, two SLE and one OND).Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.

ABSTRACT

Background: Serum autoantibodies against the water channel aquaporin-4 (AQP4) are important diagnostic biomarkers and pathogenic factors for neuromyelitis optica (NMO). However, AQP4-IgG are absent in 5-40% of all NMO patients and the target of the autoimmune response in these patients is unknown. Since recent studies indicate that autoimmune responses to myelin oligodendrocyte glycoprotein (MOG) can induce an NMO-like disease in experimental animal models, we speculate that MOG might be an autoantigen in AQP4-IgG seronegative NMO. Although high-titer autoantibodies to human native MOG were mainly detected in a subgroup of pediatric acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS) patients, their role in NMO and High-risk NMO (HR-NMO; recurrent optic neuritis-rON or longitudinally extensive transverse myelitis-LETM) remains unresolved.

Results: We analyzed patients with definite NMO (n = 45), HR-NMO (n = 53), ADEM (n = 33), clinically isolated syndromes presenting with myelitis or optic neuritis (CIS, n = 32), MS (n = 71) and controls (n = 101; 24 other neurological diseases-OND, 27 systemic lupus erythematosus-SLE and 50 healthy subjects) for serum IgG to MOG and AQP4. Furthermore, we investigated whether these antibodies can mediate complement dependent cytotoxicity (CDC). AQP4-IgG was found in patients with NMO (n = 43, 96%), HR-NMO (n = 32, 60%) and in one CIS patient (3%), but was absent in ADEM, MS and controls. High-titer MOG-IgG was found in patients with ADEM (n = 14, 42%), NMO (n = 3, 7%), HR-NMO (n = 7, 13%, 5 rON and 2 LETM), CIS (n = 2, 6%), MS (n = 2, 3%) and controls (n = 3, 3%, two SLE and one OND). Two of the three MOG-IgG positive NMO patients and all seven MOG-IgG positive HR-NMO patients were negative for AQP4-IgG. Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients. Antibodies to MOG and AQP4 were predominantly of the IgG1 subtype, and were able to mediate CDC at high-titer levels.

Conclusions: We could show for the first time that a subset of AQP4-IgG seronegative patients with NMO and HR-NMO exhibit a MOG-IgG mediated immune response, whereas MOG is not a target antigen in cases with an AQP4-directed humoral immune response.

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Serum AQP4-IgG antibodies of an NMO patient activate the complement cascade in the presence of active complement resulting in the deposition of the terminal C5b-9 complement complex. A. Formation of the terminal complement complex (TCC, red) on the surface of M23 AQP4-EmGFP expressing HEK-293A cells (green) following addition of a heat- inactivated AQP4-IgG positive serum sample (titer of 1:10,240). The membrane attack complex resulted in lysis of AQP4-expressing cells (DAPI, blue). B. No antibody mediated complement activation was detectable following incubation of the same heat-inactivated AQP4-IgG positive NMO serum sample supplemented with inactive complement (TCC, red). Images are representative of similar staining patterns observed in independent complement activation assays of the 27 AQP4-IgG positive patients (Table 3). Images are shown in 20 × (A-upper panel and B) and 63 × (A-lower panel) magnification.
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Figure 3: Serum AQP4-IgG antibodies of an NMO patient activate the complement cascade in the presence of active complement resulting in the deposition of the terminal C5b-9 complement complex. A. Formation of the terminal complement complex (TCC, red) on the surface of M23 AQP4-EmGFP expressing HEK-293A cells (green) following addition of a heat- inactivated AQP4-IgG positive serum sample (titer of 1:10,240). The membrane attack complex resulted in lysis of AQP4-expressing cells (DAPI, blue). B. No antibody mediated complement activation was detectable following incubation of the same heat-inactivated AQP4-IgG positive NMO serum sample supplemented with inactive complement (TCC, red). Images are representative of similar staining patterns observed in independent complement activation assays of the 27 AQP4-IgG positive patients (Table 3). Images are shown in 20 × (A-upper panel and B) and 63 × (A-lower panel) magnification.

Mentions: Using our live cell staining immunofluorescence assay (IF) assay, we found that human AQP4-IgG are able to activate the complement cascade at high-titers, leading to the formation of the terminal complement complex (TCC). The resultant TCC was exclusively detected on the surface of AQP4-EmGFP transfected cells (Figure 3). Furthermore, NMO antibody mediated complement activation resulted in complement-dependent lysis of AQP4 transfected cells, which could be demonstrated via DAPI staining of dead cells (Figure 3). Scanning electron microscopy analysis revealed increased apoptosis characterized by a detachment of the cell layer (Figure 4). No TCC formation was observed using AQP4-IgG positive serum samples supplemented with inactive complement. Incubation of AQP4 transfected cells with active complement without serum or with serum samples of AQP4-IgG negative patients supplemented with active complement did not result in complement dependent cytotoxicity (CDC; additional file 1). To verify the antibody mediated localization of the TCC, cells were transfected using AQP4 without the EmGFP fusion protein (Figure 5). In this setting, the membrane attack complex co-localized with human AQP4-IgG. Furthermore, complement-dependent internalization of AQP4-IgG antibodies was observed.


Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders.

Mader S, Gredler V, Schanda K, Rostasy K, Dujmovic I, Pfaller K, Lutterotti A, Jarius S, Di Pauli F, Kuenz B, Ehling R, Hegen H, Deisenhammer F, Aboul-Enein F, Storch MK, Koson P, Drulovic J, Kristoferitsch W, Berger T, Reindl M - J Neuroinflammation (2011)

Serum AQP4-IgG antibodies of an NMO patient activate the complement cascade in the presence of active complement resulting in the deposition of the terminal C5b-9 complement complex. A. Formation of the terminal complement complex (TCC, red) on the surface of M23 AQP4-EmGFP expressing HEK-293A cells (green) following addition of a heat- inactivated AQP4-IgG positive serum sample (titer of 1:10,240). The membrane attack complex resulted in lysis of AQP4-expressing cells (DAPI, blue). B. No antibody mediated complement activation was detectable following incubation of the same heat-inactivated AQP4-IgG positive NMO serum sample supplemented with inactive complement (TCC, red). Images are representative of similar staining patterns observed in independent complement activation assays of the 27 AQP4-IgG positive patients (Table 3). Images are shown in 20 × (A-upper panel and B) and 63 × (A-lower panel) magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3278385&req=5

Figure 3: Serum AQP4-IgG antibodies of an NMO patient activate the complement cascade in the presence of active complement resulting in the deposition of the terminal C5b-9 complement complex. A. Formation of the terminal complement complex (TCC, red) on the surface of M23 AQP4-EmGFP expressing HEK-293A cells (green) following addition of a heat- inactivated AQP4-IgG positive serum sample (titer of 1:10,240). The membrane attack complex resulted in lysis of AQP4-expressing cells (DAPI, blue). B. No antibody mediated complement activation was detectable following incubation of the same heat-inactivated AQP4-IgG positive NMO serum sample supplemented with inactive complement (TCC, red). Images are representative of similar staining patterns observed in independent complement activation assays of the 27 AQP4-IgG positive patients (Table 3). Images are shown in 20 × (A-upper panel and B) and 63 × (A-lower panel) magnification.
Mentions: Using our live cell staining immunofluorescence assay (IF) assay, we found that human AQP4-IgG are able to activate the complement cascade at high-titers, leading to the formation of the terminal complement complex (TCC). The resultant TCC was exclusively detected on the surface of AQP4-EmGFP transfected cells (Figure 3). Furthermore, NMO antibody mediated complement activation resulted in complement-dependent lysis of AQP4 transfected cells, which could be demonstrated via DAPI staining of dead cells (Figure 3). Scanning electron microscopy analysis revealed increased apoptosis characterized by a detachment of the cell layer (Figure 4). No TCC formation was observed using AQP4-IgG positive serum samples supplemented with inactive complement. Incubation of AQP4 transfected cells with active complement without serum or with serum samples of AQP4-IgG negative patients supplemented with active complement did not result in complement dependent cytotoxicity (CDC; additional file 1). To verify the antibody mediated localization of the TCC, cells were transfected using AQP4 without the EmGFP fusion protein (Figure 5). In this setting, the membrane attack complex co-localized with human AQP4-IgG. Furthermore, complement-dependent internalization of AQP4-IgG antibodies was observed.

Bottom Line: AQP4-IgG was found in patients with NMO (n = 43, 96%), HR-NMO (n = 32, 60%) and in one CIS patient (3%), but was absent in ADEM, MS and controls.High-titer MOG-IgG was found in patients with ADEM (n = 14, 42%), NMO (n = 3, 7%), HR-NMO (n = 7, 13%, 5 rON and 2 LETM), CIS (n = 2, 6%), MS (n = 2, 3%) and controls (n = 3, 3%, two SLE and one OND).Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Clinical Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.

ABSTRACT

Background: Serum autoantibodies against the water channel aquaporin-4 (AQP4) are important diagnostic biomarkers and pathogenic factors for neuromyelitis optica (NMO). However, AQP4-IgG are absent in 5-40% of all NMO patients and the target of the autoimmune response in these patients is unknown. Since recent studies indicate that autoimmune responses to myelin oligodendrocyte glycoprotein (MOG) can induce an NMO-like disease in experimental animal models, we speculate that MOG might be an autoantigen in AQP4-IgG seronegative NMO. Although high-titer autoantibodies to human native MOG were mainly detected in a subgroup of pediatric acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS) patients, their role in NMO and High-risk NMO (HR-NMO; recurrent optic neuritis-rON or longitudinally extensive transverse myelitis-LETM) remains unresolved.

Results: We analyzed patients with definite NMO (n = 45), HR-NMO (n = 53), ADEM (n = 33), clinically isolated syndromes presenting with myelitis or optic neuritis (CIS, n = 32), MS (n = 71) and controls (n = 101; 24 other neurological diseases-OND, 27 systemic lupus erythematosus-SLE and 50 healthy subjects) for serum IgG to MOG and AQP4. Furthermore, we investigated whether these antibodies can mediate complement dependent cytotoxicity (CDC). AQP4-IgG was found in patients with NMO (n = 43, 96%), HR-NMO (n = 32, 60%) and in one CIS patient (3%), but was absent in ADEM, MS and controls. High-titer MOG-IgG was found in patients with ADEM (n = 14, 42%), NMO (n = 3, 7%), HR-NMO (n = 7, 13%, 5 rON and 2 LETM), CIS (n = 2, 6%), MS (n = 2, 3%) and controls (n = 3, 3%, two SLE and one OND). Two of the three MOG-IgG positive NMO patients and all seven MOG-IgG positive HR-NMO patients were negative for AQP4-IgG. Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients. Antibodies to MOG and AQP4 were predominantly of the IgG1 subtype, and were able to mediate CDC at high-titer levels.

Conclusions: We could show for the first time that a subset of AQP4-IgG seronegative patients with NMO and HR-NMO exhibit a MOG-IgG mediated immune response, whereas MOG is not a target antigen in cases with an AQP4-directed humoral immune response.

Show MeSH
Related in: MedlinePlus