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

Serum AQP4-IgG and high-titer MOG-IgG antibodies in different disease groups

Using our assay with M23 AQP4 transfected HEK-293A cells, we detected significantly increased frequencies of serum AQP4-IgG in NMO (n = 43, 96%) and HR-NMO (n = 32, 60%; Table 1). Median AQP4-IgG titers of seropositive patients were 1:1,280 (1:40-1:40,960) in NMO and 1:1,280 (1:20-1:20,480) in HR-NMO (Figure 1). In addition, AQP4-IgG (titer 1:640) was detected in one patient with clinically isolated syndrome (CIS) presenting with myelitis. AQP4-IgG antibodies were absent in two patients with NMO (4%), 21 patients with HR-NMO (40%), 31 CIS patients (97%) and all patients with ADEM and MS as well as all controls (CTRL) including patients with systemic lupus erythematosus (SLE), other neurological diseases (OND) and healthy individuals (Table 1).

Disease	Number	Females1	Age (y)2	AQP4-IgG1,3	MOG-IgG1,3
NMO	45	41 (91%) *	50 (2-80)	43 (96%) §	3 (7%)
				1,280	2,560
				(40-40,960)	(160-2,560)
HR-NMO	53	34 (64%)	48 (13-74)	32 (60%) §	7 (13%)
				1,280	2,560
				(20-20,480)	(640-5,120)
ADEM	33	20 (61%)	12 (2-52) §	0 (0%)	14 (42%) §
				-	2,560
					(160-20,480)
CIS	32	22 (69%)	34 (5-57) +	1 (3%)	2 (6%)
				640	(640; 5,120)
MS	71	43 (61%)	40 (14-66)	0 (0%)	2 (3%)
					(160; 160)
CTRL	101	79 (78%)	43 (14-84)	0 (0%)	3 (3%)
					320
					(160-640)
p-value		0.003	< 0.001	< 0.001	< 0.001

P-value: groups were compared using¹ Chi-Square test with Fisher's exact post-hoc test and² Kruskal-Wallis test with Dunn's post-hoc test (data are shown as median with range);³ median titer level (range) of MOG-IgG or AQP4-IgG seropositive samples; * statistically different from HR-NMO, ADEM and MS; + statistically significant from NMO, HR-NMO and ADEM; § statistically significant from all other groups (p-values were corrected for multiple comparisons). Abbreviations: y = years, NMO = Neuromyelitis optica, HR-NMO = High-risk NMO, ADEM = acute disseminated encephalomyelitis, CIS = clinically isolated syndrome, MS = multiple sclerosis, CTRL = controls.

In addition to AQP4-IgG, we analyzed antibodies directed to natively folded human MOG expressed on the surface of human cells in the same set of patients (Table 1 and Figure 1). The frequency of high-titer (≥ 1:160) serum MOG-IgG antibodies was significantly increased in patients with ADEM (n = 14, 42%). However, high-titer MOG-IgG were also found in patients with NMO (n = 3, 7%), HR-NMO (n = 7, 13%), CIS (n = 2, 6%), MS (n = 2, 3%) and CTRL (3, 3%) (Table 1, Figure 1). Median MOG-IgG titers of seropositive patients were 1:2,560 (1:160-1:2,560) in NMO, 1:2,560 (1:640-1:5,120) in HR-NMO, 1:2,560 (1:160-1:20,480) in ADEM, 1:640 and 1:5,120 in CIS, 1:160 and 1:160 in MS and 1:320 (1:160-1:640) in CTRL (Figure 1 and Table 1).

The clinical characteristics of MOG-IgG positive patients with NMO, HR-NMO and CIS are shown in Table 2. The MOG-IgG positive NMO patients consisted of two AQP4-IgG seronegative patients (a two year old female child and a 56 year old male), both with a MOG-IgG titer of 1:2,560, and one patient (a 39 year old woman) who was double positive for both, MOG-IgG (titer 1:160) and AQP4-IgG (titer 1:1,280). Within the HR-NMO group, seven of 21 (33%) AQP4-IgG negative patients were positive for high-titer MOG-IgG (Table 1 and 2). These seven patients included five patients with recurrent ON and two patients with monophasic LETM. The spinal magnetic resonance image (MRI) of a high-titer MOG-IgG positive patient presenting with LETM (patient number 10, Table 2) is shown in Figure 2. Both MOG-IgG seropositive CIS patients presented with monophasic ON and were negative for AQP4-IgG (Table 2).

No./Sex/Age (y)	Diagnosis	MOG-IgG titer	AQP4-IgG titer	relapses	ON	myelitis	LETM	Cerebral MRI lesions	MOG -TCC
1/F/2	NMO	1:2,560	negative	2	yes	yes	yes	no	pos
2/F/39	NMO	1:160	1:1,280	4	yes	yes	yes	no	n.a
3/M/56	NMO	1:2,560	negative	2	yes	yes	yes	n.a	pos
4/F/28	HR-NMO (rON)	1:5,120	negative	2	yes	no	no	no	pos
5/M/13	HR-NMO (rON)	1:5,120	negative	3	yes	no	no	no	pos
6/F/48	HR-NMO (rON)	1:2,560	negative	16	yes	no	no	no	pos
7/F/46	HR-NMO (rON)	1:640	negative	3	yes	no	no	no	neg
8/M/32*	HR-NMO (rON)	1:640	negative	11	yes	no	no	no	neg
9/F/43	HR-NMO (LETM)	1:2,560	negative	1	no	yes	yes	no	pos
10/F/49*	HR-NMO (LETM)	1:640	negative	1	no	yes	yes	no	pos
11/F/5	CIS (ON)	1:5,120	negative	1	yes	no	no	no	pos
12/M/13	CIS (ON)	1:640	negative	1	yes	no	no	no	neg

Abbreviations: No. = patient number, F = female, M = male, y = years, MRI = magnetic resonance image, NMO = Neuromyelitis optica, HR-NMO = High-risk NMO, rON = recurrent optic neuritis, ON = optic neuritis, LETM = longitudinally extensive transverse myelitis, CIS = clinically isolated syndrome, MOG-TCC = MOG-IgG dependent terminal complement complex, n.a = not available. * paired serum and CSF samples were available from patient 8 and 10. The corresponding CSF was found to be negative for AQP4-IgG as well as for MOG-IgG (CSF was analyzed undiluted and diluted 1:2 as previously described [48]).

Furthermore, MOG-IgG was detected at threshold levels (1:160) in two of 71 MS patients (secondary progressive MS and pediatric MS). Within the CTRL cohort, MOG-IgG was observed in two of 27 SLE patients (1:320 and 1:160) and one of 24 OND patients (1:640, pediatric patient with genetically confirmed citrullinemia, presenting with encephalopathy and multifocal neurological deficits [24]), whereas all 50 healthy controls were MOG-IgG negative.

Analysis of AQP4-IgG and MOG-IgG mediated complement activation using MOG or AQP4 transfected HEK-293A cells

We additionally analyzed a subset of 15 AQP4-IgG positive samples for the presence of IgG1-IgG4 isotypes, and found that AQP4-IgG antibodies consisted primarily of the IgG1 isotype in 13 patients (87%), while two patients presented with IgG1 and IgG3 antibodies (13%). In contrast to anti-AQP4 autoantibodies, analysis of IgG1-IgG4 isotypes revealed that human serum MOG-IgG antibodies of 15 investigated patients consisted only of the IgG1 isotype.

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.

MOG-IgG antibodies were able to induce the complement cascade in vitro in the same manner as shown for AQP4-IgG antibodies. Using MOG transfected cells with and without EmGFP fusion protein, we could clearly show a co-localization of the TCC with MOG-EmGFP (Figure 6). The membrane attack complex resulted in an internalization of anti-MOG antibodies and complement-mediated lysis of MOG transfected cells (Figure 6, additional file 2). In order to exclude an unspecific activation of complement by MOG itself [31], MOG transfected cells were incubated with active complement in the absence of serum (additional file 2). In contrast to high-titer MOG-IgG positive serum samples of ADEM, NMO, HR-NMO and CIS patients, low-titer and MOG-IgG negative samples did not lead to CDC in the presence of active complement (additional file 2).

AQP4-IgG and MOG-IgG directed complement-mediated cytotoxicity in patients with CNS demyelinating diseases

Overall, AQP4-IgG mediated complement activation was observed exclusively in 27 patients positive for AQP4-IgG with a median titer of 1:1,280 (ranging from 1:160 to 1:20,480; Table 3 and additional file 3), consisting of NMO, HR-NMO and one CIS patient. In contrast, samples of patients with lower AQP4-IgG antibody titers and all investigated serum samples of AQP4-IgG negative CNS demyelinating diseases and controls were not able to activate the complement cascade on the surface of AQP4-expressing HEK-293A cells (Table 3 and additional file 3).

Similarly, assembly of the TCC was observed on the surface of MOG transfected cells after incubation with serum samples having a median MOG-IgG titer level of 1:2,560 (ranging from 1:640 to 1:20,480), as shown in Table 3 and additional file 3. Within the group of subjects investigated for CDC, MOG-IgG dependent TCC formation was found in 2/23 (9%) definite NMO, 5/33 (15%) HR-NMO, 8/19 (42%) ADEM, 1/14 (7%) CIS and 1/14 (7%) CTRL (pediatric patient with genetically confirmed citrullinemia with a MOG-IgG serum titer of 1:640). MOG-IgG mediated complement activation did not correlate with clinical parameters (data not shown), but was associated with a younger age of the investigated patients (Table 3). In contrast, subjects with lower MOG-IgG titers and all MOG-IgG negative patients (NMO, HR-NMO, ADEM, CIS, MS and CTRL) did not activate the complement cascade on the surface of MOG transfected cells.

Patients and serum samples

The following patients were recruited from Austria (n = 295), Germany (n = 19), Slovakia (n = 2) and Serbia (n = 19) (Table 1): (1) a total of 45 NMO patients diagnosed according to the revised diagnostic criteria from Wingerchuk et al., 2006 [3], (2) 53 patients with a high risk of developing NMO (HR-NMO) including 28 monophasic LETM, 13 recurrent LETM and 12 recurrent ON subjects [3, 10], (3) 33 patients fulfilling the diagnostic criteria for ADEM [46], (4) 32 CIS patients comprising 19 myelitis (59%) and 13 ON (41%), (5) 71 patients with MS according to the revised "McDonald Criteria" 2005 [47] including 44 patients with relapsing remitting MS, 8 patients with primary progressive MS and 19 patients with secondary progressive MS, (6) 101 controls including 24 patients with OND (stroke, Parkinson's disease, epileptic seizure, radiculopathy, insomnia, sleep apnoea syndrome, CNS lymphoma, traumatic brain injury, myasthenia gravis, chronic inflammatory demyelinating polyneuropathy, vestibular neuritis, orthostatic syncope, psychogenic neurological symptoms, CNS vasculitis, hereditary neuropathy, analgesic-induced headache, neuroborreliosis, viral encephalitis, chronic tension-type headache, glioblastoma multiforme), 27 patients with SLE and 50 healthy blood donors obtained from the central institute for blood transfusion (Central Institute for Blood Transfusion and Immunological Department, Innsbruck University Hospital).

External serum samples were shipped on dry ice to Innsbruck and all samples were stored at -20°C until analysis. The present study was approved by the ethical committee of Innsbruck Medical University (study no. UN3041 257/4.8, 21.09.2007) and all Austrian patients or parents/legal guardians gave written informed consent to the study protocol. All Serbian and Slovakian patients gave their informed consent for serum sampling and this study was approved by the Institutional Review Board of the Clinic of Neurology, Clinical Center of Serbia, Belgrade. The Slovakian patients signed the translated informed consent form of the Innsbruck Medical University. All German samples were tested anonymously as requested by the institutional review board of the University of Heidelberg.

AQP4-IgG and MOG-IgG immunofluorescence assays

Analysis of M23 AQP4-IgG was performed using a live cell staining IF assay as recently described [9, 33, 48].

HEK-293A cells (ATCC, LGC Standards GmbH, Wesel, Germany) were transiently transfected (Fugene 6 transfection reagent, Roche, Mannheim, Germany) using the Vivid Colours™ pcDNA™ 6.2C-EmGFP-GW/TOPO plasmid (Invitrogen, Carlsbad, CA, USA), expressing M23 AQP4 fused C-terminally to an emerald green fluorescence protein (EmGFP). The AQP4-IgG IF assay was performed by blocking the transfected cells with 4 μg/ml goat IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS/10% FCS (Sigma-Aldrich), subsequently incubating the cells with pre-absorbed serum samples (rabbit liver powder, Sigma-Aldrich) at a 1:20 and 1:40 dilution for one hour at 4°C. Bound antibodies were detected using Cy™3-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) for 30 minutes at room temperature. Dead cells were excluded by DAPI staining (Sigma-Aldrich). The AQP4-IgG status was determined using a fluorescence microscope (Leica DMI 4000B, Wetzlar, Germany). Each serum sample was individually evaluated by three independent, clinically blinded investigators (SM, KS and VG), yielding a concordance rate of 100%.

In order to determine AQP4-IgG titer levels, AQP4-IgG seropositive samples were further diluted until loss of specific antibody staining. AQP4-IgG was purified from a NMO patient's plasma exchange material as recently described [33] and served as positive control for each assay.

Serum MOG-IgG was determined in pre-absorbed samples using HEK-293A cells transiently transfected with human MOG cloned into the mammalian expression vector Vivid Colours™ pcDNA™ 6.2 C-EmGFP/TOPO (Invitrogen), expressing MOG fused C-terminally to EmGFP as previously reported [24]. Serum MOG-IgG was detected on the surface of MOG expressing cells, using Cy™3-conjugated goat anti-human IgG antibody. Titer levels were determined for MOG-IgG positive samples by serial dilution of serum until loss of signal. Based on our previous results, the cut-off value of high-titer MOG-IgG antibodies was defined as ≥ 1:160, with 100% specificity compared to healthy controls [24]. Consequently, in this study patients with titers ≥ 1:160 are defined as being "high-titer positive". In contrast, patients with "low serum antibody reactivity" (1:20-1:80) as well as serum antibody negative samples (with no detectable antibodies, titer = 0) were summarized as "negative" cohort, to simplify the data sets. However, titer levels below 1:160 are mentioned whenever relevant and illustrated in Figure 1. Additionally, antibodies purified from the plasma exchange material of a high-titer MOG-IgG positive ADEM patient were added as a quality control to each assay. Dead cells were excluded by DAPI staining and the presence and titer levels of MOG-IgG were analyzed by three clinically blinded and experienced investigators (SM, KS and VG).

In order to exclude unspecific background staining, we additionally performed serum antibody stainings using untransfected HEK-293A cells for both IF assays, transfected cells expressing the fusion protein (EmGFP) as a control for the AQP4-IgG assay as well as CD2-EmGFP transfected cells (another protein of the immunoglobulin superfamily) for the MOG-IgG assay. Non-specific background binding was clearly distinguishable from a specific antibody staining in our immunofluorescence setting.

Furthermore, MOG-IgG and AQP4-IgG seropositive and seronegative control samples are regularly retested for antibody titer levels to ensure the quality of the testing system. Titer levels remain constant in the serum samples which are stored at -20°C, even two years after first analysis.

Determination of IgG1-IgG4 isotypes

Serum antibodies to MOG and AQP4 were analyzed in a subgroup of 15 patients for IgG1-IgG4 isotypes via our live cell staining IF assay using MOG or AQP4 transfected cells. After blocking with goat IgG, the transfected cells were incubated with the pre-absorbed serum samples (1:20 and 1:40 dilution) for one hour. Subsequently, cells were washed and incubated with mouse monoclonal anti-human IgG1-IgG4 isotype antibodies for 30 minutes (Sigma-Aldrich, 1:100 dilution in PBS/10% FCS), followed by detection using Alexa Fluor^® 546 goat anti-mouse IgG (Invitrogen) for 30 minutes. Dead cells were excluded by DAPI staining and analysis was performed by three independent investigators (SM, KS and VG).

Antibody mediated terminal complement complex (TCC) in cells expressing AQP4 or MOG

Antibody mediated complement activation was investigated in 23 NMO, 33 HR-NMO, 19 ADEM, 14 CIS, 10 MS and 14 CTRL. The selection of patients for the analysis of complement-mediated cytotoxicity was based on the availability of serum samples and the use of samples which are representative for our entire study population. Briefly, serum samples and human complement (Sigma-Aldrich) were heat-inactivated at 56°C for 45 minutes. Inactivated serum samples were diluted 1:10 in serum-free X-VIVO 15 medium (Lonza, Verviers, Belgium) and pre-absorbed with rabbit liver powder. Cells expressing either MOG or AQP4 were washed three times with X-VIVO 15 medium and subsequently incubated with heat-inactivated, pre-absorbed serum samples and 20% active versus 20% heat-inactivated human complement for 90 minutes at 37°C. After washing the cells three times with 100 μl X-VIVO 15 medium, detection of TCC formation was performed by adding the murine-monoclonal anti-human SC5b-9 (Quidel, San Diego, CA, USA; diluted 1:200 in X-VIVO 15) for one hour at 4°C. Following a 30 minutes incubation with the fluorescence labelled Alexa Fluor^® 546 goat anti-mouse IgG antibody (1:300, Invitrogen), cells were washed with PBS/10% FCS and dead cells were visualized by DAPI staining. All samples were assessed for the presence of the surface membrane attack complex by three independent investigators blinded for clinical information as well as the design of the assay concerning usage of active/inactive complement (SM, KS and VG).

To analyze the co-localization of the TCC and serum MOG-IgG or AQP4-IgG, we used HEK-293A cells expressing MOG or AQP4 without EmGFP fusion protein.

To obtain M23 AQP4 without EmGFP fusion protein, the M23 AQP4 isoform was cloned into the pcDNA3.1 Directional TOPO Expression vector (Invitrogen) [9]. In order to conduct experiments with transfected cells expressing MOG without EmGFP fusion protein, we cloned MOG into the pCMV vector (Invitrogen).

Briefly, after incubating the cells with heat-inactivated samples and active versus inactive complement (90 minutes, 37°C), the cells were washed (X-VIVO 15) and stained with the murine-monoclonal anti-human SC5b-9 (Quidel, diluted 1:200 in X-VIVO 15 medium, one hour, room temperature) as described above. Following three washing steps, the Alexa Fluor^® 488 goat anti-mouse IgG antibody and Cy™3-conjugated goat anti-human IgG antibody were diluted in X-VIVO 15 medium and incubated for 30 minutes. Co-staining of AQP4-IgG and MOG-IgG antibodies (red) and TCC (green) was investigated in a blinded fashion (SM, KS and VG), and dead cells were visualized by DAPI staining. Control experiments using active complement in the absence of serum showed no TCC formation on MOG or AQP4 expressing cells (additional file 1 and additional file 2). Additionally, no cross reaction of the antibodies to other species than stated was observed.

Scanning electron microscopy (SEM)

AQP4-IgG mediated complement activation was confirmed via SEM. Briefly, HEK-293A cells were seeded on poly-L-lysine (Sigma-Aldrich) coated glass slides (Menzel, Braunschweig, Germany) and transiently transfected with the AQP4-EmGFP vector. Thereafter, serum samples supplemented with either active or inactive complement were added to the cells. Following incubation at 37°C for 90 minutes, the cells were washed with PBS and fixed in glutaraldehyde (2.5%, v/v in 0.1 m PBS, pH 7.4). After incubation for 30 minutes at room temperature, the fixative was replaced by fresh fixative, and incubated for another two hours at room temperature. Subsequently, complement activation was investigated via SEM according to standard procedures [49]. Samples were viewed for complement activation in a blinded fashion using the field-emission SEM DSM982-Gemini (ZEISS, Oberkochen, Germany).

Statistical analysis

Statistical analysis (means, medians, range, standard deviations) and significance of group differences were done using IBM SPSS software (release 18.0, SPSS Inc., USA) or GraphPad Prism 5 (GraphPad, San Diego, USA). Between-group comparisons were performed with Kruskal-Wallis test, Dunn's multiple comparison post-hoc test, Mann-Whitney U test, Fisher's exact test and Chi-square test as appropriate. Correlation of parameters was analyzed with Spearman's non-parametric correlation. Statistical significance was defined as two-sided p-value less than 0.05 and Bonferroni's correction was applied for multiple comparisons when appropriate.