Plasmid constructs

pcDNA3.1-hygro was purchased from Invitrogen Ltd, CA, USA and pEGFP-N1 was purchased from Clontech Laboratories, CA, USA. cDNAs encoding human AChR subunits α- (P3A isoform), β-, γ-, δ- and ε-, (Beeson et al., 1993) were cloned into pcDNA3.1-hygro. cDNA encoding full length human MuSK (McConville et al., 2004) was also cloned into pcDNA3.1-hygro. Human rapsyn cDNA, AChR α-subunit or MuSK were also cloned into pEGFP-N1 so that the expressed proteins were tagged intracellularly with enhanced green fluorescent protein (EGFP) (Cossins et al., 2006).

Maintenance and transfection of human embryonic kidney cells

The human embryonic kidney (HEK) 293 cell-line was grown in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% FCS (TCS Cellworks Ltd, Buckingham, UK) and 100 units/ml each of penicillin G and streptomycin (Invitrogen) at 37°C in an atmosphere of 5% CO₂ to 50% confluence on 13 mm glass coverslips placed in 6-well cell culture plates. Cells were transiently co-transfected using polyethylenimine (PEI) with adult or foetal AChR and rapsyn-EGFP (with the α-, β-, δ- and ε- or γ-subunits and rapsyn-EGFP in the ratio of 2 : 1 : 1 : 1 : 1). Rapsyn was used so that the AChRs formed dense clusters on the cell surface (Sanes and Lichtman, 2001), and the EGFP allowed these clusters to be visualized. For cells with unclustered AChRs, rapsyn was omitted and the α-subunit was tagged with EGFP. For mock transfections, cells were transfected only with pcDNA 3.1 hygro and EGFP. MuSK DNA tagged with EGFP was used instead of the AChR subunits and rapsyn for the MuSK transfection. Later, cells were also co-transfected with AChR subunits, rapsyn, MuSK and the recently identified MuSK interacting cytoplasmic docking protein 7 (Dok-7) (Beeson et al., 2006; Okada et al., 2006) (hereafter described as ARMD co-transfection). The expression of the EGFP-tagged proteins was monitored using an Axion 200 inverted Zeiss fluorescence microscope.

Immunofluorescence staining of the cells and microscopy

Immunofluorescence staining of transfected HEK 293 cells was performed 48 h after transfection. The coverslips were transferred into 24-well cell culture plates and rinsed gently with DMEM-N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) (HEPES) buffer (DMEM containing 20 mM HEPES). Cells were incubated with anti-AChR monoclonal antibody [mAb; anti-β-subunit, B3; (Jacobson et al., 1999)], or with patient or control sera/plasmas (1 : 20), in 1% bovine serum albumin (BSA) DMEM-HEPES buffer, for 1 h at room temperature (RT). Cells were washed three times with DMEM-HEPES buffer and fixed immediately with 3% formaldehyde in phosphate buffered saline (PBS) for 15 min at RT. After rinsing with PBS, cells were labelled as follows: (i) for the detection of IgG or IgM antibodies, cells were incubated with anti-mouse IgG-, anti-human IgG- or anti-human IgM-Alexa Fluor 568-conjugated secondary antibody (Invitrogen-Molecular Probes, Paisley, UK) at 1 : 750 in DMEM-HEPES buffer +1%BSA, for 45 min at RT; (ii) for the identification of IgG subclasses, cells were incubated with mouse anti-human IgG1 or mouse anti-human IgG4 (Binding site, Birmingham, UK) at 1 : 50 in HEPES buffered DMEM with 1% BSA, for 1 h at RT; after three washes, cells were labelled with goat anti-mouse isotype specific IgG-Alexa Fluor 568-conjugated antibody (Invitrogen-Molecular Probes, Paisley, UK). After both stainings, cells were washed four times in PBS, nuclei counterstained with DAPI (4′,6′-diamidino-2-phenylindole dichloride) and the coverslips mounted on slides in fluorescence mounting medium (DakoCytomation, Cambridge, UK). They were stored at 4°C and protected from light for ~24 h before examination and imaging on a fluorescence microscope with a MacProbe v4.3 digital image system. All the photographs were taken under similar conditions.

FACS analysis

For flow cytometry the cells were cultured in 175 ml flasks and transfected as described above, but without EGFP. Approximately 48 h later, cells were detached from the flasks using Trypsin/EDTA, centrifuged, counted and resuspended in DMEM at 10⁷cells/ml. Hundred microlitre of the cells (10⁶) were added to 100 μl of plasma sample diluted at 1 : 5 in DMEM + HEPES + 1% FCS + 5 mM EDTA + 0.02% sodium azide. The cells were incubated for 1 h at RT rotating gently, and then washed three times in FACS buffer (PBS + HEPES + 1%FCS + 5 mM EDTA + 0.02% sodium azide). To detect the bound antibody, cells were rotated with 100 μl of goat anti-human IgG-Alexa Fluor 488 at 1 : 50 for 45 min at RT, protected from light. After three washes, cells were resuspended in 400 μl of PBS + 5 mM EDTA, transferred into FACS tubes and analysed immediately on a FACScan (Becton Dickinson, NJ, USA). Data were acquired and analysed using CellQuest software. A population of 10⁴ cells was gated to exclude cell debris and to concentrate the analysis on a defined population of cells with similar characteristics. Cells stained only with the secondary antibody were used to test the fluorescence intensity of unstained cells, and cells stained with plasma samples with very high titre on radioimmunoprecipitation assay and high score on the cell staining were used as positive controls. The results are expressed as the percentage of gated cells positively labelled by IgG-Alexa Fluor 488.

Detection of C3b and C5b-9 deposition

HEK cells expressing AChR clusters, MuSK or ARMD were sensitized with heat-inactivated sera (30 min at 56°C) from the above patients at 1 : 20 dilution for 30 min at 37°C and then washed briefly. Freshly frozen serum from a healthy donor was used as a source of complement. This serum was thawed gradually and first incubated with HEK 293 cells on ice for 30–45 min to remove natural antibodies and reduce subsequent non-specific complement activation. The pre-adsorbed serum was applied to the sensitized cells at 1 : 20 dilution for 30 min at 37°C. The cells were then fixed in 3% formaldehyde and all further steps were carried out on ice to prevent the loss of C3 fragments from the cell surface. The cells were incubated with a polyclonal rabbit anti-human C3c antibody that detects C3b (DakoCytomation, 1 : 500, 30 min at 4°C) or polyclonal rabbit anti-human C5b-9 (membrane attack complex, MAC) or a rabbit isotype control (Zymed Laboratories, CA, USA, provided ready for use), followed by Alexa Fluor-568 goat anti-rabbit IgG antibody (Invitrogen; 1 : 750, for 45 min at 4°C). Coverslips were mounted using fluorescence mounting medium (DakoCytomation) containing DAPI (1 : 1000). HEK cells were found to express significant amounts of complement regulator proteins, CD55, CD59 and CD46 during the initial experiments. Therefore, non-complement-activating monoclonal antibodies against CD55 and CD59 were used to block the functional effects of these complement regulators (2–6 h, 37°C) prior to the initial serum sensitization (Bodian et al., 1997; Harris et al., 2000).

Immunofluorescence analysis

For simplicity, AChR-EGFP and AChR/Rapsyn-EGFP are designated ‘AChR’ and ‘AChR clusters’, respectively. Similarly, all the Alexa Fluor 568-labelled antibodies (anti-mouse IgG or anti-human IgG or IgM) will be identified as mouse IgG or human IgG or IgM. As expected, the EGFP and AChR expression levels varied noticeably between cells, so we checked all coverslips in their entirety. They were coded and scored for the frequencies and intensities of surface antibody binding and for the pattern of labelling of AChR (diffuse or in microaggregates) and AChR clusters (macroaggregates). We also recorded any co-localization of the red-labelled Igs with the EGFP-labelled AChR or AChR clusters. We used the following scoring system systematically for every coverslip: (0) = no labelling; (0.5) = very weak labelling of very few transfected cells with no obvious co-localization; (1) = weak labelling of some of the transfected cells, with co-localization; (2) = moderate labelling of some (~20–50%) of the transfected cells, with precise co-localization; (3) = moderate/strong labelling of ~50–80% of the transfected cells, with perfect co-localization; (4) = strong labelling of virtually all transfected cells, with perfect co-localization. The scores for each serum were usually identical, the same values were usually obtained by two independent observers, and none of them varied more than one point. The final score represents the median of these observations for three independent experiments for each of the sera. On the basis of the results of control samples, those scoring consistently 1 or more were classified as positive.

To test whether the antibodies were binding exclusively to the bungarotoxin binding site, we retested 34 MG sera that had given positive binding to clustered adult AChR after pre-incubating the cells with α-bungarotoxin (α-BuTx; 1 μg/ml; 15 min).

To test the specificity of the antibody binding, we retested 7 MG samples (6 SNMG and one AChR-MG) all positive on clustered AChR, after pre-absorbing the serum samples with recombinant proteins representing the α-subunit or δ-subunit of AChR (Jacobson et al., 1999) or the four extracellular domains of MuSK (McConville et al., 2004) (three cycles of pre-absorption of 30 min each, using a total amount of 3 μg of recombinant protein per 1 μl of serum in 250 μl of 1% BSA DMEM-HEPES buffer).

Immunofluorescence for complement activation in thymic tissue

Thymic sections were processed as published elsewhere (Leite et al., 2005). Briefly, 5 µm thymic sections from routine formalin-fixed, paraffin-embedded blocks were de-waxed and rehydrated and then either microwaved in Target Retrieval Solution (DakoCytomation, Glostrup, Denmark) for cytokeratin, CD3, CD1a, CD20 or desmin antibodies, or pre-treated with protease Type XXIV [Sigma, UK; 0.0125% solution (w/v) in PBS] for C3c and desmin antibodies.

Pre-treated sections were blocked with a peroxidase-blocking reagent (DakoCytomation) and incubated with antibodies to human Cytokeratin, CD3, CD1a, CD20 or Desmin. The binding was detected with the peroxidase-based Envision⁺ (DakoCytomation) method, counterstained with haematoxylin and mounted (Leite et al., 2005). For double immunofluorescence labelling, pre-treated sections were incubated with a mixture of the two primary antibody dilutions (anti-C3c and anti-cytokeratin, anti-C3c and anti-desmin or anti-desmin and anti-CD20) and then incubated with isotype-specific secondary antibodies conjugated to Alexa Fluor 488 or 568 (Molecular Probes, Leiden, The Netherlands); slides were mounted, and nuclei counterstained with DAPI in fluorescence mounting medium (DakoCytomation).

All slides were coded and analysed systematically by a single blinded observer (M.I.L.): entire sections labelled for cytokeratin or CD3 were used to measure areas of total thymic tissue and the extra-parenchymal infiltrates (Leite et al., 2005). Myoid cells were counted over two entire sections from each case (one double-stained for desmin/CD20 and the other for desmin/cytokeratin); counts were averaged to calculate the total numbers and frequencies of myoid cells. We also counted separately the myoid cells located either in the normal medulla or adjacent to or within the infiltrates (designated ‘exposed’ to infiltrates) in all the sections double labelled for desmin/cytokeratin, and calculated the percentage of myoid cells in those thymic compartments. In the staining for complement components C3c and C3b, we recorded and quantified the labelled myoid cells according to the thymic compartments in which they were found. More detailed staining methods, positive and negative controls used as well as microscope and imaging analysis are published elsewhere (Leite et al., 2005).

Binding of IgG to AChRs expressed on the HEK cell surface

We tested all sera for binding to AChR expressed on transfected HEK cells comparing with binding of control sera. The AChR in these experiments was tagged with EGFP so that the cells expressing AChR could be identified. Figure 1B shows examples of binding of IgG antibodies from each group. Binding of each serum was scored from 0 to 4 with the observers blind to the antibody status of the samples, except for the AChR-MGhigh sera used as positive controls. Binding of control sera to the AChR expressing cells scored 0–0.5 (mean + 3SD = 0.32), and values of 1 or greater were considered positive, therefore. As summarized in Table 1, all of the AChR-MGhigh samples and 7/17 of the AChR-MGlow sera were positive (both significantly different from control values). Interestingly, both the MuSK-MG and SNMG cohorts also contained a few sera that were weakly positive for binding to AChR, although the results in these two groups were not significantly different from controls (Fig. 1C).

Table 1

Frequency and characteristics of antibodies that bind to AChR or to MuSK expressed on HEK cells

	AChR-MG high (N = 10)	AChR-MG low (N = 17)	MuSK-MG (N = 24)	SNMG (N = 24)	Controls (N = 28)
AChR-expressing HEK
Binding to unclustered AChR
IgG	10	7	2	4	0
IgM	1	2	2	4	0
Binding to clustered AChR
IgG	10	17	8	14	0
IgM	1	2	2	4	0
Binding to clustered AChR in additional SNMG seraa
(number tested)				(14)
IgG				11
IgG subclasses of antibodies to clustered AChR
(number tested)	(5)	(15)	(3)	(13)	(4)
IgG1	5	13	3	11	0
IgG4	0	2	0	0	0
Complement activation on clustered AChR-expressing cells
(number tested)	(5)	(13)		(13)	(5)
C3b deposition	5	8		3	0
MuSK-expressing HEK
Binding to MuSK
IgG	0	3	24	0	0
IgM	0	3	0	1	0
IgG subclasses of antibodies to MuSK
(number tested)			(21)		(2)
IgG1			17		0
IgG4			21		0
IgG4 > IgG1			21		–
Complement activation on MuSK-expressing cells
(number tested)			(21)		(4)
C3b deposition			12		0

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^aAdditional SNMG sera from 14 patients with demographic and clinical characteristics similar to those of the 24 SNMG patients described in Supplementary Table 1.

Binding of IgG and IgM to clustered AChRs expressed on the HEK cell surface

For these experiments, we transfected the cells with AChR subunits and rapsyn-EGFP so that clusters of AChR/rapsyn could be identified by EGFP. Examples are shown in Fig. 2A and the scores in Fig. 2B. Again, control sera scored 0–0.5 (mean + 3SD = 0.95) and sera scoring 1 or more were considered positive. The AChR-MG sera all bound strongly to the clusters, which could easily be seen on the cell surface (Fig. 2A). In addition, many SNMG sera bound appreciably to the clustered AChRs (Fig. 2A and ​andB,B, Table 1). When the scores were compared with those for unclustered AChR (Fig. 2C), the results were significantly higher for binding to clustered AChR for AChR-MGhigh, AChR-MGlow and SNMG sera. Whereas, only 4/24 (17%) SNMG sera scored 1 or greater on unclustered AChR, 14/24 (58%) were positive on clustered AChR (P < 0.001 Fisher's exact test; Table 1).

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Fig. 2

AChR clustering enhances detection of AChR antibodies. (A) Binding of IgG antibodies (red) to adult AChR clustered by rapsyn-EGFP on HEK cell surface (green) with scores shown on the right. (B) Scores for all samples tested. Median values are shown, and number of sera tested in brackets. (C) Comparison between IgG binding scores to unclustered and clustered AChR for all sera tested. Columns show means +SEM. There were differences between binding to clustered and unclustered AChR for AChR-MGhigh, AChR-MGlow and SNMG sera (all P < 0.001).

It was of interest to see if the high density of AChR in the clusters might detect binding of low-affinity IgM antibodies, whose presence in MG sera has been controversial. Very few MG sera had IgM antibodies (Table 1), and overall the scores were not different from those of the healthy controls (Supplementary Fig. 1 online).

To demonstrate the binding objectively, we performed FACS analysis on cells treated with selected sera (Fig. 3A). Again some of the SNMG sera bound weakly to the unclustered AChR but most SNMG sera showed significantly more (P < 0.01) binding to clustered AChR than to unclustered AChR (Fig. 3B). There was a strongly positive correlation between the visual scores for both clustered and unclustered AChR and the corresponding FACS results (Fig. 3C).

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Fig. 3

(A) FACS analysis of binding demonstrating the gating, based on untreated cells, and showing some example profiles. (B) Scatter plots of FACS analyses, comparing IgG binding (percentage of gated) to cells expressing unclustered or clustered AChR. The cut-off was based on the mean +2 SDs of results from seven healthy controls binding to clustered AChR. (C) Correlation between positive cells (percentage of gated) on FACS and binding scores obtained by visual inspection for the 39 sera tested.

Binding of IgG antibodies to adult or foetal AChR, and to the α-Butx binding site on the AChR

All of the experiments reported above used the adult form of the AChR, but it is possible that some sera are only reactive with foetal AChR. None of those samples that were negative on clustered adult AChR were positive for binding to clustered foetal AChR, and 36/39 of those that were positive bound equally to both forms. However, two AChR-MG and one SNMG sera showed strong binding only to adult AChR (e.g. Supplementary Fig. 2 online).

Since there have been concerns that the radioimmunoprecipitation assay fails to detect antibodies that bind to the ACh binding site on the AChR, which is occupied by ¹²⁵I-α-bungarotoxin, we tested whether pre-incubation of the cells in α-Butx would prevent the binding of the SNMG antibodies. The only serum that did not bind in the presence of α-Butx (data not shown) was from a SNMG patient who had no IgG binding to unclustered AChR but scored four for IgG binding to clustered AChR, and recognized adult but not foetal AChR. The serum also scored 2 for IgM binding to either clustered or unclustered AChR. Interestingly, this serum was one whose IgM fraction strongly inhibited adult AChR currents in transfected HEK or CN21 cells (SNMG1 in Spreadbury et al., 2005).

Absorption of AChR antibodies with recombinant AChR subunits

To confirm the specificity of the immunostaining, and to see whether the antibodies in SNMG, like many in AChR-MG, bound the AChR α-subunit, we performed a limited number of absorption studies, using purified recombinant proteins representing the extracellular domains of the AChR α- and δ-subunits and MuSK as a control. Supplementary Table 2 online shows the results. There was limited absorption of the AChR-MGhigh serum with recombinant subunits, probably because there was an excess of AChR antibodies under the conditions that we tested and because typical AChR antibodies bind poorly to recombinant subunits. Binding of SNMG1, that was specific for the ε-containing adult form of the AChR, was not reduced by any of the three proteins, whereas the other SNMG sera, that bound to both adult and foetal AChR, showed reduced binding in the presence of α-subunit, but not in the presence of δ-subunit or MuSK, suggesting that they are mainly directed against the α-subunit and can recognize the recombinant α-subunit polypeptide.

IgG subclass and complement-fixation by SNMG antibodies

The cell-based assay described here offers a highly suitable substrate for determining whether the antibodies are able to activate complement when bound to their antigenic target in a native conformation. We found that the majority of SNMG antibodies were the complement-activating IgG1 rather than IgG4 (Fig. 4A, Table 1), similar to those of AChR-MG patients. Moreover, in the presence of fresh serum from a healthy control donor, 3 out of 13 SNMG samples (tested after heat-inactivation to remove any intrinsic complement activity) were capable of activating complement C3 and 6/13 SNMG sera activated the membrane attack complex C5-9 (MAC), demonstrating their potential to cause complement-mediated damage to the neuromuscular junction (Fig. 4B and C, Table 1), although, not unexpectedly, the scores for C3b or MAC deposition for SNMG sera were lower than those for AChR-MGlow or AChR-MGhigh sera (data not shown). Figure 4C shows the scores for the IgG subclasses, C3b and MAC deposition for nine SNMG sera tested in this manner.

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Fig. 4

Characterization of IgG antibodies to clustered adult AChR in AChR-MGlow and in SNMG samples. (A) Merged images show predominantly IgG1 (orange), and little IgG4 AChR antibody. (B) In the presence of fresh human serum and anti-human C3b or anti-human C5-9 (membrane attack complex, MAC), activated complement deposits were found in both AChR-MG and SNMG samples. Shown are C3b deposits for one AChR-MG serum, and C3b or MAC deposits for three SNMG sera. (C) Scores of IgG1 and IgG4 binding, and detection of C3b and MAC deposits in nine SNMG samples tested. Lines show median values and number of samples tested are in brackets. Sera from healthy individuals did not show any evidence of complement deposition.

Binding of sera to MuSK expressed on cell lines

We performed similar experiments with binding of the sera to MuSK-EGFP-transfected HEK cells. In this case we did not use any clustering protein, but MuSK expression was very high. All MuSK-MG sera were positive. None of the AChR-MGhigh sera showed detectable binding but three AChR-MGlow sera gave low positive results (Fig. 5A, Table 1). Interestingly, although the MuSK-MG antibodies were largely IgG4 (McConville et al., 2004), most sera also included IgG1 antibodies (Table 1, Fig. 5B and D) and complement activation was demonstrated by high scores for C3b deposition (Fig. 5C and D).

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Fig. 5

Detection of MuSK-MG antibodies binding to MuSK-EGFP transfected HEK cells and their characterization. (A) Scores of IgG binding to MuSK of all serum samples. (B) Merged pictures of IgG1 and IgG4 binding (yellow/orange) showing predominantly IgG4 antibodies but some IgG1. (C) C3b deposition (yellow/orange) in the presence of fresh human serum. (D) Scores of IgM, IgG1 and IgG4 antibody binding and scores of C3b deposition. Lines show median values and number of samples tested are in brackets.

Finally, we asked whether co-expressing ARMD would increase binding of SNMG sera. Four out of 10 SNMG samples that had not bound to rapysyn-clustered AChR showed weak binding (Score 1) when the cells were transfected to express the combination of proteins, suggesting that MuSK and Dok-7 might influence the AChR density or conformation. None of these samples showed detectable binding to cells transfected with MuSK or Dok-7 alone (data not shown).

We are grateful to the Medical Research Council, the Muscular Dystrophy Campaign (J.C., D.B., N.W. and A.V.) and The Wellcome Trust (Programme no. 068590, BPM) for support. M.I.L. was supported by Fundação para a Ciência e a Tecnologia, Portugal. S.J. was supported by the Muscular Dystrophy Campaign and the Myasthenia Gravis Association, UK. S.V. was supported by the Patrick Berthoud Charitable Trust, UK. A.V. and the Department of Clinical Neurology in Oxford receive royalties and payments for antibody tests.