Objective Multifocal motor neuropathy (MMN) and the Guillain-Barré syndrome (GBS) are immune-mediated motor neuropathies with antibodies against the ganglioside GM1. In GBS, these antibodies are induced by molecular mimicry, but in MMN their origin is elusive.
Methods We compared the light-chain use of anti-GM1 IgM antibodies in serum from 42 patients with MMN and 23 patients with GBS by ELISA.
Results Exclusive use of either κ or λ light chains was found in 38 (90%) patients with MMN and 9 (39%) with GBS (p<0.001).
Conclusions Anti-GM1 IgM antibodies in most patients with MMN are produced by only a single or very limited number of B-cell clones, whereas in most patients with GBS, anti-GM1 IgM antibodies are most likely polyclonal.
- GUILLAIN-BARRE SYNDROME
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The presence of antibodies against the ganglioside GM1, which is highly expressed in motor nerves, is a characteristic of multifocal motor neuropathy (MMN) and variants of the Guillain-Barré syndrome (GBS).1 Studies in animal models have shown that anti-GM1 antibodies have various pathogenic effects resulting in focal motor nerve dysfunction at the nodes of Ranvier. In GBS, these antibodies are part of the immune response against a recent infection with Campylobacter jejuni or other microbes. Lipo-oligosaccharide from C. jejuni shows molecular mimicry with GM1 and provides the antigenic trigger for clonal proliferation of cross-reactive B cells.2 The anti-GM1 antibodies usually disappear within weeks to months from onset at the time the patients recover. In MMN, such recent infections preceding the onset of weakness have not been reported. A second difference with GBS is that MMN has a chronic-progressive clinical course with a lifelong production of anti-GM1 antibodies. The mechanism responsible for the production of anti-GM1 antibodies in patients with MMN is unknown. One possibility is that the antibodies are produced by a single clone of B cells, as may occur in patients with polyneuropathy associated with monoclonal gammopathy of undetermined significance (MGUS).3 Antibodies produced by a single B-cell clone all share the same type of immunoglobulin (Ig) light chain, being either κ or λ. This feature of exclusive light-chain usage can be used to discriminate between antibodies produced by a monoclonal or polyclonal population of B cells. We hypothesise that in MMN a monoclonal population of B cells is responsible for the sustained production of anti-GM1 antibodies and the related chronic-progressive disease course. In the current study, the light-chain profile of anti-GM1 antibodies was determined in serum from patients with MMN or GBS, to provide an indication of the monoclonal or polyclonal origin.
Patients and methods
Patients and serum samples
Serum samples from 42 patients with MMN and 23 patients with GBS with elevated titres of anti-GM1 IgM antibodies and negative for anti-GM1 IgA and IgG antibodies were obtained before treatment with intravenous Igs. All patients fulfilled the diagnostic criteria as published previously.4 ,5
Serum samples of two patients with a previously described MGUS polyneuropathy and monoclonal anti-GM1 IgM antibodies with either κ or λ Ig light chains were used as positive controls.3 Serum samples from nine healthy controls without elevated titres of anti-GM1 IgM antibodies were used as negative controls. All serum samples were stored at −80°C until use.
Serum anti-GM1 IgM antibody titres and light-chain use were determined using the validated ELISA of the Inflammatory Neuropathy Cause and Treatment (INCAT) group,6 using the peroxidase-conjugated goat-antihuman IgM, goat-antihuman κ Ig (Sigma, A7164) and goat-antihuman λ Ig (Sigma, A5175). The concentration of anti-GM1 IgM light chains was expressed as the equivalent of standard dilution series of a monoclonal IgG1 κ and a monoclonal IgG1 λ fraction purified from the plasma of myeloma patients with a purity of at least 95% (Sigma I5154 and Sigma I5029). Detection limits were 0.013 μg/mL for κ light chains (IgK) and 0.006 μg/mL for λ light chains (IgL) and there was no cross-reactivity. A serum was considered to contain anti-GM1 IgM antibodies using only a single type of light chain when the concentration of one of the light chains was below and the other above these detection limits. If both IgK and IgL signals were higher than the detection limits, the observed concentrations were used to calculate an IgK/IgL ratio.
Differences in proportions were tested by the χ2 analysis using 2×2 tables. p Values <0.05 were considered statistically significant.
Serum anti-GM1 IgM antibody titres determined in the sera from patients with MMN and GBS are shown in table 1.
The sera obtained from the nine healthy controls were all negative for anti-GM1 IgM, IgK and IgL. The anti-GM1 light chains in serum from the two positive controls with polyneuropathy associated with IgM monoclonal gammopathy were the same as the light chain of the M-protein. The positive control patient P1 with an IgM λ M-protein had a concentration of anti-GM1 IgL in serum of 0.089 µg/mL and no detectable anti-GM1 IgK. The positive control patient P2 with an IgM κ M-protein had a concentration of anti-GM1 IgK in serum of 0.71 µg/mL and no detectable anti-GM1 IgL.
Patients with GBS
Serum anti-GM1 IgM antibodies in 9 (39%) of 23 patients with GBS expressed only a single light chain. The exclusive use of IgK was found in four patients (concentration range 0.216–1.329 µg/mL), and the exclusive use of IgL in five patients (concentration range 0.01–0.047 µg/mL). In addition, three patients had a serum IgK/IgL ratio of >10 (IgK 0.536 μg/mL, IgL 0.01 µg/mL, ratio 53.6; IgK 0.235 μg/mL, IgL 0.01 μg/mL, ratio 23.5; IgK 0.757 μg/mL, IgL 0.041 μg/mL, ratio 18.46) and no patients had an IgK/IgL ratio of <0.1. The remaining 11 patients had anti-GM1 IgM antibodies expressing both light chains with an IgK/IgL ratio between 0.1 and 10.
Patients with MMN
In 38 (90%) of 42 patients with MMN, anti-GM1 antibodies of only one type of light chain were detected. Exclusive anti-GM1 IgK antibodies were found in 20 patients (concentration range 0.089–2.179 µg/mL), and exclusive anti-GM1 IgL antibodies in 18 patients (concentration range 0.01–0.334 µg/mL). In addition, two patients had a serum IgK/IgL concentration ratio of <0.1 (IgK 0.01 µg/mL, IgL 0.341 µg/mL, ratio 0.03 and IgK 0.079 µg/mL, IgL 0.89 µg/mL, ratio 0.09). The remaining two patients with MMN had anti-GM1 antibodies with both IgK and IgL light chains and a IgK/IgL ratio between 0.1 and 10.
The concentrations of the anti-GM1 IgK and IgL in the sera from patients with MMN and GBS are shown in figure 1.
Patients with MMN more frequently had serum anti-GM1 IgM antibodies using a single light chain than patients with GBS (p<0.001).
In this study, we demonstrated that the majority of patients with MMN produce serum anti-GM1 IgM antibodies with only one type of Ig light chain. This finding suggests that in MMN these antibodies are produced by only a single or very few anti-GM1 B-cell clones. In contrast, the majority of patients with GBS produced anti-GM1 IgM antibodies with a mixture of κ and λ light chains, compatible with a polyclonal origin of the antibodies.
In our view, the observed differences cannot be explained by methodological limitations. The ELISA was sensitive enough to detect both κ and λ light chains at very low concentrations. In some patients, only anti-GM1 κ antibodies were demonstrated, in others only anti-GM1 λ, excluding the possibility of preferential detection of one of the light chains in ELISA. This variation in light-chain usage between patients also excludes that GM1 is predominantly recognised by one of the light chains. Antibody titres did not differ between patients with MMN and GBS, which rules out the possibility of higher detection rates of light chains in patients with GBS. A single type of anti-GM1 light chain was demonstrated in 90% of patients with MMN and 39% of patients with GBS. Not excluded is the possibility that in these patients the antibodies are produced not by a single clone, but by a set of subclones, each producing antibodies of the same light chain. Additional studies are required to define the number of subclones. The current study demonstrates that this situation of a highly restricted clonality was more frequent in MMN than in GBS (p<0.001). In addition, two patients with MMN had an anti-GM1 IgK/IgL ratio of >10 or <0.1, further suggesting a difference in the clonality of the anti-GM1 antibody response in GBS and MMN.
Antibody responses against infections are usually transient as occurs in patients with GBS.2 Instead, the anti-GM1 IgM antibodies in patients with MMN are produced lifelong by a single or highly restricted number of B-cell clones. Autoimmune reactions with polyclonal B-cell activation resulting from infections, for example, postviral cold haemoglobinuria,7 have been described previously. In contrast, monoclonal or oligoclonal B-cell activation has been reported in chronic autoimmune diseases such as chronic immune thrombocytopaenic purpura.8 ,9 However, it is not known whether autoantibody clonality differentiates between autoimmune reactions induced by molecular mimicry or more classic autoimmune disease. In support of the restricted number of B-cell clones in MMN are two patients with MMN and a M-protein with the same light chain use as the anti-GM1 antibodies. We recently found that patients with MMN show an increased frequency of IgM monoclonal gammopathy compared with healthy controls (Vlam L, Piepers S, Cats EA et al, unpublished data). In addition, monoclonal anti-GM1 antibodies have previously been demonstrated in patients with chronic neuropathy and monoclonal gammopathy,3 ,10 and patients with B-cell lymphoma may develop a neuropathy similar to MMN.11–13 The concentration of anti-GM1 antibodies in serum is probably too low to be detected by electrophoresis and immunofixation. Previous purification efforts of anti-GM1 antibodies from serum of patients with MMN yielded data compatible with a monoclonal pattern.10 ,14 ,15
The finding that single GM1-specific B-cell clones may proliferate in MMN may, despite the unknown underlying mechanism, provide clues for treatment strategies. Further characterisation of GM1-specific B cells may allow the development of selective depletion or inactivation strategies.
Contributors EAC, W-LvdP, APT-G, FPD, LHvdB, BCJ met the following four criteria: (1) substantial contributions to the conception or design of the work; or the acquisition, analysis or interpretation of data for the work. (2) Drafting the work or revising it critically for important intellectual content. (3) Final approval of the version to be published. (4) Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. EAC and BCJ conducted the statistical analysis.
Competing interests EAC received a travel grant from Baxter. W-LvdP received research support from the Prinses Beatrix Fonds and received a travel grant from Baxter. LHvdB received research support from the Prinses Beatrix Spierfonds and received a travel grant and personal payment for a scientific presentation from Baxter. BCJ received research support from the Netherlands Organization for Health Research and Development, Erasmus MC, Prinses Beatrix Spierfonds and GBS-CIDP Foundation International, as well as travel support from Baxter Biopharmaceutics.
Patient consent Obtained.
Ethics approval The study was approved by the Ethics Committees of the participating hospitals.
Provenance and peer review Not commissioned; externally peer reviewed.
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