Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP: Clinical relevance of IgG isotype

Abstract

Objective: To assess the prevalence and isotypes of anti-nodal/paranodal antibodies to nodal/paranodal proteins in a large chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) cohort, compare clinical features in seronegative vs seropositive patients, and gather evidence of their isotype-specific pathogenic role.

Methods: Antibodies to neurofascin-155 (Nfasc155), neurofascin-140/186 (Nfasc140/186), contactin-1 (CNTN1), and contactin-associated protein 1 (Caspr1) were detected with ELISA and/or cell-based assay. Antibody pathogenicity was tested by immunohistochemistry on skin biopsy, intraneural injection, and cell aggregation assay.

Results: Of 342 patients with CIDP, 19 (5.5%) had antibodies against Nfasc155 (n = 9), Nfasc140/186 and Nfasc155 (n = 1), CNTN1 (n = 3), and Caspr1 (n = 6). Antibodies were absent from healthy and disease controls, including neuropathies of different causes, and were mostly detected in patients with European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) definite CIDP (n = 18). Predominant antibody isotypes were immunoglobulin G (IgG)4 (n = 13), IgG3 (n = 2), IgG1 (n = 2), or undetectable (n = 2). IgG4 antibody-associated phenotypes included onset before 30 years, severe neuropathy, subacute onset, tremor, sensory ataxia, and poor response to intravenous immunoglobulin (IVIG). Immunosuppressive treatments, including rituximab, cyclophosphamide, and methotrexate, proved effective if started early in IVIG-resistant IgG4-seropositive cases. Five patients with an IgG1, IgG3, or undetectable isotype showed clinical features indistinguishable from seronegative patients, including good response to IVIG. IgG4 autoantibodies were associated with morphological changes at paranodes in patients' skin biopsies. We also provided preliminary evidence from a single patient about the pathogenicity of anti-Caspr1 IgG4, showing their ability to penetrate paranodal regions and disrupt the integrity of the Nfasc155/CNTN1/Caspr1 complex.

Conclusions: Our findings confirm previous data on the tight clinico-serological correlation between antibodies to nodal/paranodal proteins and CIDP. Despite the low prevalence, testing for their presence and isotype could ultimately be part of the diagnostic workup in suspected inflammatory demyelinating neuropathy to improve diagnostic accuracy and guide treatment.

Classification of evidence: This study provides Class III evidence that antibodies to nodal/paranodal proteins identify patients with CIDP (sensitivity 6%, specificity 100%).

Figure 1. Reactivity to Nfasc155, CNTN1, and Caspr1 in CIDP by ELISA and CBA

(A) Serum samples from patients with CIDP (n = 342), MMN (n = 13), GBS (n = 31), genetic PN (n = 18), other noninflammatory PN (n = 52), MS (n = 60) and from HCs (n = 60) were tested for autoantibodies to human Nfasc155 (left) and CNTN1 (right) by ELISA. OD are shown after subtraction of the baseline OD reading to bovine serum albumin. The red line represents the mean OD in HCs plus 3 standard deviations. (B) IgG isotype in Nfasc155- and CNTN1-positive patients. (C) The sera (here case 14) were tested on living HEK cells transfected with CNTN1 and Caspr1 (red) and then revealed with mouse antihuman IgG1, IgG2, IgG3, or IgG4 (green) as indicated. Nuclei were stained with DAPI (blue). (D) These are teased fibers from mouse sciatic nerves immunostained for CNTN1 (red) and the serum from case 14, then revealed with mouse antihuman IgG1, IgG2, IgG3, or IgG4 (green) as indicated. IgG1 and IgG4 from this patient reacted against Caspr1 and bound to the paranodal regions. Scale bars: 10 μm. Caspr1 = contactin-associated protein 1; CBA = cell-based assay; CIDP = chronic inflammatory demyelinating polyradiculoneuropathy; CNTN1 = contactin-1; GBS = Guillain-Barré syndrome; HC = healthy control; HEK = human embryonic kidney; MMN = multifocal motor neuropathy; Nfasc155 = neurofascin-155; OD = Optical density; PN = peripheral neuropathy.

Figure 2. Morphological alterations of the nodes of Ranvier in patients with CIDP with IgG4 autoantibodies

We evaluated skin biopsies from 3 patients with IgG4 anti-Nfasc155 antibodies, 1 patient with IgG3/IgG4 anti-CNTN1 antibodies, 1 patient with IgG4 anti-Caspr1 antibodies, 1 patient with undetectable isotype IgG anti-Nfasc155, and 6 seronegative patients with CIDP. Analysis of myelinated fibers showed elongation of the nodes of Ranvier and loss of paranodal Nfasc155 staining in skin biopsies from patients with anti-Nfasc155 (C) and Caspr1 (E) IgG4. Moderate elongation of the nodes of Ranvier and loss of Nfasc155 paranodal staining were also observed in myelinated fibers of a CNTN1 IgG3/IgG4-positive patient (D). Contrarily, we did not observe similar changes in the patient with undetectable isotype IgG anti-Nfasc155 antibodies (F), in seronegative patients with CIDP (B), or in HCs (A). A complete loss of Caspr1 staining was observed in biopsies from patients with IgG4 antibodies to paranodal proteins (I, L, M), but not in an Nfasc155 seropositive patient with an undetectable isotype (N) and in seronegative CIDP (H) or healthy patients (G). Caspr1 = contactin-associated protein 1; CIDP = chronic inflammatory demyelinating polyradiculoneuropathy; CNTN1 = contactin-1; HC = healthy control; Nfasc155 = neurofascin-155.

Figure 3. IgG4 to Caspr1 disrupt the interaction between Nfasc155 and CNTN1/Caspr1

(A–D) HEK cells transfected with CNTN1 and Caspr1 (green) or Nfasc155 (red) were incubated together for 2 hours in the presence of control IgG4 (B), anti-Caspr1 IgG1 (C), or anti-Caspr1 IgG4 (D). As negative controls, HEK cells transfected with Nfasc155 (red) were incubated with cells transfected with GFP (A). Anti-Caspr1 IgG4, but not anti-Caspr1 IgG1, abrogated the aggregation of Nfasc155-transfected cells with CNTN1/Caspr1. Scale bar: 10 μm. (E–F) The graphs represent the relative frequency of green cells per aggregates (n = 4 experiments for each condition). The percentage of cell clusters with contacts between green and red cells was quantified (F). The percentage of contacts was significantly decreased in the presence of anti-Caspr1 IgG4 (p < 0.005 by unpaired 2-tailed Student t tests and by one-way ANOVA, followed by Bonferroni post hoc tests). Bars represent mean and SEM. ANOVA = analysis of variance; Caspr1 = contactin-associated protein 1; CNTN1 = contactin-1; GFP = green fluorescent protein; HEK = human embryonic kidney; Nfasc155 = neurofascin-155.

Figure 4. Anti-Caspr1 IgG4, but not IgG1, invades the paranodal regions

(A–C) Sciatic nerve fibers were incubated in vitro with purified control IgG4 (A), anti-Caspr1 IgG1 (B), or anti-Caspr1 IgG4 (C) for 3 hours and immunolabeled for IgG (green) and CNTN1 (red). (D–F) Sciatic nerves were fixed 1 or 3 days after intraneural injections of purified control IgG4 (D), anti-Caspr1 IgG4 (E–F), immunolabeled for CNTN1 (red), and human IgG (green). Note that only anti-Caspr1 IgG4 penetrated the paranodes. One or 3 days after injection, IgG4 antibodies were detected at the paranode borders (arrows). Images are representative of 3 independent experiments. Scale bar: 10 μm. Caspr1 = contactin-associated protein 1; CNTN1 = contactin-1.