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Autoantibodies against vinculin in patients with chronic inflammatory demyelinating polyneuropathy
Journal of Neuroimmunology 287 (2015) 9–15
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Autoantibodies against vinculin in patients with chronic inflammatory demyelinating polyneuropathy Minako Beppu a,b,⁎, Setsu Sawai b, Mamoru Satoh b, Masahiro Mori a, Takahiro Kazami b, Sonoko Misawa a, Kazumoto Shibuya a, Masumi Ishibashi b, Kazuyuki Sogawa b, Sayaka Kado c, Yoshio Kodera d, Fumio Nomura b, Satoshi Kuwabara a a
Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan c Chemical Analysis Center, Chiba University, 1–33 Yayoicho, Inage-ku, Chiba 263-8522, Japan d Department of Physics, School of Science, Kitasato University, 1-15-1, Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan b
a r t i c l e
i n f o
Article history: Received 19 March 2015 Received in revised form 19 July 2015 Accepted 24 July 2015 Available online xxxx Keywords: Chronic inflammatory demyelinating polyneuropathy Autoantibody Vinculin Epitope
a b s t r a c t To identify the target molecules of chronic inflammatory demyelinating polyneuropathy (CIDP), we used proteomic-based approach in the extracted proteins from porcine cauda equina. Two of 31 CIDP patients had markedly elevated serum autoantibodies against vinculin, a cell adhesion protein. Both of the patients with anti-vinculin antibodies had similar clinical manifestation, which are compatible with those of “typical” CIDP. Immunocytochemistry showed that vinculin was stained at the myelin sheath of the sciatic nerves by serum samples. Our results suggest that vinculin is a possible immunological target molecule in a subpopulation of typical CIDP patients. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune disease of the peripheral nervous system (PNS) characterized by progressive loss of distal sensorimotor function but otherwise presenting with substantial variability in symptom expression, treatment response, and prognosis (Barohn et al., 1989; Hughes et al., 1992; McCombe et al., 1987). The disease is classified as either “typical CIDP” or “atypical CIDP” according to clinical criteria proposed by the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS); the latter category further subdivided into distal acquired demyelinating symmetric and multifocal acquired sensory and motor (MADSAM) neuropathy and focal, pure motor, and pure sensory subtypes (Van den Bergh et al., 2010). In a previous report, CIDP was shown to exhibit several distinct distribution patterns of demyelinating lesions (Kuwabara et al., 2002). The differences in clinical features
Abbreviations: CIDP, chronic inflammatory demyelinating polyneuropathy; MADSAM, multifocal acquired demyelinating sensory and motor neuropathy; GBS, Guillain–Barré syndrome; DADS, distal acquired demyelinating symmetric; 2-DE, 2-dimensional electrophoresis; BNB, blood–nerve barrier; BBB, blood–brain barrier. ⁎ Corresponding author at: Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] (M. Beppu).
http://dx.doi.org/10.1016/j.jneuroim.2015.07.012 0165-5728/© 2015 Elsevier B.V. All rights reserved.
among CIDP subtypes suggest the different pathophysiology in each subtype (Kuwabara and Misawa.2011). The beneficial effects of plasma exchange (Eftimov et al., 2009) suggest an antibody-mediated demyelination in CIDP. Deposits containing complement-fixing Immunoglobulins G (IgG) and M (IgM) were observed on the peripheral myelin sheaths of CIDP patients (Dalakas and Engel, 1980), and B cells from CIDP patients presented lower expression of Fcγ receptor 2-b (FcγR2b), an inhibitory receptor that prevents B cells from differentiating into IgG-positive plasma cells (Tackenberg et al., 2009). Recent studies regarding autoantibodies in CIDP have focused on glycolipids and proteins expressed on the myelin sheath, node of Ranvier, and paranodal membrane (Devaux et al., 2012; Koski et al., 1985). Some CIDP patients produce serum autoantibodies against ganglioside GM1 (Ilyas et al., 1992), LM1 (Fredman et al., 1991), or LM1-containing ganglioside complexes (Kuwahara et al., 2011). CIDP patients also occasionally express autoantibodies to peripheral nerve myelin proteins (P0, PMP22, and P2) (Khalili-Shirazi et al., 1993; Sanvito et al., 2009; Yan et al., 2001). At the paranodal axoglial junction, 4% of CIDP patients had anti-neurofascin 155 antibody as indicated by ELISA (Ng et al., 2012), and 3 of 46 patients had antibodies to contactin-1 (Querol et al., 2013). Thus, each autoantibody has been detected in only a small minority of CIDP patients, consistent with pathogenic heterogeneity. However, most previous studies have used a
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speculative approach, focusing on one or a few possible candidates. In a previous study, we used a proteomics-based approach to search for molecules reactive to serum antibodies from patients with the closely related Guillain–Barré syndrome (GBS), and moesin was detected as a novel target antigen (Sawai et al., 2014). In this study, we identified a novel autoantibody in patients with a particular subtype of CIDP using proteomic analysis. 2. Materials and methods 2.1. Patients and serum sample preparation Peripheral blood samples were obtained from 31 patients (19 men), who fulfilled the EFNS/PNS diagnostic criteria for CIDP. According to the clinical criteria of the EFNS/PNS, we classified patients into the following two subgroups: typical CIDP and MADSAM neuropathy (Van den Bergh et al., 2010); patients with other clinical subtypes of atypical CIDP were excluded from this study because small number of patients (0 or 1). 22 patients were diagnosed as typical type and nine patients as MADSAM. According to the electrodiagnostic classification for distribution pattern of demyelination (Kuwabara et al., 2002), patients were classified as having “distal”, “intermediate”, or “diffuse” demyelination. All serum samples were obtained in the untreated state. All subjects gave informed consent, and all procedures were approved by the Ethics Committee of the Chiba University School of Medicine. Studies were performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Peripheral blood was also obtained from 26 age-matched normal individuals (17 men), 13 GBS patients (eight men) and 14 multiple sclerosis (MS) patients (three men), nine neuromyelitis optica (NMO) patients (one man), eight myasthenia gravis (MG) (three men) and four encephalitis patients (three men) . According to electrodiagnostic criteria (Ho et al., 1995), 13 GBS patients were classified as having acute inflammatory demyelinating polyneuropathy (AIDP; n = 7) or acute motor axonal neuropathy (AMAN; n = 4), or were unclassified (n = 2). Peripheral blood samples were centrifuged at 3000 g for 10 min to obtain sera. Serum samples were stored in aliquots at −80 °C until further use. 2.2. Protein extraction from swine cauda equine Tissue from porcine cauda equina was frozen in liquid nitrogen and ground into powder. The samples were homogenized in 20 fold volumes of solution containing 7 M urea, 2 M thiourea, and 2% (w/v) CHAPS and then vortexed to ensure a homogeneous suspension. The suspension was sonicated for 30 s twice and then ultracentrifuged at 100,000 g for 60 min at 4 °C. The clear supernatant was refrigerated at − 80 °C until subjected to agarose two-dimensional electrophoresis (2-DE). 2.3. Agarose 2-DE and immunoblotting Agarose gels for first-dimension isoelectric focusing (IEF) were prepared as previously described (Oh-Ishi et al., 2000). Gels were cast in a glass tube 2 mm in diameter and 70 mm in length. Polyacrylamide gels for the second electrophoresis were 10%–20% gradient slab gels measuring 70 mm × 80 mm × 1.0 mm (DRC Co., Ltd., Tokyo, Japan). Agarose 2-DE was performed as previously described (Kawashima et al., 2009). The extracted protein samples from porcine cauda equina were applied to the agarose gels and separated by IEF at 4 °C and 6000 Vh. After in-gel protein fixation for 5 min in 10% trichloroacetic acid (TCA) and 5% sulfosalicylic acid, agarose gels were placed on top of polyacrylamide gels and SDS-PAGE was performed. Separated proteins were electrophoretically transferred from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes (0.2-μm thickness, Millipore, Billerica,
MA, USA). The membranes were blocked overnight in Tris buffered saline plus 0.1% Tween 20 (TBS-T) containing 0.5% low-fat skim milk. The blocked membranes were incubated for 1 h at room temperature (RT) with patient or control serum diluted 1:500 in TBS-T plus 0.5% skim milk, washed three times (5 min/wash) in TBS-T, and then incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat antihuman IgG antibodies (Jackson ImmunoResearch Laboratories, PA, USA) diluted to 1:10,000 in TBS-T plus 0.5% skim milk. Immunolabeled membranes were washed 3 times (5 min/wash) in TBS-T and incubated in Western blotting detection reagent (Pierce ECL Plus Western Blotting Substrate, NCI 32132JP, Thermo scientific, Rockford, IL, USA). Stained bands were detected using a LPR-400EX chemiluminescence imager (Taitec, Tokyo, Japan). The immunoreactive protein spots were compared to the total population of spots stained with Coomassie Brilliant Blue (CBB). 2.4. In-gel digestion and identification of proteins using mass spectrometry (MS) In-gel digestion and MS identification of proteins were performed by previously described methods (Satoh et al., 2005). Protein spots in CBBstained 2-DE gels corresponding to the immunoreactive spots in serumtreated gels were isolated, destained in 50% (v/v) CH3CN containing 50 mM NH4HCO3, and washed with deionized water. The gel pieces were dehydrated in 100% CH3CN for approximately 15 min and dried for 60 min in a TOMY CC-105 microcentrifugal vacuum concentrator (Tomy Seiko, Tokyo, Japan). Dried gel pieces were then rehydrated and immersed for 45 min at 4 °C in 10–30 μL of 25 mM NH4HCO3 containing 50 ng/μL proteomics grade trypsin (Roche Diagnostics, Mannheim, Germany). Gel pieces were then incubated for 24 h at 37 °C in siliconized plastic tubes containing minimal volumes (10–20 μL) of 25 mM NH4HCO3 buffer under slow rotation to allow digested peptide fragments to diffuse out of the gel into the surrounding solution. Peptide fragments remaining in the gel pieces were recovered by incubation for 20 min at RT in a minimal volume of 5% (v/v) formic acid containing 50% (v/v) CH3CN. The two peptide-containing solutions were pooled in tubes for MS. Prior to MS measurements, digested peptides were desalted and selectively enriched with C18-StageTips (Rappsilber et al., 2007). Digested peptides were injected onto a trap column (C18, 0.3 × 5 mm, Dionex, CA, USA) and an analytical column (C18, 0.075 × 120 mm, Nikkyo Technos, Tokyo, Japan) coupled to an Ultimate 3000 HPLC system (DIONEX, CA, USA). The flow rate was 300 nL/min and the solvent composition of the mobile phase was programmed to change in 120-min cycles with varying ratios of solvent A [2% (v/v) CH3 CN and 0.1% (v/v) HCOOH] to solvent B [90% (v/v) CH 3 CN and 0.1% (v/v) HCOOH] as follows: 5%–10% B over 5 min, 10%–13.5% B over 35 min, 13.5%–35% B over 65 min, 35%–90% B over 4 min, 90% B for 0.5 min, 90%–5% B over 0.5 min and 5% B for 10 min. Purified peptides eluted from the HPLC column were introduced into a LTQ Orbitrap XL hybrid ion trap-Fourier transform mass spectrometer (Thermo Scientific, San Jose, CA, USA). The MS and MS/MS peptide spectra were acquired in a datadependent manner according to the manufacturer's operating specifications. The Mascot search engine (version 2.2.6, Matrix Science, London, UK) was used to identify proteins from the MS and MS/MS peptide spectra. Peptide mass data were matched by searching the UniProtKB mammalia database (SwissProt 2014; 66,397 sequences, ftp://ftp.uniprot.org/pub/databases/uniprot/). The database search parameters were as follows: peptide mass tolerance of 2 ppm; fragment tolerance of 0.6 Da; enzyme set as trypsin; one missed cleavage allowed; and methionine oxidation set as variable modifications. The minimum criterion for protein identification was set to a false discovery rate (FDR) of b 1%. The FDR was estimated by searching the data against a randomized decoy database created by the Mascot Perl program supplied by Matrix Science.
M. Beppu et al. / Journal of Neuroimmunology 287 (2015) 9–15
2.5. Western blot validation using human recombinant vinculin protein Full-length human recombinant vinculin (TP304576, OriGene, Rockville, MD, USA) was separated (100 ng/gel lane) on 7.5% polyacrylamide gels and transferred to PVDF membranes (0.45-μm thickness; Millipore, Billerica, MA, USA). The membranes were blocked in TBS-T plus 0.5% low-fat milk for 2 h and incubated with serum samples at R/T diluted to 1:500 in TBS-T plus 0.5% skim milk. Immunolabeled membranes were washed three times (10 min/wash) in TBS-T and incubated with HRP-conjugated goat antihuman IgG antibodies (109-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted to 1:5,000 in TBS-T plus 0.5% skim milk. The immunolabeled membrane was then washed three times (10 min/wash) in TBS-T. The presence of antivinculin IgG antibodies was detected using a LPR-400EX chemiluminescence imager (Taitec, Tokyo, Japan) with ECL Plus enhanced chemiluminescence detection reagent (Thermo scientific). Following 5-min incubation in ECL plus, densitometric measurements of the Western blot band images were performed using LumiVision Imager imaging analysis software (Taitec) with 5-min exposure. The intensity cutoff value for vinculin immunobinding was two standard deviations above the healthy control sample mean. 2.6. Immunohistochemistry and immunocytochemistry The sciatic nerves of male mice were collected and fixed for 30 min with 4% paraformaldehyde in phosphate-buffered saline (PBS). Fixed nerves were cryoprotected with 30% sucrose in PBS, pH 7.4, overnight at 4 °C as described previously (Ishibashi et al., 2004). After flat embedding in rectangular molds with OCT medium, nerves were sectioned longitudinally at 8 μm. Cryosections were permeabilized for 1 h in 0.1 M PB, pH 7.4, containing 0.3% Triton X-100 and 10% goat serum (PBTGS). For double-labeling experiments, sections were incubated overnight at 4 °C with patient serum (1:4000) and mouse monoclonal antibody against vinculin (1:200; ab18058, Abcam, Cambridge, UK) diluted in PBTGS. Immunolabeled sections were thoroughly rinsed in PBS, followed by application of Alexa Fluor 488-labeled goat antihuman IgG or Alexa Fluor 568-labeled goat antimouse IgG (1:1000; A-11013, A-21124, Life Technologies, USA). Immunocytochemical staining was performed as described previously (Tomonaga et al., 2003). Primary cultured human Schwann cells (#1700, Sciencell, Carlsbad, CA, USA) were fixed with 4% paraformaldehyde in PBS for 10 min at RT and permeabilized with 0.5% Triton X-100 in PBS. After blocking in PBS containing 10% FBS, cells were stained with a monoclonal mouse antihuman vinculin antibody (1:500; Abcam) diluted in PBS containing 3% bovine serum albumin (BSA) (positive control) or with patient serum diluted 1:500 in PBS with 3% BSA. The vinculin antibody was visualized with an Alexa Fluor 568-conjugated goat antimouse IgG and human serum IgGs with an Alexa Fluor 488 goat antihuman IgG (1:1000; Life Technologies, USA). The stained samples were viewed under an Axio Imager Z1 microscope or LSM510 confocal microscope (Carl Zeiss, Jena, Germany), and the images were captured using AxioVision software or LSM510 software (Carl Zeiss). 3. Results 3.1. Proteomic analysis revealed elevated serum vinculin autoantibody titers in some CIDP patients Seven immunoreactive spots appeared in 2-DE Western blots using the serum from seven CIDP patients. We chose five spots not appearing in blots probed with sera from control individuals (Fig. 1). The proteins were identified as vinculin, neurofilament medium polypeptide, neurofilament light polypeptide, and aspartate aminotransferase (Table 1). Of the five candidate proteins, only vinculin is a plasma membrane protein according to our database analysis (SwissProt 2014, ftp://ftp.uniprot.org/ pub/databases/uniprot/).
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We then examined the specificity of anti-vinculin autoantibodies to CIDP (Fig. 2). Of 31 CIDP patients tested, serum samples from four demonstrated intense immunolabeling of human recombinant vinculin (as indicated by a vinculin band density on Western blots greater than two standard deviations above the mean of healthy controls). Furthermore, two typical CIDP patients expressed significantly higher serum anti-vinculin antibody titers, both males in their thirties with distal demyelination and good clinical course. Only one of the 13 GBS patients (classified as AIDP), one of the 26 controls, two of the eight multiple sclerosis patients, and one of four encephalitis patients had immunoreactivity to vinculin, but the reactivity was much lower compared with that of the two vinculin-positive CIDP patients (Fig. 2). 3.2. Vinculin is an immune target at the myelin sheath To investigate antibody binding region and localization of vinculin in peripheral nerve, we immunostained mouse sciatic nerve with serum IgG and anti-vinculin monoclonal antibody. Anti-vinculin antibody stained mainly outer membrane including myelin sheath and nodal area (Fig. 3). Serum IgG from CIDP patients with high vinculin titers stained diffusely myelin sheath and axon including nodal area (Fig. 3), whereas sections incubated with control serum showed no significant immunoreactivity. Sera from patients contained various IgG binding to many kinds of antigens, therefore serum stained the sciatic nerve widely compared with anti-vinculin monoclonal antibody. However, staining with patient serum IgG colocalized with that of an anti-vinculin monoclonal antibody on myelin sheath and nodal area (Fig. 3). 3.3. Viculin is an immune target at the Schwann cell Sera IgG of the two CIDP patients reacted to vinculin which was expressed in cytoplasm of Schwann cell primary culture with dot-like pattern (Fig. 4). In contrast, sera from controls did not stain Schwann cells. After preincubation with Serum from patients and vinculin, the reaction disappeared (supple Fig.). 4. Discussion Vinculin is a 117-kDa protein associated with integrin-mediated cell-matrix adhesion and cadherin-mediated cell–cell junctions. The human VCL shared high homology with porcine (99.2%) and mice (90.2%) by blast search. Therefore we have considered that the results were not dependent on the animal species. This high molecular weight protein is difficult to detect by conventional 2-DE (immobilized pH gradient 2-DE method); therefore, we used agarose gel first-dimension IEF (Oh-Ishi et al., 2000) to enhance the detection. Using this approach, combined with MS-based protein identification, immunoblotting, immunohistochemistry, and immunocytochemistry, we found two typical CIDP patients with elevated serum anti-vinculin autoantibody that was not detected in the other CIDP patients, multiple sclerosis or GBS patients, and normal controls. We suggest that anti-vinculin antibodies are involved in the pathophysiology of a certain population of typical CIDP caused by autoimmunity against PNS vinculin. Vinculin is present either as the active or inactive form. The active form of vinculin localizes to focal adhesions at the membrane whereas the inactive form resides in the cytoplasm (Carisey and Ballesterm., 2011). Vinculin is a critical focal adhesion protein in Schwann cells (Weiner et al., 2001) and has also been detected in isolated myelin preparations from rodent brains (Bacon et al., 2007). In our study, vinculin was localized at the myelin sheath and nodal area of sciatic nerve a. These results suggest that vinculin forms focal adhesions between Schwann cells and the axolemma and inner mesaxon and that disruption of these contacts by vinculin autoantibodies may trigger demyelination. Vinculin is also expressed in endothelial cells in the peripheral nerve (Massa et al., 1995) where it is involved cell–cell contacts that form
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Fig. 1. Identification of protein spots immunoreactive to serum from chronic inflammatory demyelinating polyneuropathy (CIDP) patients using two-dimensional electrophoresis and Western blotting. (A) A polyvinylidene difluoride (PVDF) membrane blotted with separated proteins extracted from pig cauda equina were stained with Coomassie Brilliant Blue. (B, C) A PVDF membrane reacted with 1:10,000-diluted serum from a typical CIDP patient, (D) multifocal acquired sensory and motor neuropathy patient, and (E) a normal control subject. Spots 1–5 appeared only in CIDP patients. Spot 6 appeared in the patients and a normal control.
the blood–nerve barrier (BNB). The disruption of the peripheral blood– nerve barrier (BNB) by vinculin autoantibodies may allow immunoglobulins and other inflammatory mediators access to peripheral nerves, resulting in inflammatory polyneuropathy. In our study, the two patients with elevated anti-vinculin autoantibody titers had a similar disease phenotype, namely distal demyelination
and good response to treatment. Electrophysiological studies have demonstrated that demyelination in typical CIDP patients occurs predominantly around the distal nerve terminals (Kuwabara et al., 2014). At distal nerve terminals, the BNB is anatomically deficient (Olsson, 1968; Olsson, 1990); therefore, autoantibodies may bind freely to target antigens. In addition, typical CIDP patients also show good response to
Table 1 Mass spectrometric identification of antigens reacted with serum of patients with CIDP. Spot IDa
Top-ranked candidate
Molecular weight (kDa)
Swiss-Prot accession no.
Mowse score
MS/MSb
Homology to human proteinsc
Number of patients' serum IgG showed immunoreactivity to the spot
1 2 3 4 5
Not detectedd Vinculin Neurofilament light polypeptide Neurofilament medium polypeptide Aspartate aminotransferase
– 124 62 51 46
– P26234 P02547 P08552 P00503
– 582 2423 220 112
– 34 44 32 12
– 99% 95% 93% 93%
3/7 1/7 4/7 3/7 1/7
a b c d
Spot labels as shown in Fig. 1. Number of peptide fragments yielding informative MS/MS data (number of unique peptides). Homology to human proteins by BLAST search. Candidates were not detected as pig proteins.
M. Beppu et al. / Journal of Neuroimmunology 287 (2015) 9–15
Fig. 2. Anti-vinculin autoantibody titers in serum samples from chronic inflammatory demyelinating polyneuropathy (CIDP) patients and healthy controls. (A) Western blot analysis of serum samples from patients with typical CIDP, multifocal acquired sensory and motor neuropathy, Guillain–Barré syndrome, or multiple sclerosis and from normal controls. (B) Densitometry of Western blotting revealed that four individual serum samples from patients with CIDP had anti-vinculin autoantibody titers above the cutoff value (dashed line, +2 standard deviations above healthy control mean). The patients with MS, NMO, MG and encephalitis were included in ‘Other Neurological Diseases (OND)’ group.
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intravenous immunoglobulin therapy, consistent with the autoantibodymediated pathogenesis of typical CIDP. In addition to peripheral nerve terminals, some CIDP patients show involvement of the distal and intermediate nerve trunk (Kuwabara et al., 2014), suggesting that other humoral factors may act to cause more extensive BNB breakdown. In a recent study, the sera obtained from typical CIDP patients but not that from atypical patients was found to decrease the levels of claudin-5, a tight junction protein, in peripheral nerve microvascular endothelial cells, suggesting that humoral factors in the sera of typical CIDP patients may cause more severe BNB damage (Shimizu et al., 2014). Another study demonstrated elevated TNF-α concentrations only in CIDP patients with diffuse demyelination (Kuwabara et al., 2002). Therefore, TNF-α may also contribute to BNB breakdown in CIDP. Only the serum IgG of CIDP patients reacted with Schwann cells, and immunostaining pattern colocalized with that of anti-vinculin. Kwa et al. (2003) found that immunostaining by sera from CIDP patients was concentrated at the distal tips of Schwann cells, a pattern more restricted than that observed in our study. However, the heterogeneity of CIDP may account for this difference. In most studies for examining autoantibodies in CIDP, including the current study, specific autoantibodies have been observed in only a small subfraction of patients. Nonetheless, the specific autoantibody expressed may determine the particular disease phenotype (Querol et al., 2013; Querol et al., 2014). Activated form of vinculin is located on the part of plasma membrane. However, many studies showed vinculin to be a cytosolic protein. It remains unclear whether vinculin is expressed on the cell surface and how antigens come into contact with immune cells. In summary, we identified elevated autoantibodies to vinculin in a small proportion of typical CIDP patients. Further examination of the role of vinculin autoimmunity in the pathogenesis of typical CIDP is required. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2015.07.012.
Fig. 3. Tissue localization of anti-vinculin and serum immunofluorescence staining of mouse sciatic nerve. (A−C) Immunostaining pattern of mouse sciatic nerve incubated with serum IgG from CIDP patients or (E−F) healthy controls. Bar: 20 μm.
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Fig. 4. Subcellular localization of anti-vinculin and serum immunofluorescence staining of human primary Schwann cells. (A−C) Immunostaining pattern of Schwann cells incubated with serum IgG from chronic inflammatory demyelinating polyneuropathy patients (Magnified images shown in left lower corners) or (E−F) healthy controls. Bar: 20 μm.
Disclosure of conflict of interest Dr Beppu, Dr Sawai, Dr Satoh, Ms Ishibashi, Dr Kazami, Dr Misawa, Dr Shibuya, Dr Sogawa, Dr Mori, Dr Kodera, Dr Nomura and Dr Kuwabara report no disclosures.
Acknowledgment This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. (Grant Number 25860699).
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Sawai, S., Satoh, M., Mori, M., Misawa, S., Sogawa, K., Kazami, T., Ishibashi, M., Beppu, M., Shibuya, K., Ishige, T., Sekiguchi, Y., Noda, K., Sato, K., Matsushita, K., Kodera, Y., Nomura, F., Kuwabara, S., 2014. Moesin is a possible target molecule for cytomegalovirus-related Guillain–Barre syndrome. Neurology 83, 113–117. Shimizu, F., Sawai, S., Sano, Y., Beppu, M., Misawa, S., Nishihara, H., Koga, M., Kuwabara, S., Kanda, T., 2014. Severity and patterns of blood–nerve barrier breakdown in patients with chronic inflammatory demyelinating polyradiculoneuropathy: correlations with clinical subtypes. PLoS One 9, e104205. Tackenberg, B., Jelcic, I., Baerenwaldt, A., Oertel, W.H., Sommer, N., Nimmerjahn, F., Lunemann, J.D., 2009. Impaired inhibitory Fcgamma receptor IIB expression on B cells in chronic inflammatory demyelinating polyneuropathy. Proc. Natl. Acad. Sci. U. S. A. 106, 4788–4792. Tomonaga, T., Matsushita, K., Yamaguchi, S., Oohashi, T., Shimada, H., Ochiai, T., Yoda, K., Nomura, F., 2003. Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res. 63, 3511–3516. Van Den Bergh, P.Y., Hadden, R.D., Bouche, P., Cornblath, D.R., Hahn, A., Illa, I., Koski, C.L., Leger, J.M., Nobile-Orazio, E., Pollard, J., Sommer, C., Van Doorn, P.A., Van Schaik, I.N., 2010. European Federation of Neurological Societies/Peripheral Nerve Society guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society — first revision. Eur. J. Neurol. 17, 356–363. Weiner, J.A., Fukushima, N., Contos, J.J., Scherer, S.S., Chun, J., 2001. Regulation of Schwann cell morphology and adhesion by receptor-mediated lysophosphatidic acid signaling. J. Neurosci. 21, 7069–7078. Yan, W.X., Archelos, J.J., Hartung, H.P., Pollard, J.D., 2001. P0 protein is a target antigen in chronic inflammatory demyelinating polyradiculoneuropathy. Ann. Neurol. 50, 286–292.
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Autoantibodies against vinculin in patients with chronic inflammatory demyelinating polyneuropathy Minako Beppu a,b,⁎, Setsu Sawai b, Mamoru Satoh b, Masahiro Mori a, Takahiro Kazami b, Sonoko Misawa a, Kazumoto Shibuya a, Masumi Ishibashi b, Kazuyuki Sogawa b, Sayaka Kado c, Yoshio Kodera d, Fumio Nomura b, Satoshi Kuwabara a a
Department of Neurology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan c Chemical Analysis Center, Chiba University, 1–33 Yayoicho, Inage-ku, Chiba 263-8522, Japan d Department of Physics, School of Science, Kitasato University, 1-15-1, Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan b
a r t i c l e
i n f o
Article history: Received 19 March 2015 Received in revised form 19 July 2015 Accepted 24 July 2015 Available online xxxx Keywords: Chronic inflammatory demyelinating polyneuropathy Autoantibody Vinculin Epitope
a b s t r a c t To identify the target molecules of chronic inflammatory demyelinating polyneuropathy (CIDP), we used proteomic-based approach in the extracted proteins from porcine cauda equina. Two of 31 CIDP patients had markedly elevated serum autoantibodies against vinculin, a cell adhesion protein. Both of the patients with anti-vinculin antibodies had similar clinical manifestation, which are compatible with those of “typical” CIDP. Immunocytochemistry showed that vinculin was stained at the myelin sheath of the sciatic nerves by serum samples. Our results suggest that vinculin is a possible immunological target molecule in a subpopulation of typical CIDP patients. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune disease of the peripheral nervous system (PNS) characterized by progressive loss of distal sensorimotor function but otherwise presenting with substantial variability in symptom expression, treatment response, and prognosis (Barohn et al., 1989; Hughes et al., 1992; McCombe et al., 1987). The disease is classified as either “typical CIDP” or “atypical CIDP” according to clinical criteria proposed by the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS); the latter category further subdivided into distal acquired demyelinating symmetric and multifocal acquired sensory and motor (MADSAM) neuropathy and focal, pure motor, and pure sensory subtypes (Van den Bergh et al., 2010). In a previous report, CIDP was shown to exhibit several distinct distribution patterns of demyelinating lesions (Kuwabara et al., 2002). The differences in clinical features
Abbreviations: CIDP, chronic inflammatory demyelinating polyneuropathy; MADSAM, multifocal acquired demyelinating sensory and motor neuropathy; GBS, Guillain–Barré syndrome; DADS, distal acquired demyelinating symmetric; 2-DE, 2-dimensional electrophoresis; BNB, blood–nerve barrier; BBB, blood–brain barrier. ⁎ Corresponding author at: Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] (M. Beppu).
http://dx.doi.org/10.1016/j.jneuroim.2015.07.012 0165-5728/© 2015 Elsevier B.V. All rights reserved.
among CIDP subtypes suggest the different pathophysiology in each subtype (Kuwabara and Misawa.2011). The beneficial effects of plasma exchange (Eftimov et al., 2009) suggest an antibody-mediated demyelination in CIDP. Deposits containing complement-fixing Immunoglobulins G (IgG) and M (IgM) were observed on the peripheral myelin sheaths of CIDP patients (Dalakas and Engel, 1980), and B cells from CIDP patients presented lower expression of Fcγ receptor 2-b (FcγR2b), an inhibitory receptor that prevents B cells from differentiating into IgG-positive plasma cells (Tackenberg et al., 2009). Recent studies regarding autoantibodies in CIDP have focused on glycolipids and proteins expressed on the myelin sheath, node of Ranvier, and paranodal membrane (Devaux et al., 2012; Koski et al., 1985). Some CIDP patients produce serum autoantibodies against ganglioside GM1 (Ilyas et al., 1992), LM1 (Fredman et al., 1991), or LM1-containing ganglioside complexes (Kuwahara et al., 2011). CIDP patients also occasionally express autoantibodies to peripheral nerve myelin proteins (P0, PMP22, and P2) (Khalili-Shirazi et al., 1993; Sanvito et al., 2009; Yan et al., 2001). At the paranodal axoglial junction, 4% of CIDP patients had anti-neurofascin 155 antibody as indicated by ELISA (Ng et al., 2012), and 3 of 46 patients had antibodies to contactin-1 (Querol et al., 2013). Thus, each autoantibody has been detected in only a small minority of CIDP patients, consistent with pathogenic heterogeneity. However, most previous studies have used a
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speculative approach, focusing on one or a few possible candidates. In a previous study, we used a proteomics-based approach to search for molecules reactive to serum antibodies from patients with the closely related Guillain–Barré syndrome (GBS), and moesin was detected as a novel target antigen (Sawai et al., 2014). In this study, we identified a novel autoantibody in patients with a particular subtype of CIDP using proteomic analysis. 2. Materials and methods 2.1. Patients and serum sample preparation Peripheral blood samples were obtained from 31 patients (19 men), who fulfilled the EFNS/PNS diagnostic criteria for CIDP. According to the clinical criteria of the EFNS/PNS, we classified patients into the following two subgroups: typical CIDP and MADSAM neuropathy (Van den Bergh et al., 2010); patients with other clinical subtypes of atypical CIDP were excluded from this study because small number of patients (0 or 1). 22 patients were diagnosed as typical type and nine patients as MADSAM. According to the electrodiagnostic classification for distribution pattern of demyelination (Kuwabara et al., 2002), patients were classified as having “distal”, “intermediate”, or “diffuse” demyelination. All serum samples were obtained in the untreated state. All subjects gave informed consent, and all procedures were approved by the Ethics Committee of the Chiba University School of Medicine. Studies were performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Peripheral blood was also obtained from 26 age-matched normal individuals (17 men), 13 GBS patients (eight men) and 14 multiple sclerosis (MS) patients (three men), nine neuromyelitis optica (NMO) patients (one man), eight myasthenia gravis (MG) (three men) and four encephalitis patients (three men) . According to electrodiagnostic criteria (Ho et al., 1995), 13 GBS patients were classified as having acute inflammatory demyelinating polyneuropathy (AIDP; n = 7) or acute motor axonal neuropathy (AMAN; n = 4), or were unclassified (n = 2). Peripheral blood samples were centrifuged at 3000 g for 10 min to obtain sera. Serum samples were stored in aliquots at −80 °C until further use. 2.2. Protein extraction from swine cauda equine Tissue from porcine cauda equina was frozen in liquid nitrogen and ground into powder. The samples were homogenized in 20 fold volumes of solution containing 7 M urea, 2 M thiourea, and 2% (w/v) CHAPS and then vortexed to ensure a homogeneous suspension. The suspension was sonicated for 30 s twice and then ultracentrifuged at 100,000 g for 60 min at 4 °C. The clear supernatant was refrigerated at − 80 °C until subjected to agarose two-dimensional electrophoresis (2-DE). 2.3. Agarose 2-DE and immunoblotting Agarose gels for first-dimension isoelectric focusing (IEF) were prepared as previously described (Oh-Ishi et al., 2000). Gels were cast in a glass tube 2 mm in diameter and 70 mm in length. Polyacrylamide gels for the second electrophoresis were 10%–20% gradient slab gels measuring 70 mm × 80 mm × 1.0 mm (DRC Co., Ltd., Tokyo, Japan). Agarose 2-DE was performed as previously described (Kawashima et al., 2009). The extracted protein samples from porcine cauda equina were applied to the agarose gels and separated by IEF at 4 °C and 6000 Vh. After in-gel protein fixation for 5 min in 10% trichloroacetic acid (TCA) and 5% sulfosalicylic acid, agarose gels were placed on top of polyacrylamide gels and SDS-PAGE was performed. Separated proteins were electrophoretically transferred from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes (0.2-μm thickness, Millipore, Billerica,
MA, USA). The membranes were blocked overnight in Tris buffered saline plus 0.1% Tween 20 (TBS-T) containing 0.5% low-fat skim milk. The blocked membranes were incubated for 1 h at room temperature (RT) with patient or control serum diluted 1:500 in TBS-T plus 0.5% skim milk, washed three times (5 min/wash) in TBS-T, and then incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat antihuman IgG antibodies (Jackson ImmunoResearch Laboratories, PA, USA) diluted to 1:10,000 in TBS-T plus 0.5% skim milk. Immunolabeled membranes were washed 3 times (5 min/wash) in TBS-T and incubated in Western blotting detection reagent (Pierce ECL Plus Western Blotting Substrate, NCI 32132JP, Thermo scientific, Rockford, IL, USA). Stained bands were detected using a LPR-400EX chemiluminescence imager (Taitec, Tokyo, Japan). The immunoreactive protein spots were compared to the total population of spots stained with Coomassie Brilliant Blue (CBB). 2.4. In-gel digestion and identification of proteins using mass spectrometry (MS) In-gel digestion and MS identification of proteins were performed by previously described methods (Satoh et al., 2005). Protein spots in CBBstained 2-DE gels corresponding to the immunoreactive spots in serumtreated gels were isolated, destained in 50% (v/v) CH3CN containing 50 mM NH4HCO3, and washed with deionized water. The gel pieces were dehydrated in 100% CH3CN for approximately 15 min and dried for 60 min in a TOMY CC-105 microcentrifugal vacuum concentrator (Tomy Seiko, Tokyo, Japan). Dried gel pieces were then rehydrated and immersed for 45 min at 4 °C in 10–30 μL of 25 mM NH4HCO3 containing 50 ng/μL proteomics grade trypsin (Roche Diagnostics, Mannheim, Germany). Gel pieces were then incubated for 24 h at 37 °C in siliconized plastic tubes containing minimal volumes (10–20 μL) of 25 mM NH4HCO3 buffer under slow rotation to allow digested peptide fragments to diffuse out of the gel into the surrounding solution. Peptide fragments remaining in the gel pieces were recovered by incubation for 20 min at RT in a minimal volume of 5% (v/v) formic acid containing 50% (v/v) CH3CN. The two peptide-containing solutions were pooled in tubes for MS. Prior to MS measurements, digested peptides were desalted and selectively enriched with C18-StageTips (Rappsilber et al., 2007). Digested peptides were injected onto a trap column (C18, 0.3 × 5 mm, Dionex, CA, USA) and an analytical column (C18, 0.075 × 120 mm, Nikkyo Technos, Tokyo, Japan) coupled to an Ultimate 3000 HPLC system (DIONEX, CA, USA). The flow rate was 300 nL/min and the solvent composition of the mobile phase was programmed to change in 120-min cycles with varying ratios of solvent A [2% (v/v) CH3 CN and 0.1% (v/v) HCOOH] to solvent B [90% (v/v) CH 3 CN and 0.1% (v/v) HCOOH] as follows: 5%–10% B over 5 min, 10%–13.5% B over 35 min, 13.5%–35% B over 65 min, 35%–90% B over 4 min, 90% B for 0.5 min, 90%–5% B over 0.5 min and 5% B for 10 min. Purified peptides eluted from the HPLC column were introduced into a LTQ Orbitrap XL hybrid ion trap-Fourier transform mass spectrometer (Thermo Scientific, San Jose, CA, USA). The MS and MS/MS peptide spectra were acquired in a datadependent manner according to the manufacturer's operating specifications. The Mascot search engine (version 2.2.6, Matrix Science, London, UK) was used to identify proteins from the MS and MS/MS peptide spectra. Peptide mass data were matched by searching the UniProtKB mammalia database (SwissProt 2014; 66,397 sequences, ftp://ftp.uniprot.org/pub/databases/uniprot/). The database search parameters were as follows: peptide mass tolerance of 2 ppm; fragment tolerance of 0.6 Da; enzyme set as trypsin; one missed cleavage allowed; and methionine oxidation set as variable modifications. The minimum criterion for protein identification was set to a false discovery rate (FDR) of b 1%. The FDR was estimated by searching the data against a randomized decoy database created by the Mascot Perl program supplied by Matrix Science.
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2.5. Western blot validation using human recombinant vinculin protein Full-length human recombinant vinculin (TP304576, OriGene, Rockville, MD, USA) was separated (100 ng/gel lane) on 7.5% polyacrylamide gels and transferred to PVDF membranes (0.45-μm thickness; Millipore, Billerica, MA, USA). The membranes were blocked in TBS-T plus 0.5% low-fat milk for 2 h and incubated with serum samples at R/T diluted to 1:500 in TBS-T plus 0.5% skim milk. Immunolabeled membranes were washed three times (10 min/wash) in TBS-T and incubated with HRP-conjugated goat antihuman IgG antibodies (109-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted to 1:5,000 in TBS-T plus 0.5% skim milk. The immunolabeled membrane was then washed three times (10 min/wash) in TBS-T. The presence of antivinculin IgG antibodies was detected using a LPR-400EX chemiluminescence imager (Taitec, Tokyo, Japan) with ECL Plus enhanced chemiluminescence detection reagent (Thermo scientific). Following 5-min incubation in ECL plus, densitometric measurements of the Western blot band images were performed using LumiVision Imager imaging analysis software (Taitec) with 5-min exposure. The intensity cutoff value for vinculin immunobinding was two standard deviations above the healthy control sample mean. 2.6. Immunohistochemistry and immunocytochemistry The sciatic nerves of male mice were collected and fixed for 30 min with 4% paraformaldehyde in phosphate-buffered saline (PBS). Fixed nerves were cryoprotected with 30% sucrose in PBS, pH 7.4, overnight at 4 °C as described previously (Ishibashi et al., 2004). After flat embedding in rectangular molds with OCT medium, nerves were sectioned longitudinally at 8 μm. Cryosections were permeabilized for 1 h in 0.1 M PB, pH 7.4, containing 0.3% Triton X-100 and 10% goat serum (PBTGS). For double-labeling experiments, sections were incubated overnight at 4 °C with patient serum (1:4000) and mouse monoclonal antibody against vinculin (1:200; ab18058, Abcam, Cambridge, UK) diluted in PBTGS. Immunolabeled sections were thoroughly rinsed in PBS, followed by application of Alexa Fluor 488-labeled goat antihuman IgG or Alexa Fluor 568-labeled goat antimouse IgG (1:1000; A-11013, A-21124, Life Technologies, USA). Immunocytochemical staining was performed as described previously (Tomonaga et al., 2003). Primary cultured human Schwann cells (#1700, Sciencell, Carlsbad, CA, USA) were fixed with 4% paraformaldehyde in PBS for 10 min at RT and permeabilized with 0.5% Triton X-100 in PBS. After blocking in PBS containing 10% FBS, cells were stained with a monoclonal mouse antihuman vinculin antibody (1:500; Abcam) diluted in PBS containing 3% bovine serum albumin (BSA) (positive control) or with patient serum diluted 1:500 in PBS with 3% BSA. The vinculin antibody was visualized with an Alexa Fluor 568-conjugated goat antimouse IgG and human serum IgGs with an Alexa Fluor 488 goat antihuman IgG (1:1000; Life Technologies, USA). The stained samples were viewed under an Axio Imager Z1 microscope or LSM510 confocal microscope (Carl Zeiss, Jena, Germany), and the images were captured using AxioVision software or LSM510 software (Carl Zeiss). 3. Results 3.1. Proteomic analysis revealed elevated serum vinculin autoantibody titers in some CIDP patients Seven immunoreactive spots appeared in 2-DE Western blots using the serum from seven CIDP patients. We chose five spots not appearing in blots probed with sera from control individuals (Fig. 1). The proteins were identified as vinculin, neurofilament medium polypeptide, neurofilament light polypeptide, and aspartate aminotransferase (Table 1). Of the five candidate proteins, only vinculin is a plasma membrane protein according to our database analysis (SwissProt 2014, ftp://ftp.uniprot.org/ pub/databases/uniprot/).
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We then examined the specificity of anti-vinculin autoantibodies to CIDP (Fig. 2). Of 31 CIDP patients tested, serum samples from four demonstrated intense immunolabeling of human recombinant vinculin (as indicated by a vinculin band density on Western blots greater than two standard deviations above the mean of healthy controls). Furthermore, two typical CIDP patients expressed significantly higher serum anti-vinculin antibody titers, both males in their thirties with distal demyelination and good clinical course. Only one of the 13 GBS patients (classified as AIDP), one of the 26 controls, two of the eight multiple sclerosis patients, and one of four encephalitis patients had immunoreactivity to vinculin, but the reactivity was much lower compared with that of the two vinculin-positive CIDP patients (Fig. 2). 3.2. Vinculin is an immune target at the myelin sheath To investigate antibody binding region and localization of vinculin in peripheral nerve, we immunostained mouse sciatic nerve with serum IgG and anti-vinculin monoclonal antibody. Anti-vinculin antibody stained mainly outer membrane including myelin sheath and nodal area (Fig. 3). Serum IgG from CIDP patients with high vinculin titers stained diffusely myelin sheath and axon including nodal area (Fig. 3), whereas sections incubated with control serum showed no significant immunoreactivity. Sera from patients contained various IgG binding to many kinds of antigens, therefore serum stained the sciatic nerve widely compared with anti-vinculin monoclonal antibody. However, staining with patient serum IgG colocalized with that of an anti-vinculin monoclonal antibody on myelin sheath and nodal area (Fig. 3). 3.3. Viculin is an immune target at the Schwann cell Sera IgG of the two CIDP patients reacted to vinculin which was expressed in cytoplasm of Schwann cell primary culture with dot-like pattern (Fig. 4). In contrast, sera from controls did not stain Schwann cells. After preincubation with Serum from patients and vinculin, the reaction disappeared (supple Fig.). 4. Discussion Vinculin is a 117-kDa protein associated with integrin-mediated cell-matrix adhesion and cadherin-mediated cell–cell junctions. The human VCL shared high homology with porcine (99.2%) and mice (90.2%) by blast search. Therefore we have considered that the results were not dependent on the animal species. This high molecular weight protein is difficult to detect by conventional 2-DE (immobilized pH gradient 2-DE method); therefore, we used agarose gel first-dimension IEF (Oh-Ishi et al., 2000) to enhance the detection. Using this approach, combined with MS-based protein identification, immunoblotting, immunohistochemistry, and immunocytochemistry, we found two typical CIDP patients with elevated serum anti-vinculin autoantibody that was not detected in the other CIDP patients, multiple sclerosis or GBS patients, and normal controls. We suggest that anti-vinculin antibodies are involved in the pathophysiology of a certain population of typical CIDP caused by autoimmunity against PNS vinculin. Vinculin is present either as the active or inactive form. The active form of vinculin localizes to focal adhesions at the membrane whereas the inactive form resides in the cytoplasm (Carisey and Ballesterm., 2011). Vinculin is a critical focal adhesion protein in Schwann cells (Weiner et al., 2001) and has also been detected in isolated myelin preparations from rodent brains (Bacon et al., 2007). In our study, vinculin was localized at the myelin sheath and nodal area of sciatic nerve a. These results suggest that vinculin forms focal adhesions between Schwann cells and the axolemma and inner mesaxon and that disruption of these contacts by vinculin autoantibodies may trigger demyelination. Vinculin is also expressed in endothelial cells in the peripheral nerve (Massa et al., 1995) where it is involved cell–cell contacts that form
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Fig. 1. Identification of protein spots immunoreactive to serum from chronic inflammatory demyelinating polyneuropathy (CIDP) patients using two-dimensional electrophoresis and Western blotting. (A) A polyvinylidene difluoride (PVDF) membrane blotted with separated proteins extracted from pig cauda equina were stained with Coomassie Brilliant Blue. (B, C) A PVDF membrane reacted with 1:10,000-diluted serum from a typical CIDP patient, (D) multifocal acquired sensory and motor neuropathy patient, and (E) a normal control subject. Spots 1–5 appeared only in CIDP patients. Spot 6 appeared in the patients and a normal control.
the blood–nerve barrier (BNB). The disruption of the peripheral blood– nerve barrier (BNB) by vinculin autoantibodies may allow immunoglobulins and other inflammatory mediators access to peripheral nerves, resulting in inflammatory polyneuropathy. In our study, the two patients with elevated anti-vinculin autoantibody titers had a similar disease phenotype, namely distal demyelination
and good response to treatment. Electrophysiological studies have demonstrated that demyelination in typical CIDP patients occurs predominantly around the distal nerve terminals (Kuwabara et al., 2014). At distal nerve terminals, the BNB is anatomically deficient (Olsson, 1968; Olsson, 1990); therefore, autoantibodies may bind freely to target antigens. In addition, typical CIDP patients also show good response to
Table 1 Mass spectrometric identification of antigens reacted with serum of patients with CIDP. Spot IDa
Top-ranked candidate
Molecular weight (kDa)
Swiss-Prot accession no.
Mowse score
MS/MSb
Homology to human proteinsc
Number of patients' serum IgG showed immunoreactivity to the spot
1 2 3 4 5
Not detectedd Vinculin Neurofilament light polypeptide Neurofilament medium polypeptide Aspartate aminotransferase
– 124 62 51 46
– P26234 P02547 P08552 P00503
– 582 2423 220 112
– 34 44 32 12
– 99% 95% 93% 93%
3/7 1/7 4/7 3/7 1/7
a b c d
Spot labels as shown in Fig. 1. Number of peptide fragments yielding informative MS/MS data (number of unique peptides). Homology to human proteins by BLAST search. Candidates were not detected as pig proteins.
M. Beppu et al. / Journal of Neuroimmunology 287 (2015) 9–15
Fig. 2. Anti-vinculin autoantibody titers in serum samples from chronic inflammatory demyelinating polyneuropathy (CIDP) patients and healthy controls. (A) Western blot analysis of serum samples from patients with typical CIDP, multifocal acquired sensory and motor neuropathy, Guillain–Barré syndrome, or multiple sclerosis and from normal controls. (B) Densitometry of Western blotting revealed that four individual serum samples from patients with CIDP had anti-vinculin autoantibody titers above the cutoff value (dashed line, +2 standard deviations above healthy control mean). The patients with MS, NMO, MG and encephalitis were included in ‘Other Neurological Diseases (OND)’ group.
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intravenous immunoglobulin therapy, consistent with the autoantibodymediated pathogenesis of typical CIDP. In addition to peripheral nerve terminals, some CIDP patients show involvement of the distal and intermediate nerve trunk (Kuwabara et al., 2014), suggesting that other humoral factors may act to cause more extensive BNB breakdown. In a recent study, the sera obtained from typical CIDP patients but not that from atypical patients was found to decrease the levels of claudin-5, a tight junction protein, in peripheral nerve microvascular endothelial cells, suggesting that humoral factors in the sera of typical CIDP patients may cause more severe BNB damage (Shimizu et al., 2014). Another study demonstrated elevated TNF-α concentrations only in CIDP patients with diffuse demyelination (Kuwabara et al., 2002). Therefore, TNF-α may also contribute to BNB breakdown in CIDP. Only the serum IgG of CIDP patients reacted with Schwann cells, and immunostaining pattern colocalized with that of anti-vinculin. Kwa et al. (2003) found that immunostaining by sera from CIDP patients was concentrated at the distal tips of Schwann cells, a pattern more restricted than that observed in our study. However, the heterogeneity of CIDP may account for this difference. In most studies for examining autoantibodies in CIDP, including the current study, specific autoantibodies have been observed in only a small subfraction of patients. Nonetheless, the specific autoantibody expressed may determine the particular disease phenotype (Querol et al., 2013; Querol et al., 2014). Activated form of vinculin is located on the part of plasma membrane. However, many studies showed vinculin to be a cytosolic protein. It remains unclear whether vinculin is expressed on the cell surface and how antigens come into contact with immune cells. In summary, we identified elevated autoantibodies to vinculin in a small proportion of typical CIDP patients. Further examination of the role of vinculin autoimmunity in the pathogenesis of typical CIDP is required. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2015.07.012.
Fig. 3. Tissue localization of anti-vinculin and serum immunofluorescence staining of mouse sciatic nerve. (A−C) Immunostaining pattern of mouse sciatic nerve incubated with serum IgG from CIDP patients or (E−F) healthy controls. Bar: 20 μm.
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M. Beppu et al. / Journal of Neuroimmunology 287 (2015) 9–15
Fig. 4. Subcellular localization of anti-vinculin and serum immunofluorescence staining of human primary Schwann cells. (A−C) Immunostaining pattern of Schwann cells incubated with serum IgG from chronic inflammatory demyelinating polyneuropathy patients (Magnified images shown in left lower corners) or (E−F) healthy controls. Bar: 20 μm.
Disclosure of conflict of interest Dr Beppu, Dr Sawai, Dr Satoh, Ms Ishibashi, Dr Kazami, Dr Misawa, Dr Shibuya, Dr Sogawa, Dr Mori, Dr Kodera, Dr Nomura and Dr Kuwabara report no disclosures.
Acknowledgment This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. (Grant Number 25860699).
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