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The Journal of Immunology, 2007, 178: 6533-6539.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Peripherin Is a Relevant Neuroendocrine Autoantigen Recognized by Islet-Infiltrating B Lymphocytes1

Maria Carmen Puertas*, Jorge Carrillo*, Xavier Pastor*, Rosa Maria Ampudia*, Raquel Planas*, Aurora Alba*, Roxana Bruno*, Ricardo Pujol-Borrell*, Josep Maria Estanyol{dagger}, Marta Vives-Pi* and Joan Verdaguer2,*,{ddagger}

* Laboratory of Immunobiology for Research and Diagnosis and Center for Transfusion and Tissue Bank; Institut d’Investigacio Germans Trias i Pujol, Badalona, Barcelona, Spain; {dagger} Plataforma de proteomica, Facultat de Medicina, Hospital Clínic, Barcelona, Spain; and {ddagger} Immunology Unit, Department of Ciencies Mediques Basiques, Faculty of Medicine, University of Lleida, Lleida, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Most of our knowledge of the antigenic repertoire of autoreactive B lymphocytes in type 1 diabetes (T1D) comes from studies on the antigenic specificity of both circulating islet-reactive autoantibodies and peripheral B lymphocyte hybridomas generated from human blood or rodent spleen. In a recent study, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis, but with different degrees of susceptibility to T1D, to characterize the antigenic specificity of islet-infiltrating B lymphocytes during progression of the disease. We found that many hybridomas produced mAbs restricted to the peripheral nervous system (PNS), thus indicating an active B lymphocyte response against PNS elements in the pancreatic islet during disease development. The aim of this study was to identify the autoantigen recognized by these anti-PNS mAbs. Our results showed that peripherin is the autoantigen recognized by all anti-PNS mAbs, and, therefore, a relevant neuroendocrine autoantigen targeted by islet-infiltrating B lymphocytes. Moreover, we discovered that the immune dominant epitope of this B lymphocyte immune response is found at the C-terminal end of Per58 and Per61 isoforms. In conclusion, our study strongly suggests that peripherin is a major autoantigen targeted during T1D development and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 diabetes (T1D)3 is a polygenic disease caused by the loss of insulin-producing beta cells, as a consequence of an autoimmune process mediated by the patient’s own immune system (1). Currently, the NOD mouse is one of the best animal models to study this disease, because it spontaneously develops a form of autoimmune diabetes that resembles human T1D (2, 3). Studies using this animal model have shown that beta cell damage is mainly caused by T lymphocytes (4). However, other cells of the immune system also play a relevant role in the development of the disease. In this regard, it is believed that B lymphocytes may act both as APCs for autoreactive T lymphocytes and/or as producers of autoantibodies against several beta cell autoantigens (5, 6).

The autoimmune response of beta cell-reactive T and B lymphocytes in T1D patients and in NOD mice is directed to a wide range of autoantigens (i.e., glutamic acid decarboxylase insulin, islet cell autoantigen 69, S100beta, carboxypeptidase H, heat shock protein 60, IA-2, glial fibrillary acidic protein peripherin, and/or islet-specific glucose-6-phosphatase catalytic subunit-related protein). Most of these autoantigens are found not only in pancreatic beta cells, but also in cells from the nervous system (7). In fact, islet beta cells and neurons share several phenotypical similarities, thus suggesting the same ectodermic origin (8, 9). Moreover, the hypothesis of a sole autoimmune response against islet beta cells in T1D is controversial because several studies have demonstrated that the pancreatic nervous system is also affected by the autoimmune response during the development of T1D (10, 11, 12). In this regard, it has been hypothesized that this response to the pancreatic nervous system may precede the attack on pancreatic beta cells.

During the development of T1D, B lymphocytes migrate to the pancreatic islets but their exact role remains poorly understood. At present, very few studies have analyzed the nature of the B lymphocyte population that infiltrates the islets. Although the results obtained from these studies are often divergent (13, 14, 15, 46), they all suggest that islet-infiltrating B lymphocytes play a crucial role in T1D.

To elucidate the antigenic repertoire of islet-infiltrating B lymphocytes, we generated hybridoma cell lines of infiltrating B lymphocytes from different mouse strains developing insulitis (16). We found that many hybridomas (56%) produced mAbs restricted to the peripheral nervous system (PNS) elements. Ig class analyses indicated that these islet-derived hybridomas came from B lymphocytes that had undergone Ig class switch recombination. Therefore, islet-associated B lymphocytes are involved in active, Th-driven immune responses against local antigenic targets.

The aim of the present study was to identify the autoantigen recognized by these mAbs. Interestingly, we discovered that peripherin is the autoantigen recognized by all mAbs [21/21]. Despite being an intermediate filament protein predominantly found in PNS, it has been demonstrated that peripherin is also produced by beta cells. Three peripherin isoforms have been described: Per58, Per61, and Per56. In our study, we have observed that all mAbs recognize Per58 and Per61 isoforms, whereas only one [1/21] also recognizes Per56 isoform. Because the only difference between Per58 and Per61, compared with Per56, is the last 21 C-terminal amino acids, our results indicate that the epitope recognized by most mAbs is found at the C-terminal end of both Per58 and Per61 isoforms. In conclusion, peripherin may be a crucial autoantigen during the development of T1D.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Breeding pairs of NOD mice were purchased from The Jackson Laboratory and maintained by brother-sister mating at our animal facility under specific pathogen-free conditions. Institutional guidelines for animal welfare, according to European Laws and European Community Directives, were followed.

Cell lines

Previously generated islet-infiltrating B cell-derived hybridomas showing neuronal staining pattern over tissue cryosections (16) were expanded and culture supernatants collected for further studies. The 21 hybridomas come from 10 independent procedures conducted using islet-infiltrating mononuclear cells from pools of pancreatic islets isolated from two to five mice. Two of these hybridoma cell lines derived from islet-infiltrating B lymphocytes of NOD mice, three from 8.3-NOD, seven from (NOD x NOR)F1, and nine from 8.3-(NOD x NOR)F1 mice (Table I). Neuroblastoma cell line N1E-115, insulinoma NIT-1, and control fibroblast SV-T2 were obtained from the American Type Culture Collection. The human adrenal carcinoma cell line, SW13 vim(–), which lacks cytoplasmic intermediate filaments, was provided by Dr. J. Robertson (Centre for Research in Neurodegenerative Diseases, Toronto, Canada) (17).


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  		Table I. Mouse strain source of hybridoma cell linesa

 
Immunofluorescence staining

Cells were cultured on slides overnight at 37°C in 5% CO2 to allow them to attach to the glass. Slides were then rinsed in PBS and later cells were fixed in 4% formaldehyde and permeabilized in 0.1% saponin. After washing with PBS, cells were incubated with hybridoma supernatants overnight at 4°C, washed with PBS, and labeled with Alexa 488-conjugated goat anti-mouse IgG2b (Molecular Probes). The isotype control that was used was irrelevant IgG2b mouse monoclonal.

Cryosections (5 µm) from pancreases of NOD.RAG-2–/– mice were air dried for 30 min, incubated at 4°C overnight with 30 µl of each hybridoma supernatant, washed, and then incubated with the secondary Ab (goat anti-mouse IgM+G+A-FITC, from Southern Biotechnology Associates) at 4°C for 45 min. Double staining was achieved with two more incubation steps with a guinea pig anti-pig insulin (Sigma-Aldrich) and subsequently with a goat anti-guinea pig IgG-TRITC (ICN Biomedicals).

Protein extraction and Western blot analysis

Cultured cells or freshly isolated pancreatic islets from 4-wk-old NOD mice (18) were washed twice in PBS before protein extraction with ProteoExtract Subcellular Proteome Extraction kit (Calbiochem). We obtained four protein fractions from cytosol, membrane/organelle, nucleus and cytoskeleton compartments (i.e., F1, F2, F3, and F4, respectively). Then, we loaded 10 µg of protein/well and electrophoresed in 4–12% SDS gels (Invitrogen Life Technologies), and subsequently electrotransferred to nitrocellulose membranes (Amersham Biosciences). Right protein transference was always assessed by Ponceau S staining (Sigma-Aldrich) before membrane blocking for 2 h using 2.5% fat-free milk powder and 0.1% Tween 20 (Sigma-Aldrich) in PBS. After several washes with PBS, membranes were incubated overnight at 4°C with hybridoma supernatants, diluted 1/10 in PBS containing 1% milk powder, and 0.1% Tween 20. Next, the membranes were washed in PBS and incubated with peroxidase-conjugated goat anti-mouse Ig (BD Pharmingen) diluted to 1/1000 for 1 h. After several washes, the membranes were developed with the chemiluminescent substrate (SuperSignal; Pierce) and exposed to autoradiography film (Hyperfilm ECL; Amersham Biosciences). For positive controls, we used goat anti-peripherin (C-19; Santa Cruz Biotechnology; generated against a peptide mapping at the C-terminal region of peripherin); rabbit anti-peripherin (Covance; raised against the entire protein) detected with peroxidase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) or peroxidase-conjugated donkey anti-rabbit IgG (Pierce).

Two-dimensional (2-D) electrophoresis

For 2-D protein separation, 50 µg of protein extract were acetone precipitated and resuspended in 7 M urea 2 M tio-urea 4% CHAPS 65 mM DTT. Then, samples were loaded into immobilized pH gradient (IPG) strips (Bio-Rad) during rehydration, according to the manufacturer’s instructions. Isoelectric focusing was performed into a Protean IEF Cell (Bio-Rad), according to IPG pH range. Before running the second dimension in 4–12% SDS gels (ZOOM Gel; Invitrogen Life Technologies), IPG strips were equilibrated in SDS-containing buffer. Finally, the obtained 2-D SDS gels were immunoblotted as previously described, or alternatively silver stained (SilverQuest; Invitrogen Life Technologies) for spot identification by mass spectrometry.

In-gel digestion and mass spectrometry

Spots from silver-stained 2D gels were cut and digested overnight with 100–150 ng of trypsin (Promega) at 37°C using a Montage In-Gel Digest_ZP (Millipore) and following the standard manufacturer’s protocol. Peptides were dried and resuspended in water 0.1% trifluoroacetic acid. A half microliter of them were mixed with an equal amount of a-Cyano-4-hydroxycinnamic acid (3 mg/ml) and analyzed with a MALDI-ToF Voyager DE Pro mass spectrometer (Applied Biosystems) operated in delayed extraction reflector mode at 20 kV as accelerating voltage, 90 ns of pulse delay time, 75% of a grid voltage, and a guide wire voltage of 0.005%. Spectra were accumulated for 100 laser shots and analyzed using MSFit software of Protein Prospector version v 3.2.1 using Swissprot as database search.

Transient transfections

The mouse peripherin isoform cDNAs, Per56, Per58, and Per61, cloned into the mammalian expression vector pRcCMV, were obtained from Dr. J. Robertson (17). SW13 vim(–) cells were transiently transfected using Lipofectamine 2000 (Invitrogen Life Technologies). Forty-eight hours after transfection, cells were harvested and protein extraction and Western blot analysis were performed as previously described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
All mAbs targeting PNS elements recognize a 58-kDa protein produced in neuroblastoma and insulinoma cell lines

For this study, we selected 21 islet-derived Ab-secreting hybridomas originated from islet-infiltrating B lymphocytes with specificity for pancreatic nervous system elements (referred to in Ref. 16 as the G neuronal staining pattern (Table I and Fig. 1A). Please notice that in the previous manuscript, 25 Ab-secreting hybridomas with this same staining pattern were analyzed. In the present manuscript, 4 of them are not described. This is due to the fact that one of them was unstable and lost its capacity to produce Abs, whereas the remaining 3 share the same Ig V region sequence with other hybridomas already analyzed in this study). We chose mAb 228E1 as the representative of this group to perform the first studies. Because the nervous system represents a very small portion of the pancreatic tissue, we tested the mouse neuroblastoma N1E-115 cell line as a putative Ag source. Immunofluorescence staining on N1E-115 cells indicated that the Ag recognized by the 228E1 mAb was present in this neuroblastoma cell line. The stain was a reticular pattern characteristic of an intermediate filament network (Fig. 1B). The same staining pattern was observed when testing neuroblastoma cell lines from rats or humans (data not shown).


Figure 1
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  		FIGURE 1. The autoantigen recognized by the 228E1 mAb is present in the N1E-115 neuroblastoma cell line. A, Characteristic immunofluorescence staining pattern of mAbs with specificity for pancreatic nervous system elements on cryosections from the pancreas of the NOD.RAG-2–/– mouse (referred to in Ref. 16 as the G neuronal staining pattern). Islet beta cells (anti-insulin staining in red) surrounded by the PNS (stained with 228E1 MoAb in green). Bar, 100 µm. B, Neuroblastoma N1E-115 cells were cultured on glass slides. After fixation and permeabilization, the cells were incubated with the 228E1 hybridoma supernatant or a mouse IgG2b monoclonal control Ab. Fluorescent immunodetection was performed with an Alexa 488-conjugated anti-mouse IgG2b Ab.

 
Following the identification of N1E-115 cells as Ag source, we determined the molecular characteristics of the autoantigen. We split protein extraction of N1E-115 cells using a protocol based on sequential extraction buffers with different solubilizing properties. This allowed us to divide the cellular proteome into cytosol, membrane/organelle, nuclear, and cytoskeleton fractions (hereinafter F1, F2, F3, and F4, respectively). After SDS-PAGE and Western blot analysis using 228E1 mAb, we detected a 58-kDa protein of the cytoskeleton fraction (Fig. 2A). Next, we conducted the same studies on NIT-1 insulinoma and pancreatic islet extracts to determine whether 228E1 mAb also recognized a protein present on islet beta cells. In both cases, the same 58-kDa protein was detected (Fig. 2B).


Figure 2
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  		FIGURE 2. The Ag targeted by the 228E1 mAb is a 58-kDa cytoskeleton protein found in neuroblastoma and insulinoma cell lines. A, Western blot analysis with the 228E1 mAb on four different protein extraction fractions from the N1E-115 neuroblastoma cell line. F1, F2, F3, and F4 refer to cytosol fraction, membrane/organelle fraction, nucleic fraction, and cytoskeleton fraction, respectively. The 228E1 hybridoma supernatant targets a 58-kDa protein mostly found in the cytoskeleton fraction (F4). B, Comparative Western blot analysis with the 228E1 mAb on cytoskeleton protein fractions (F4) from the N1E-115 neuroblastoma, NIT-1 insulinoma, SV-T2 fibroblast-derived cell lines, and from islets of 4-wk-old NOD mice. The cytoskeleton protein fraction from the SV-T2 fibroblastic cell line was used as negative control. The results indicate a neuroendocrine origin of the autoantigen targeted by the 228E1 mAb.

 
We also performed Western blot analysis using the cytoskeleton protein fraction from N1E-115 cells to determine whether the other mAbs with specificity for PNS recognized the same specificity. As shown in Fig. 3, we observed a band of 58 kDa in all 20 cases, strongly suggesting that all mAbs targeting nervous system elements recognized the same autoantigen. A second band was detected in the immunoblotting in three cases, indicating that mAbs may react with different isoforms of the same protein and/or with other Ags found in the N1E-115 extract.


Figure 3
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  		FIGURE 3. All mAbs that target nervous system elements recognize the same cytoskeleton 58-kDa autoantigen. The neuroblastoma N1E-115 cytoskeleton protein fraction was loaded into SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were then incubated with each of the 21 hybridoma supernatants previously classified as PNS specific according to their staining pattern (referred to in Ref. 16 as the neuronal staining pattern). Complete culture medium (MCC) and the islet-specific 116A1 mAb were used as negative controls. The same 58-kDa band was detected in the immunoblotting of all tested supernatants save in the negative controls.

 
Peripherin is the 58-kDa candidate autoantigen

To further characterize the 58-kDa autoantigen, we performed 2-D gel electrophoresis (using the cytoskeleton protein fraction from N1E-115), followed by Western blot analysis with the 228E1 mAb. The results indicated that the 58-kDa autoantigen had an isoelectric point of 5.6 with a microheterogenicity probably caused by posttranslational modifications (Fig. 4, A and B). Similar results were obtained when 2-D was conducted with the protein extract of the NIT-1 insulinoma cell line (data not shown). To determine the molecular nature of 58-kDa protein, we carefully cut away the spot detected by immunoblotting from a silver-stained 2-D electrophoresis gel and then we subjected the sample to in-gel digestion with trypsin and mass spectrometry analysis. Mass spectrometry analysis assigned 24 different peptides (64% of the peptides analyzed) to the mouse peripherin protein (accession no. P15331, SwissProt), resulting in a molecular weight search score of 1.2 x 1011 (Fig. 4C). Combined, these peptides covered 46% of the whole protein sequence (Table II), thus indicating that peripherin was the protein targeted by the 228E1 mAb.


Figure 4
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  		FIGURE 4. Identification of peripherin as the 58-kDa autoantigen. A, Two-dimensional gel electrophoresis of a cytoskeleton protein fraction from the neuroblastoma N1E-115 cell line. The most intense spot after Coomassie blue staining corresponds to vimentin (Vi); 53.6 kDa and 5.06 isoelectric point, as assessed by mass spectrometry. B, Immunoblotting from the previous 2-D gel incubated with 228E1 hybridoma supernatant. The "spot" corresponds to the protein recognized by the mAb. C, Mass spectrometry of major peptide monoisotopic masses obtained after analyzing the spot digested with trypsin. Peptide masses associated with peripherin appear encircled.

 

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  		Table II. Identification of peripherin peptides by mass spectrometrya

 
Most anti-PNS mAbs recognize a dominant epitope mapping at the C-terminal region of the Per58 and Per61 peripherin isoforms

After discovering that peripherin was the Ag targeted by the 228E1 mAb, we analyzed whether the remaining 20 mAbs with specificity for PNS also recognized peripherin. We analyzed the reactivity of these mAbs on SW13 vim(–) cells, which lack the endogenous intermediate filament network, transfected with mouse peripherin cDNA. Three peripherin protein isoforms (19) produced by alternative mRNA splicing from a unique mouse peripherin gene have been described. Per58 isoform is encoded by all nine exons of the peripherin gene, whereas Per61 includes intron 4 in the central rod. A replacement of the C-terminal 21 aa with a unique 8 aa sequence (Fig. 5A) differentiates Per56 from Per58 and Per61 isoforms. In vivo production levels of Per58 are very high, whereas those of Per61 and Per56 are very low (20). Therefore, to investigate which peripherin isoform was targeted by our mAbs, three mammalian expression plasmids encoding the peripherin isoforms Per58, Per56, or Per61 were transfected into SW13 vim(–) cells, and the reactivity of mAbs was analyzed (Fig. 5, B and C). Interestingly, 20 of the 21 mAbs reacted with Per58 and Per61 peripherin isoforms, but not with Per56. This same pattern was shown by a polyclonal Ab generated against the C-terminal peptide of the Per58 isoform (Fig. 5C). Thus, the results strongly suggest that the B lymphocyte response against peripherin is directed against an immune dominant epitope mapping at the C-terminal end of the Per58 and Per61 isoforms.


Figure 5
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  		FIGURE 5. Most anti-peripherin mAbs target an epitope mapping at the C-terminal region of Per58 and Per61 peripherin isoforms. A, Schematic representation of the three peripherin isoforms, generated by alternative splicing of the same mRNA. Per58 is the major constitutive form; Per61 results from the insertion of 32 aa in the rod part, corresponding to the I4 intron of the protein; Per56 results from the use of a cryptic acceptor site at the beginning of the last exon, leading to a deletion and a change in the reading frame, thus replacing the C-terminal 21 aa with a different 8-aa sequence. B, SW13 vim(–) cells were transiently transfected with cDNA encoding each peripherin isoform. Peripherin production was assessed by Western blot analysis on cytoskeleton protein fractions (fraction F4) of transfected SW13 vim(–) cells using a polyclonal anti-peripherin Ab (anti-peripherin Ab generated against the full-length protein, which recognize all three peripherin isoforms). Production of all three isoforms was detected in transfected SW13 vim(–) cells. C, All 21 anti-peripherin mAbs targeted Per58 and Per61, whereas only one [1/21] also reacted with the Per56 peripherin isoform, thus indicating that most mAbs bind to an epitope that maps at the C-terminal region of the Per58 and Per61 isoforms. We show the Western blot analysis on cytoskeleton protein fractions of transfected SW13 vim(–) cells using the 228E1 mAb, which reacts with Per58 and Per61, but not with Per56 peripherin isoforms. As a positive control, we used a goat anti-peripherin Ab (C-19) raised against a peptide mapping at the C-terminal region of peripherin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In a previous study, we generated hybridoma cell lines from different mouse strains developing insulitis (NOD, 8.3-NOD, (NOD x NOR)F1, and 8.3-(NOD x NOR)F1 mice; Ref. 16) to analyze the antigenic repertoire of islet-infiltrating B lymphocytes to different degrees of susceptibility to T1D. Surprisingly, the characterization of the tissue specificity of the Abs secreted by these cells revealed that a predominant fraction of these hybridomas produced Abs specific for PNS (~16% Ab-secreting hybridomas obtained from islet-infiltrating B lymphocytes of NOD and 8.3-NOD mice, and ~45% in (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice displayed the same the G neuronal staining pattern). This finding indicated a predominant active B lymphocyte response in islets against PNS elements, especially in those mice with more resistance to the disease development. In this study, we demonstrate that all these mAbs selectively target peripherin, thus suggesting that a relevant population of islet-infiltrating B lymphocytes directs their immune response against this neuroendocrine autoantigen.

Peripherin is a cytoskeleton class III intermediate filament with a neuroendocrine tissue expression pattern. In mice, humans, and other mammals, the production of peripherin is limited to the PNS and subsets of CNS neurons (e.g., the spinal motor neurons, neurons of sensory origin, and small interneurons in the cortex and hippocampus) (20). Pathologically, peripherin has been found to be associated with pathological aggregates in the motor neurons of patients with amyotrophic lateral sclerosis (21, 22, 23), and in the murine model of this disease, i.e., the transgenic mice expressing a mutant of the superoxide dismutase-1 (SOD1G37R) (24, 25).

In addition to this neurological specificity, peripherin is also produced in islet beta cells. Peripherin can be found in islet beta cells of mouse embryonic pancreas using histochemistry procedures, but its production in the islets of pancreas of adult mice decreases until becoming undetectable using these same procedures (9). In our previous study (16), hybridoma screening was conducted on cryosections of pancreata from adult NOD.RAG-2–/– mice. Hence, when we screened anti-peripherin mAbs, we detected reactivity in the pancreatic nervous system, but not in islets; thus, in accordance with this neuronal staining pattern, instead of anti-neuroendocrine Abs, anti-peripherin mAbs were classified as anti-neuronal Abs.

In the early 90s, peripherin was described as an autoantigen linked to T1D in NOD mice (26, 27). These studies reported the existence of autoantibodies and T lymphocyte responses against peripherin at the early age of 6 wk and during progressive disease to overt clinical diabetes. The generation of a NOD spleen-derived Ab-secreting hybridoma specific for peripherin indicated an Ag-driven selection (28), and thus an active B lymphocyte response against this autoantigen. However, peripherin was considered a minor T1D autoantigen because several studies indicated that T1D development could be prevented in NOD mice by administering other beta cell autoantigens (such as glutamic acid decarboxylase, insulin, and heat shock protein 60) (27, 29, 30, 31), whereas intrathymic and i.v. injections of peripherin did not have such a protective effect (27).

Shortly after birth, the pancreas of mice undergoes tissue remodeling to reach adult morphology (32, 33, 34, 35, 36, 37). As a consequence, massive physiological destruction of pancreatic beta cells and autonomous nervous system cells occurs. It has been hypothesized that a defect in the clearance of apoptotic nervous and beta cells during this period of time may be crucial in the development of the autoimmune disease in NOD mice (38, 39). Peripherin is a key factor in neuronal development (20), and its production, unlike other neurofilaments, is up-regulated after nerve injury and by proinflammatory cytokines, such as IL-6 (40, 41). Thus, we hypothesize that a defect in the clearance process causes an incipient inflammation that up-regulates the production of peripherin in the pancreatic nervous system and/or beta cells and favors the autoreactive response in this proinflammatory context.

The study by Boitard et al. (26) showed that several anti-I-Ag7 Abs react against peripherin. This indicates the existence of cross-reactivity between the autoantigen and the MHC H-2 I-Ag7 molecules, suggesting that it may contribute to the lack of tolerance to islets beta cells in NOD mice. Consequently, we analyzed whether our anti-peripherin mAbs reacted against I-Ag7. The results indicated that none of our Abs reacted against I-Ag7 molecules (data not shown). In fact, almost all mAbs [20/21] targeted linear epitope mapping at the C-terminal region of the peripherin protein, which has no sequence homology with the NOD class II MHC molecule or with other intermediate filament proteins.

At present, no data has been published on the detection of peripherin autoantibodies in T1D patients. This is surprising, because peripherin was one of the first autoantigens related to diabetes in NOD mice. The lack of published data may be due to the molecular properties of the protein (peripherin, like other intermediate filaments, is extremely insoluble), and to the fact that the first studies indicated that peripherin was not the initial autoantigen in T1D in NOD mice. The findings indicating that most mAbs are directed against the C-terminal sequence of peripherin will allow the use of a soluble peptide for better determining the reactivity of peripherin in T1D patients and in NOD mice in future studies. Therefore, taking into account new data reported in this study, it will be interesting to evaluate the existence of peripherin autoantibodies in both T1D and in patients with diabetic neuropathy.

The presence of an humoral immune response to peripherin in diabetogenesis strongly suggests the existence also of a T lymphocyte-reactive response to this autoantigen, that in the long term could actively participate in the destruction of both PNS and islet beta cells.

Lastly, our results suggest a possible relation between the presence of anti-peripherin B lymphocytes and resistance to T1D. The (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice are considered to be good models of resistance to T1D development, because they suffer severe insulitis but only a few develop T1D (42, 43). Thus, the fact that the percentage of Ab-secreting hybridomas against peripherin was clearly higher in insulitis-prone but diabetes-resistant (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice suggests that anti-peripherin B lymphocytes may be involved in the modulation of the autoimmune response in situ. In a recent publication, Razavi et al. (44) demonstrated that the loss of sensory neurons that innervate pancreatic islets in NOD mice prevents insulitis and diabetes in those mice. Interestingly, peripherin knockout mice display a substantial reduction of unmyelinated sensory fibers, thus indicating that peripherin is required for their adequate development (45). Whether the response to peripherin may contribute to modulating the function of the sensory fibers, that in the long term would help to prevent T1D development in (NOD x NOR)F1 and 8.3-(NOD x NOR)F1 mice, is an attractive hypothesis to explore.

We conclude that peripherin is a neuroendocrine autoantigen targeted by a sizable proportion of islet-infiltrating B lymphocytes and that the immunodominant epitope recognized by these B lymphocytes maps at the C-terminal region of this protein. Thus, our study demonstrates that peripherin is a relevant autoantigen in diabetogenesis and poses a new question on why peripherin-specific B lymphocytes are mainly attracted to the islet during the development of T1D.


   Acknowledgments
 
We thank Dr. Janice Robertson for providing the mouse peripherin isoform cDNAs and SW13 vim(–) cells. We thank Dr. O. Bachs for his technical advice. We also thank Dr. P. Santamaria for reading the manuscript and D. Cullell-Young for editorial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Fondo de Investigaciones Sanitarias of the Spanish National Institute of Health (FIS 03/0775). M.C.P. was supported by a Severo Ochoa fellowship from the Ferrer Foundation (Barcelona, Spain). A.A. and R.P. were supported by a BEFI Predoctoral Fellowship (01/9065 and 05/0418, respectively) from the Instituto Carlos III of the Spanish National Institute of Health. R.M.A. and X.P. were supported by the Juvenile Diabetes Research Foundation 1-2002-724 Research Grant. M.V.-P. is a researcher at the Fondo de Investigaciones Sanitarias of the Spanish National Institute of Health and the Department of Health of Catalan Government. J.V. is an associate professor of the Serra-Hunter Programme from the Catalan Government. Back

2 Address correspondence and reprint requests to Dr. Joan Verdaguer, Unitat d’Immunologia, Departament de Ciencies Mediques Basiques, Facultat de Medicina, Universitat de Lleida, Carrer Montserrat Roig 2, 25008 Lleida, Spain. E-mail address: joan.verdaguer{at}cmb.udl.es Back

3 Abbreviations used in this paper: T1D, type 1 diabetes; PNS, peripheral nervous system; 2-D, two dimensional; IPG, immobilized pH gradient. Back

Received for publication September 25, 2006. Accepted for publication March 7, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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