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Clinical Immunology (2008) 128, 269–276

Demonstration of autoantibody binding to muscarinicacetylcholine receptors in the salivary gland in primarySjögren's syndromeLászló Kovács a,⁎, Erzsébet Fehér b, Ibolya Bodnár c, Ilona Marczinovits d,György M. Nagy c, János Somos e, Viktória Boda e

a 1st Department of Internal Medicine, Saint George Hospital of Fejér County, Székesfehérvár, Hungaryb Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungaryc Neuroendocrine Research Laboratory, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungaryd Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Szeged, Hungarye Department of Oral Surgery, Saint George Hospital of Fejér County, Székesfehérvár, Hungary

Received 23 November 2007; accepted with revision 5 April 2008Available online 27 May 2008

⁎ Corresponding author. 1st DepartFaculty of Medicine, Albert Szent-Györof Szeged., Korányi fasor 8-10, Szeged,545 185.

E-mail address: kovl@in1st.szote.u

1521-6616/$ – see front matter © 200doi:10.1016/j.clim.2008.04.001

Abstract A significant pathogenetic role of antimuscarinic acetylcholine receptor-3 (anti-m3AChR) autoantibodies in primary Sjögren's syndrome (pSS) has been suggested. However, thebinding of these antibodies to the receptors in the target tissues has not yet been demonstrated.In this study, the binding characteristics of pSS sera and anti-m3AChR-monospecific sera affinity-purified from pSS patients to labial salivary gland samples from healthy subjects were studiedwith light- and electron microscopy. Furthermore, the ultrastructural localisation of in vivodeposited antibodies in pSS salivary glands was also investigated. Light microscopicimmunohistochemistry revealed the binding of the anti-m3AChR-specific sera to the membraneof acinar cells. Similar reaction end-products were observed in the pSS salivary gland epithelialcell membranes. With electron microscopy, the autoantibody binding was observed to belocalised to the junctions of epithelial cell membranes with nerve endings, both in normal andpSS glands. The results indicate that anti-m3AChR antibodies bind to the receptors in the salivaryglands.© 2008 Elsevier Inc. All rights reserved.

KEYWORDSPrimary Sjögren'ssyndrome;Muscarinic acetylcholinereceptor;Antimuscarinic receptorautoantibody;Immunohistochemistry;Immuno-electronmicroscopy;Ultrastructurallocalisation

ment of Internal Medicine,gyi Clinical Centre, UniversityH-6720 Hungary. Fax: +36 62

-szeged.hu (L. Kovács).

8 Elsevier Inc. All rights reserved

Introduction

Primary Sjögren's syndrome (pSS) is a systemic autoimmunedisease characterised primarily by dry eyes and mouth as aconsequence of inflammation and subsequent destructionof the lacrimal and salivary glands. Further exocrine glandsand several non-glandular organs are frequently affected inthis disease. The pathogenesis of pSS involves a predominantly

.

270 L. Kovács et al.

T-lymphocytic infiltration of the involved exocrine glands, anda marked B-lymphocyte hyperactivity resulting in polyclonalhypergammaglobulinaemia, the production of a variety ofautoantibodies, and eventually the development of B-celllymphoma with a much higher prevalence in pSS than in thegeneral population [1].

In addition to a complex network of pathological immu-nological processes, dysfunction of the autonomic nervoussystem has also been proposed as a contributor to thedevelopment of various signs and symptoms of the syn-drome. In particular, autoantibodies reactive with themuscarinic acetylcholine receptor subtype-3 (m3AChR), amembrane protein that transmits the stimulatory parasym-pathetic neural stimuli to the secretory acinar epithelialcells of the salivary and the lacrimal glands, have become ofparticular interest in recent years [2]. It has been demon-strated that immunoglobulin G (IgG) from pSS patients isable to elicit a secretory dysfunction via a specific bindingto the m3AChR when transferred to rodents [3], and to exertan inhibitory effect on the parasympathetic neurotransmis-sion in isolated rabbit urinary bladder strips [4]. Recently,we developed an ELISA system for the detection of anti-muscarinic receptor antibodies with the use of a recombi-nant fusion protein containing an immunodominant epitopepeptide of the receptor (m3AChR213–228) and glutathione-S-transferase (GST) as an antigen [5]. With this method, wehave demonstrated a prevalence of 90% of anti-m3AChRantibodies among pSS patients, an occurrence significantlyhigher than that in the control groups of patients withvarious systemic autoimmune diseases or with sicca syn-drome from causes other than pSS [6].

Despite these results, the precise functional role of anti-m3AChR autoantibodies in pSS has not yet been determined.Although circulating antibodies reactive with an m3AChR-specific epitope are detectable in the patients, and seem toexert a physiological action in certain experimentalmodels, itis not yet knownwhether these antibodies actually bind to thenative m3AChR in the target organs in human pSS. In order toprovide data for an assessment of the pathogenetic role of theanti-m3AChR antibodies in pSS,we have investigatedwhetherthese autoantibodies are detectable in salivary gland tissuespecimens and characterised their exact binding localisation.

Patients and methods

Serum and labial salivary gland samples

Sera from 4 pSS patients (3 females) were used in the ex-periments. All the patients fulfilled the American–EuropeanConsensus Criteria for the Classification of pSS [7]. The meanage of the patients was 54 years (range: 47–62). Labial sali-vary gland biopsy was performed in 3 of these patients(2 females), and that part of the sample not used for theroutine diagnostic histologic evaluation was used in thisstudy. Minor salivary gland samples from normal labial tissuewere also obtained from two otherwise healthy personsduring the surgical removal of a labial mucocele (healthycontrols). Negative control sera were obtained from healthyblood donors. Informed consent was obtained from all partic-ipants, and the study was approved by the local institutionalethics committee.

Affinity purification of m3AChR-specific antibodies

In order to obtain affinity-purified sera specific to anti-m3AChR213–228, we prepared a chromatographic column inwhich the m3AChR213–228–GST fusion protein was covalentlyattached to glutathione immobilised on a matrix (cross-linked 4% beaded agarose (GST Orientation Kit; Pearce). Thepurification process followed the manufacturer's instruc-tions. Briefly, the fusion protein dissolved in 0.1 M phosphatebuffered saline pH 7.4 (PBS) was incubated with the columnfor 45 min. The bound fusion protein was cross-linked to theimmobilised glutathione with disuccinimidyl suberate solu-tion. The non-specific amine-containing binding sites wereblocked with 0.1 M ethanolamine buffer pH 8.2, and thefusion proteins left unbound to the column were removed bywashing the column with glutathione dissolved in a neutra-lisation buffer. Subsequently, pooled sera of the 4 pSSpatients were loaded onto the column and incubated for1 h at room temperature. The column was washed with PBS,and the bound m3AChR213–228 -specific antibodies were theneluted with glycine buffer pH 2.8. The eluate was extensivelydialysed against PBS. The protein contents of the variousfractions (bound and unbound) were measured by theBradford method [8]. The bound fraction used for theexperiments proved to have a protein content of 70 μg/ml.The efficacy of the purification process was checked withELISA [5] using the m3AChR213–228–GST fusion protein as anantigen. These measurements demonstrated the presence ofm3AChR213–228-specific antibodies in the purified serum.

Light microscopic immunohistochemistry

Labial biopsy samples were fixed in Zamboni's fixative (4%paraformaldehyde, 0.1% glutaraldehyde and 0.19% picricacid in PBS). Twenty μm thick serial sections were cut bymeans of a cryostat.

1. Indirect immunohistochemistry: The sections from the 2healthy controls were then incubated with the sera frompSS patients (diluted 1:200 in PBS). After incubation for48 h, the sections were incubated for 1 h in peroxidase-labelled anti-human IgG (SigmaAldrich) diluted 1:666 inPBS. As the final step, the sections were exposed to asolution containing 3,3-diaminobenzidine (DAB) and0.003% H2O2 for 15 min at room temperature. Betweeneach transfer of solutions, the sections were always rinsedseveral times with PBS. The stained sections weremounted on gel-dipped slides.

2. Direct immunohistochemistry: For the sections from the 3pSS patients, a similar procedure was applied, with theexception of the addition of the primary antisera, whichstep was omitted from these experiments. Thus, thelabial salivary gland samples were reacted directly withthe anti-human IgG solution.

Electron microscopic immunocytochemistry

These procedures were performed similarly as in ourprevious work [9] with some modifications. The labial biopsysamples were fixed in Zamboni's fixative. After washing, thetissue specimens were placed overnight in glutaraldehyde-

271Demonstration of autoantibody binding to mAChR in the salivary gland in primary Sjögren's syndrome

free fixative containing 10% sucrose at 4 °C. Fortymicrometer thick sections were cut by Vibroslices in PBScontaining 20% sucrose, plunged rapidly into liquidnitrogen and rinsed in PBS in several changes. The seraof pSS patients (diluted 1:500 in PBS) were incubated withthe labial biopsy samples from the healthy subjects for 24or 48 h. An avidin–biotin immunoperoxidase kit (Vectas-tain ABC, Vector Laboratories, Peterborough, UK) wasused to visualise the immunoreactions. This kit containsDAB as a chromogen. Washing steps of 3×5 min wereperformed between each step. Subsequently, the speci-mens were postfixed in 1% osmium tetroxide for 60 min,dehydrated and embedded in Epon. Ultrathin sectionswere cut, stained with uranyl acetate and lead citrate andexamined under a Jeol 100 electron microscope. Thelabial biopsy samples from pSS patients were investigatedby direct immunocytochemistry. These experiments wereperformed similarly to those described earlier in thissection, with the exception that a primary antiserum wasnot used.

Control experiments

In order to confirm the specificity of the antibody binding tothe m3AChR during the indirect light microscopic immuno-histochemistry and the indirect electron microscopic immu-nocytochemistry, the following modifications of the originalprocedures were applied:

1. Omission of the primary antiserum.2. The application of sera from healthy subjects as primary

antisera diluted 1:200 and 1:500 in PBS for the lightmicroscopic and the electron microscopic examinations,respectively (negative control sera).

3. The use of an affinity-purified anti-m3AChR213–228 mono-specific serum (prepared from the pooled sera of 4 pSSpatients as described above) as a primary antiserum,diluted 1:10 and 1:200 in PBS for the light microscopic andthe electron microscopic examinations, respectively.

4. The pre-absorption of the anti-m3AChR213–228 monospe-cific serum with the antigenic peptide (inhibition experi-ments). During these procedures, the anti-m3AChR213–228

monospecific serum was pre-incubated with them3AChR213–228 synthetic peptide (1 mg/ml in PBS) for1 h at room temperature and, subsequently, at +4 °Covernight. The inhibitor peptide was synthesised by solid-phase technique, utilising tBoc chemistry as describedpreviously [5]. The subsequent steps were identical tothose performed without the pre-absorption of themonospecific serum with the inhibitor. The binding ofthe serum pre-incubated with the inhibitor peptide wascompared with that of the serum without pre-incubation.The inhibition experiments were carried out only in theelectron microscopic immunocytochemistry system.

5. During the electron microscopic studies, a polyclonal anti-vasoactive intestinal polypeptide (VIP) serum (1:1000;Peninsula Lab Inc., Bachem, USA) was also tested as aprimary antiserum, because VIP is co-localised withacetylcholine in the cholinergic nerve endings [10], andit is therefore suitable as a control for the localisation ofcholinergic axon terminals.

Results

Light microscopic studies

Indirect immunohistochemistryIncubation of normal minor salivary gland tissue samples withthe whole, non-purified sera of the pSS patients revealedmultiple reaction end-products predominantly along thebasolateral membrane of acinar and ductal cells, but alsointracellularly (Fig. 1A). In addition to the specific signals,staining that could be regarded as a consequence of non-specific binding was also observed. When the normal tissuesamples were reacted with the anti-m3AChR213–228 mono-specific sera, in a background of a markedly weaker overallstaining intensity, clearly identifiable, specific, dot-like orshort linear reaction end-products could be visualised, whichwere observed only at the basolateral membrane of theepithelial cells (Fig. 1B). This is consistent with theanatomical localisation of autonomic nerve endings innervat-ing the epithelial cells. No such reaction was found in thecontrol sections, i.e. with the sera of the healthy subjects(Fig. 1C) or when a primary antibody was omitted during theincubation of the salivary gland samples.

Direct immunohistochemistryWhen in vivo deposited antibodies were investigated in theminor salivary gland tissue samples obtained from the pSSpatients, multiple binding reactions were revealed. Immu-noglobulin binding was visualised along the surface of theacinar and ductal epithelial cells, including circumscript, cellmembrane-bound immunoreactivities (Fig. 2A), and, withless density and intensity, also in the cytoplasm of these cells(Fig. 2B). Furthermore, specific signals were also observedalong the endothelial cells of the arterioles and venules. Asexpected and reported previously [11], these observationsreflect that immunoglobulin deposition occurs at multiplesites in the inflamed salivary gland of pSS patients.

Electron microscopic investigations

As noted above, the light microscopic immunohistochemistryexaminations indicated that 1) a serum fraction from pSSpatients monospecific to anti-m3AChR recognises an antigenlocated at the basolateral membrane of the salivary glandacinar cells, and 2) in vivo deposited IgG antibodies aredetectable in multiple locations in the salivary glands of pSSpatients. In order to assess the binding localisation of thesemembrane-bound antibodies more precisely, with specialattention to the regions adjacent to cholinergic nerve terminals,we decided to apply ultrastructural imaging methods, and inparticular immuno-electron microscopic investigations.

Indirect immuno-electron microscopic investigationsWhen normal minor salivary gland samples were incubatedwith whole pSS sera, specific binding reactions were visualisedalong the junctions of the acinar epithelial cells and adjacentaxon terminals (Figs. 3A, 3B). The reaction end-productsappeared as bead-like clusters of fine, granular deposits atsites corresponding to the membrane regions at the interfaceof nerve varicosities and epithelial cells. Similar immuno-reactivities were observed on myoepithelial cell membranes(Fig. 3C). Weak immunoreactivities were also seen at the

Figure 2 Light microscopic images of a minor salivary glandsample from a pSS patient reacted with anti-human IgG (directimmunohistochemistry). Panel A. Multiple reaction end-productscorresponding to the membrane of acinar cells (arrows) surround-ing dilated acini. The reaction end-products are organised asdark, bead-like clusters of granular deposits approximately 200 to400 nm in diameter. Panel B. In addition to the membrane-bound immunolabelling similar to that seen in Panel A (thin blackarrows), extensive, often confluent cytoplasmic staining is alsoobserved (thick arrows) in the acinar cells. The acinar cells appearatrophic. Within this cytoplasmic staining, occasional dot-likereaction end-products are also detectable, corresponding tomoderate, specific immunoreactivities (white arrow). Magnifica-tion: 40×.

Figure 1 Light microscopic immunohistochemistry images ofnormal salivary glands reactedwith the serum of a pSS patient (A),an anti-m3AChR213–228 monospecific affinity-purified serumpooled from 4 pSS patients (B), or serum of a healthy controlsubject (C). The arrows indicate specific reaction end-products atthe membrane of acinar cells. In Panel A, cytoplasmic stainingand non-specific binding to connective tissue are also observed,and the membrane-bound reaction end-products appear as dot-like or short linear immunoreactivities. In Panel B, the back-ground staining is markedly less intense and the specific reactionend-products appear as scattered, segmental membrane-boundsignals (arrows). No specific staining is observed in Panel C.Magnification: 40×.

272 L. Kovács et al.

secretory granules in the cytoplasm of the acinar cells. Theincubation of the normal salivary gland tissue with the anti-m3AChR213–228 monospecific sera revealed specific immunor-eactivities at structures identical to the above-mentionedmembrane regions, but in smaller numbers and intensity thanwith the non-purified sera (Fig. 3D). Axon terminals ofidentical electron microscopic appearance were identifiedwith the anti-VIP serum (Fig. 3E) as the nerve endingsassociated with the specific immunoreactivities described

above. The normal sera yielded only rare, scattered reactionsin the regions corresponding to the junctions betweenepithelial cells and nerve varicosities. These signals wereclearly less densely observable than those with the positivesera. Pre-incubation of the anti-m3AChR213–228 monospecificsera with the antigenic peptide completely abolished thebinding of the serum to the tissue specimens (Fig. 3F).

Direct immuno-electron microscopic investigationsThe binding localisation of autoantibodies deposited in theminor salivary glands in vivo were visualised by directlyreacting the labial biopsy samples obtained from the pSSpatients with anti-human IgG. Particular attention was paidto the precise subcellular localisation of the reaction end-products. Immunoglobulin deposition was detected along theacinar cell membrane and sparsely also in an intracytoplas-matic distribution associated with some of the secretorygranules (Fig. 4A). Of note, clusters of clear, specific reaction

Figure 3 Electron micrographs of normal salivary gland sections reacted with the serum of a pSS patient (A–C), an affinity-purifiedanti-m3AChR213–228-specific serum (D), an anti-VIP serum (E), or an anti-m3AChR213–228-specific serum pre-incubated with them3AChR213–228 peptide (inhibition experiment) (F). Panel A. The junction of an epithelial cell with a nerve ending. Two axon terminals(indicated with black squares) containing synaptic vesicles are represented on the right side of the picture. On the left side, there is anepithelial cell containing intracytoplasmic secretory granules (asterisks). Along the interface of the axolemma and the acinar cellmembrane, specifically labelled clusters of dot-like immunoreactivities are observed in a discontinuous distribution (arrows). Scalebar: 500 nm. Panel B. Higher magnification of the image in Panel A. Corresponding to the interdigitating pattern of the junction of thenerve varicosity and the postsynaptic region of the epithelial cell, the cytoplasm of the epithelial cell appears in multiple locations inthis section, indicated with asterisks. The synaptic vesicles in the axon terminals (squares) are highlighted with a thin arrow. Thespecific immunoreaction indicative of the binding of the pSS sera to the epithelial cell membrane regions adjacent to the nerveendings is highlighted with thick arrows. Scale bar: 200 nm. Panel C. Two axon terminals (indicated with asterisks) containing synapticvesicles (thin arrows); the left one is adjacent to an acinar epithelial cell (a) containing secretory granules, while the right one islinked to a myoepithelial cell (m). Specific immunoperoxidase reaction end-products corresponding to the postsynaptic membrane ofboth salivary gland cell types are highlighted with thick arrows. Scale bar: 1 μm. Panel D. The arrows indicate the immunoreactivitieslocated at the interface of the axon terminal and the adjacent epithelial cell. Scale bar: 500 nm. Panel E. In the upper part of theimage, there are two nerve varicosities containing large, round reaction end-products (arrows) corresponding to VIP-containingsynaptic vesicles. Scale bar: 1 μm. Panel F A nerve ending indicated with an asterisk in the vicinity of an acinar cell (a). The arrowspoint at synaptic vesicles. No specific immunolabelling can be observed. Scale bar: 1 μm.

273Demonstration of autoantibody binding to mAChR in the salivary gland in primary Sjögren's syndrome

end-products were observed at the acinar epithelial cellmembranes at the junctions of epithelial cells and nervevaricosities, indicating that antibodies reactive with post-

synaptic membrane receptors had been bound to theirantigenic targets in vivo (Fig. 4B). A further marked findingwas that, in contrast with the normal salivary gland samples,

Figure 4 Electron microscopic immunocytochemistry images of a pSS minor salivary gland sample reacted directly with anti-humanIgG. Panel A. An acinar epithelial cell (a) containing confluent secretory granules (asterisk) and an adjacent nerve ending in the upperright part of the image. An example of the immunoreactivities on the epithelial cell membrane at the connections with the axonterminal is highlighted with a black arrow. Weak immunolabelling of some secretory granules is also observed (white arrows). Scalebar: 500 nm. Panel B. Higher magnification of the axon terminal adjacent to the epithelial cell. The acinar cell cytoplasm (star)contains mytochondria (Myt). The arrows point at reaction end-products on the epithelial cell membrane at the interface with theaxon terminal. Two further specific reaction end-products corresponding to synaptic vesicles and axolemmal segments are alsoobserved (arrowheads). Scale bar: 500 nm.

274 L. Kovács et al.

fusion of the secretory granules was observed in the pSSsamples (Fig. 4A).

Discussion

Since their initial description in 1996 [12], antimuscarinicreceptor antibodies have become of considerable interest.As the exocrine dysfunction characteristic of pSS could notbe fully ascribed to the tissue damage in the salivary glands[13], antimuscarinic receptor antibodies have emerged as apotential link between immune-mediated inflammatoryprocesses in the involved exocrine glands and the secretorydysfunction [14]. This concept was supported by the findingsthat IgG from pSS patients is able to inhibit the cholinergicneurotransmission in isolated rabbit urinary bladder strips [4]or in epithelial cells of a human salivary ductal cell line [14].We have demonstrated the presence of IgG class antibodiesreactive with a 16-mer peptide located at the secondextracellular loop of the human m3AChR (m3AChR213–228)predicted as an immunodominant epitope of the receptor[5]. With our ELISA system, we were able to differentiatebetween pSS patients and various control groups withconsiderably high specificity and sensitivity [6]. However,these findings do not automatically imply that the nativem3AChR located in the inflamed exocrine gland is theautoantigenic target, and that these circulating antibodiesactually bind to the receptor in the patients. Furthermore,Western blotting studies that aimed at a demonstration ofthe binding specificities of pSS IgG with membrane extractsfrom lacrimal glands [15] or from Chinese hamster ovary cellstransfected with the human m3AchR [16] have yielded

conflicting results. The precise antigenic target of anti-m3AChR antibodies has therefore remained uncertain.

According to a recently published set of criteria [17], onecondition that an antibody has to fulfil in order to be regardedas pathogenic is the demonstration of the reaction of thisantibody with the target antigen. The studies presented hereprovide data that seem to support the pathogenetic role ofanti-m3AChR antibodies in pSS. Our light microscopic investi-gations indicate that a serum fraction from pSS patientsmonospecific to m3AChR213–228 selectively binds to thebasolateral membrane of minor salivary gland acinar cells ina discontinuous pattern, forming dot-like or short linearreaction end-products. The specificity of the monospecificserum in its binding to the m3AChR is verified by its highlypositive reaction with ELISA, using the m3AChR213–228 asantigen. The binding specificity of the anti-m3AChR213–228

antibodies has also been checked via Western blotting andinhibition ELISA with the synthetic peptide as an inhibitor [5].The fact that an anti-m3AChR-specific serum demonstrates aspecific binding to the acinar cells suggests that the antigenictarget in the reaction end-products is the m3AChR.

To verify the exact antigenic target of the pSS IgG, weproceeded to ultrastructural studies with immuno-electronmicroscopic examinations. These studies confirmed that thepositive reaction end-products visualised by light micro-scopic immunohistochemistry correspond to the localisationof the m3AChR. Specific signals were revealed at the cellmembrane of the acinar cells in regions of the interfacebetween the epithelial cells and the adjacent nerve endings.This localisation and morphologic appearance were comple-tely identical to those seen with VIP, a marker of cholinergic

275Demonstration of autoantibody binding to mAChR in the salivary gland in primary Sjögren's syndrome

axon terminals, which strongly suggests that the localisationof the antigen-antibody reaction corresponds to the choli-nergic postsynaptic membrane. Furthermore, the binding ofthe anti-m3AChR monospecific serum was completelyblocked by the pre-incubation of this serum with theantigenic peptide. Thus, both the binding localisation andthe antigenic specificity of the primary sera used in thestudies lend support to the conclusion that antibodies thatbind to salivary gland m3AChR are present in the sera of pSSpatients. Positive reactions with similar electron microscopicappearance were observed in minor salivary gland tissuesamples obtained from the pSS patients, which implies thatanti-m3AChR antibodies have already bound to their corre-sponding antigenic targets in vivo.

The fact that the anti-m3AChR213–228-specific serumspecifically bound to the native m3AChR in the salivarygland confirms the physiological relevance of this particularpeptide as an immunodominant epitope of the m3AChR. Thedetermination of the antigenic determinant targeted by thecirculating anti-m3AChR antibodies in pSS patients has beena subject of debate, and a number of different amino acidsequences have been tested in previous studies[5,12,15,16,18]. Our present findings seem to confirm theappropriateness of our previous epitope prediction [5], andmay aid in a more precise characterisation of the function-ally relevant antigenic epitope of the m3AChR in pSS.

There are many similarities between our light microscopicimmunohistochemistry findings and the results of Beroukaset al [19], who examined normal and pSS salivary glands withindirect immunofluorescence and confocal laser microscopyusing a goat polyclonal antibody raised against the COOH-terminus of the human m3AChR to identify the localisation ofthe m3AChR-s. They observed “beads of discontinuous punc-tate staining, approximately 0.4 μm in size” associated withthe membrane of the acinar and the ductal (both intercalatedand striated) epithelial cells. This lightmicroscopic appearanceis very similar both in localisation and in size to the bead-like clusters of immunoreactivities described in the presentreport. This is indirect support for the relevance of our findings,even if we note that the epitope specificity of the anti-m3AChR antibody used by Beroukas et al is different fromthat of our monospecific serum purified from pSS patients, asthat epitope is located at the intracellular terminal of thereceptor.

Although it was not specifically an aim of this study toanalyse the electron microscopic appearance of pSS salivarygland cells, it was conspicuous that the pSS acinar cells weredifferent from the healthy cells in that the secretory granulesdisplayed extensive fusion. The functional significance of thisphenomenon is not yet certain, but it is interesting to note thatin a recent electron microscopic study on the ultrastructuralchanges in non-obese diabetic (NOD) mice, a murine model ofpSS, similar elements were observed and were depicted as“cytoplasmic fusion of secretory vesicles to multivesicularaggregates” [20]. It was hypothesised in that manuscript thatthis fusion might be a consequence of an altered cholinergicagonist stimulation, a theory that needs further verification.

Our approach of localising an autoantigen in the targettissue is very similar to that applied by Schulze et al, whoidentified the muscarinic acetylcholine receptor subtype-2(m2AChR) as an autoantigen in idiopathic dilated cardiomyo-pathy by using affinity-purified anti-m2AChR antibodies

which bound to the cell membrane of human heart bioptatesand cultured cardiomyocytes [21]. Other authors havereported on the ultrastructural localisation of autoantibo-dies to their respective intradermal autoantigens in pem-phigus vulgaris [22] or bullous pemphigoid [23] usingimmuno-electron microscopy. Their observations indicatedthat the method that we used is suitable for the clarificationof the pathogenetic role of circulating autoantibodies aseffectors in the immune processes in the involved organs.

In summary, we have demonstrated that m3AChR auto-antibodies in the sera of pSS patients are able to recognise andbind to the receptors in normal human salivary glands, and thatthis antigen recognition and binding occurs in vivo in pSSpatients. It may be a subject of further investigations toevaluate the functional importance of these m3AChR-bindingantibodies in the elicitation of the secretory dysfunction in pSSpatients.

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