New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome

Abstract

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca²⁺ concentrations [(Ca²⁺)i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca²⁺)i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca²⁺)i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes.

Introduction

Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands, including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of autoantibodies, such as anti-SS-A and SS-B antibodies, are detected in patients with SS. However, no SS-specific pathological autoantibodies have yet been found in this condition [1].

Data from recent studies have suggested that some patients with SS carry inhibitory autoantibodies directed against muscarinic acetylcholine receptors, especially M3 muscarinic acetylcholine receptor (M3R) [1]. To date, five subtypes of muscarinic acetylcholine receptors (M1R–M5R) have been identified, and M3R is expressed in exocrine glands and plays crucial roles in exocrine secretion. Acetylcholine binds to and activates M3R on salivary gland cells, causing a rise in intracellular Ca²⁺ via inositol 1, 4, 5-trisphosphate (IP3) and IP3 receptors. Consequently, the rise in intracellular Ca²⁺ activates apical membrane Cl^– channels and induces salivary secretion [1]. Activation of M3R also induces trafficking of aquaporin 5 (AQP5) to the apical membrane from the cytoplasm, which causes rapid transport of water across the cell membrane [2]. M3R has four extracellular domains: the N-terminal region and the first, second and third extracellular loops. Among these domains, the second extracellular loop is critical for receptor activation by agonists [3]. Therefore, the second extracellular loop of M3R has been the focus of our interest, and we report a subgroup of SS patients who had anti-M3R antibodies that recognized the second extracellular loop of M3R [4,5]. Although these data indicate that the second extracellular loop is the target antigen, the precise epitopes are currently unknown. A recent study reported that the third extracellular loop represents a functional epitope bound by IgG derived from SS patients [6].

The present study was designed to clarify the precise B cell epitopes of M3R and the function of anti-M3R antibodies. For this purpose, we screened sera of SS patients for anti-M3R autoantibodies against all four extracellular domains of M3R by enzyme-linked immunosorbent assay (ELISA) using synthetic peptide antigens and performed functional assays of these antibodies using human salivary gland (HSG) cells. We assessed the correlation between epitopes and function and various clinical features.

Materials and methods

Study population

Serum samples were collected from 42 Japanese patients with SS (15 with primary SS and 27 with secondary SS) who had been followed-up at the Division of Rheumatology, University of Tsukuba Hospital, Ibaraki, Japan. All patients with SS satisfied the Japanese Ministry of Health criteria for the diagnosis of SS. These criteria included four clinicopathological findings: lymphocytic infiltration of the salivary or lacrimal glands, dysfunction of salivary secretion, keratoconjunctivitis sicca and presence of anti-SS-A or SS-B antibodies. The diagnosis of SS was based on the presence of two or more of the above items. We also recruited 42 healthy controls (HC). Approval for this study was obtained from the local ethics committee and signed informed consent was obtained from each subject.

Synthesis of peptide antigens

We synthesized different peptides encoding the extracellular domains of human-M3R. The N-terminal of human-M3R has a 66-mer amino acid sequence, and accordingly we divided this domain into three segments. The sequences were: MTLHNNSTTSPLFPNISSSWIHSPSDAGLP for N-terminal 1, IHSPSDAGLPPGTVTHFGSYNVSRAAGNFS for N-terminal 2 and NVSRAAGNFSSPDGTTDDPLGGHTVWQV for N-terminal 3 (Sigma-Aldrich Japan, Ishikari, Japan). These three peptides were mixed and used for the peptide antigens of the N-terminal region. We also synthesized three peptides corresponding to the sequences of the three extracellular loops of human-M3R, the sequences of which were FTTYIIMNRWALGNLACDLW for the first extracellular loop, KRTVPPGECFIQFLSEPTITFGTAI for the second and VLVNTFCDSCIPKTFWNLGY for the third (Sigma-Aldrich Japan). As a control peptide, we synthesized a peptide corresponding to the sequences of the third extracellular loop of human-M5 muscarinic acetylcholine receptor (M5R), the sequences of which were STFCDKCVPVTLWH (Sigma-Aldrich Japan). As a negative peptide, we also synthesized a 25-mer peptide whose sequence was SGSGSGSGSGSGSGSGSGSGSGSGS (Sigma-Aldrich Japan).

ELISA

Peptide solution (100 µl/well at 10 µg/ml) in 0·1 M Na₂CO₃ buffer, pH 9·6, was adsorbed onto a Nunc-Immuno plate (Nalge Nunc International, Rochester, NY, USA) overnight at 4°C, and blocked with 5% bovine serum albumin (NSA) (Wako Pure Chemical Industries, Osaka, Japan) in phosphate-buffered saline (PBS) for 1 h at 37°C. For the dose-dependent curve, serum from anti-M3R antibodies positive SS and from HC were diluted at 1:25, 1:50, 1:100, 1:200, 1:400, 1:800 and 1:1600 in blocking buffer, and incubated for 2 h at 37°C. Serum to be examined at 1:50 dilution in blocking buffer was also incubated for 2 h at 37°C. The plates were then washed six times with 0·05% Tween20 in PBS, and 100 µl of solution of alkaline phosphatase-conjugated goat anti-human IgG (Fc; American Qualex, San Clemente, CA, USA) diluted 1:1000 in PBS was added for 1 h at room temperature. After nine washes, 100 µl of p-nitrophenyl phosphate (Sigma) solution was added at a final concentration of 1 mg/ml as alkaline phosphatase substrate. Plates were incubated for 30 min at room temperature in the dark, and the absorbance at 405 nm was measured by plate spectrophotometry. Measurements were performed in triplicate and standardized between experiments by using the absorbance value of the positive control.

Measurement of salivary secretion

We assessed salivary secretion by the gum test. In this test, the volume of saliva is measured after chewing gum for 10 min.

Histopathological examination

Histopathological findings of the labial salivary glands were classified according to Greenspan grading [7].

Expression of M3R mRNA in HSG cells

Total RNA was extracted from HSG cells and cDNA was synthesized by cDNA synthesis kit (Fermentas International, Burlington, Ontario, Canada). Polymerase chain reaction (PCR) was performed with cDNA using the human-M3R-specific primers [2]. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified to assess the cDNA yield.

Expression of M3R proteins on the cell surface of HSG cells

For immunofluorescent analysis, HSG cells were precultured in two-well chamber slides for 48 h. Without fixation, HSG cells were incubated with the first antibodies: anti-human M3R antibody (goat IgG, polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat control IgG (Invitrogen Corporation, Carlsbad, CA, USA) for 2 h. After washing, HSG cells were incubated with the second antibodies: fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat IgG antibodies (IgG; MP Biomedicals, Irvine, CA, USA). Stained HSG cells were observed by fluorescence microscope.

Functional assays

HSG cells (15 000 cells/well) were precultured in 96-well plates for fluorescence assays at 37°C for 48 h. Then, the cells were preincubated with IgG fractions separated from sera of anti-M3R antibodies positive for five SS patients, anti-M3R antibodies negative for one SS patient, and HC by using protein G column (1·0 mg/ml) for 12 h. The referral of the anti-M3R antibodies positive or negative sera was on the basis of our ELISA results. IgG was washed off and the HSG cells were loaded with Fluo-3, which was a fluorescence probe for calcium, for 1 h. Fluo-3 was washed off, and then the HSG cells were analysed. For the Ca²⁺ influx assay, the HSG cells were stimulated with cevimeline hydrochloride, which was a M3R specific agonist at a final concentration of 20 mM. Changes in intracellular calcium concentrations [(Ca²⁺)i] in HSG cells were measured by fluorescence plate reader. Maximum changes of (Ca²⁺)i [peak (Ca²⁺)i – baseline (Ca²⁺)i] in IgG from SS patients or without IgG were shown as ratiometric data compared to maximum change of (Ca²⁺)i in HC [2].

Statistical analysis

Differences between groups were examined for statistical significance using the Mann–Whitney U-test, while differences in frequencies were analysed by Fisher's exact probability test. A P-value less than 0·05 was considered as the statistically significant difference.

Results

Anti-M3R antibodies in patients with SS and control subjects

The average age of SS patients was 53·1 ± 13·2 years, that of HC was 33·1 ± 8·7 years (P < 0·05, Mann–Whitney U-test). All 42 SS patients were female, 22 of HC female and 20 of HC male. Among 27 patients with secondary SS, 11 were complicated with rheumatoid arthritis (RA), 11 with systemic lupus erythematosus (SLE), two with mixed connective tissue disease (MCTD) and three with other autoimmune diseases.

Anti-M3R antibodies were really specific for each M3R peptide, because the binding activities of sera from SS patients were dose-dependent and were not in the control sera from healthy subjects. Furthermore, sera from anti-M3R antibodies positive SS did not recognize the peptide corresponding to the sequences of the third extracellular loop of human-M5R (Fig. 1a).

Fig. 1.

(a) The dose-dependent curve on anti-M3 muscarinic acetylcholine receptor (M3R) antibodies. M3R and M5R peptide-specific absorbance values at 405 nm (Δabsorbance) were calculated for each serum sample by subtracting the absorbance value of the negative peptide from that of the peptides encoding the extracellular domains of human-M3R and M5R. The clear dose–response of M3R peptide-specific absorbance to changes on serum concentrations was shown in serum from anti-M3R antibody-positive Sjögren's syndrome (SS), but not in serum from healthy controls (C). The clear dose–response of the third extracellular loop of M5R-specific absorbance to changes on serum concentrations was not shown in serum from anti-M3R antibody-positive SS. (b) Anti-M3R antibodies in patients with SS and control subjects. M3R peptide-specific absorbance values at 405 nm (Δabsorbance) in Sjögren's syndrome (SS) and healthy controls (C). The cut-off level between negative and positive values was the mean ± 2 standard deviation value of the normal control (grey line). The prevalence and titres of anti-M3R antibodies against all extracellular domains were significantly higher in patients with SS than control subjects (Fisher's exact probability test for prevalence, Mann–Whitney U-test for titres). N: N-terminal region; 1st: first extracellular loop; 2nd: second extracellular loop; 3rd: third extracellular loop.

Antibodies to the N-terminal region were detected in 42·9% (18 of 42) of SS patients but in only 4·8% (two of 42) of the control (P < 0·05, Fisher's exact probability test). Antibodies to the first extracellular loop were detected in 47·6% (20 of 42) of SS and 7·1% (three of 42) of the control (P < 0·05, Fisher's exact probability test). Antibodies to the second extracellular loop were detected in 54·8% (23 of 42) of SS and 2·4% (one of 42) of the control (P < 0·05, Fisher's exact probability test). Antibodies to the third extracellular loop were detected in 45·2% (19 of 42) of SS and 2·4% (one of 42) of the control (P < 0·05, Fisher's exact probability test). The frequencies and titres of anti-M3R antibodies against all extracellular domains were significantly higher in SS patients than the control (P < 0·05, Fisher's exact probability test for frequencies, Mann–Whitney U-test for titres) (Fig. 1b).

B cell epitopes on the M3R

Table 1 lists the epitopes of anti-M3R antibodies in patients with SS. Of the 42 SS patients, 28 had anti-M3R antibodies reactive to at least one B cell epitope on the M3R, while the other 14 SS patients did not have any anti-M3R antibodies. Antibodies to one B cell epitope on the M3R (N-terminal, first, second and third extracellular loops) were detected in one, two, two and one of 28 SS patients, respectively. Antibodies reactive to two B cell epitopes (N-terminal and first extracellular loop, N-terminal and second extracellular loop, first and second extracellular loop, second and third extracellular loop) were detected in one, one, two and two SS patients, respectively. Two SS patients showed the presence of antibodies to three B cell epitopes (N-terminal and second and third extracellular loop, first and second and third extracellular loop). In 50% of the SS patients (14 of 28), antibodies reactive to all four B cell epitopes were detected. Based on these results, we concluded that anti-M3R antibodies had several B cell epitopes on the extracellular domains of M3R, and that some SS patients carried anti-M3R antibodies that recognized several extracellular domains of M3R.

Table 1.

B cell epitopes on the M3 muscarinic acetylcholine receptor (M3R).

	B cell epitopes on the M3R
Number of B cell epitopes on the M3R	N	1st	2nd	3rd	Number of cases
1	+	−	−	−	1
	−	+		−	2
	−	−	+	−	2
	−	−	−	+	1
2	+	+	−	−	1
	+	−	+	−	1
	−	+	+	−	2
	−	−	+	+	2
3	+	−	+	+	1
	−	+	+	+	1
4	+	+	+	+	14
Total number of cases					28

N, N-terminal; 1st, first extracellular loop; 2nd, second extracellular loop; 3rd, third extracellular loop; +, positive for anti-M3R antibodies; −, negative for anti-M3R antibodies.

Correlation between anti-M3R antibodies and various clinicopathological features

Disease duration of SS was shorter among anti-M3R antibody-positive SS (7·3 ± 7·6 years) than -negative SS (15·5 ± 11·1 years, P < 0·05, Mann–Whitney U-test). The positivity for anti-SS-A antibody and the IgG value in serum was more prevalent and higher among anti-M3R antibody-positive SS than -negative SS (P < 0·05, Fisher's exact probability test and Mann–Whitney U-test). In contrast, there were no differences in age, positivity for anti-SS-B antibody and rheumatoid factor, tear volume by Schirmer test, saliva volume by gum test, extra-glandular involvement and Greenspan grading between anti-M3R antibody-positive and -negative SS (Table 2). There is no significant relationship between each B cell epitope and clinical characteristics such as saliva secretion.

Table 2.

Clinicopathological features in anti-M3 muscarinic acetylcholine receptor (M3R) antibody-positive and -negative Sjögren's syndrome (SS) patients.

	Positive SS n = 28	Negative SS n = 28	P-value
Primary/secondary	12/16	3/11	n.s.
Age (years)	51·4 ± 12·1	56·4 ± 12·1	n.s.
Disease duration (years)	7·3 ± 7·6	15·5 ± 11·1	P < 0·05
Anti-SSA (%)	92·9	57·1	P < 0·05
Anti-SSB (%)	21·4	14·3	n.s.
Rheumatoid factor (%)	46·4	50·0	n.s.
IgG (mg/dl)	2013 ± 767	1427 ± 515	P < 0·05
Extra-glandular involvements in primary SS (%)	83·3	66·7	n.s.
Schirmer test (mm/5 min)	4·4 ± 6·2	4·2 ± 5·0	n.s.
Gum test (ml/10 min)	8·3 ± 7·8	8·8 ± 5·1	n.s.
Histological examination (Greenspan grading)	3·1 ± 0·5	3·0 ± 0·8	n.s.

n.s., not statistically significant.

Expression of M3R mRNA and proteins in HSG cells

PCR products revealed the expression of M3R mRNA in HSG cells used in the present study. The expected PCR product for M3R was detected at 201 base pairs (bp) (Fig. 2a). Moreover, M3R proteins were detected on HSG cells stained with anti-human M3R antibody, whereas they were not found with control IgG (Fig. 2b). These results indicated that HSG cells expressed M3R molecules on their surface.

Fig. 2.

(a) Expression of M3 muscarinic acetylcholine receptor (M3R) mRNA in human salivary gland (HSG) cells. (b) Expression of M3R proteins on the surface of HSG cells detected by immunofluorescent analysis.

Functional roles of anti-M3R antibodies

IgG derived from two SS patients positive for anti-M3R antibodies to the second extracellular loop inhibited the increase in (Ca²⁺)i induced by cevimeline hydrochloride 16% and 25%, respectively (P < 0·05, versus IgG derived from HC, Mann–Whitney U-test) (Figs 3c,d and 4). In contrast, IgG derived from SS patients positive for antibodies to the N-terminal and the first extracellular loop enhanced the increase in (Ca²⁺)i induced by cevimeline hydrochloride 14% and 15%, respectively (P < 0·05, versus IgG derived from HC, Mann–Whitney U-test) (Figs 3a,b and 4). IgG derived from a SS patient positive for antibodies to the third extracellular loop had no effect on (Ca²⁺)I, as well as IgG derived from an anti-M3R antibody-negative SS patient (Figs 3e and 4).

Fig. 3.

Functional analysis of anti-M3 muscarinic acetylcholine receptor (M3R) antibodies in Sjögren's syndrome (SS) patients. (a,b) Immunoglobulin G (IgG) derived from SS patient with anti-M3R antibodies to the N-terminal region and the first extracellular loop enhanced the increase in (Ca²⁺)i induced by cevimeline hydrochloride 14% and 15%, respectively, compared to IgG from healthy control (HC). The traces were representative traces, which were performed in triplicate, and three independent experiments with each IgG. Human salivary gland (HSG) cells were stimulated with cevimeline hydrochloride (20 mM) at 10 s. IgG^-; without IgG, HC; IgG derived from healthy control, M3R^-SS; IgG derived from SS patient negative for anti-M3R antibodies, N⁺SS; IgG derived from SS patient positive for anti-M3R antibodies to the N-terminal region; 1st⁺SS: IgG derived from SS patient positive for anti-M3R antibodies to the first extracellular loop. (c,d) IgG derived from two SS patients positive for antibodies to the second extracellular loop inhibited the increase in (Ca²⁺)i induced by cevimeline hydrochloride 16% and 25%, respectively, compared to IgG from HC. The traces were representative traces, which were performed in triplicate, and three independent experiments with each IgG; 2nd⁺SS: IgG derived from SS patient positive for anti-M3R antibodies to the second extracellular loop. (e) IgG derived from SS patient positive for antibodies to the third extracellular loop had no effect on the increase in (Ca²⁺)i induced by cevimeline hydrochloride. The traces were representative traces, which were performed in triplicate, and three independent experiments with each IgG; 3rd⁺SS: IgG derived from SS patient positive for antibodies to the third extracellular loop.

Fig. 4.

Summary of B cell epitopes on M3 muscarinic acetylcholine receptor (M3R) and the function of anti-M3R antibodies in Sjögren's syndrome (SS) patients. Mean ± standard deviation values of maximum change in (Ca²⁺)i [peak (Ca²⁺)i – (Ca²⁺)i] induced by cevimeline hydrochloride in immunoglobulin G (IgG) from SS patients or without IgG were shown as a ratio compared to maximum change of (Ca²⁺)i in healthy control (HC). The maximum change in HC was described as 1·0. Data were averaged of triplicate and three independent experiments. *P < 0·05 versus IgG derived from HC, Mann–Whitney U-test, HC, IgG^-, M3R^-SS, N⁺SS, 1st⁺SS, 2nd⁺SS and 3rd⁺SS; the same as in Fig. 3.

Discussion

Recently, anti-M3R antibodies have been the focus of interest in rheumatology because of their potential pathogenic role, use as diagnostic markers and being therapeutic targets in patients with SS [1]. Several methods have been used to detect anti-M3R antibodies in SS patients [1]. In functional assays using smooth muscles, IgG fractions from patients with SS (SS-IgG) inhibited carbachol-evoked or nerve-evoked bladder or colon contractions [8,9]. In salivary gland cells, SS-IgG inhibited the rise in (Ca²⁺)i induced by carbachol, and also inhibited pilocarpine-induced AQP5 trafficking to the apical membrane from the cytoplasm [2]. The inhibitory actions of SS-IgG on the rise in (Ca²⁺)i was acutely reversible [10]. Anti-M3R antibodies from SS patients can be detected by immunofluorescent analysis using rat lacrimal glands [11], and by flow cytometry using the M3R-transfected Chinese hamster ovary (CHO) cell line [12]. Moreover, anti-M3R antibodies in sera of SS patients were detected by ELISA using synthetic peptides or recombinant proteins of the second extracellular loop of M3R [13]. We have reported previously the presence of anti-M3R antibodies in a group of patients with SS, which recognized the second extracellular loop by ELISA using synthetic peptides [4,5].

In the present study, we established a standard method to detect anti-M3R antibodies that can be used for screening large patient populations. Functional assays and flow cytometry are too laborious for routine use. Although ELISA is easy, the results from some ELISA systems used for screening anti-M3R antibodies differ widely with regard to the prevalence of anti-M3R antibodies (from 11 to 90%) [4,14]. Furthermore, Cavill et al.[15] reported failure to detect anti-M3R antibodies by ELISA using synthetic peptides. In the present study, we reported higher frequencies and titres of anti-M3R antibodies against all extracellular domains in SS patients than the control. The prevalence of anti-M3R antibodies against the second extracellular loop in SS (55%) determined in the present study was much higher than that reported in our previous study (11%) [4]. The reason for this difference is probably related to the change in the methodology, such as increased sensitivity resulting from purity of the synthetic peptides, modification of the washing procedure or other factors introduced in the modified ELISA system.

In the present study, we also determined the precise B cell epitopes of M3R molecules. B cell epitopes in the present study are areas including peptides recognized by anti-M3R antibodies, although we do not know whether or not these linear peptides are really conformational epitopes. However, we showed that anti-M3R antibodies against these linear epitopes exactly influenced Ca influx via M3R in HSG cells. Therefore, we suggest that these linear peptides might consist of the conformational epitopes on the M3R. Several B cell epitopes were identified on the extracellular domains, and some SS patients were reactive to several extracellular domains other than the second extracellular loop. The second extracellular loop of M3R has been the focus of our interest in epitopes and function of anti-M3R antibodies [4,5,9,10]. Recently, Koo et al.[6] reported that the third extracellular loop represents a functional epitope bound by SS-IgG. In contrast to these results, we found in the present study that antibodies to the second extracellular loop of M3R inhibited the increase of (Ca²⁺)i induced by cevimeline hydrochloride in a functional assay using HSG cells. This inhibitory effect of anti-M3R antibodies might explain the reduction in salivary secretion in some SS patients. Our data also demonstrated that antibodies against the third extracellular loop did not have an effect on the increase in (Ca²⁺)i, while antibodies against the N-terminal and first extracellular loop enhanced the increase in (Ca²⁺)i. These results indicate that the effects of anti-M3R antibodies on the secretion of saliva could be different from these epitopes, although further experiments using antibodies from more patients are necessary.

Although the molecular mechanisms on the difference among individual B cell epitopes have not been elucidated, we could propose the following three possibilities. The first is that antibodies against the second extracellular domain of M3R directly inhibit the intracellular signal pathway, resulting in the decrease of Ca²⁺ influx and reduction of saliva. In contrast, antibodies against N-terminal region and the first extracellular domain of M3R might enhance the intracellular signalling and increase of Ca²⁺ influx. The second is that anti-M3R antibodies binding to the second extracellular domain could inhibit the M3R agonist, and then antibodies suppress indirectly the stimulation of Ca²⁺ influx. The third is that anti-M3R antibodies influence the expression of M3R molecules on HSG. Some antibodies which target the N-terminal region or the first extracellular loop of M3R may be able to up-regulate expression of M3R and enhance Ca²⁺ influx, whereas the other antibodies against the second extracellular domain might down-regulate the expression of M3R on HSG, resulting in a reduction of Ca²⁺ influx. It has been reported that the expression of M3R in salivary glands could be affected by anti-M3R antibodies in patients with SS [1]. Further experiments on the effect of anti-M3R antibodies on M3R signalling, binding to the M3R agonist, and the influence on M3R expression should shed light on the mechanism of the different functions of anti-M3R antibodies.

We have reported previously the presence of anti-M3R antibodies that recognized the second extracellular loop in SS patients but not in patients with RA or SLE, suggesting that anti-M3R antibodies could be used potentially as diagnostic markers for SS [4]. However, Kovacs et al.[14] reported the detection of anti-M3R antibodies in 35% of their RA patients and 32% of SLE. These conflicting results emphasize the need to examine the precise prevalence of anti-M3R antibodies in other autoimmune diseases using our modified ELISA system.

The correlation between anti-M3R antibodies and clinical features is still unclear. The previous study reported leukopenia was more common in anti-M3R antibody-positive than in -negative patients with primary SS [14]. Our observations in the present study showed that positivity for anti-SS-A antibody and IgG values in serum was more prevalent and higher in anti-M3R antibody-positive SS patients than -negative SS patients. The disease duration of SS was shorter among anti-M3R antibody-positive SS than -negative SS; however, there was no difference in other clinical and histological features between anti-M3R antibody-positive and -negative SS patients. We could not detect any significant relationship between each B cell epitope and clinical characteristics such as saliva secretion.

In conclusion, these findings support the notion of presence of several B cell epitopes on M3R in SS patients, and that some SS patients are reactive to several extracellular domains of the M3R. It is possible that some anti-M3R antibodies alter salivary secretion in SS via M3R, and in particular antibodies against the second extracellular loop of the M3R could suppress the increase in (Ca²⁺)i induced by M3R agonists, resulting in reduction of salivary secretion. Therefore, anti-M3R antibodies might play pathogenic roles in salivary secretion abnormalities characteristic of patients with SS.

Disclosure

None of the authors has any conflict of interest with the subject matter or materials discussed in the manuscript.