Clinical associations of autoantibodies to human muscarinic acetylcholine receptor 3213228 in primary Sjögren's syndrome
L. Kovács,
I. Marczinovits1,
A. György3,
G. K. Tóth2,
L. Dorgai3,
J. Pál4,
J. Molnár1 and
G. Pokorny
Department of Rheumatology, 1 Department of Physiology and 2 Department of Medical Chemistry, Szeged, University of Szeged, 3 Bay Zoltán Foundation for Applied Research, Institute for Biotechnology, Szeged and 4 Hungarian Academy of Sciences, Clinical Neuroscience Research Group, Pécs, Hungary.
Correspondence to: L. Kovács. E-mail: kovl{at}in1st.szote.u-szeged.hu
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Abstract
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Objectives. The authors have previously identified a peptide of the human muscarinic acetylcholine receptor-3 (m3AChR) as a suitable antigen for the immunodetection of antimuscarinic acetylcholine receptor autoantibodies in primary Sjögren's syndrome (pSS). The aim of this study was to assess the clinical correlations and disease specificity of these antibodies.
Methods. Seventy-three pSS, 40 rheumatoid arthritis (RA), 19 systemic lupus erythematosus (SLE), 14 secondary Sjögren's syndrome (sSS) patients, 22 subjects in whom pSS was suspected but in whom the diagnosis not could eventually be established (suspSS) and 40 healthy subjects were investigated. An enzyme-linked immunosorbent assay system developed by the authors using a 16-mer peptide of the m3AChR (m3AChR213228) in a recombinant fusion peptide form was used as the antigen.
Results. Anti-m3AChR213228 antibody positivity was observed in 66 (90%) of the pSS patients. The antibody levels correlated positively with the number of extraglandular organ manifestations. Both the mean antibody levels and the occurrence of anti-m3AChR213228 positivity were significantly higher in pSS than in the comparison groups. The test discriminated the pSS patients from the various comparison groups with specificities of 65, 68, 71 and 50% for RA, SLE, sSS and suspSS, respectively.
Conclusions. The presence of m3AChR213228 antibodies is a common feature in pSS. Although it is significantly more common in pSS than in the comparison groups, anti-m3AChR213228 positivity is not exclusive to pSS.
KEY WORDS: Antimuscarinic acetylcholine receptor-3 autoantibody, Disease specificity, Muscarinic acetylcholine receptor-3213228, Primary Sjögren's syndrome
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Introduction
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Sjögren's syndrome (SS) is a systemic autoimmune connective tissue disease characterized by the obligatory manifestations of keratoconjunctivitis sicca and xerostomia, and, in the majority of patients, by various other extraglandular organ involvements. The disease is classified as primary Sjögren's syndrome (pSS) if no other systemic autoimmune disease is present, and as secondary Sjögren's syndrome (sSS) if the symptoms of ocular and oral dryness evolve in a patient with an established diagnosis of another connective tissue disease, most commonly rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).
A prominent feature of pSS is the presence of B-lymphocyte hyperactivity, leading to the presence of a variety of autoantibodies. In addition to the classical anti-SSA and anti-SSB antibodies, other novel antibodies have recently been detected. One of these is the autoantibody reactive with the muscarinic acetylcholine receptor subtype-3 (m3AChR) [14]. This receptor mediates the secretagogue cholinergic stimuli in the lachrymal and salivary glands, and studies on animal models of SS have indicated that antibodies from the sera of pSS patients reacting with the m3AChR are essential for the elicitation of a glandular dysfunction [2, 5].
We recently developed an enzyme-linked immunosorbent assay (ELISA) system for the detection of antibodies to a 16-mer peptide sequence of the human m3AChR (m3AChR213228 [6]). On application of this peptide sequence of the second extracellular loop (the ligand-binding region) of the human m3AChR, produced in recombinant form fused with glutathione-S-transferase (GST), the ELISA method differentiated the pSS patients from the healthy controls in a reliable and highly sensitive way. In the work presented here, we tested the sera of a larger cohort of pSS patients and of various comparison groups in order to determine the prevalence, clinical associations and disease specificity of m3AChR213228 antibodies.
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Patients and methods
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Study populations
Seventy-three pSS patients (70 females, mean age 55 yr, range 3082) were included in this study. They all met the AmericanEuropean classification criteria for pSS [7]. Five comparison groups were included: (group 1) 40 patients with RA (36 females, mean age 56 yr, range 2780), classified according to the American College of Rheumatology (ACR) criteria for RA [8]; (group 2) 19 SLE patients (16 females, mean age 40 yr, range 2673), fulfilling the ACR classification criteria for SLE [9]; (group 3) 14 sSS patients (all females, mean age 53 yr, range 3263), who met the AmericanEuropean classification criteria for sSS [7]; (group 4) 22 patients (21 females, mean age 52 yr, range 2178) who were referred to our department because of suspected SS but in whom, after subsequent detailed examinations, the diagnosis of pSS could not be established (suspSS group); and (group 5) 40 healthy blood donors (all females, mean age 49 yr, range 2362 yr). All of the patients in the suspSS group had either subjective plus objective xerophthalmia and xerostomia (a Schirmer test result of
5 mm/5 min and a Saxon test result of <2.7 ml/2 min), and/or a combination of other clinical features suggestive of pSS, including bilateral chronic parotid gland enlargement, purpura or arthralgia, with laboratory abnormalities including antinuclear antibody (ANA; 12 patients), anti-SSA (four patients) or anti-SSB (three patients) antibodies or rheumatoid factor (eight patients). The clinical diagnoses in this group included hypothyroidism, fibromyalgia, depression, hepatitis C virus-associated sicca complex or drug-induced xerostomia/xerophthalmia. Patients were not eligible for entry into group 1 or 2 if they had any signs or symptoms giving rise to the suspicion of sSS (oral or ocular dryness reported in response to specific questions, or abnormal Schirmer's or Saxon's test results). In the sSS group, the diagnoses associated with SS were RA (n = 7), SLE (n = 5) and systemic sclerosis (n = 2). The study was approved by the Medical Ethics Committee of the University of Szeged. The authors declare that the patients gave informed consent to the examinations detailed in this work.
ELISA method
A recombinant protein containing the 16-mer peptide sequence KRTVPPGECFIQFLSE (KRSE, m3AChR213228) fused with GST (GST-KRSE) was produced in Escherichia coli as described previously [10, 11]. Microtitre plates were coated with 100 µl of 10 µg/ml GST-KRSE in 0.1 M Na2CO3 buffer, pH 9.6, containing 10 mM dithiothreitol overnight at 4°C. For every sample, GST alone was also coated in parallel with the GST-KRSE, and the quantities of antigens used for coating were equalized to the GST portion of the fusion partner. GST was therefore applied at a concentration of 9 µg/ml in the same coating buffer. Subsequently, the plates were washed with phosphate-buffered saline and blocked with SuperBlock Blocking Buffer (Pierce) for 1 h at room temperature. The plates were next washed, and the serum samples were added at a dilution of 1:100 in blocking solution, followed by incubation for 2 h at 37°C. After washing, incubation followed with horseradish peroxidase-labelled goat anti-human IgG antibody (1:10 000; Sigma) in blocking solution. After thorough washing, the reaction was developed with o-phenylenediamine in phosphatecitric acid buffer, pH 5.0, for 30 min at room temperature in the dark. The optical density (OD) values were measured at 492 nm. The peptide-specific OD value for each serum was calculated by subtracting the OD value of the GST from that of the GST-KRSE of the corresponding sample. The cut-off level between normal and positive values was taken as the mean + 2 S.D. in the healthy control group; this calculation resulted in a cut-off OD of 0.24.
Statistical methods
Correlations between the anti-m3AChR213228 antibody levels (quantified with the OD values) and continuous variables (e.g. age and disease duration) were assessed with Pearson's correlation test, while the Spearman signed rank test was used to investigate an association between the OD values and categorical variables (the presence or absence of organ manifestations or serological positivities). To investigate a correlation between the anti-m3AChR213228 levels and the number of extraglandular organ manifestations, the JonckheereTerpstra test was applied, as this was considered most appropriate for the testing of whether the distributions differ in a specified direction (i.e. whether an increasing number of organ manifestations is associated with higher amounts of anti-m3AChR213228 antibodies). Comparisons between the pSS group and the other patient groups were performed with an analysis of variance (ANOVA) test, with Dunnett's multiple comparison test as a post hoc test (differences between means), or with
2 tests (differences between the frequency distribution of occurrences).
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Results
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Sixty-six of the 73 pSS patients (90%) proved to be anti-m3AChR213228 antibody-positive. The various extraglandular organ manifestations and other clinical data, together with the serological abnormalities in the anti-m3AChR213228-positive and -negative groups and in the overall group of pSS patients are shown in Table 1. The antibody concentrations were not associated with the age of the patients or the disease duration, or with the severity of the glandular insufficiency, as assessed by the stimulated whole saliva production measured with the Saxon test. However, an increasing number of extraglandular organ manifestations in a given patient correlated positively with the concentration of anti-m3AChR213228 antibodies (P<0.05; Fig. 1). In fact, each of the organ involvements was more common in the anti-m3AChR213228-positive patients than in those without this antibody, although this difference was statistically significant only in the case of leucopenia. As the m3 subtype of the AChR is also the functionally dominant receptor in the gastrointestinal and the urinary tracts [12, 13], we examined whether there is a relationship between the amount of anti-m3AChR213228 antibodies and the degree of parasympathetic dysfunction in these organs, as measured in our previous studies [14]. No correlation was found (data not shown).
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TABLE 1. Demographic features and the presence of extraglandular organ manifestations and serological variables in anti-m3AChR213228-positive and anti-m3AChR213228-negative pSS patients and in the overall cohort
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FIG. 1. Box and whisker plots representing anti-m3AChR213228 antibody levels in pSS patients with various numbers of extraglandular organ manifestations. An increasing number of extraglandular organ manifestations correlated positively with the mean anti-m3AChR213228 antibody titres (P<0.05). Horizontal lines inside the box show median OD values; boundaries of the box show 25th and 75th percentiles; whiskers represent minimum and maximum values that are not extreme or outlier values. The numbers above the horizontal axis indicate the numbers of patients in each subgroup.
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The frequencies of anti-m3AChR213228 positivity in the five comparison groups are demonstrated in Table 2, together with the mean antibody concentrations. The distribution of the antibody levels is presented in Fig. 2. Both the mean antibody concentrations and the frequencies of anti-m3AChR213228 positivity were significantly lower in each of the comparison groups than in the pSS group. The sensitivity of the anti-m3AChR213228 measurement to pSS proved to be 90%. The specificity values are to be seen in Table 2. The anti-m3AChR213228 testing differentiates between the pSS and the various non-pSS patient groups with specificities varying in the interval 5071%. The likelihood ratios (Table 2) indicate that a negative anti-m3AChR213228 result decreases the probability of pSS to 0.130.19 in the various patient comparison groups, while a positive test result leads to a 1.81- to 3.16-fold increase in the likelihood of pSS.
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TABLE 2. Results of the anti-m3AChR213228 ELISA in the six groups examined, and the statistical parameters assessing the potential of the method to discriminate between pSS and the other comparison groups
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FIG. 2. Scattergram demonstrating the distribution of the OD values measured with the anti-m3AChR213228 ELISA in the different study groups. The cut-off value is indicated by the horizontal line. The number of patients with positive test results and the statistical comparison between the groups are shown in Table 2.
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Discussion
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In recent years, novel autoantibodies that react with the m3AChR have been identified in pSS. These antibodies have been demonstrated to play an essential role in the elicitation of the glandular dysfunction in the NOD mouse model of pSS [2, 5], possibly via binding to and exerting an inhibitory effect on the receptor [15, 16]. We recently developed an ELISA system which enables us to measure m3AChR213228 antibody levels on a large scale [6]. To our knowledge, this is the first report on the clinical associations of anti-m3AChR autoantibodies in pSS.
The data we obtained from a relatively numerous cohort of pSS patients indicate that these antibodies are present in the vast majority of pSS patients. There are some indications in our results that the presence or increasing concentrations of anti-m3AChR213228 antibodies may be associated with more extraglandular manifestations in the given patient, i.e. with a more systemic form of pSS. In this regard, it may be interesting that, similarly to the present results, we previously found that each extraglandular organ involvement occurred with higher frequency in a subgroup of pSS patients who demonstrated an impaired microvascular response to cholinergic stimulation than in those in whom a normal response was observed [17]. However, as very few statistically significant differences have been revealed, this possibility needs further confirmation.
An intriguing question with regard to antimuscarinic autoantibodies is their in vivo role in the elicitation of the glandular dysfunction in pSS. We therefore tested whether there is a correlation between the level of anti-m3AChR213228 antibodies and the degree of salivary gland dysfunction, but we failed to demonstrate a direct relationship. However, in view of the high prevalence of anti-m3AChR213228 antibodies in pSS, together with the results of previous experiments [2, 4, 5, 17], and the physiological importance of the m3AChR in the regulation of the glandular function, we consider that the question of whether these autoantibodies play some role in the pathogenesis of pSS remains justified.
The pSS group was significantly different from each of the comparison groups in terms of both the frequency of anti-m3AChR213228 positivity and the mean anti-m3AChR213228 levels. However, the potential of anti-m3AChR213228 antibodies for clinical discrimination between pSS and the examined comparison groups of patients with the clinical conditions that most commonly cause differential diagnostic difficulties in clinical practice proved inappropriately low in the present setting. Despite the statistically highly significant differences in the various groups, the number of anti-m3AChR213228-positive patients was sufficiently high in all the comparison groups to give the test modest specificity. An analogous approach to the assessment of the clinical value of the test is the calculation of likelihood ratios. This indicates that a negative test result is relatively strong evidence against the diagnosis of pSS, while a positive test result itself is not sufficiently helpful. This is still true even if we consider that the members of the suspSS group were selected in a fairly strict way. These patients exhibited many features resembling pSS, including not only a subjective, but also an objective glandular dysfunction, compatible in severity with that required for the diagnosis of pSS in the AmericanEuropean classification criteria [7]. Many of them were ANA-positive and some of them also had anti-SSA/SSB antibodies. However, none of them could be considered to have pSS, either as concerns the number of classification criteria or by clinical judgement.
The marked difference between the pSS and sSS groups with regard to anti-m3AChR213228 antibodies may support the view that these are diseases with different pathology and pathogenesis [18]. However, with regard to the data in the literature, this finding requires confirmation. The presence of antibodies to the human m3AChR in an evaluable number of patients with diseases other than pSS has been reported in only one publication [3]. In that study, 14 of 17 sSS patients were found to be anti-m3AchR-positive. A further difference between the results of that study and ours is that the prevalence of anti-m3AChR antibodies in a control group termed non-SS dry eye patients was very low. In addition to the fact that a different (although partly overlapping) sequence of the human m3AChR was used as antigen, tested with a different method (synthetic peptide ELISA), the control patient selection was probably also different from ours, as evident from the above considerations about the suspSS group. The involvement of more comparison patients and more specific determination of the control groups may help answer the questions raised by the present preliminary study concerning the disease-specificity of anti-m3AChR antibodies.
In conclusion, we have revealed a high prevalence of anti-m3AChR213228 antibodies in pSS. In our opinion, an attempt should be made to increase the specificity of the applied ELISA test in order to assess whether anti-m3AChR testing may be of clinical benefit as a differential diagnostic aid in pSS.
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Acknowledgments
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This work was supported by the Hungarian Scientific Research Fund (OTKA grant T038303), by the Hungarian Ministry of Health (ETT 214/2001) and by a PhD Fellowship grant from the Bay Zoltán Foundation for Applied Research to A.G.
The authors have declared no conflicts of interest.
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References
|
---|
- Bacman S, Sterin-Borda L, José Camusso J, Arana R, Hubscher O, Borda E. Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjögren's syndrome. Clin Exp Immunol 1996;104:4549.[CrossRef][ISI][Medline]
- Robinson CP, Brayer J, Yamachika S et al. Transfer of human serum IgG to nonobese diabetic Igµnull mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren's syndrome. Proc Natl Acad Sci USA 1998;95:753843.[Abstract/Free Full Text]
- Bacman S, Berra A, Sterin-Borda L, Borda E. Muscarinic acetylcholine receptor antibodies as a new marker of dry eye Sjögren's syndrome. Invest. Ophthalmol Vis Sci 2001;42: 3217.[Abstract/Free Full Text]
- Waterman SA, Gordon TP, Rischmueller M. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjögren's syndrome. Arthritis Rheum 2000;43:164754.[CrossRef][ISI][Medline]
- Nguyen KHT, Brayer J, Cha S et al. Evidence for antimuscarinic acetylcholine receptor antibody-mediated secretory dysfunction in NOD mice. Arthritis Rheum 2000;43:2297306.[CrossRef][ISI][Medline]
- Marczinovits I, Kovacs L, Gyorgy A et al. A peptide of human muscarinic acetylcholine receptor 3 is antigenic in primary Sjögren's syndrome. J Autoimmun 2005;24:4754.[CrossRef][ISI][Medline]
- Vitali C, Bombardieri S, Jonsson R et al. Classification criteria for Sjögren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002;61:5548.[Abstract/Free Full Text]
- Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1998;31:31524.[CrossRef][Medline]
- Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:12717.[ISI][Medline]
- Molnár J, Marczinovits I, Kiss M et al. Recombinant antigens by fusion of antigenic epitopes to a GST partner. Prog Biotechnol 1998;15:6916.
- Marczinovits I, Somogyi CS, Patthy A, Németh P, Molnár J. An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli. J Biotechnol 1997;56:818.[CrossRef][ISI][Medline]
- Stengel PW, Yamada M, Wess J, Cohen ML. M3 receptor knockout mice muscarinic receptor function in atria, stomach fundus, urinary bladder and trachea. Am J Physiol Regul Integr Comp Physiol 2002;282:R14439.[Abstract/Free Full Text]
- Chess-Williams R, Chapple CR, Yamanishi T, Yasuda K, Sellers DJ. The minor population of M3 receptors mediate contraction of human detrusor muscle in vitro. J Auton Pharmacol 2001;21:2438.[CrossRef][ISI][Medline]
- Kovács L, Papós M, Takács R et al. Autonomic nervous system dysfunction involving the gastrointestinal and urinary tracts in primary Sjögren's syndrome. Clin Exp Rheumatol 2003;21:697703.[ISI][Medline]
- Cavill D, Waterman SA, Gordon TP. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sjögren's syndrome. Scand J Immunol 2004;59:2616.[CrossRef][ISI][Medline]
- Li J, Ha YM, Ku NY et al. Inhibitory effects of autoantibodies on the muscarinic receptor in Sjögren's syndrome. Lab Invest 2004;84:14308.[CrossRef][ISI][Medline]
- Kovács L, Török T, Bari F et al. Impaired microvascular response to cholinergic stimuli in primary Sjögren's syndrome. Ann Rheum Dis 2000;59:4853.[Abstract/Free Full Text]
- Manoussakis MN, Moutsopoulos HM. Sjögren's syndrome: autoimmune epitheliitis. Baillieres Best Pract Res Clin Rheumatol 2000;1:7395.[CrossRef]
Submitted 21 December 2004;
revised version accepted 1 April 2005.