Rheumatology 2005; 1 of 5doi:10.1093/rheumatology/keh672
Clinical associations of autoantibodies to human
muscarinic acetylcholine receptor 3213–228
in primary Sjo ¨ gren’s syndrome
L. Kova ´ cs, I. Marczinovits1, A. Gyo ¨ rgy3, G. K. To ´ th2, L. Dorgai3, J. Pa ´ l4,
J. Molna ´ r1and G. Pokorny
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 Sjo ¨ gren’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 Sjo ¨ gren’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 (m3AChR213–228) in a recombinant fusion peptide form was used as the antigen.
Results. Anti-m3AChR213–228antibody 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-m3AChR213–228positivity 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,
Conclusions. The presence of m3AChR213–228antibodies is a common feature in pSS. Although it is significantly more common
in pSS than in the comparison groups, anti-m3AChR213–228positivity is not exclusive to pSS.
KEY WORDS: Antimuscarinic acetylcholine receptor-3 autoantibody, Disease specificity, Muscarinic acetylcholine receptor-3213–228,
Primary Sjo ¨ gren’s syndrome.
Sjo ¨ gren’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 Sjo ¨ gren’s syndrome (pSS) if no
other systemic autoimmune disease is present, and as secondary
Sjo ¨ gren’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 autoanti-
bodies. 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) [1–4]. 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 (m3AChR213–228). 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 deter-
mine the prevalence, clinical associations and disease specificity
Patients and methods
Seventy-three pSS patients (70 females, mean age 55yr, range
30–82) were includedin this
American–European classification criteria for pSS . Five
comparison groups were included: (group 1) 40 patients with
RA (36 females, mean age 56yr, range 27–80), classified according
to the American College of Rheumatology (ACR) criteria for
RA ; (group 2) 19 SLE patients (16 females, mean age 40yr,
range 26–73), fulfilling the ACR classification criteria for SLE ;
(group 3) 14 sSS patients (all females, mean age 53yr, range
32–63), who met the American–European classification criteria for
sSS ; (group 4) 22 patients (21 females, mean age 52yr, range
21–78) 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
Correspondence to: L. Kova ´ cs. E-mail: firstname.lastname@example.org
Department of Rheumatology,1Department of Physiology and2Department of Medical Chemistry, Szeged, University of Szeged,3Bay Zolta ´ n Foundation
for Applied Research, Institute for Biotechnology, Szeged and4Hungarian Academy of Sciences, Clinical Neuroscience Research Group, Pe ´ cs, Hungary.
Submitted 21 December 2004; revised version accepted 1 April 2005.
? The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: email@example.com
1 of 5
Rheumatology Advance Access published May 11, 2005
by guest on June 2, 2013
(group 5) 40 healthy blood donors (all females, mean age
49yr, range 23–62yr). All of the patients in the suspSS group had
either subjective plus objective xerophthalmia and xerostomia
(a Schirmer test result of ?5mm/5min and a Saxon test result
of <2.7ml/2min), 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 rheuma-
toid 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 (n¼7),
SLE (n¼5) and systemic sclerosis (n¼2). 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.
A recombinant protein containing the 16-mer peptide sequence
KRTVPPGECFIQFLSE (KRSE, m3AChR213–228) fused with
GST (GST-KRSE) was produced in Escherichia coli as described
previously [10, 11]. Microtitre plates were coated with 100?l of
10mg/ml GST-KRSE in 0.1 M Na2CO3buffer, pH 9.6, containing
10mM 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?g/ml in the same coating buffer. Subsequently,
the plates were washed with phosphate-buffered saline and blocked
with SuperBlock Blocking Buffer (Pierce) for 1h 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 2h at 37?C. After washing, incubation followed
with horseradish peroxidase-labelled
antibody (1:10000; Sigma) in blocking solution. After thorough
washing, the reaction was developed with o-phenylenediamine
in phosphate–citric acid buffer, pH 5.0, for 30min at room
temperature in the dark. The optical density (OD) values were
measured at 492nm. 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 meanþ2 S.D.
in the healthy control group; this calculation resulted in a cut-off
OD of 0.24.
Correlations between the anti-m3AChR213–228antibody 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-m3AChR213–228levels and the number of extraglandular
organ manifestations, the Jonckheere–Terpstra 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-m3AChR213–228antibodies). 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 ?2tests (differences between the frequency
distribution of occurrences).
Sixty-six of the 73 pSS patients (90%) proved to be anti-
m3AChR213–228antibody-positive. The various extraglandular
organ manifestations and other clinical data, together with the
serological abnormalities in the anti-m3AChR213–228-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-m3AChR213–228antibodies (P<0.05; Fig. 1).
In fact, each of the organ involvements was more common in
TABLE 1. Demographic features and the presence of extraglandular organ manifestations and serological variables in anti-m3AChR213–228-positive and
anti-m3AChR213–228-negative pSS patients and in the overall cohort
Clinical or serological variableAnti-m3AChR213–228-positive (n¼66)
Age (yr; mean)
Disease duration (yr; mean)
Saxon test (ml/2min; mean)
Numbers indicate the numbers of patients involved, with the percentages in parentheses, unless otherwise stated.
Renal involvement: renal tubular acidosis with or without biopsy-proven chronic tubulointerstitial nephritis.
Vasculitis: palpable purpura or other, biopsy-proven skin vasculitic lesion.
Leucopenia was significantly more common in the anti-m3AChR213–228-positive pSS patients than in those without this antibody.
2 of 5L. Kova ´ cs et al.
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the anti-m3AChR213–228-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-m3AChR213–228anti-
bodies and the degree of parasympathetic dysfunction in these
organs, as measured in our previous studies . No correlation
was found (data not shown).
The frequencies of anti-m3AChR213–228positivity 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
positivity were significantly lower in each of the comparison
groups than in the pSS group. The sensitivity of the anti-
m3AChR213–228measurement to pSS proved to be 90%. The
specificity values are to be seen in Table 2. The anti-
m3AChR213–228testing differentiates between the pSS and the
various non-pSS patient groups with specificities varying in
the interval 50–71%. The likelihood ratios (Table 2) indicate that
a negative anti-m3AChR213–228result decreases the probability
of pSS to 0.13–0.19 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.
In recent years, novel autoantibodies that react with the m3AChR
have been identified in pSS. These antibodies have been demon-
strated 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 m3AChR213–228antibody levels on a large scale .
Toour knowledge,this isthe first report onthe 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-
m3AChR213–228antibodies may be associated with more extra-
glandular 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 . However, as very
few statistically significant differences have been revealed, this
possibility needs further confirmation.
An intriguing question with regard to antimuscarinic autoanti-
bodies 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-m3AChR213–228antibodies
and the degree of salivary gland dysfunction, but we failed to
demonstrate a direct relationship. However, in view of the high
prevalence of anti-m3AChR213–228antibodies 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
No of extraglandular manifestations
Anti-m3AChR-213-228 levels (OD)
n=6 n=17 n=28 n=13n=6 n=1 n=2
FIG. 1. Box and whisker plots representing anti-m3AChR213–228
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-m3AChR213–228antibody 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.
TABLE 2. Results of the anti-m3AChR213–228ELISA in the six groups examined, and the statistical parameters assessing the potential of the method
to discriminate between pSS and the other comparison groups
Anti-m3AChR213–228levels (mean OD)
No. of anti-m3AChR213–228-positive patients (%)
Positive likelihood ratio
Negative likelihood ratio
The mean OD values and the prevalences of anti-m3AChR213–228antibody positivity were significantly lower in each of the comparison groups than
in the pSS patients.
n.a., not applicable.
Clinical associations of human mAChR autoantibodies in primary SS 3 of 5
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The pSS group was significantly different from each of the
comparison groups in terms of both the frequency of anti-
m3AChR213–228positivity and the mean anti-m3AChR213–228
levels. However, the potential of anti-m3AChR213–228antibodies
for clinical discrimination between pSS and the examined com-
parison 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-m3AChR213–228-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 American–European classification
criteria . 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-m3AChR213–228antibodies may support
the view that these are diseases with different pathology and
pathogenesis . 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 . 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 consider-
ations 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
In conclusion, we have revealed a high prevalence of anti-
m3AChR213–228antibodies 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.
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 Zolta ´ n Foundation for Applied Research to A.G.
The authors have declared no conflicts of interest.
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? Antimuscarinic acetylcholine receptor-
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? Mean levels and occurrences are higher
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FIG. 2. Scattergram demonstrating the distribution of the OD
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