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Detection of antitype 3 muscarinic acetylcholine receptor autoantibodies in the sera of Sjgren's syndrome patients by use of a transfected cell line assay.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 8, August 2004, pp 2615–2621
DOI 10.1002/art.20371
© 2004, American College of Rheumatology
Detection of Anti–Type 3 Muscarinic Acetylcholine Receptor
Autoantibodies in the Sera of Sjögren’s Syndrome Patients
by Use of a Transfected Cell Line Assay
Juehua Gao,1 Seunghee Cha,1 Roland Jonsson,2 Jeffrey Opalko,3 and Ammon B. Peck1
M3R autoantibodies bound to CHO-transfected cells
revealed the presence of anti-M3R autoantibodies in SS
patients (9 of 11) but not in healthy controls (0 of 11).
Although the anti-M3R autoantibodies detected in patient sera were of multiple isotypes, the most consistently detected were IgG1, IgG3, and IgA.
Conclusion. Using a newly constructed cell line
expressing human M3R, anti-M3R autoantibodies were
easily detected in sera from SS patients. These autoantibodies were skewed toward the IgG1, IgG3, and IgA
isotypes, probably recognizing a tertiary epitope created
by extracellular domains of the receptor protein. AntiM3R autoantibodies represent a highly promising clinical marker for the identification of SS.
Objective. Sjögren’s syndrome (SS) is an autoimmune disease affecting primarily the salivary and
lacrimal glands, leading to dry mouth and dry eyes.
Recent studies have suggested that autoantibodies reactive with the type 3 muscarinic acetylcholine receptors
(M3Rs) expressed on salivary and lacrimal gland cells
may be highly specific for SS. To test this hypothesis, we
constructed a cell line expressing the human M3R gene
in order to screen for anti-M3R autoantibodies in sera
from SS patients.
Methods. Complementary DNA encoding the
open-reading frame (ORF) of the human M3R gene was
amplified, ligated into the pcDNA5/FRT/V5-His-TOPO
TA vector, and then used to transform Escherichia coli
bacteria. Plasmid DNA containing the M3R ORF with
the correct orientation was transfected into Flp-In Chinese hamster ovary (CHO) cells using Flp
recombinase–mediated site-specific recombination. An
M3R-transfected CHO cell line, selected and propagated in hygromycin, was used to detect anti-M3R
autoantibodies in SS patient and healthy control sera by
flow cytometry.
Results. Testing of sera for the presence of anti-
Sjögren’s syndrome (SS) is an autoimmune disease characterized by a progressive and chronic mononuclear cell infiltration of the exocrine glands, in particular, the lacrimal and salivary glands, resulting in
symptoms of dry eyes and dry mouth (1). The identification of SS in a given patient has been historically
somewhat arbitrary because of the use of multiple
classification criteria for clinical diagnosis worldwide (2);
however, a recent joint effort by American and European researchers has now established a more standardized set of diagnostic markers (3). Although the demonstration of either histologic evidence of inflammation on
biopsy of minor salivary glands and/or serologic evidence of circulating autoantibodies against the nuclear
antigens SSA/Ro and/or SSB/La is recommended, this
new standardized classification scheme still relies on
subjective criteria and evidence of abnormal ocular and
salivary gland function.
A variety of autoantibodies have been reported
to be present in the sera of patients with primary and
secondary SS, including anticholinergic autoantibody
(4). Recent studies from our laboratory (5,6) have
provided evidence that antibodies reactive with the type
Supported in part by PHS grants from the National Institute
of Dental and Craniofacial Research (DE-05152 and DE-55304) and
the National Institute of Allergy and Infectious Diseases (AI-47483).
1
Juehua Gao, MD, Seunghee Cha, DDS, PhD, Ammon B.
Peck, PhD: University of Florida, Gainesville; 2Roland Jonsson,
DMD, PhD: University of Bergen, Bergen, Norway; 3Jeffrey Opalko,
MS: Ixion Biotechnology, Inc., Alachua, Florida.
Dr. Peck is a scientific consultant to, and has a financial
interest in, Ixion Biotechnology, Inc. Drs. Gao, Cha, and Peck are
coinventors of a pending patent related to technology used in these
studies and may benefit from royalties paid to the University of Florida
in relation to any future commercialization.
Address correspondence and reprint requests to Ammon B.
Peck, PhD, Department of Pathology, Immunology, and Laboratory
Medicine, College of Medicine, PO Box 100275, University of Florida,
Gainesville, FL 32610. E-mail: peck@pathology.ufl.edu.
Submitted for publication September 2, 2003; accepted in
revised form March 29, 2004.
2615
2616
GAO ET AL
3 muscarinic acetylcholine receptor (M3R) may be the
primary underlying cause for the loss of secretory function that leads to dry mouth, a common complaint of SS
patients. Unlike the many intracellular antigens that give
rise to autoantibodies, the M3R is a membrane-bound
protein involved in the parasympathetic neurostimulation of exocrine and some nonexocrine cells. Although
the evidence is indirect, studies showing that sera from
patients with either primary or secondary SS can inhibit
smooth muscle contraction in isolated strips of bladder
support the concept that autoantibodies can interfere
with parasympathetic neurotransmission (7).
A persistent problem in identifying SS is that the
autoantibodies currently being detected in patient sera
are not specific for SS per se, and their relevance to
disease remains unclear, despite the fact that the majority of classification criteria rely on their detection. For
example, only 40–60% of patients with primary SS have
detectable anti-SSB/La autoantibodies while 50–60%
have anti-SSA/Ro; however, these autoantibodies can be
prevalent in other connective tissue disorders, such as
systemic lupus erythematosus (SLE), as well (8). Furthermore, the frequencies of anti-SSA/Ro and antiSSB/La antibodies in SS patients often depend on the
detection methods and setting of the study.
Identification of autoantibodies against M3Rs in
the sera of SS patients, together with studies showing the
probable importance of these autoantibodies in the
disease pathogenesis, has raised interest in attempts to
measure anti-M3R autoantibodies in patient sera. To
this end, studies using synthetic peptides homologous to
the M3R have proved disappointing, especially by failing
to show specificity (9) despite a report on the positive
reactivity of sera toward a 25-mer synthesized peptide
(10). This raises the possibility that anti-M3R autoantibodies recognize an epitope created by the tertiary
structure of the transmembrane segments. In the present
study, we developed a test system in which the human
M3R gene was cloned and expressed in the Chinese
hamster ovary (CHO) cell line in order to maintain
inherent folding of the membrane-associated protein.
We used this transfected cell line to examine the ability
to detect anti-M3R autoantibodies in the sera of patients
with primary and secondary SS.
PATIENTS AND METHODS
Serum specimens. Sera used in this study were obtained and prepared by one of us (RJ) from either normal
healthy individuals or SS patients living in Norway. Primary
and secondary SS were diagnosed using the European classi-
fication criteria for SS (11) and the American College of
Rheumatology (ACR; formerly, the American Rheumatism
Association) criteria for SLE (12) and for rheumatoid arthritis
(RA) (13). Sera were collected under a research protocol
approved by the Regional Committee for Medical Research
Ethics of Western Norway. The current studies were performed under University of Florida Institutional Review
Board–approved protocol number 605-2000.
Amplification of M3R from Jurkat cells. The coding
region for human M3R was amplified by reverse transcriptase–
polymerase chain reaction (RT-PCR) using messenger RNA
isolated from 1 ⫻ 106 Jurkat cells (TIB-152; American Type
Culture Collection, Rockville, MD) a cell line known to
express the M3R (14). The PCR was performed as described
elsewhere (6), with synthesized oligonucleotide primers 5⬘CGGAATTCGAGTCACAATGACCTTGCACAA-3⬘ (forward) and 5⬘-CAAGGCCTGCTCGGGTGC-3⬘ (reverse). A
1.7-kbp sequence encoding the M3R open-reading frame
(ORF) was purified using a gel extraction kit from Qiagen
(Valencia, CA) and quantified by spectrophotometric analysis
(optical density measured at 260 nm).
Construction of the M3R cloning vector. The isolated
PCR product was ligated into the pcDNA5/FRT/V5-HisTOPO TA cloning vector (Invitrogen, Carlsbad, CA) containing the ampicillin resistance gene. Ligation and transformation
of Escherichia coli were performed according to the manufacturer’s protocol. Several transformed colonies were selected,
and each colony was grown overnight in 3 ml of Luria-Bertani
broth supplemented with 50 ␮g/ml of ampicillin in a shaking
incubator (250 revolutions per minute) at 37°C. Plasmid DNA
was extracted (Mini-Preps DNA Purification kit; Qiagen), and
a restriction enzyme digestion was performed with Nhe I and
Bfr I (Roche Diagnostics, Mannheim, Germany) to identify
which plasmids possessed the ORF of the M3R gene in the
correct orientation.
Transfection of Flp-In CHO cells with M3R. The
Flp-In CHO cell line (Invitrogen) was maintained in UltraCHO Medium (catalog no. R758-07; BioWhittaker, Walkersville, MD) supplemented with 0.1% Zeocin (Research Products International, Mount Prospect, IL). Flp-In CHO cells in
growth phase were cotransfected with the recombinant
pcDNA5/FRT/V5-His-TOPO TA vector containing the M3R
gene and the pOG44 plasmid expressing the Flp recombinase
gene, as described in the manufacturer’s instructions (Invitrogen). Flp-In CHO cells were incubated for 24 hours to allow
for expression of the hygromycin resistance gene, then selected
in growth medium ProCHO 4 (BioWhittaker) supplemented
with 0.80 mg/ml of hygromycin B (Research Products International) and 5% fetal bovine serum.
M3R expression on transfected Flp-In CHO cells
detected by anti-M3R antibody and Western blotting. Transfected and nontransfected Flp-In CHO cells were grown to
near confluence on glass-bottomed microwell dishes (catalog
no. P35GCol-1.5-14C; MatTek, Ashland, MA), fixed in 3.7%
formalin, washed thrice with phosphate buffered saline (PBS),
and the plates were stored at 4°C until used. Expression of the
M3R protein was determined by incubating the fixed cells first
in rabbit anti-human M3R antibody (catalog no. AS-3741G;
Research and Diagnostic Antibodies, Berkeley, CA) at 1:100
dilution for 30 minutes, then with fluorescein isothiocyanate
(FITC)–conjugated goat anti-rabbit IgG (catalog no. A11034;
ANTI–TYPE 3 MUSCARINIC RECEPTOR AUTOANTIBODIES IN SS PATIENTS
Molecular Probes, Eugene, OR) for 30 minutes. The cells were
washed 5 times following each antibody treatment.
In addition, Flp-In CHO cells were collected, pelleted
by centrifugation, and the cell pellet was lysed by adding 1 ml
of 50 mM Tris buffer (pH 7.5 using HCl). This mixture was
sequentially frozen in an ethanol/dry ice bath and thawed in
warm water 3 times. The lysate was then drawn through
18-gauge and 26-gauge needles to mechanically dissociate any
aggregated material. Membrane fractions were prepared by
centrifugation of the lysate first at 500g for 5 minutes, then the
supernatant at 40,000g for 20 minutes at 4°C. The pelleted
membrane fraction was washed twice with 50 mM Tris HCl
buffer (pH 7.5), with each wash followed by centrifugation at
40,000g for 15 minutes at 4°C. Following the second wash, the
pellet was resuspended in 1 ml of Tris HCl buffer (pH 7.5).
The membrane proteins from each membrane fraction were
size-separated using 12% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes, and the M3R-His–tagged fusion protein
was visualized with alkaline phosphatase–conjugated anti-His
antibody at a dilution of 1:2,000 and nitroblue tetrazolium/
BCIP solution (Sigma, St. Louis, MO).
Detection of anti-M3R autoantibodies in sera using
transfected Flp-In CHO cells. Nontransfected and pcDNA5/
FRT/V5-His M3R–transfected Flp-In CHO cells were collected from culture, washed once with PBS, and resuspended
in fluorescence-activated cell sorter (FACS) buffer (PBS, 2%
bovine serum albumin, 0.01% NaN3) at a density of 1 ⫻ 107
cells/ml. Aliquots of cells were incubated for 2 hours at 4°C
with 5 ␮l of sera from SS patients or healthy donors. Cells were
washed once with FACS buffer and stained with either FITCconjugated goat anti-human IgG (PharMingen, San Diego,
CA) or FITC-conjugated goat anti-human IgA, IgG1, IgG2,
IgG3, IgG4, IgE, and IgM (Accurate, Westbury, NY) for 30
minutes at 4°C. After a final wash with FACS buffer, the cells
were resuspended and analyzed using a FACScan cytometer
(Becton Dickinson, Mountain View, CA).
RESULTS
Construction of a transfected cell line stably
expressing human M3R. For this study, we constructed
a cell line that is transfected with the human M3R gene
expressed from a vector system incorporated directly
into the cells’ genomes. To accomplish this, cDNA of the
ORF for the human M3R gene was generated by PCR,
ligated into the pcDNA5/FRT/V5-His-TOPO-TA vector, and used to transform E coli. Following sequencing
of the insert for fidelity and orientation, genetically
manipulated Flp-In CHO cells were cotransfected with
the recombinant human M3R-pcDNA5/FRT/V5-HisTOPO-TA plasmid and the Flp recombinase–containing
pOG44 plasmid for generation of a stably transfected
cell line.
To determine if the transfected cells expressed
human M3R as a membrane protein, an aliquot each of
the transfected and nontransfected cells was stained with
2617
Figure 1. Expression of human type 3 muscarinic acetylcholine receptors (M3Rs) in transfected Flp-In Chinese hamster ovary (CHO) cells.
Expression of human M3R fusion protein in transfected Flp-In CHO
cells was confirmed by A, staining with anti–human M3R antibody and
B, Western blotting. Transfected and nontransfected cells growing as
monolayers were fixed in formalin then incubated in rabbit anti-human
M3R antibody, and were detected with a fluorescein isothiocyanate–
conjugated goat anti-rabbit IgG antibody. For Western blots, membrane proteins from lysed cells were separated on a 12% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis gel, transferred to
a nitrocellulose membrane, and probed with alkaline phosphatase–
conjugated anti-His antibody. A 65-kd protein band was seen in lysates
of transfected (T) Flp-In CHO cells, but not in lysates of the
nontransfected (N-T) Flp-In CHO cells. MWr ⫽ relative molecular
weight.
anti–human M3R antibody (Figure 1A). In addition,
membrane fractions were prepared from both transfected and nontransfected Flp-In CHO cells undergoing
expansion as suspension cultures. Proteins from the
membrane preparations were separated by electrophoresis and screened by Western blotting using an anti-His
antibody (Figure 1B). Transfected Flp-In CHO cells
stained positively with the anti-M3R antibody. Western
blots of membrane preparations showed the expected
65-kd protein band in the human M3R–transfected
Flp-In CHO cells, but not in the nontransfected Flp-In
CHO cells. Thus, we constructed a system consisting of
a parental CHO cell line that did not express a muscarinic acetylcholine receptor (control) plus a CHO cell
line that constitutively expressed the human M3R (experimental).
Detection of M3R autoantibodies and isotypes in
sera from SS patients. Sera collected from patients with
primary SS (n ⫽ 5), patients with secondary SS (n ⫽ 6),
and normal healthy individuals (n ⫽ 11) were examined
for the presence of detectable anti-M3R autoantibodies
using the human M3R–transfected Flp-In CHO system.
2618
Figure 2. Representative flow cytometric analysis of human type 3
muscarinic acetylcholine receptor (M3R) autoantibody in the sera of
patients with Sjögren’s syndrome (SS). Anti-M3R autoantibodies were
detected in all sera from patients with secondary (2°) SS diagnosed as
having systemic lupus erythematosus (SLE) and in a majority of sera
from patients with primary (1°) SS when incubated with Flp-In Chinese
hamster ovary (CHO) cells transfected with the human M3R (hM3R)
gene, but not with nontransfected control Flp-In CHO cells. Also
presented are the results using a serum from a patient with primary SS
that failed to show detectable autoantibody. All analyses include a
comparison with sera from normal healthy individuals as controls.
Letters and numbers in parentheses are patient identification codes.
Patients were classified using the American–European
Consensus Group criteria for SS and the ACR criteria
for SLE and RA (11–13). Individual sera were incubated
with either 1 ⫻ 106 human M3R–transfected or 1 ⫻ 106
nontransfected Flp-In CHO cells, followed by treatment
with FITC-conjugated goat anti-human IgG secondary
antibody. Flow cytometric analyses of each reaction pair,
as presented in part in Figure 2, indicated that 3 of 5 sera
from patients with primary SS, 6 of 6 sera from patients
with secondary SS, but 0 of 11 sera from normal healthy
controls reacted with the human M3R–transfected
Flp-In CHO cells. No sera reacted with the nontransfected Flp-In CHO cells.
To determine if the present assay system can
GAO ET AL
distinguish the individual isotypes of anti–human M3R
autoantibodies in human sera, SS patient sera were
incubated with either 1 ⫻ 106 human M3R–transfected
or 1 ⫻ 106 nontransfected Flp-In CHO cells, followed by
treatment with FITC-conjugated goat anti-human Ig
isotype-specific secondary antibody. Flow cytometric
analyses revealed that anti–human M3R autoantibodies
of any Ig isotype could be present in any individual
serum, but that antibodies of the IgG1, IgG3, and IgA
isotypes were detected most consistently (Figure 3).
However, in some patients, levels of IgG4 isotype autoantibody were also significant. Little or no IgE was
detected, and neither IgM nor IgG2 isotypes proved
consistently significant. No functional studies have been
completed using the individual isotype autoantibodies.
Stability of the human M3R–transfected Flp-in
CHO cells in expressing M3R protein. To determine the
long-term stability of this human M3R–transfected
Flp-In CHO cell system for expressing membraneassociated human M3R protein, flow cytometric analyses using each of the sera from the SS patients and
normal healthy individuals were performed at weekly
intervals over a 10-week period. In all cases, no differences in responses were observed, suggesting good stability in the expression of human M3R by the Flp-In
system, as well as reproducibility in the detection of
anti–human M3R autoantibodies (data not shown).
DISCUSSION
In the present study, we present preliminary data
indicating a high prevalence of anti-M3R autoantibodies
in the sera of patients classified as having SS according
to the American–European Consensus Group criteria,
but not in the sera of normal healthy individuals. Detection of anti-M3R autoantibodies in SS patients was
facilitated by the construction of a transgenic cell line
expressing the human M3R protein, as proposed previously by Konttinen et al (15).
Unlike our earlier M3R-transfected cell lines that
were constructed with the rodent M3R gene (6), the
model presented in the current study expresses not only
the human M3R gene, but also the M3R protein from a
gene incorporated within the cell line’s genome, as
opposed to an epigenetic element. This has been made
possible through the use of the commercially available
Flp-In CHO cell system and has resulted in a stable
M3R gene-expression system. Because this system was
designed to use flow cytometric analysis, we were able to
evaluate additional features, for example, the isotypes of
the anti-M3R autoantibodies present within individual
Figure 3. Isotype analysis of human type 3 muscarinic acetylcholine receptor (M3R) autoantibodies in the sera of Sjögren’s syndrome (SS) patients. Transfected and nontransfected Flp-In Chinese
hamster ovary (CHO) cells were incubated with sera from patients and normal healthy controls for 2 hours at 4°C. Cells were washed and counterstained with fluorescein isothiocyanate–conjugated
goat anti-human IgA, IgG1, IgG2, IgG3, IgG4, IgE, or IgM for an additional 30 minutes. After 2 washes with fluorescence-activated cell sorter (FACS) buffer, the cells were resuspended in FACS
buffer and analyzed using a FACScan flow cytometer. Isotype analysis from 3 patients with primary (1°) SS and 2 with secondary (2°) SS with systemic lupus erythematosus (SLE) and/or rheumatoid
arthritis (RA) are shown. Anti–human M3R autoantibodies belonging to the IgG1, IgG3, and IgA isotypes were detected consistently. Other isotypes were occasionally observed. Numbers in
parentheses are patient identification codes.
2620
sera. The M3R-transfected CHO cell system establishes
the methodology for a quick and simple diagnostic test
with both a positive-expressing cell line and a negativeexpressing cell line. Theoretically, the Flp-In system
allows site-specific integration, which results in all cells
being isogenic after selection, and thus, no subcloning is
required for development of pure populations. However, not all cells appear to express M3R at the same
time or the same intensity. This may suggest a heterogeneity in protein expression related to the cell cycle, as
implied by the results shown in Figure 1A, where the
larger cells undergoing division appeared not to stain
with anti–human M3R antibody. Further investigation
of this feature is required.
In previous studies using the NOD mouse and its
congenic strains as a model of human SS (6), we were
able to dissect the pathogenesis and clinical onset of
disease into 3 distinct, but interdependent, phases. The
first phase involves a series of physiologic and biochemical changes in the exocrine tissues that are independent
of the immune system. The second phase involves a
progressive immune attack against the exocrine tissues,
apparently in response to the cellular damage resulting
from the phase 1 events, that is characterized by lymphocytic infiltration of the exocrine glands. The third
phase is the loss of secretory function, an event that is
dependent on the production of IgG anti-M3R antibodies (16). A correlation between the appearance of antiM3R autoantibodies and the development of clinical
disease, which is under active investigation, has led to
recent attempts to develop a simple test using anti-M3R
autoantibodies present in patient sera as a disease
marker (6,7,9,10). Blockage of neurosecretory pathways
by anti-M3R antibodies could explain not only the
interference with the receptor and postreceptor signaling pathways, which manifest as secretory dysfunction of
the salivary and lacrimal glands, but also the many other
complications seen in SS patients (e.g., mucosal dryness,
arthralgia, fatigue, and fibromyalgia).
Although the number of samples tested in the
preliminary analysis presented herein is relatively small,
two observations are of interest. First, the vast majority
of the autoantibodies most consistently detected were of
the IgG1, IgG3, and IgA isotypes. This is consistent with
our earlier observations in the NOD mouse model, in
which the anti-M3R autoantibodies were of the IgG1
isotype and knocking out the interleukin-4 gene (thereby
preventing isotype switching to IgG1) eliminated the
subsequent manifestation of clinical disease. This is also
consistent with discussions by other investigators sug-
GAO ET AL
gesting that the pathogenic IgG autoantibodies in SS
patients might be of the IgG1 and IgG3 isotypes.
The second observation of interest is that antiM3R autoantibodies could not be detected in sera from
2 of the 5 patients classified as having primary SS, yet all
6 sera from patients with secondary SS showed positive
reactions. This raises several interesting possibilities,
including 1) the diagnostic criteria used to classify
primary SS remain inexact, 2) the patients with primary
SS included in the present study may be in different
stages of the disease, and anti-M3R autoantibodies are
below the level of detection in some of these stages,
and/or 3) not all SS patients have detectable levels of
anti-M3R autoantibodies. With the recent consensus
about the criteria to be used to classify SS patients (3), it
is possible that the group of patients classified as having
primary SS may be narrowed, and this could have an
impact on the results of using this human M3R–
transfected Flp-In CHO system for analytic and clinical
testing. Future studies will focus on determining the
association between the detection of anti-M3R autoantibodies and the prediction of the disease and its severity. Such an expanded study is required for more conclusive findings; nonetheless, our attempts to develop a
simple, nonsurgical, SS-specific diagnostic test as presented herein should be a welcome advancement for the
patient, physician, and clinical laboratory.
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