Detection of antitype 3 muscarinic acetylcholine receptor autoantibodies in the sera of Sjgren's syndrome patients by use of a transfected cell line assay.код для вставкиСкачать
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: email@example.com. 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. 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