The University of Liverpool, Clinical Dental Sciences, Liverpool, UK.
Correspondence to: L. J. Dawson. E-mail: ldawson{at}liv.ac.uk
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Abstract |
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Methods. IgG was purified by affinity chromatography using protein G from sera collected from 15 patients with pSS. Antibody detection was by Western blotting, whole-cell enzyme-linked immunsorbent assay (ELISA) and immunoblotting. The antisecretory activity of the IgG faction was determined using fura-2 microfluorimetry to measure changes in intracellular Ca2+ activity ([Ca2+]i) in human and mouse salivary gland acinar cells and in Chinese hamster ovary (CHO) cells transfected with human M3 receptors (CHO-M3).
Results. We found no specific M3 receptor recognition by the IgG fraction obtained from pSS patients using either Western blotting or ELISA or immunoblot techniques in which epitope conformation were preserved. Chronic exposure to pSS IgG had no effect on agonist-evoked Ca2+ signals measured in human or mouse submandibular acinar cells or in CHO-M3 cells. However, acute application of IgG from Sjögren's syndrome patients produced a rapidly reversible reduction in the agonist-stimulated elevation in [Ca2+]i.
Conclusion. These data represent the first demonstration of salivary acinar cell inhibition by pSS IgG; however, this inhibition was found to be reversible. Our data also show that pSS IgG binding to M3R cannot be visualized by conventional immunological approaches.
KEY WORDS: Sjögren's syndrome, Anti-muscarinic M3 antibodies, Human salivary acinar cells, Calcium
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Introduction |
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Fluid secretion by salivary acinar cells is controlled by parasympathetic nerves which release acetylcholine (ACh). Recent experiments with knock-out mice have confirmed that fluid secretion from salivary acinar cells is predominantly mediated by activation of muscarinic type-3 receptors (M3R) [6, 7]. These receptors are G-protein linked and their activation triggers a second-messenger cascade which culminates in a rise in intracellular calcium ([Ca2+]i) and activation of the K+ and Cl channels that drive fluid secretion [6].
Blockade of M3R would be an obvious mechanism to account for the salivary gland hypofunction associated with pSS and blockade of M3R by anti-M3R autoantibodies would provide a link to the immune response. Recent work from several groups utilizing a variety of techniques including immunohistochemistry [8], Western blotting [9], radioligand binding [10, 11], enzyme-linked immunosorbent assay (ELISA) [12] and bioassay [11, 13, 14], suggests that patients with pSS may have antibodies present in their IgG fraction that recognize M3R.
Whilst these studies may at first sight appear compelling, much of the evidence they contain is circumstantial. For example, many of the studies reporting the presence of anti-M3R have relied on a single antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) [2, 13, 15] which discriminates poorly between muscarinic receptor subtypes [16, 17]. Therefore there is no conclusive demonstration that pSS IgG antibodies interact specifically with M3R compared with other, minor, muscarinic populations on the surface of salivary acinar cells such as M1R [15, 18] or M4R [19]. Furthermore, demonstration of antibodyantigen interaction on membrane fragments does not automatically imply that an antibody has a pathophysiological role. Those studies which have shown an effect of pSS IgG antibodies on physiological function have been directed towards whole animals [11], mouse bladder detrusor [13] or mouse colon [14]. There are therefore no data demonstrating that pSS IgG antibodies have any effect on the function of salivary acinar cells.
We have used conventional immunological techniques to determine whether anti-M3R antibodies may be easily detected in the serum of pSS patients and a sensitive bioassay to determine whether the IgG fraction from patients with pSS has a direct effect on stimulus secretion coupling in salivary gland acinar cells.
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Methods |
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The acinar cell culture medium contained: serum-free 50:50 Dulbecco's MEM:F12 medium plus antibiotics and antimycotics (Life Technologies UK).
The Chinese hamster ovary (CHO) cell culture medium contained: 50:50 Dulbecco's MEM:F12 medium with 1% (v/v) fetal calf serum (Gibco).
The homogenization buffer contained: 10 mM Tris, 10 mM EDTA (pH 7.4) + 50 µg/mg cells protease inhibitors (Sigma).
Cell membrane preparation
Native CHO cells and CHO cells that had been stably and functionally transfected with the human M3R gene [20] were grown at 37°C and 5% CO2 in CHO cell media until approaching confluence. A crude membrane preparation was prepared as described previously [21]. Briefly: while still within the culture flasks the cells were washed gently in phosphate-buffered saline (PBS) at 4°C (3x). Washed cells were then harvested by scraping and resuspended in 10 ml of ice-cold homogenization buffer. Cells were homogenized with a 1 x 12 s pulse using an Ultraturrax (setting 4) and centrifuged at 40 000 g for 30 min at 4°C. Following total protein determination by the bicinchoninic acid method [22], cell membranes were resuspended at 1 mg/ml in ice-cold Na-HEPES + protease inhibitors and stored at 80°C until needed.
Serum collection
Following ethical committee approval (South Sefton Ethics Committee approval: EC.38.02) and informed consent, 10 ml of whole blood was collected from female patients aged between 40 and 60 yr with pSS and age- and sex-matched healthy controls. In the case of the pSS patients the collection was conducted during their routine clinic attendance (all patients with pSS fulfilled the current unified criteria for the diagnosis of pSS [23, 24], having both subjective signs of xerostomia and xerophthalmia as well as positive labial gland biopsies and Ro and La antibodies). Once collected the blood was allowed to clot and then centrifuged at 1000 g for 5 min. The serum was then aliquoted and stored at 20°C until required.
IgG preparation
1 ml of test serum was diluted 1:4 with ice-cold 0.1 M phosphate buffer (pH 7.0) and passed through a sterile 0.2 mM filter (Acrodisk) to remove particulates. The serum solution was passed through a protein G column (Pharmacia Hi-Trap protein G). The IgG fractions were then eluted from the column using 0.1 M glycine-HCl buffer (pH 2.7). The eluted IgG fraction was detected using a UV monitor (Pharmacia) connected to a chart recorder and the fraction corresponding to the elution peak was collected into a tube containing 400 µl of 1 M Tris-HCl buffer (pH 9.0). The elution buffer was then exchanged for Na-HEPES by dialysis (size 8, 1214 kDa) overnight at 4°C. The total protein content of each sample was determined using the bicinchoninic acid method [22] and the IgG fractions were stored at 20°C until needed. All purified IgG samples possessed a single band when analysed by non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (data not shown).
Western blotting
Proteins (25 µg and 50 µg) from the CHO and CHO-M3 membrane preparations were subjected to SDS-PAGE analysis [25] using a 7.5% separating gel with a 4% stacking gel (Bio-Rad). Proteins were visualized with Coomassie Blue R250 solution (Bio-Rad) and transferred to nitrocellulose using the Trans-Blot® SD semi-dry electrophoretic transfer cell (Bio-Rad), according to the manufacturer's directions. The nitrocellulose blots were blocked overnight with 0.5% w/v casein (Vector Labs) at room temperature (RT) and then incubated with 2 mg/ml pSS or control IgG for 1 h at RT. These blots were then washed (3x) for 10 min with 0.5% w/v casein (Vector Labs) followed by incubation with secondary antibody (Vectastain ABC kit) for 30 min according to the manufacturer's directions. Detection was performed using Sigma FastTM 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets (Sigma) according to the manufacturer's directions. This reaction was stopped with multiple water washes following band development. Control anti-M3, raised against the second intracellular loop of M3R, was a kind gift from Dr Andrew Tobin, University of Leicester, UK [21].
Whole-cell ELISA
Actively dividing CHO and CHO-M3 cells were harvested at 80% confluence using 0.02% EDTA solution (Sigma). Harvested cells were washed in PBS and suspended in CHO culture medium at a concentration of 1 x 106 cells/ml. 50 µl of CHO or CHO-M3 cell suspension (5 x 104 cells/well) was placed into each well on a 96-well ELISA plate (Griener) and incubated overnight at 37°C in 5% CO2. We observed no difference in cell growth overnight between CHO and CHO-M3 cells. The live cells were washed (3x) using a plate washer (Multiwash II) and blocked with 1% albumin (Fraction V sigma) in Na-HEPES for 1 h at RT. CHO or CHO-M3 cells were exposed to 2 mg/ml of pSS patient IgG for 1 h at RT, washed (3x) with Na-HEPES/1% albumin and treated with biotinylated anti-human IgG (Vector Labs) and developed using ABTS (Sigma) and read at 405 nm. Positive controls for these experiments were not possible because a well-characterized anti-M3R for a recognizing an extracellular epitope of M3R was not available.
Spot blots
Following sonication to evenly disperse the membrane fragments, 25, 10, 5 and 2 µg of CHO and CHO-M3 membrane protein were spotted onto nitrocellulose via vacuum. Membranes were blocked with 0.5% w/v casein (Vector Labs) overnight at RT. Blots were then exposed to patient and control IgG (2 mg/ml) for 30 min, followed by treatment with biotinylated anti-human IgG (Vector Labs) and developed using Opti-4CN (Biorad). Goat anti-M3R (C-20, Santa-Cruz Biotec sc-7474) and biotinylated anti-goat (Vector Labs) were used as a positive control.
Mouse acinar cell collection
Adult male CD1 mice were killed by cervical dislocation, and submandibular acinar cells were isolated by collagenase (Worthington Diagnostic USA) digestion in extracellular media containing 1 mM Ca2+, as described previously [26].
Human acinar cell collection
Following ethical approval (South Sefton Ethics Committee approval: EC.38.02) and informed consent small portions of human submandibular glands were collected, at the time of surgery, from patients whose submandibular glands were removed as a necessary part of routine head and neck surgery. The gland portion collected for research did not interfere with the subsequent diagnosis and treatment of the patient. Once harvested, the samples were immediately placed in ice-cold acinar cell culture medium and arrived in the laboratory within 1 h of removal. In the laboratory the harvested tissue was further sectioned, so that a representative portion could be sent for routine histology. All tissue used for experiments was retrospectively confirmed as histologically normal (data not shown). Acinar cells were isolated from the experimental tissue in an identical fashion to mouse acinar cells.
Acinar cell preparation
Following dispersal, cells were suspended in acinar cell culture medium and placed onto circular glass coverslips (22 mm diameter) coated with a thin (1 mm) layer of a basement membrane matrix (Matrigel, Becton Dickinson, UK) [27]. Each coverslip was placed into one well of a six-well plate and covered with acinar cell culture medium and kept overnight at 37°C with 5% CO2.
Microfluorimetry
Cells were removed from culture immediately before an experiment and loaded with Fura-2 by incubation for 20 min in the presence of 2 mM of cell-permeable fura-2 acetoxymethylester (Fura-2 AM, Molecular Probes). The acinar cell-coated coverslips formed the base of a perfusion chamber placed on to the stage of an inverted microscope (TMD 100, Nikon, Kingston, Surrey, UK). All experiments were carried out at 24±2°C. Measurements were made using 1000x magnification on single cells, either completely isolated or as part of a small (two to eights cells) clump. Cells were superfused continuously at 0.5 ml/min from one of several parallel superfusion pipettes. In order to accommodate the small volumes of pSS IgG available, chronic exposure to pSS IgG was achieved by stopping the perfusion system for 30 min and adding a known concentration of IgG to the perfusion chamber. Acute application of pSS IgG was achieved by introducing a micropipette to within 100 µm of the test cell so that a small volume of perfusate could be delivered to the cell surface. Preliminary experiments (data not shown) showed that when this system was activated, cells responded only to the contents of the micropipette and not to that of the primary perfusion system.
The ratio of light emitted at 510 nm following excitation at 340 nm to that emitted following excitation at 380 nm was measured using a Cairn (Cairn Research Ltd, Faversham, Kent, UK) spectrophotometer (excitation was at 96 Hz, data were averaged online and collected at 4 Hz). Intracellular Ca2+ activity was calculated from this ratio using the Grynkiewiez equation and custom-written software.
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Results |
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Spot blotting
In addition to Western blotting and ELISA, all IgGs were screened with a simple CHO and CHO-M3 cell membrane-based spot blotting assay. This method of attaching the CHO/CHO-M3 membranes to nitrocellulose should maintain M3R conformation in a high proportion of membrane fragments. Figure 3 shows recognition of M3R by a commercial affinity purified goat polyclonal anti-M3R raised against the C-terminus of M3R (C-20, Santa-Cruz Biotec), demonstrating that M3R can be detected with this technique in a CHO background. However, no reproducible recognition was seen in any (16/16) of the pSS IgGs or in 6/6 of the control IgG samples.
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Discussion |
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There is a significant difference between the initial reports on anti-M3R autoantibodies in pSS and equivalent studies into Grave's disease. Whereas autoantibodies to the TSH receptor may only be detected under conditions where the conformation of the binding epitope was preserved [3032], pSS antibodies were apparently detected under conditions where the conformation of the epitope was not preserved, namely Western blotting [9] using crude rat lachrymal gland membrane preparations as a source of M3R and ELISA using a 25-mer peptide, corresponding to the sequence of the second extracellular loop of M3R [12]. Close examination of these early studies reveals flaws which throw doubt on this conclusion. The report by Bacman et al. [9] showed, using Western blotting, that pSS IgG recognized a rat membrane protein with a molecular mass of 70 kDa. Whilst this is in good agreement with the molecular mass of M3R predicted on the basis of the amino acid sequence (approximately 65 kDa), it is difficult to reconcile with the molecular mass of human M3R (102 kDa) expressed in CHO cells following post-transcriptional modification [21]. Given that the identity shared between human and rat M3R is 92%, and they differ in size by only 1 amino acid, the rat protein is likely to be the same size as the human and the data shown by Bacman et al. [9] may not therefore represent binding to M3R. The ELISA data demonstrating binding of pSS IgG to a 25-mer peptide, corresponding to the sequence of the second extracellular loop of M3R [12] is similarly flawed (as indicated by Cavill et al. [33]) because the sequence to which pSS IgG was shown to bind is not from M3R but is the second extracellular loop of M4R [33, 34]. A subsequent ELISA study using the correct sequence of the second extracellular loop of M3R concluded that these peptides were not an appropriate tool with which to detect anti-M3R antibodies because of the loss of conformational epitopes [33]. This latter conclusion would explain our own findings, as we were also unable to detect anti-M3R autoantibodies in the serum of pSS patients using Western blotting (Fig. 1).
To enhance our chances of detecting the binding of pSS IgG to M3R we used techniques in which epitope conformation is preserved. We were unable to detect such binding in whole-cell ELISA using CHO cells stably transfected with M3R (CHO-M3R). We cannot show a positive antibody control for M3R in these studies because there are no well-characterized anti-M3R antibodies to extracellular domains of M3R. CHO cells transfected with human thyrotropin receptor were used successfully to detect the thyroid stimulating autoantibodies (TSA) of Graves disease [35]. The validity of these studies depends on correct insertion of the transfected protein into the membrane [36]. The transfection of M3R into CHO cells is functional (Fig. 4) which indicates that the receptor has the appropriate conformational insertion. One possible reason for our lack of positive findings, using whole-cell ELISA, is that following prolonged exposure to the pSS IgG, the M3R on the surface of the live CHO-M3 was internalized. However, preliminary experiments (data not shown) in which either CHO-M3 or mouse submandibular acinar cells were exposed to up to 4 mg/ml pSS IgG, for up to 24 h, failed to show an effect on the ACh-evoked rise in [Ca2+]i. In addition we routinely monitored the functionality of M3R in CHO-M3R cells cultured under conditions identical to those used for the ELISA experiments and found no change in the response of these cells to agonist stimulation. Taken together these data strongly suggest that the reason for our negative findings, for the whole-cell ELISA experiments, is not internalization of M3R but more likely it is a reflection of the inability of our whole-cell ELISA assay to detect anti-M3R antibodies. We were also unable to detect any anti-M3R activity by pSS IgG using a simple immunospot blot (Fig. 3) in which we would have expected a significant proportion of the M3Rs on the bound membrane fragments to be exposed.
We do not conclude from these findings that autoantibodies to M3R are not present in pSS IgG but rather that, despite some previous reports to the contrary [9, 12], they are difficult to detect using routine immunological techniques [33]. One possible reason why there is no detectable binding of pSS IgG to M3R is that anti-M3R antibodies are present in the peripheral circulation at very low concentrations. The comparison with Grave's disease may again be appropriate; TSA has been shown to circulate at nanogram [31] concentrations which may be below the threshold for detection using our whole-cell ELISA or immunoblotting techniques. The most effective technique for the detection of TSA activity is bioassay [35], and in pSS the most compelling evidence for the existence of anti-M3R autoantibodies has been obtained using bioassays [11, 13, 14]. However, prior to this study, none of the bioassays used for detection of M3R autoantibodies have utilized the tissue most affected by pSS, namely salivary gland acinar cells.
We measured the ACh-evoked change in [Ca2+]i in human and mouse submandibular acinar cells and in CHO-M3 cells before and after a 30-min exposure to pSS IgG. Each cell type displayed characteristic responses to a range of ACh concentrations that was unchanged by exposure to pSS IgG (Figs 4, 5 and 6). We did not think it necessary to simultaneously perfuse pSS IgG and ACh in these experiments because we assumed antibodyreceptor binding to be essentially irreversible. Furthermore, perfusion of the cells with pSS IgG would have required prohibitively large volumes of pSS IgG. Nevertheless, when pre-exposure to pSS IgG was found to be without effect we modified the perfusion apparatus to accommodate small volumes of pSS IgG and permit simultaneous perfusion of pSS IgG and carbachol. Under these conditions we found that application of pSS IgG inhibited the carbachol-dependent Ca2+ signal (Fig. 7). The effect of pSS IgG on the Ca2+ signal was dependent on the continued presence of pSS IgG and the Ca2+ signal rapidly recovered following removal of pSS IgG. These data are the first demonstration that pSS IgG has an antisecretory effect on intact salivary acinar cells. The rapid recovery of [Ca2+]i following removal of pSS IgG is in contradiction to previous reports of an irreversible action of pSS IgG [9, 10]; however, this observation may explain why anti-M3R activity was not detected using conventional immunological approaches. At present, this effect has manifest in 100% of pSS patients tested and in none of the age- and sex-matched controls. Although we feel that the most likely target for the pSS IgG is the M3R further work is needed to be certain. Therefore work is currently in progress to confirm whether or not the antisecretory activity of pSS IgG is due to anti-M3R antibody activity.
In conclusion pSS is an autoimmune disease for which we lack a convincing demonstration of a disease-specific autoantibody [37]. Identification of such an antibody would be an important step forward not only in understanding the aetiology of the condition, but also because an easily detectable antibody of high specificity and incidence in pSS patients would revolutionize diagnosis, management and treatment of the condition. In this study we have demonstrated that anti-M3R activity by pSS IgG cannot be detected by conventional immunological approaches. This could be because pSS IgG is present in the peripheral circulation only at low levels or perhaps because a necessary tissue factor is not present in our experiments [36]. Alternatively, at room temperature, our highly sensitive salivary acinar cell bioassay has revealed a novel, reversible, interaction between pSS IgG and the Ca2+ signal which would be invisible to conventional immunological approaches that depend on irreversible binding of antibody and receptor.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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