SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Sjögren's syndrome (SS) is an autoimmune condition affecting salivary glands, for which a clearly defined pathogenic autoantibody has yet to be identified. Autoantibodies that bind to the muscarinic M3 receptors (M3R), which regulate fluid secretion in salivary glands, have been proposed in this context. However, there are no previous data that directly show antisecretory activity. This study was undertaken to investigate and characterize the antisecretory activity of anti-M3R.

Methods

Microfluorimetric Ca2+ imaging and patch clamp electrophysiologic techniques were used to measure the secretagogue-evoked increase in [Ca2+]i and consequent activation of Ca2+-dependent ion channels in individual mouse and human submandibular acinar cells. Together, these techniques form a sensitive bioassay that was used to determine whether IgG isolated from patients with primary SS and from control subjects has antisecretory activity.

Results

IgG (2 mg/ml) from patients with primary SS reduced the carbachol-evoked increase in [Ca2+]i in both mouse and human acinar cells by ∼50%. IgG from control subjects had no effect on the Ca2+ signal. Furthermore, the inhibitory action of primary SS patient IgG on the Ca2+ signal was acutely reversible. We repeated our observations using rabbit serum containing antibodies raised against the second extracellular loop of M3R and found an identical pattern of acutely reversible inhibition. Anti-M3R–positive serum had no effect on Ca2+-dependent ion channel activation evoked by the direct intracellular infusion of inositol 1,4,5-triphosphate.

Conclusion

These observations show for the first time that IgG from patients with primary SS contains autoantibodies capable of damaging saliva production and contributing to xerostomia. The unusual but not unprecedented acute reversibility of the effects of anti-M3 autoantibodies is the subject of further research.

The classic clinical feature of primary Sjögren's syndrome (SS) is dryness of the mouth, secondary to hypofunction of the salivary glands. Salivary secretion by salivary acinar cells follows cholinergic stimulation of muscarinic type 3 receptors (M3R) (1–3). Recent data have suggested that putative antibodies directed against M3R (anti-M3R), present in the IgG serum fraction of patients with primary SS, may contribute to the hypofunction of the salivary glands (4–8) and be responsible for a host of extraglandular features, such as bladder (9, 10) and colon (11) irritability, Adie tonic pupil (12), and altered microvascular responses (13).

Overall, data supporting a definite pathologic role of anti-M3R in SS remain unclear (for review, see ref. 14), and there are few data that provide direct evidence of a role of anti-M3R in salivary gland hypofunction. Early reports showing binding of anti-M3R to salivary acinar cell membranes (5) have not been substantiated (4). Immunohistochemical evidence of increased expression of M3R on the surface of human labial acinar cells isolated from patients with primary SS (6) provides indirect support of a possible pathologic role of anti-M3R in salivary glands, but these cellular changes could also be the result of increased cholinesterase activity in the salivary glands of patients with primary SS (15, 16), or inhibition of neurotransmitter release due to the action of cytokines (17).

An unequivocal demonstration of IgG binding to M3R in primary SS would clearly strengthen the claim that patients with this disease express anti-M3R autoantibodies. However, these data would not themselves confirm a pathologic role of such autoantibodies in salivary gland hypofunction. There are some indications that IgG from patients with primary SS could have antisecretory activity since, for example, infusion of primary SS IgG into Igμnull NOD mice caused reversible salivary gland hypofunction (8) and, in healthy mice, caused alteration of bladder contractility (10). Nevertheless, the most simple and direct way to demonstrate a possible pathologic role of anti-M3R antibodies in primary SS would be to demonstrate that these antibodies and primary SS IgG impair secretory function in salivary gland acinar cells.

Data from previous studies have indicated that anti-M3R cannot be detected with linear peptides corresponding to M3R (18), nor can they be reliably detected using cell membranes expressing M3R (4, 14). To date, the “gold standard” for anti-M3R detection has been bioassay using the inhibition of bladder (9) or colon (11) smooth muscle as a detection system. Recently, we have demonstrated that microfluorimetric measurement of agonist-evoked changes in [Ca2+]i in isolated salivary acinar cells may also be used as a sensitive bioassay to detect the presence of putative anti-M3R antibodies in primary SS IgG. We have now used this technique in conjunction with patch clamp electrophysiology to more fully characterize the antisecretory activity of primary SS IgG (4) in both mouse and human acinar cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Solutions.

The extracellular bathing solution contained 140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl2, 1 mM CaCl2, and 10 mM glucose, buffered to pH 7.4 with 10 mM HEPES. The acinar cell culture medium contained serum-free 50:50 Dulbecco's modified Eagle's medium–Ham's F-12 plus antibiotics and antimycotics (Life Technologies, Paisley, UK). The patch clamp pipette contained 140 mM KCl, 1.13 mM MgCl2, 10 mM glucose, 0.5 mM EGTA, and 1 mM ATP, buffered to pH 7.4 with 10 mM HEPES (with or without 1 μM inositol 1,4,5-triphosphate [IP3]). Carbachol was used as the muscarinic agonist in order to avoid any effects from serum cholinesterases (15, 16).

Serum collection.

After ethics committee approval (South Sefton Ethics Committee; EC.38.02) and informed consent were obtained, 10 ml of whole blood was collected from female patients ages 40–60 years with primary SS and from age- and sex-matched healthy controls. In the case of the patients with primary SS, the collection was conducted during their routine clinic attendance. All patients with primary SS fulfilled the current unified criteria for the diagnosis of the disease (19, 20), having subjective signs of xerostomia and xerophthalmia as well as positive labial gland biopsy findings and Ro and La antibodies. After blood was collected, it was allowed to clot and then centrifuged for 5 minutes at 1,000g. The serum was then aliquoted and stored at −20°C until used.

Preparation of human IgG.

Test serum (1 ml) was diluted 1:4 with ice-cold 0.1M phosphate buffer (pH 7.0) and passed through a sterile 0.2 μM filter (Acrodisk; Pall Life Sciences, Farlington, UK) to remove particulates. The serum solution was passed through a protein G column (Hi-Trap; Pharmacia, St. Albans, UK). The IgG fractions were then eluted from the column using 0.1M glycine HCl buffer (pH 2.7). The eluted IgG fraction was detected using an ultraviolet monitor (Pharmacia) connected to a chart recorder, and the fraction corresponding to the elution peak was collected into a tube containing 400 μl of 1M Tris HCl buffer (pH 9.0). The elution buffer was then exchanged for Na-HEPES by dialysis (size 8; 12–14 kd) overnight at 4°C. The total protein content of each sample was determined using the bicinchoninic acid method (21), and the IgG fractions were stored at −20°C until used. All purified IgG samples were found to possess a single band when analyzed by nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (results not shown).

Rabbit serum.

Polyclonal IgG was raised against amino acid residues 213–237 (KRTVPPGECFIQFLSEPTITFGTAI) of the second extracellular loop of human M3R in a rabbit. The active component of the serum has been shown to comprise anti-M3R antibodies that recognize a short (10–amino acid) peptide sequence (EPTITFGTAI) located at the COOH terminus of the second extracellular loop. This serum (22) was a kind gift from Professor Tom Gordon (Flinders University, Adelaide, South Australia, Australia).

Collection of mouse acinar cells.

Adult male CD1 mice were killed by cervical dislocation, and submandibular acinar cells were isolated by collagenase (Worthington, Freehold, NJ) digestion in extracellular media containing 1 mM Ca2+, as described previously (23).

Collection of human acinar cells.

After ethics committee approval (South Sefton Ethics Committee; EC.38.02) and informed consent were obtained, 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. Collection of the gland portion 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 media and delivered to the laboratory within 1 hour of removal. In the laboratory, the harvested tissue was further sectioned, so that a representative portion could be sent for routine histologic analysis. All tissue used for experiments was retrospectively confirmed as being histologically normal (results not shown). Acinar cells were isolated from the experimental tissue in an identical manner to mouse acinar cells.

Preparation of acinar cells.

Following dispersal, cells were suspended in acinar cell culture media and placed onto circular glass coverslips (22 mm diameter) coated with a thin (∼1 mm) layer of a basement membrane matrix (Matrigel; Becton Dickinson, Oxford, UK) (24). Each coverslip was placed into 1 well of a 6-well plate, covered with acinar cell culture media, and kept overnight at 37oC with 5% CO2.

Microfluorimetry.

Cells were removed from culture immediately before each experiment and loaded with Fura 2 by incubation for 20 minutes in the presence of 2 μM cell-permeable Fura 2 acetoxymethylester (Fura 2 AM; Molecular Probes, Eugene, OR). The acinar cell–coated coverslips formed the base of a perfusion chamber placed onto the stage of an inverted microscope (TMD 100; Nikon, Kingston, UK). All experiments were carried out at 24°C (±2°C). Measurements were made using 1,000× magnification on single cells, either completely isolated or as part of a small cell clump (2–8 cells). Cells were superfused continuously at 0.5 ml/minute from one of several parallel superfusion pipettes. In order to accommodate the small volumes of primary SS IgG available, application of primary SS 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, as described previously (4). Prior to each experiment the effective agonist concentration range was determined, so that the optimal agonist concentration could be used during the experiment.

A monochromater provided excitation light at 340 nm and 380 nm, and images of light emitted at 510 nm were captured using a CCD camera. Pairs of images were captured every 120–500 msec, depending on the degree of time averaging used. When shown, [Ca2+]i was calculated from this ratio, using the Grynkiewiez equation.

Patch clamp.

Cells were removed from culture immediately before each experiment. The coverslips formed the base of the perfusion chamber and were placed on the stage of an inverted microscope (CM; Olympus UK, Southall, UK). Cells were superfused continuously at 0.5 ml/minute from one of several parallel superfusion pipettes. All experiments were carried out at 24oC (±2°C).

The patch clamp whole-cell configuration was achieved with single cells, using 2–4 MΩ patch clamp pipettes pulled from borosilicate glass capillaries (Assistant Haematocrit, Sondheim, Germany) with a DMZ universal puller (Zeitz Instruments, Munich, Germany). Access resistance through the patch pipette was ∼3 times that of the pipette itself. Cells were voltage-clamped to −40 mV using an axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA). K+ and Cl currents were measured separately by pulsing to 0 mV and −80 mV, respectively, for 100 msec, twice a second. Currents were digitized using the CED 1401 interface (Cambridge Electronics Design, Cambridge, UK) and stored and analyzed using a PC with custom-written software (25).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Effect of primary SS IgG on microfluorimetric [Ca2+]i measurements.

The data presented in Figure 1B show that application of primary SS IgG (2 mg/ml) to mouse submandibular acinar cells, in the continued presence of the muscarinic receptor agonist carbachol, caused a rapid reduction in agonist-stimulated [Ca2+]i. The reduction in [Ca2+]i persisted for as long as the cells were exposed to primary SS IgG, and [Ca2+]i returned to stimulated levels immediately following removal of primary SS IgG. The application of IgG from an age- and sex-matched control subject had no effect on the carbachol-evoked rise in [Ca2+]i (Figure 1A). These data confirm our previously published preliminary observations (4) and reiterate that the acute reversibility of the effect of primary SS IgG most likely accounts for the difficulty in detecting anti-M3R using conventional immunologic techniques that depend on irreversible binding of antibody to receptor (4).

thumbnail image

Figure 1. Acute reversibility of the inhibition of the carbachol-evoked change in [Ca2+]i in mouse submandibular acinar cells by IgG from patients with primary Sjögren's syndrome (PSS). Shown are results of a representative experiment demonstrating that in mouse submandibular acinar cells, application of 2 mg/ml IgG from a control subject (age- and sex-matched to the patients with primary SS) did not affect the carbachol (CCh; 500 nM)–evoked increase in [Ca2+]i (A), whereas application of 2 mg/ml IgG from a patient with primary SS caused acute inhibition of the carbachol-evoked Ca2+ signal, which persisted only for the duration of exposure to IgG (B). Cells in this and all subsequent equivalent experiments were perfused with IgG via a microperfusion system situated ∼100 μm from the cell. Carbachol was added to the IgG in the microperfusion system to maintain a constant concentration of carbachol throughout the experiment. Preliminary experiments using carbachol alone in the microperfusion system (data not shown) showed that use of the microperfusion system did not itself alter the carbachol-evoked increase in [Ca2+]i.

Download figure to PowerPoint

Examination of data on a patient-by-patient basis indicated that IgG from 4 of 5 patients with primary SS showed clear, reproducible inhibitory effects, while that from 1 of 5 patients produced no detectable inhibition. The mean ± SEM data obtained using the 4 primary SS IgG samples that caused inhibition (12 experiments) showed a 55 ± 7% reduction in the agonist-stimulated increase in [Ca2+]i, which was significantly different (P < 0.01) from the 2 ± 2% inhibition seen using IgG from control subjects (n = 3 experiments) (Figure 2A). The data in Figure 2B show the concentration-dependence of the inhibitory effect of primary SS IgG from an individual patient. The mean ± SEM percentage inhibition was 51 ± 8%, 26 ± 12%, and 0% with primary SS IgG concentrations of 2 mg/ml (n = 6), 1 mg/ml (n = 8), and 0.8 mg/ml (n = 2), respectively.

thumbnail image

Figure 2. Percentage inhibition of [Ca2+]i and concentration-dependence of the effect of IgG from patients with primary Sjögren's syndrome (pSS) in mouse submandibular acinar cells. A, Percentage inhibition of the carbachol (500 nM)–evoked change in [Ca2+]i levels in mouse submandibular acinar cells, induced by exposure to IgG (2 mg/ml) from patients with primary SS and control subjects. IgG was collected from 4 patients with primary SS and 3 controls. Primary SS IgG caused a 54.8 ± 6.8% reduction (n = 12 experiments) in the 340:380 ratio (see Materials and Methods), which was significantly (P < 0.01) greater than the 2 ± 2% reduction seen following exposure to control IgG (n = 3 experiments). B, IgG concentration–dependence of the inhibition of the carbachol-evoked change in [Ca2+]i levels induced by IgG from a single patient with primary SS. The IgG concentrations tested were 0.8 mg/ml (2 experiments), 1 mg/ml (6 experiments), and 2 mg/ml (6 experiments), with resultant inhibition of 0%, 26 ± 12%, and 51 ± 8%, respectively. Values are the mean ± SEM.

Download figure to PowerPoint

We observed an identical picture of response and inhibition using carbachol and primary SS IgG on human submandibular gland acinar cells (Figure 3B). On average, primary SS IgG caused 57 ± 17% inhibition of the carbachol-stimulated [Ca2+]i responses following cholinergic stimulation in human salivary acinar cells (3 experiments). The data shown in Figure 3A demonstrate that control IgG had no effect and that the inhibitory action of primary SS IgG was as acutely reversible when applied to human acinar cells as it was when applied to mouse acinar cells.

thumbnail image

Figure 3. Acute reversibility of the inhibition of the carbachol-evoked change in [Ca2+]i in human submandibular acinar cells by primary SS IgG. Shown are results of a representative experiment demonstrating that in human submandibular acinar cells, application of 2 mg/ml IgG from a control subject (age- and sex-matched to the patients with primary SS) did not affect the carbachol (500 nM)–evoked increase in [Ca2+]i (represented by the 340:380 ratio [see Materials and Methods]) (A), whereas application of 2 mg/ml IgG from a patient with primary SS caused acute inhibition of the carbachol-evoked Ca2+ signal, which persisted only for the duration of exposure to IgG (B). Primary SS IgG caused a 57 ± 17% inhibition of the carbachol-evoked change in the 340:380 ratio (mean ± SEM of 3 experiments). See Figure 1 for definitions.

Download figure to PowerPoint

Effect of primary SS IgG on patch clamp recordings.

The agonist-evoked changes in [Ca2+]i levels in acinar cells serve to activate the Ca2+-dependent ion channels that drive fluid secretion (3). The patch clamp whole cell pulse protocol technique allows simultaneous measurement of changes in conductance in both the Ca2+-activated K+ and the Ca2+-activated Cl channels (25). The patch clamp technique is also particularly sensitive to local, oscillatory changes in [Ca2+]i at the apical pole of the cell, which manifest as rapid, oscillatory changes Cl channel activity (26, 27). Such a response to carbachol, when applied to mouse submandibular acinar cells, is seen in Figure 4. Application of IgG from a control subject (3 experiments) had no effect on the agonist-evoked changes in either Cl or K+ conductance (Figure 4A). However, primary SS IgG (3 experiments) resulted in an acutely reversible reduction in both the frequency and the magnitude of the oscillatory changes in Cl conductance (Figure 4B). These data indicate that the inhibition of Ca2+ signaling caused by primary SS IgG is sufficient to inhibit the ion channel activity that underlies fluid secretion in salivary acinar cells.

thumbnail image

Figure 4. Patch clamp traces showing the effect of primary SS IgG on carbachol-evoked changes in Cl channel currents in mouse submandibular acinar cells. The patterns obtained without stimulation were oscillatory in nature and typical for these experiments (n = 3). Application of control IgG (2 mg/ml) had no effect on either the K+ or the Cl current (A), while primary SS IgG caused an ∼50% decrease in both the oscillatory frequency and the magnitude of the Cl current, which persisted only for the duration of exposure to primary SS IgG (B). See Figure 1 for definitions.

Download figure to PowerPoint

Effect of anti-M3R rabbit serum on microfluorimetric [Ca2+]i measurements.

Our data are consistent with the notion that primary SS IgG contains an antimuscarinic antibody directed against M3R (4–8, 10, 11, 18, 28–35). However, the reversibility of the inhibition caused by primary SS IgG is unusual for an antibody-mediated effect. Therefore, we repeated our experiments using rabbit serum containing functional anti-M3R antibodies against the second extracellular loop of human M3R (amino acids 213–237) (22).

The data shown in Figure 5B demonstrate that anti-M3R–containing serum (1:100 dilution) inhibited the carbachol-evoked rise in [Ca2+]i in human submandibular acinar cells. This inhibition was acutely reversed following removal of the serum. Both inhibition and the reversal of inhibition following removal of serum were observed in every experiment. Control experiments (n = 6) (Figure 5A), performed using serum from the same animal obtained before immunologic challenge, revealed no inhibition. Averaged data (Figure 6A) revealed a mean ± SEM 57 ± 8% inhibition of the carbachol-evoked rise in [Ca2+]i (cells from 4 subjects, 9 experiments), which was significantly different (P < 0.01) from the 4 ± 2% inhibition induced by the preimmune sera from 4 subjects (6 experiments). Figure 6B shows the concentration-dependence of the inhibitory response of anti-M3R–positive sera, with the resultant percentage inhibitions of 69 ± 3%, 57 ± 8%, 56 ± 14%, and 0% with serum dilutions of 1:25 (n = 2), 1:100 (n = 9), 1:500 (n = 3), and 1:1,000 (n = 3), respectively. These data show a very narrow range of concentration-dependence, which is similar to that observed with primary SS IgG and consistent with results of previous studies using this anti-M3R–positive serum in a smooth muscle assay (22).

thumbnail image

Figure 5. Acute reversibility of the inhibition of the carbachol (CCh)–evoked change in [Ca2+]i in human submandibular acinar cells by rabbit anti–muscarinic type 3 receptor (anti-M3R)–positive serum. Shown are results of a representative experiment demonstrating that in human submandibular acinar cells, application of rabbit preimmune serum (diluted 1:100) did not affect the carbachol (100 nM)–evoked increase in [Ca2+]i represented by the 340:380 ratio (see Materials and Methods) (A), whereas application of rabbit serum containing anti-M3R antibodies (raised against a 25-mer peptide corresponding to amino acids 213–237 of the second extracellular loop of human M3R [21]) caused acute inhibition of the carbachol-evoked Ca2+ signal, which persisted only for the duration of exposure to the serum (B).

Download figure to PowerPoint

thumbnail image

Figure 6. Percentage inhibition of [Ca2+]i and concentration-dependence of the effect of anti–muscarinic type 3 receptor (anti-M3R)–containing rabbit serum in human submandibular acinar cells. A, Percentage inhibition of the carbachol (100 nM)–evoked change in [Ca2+]i levels in human submandibular acinar cells induced by exposure to rabbit serum containing anti-M3R antibodies raised against a 25-mer peptide corresponding to amino acids 213–237 of the second extracellular loop of human M3R (diluted 1:100) (21) and to rabbit preimmune serum (diluted 1:100). Anti-M3R–positive serum caused a 57 ± 8% reduction in the 340:380 ratio (see Materials and Methods) (n = 9 experiments using cells from 4 subjects), which was significantly (P < 0.01) greater than the 4 ± 2% reduction (n = 6 experiments using cells from 4 subjects) seen following exposure to preimmune serum. B, Anti-M3R concentration–dependence of the inhibition of the carbachol-evoked change in [Ca2+]i levels induced by anti-M3R–positive serum. The anti-M3R dilutions tested were 1:25 (2 experiments), 1:100 (9 experiments), 1:500 (3 experiments), and 1:1,000 (3 experiments), with resultant inhibition of 69 ± 3%, 57 ± 8%, 56 ± 14%, and 0%, respectively. Values are the mean ± SEM.

Download figure to PowerPoint

Effect of anti-M3R–positive sera on patch clamp recordings.

The patch clamp technique allows intracellular second messengers, such as IP3, to be directly introduced into the cell (36, 37). Thus, it is possible to mobilize Ca2+ and activate the K+ and Cl channels independently of muscarinic receptor activation (36, 37), and so determine that the inhibitory effect of the anti-M3R–containing sera was mediated through M3R blockage by anti-M3R, rather than by some nonspecific action on the Ca2+ signaling process itself. In the experiment shown in Figure 7, IP3 caused a sustained increase in the Ca2+-dependent K+ current, which was not inhibited by repeated application of anti-M3R–positive serum (diluted 1:100). No effect of anti-M3R–positive serum on IP3-evoked changes in ion current activity was observed in 4 similar experiments.

thumbnail image

Figure 7. Patch clamp tracings showing the effect of anti–muscarinic type 3 receptor (anti-M3R)–containing rabbit serum on the inositol 1,4,5-triphosphate (IP3)– and ATP-evoked K+ and Cl currents in mouse submandibular acinar cells. Shown is a representative patch clamp recording (n = 4) of the changes in the K+ and Cl currents in mouse submandibular acinar cells following the introduction of 1 μM IP3 through the patch pipette. Application of anti-M3R–containing rabbit serum (1:100) (21) had no effect on the K+ and Cl currents.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Primary Sjögren's syndrome is classified as an autoimmune disease despite the lack of any convincing demonstration of a pathogenic autoantibody (38, 39). There are 3 criteria that should be satisfied in order to provide a clear indication of a role of an autoantibody in the pathology of an autoimmune disease (40), namely, that 1) the antibody interferes with the function of the target tissue, 2) passive transfer of the antibody causes the disease in the recipient, and 3) antibody is present at significant levels only in those individuals who have disease.

It is not possible, at present, to satisfy all of these criteria with respect to primary SS and anti-M3R antibodies (for review, see ref. 14). There is some evidence of passive transfer of disease following infusion of human SS IgG into mice (8, 10). Where M3R binding activity has been detected in serum from SS patients, it has been shown to be present at very high incidence (80–90%) (5, 9, 32, 41). The specificity for SS remains uncertain, however, because IgG antibodies with antimuscarinic activity have also been detected in association with scleroderma (42) (for review, see ref. 14). Nevertheless, our data do significantly advance the case for a role of anti-M3R antibodies in SS, because they provide the first unequivocal demonstration that anti-M3R antibodies interfere with the function of the target tissue, salivary gland acinar cells.

Binding of acetylcholine to muscarinic M3 receptors is the trigger for increased [Ca2+]i, which in turn activates K+ and Cl channels, which drive fluid secretion in acinar cells (3). There can be no fluid secretion without Ca2+ mobilization. Our data show that primary SS IgG inhibited agonist-evoked Ca2+ mobilization by ∼50%, sufficient to reduce consequent ion channel activation in both mouse and human submandibular acinar cells (Figures 1–4). Exposure of the cells to a known anti-M3R had a strikingly similar effect (Figures 5 and 6). The same maximum inhibition (∼50%) was seen in both human and mouse cells challenged with primary SS IgG, and in mouse cells challenged with anti-M3R.

We cannot speculate at this point whether the magnitude of the inhibition is of itself of any significance. The concentration of muscarinic agonist in these experiments was optimized to give a stable elevated, cell-wide Ca2+ signal so that the effect of primary SS IgG might be seen most clearly. Salivary gland acinar cells are capable of a range of Ca2+ signals depending both on the magnitude of the stimulus and on the metabolic state of the cell (26, 37, 43), and it is possible that primary SS IgG could completely abolish the response to less vigorous stimulation. The complex processes that underlie Ca2+ mobilization are also capable of massive amplification of the stimulatory stimulus (3), which could underlie the very narrow apparent concentration-dependence of both primary SS IgG and anti-M3R (Figures 2B and 6B). It is therefore difficult to extrapolate quantitatively from these data to the in vivo situation, where the concentration of both antibody and acetylcholine is unknown. What is clear is that the antisecretory activity of the anti-M3R is mediated by binding to the M3 receptor, because anti-M3R had no effect on increased ion channel activity stimulated directly by infusion of IP3 into the cell (Figure 7).

The inhibition caused by primary SS IgG was reversed as soon as the perfusion of IgG was stopped. This effect was highly reproducible in both mouse and human submandibular acinar cells. This could account for our previously published observation (4) that preincubation of primary SS IgG with acinar cells did not affect the response of the cells to subsequent challenge with carbachol. Furthermore, the inhibitory action of a known anti-M3R was also reversed immediately when the antibody was removed. Investigators at the laboratory of Professor Tom Gordon, who raised the antibody, found that the inhibitory activity was irreversible using a smooth muscle assay (9, 22). A reduction in agonist-evoked Ca2+ signaling following preincubation of SS IgG with epithelial cells of salivary duct origin (HSG cells) has also been reported (7).

Together with our data, these observations provide evidence of a difference in binding of anti-M3R to the muscarinic receptor in acinar cells compared with binding to the muscarinic receptor in smooth muscle and possibly elsewhere. This possibility is supported by data obtained from studies in the NOD mouse, which also suggested that antibody interaction with salivary acinar cells was reversible (8). Most antibody binding is not rapidly reversible. However, similar atypical behavior has been observed with antibodies against the nicotinic receptor in myasthenia gravis, where experimental conditions were shown to markedly influence antibody binding (44).

Our results show that primary SS IgG and a known anti-M3R antibody have an identical and unusual pattern of inhibition of agonist-evoked Ca2+ signals. There can be no doubt that primary SS IgG has antisecretory activity, and it is likely that this activity is mediated through an anti-M3R IgG antibody.

Salivary gland secretion is triggered by muscarinic M3 receptor activation. The presence of anti-M3R antibody in SS patients is therefore likely to be at least a contributing factor in glandular hypofunction in these patients. Furthermore, the reversibility of the interaction could have important implications regarding the development of therapeutic interventions for SS.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We would like to thank Professor Tom Gordon for helpful discussions.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Nakamura T, Matsui M, Uchida K, Futatsugi A, Kusakawa S, Matsumoto N, et al. M3 muscarinic acetylcholine receptor plays a critical role in parasympathetic control of salivation in mice. J Physiol 2004; 558: 56175.
  • 2
    Matsui M, Motomura D, Karasawa H, Fujikawa T, Jiang J, Komiya Y, et al. Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype. Proc Natl Acad Sci U S A 2000; 97: 957984.
  • 3
    Smith PM. Mechanisms of secretion by salivary glands. In: EdgarWM, O'MullaneD, editors. Saliva and oral health. London: BDJ; 1996. p. 925.
  • 4
    Dawson LJ, Allison HE, Stanbury J, Fitzgerald D, Smith PM. Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conventional immunological approaches. Rheumatology (Oxford) 2004; 43: 148895.
  • 5
    Bacman S, Sterin-Borda L, Camusso JJ, Arana R, Hubscher O, Borda E. Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjogren's syndrome. Clin Exp Immunol 1996; 104: 4549.
  • 6
    Beroukas D, Goodfellow R, Hiscock J, Jonsson R, Gordon TP, Waterman SA. Up-regulation of M3-muscarinic receptors in labial salivary gland acini in primary Sjogren's syndrome. Lab Invest 2002; 82: 20310.
  • 7
    Li J, Ha YM, Ku NY, Choi SY, Lee SJ, Oh SB, et al. Inhibitory effects of autoantibodies on the muscarinic receptors in Sjogren's syndrome. Lab Invest 2004; 84: 14308.
  • 8
    Robinson CP, Brayer J, Yamachika S, Esch TR, Peck AB, Stewart CA, et al. Transfer of human serum IgG to nonobese diabetic Igμ null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjogren's syndrome. Proc Natl Acad Sci U S A 1998; 95: 753843.
  • 9
    Waterman SA, Gordon TP, Rischmueller M. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjögren's syndrome. Arthritis Rheum 2000; 43: 164754.
  • 10
    Wang F, Jackson MW, Maughan V, Cavill D, Smith AJ, Waterman SA, et al. Passive transfer of Sjögren's syndrome IgG produces the pathophysiology of overactive bladder. Arthritis Rheum 2004; 50: 363745.
  • 11
    Cavill D, Waterman SA, Gordon TP. Antiidiotypic antibodies neutralize autoantibodies that inhibit cholinergic neurotransmission. Arthritis Rheum 2003; 48: 3597602.
  • 12
    Bachmeyer C, Zuber M, Dupont S, Blanche P, Dhote R, Mas JL. Adie syndrome as the initial sign of primary Sjogren syndrome. Am J Ophthalmol 1997; 123: 6912.
  • 13
    Kovacs L, Torok T, Bari F, Keri Z, Kovacs A, Makula E, et al. Impaired microvascular response to cholinergic stimuli in primary Sjogren's syndrome. Ann Rheum Dis 2000; 59: 4853.
  • 14
    Dawson L, Tobin A, Smith P, Gordon T. Antimuscarinic antibodies in Sjögren's syndrome: where are we, and where are we going? [review]. Arthritis Rheum 2005; 52: 298495.
  • 15
    Dawson LJ, Christmas SE, Smith PM. An investigation of interactions between the immune system and stimulus-secretion coupling in mouse submandibular acinar cells: a possible mechanism to account for reduced salivary flow rates associated with the onset of Sjogren's syndrome. Rheumatology (Oxford) 2000; 39: 122633.
  • 16
    Dawson LJ, Caulfield VL, Stanbury JB, Field AE, Christmas SE, Smith PM. Hydroxychloroquine therapy in patients with primary Sjogren's syndrome may improve salivary gland hypofunction by inhibition of glandular cholinesterase. Rheumatology (Oxford) 2005; 44: 44955.
  • 17
    Zoukhri D, Kublin CL. Impaired neurotransmitter release from lacrimal and salivary gland nerves of a murine model of Sjogren's syndrome. Invest Ophthalmol Vis Sci 2001; 42: 92532.
  • 18
    Cavill D, Waterman SA, Gordon TP. Failure to detect antibodies to extracellular loop peptides of the muscarinic M3 receptor in primary Sjogren's syndrome. J Rheumatol 2002; 29: 13424.
  • 19
    Vitali C. Classification criteria for Sjogren's syndrome. Ann Rheum Dis 2003; 62: 945.
  • 20
    Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al, and the European Study Group on Classification Criteria for Sjogren's Syndrome. Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis 2002; 61: 5548.
  • 21
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid (published erratum appears in Anal Biochem 1987;163:279). Anal Biochem 1985; 150: 7685.
  • 22
    Cavill D, Waterman SA, Gordon TP. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sjogren's syndrome. Scand J Immunol 2004; 59: 2616.
  • 23
    Smith PM, Gallacher DV. Acetylcholine- and caffeine-evoked repetitive transient Ca2+-activated K+ and C1− currents in mouse submandibular cells. J Physiol 1992; 449: 10920.
  • 24
    Hann LE, Kelleher RS, Sullivan DA. Influence of culture conditions on the androgen control of secretory component production by acinar cells from the rat lacrimal gland. Invest Ophthalmol Vis Sci 1991; 32: 261021.
  • 25
    Smith PM. Patch-clamp whole-cell pulse protocol measurements using a microcomputer. J Physiol 1992; 446: 72P.
  • 26
    Harmer AR, Smith PM, Gallacher DV. Local and global calcium signals and fluid and electrolyte secretion in mouse submandibular acinar cells. Am J Physiol Gastrointest Liver Physiol 2005; 288: G11824.
  • 27
    Thorn P, Lawrie AM, Smith PM, Gallacher DV, Petersen OH. Ca2+ oscillations in pancreatic acinar cells: spatiotemporal relationships and functional implications. Cell Calcium 1993; 14: 74657.
  • 28
    Bacman S, Berra A, Sterin-Borda L, Borda E. Muscarinic acetylcholine receptor antibodies as a new marker of dry eye Sjogren syndrome. Invest Ophthalmol Vis Sci 2001; 42: 3217.
  • 29
    Bacman S, Perez Leiros C, Sterin-Borda L, Hubscher O, Arana R, Borda E. Autoantibodies against lacrimal gland M3 muscarinic acetylcholine receptors in patients with primary Sjogren's syndrome. Invest Ophthalmol Vis Sci 1998; 39: 1516.
  • 30
    Bacman SR, Berra A, Sterin-Borda L, Borda ES. Human primary Sjogren's syndrome autoantibodies as mediators of nitric oxide release coupled to lacrimal gland muscarinic acetylcholine receptors. Curr Eye Res 1998; 17: 113542.
  • 31
    Gordon TP, Bolstad AI, Rischmueller M, Jonsson R, Waterman SA. Autoantibodies in primary Sjogren's syndrome: new insights into mechanisms of autoantibody diversification and disease pathogenesis. Autoimmunity 2001; 34: 12332.
  • 32
    Kovacs L, Marczinovits I, Gyorgy A, Toth GK, Dorgai L, Pal J, et al. Clinical associations of autoantibodies to human muscarinic acetylcholine receptor 3(213-228) in primary Sjogren's syndrome. Rheumatology (Oxford) 2005; 44: 10215.
  • 33
    Perez Leiros C, Sterin-Borda L, Hubscher O, Arana R, Borda ES. Activation of nitric oxide signaling through muscarinic receptors in submandibular glands by primary Sjogren syndrome antibodies. Clin Immunol 1999; 90: 1905.
  • 34
    Nguyen KH, Brayer J, Cha S, Diggs S, Yasunari U, Hilal G, et al. Evidence for antimuscarinic acetylcholine receptor antibody–mediated secretory dysfunction in NOD mice. Arthritis Rheum 2000; 43: 2297306.
  • 35
    Gao J, Cha S, Jonsson R, Opalko J, Peck AB. Detection of anti-type 3 muscarinic acetylcholine receptor autoantibodies in the sera of Sjogren's syndrome patients by use of a transfected cell line assay. Arthritis Rheum 2004; 50: 261521.
  • 36
    Smith PM. Ins(1,3,4,5)P4 promotes sustained activation of the Ca2+-dependent Cl− current in isolated mouse lacrimal cells. Biochem J 1992; 283: 2730.
  • 37
    Harmer AR, Gallacher DV, Smith PM. Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid-adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells. Biochem J 2001; 353: 55560.
  • 38
    Scully C. Sjogren's syndrome: clinical and laboratory features, immunopathogenesis, and management. Oral Surg Oral Med Oral Pathol 1986; 62: 51023.
  • 39
    Fox PC, Speight PM. Current concepts of autoimmune exocrinopathy: immunologic mechanisms in the salivary pathology of Sjogren's syndrome. Crit Rev Oral Biol Med 1996; 7: 14458.
  • 40
    Drachman DB. Autonomic “myasthenia”: the case for an autoimmune pathogenesis. J Clin Invest 2003; 111: 7979.
  • 41
    Marczinovits I, Kovacs L, Gyorgy A, Toth GK, Dorgai L, Molnar J, et al. A peptide of human muscarinic acetylcholine receptor 3 is antigenic in primary Sjogren's syndrome. J Autoimmun 2005; 24: 4754.
  • 42
    Goldblatt F, Gordon TP, Waterman SA. Antibody-mediated gastrointestinal dysmotility in scleroderma. Gastroenterology 2002; 123: 114450.
  • 43
    Harmer AR, Gallacher DV, Smith PM. Correlations between the functional integrity of the endoplasmic reticulum and polarized Ca2+ signalling in mouse lacrimal acinar cells: a role for inositol 1,3,4,5-tetrakisphosphate. Biochem J 2002; 367: 13743.
  • 44
    Jahn K, Franke C, Bufler J. Mechanism of block of nicotinic acetylcholine receptor channels by purified IgG from seropositive patients with myasthenia gravis. Neurology 2000; 54: 4749.