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.
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- MATERIALS AND METHODS
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.