ARTHRITIS & RHEUMATISMVol. 52, No. 10, October 2005, pp 2984–2995DOI 10.1002/art.21347© 2005, American College of Rheumatology

REVIEW

Antimuscarinic Antibodies in Sjogren’s Syndrome

Where Are We, and Where Are We Going?

Luke Dawson,1 Andrew Tobin,1 Peter Smith,1 and Tom Gordon2

Introduction

Sjogren’s syndrome (SS) has been described as“an autoimmune disease of the exocrine glands, whichparticularly involves the salivary and lacrimal glands.The secretory tissues in the affected glands are progres-sively destroyed and replaced by a lymphoreticular cellinfiltrate. This process may occur as an isolated phenom-enon, in which case it is termed primary Sjogren’ssyndrome, or in conjunction with a connective tissue orcollagen disease, in which case it is referred to assecondary Sjogren’s syndrome” (1). Implicit within thisdescription is the concept that the characteristic salivarygland hypofunction of SS is the direct consequence ofimmune-mediated destruction of the secretory acinartissue. The assumption that lack of glandular function isthe direct consequence of tissue loss has become soingrained that it has directed SS research for more than60 years.

More recently, our understanding of the pathol-ogy underlying the glandular hypofunction associatedwith SS has undergone a dramatic change. Two keyobservations have led to this change: 1) many patientswith SS have within their salivary glands large amountsof acinar tissue that is unable to function in vivo, asdemonstrated by the lack of salivary flow (2–4), and 2)data from work on salivary acinar cells isolated frompatients with SS demonstrates that the remaining tissueis functional in vitro (5,6), but with a reduced sensitivityto threshold levels of muscarinic stimulation (5). In

addition, it is well established that glandular atrophy isthe long-term consequence of diminished function andthat, in the elderly, it is possible to lose significantamounts of glandular tissue without affecting salivaryflow (7–9). Overall, these findings strongly suggest thatthe lack of glandular function in many patients with SS isthe result of a perturbation of acinar function, ultimatelyfollowed by atrophy (2–4,10).

In other autoimmune diseases in which there areclearly defined organ targets of the autoimmune re-sponse, such as myasthenia gravis and Graves’ disease,pathogenic autoantibodies have been identified (11,12).Although SS is classified as an autoimmune disease, nospecific pathologic autoantibody has been found (3,13).However, data from recent studies have suggested thatpatients with primary SS and patients with secondary SSmay have inhibitory autoantibodies directed againstmuscarinic receptors (14–16). The presence of inhibi-tory autoantibodies directed against salivary gland mus-carinic receptors would serve to unite the pathologiesunderlying the glandular hypofunction of both primaryand secondary SS, explaining why the degree of glandu-lar hypofunction is equivalent in the two (17). Further-more, perturbation of muscarinic receptor function bythe presence of antimuscarinic antibodies would accountin large part for some of the reported extraglandularfeatures of SS, such as bladder irritability (15,18,19),impairment of esophageal motor function (20) andmicrovascular responses to cholinergic stimulation (21),Adie pupil (22), and variable heart rate (23).

Identification of a pathogenic autoantibodywould revolutionize the management of SS, in that 1)antibody identification could form the basis of a diag-nostic test, 2) removal of the antibody could form thebasis of a therapy akin to that for myasthenia gravis, and3) better understanding of the pathology underlying theglandular hypofunction could lead to the developmentof disease-modifying treatments. In the present report,

1Luke Dawson, BSc, BDS, PhD, Andrew Tobin, BSc, PhD,Peter Smith, BSc, PhD: University of Liverpool, Liverpool, UK; 2TomGordon, MD, PhD: Flinders Medical Centre, Bedford Park, SouthAustralia, Australia.

Address correspondence and reprint requests to Luke Daw-son, BSc, BDS, PhD, University of Liverpool, Room 1.10, EdwardsBuilding, Daulby Street, Liverpool Merseyside L69 3GN, UK. E-mail:ldawson@liv.ac.uk.

Submitted for publication March 23, 2005; accepted in revisedform June 30, 2005.

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we critically evaluate the evidence for the existence ofpathogenic antimuscarinic autoantibodies in SS.

Muscarinic receptors

Early pharmacologic data demonstrated that ace-tylcholine (ACh) could mediate physiologic responses,such as smooth muscle contraction, glandular secretion,and cardiac rate, through a family of muscarinic receptorsubtypes (24). However, it was not until the cloning ofthe first muscarinic receptors, as reported by Kubo andcolleagues in 1986 (25,26), that the full extent of themuscarinic receptor gene family was realized (27). It isnow clear that the muscarinic receptor family is encodedby 5 separate genes (28,29). The fact that the codingregion of these genes lacks an intron, a feature charac-teristic of a number of other G protein–coupled receptorfamilies, means that there are just 5 muscarinic geneproducts, designated M1–M5 (30).

Because the muscarinic receptor gene family wascloned within a short period of time by investigators at 3different laboratories, there was initially some confusionregarding the designation of the sequences (comparerefs. 27 and 28). This confusion has now been resolved,and the accession numbers shown in Table 1 refer to the

human muscarinic receptor subtypes held in GenBankand conform to the designations given by Bonner (27).

The cloning of the muscarinic receptor genes hasallowed for their isolated expression in immortalizedcells followed by analysis of the pharmacologic andsignaling properties of each of the receptor subtypes.These cloning studies have highlighted the inadequacyof the pharmacologic tools available to study muscarinicreceptors. Table 2 shows the binding affinity constants ofsome of the more commonly used antagonists. The dataillustrate the lack of specificity of these ligands; forexample, pirenzepine, the widely used “selective” antag-onist for the M1 receptor (M1R), has a respectable45-fold selectivity compared with that for the M2R, butonly 17.9-fold and 3.5-fold selectivity compared with thatfor M3R and M4R, respectively (30). Similarly, darife-nacin, which is considered to be M3R selective, has only10-fold selectivity for the M3R receptor over the M1R.

The poor selectivity of the muscarinic receptorligands makes the identity of a muscarinic receptorsubtype in a tissue or cell line difficult to establish. Toaccurately define the pharmacologic identity of a mus-carinic receptor response, the antagonist affinity con-stants ideally should be determined for a number ofantagonists and compared against the affinity constantsreported for the individual receptors analyzed in clonedexpression systems (see ref. 31). This can often bepractically difficult due to limited availability of tissue orcells; certainly the process is time-consuming and expen-sive, and it may ultimately yield ambiguous results due toexperimental variation or vagaries associated with aparticular biologic sample.

There are, however, good examples in which thisprinciple has been applied, for instance, in the determi-nation of the muscarinic receptor population expressedin rat parotid gland and the parotid cell line PAR-5 (32).The affinities for a number of muscarinic selectiveantagonists were determined and compared against the

Table 1. Accession numbers for members of the human muscarinicreceptor gene family

ReceptorNucleotide accession

nos.Protein accession

nos.No. of

amino acids

M1 NM_000738 NP_000729 460X15263 CAA33334

M2 AF498916 AAM18939 466X15264 CAA33335

M3 NM_000740 NP_000731 590X15266 CAA33337

M4 NM_000741 NP_000732 479X15265 CAA33336

M5 NM_012125 NP_036257 532

Table 2. Antagonist affinities (log affinity values) for the various muscarinic receptor subtypes*

Antagonist

Muscarinic receptor

M1 M2 M3 M4 M5

Pirenzepine 8.15 6.5 (44.6) 6.9 (17.9) 7.6 (3.54) 6.65 (31.6)AF-DX 384 7.4 (15.8) 8.6 7.5 (12.6) 8.35 (2.0) 6.3 (200)Darifenacin 7.65 (10.0) 7.2 (6.3) 8.65 7.85 (6.4) 8.05 (4.0)Methoctramine 7.45 (4.0) 8.05 6.6 (28.2) 7.75 (2.0) 7.05 (10.0)4-DAMP 8.9 (1.6) 8.1 (10) 9.1 8.9 (1.6) 8.95 (1.4)

* Values in boldface are the highest affinity value for the particular antagonist. Values in parentheses arethe fold decrease in affinity compared with the value in boldface. 4-DAMP � 4-diphenylacetoxy-N-methylpiperidine methiodide. Adapted, with permission, from ref. 30.

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affinities obtained for the expressed recombinant recep-tors. In those experiments the affinity of the “specific”M3 antagonist 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) was found to be in the nM range(32). However, it can be seen from Table 2 (and in ref.32) that 4-DAMP has poor selectivity for the M3R. Itwas therefore important to also include other muscarinicantagonists to develop a picture of a range of antagonistaffinities. In the study by Bockman et al (32), theaffinities of 4-DAMP, pirenzepine, methoctramine, anddarifenacin (among others) were determined in parotidgland and PAR-5 cells and found to be consistent withthat for the M3R.

To circumvent some of these pharmacologic lim-itations, a series of antibodies have been produced toallow for the detection of specific receptor subtypesusing immunologic methods such as Western blotting,immunoprecipitation, and immunocytochemistry. Thefirst of these antibodies were raised by Li, Wall, Wolfe,and colleagues, who, in a series of elegant studies,combined the selectivity of antimuscarinic receptor an-tibodies with the high-affinity binding of a radiolabeledmuscarinic antagonist, N-methylscopolamine (33–35).This approach led to a highly sensitive and quantitativeassay for muscarinic receptors in tissues and cell lines(35). Using this technique, the preponderance of M2Rover M3R and M1R subtypes in lung, ileum, and bladderwas confirmed (35). Similar studies on rabbit submaxil-lary glands demonstrated that in this species the M1Rand M3R were expressed in equal proportions, whereasin the rat parotid gland the M3R was found to represent93% of the muscarinic receptor population (31).

Although these antibodies have been extremelyuseful in defining muscarinic receptor populations, re-searchers need to be cautious. Over the past 5 yearsmany of the muscarinic receptor–specific antibodieshave become commercially available, but unfortunately,in our experience the majority of these antibodies havenot been selective for the receptors to which they havebeen raised. It is imperative that researchers obtainreceptors cloned by recombinant expression so that theselectivity of the antibody can be tested. We havesuccessfully raised a number of polyclonal (36,37) andmonoclonal antibodies (Tobin AB, et al: unpublishedobservations) to the M3R and have rigorously tested thespecificity of these antibodies on cloned receptors be-fore using them on tissue samples (Figure 1). Similarstudies carried out with commercially available antibod-ies have (with only a few exceptions) led us to reject theantibodies.

Early cloning studies revealed a number of con-

sensus glycosylation sites in the N-terminal extracellulardomain of the muscarinic receptors (27). When thereceptors are resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), theglycosylation status of the receptor causes the receptorto run at a higher apparent molecular weight than thatpredicted from the amino acid backbone. Hence, thehuman M3R has a predicted molecular weight of 66 kdbut runs on SDS-PAGE as a broad band at a molecularweight of 100–110 kd (Figure 1). Similar shifts inmolecular weight have been reported for nearly all Gprotein–coupled receptors studied to date, including the�2-adrenergic receptor (38), angiotensin AT1A receptor(39), cholecystokinin receptor (40), substance P recep-tors (41), and vasopressin V1a receptor (42). Interest-ingly, mutation of the glycosylation sites in the M2R hadno effect on receptor expression or function (43). Hence,the role of glycosylation in relation to the muscarinicreceptor family is uncertain.

Fluid secretion by salivary acinar cells

The textbook description (44) of the control ofsecretion outlines a linear sequence of events, as follows:ACh binds to and activates M3R, the G protein–coupledmuscarinic type 3 ACh receptor (45,46) (action 1 in

Figure 1. Western blot identification of the human M3 muscarinicreceptor (M3R). Chinese hamster ovary cells, nontransfected (NT) ortransfected with human M3, were grown in a 6-well culture dish andsolubilized with a detergent-based Tris buffer (36). Samples from thelysate (1 �g protein/lane) were resolved by sodium dodecyl sulfate–8%polyacrylamide gel electrophoresis. The proteins were then transferredto a nitrocellulose membrane, which was probed with an M3R-specificpolyclonal antibody (36). The receptor reacted as a broad band at100–110 kd. Note the nonreceptor bands at 96 kd and 212 kd, whichcould be mistaken for the receptor.

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Figure 2). The activated G protein stimulates phospho-lipase C to generate inositol 1,4,5-trisphosphate (IP3)(action 2 in Figure 2), which binds to IP3 receptors onintracellular Ca2� stores, causing them to release Ca2�

(action 3 in Figure 2). The rise in intracellular Ca2�

activates apical membrane Cl� channels (47) (action 5 inFigure 2). Na� follows Cl� across the cell in order tomaintain electrochemical neutrality, and the osmoticeffect of moving Na� and Cl� across the acinus dragswater into the lumen (action 6 in Figure 2).

While accurate in every particular, this descrip-tion is too simplistic and, above all, too static to be auseful model in any meaningful exploration of thepotential role of antimuscarinic antibodies in salivarysecretion. The reasons for this are described below:

1. The size and shape of the Ca2� signal dependalso on a multiplicity of factors that are not directlyregulated by activation of the muscarinic receptors,including cyclic ADP ribose and cyclic AMP (48) (action4 in Figure 2).

Figure 2. Control of fluid secretion in salivary acinar cells. Binding of acetylcholine (ACh) to the Gprotein–linked M3 muscarinic ACh receptor (action 1) stimulates phospholipase C to generate inositol1,4,5-trisphosphate (IP3) (action 2). IP3 binds to and opens the IP3 receptor on the endoplasmic reticulum at theapical pole of the cell, causing the release of Ca2� (action 3). Release of Ca2� stimulates Ca2�-induced Ca releasevia the IP3 receptor and the ryanodine receptor, which both amplifies and propagates the Ca2� signal (action 4).Increased [Ca2�]i activates the apical membrane Cl� channel (action 5) and the basolateral K� channel. Effluxof Cl� into the acinus lumen draws Na� across the cells to maintain electroneutrality, and the resulting osmoticgradient generates fluid secretion (action 6). cADPr � cyclic ADP ribose.

ANTIMUSCARINIC ANTIBODIES IN SS 2987

2. Salivary acinar cells are structurally (49) andfunctionally (50) polarized, and secretagogue-evokedCa2� signals originate in and may be restricted to theapical pole of the cell (50) (see Figure 2). A small Ca2�

signal at the right location will be a much more effectivetrigger for secretion than a large Ca2� signal that isdistributed across the cell (50). Factors that affect cel-lular polarization, e.g., anything that affects the cyto-skeleton, will therefore also alter the functionality of theCa2� signal (51,52).

3. Ca2� signals do not simply diffuse across thecell, but are instead actively amplified and propagated bya process of Ca2�-induced Ca2� release (CICR) (53)(action 4 in Figure 2). CICR, as the name suggests, is theprocess whereby Ca2� causes its own release. The Ca2�

release channels, the IP3 receptor and the ryanodinereceptor, both gate the endoplasmic reticulum Ca2�

store, and both are Ca2� sensitive (54). Ca2� mobiliza-tion is therefore an explosive example of positive feed-back. Factors that affect the propagation of the Ca2�

signal, e.g., anything that modifies Ca2� removal fromthe cytoplasm (44) including anything that alters mito-chondrial metabolism (55), will also alter the Ca2�

signal. Therefore, in light of what is known about theprocess of Ca2� mobilization, it is not sufficient todemonstrate that cells are capable of generating achange in enzyme activity or an increase in a secondmessenger, but rather it must be shown that they arecapable of generating the appropriate Ca2� signal.

There are few data available on the secretoryphysiology of salivary acinar cells from patients with SS.It is known that the degree of nervous innervation isunaffected in the human disease (6,56). However, anincrease in acinar cell expression of M3R has beendemonstrated (57). Such an increase in M3R would beexpected to result in acinar cell hyperfunction, a phe-nomenon that has been observed in the early stages ofdisease both in the MRL/lpr mouse model of SS (58) andin a very small number of human patients (59). Con-versely, detailed in vitro experiments using labial glandsisolated from patients with SS have demonstrated thatwith maximal doses of ACh, the [Ca2�]i response wasnot significantly different from that of labial gland acinarcells isolated from control subjects (5,6). However, withsubmaximal concentrations of ACh, there was a signifi-cant reduction in the 50% maximum response concen-tration, consistent with the notion of a significant changein the number or function of muscarinic receptors in thelabial glands (5) that could be related to the activity ofantimuscarinic antibodies.

Antimuscarinic antibodies and SS

Effects on smooth muscle function. It is wellestablished from radioligand binding studies that, inboth the bladder and the colon, the muscarinic receptorpopulation primarily comprises the M2R and M3R sub-types, in a mixture of �80%:20% (60). Although M2R isthe predominant receptor subtype present, work withknockout mice has confirmed that M3R is the receptorsubtype that mediates the majority of the contractileresponse in smooth muscle (46,61). Accordingly, ifpresent, antimuscarinic antibodies should be able todisrupt the function of the smooth muscle within thebladder and the colon and could therefore account forthe extraglandular symptoms of SS.

Data from in vitro studies indicate that themajority of patients with primary or secondary SS haveIgG antibodies that are capable of inhibiting bothagonist- and nerve-evoked contractions in isolatedmouse bladder or colon muscle strips, in a noncompet-itive manner (15,62). However, these findings do notprovide conclusive proof that the SS antibodies specifi-cally interact with the M3R, since contraction in smoothmuscle is known to be mediated by a number of mech-anisms. A range of pharmacologic inhibitors were usedto isolate the role of the muscarinic receptors in musclecontraction in those experiments. Unfortunately, phar-macologic inhibitors are rarely totally specific, and this isparticularly true for muscarinic receptor antagonists, asdiscussed above. Therefore, although the results supportthe notion that antibodies against muscarinic receptorshave a role in the impaired smooth muscle functionobserved in SS, it cannot be definitively concluded that1) M3R is the only muscarinic subtype involved (al-though this is likely to be the case), 2) in vivo, theinhibition is noncompetitive, or 3) the antibody interac-tion is with the receptor itself rather than with a relatedmolecule. In spite of these limitations, the sensitivity ofbioassays using muscle strips to detect antimuscarinicantibodies (63) has allowed investigators to make somepotentially important observations about the nature anddisease specificity of antimuscarinic antibodies, as dis-cussed below.

Neutralization of antimuscarinic antibodies by anti-idiotypic antibodies. Studies have indicated that theinhibition of smooth muscle contraction can be repro-duced by the monovalent Fab fragment of IgG inpatients with SS, indicating that in smooth muscle theantimuscarinic antibody activity does not require recep-tor crosslinking (64). Furthermore, antimuscarinic anti-body activity was neutralized in vitro by antiidiotypic

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antibodies in both pooled intravenous immunoglobulin(IVIG) and IgG from healthy individuals. However,IVIG did not alter the levels of anti-Ro/La antibodies(64), raising the possibility that naturally occurring an-tiidiotypic antibodies may prevent the emergence ofpotentially pathogenic antimuscarinic autoantibodies.Moreover, administration of IVIG to patients with cir-culating antimuscarinic antibodies specifically neutral-ized the activity of these autoantibodies in vivo and wasassociated with an improvement in bladder and bowelsymptoms in a few patients (65). These preliminaryfindings further support the notion of a pathogenic rolefor antimuscarinic antibodies and offer a rationale forthe use of IVIG as a treatment of autonomic dysfunctionin patients with SS. The neutralizing antiidiotypic anti-bodies are likely to be germline encoded, and if isolatedmay prove to be a more specific agent than whole IVIGfor dysautonomia in SS.

Induction of cholinergic hyperresponsiveness bypassive transfer of antimuscarinic antibodies. Althoughthe commonly observed effect of antimuscarinic anti-bodies on smooth muscle in vivo is inhibition of contrac-tion, early work also indicated that addition of SS patientIgG itself occasionally resulted in an acute episode ofmuscle contraction that preceded the inhibition (15).Data from more recent work in which mice were injectedwith IgG from antimuscarinic antibody–positive SS pa-tients or rabbit anti-M3R antibodies (63) showed thatpassive transfer of antimuscarinic antibodies produced amarked up-regulation of postsynaptic M3R expression inbladder smooth muscle, which was associated with bothmuscle hyperresponsiveness to stimulation by eitherparasympathetic nerves or exogenous muscarinic agonistand enhanced parasympathetic responses to bladderdistention. In addition, an increase in M3R immunola-beling was demonstrated in the bronchial smooth muscleand salivary glands of these mice (66).

As a result of these data, it has been suggestedthat the in vivo effects of antimuscarinic antibodies onsmooth muscle are an initial antibody-mediated block-ade of cholinergic neurotransmission, followed by areciprocal overexpression of M3R, leading to overactiv-ity. However, up-regulation of M3R is likely to be anearly effect and, in the long term, a chronic down-regulation in receptor responsiveness would be ex-pected. This mechanism could provide an explanationfor the bronchial hyperresponsiveness and early sialor-rhea observed in patients with SS (59,67). Overall, theseresults not only reinforce the previous findings that SSpatient IgG affects the muscarinic receptors of smoothmuscle, but also fulfill an important criterion for dem-

onstrating that antimuscarinic antibodies are potentiallypathogenic (68), by showing that passive transfer of SSIgG can perturb organ function.

Specificity for SS. The data from smooth musclebioassays demonstrate that antimuscarinic antibodiesare not exclusive to SS. Functional antimuscarinic auto-antibodies have been detected by smooth muscle bioas-say in 15 of 24 patients with primary SS (63%), 9 of 11patients with rheumatoid arthritis (RA) and secondarySS (82%), 0 of 6 patients with RA, 8 of 10 patients withscleroderma (80%), and 3 of 3 patients with dermato-myositis (100%) (Gordon T: unpublished observations).Therefore, they may represent a common pathogeniclink between primary and secondary SS, as well ascontribute to parasympathetic dysfunction in other sys-temic rheumatic diseases.

In summary, the pathophysiologic consequencesof antimuscarinic antibodies in vivo are likely to becomplex and may reflect a combination of directantibody-mediated effects and longer-term counterregu-latory mechanisms. While functional smooth muscleassays can be regarded as the current gold standard forthe sensitive detection of autoantibodies that inhibitcholinergic neurotransmission, they cannot be used toidentify the precise target(s) of the autoantibody, andcaution must be used in extrapolating results of theseassays to the multifactorial pathophysiology of glandularhypofunction in patients with SS.

Effects on salivary and lacrimal acinar cells.Animal experiments. The NOD mouse model is a well-established animal model for study of the salivary glandhypofunction associated with human SS (69). Antibodiescapable of immunoprecipitating muscarinic receptorshave been detected in the NOD mouse (70), suggestingthat antimuscarinic antibody activity could underlie thepathology responsible for the salivary gland hypofunc-tion. This theory is further supported by the findings that1) NOD Ig�-null mice that lack B cells do not developsalivary gland hypofunction, 2) passive transfer of NODmouse IgG or of SS patient IgG or IgG F(ab�)2 frag-ments to NOD Ig�-null mice results in the developmentof salivary gland hypofunction in the majority of recip-ient mice (although a few SS IgG fractions did causehypersecretion) (see below) (71), and 3) infusion of ananti-M3R monoclonal antibody into NOD/SCID micecauses salivary gland hypofunction (72). However, aproblem with data from animal whole-body experimentsis that they do not demonstrate the precise site ormechanism of action of the infused antibody.

Radioligand binding. Findings of radioligandbinding studies have suggested that serum from a large

ANTIMUSCARINIC ANTIBODIES IN SS 2989

proportion of patients with SS, but not from controls,contains IgG antibodies that can noncompetitively bindto the M3R of rat parotid gland (14) or rat exorbitallacrimal gland membranes (73), as well as salivary IgAantibodies that can noncompetitively bind to the M3R ofrat parotid salivary gland membranes (74). While thesedata suggest that antimuscarinic antibodies may have apathologic role in SS, they do not conclusively demon-strate that the interaction is exclusively with M3R. This isbecause experimental differentiation between the mus-carinic receptor subtypes present in a tissue is difficultsince there are no specific selective antagonists availableand, in rodent salivary glands, M3R, M4R (75), and M1R(76) are all present.

As noted above, experimental differentiation be-tween the muscarinic receptor subtypes present in atissue is difficult since there are no specific selectiveantagonists available. In only one of the radioligandbinding studies examining the ability of SS patient IgGor IgA to bind to muscarinic receptors (14,73,74) did theinvestigators attempt to use a rank order of antagoniststo characterize the site of SS IgG interaction withmuscarinic receptors (14). Unfortunately, only 4 antag-onists were used, and these included AF-DX 116, acompound known to produce variable results with M3R(77). The remainder of the studies used 4-DAMP as anM3-“selective” muscarinic antagonist (see Table 2).Therefore, it is impossible to differentiate the M3 sub-type from M1 or M4. In addition, data from studies inwhich radioligand binding has been used to detect SSIgG recognition of muscarinic receptors present onparotid gland membranes isolated from Ig�-null micesuggest that the inhibition is competitive rather thannoncompetitive (71).

Overall, the results of radioligand binding exper-iments have confirmed that lacrimal and salivary mus-carinic receptors are recognized by SS IgG antibodies.However, the precise muscarinic receptor targets andthe nature of the interaction have not been confirmedusing this approach.

Immunologic approaches. Conventional immuno-logic approaches offer the ability to determine the sitespecificity of an antigen–antibody reaction. Multipletechniques have been used to investigate the interactionbetween both salivary and lacrimal gland muscarinicreceptors and SS IgG. Data from experiments using ratlacrimal glands have suggested that SS IgG recognitionof M3R can be detected by immunofluorescence, sincethe immunofluorescent signal could be quenched bypreincubation of the SS IgG with a synthetic peptidecorresponding to the second extracellular loop of M3R

(78). Further support for this is provided by the demon-stration that the same M3R peptide could be used todetect anti-M3R antibodies in SS IgG (79) and SSsalivary IgA by enzyme-linked immunosorbent assay(ELISA) (74).

The above data would appear to strongly supportthe notion that the antimuscarinic antibodies in SS IgGrecognize M3R. However, as we have noted previously(16,80), the published sequence for the second extracel-lular loop of M3R differs from the peptide used(74,78,79). The peptide used actually corresponds to thesecond extracellular loop of M4R (Table 1). The reasonfor this confusion has been discussed above. The datafrom immunohistochemistry and ELISA experimentsthus inadvertently imply that SS IgG recognizes M4R, amuscarinic receptor subtype not associated with secre-tion by salivary acinar cells. Furthermore, in a subse-quent ELISA study using the correct sequence of thesecond extracellular loop of M3R, it was concluded thatthese peptides were not an appropriate tool for detec-tion of anti-M3R antibodies (80).

Western blotting using crude lacrimal membranefractions as a source of M3R has also been reported as asuitable method for the detection of anti-M3R antibod-ies in SS IgG (73). However, using membranes obtainedfrom Chinese hamster ovary (CHO) cells that had beenstably transfected with functional human M3R, we wereunable to detect anti-M3R activity in SS IgG by Westernblotting (16). This fundamental discrepancy between thefindings of two studies can be explained by consideringthe molecular weight of the protein recognized by SSIgG. In the earlier report (73) it was stated that SS IgGrecognized a 70-kd lacrimal membrane protein. Whilethis is in good accordance with the molecular weight ofM3R predicted on the basis of the amino acid sequence(�65 kd), it is difficult to reconcile with the molecularweight of human M3R (102 kd) expressed in CHO cellsfollowing posttranscriptional modification (16,36).Given that the homology shared between human and ratM3R is �91% and they differ in size by only 1 aminoacid, the rat protein is likely to be the same size as thehuman protein, and the rat lacrimal membrane proteinrecognized by SS IgG (73) may therefore not be M3R.

In other autoimmune diseases such as Graves’disease and myasthenia gravis, recognition of theepitope by an autoantibody is highly dependent on theconformation of the epitope (11,12,81,82). This findingcould provide an explanation of why, in the abovestudies, anti-M3R activity in SS IgG could not be reliablydetected by either ELISA or Western blotting. In anattempt to reconcile these issues we used human M3R–

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transfected CHO cells as the basis of a whole cellELISA. Under our experimental conditions, the confor-mation of the expressed M3R was maintained; neverthe-less, we were unable to definitively detect anti-M3Ractivity in the SS IgG (16). In a study using an analogousapproach, with M3R-transfected CHO cells as the basisof a flow cytometric assay, the results indicated thatanti-M3R may be present in SS IgG (83). However, thereported molecular weight of the transfected receptorexpressed by the CHO cells used in that study was only70 kd, and only a portion of the transfected CHOpopulation was recognized by SS IgG. Therefore, furtherstudies are needed to confirm these findings.

Overall, the findings from studies using immuno-logic approaches serve to confuse the issue of the sitespecificity of the SS IgG antigen–antibody interaction,rather than clarify it. However, the data do support theconcept that epitope confirmation is important. A pos-sible reason for the lack of ability to detect anti-M3R inSS IgG by immunologic approaches could be that theantibodies are present in the circulation only at lowlevels, as is well established in myasthenia gravis (11).This may explain why bioassay has proved to be the mostreliable method to date for the detection of putativeanti-M3R activity in SS IgG.

Bioassay approaches with salivary acinar cells.Compared with the volume of data obtained by musclebioassay, information from salivary gland bioassays is inits infancy. Nevertheless, SS is a disease characterized bysalivary gland hypofunction, and data demonstratingthat SS IgG can interfere with salivary cell function arevital if SS IgG is to be linked with the pathology ofsalivary gland hypofunction in the disease.

Data from a study in which HSG cells (humansalivary ductal cell line) were continuously exposed to SSIgG demonstrated that SS IgG was able to partiallyinhibit the carbachol-evoked, but not the ATP-evoked,[Ca2�]i response (84), suggesting the presence of anti-muscarinic antibody activity. Although these results arehighly supportive of those obtained using smooth musclebioassay, HSG cells are a ductal cell line while thesalivary gland cells that are responsible for fluid secre-tion and involved in SS are the salivary acinar cells (5).Using mouse submandibular acinar cells, we were ableto demonstrate that, in vitro, SS IgG partially inhibitedcarbachol-evoked [Ca2�]i in a reversible manner (16),confirming that, at least in vitro, SS IgG may also have afunctional role in salivary gland hypofunction. Unfortu-nately these data do not unequivocally pinpoint theacinar cell target recognized by SS IgG. However, pre-liminary experiments analogous to those conducted with

smooth muscle (63) indicate that human submandibularacinar cells exposed to antibodies raised against thesecond extracellular loop of human M3R precisely mimicthe effects of SS IgG, i.e., they reversibly inhibit theagonist-evoked [Ca2�]i signal (Dawson L, et al: unpub-lished observations).

The reversibility of the binding observed whenmouse or human submandibular acinar cells are exposedto SS IgG or rabbit polyclonal anti-M3R is intriguingsince it confirms the previous findings in Ig�-null mice(71) but is contradictory with data obtained from smoothmuscle bioassay (15,62,64) and radioligand binding stud-ies (14,73). In myasthenia gravis it is established that, invitro, the reversibility of the interaction between theautoantibodies and the nicotinic cholinergic receptors ishighly dependent on the length of time of exposure andthe concentration of the autoantibody (85). Therefore,the differences in the nature of the binding of SS IgGwith muscarinic receptors could be related to the exper-imental conditions and may also contribute to the equiv-ocal findings with immunologic approaches.

Alternatively it has been suggested that SS IgGmay recognize an as-yet-unidentified molecule that isclosely related to the muscarinic receptor (13,16). Aprecedent for this exists in myasthenia gravis, in whichantibodies that affect nicotinic ACh receptors, but aredirected against the closely associated molecule muscle-specific receptor tyrosine kinase (68,86), have beenidentified in 70% of patients who are seronegative forautoantibodies against nicotinic ACh receptor.

The experimental findings to date relating to theinteraction of SS IgG with glandular acinar cells must beinterpreted with caution, but the available data dodemonstrate that SS IgG can affect the function ofsalivary acinar cells both in vivo and in vitro. Recogni-tion of the salivary acinar cell epitope(s) by the autoan-tibody is likely to be conformationally dependent. How-ever, the precise epitope involved remains to beconfirmed, although the second extracellular loop ofM3R is currently the most attractive candidate.

Antimuscarinic antibodies: where are we?

There have been several attempts to define cri-teria for autoimmune diseases (87–89), and recently,Drachman suggested a set of 5 criteria that a putativeautoantibody must fulfill in order to be consideredpathogenic: 1) “Autoantibodies are present in patientswith the disease,” 2) “Antibody reacts with the targetantigen,” 3) “Passive transfer of antibody reproducesfeatures of disease,” 4) “Immunization with antigen

ANTIMUSCARINIC ANTIBODIES IN SS 2991

produces a model disease,” and 5) “Reduction in anti-body levels ameliorates the disease” (68). It seemsappropriate to consider the case for antimuscarinicantibodies in SS in relation to these criteria, and theindividual criteria are addressed below.

“Autoantibodies are present in patients with thedisease.” The available data suggest that a significantnumber of patients with primary SS and secondary SShave serum IgG antibodies that are capable of binding toand influencing the function of muscarinic receptors onsalivary acinar cells and the smooth muscle of bladderand colon, in vitro. However, due to the lack of aneffective screening assay, only a small number of subjectshave been tested. Furthermore, early data indicate thatantibodies with similar actions also occur in RA, sclero-derma, and dermatomyositis. Further work is needed toestablish the prevalence of anti-M3R in SS.

“Antibody reacts with the target antigen.” Al-though the data strongly indicate that the second extra-cellular loop of M3R is the target antigen, this has notbeen demonstrated conclusively, and the preciseepitopes are currently unknown.

“Passive transfer of antibody reproduces fea-tures of disease.” Passive transfer is arguably the mostimportant evidence to highlight the pathogenic role ofan antibody. In the case of SS IgG, results obtainedfollowing the transfer of SS IgG to mice have indicatedthat the recipient mice develop glandular hypofunction(71) and exhibit up-regulated M3R expression in bron-chioles and marked hyperresponsiveness of bladdersmooth muscle (66). However, these data should beconsidered to be preliminary and must be confirmed insubstantially larger trials.

“Immunization with antigen produces a modeldisease.” The target antigen is currently unknown, sothese crucial experiments are lacking.

“Reduction in antibody levels ameliorates thedisease.” The first stages in demonstrating this criterionhave recently been undertaken, and it was demonstratedthat 1) antimuscarinic antibody activity was neutralizedin vitro by antiidiotypic antibodies in both pooled IVIGand IgG from healthy individuals (65) and 2) adminis-tration of IVIG to patients with circulating antimusca-rinic antibodies specifically neutralized the activity ofthese autoantibodies in vivo and was associated with animprovement in bladder and bowel symptoms in a fewpatients (65). Although the data from the studies withIVIG are supportive rather than substantive, they cur-rently are the best evidence available to address thiscriterion.

Although a substantial amount of further infor-

mation is needed before it can be definitively stated thatantimuscarinic antibodies have a pathogenic role in SS,the available data strongly support this suggestion.

Where we are going

We believe future research on antimuscarinicantibodies should focus on clearly identifying theepitopes recognized by SS IgG. This information ispivotal if we are to 1) understand the antigen–antibodyinteractions of this antibody; 2) develop a simple screen-ing test to allow determination of antibody incidencewithin the SS population; 3) induce the disease inexperimental models through immunization with theantigen; and 4) neutralize antibody activity in vivo, thusfulfilling key elements of the criteria proposed by Drach-man (68). A starting point for determining possibleantigens may be derived from in vitro data showing thatcleavage of M3R by granzyme B results in novel frag-ments (90).

Diversification of the research into salivary glandhypofunction in SS is also important. There has been atendency for investigators to concentrate on a limitednumber of pathogenic mechanisms that could culminatein loss of salivary gland tissue; to this end, the roles oflymphocytes, cytokines, apoptosis, and various autoanti-bodies have all been investigated. Although this reviewhas concentrated on the evidence supporting a role forantimuscarinic antibodies in the glandular and extra-glandular symptoms of SS, it would be a gross oversim-plification to assume that these antibodies are the soleetiologic factor in salivary gland hypofunction. While itis likely that antimuscarinic antibodies have a key role inthe development of salivary gland hypofunction, otherfactors have been proposed, including cytokines, cho-linesterase (91,92), and aquaporins (93,94). The re-search challenge now is to firmly establish the role ofantimuscarinic antibodies in the pathology of SS and todetermine which of the many additional mechanismsunder consideration are pivotal and which are simplyepiphenomena.

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