Muscarinic Acetylcholine Receptor Antibodies as a New Marker of Dry Eye Sjögren Syndrome

Abstract

purpose. The authors investigated whether circulating autoantibodies against M₃ muscarinic acetylcholine receptors (mAChRs) could be a new marker for diagnosis for primary and secondary Sjögren syndrome (SS) dry eye.

methods. Enzyme-linked immunosorbent assay (ELISA) using both rat exorbital lacrimal gland acinar cell membranes and synthetic 25-mer peptide as antigens was used to determine autoantibodies against acinar cells and M₃ mAChRs. Also, nitric oxide synthase (NOS) activity was assessed to determine the biological effect of these autoantibodies in relation to the M₃ mAChR.

results. Sera from dry eye primary SS (pSS) or secondary SS (sSS) patients tested by ELISA recognized membrane lacrimal gland acinar cells antigens and the synthetic 25-mer peptide, corresponding to the second extracellular loop of human M₃ mAChRs. Moreover, the IgG fraction and the corresponding affinity-purified anti-M₃ peptide autoantibodies from the same patients were able to activate NOS coupled to lacrimal gland M₃ mAChRs. As controls, IgG and sera from women without dry eye with or without rheumatoid arthritis and from normal control subjects gave negative results on ELISA and biological assay; thus demonstrating the specificity of the reaction.

conclusions. Autoantibodies against mAChR may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes and could be a new marker to differentiate SS dry eyes from non-SS dry eyes.

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by histologic and functional alterations of exocrine glands with progressive loss of salivary and lacrimal gland secretion. ^{1 2 3} There is a marked female preponderance among patients with the disorder, with a female-to-male ratio of 9 to 1, and the disease occurs mainly in the fourth and fifth decades. ⁴ The illness may occur as a secondary phenomenon in association with a wide variety of other autoimmune disorders including rheumatoid arthritis, systemic lupus erythematosus (SLE), myositis, scleroderma, and diabetes, ^{5 6} but it also occurs independently as primary SS (pSS). 

The cardinal clinical manifestations are keratoconjunctivitis sicca and xerostomia. Keratoconjunctivitis sicca, the ocular feature of the disease, is the result of destruction of acinar cells by periductal mononuclear cell infiltrate. ⁷ The lymphoproliferation consists predominantly of CD4 T lymphocytes that transcribe IL-2, IFN-γ, and B cells that use a particular light chain. ^{9 10} The immune-mediated destruction of the lacrimal glands results in severe aqueous tear deficiency that leads to ocular surface disease and marked reduction in mucin production. ⁹  

Ocular symptoms and signs (disabling eye irritation, foreign body sensation, burning, itching, redness, photophobia, intermittent blurring of vision) and ocular surface evaluation in SS patients are identical with those observed in non-SS dry eye patients. Moreover, the clinical course has similarities, ¹¹ but the age spread of SS is broader than that of non-SS dry eye. Also, the preponderance of female patients is much greater in SS than in non-SS dry eye. ¹²  

The criteria for the diagnosis of SS continues to be controversial. ⁸ In the past, diagnosis has depended on the presence of typical clinical features and/or parotid gland swelling together with focal lymphocytic infiltration demonstrated on biopsy of minor salivary glands and lips. However, serologic findings recently have been recognized to have diagnostic values. Serologic findings include autoantibodies against SS-A/Ro and SS-B/La, ¹³ antinuclear antibodies, anti–salivary gland antibodies, and rheumatoid factor. ¹⁴ However, these autoantibodies have been associated not only with SS, but also with SLE, subacute cutaneous LE, congenital heart block, and neonatal lupus dermatitis. ¹⁵  

We have recently proposed a pathophysiological role for circulating antimuscarinic acetylcholine receptor (mAChR) autoantibodies in patients with pSS. These autoantibodies recognized and activated mAChRs in both salivary and lacrimal glands. ^{16 17 18} There is a strong regulatory action of parasympathetic stimulation on the secretion of lacrimal and salivary glands. ^{15 16 17} Lacrimal and salivary gland mAChRs are coupled to various signaling pathways, and the production of nitric oxide is of particular interest, because it is involved in several pathologic processes, including SS, where increased levels of nitrites are found in the saliva. ¹⁹ We therefore considered it to be of interest to see whether these autoantibodies were able to stimulate nitric oxide synthase (NOS) activity in the lacrimal gland through the activation of M₃ mAChRs. Furthermore, important changes in the stimulus/secretion process associated with parasympathetic stimulation have been described in an animal model of SS that could account for the reduced tear output seen in these animals. ²⁰  

The aim of this work was to study the molecular interaction between serum autoantibodies from SS patients and human M₃ mAChR, showing that the second extracellular loop of this receptor, is the main target of human SS autoantibody-mediated biological effects. Our results show that the presence of circulating antibodies directed to the second extracellular loop of human M₃ mAChRs and that these affinity-purified anti-M₃ peptide autoantibodies induce NOS activation. Also, we analyzed the distribution of anti-M₃ mAChR autoantibodies in both pSS and sSS dry eye patients compared with non-SS dry eye patients, to evaluate the relationship between the presence of such circulating autoantibodies and the evidence of dry eye in SS and non-SS patients. 

Methods

Subjects

Women (aged 35–75 years) were selected from the metropolitan area of Buenos Aires. The subjects studied were divided into five groups: group I, 20 primary Sjögren syndrome (pSS) dry eye patients; group II, 17 secondary Sjögren syndrome (sSS) dry eye patients associated with rheumatoid arthritis (RA); group III, 24 postmenopausal dry eye patients without SS; group IV, 14 RA patients with neither SS nor dry eye, and group V, 35 normal control subjects. 

Ophthamologic and Serologic Tests

The presence of anti-Ro/SS-A and anti-La/SS-B antibodies was a mandatory condition to belong to group I. The diagnosis of SS followed four or more criteria of Vitali et al. ²¹ The following ophthamologic tests were evaluated: Schirmer test; tear break-up time (BUT), rose bengal staining score, and bulbar impression cytology. Serologic tests were also performed: anti-Ro/SS-A and anti-La/SS-B antibodies, rheumatoid factor (RF), and antinuclear antibodies (ANA). Anti-Ro/SS-A and anti-La/SS-B were studied by double diffusion using human spleen extract and rabbit calf thymus extract, respectively. Both antibodies were also investigated by enzyme-linked immunsorbent assay (ELISA) using bovine purified antigens. RF was investigated by latex fixation and rose bengal test; ANA were investigated using an immunofluorescence test over cryostat sections of rat liver and mouse kidney. All the studies involving human subjects were conducted according to the Helsinki Declaration and informed consent was obtained from the subjects. 

Preparation of Rat Lacrimal Gland Acini

Exorbital lacrimal gland acini were prepared from adult female Wistar strain rats. Animals were used according to The Guide to the Care and Use of Experimental Animals (DHEW Publication, NIH 80-23). Glands were dissected away from fat, connective tissue, and lymph nodes and immersed in a tissue chamber containing Krebs-Ringer-bicarbonate (KRB) solution gassed with 5% CO₂ in oxygen and maintained at pH 7.4 and 30°C. All subsequent steps were performed at 4°C. Lacrimal glands were minced and incubated in KRB supplemented with 10 mM HEPES and 5.5 mM glucose (KRB-HEPES) and 0.5% bovine serum albumin (BSA), pH 7.4, containing collagenase (150 U/ml). Lacrimal gland lobules were subjected to gentle pipetting. The preparation then was filtered through nylon mesh (150-μm pore size), and the acini were pelleted with 2 minutes centrifugation at 50g. The pellet was then washed twice by centrifugation (50g for 2 minutes) through a 4% BSA solution made in KRB-HEPES buffer. The dispersed acini were allowed to recover for 30 minutes in 5 ml fresh KRB-HEPES buffer containing 0.5% BSA. ²²  

Preparation of Microsomal Fractions

Lacrimal gland acini were homogenized for 10 seconds twice in 50 mM phosphate buffer, pH 7.4, in an Ultra-Turrax (setting 5). The homogenate was centrifuged for 10 minutes at 1000g. The pellets were discarded, and the supernatants were centrifuged (10,000g) at 4°C for 10 minutes and then at 40,000g for 60 minutes. The resulting pellets were resuspended in the same buffer supplemented with 0.1 mM phenylmethylsulfonyl fluoride, 1 mM ethylendiamintetracetic acid (EDTA), 5 μg/ml leupeptin, and 1 μM pepstatin A as described previously ²³ and used as a membrane source for the ELISA test. Some experiments were performed with cardiac membranes prepared as previously described. ²³  

Purification of Antipeptide Antibodies by Affinity Chromatography

The IgG fraction of 10 patients from groups I and II were independently subjected to affinity chromatography on the synthesized peptide covalently linked to AffiGel 15 gel (Bio-Rad, Richmond, CA). The IgG fraction was loaded on the affinity column equilibrated with phosphate-buffered saline (PBS), and the peptide fraction was first eluted with the same buffer. Specific anti-M₃ peptide autoantibodies were then eluted with 3 M KSCN, 1 M NaCl, followed by immediate extensive dialysis against PBS. The IgG concentration of the anti-M₃ peptide antibodies was detected by radial immunodifussion assay. 

ELISA

Fifty microliters of peptide solution (20 μg/ml) in 0.1 M Na₂CO₃ buffer, pH 9.6, was used to coat microtiter plates (Nunc, Kastrup, Denmark) at 4°C overnight. After blocking the wells, diluted sera from patients of groups I, II, III, IV, and V were added in triplicate and allowed to react with the peptide for 2 hours at 37°C. After thoroughly washing the wells with 0.05% Tween 20 in PBS, 100 μl of 1:6000 biotinylated goat anti-human IgG antibodies (Sigma Chemical Co., St. Louis, MO) was added and incubated for 1 hour at 37°C. Then, a 1:6000 dilution of ExtrAvidin-alkaline phosphatase (Sigma) was allowed to react an extra 30 minutes at 37°C. After extensive washings, p-nitrophenylphosphate (1 mg/ml) was added as substrate, and the reaction was stopped at 30 minutes In addition, 50 μl of exorbital lacrimal gland acinar cell membranes (50 μg/ml) in 0.1 M Na₂CO₃ buffer, pH 9.6, was used to coat microtiter plates at 4°C overnight, and the ELISA procedure was performed as described above. In same experiments cardiac cell membranes (50 μg/ml; lacking in M₃ mAChRs) was used as coating antigen. Finally, the plates were read at 405 nm, and results for each sample were expressed as the mean ± SD of triplicate values. 

Determination of NOS Activity

Female Wistar rats were used throughout. NOS activity was measured in exorbital lacrimal glands by production of[ U-¹⁴C]citrulline from[ U-¹⁴C]arginine according to the procedure described by Bredt and Snyder ²⁴ for brain slices. Briefly, after a 10-minute preincubation in KRB solution, tissues were transferred to 500 ml of prewarmed KRB equilibrated with 5% CO₂ in oxygen in the presence of[ U-¹⁴C]arginine (0.5 μCi). When antagonists were used, appropriate concentrations of drugs were added, and tissues were incubated for 15 minutes under carbogen at 37°C before the addition of IgG or anti-M₃ mAChR peptide. Tissues were then homogenized with an Ultraturrax in 1 ml of medium containing 20 mM HEPES, pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM dithiothreitol, 1 mM leupeptin, and 0.2 mM phenylmethylsulphonyl fluoride at 4°C. After centrifugation at 2000g for 10 minutes at 4°C, supernatants were applied to 2 ml columns of Dowex AG-50 WX-8 (sodium form). [U-¹⁴C]citrulline was eluted with 3 ml water and quantified by liquid scintillation counting. Measurement of basal NOS activity in exorbital lacrimal glands by the above-mentioned procedure was 95% inhibited by 0.5 mM N ^G -monomethyl-l-arginine (l-NMMA). The results (pmol · g⁻¹ tissue wet weight) obtained from exorbital lacrimal gland were expressed as the difference between values in the absence or in the presence of l-NMMA. 

Drugs

A 25-mer peptide (K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-N-P-A-V-T-F-G-T-A-I) corresponding to the sequence of the second extracellular loop of the human M₃ mAChR and a 24-mer peptide (V-R-T-V-E-D-G-E-C-Y-I-Q-F-F-S-N-A-A-V-T-F-G-T-A) corresponding to the sequence of the second extracellular loop of the human M₂ mAChR were synthesized by the PeptidoGenetic Research Company (Livermore, CA). The synthetic peptides were synthesized by the F-moc amino acid that was activated using HOBt/DCO (l-hydroxy benzo triazole/decyclohexylcarbodiimide) strategy with an automatic peptide synthesizer (model 413A; Applied Biosystems, Foster City, CA). The peptides were desalted, purified by high-performance liquid chromatography, and subjected to amino-terminal sequence analysis by automatic Edman degradation with an Applied Biosystems (model 470) sequencer. l-NMMA was from Sigma Chemical Co., and 4-di-phenylacetoxy-N-methyl piperidine methiodide (4-DAMP) was provided by Boehringer Ingelheim Pharmaceuticals (Berlin, Germany). Stock solutions were freshly prepared in the corresponding buffer. 

Statistical Analysis

ELISA optical density values from anti-M₃ peptide antibodies were distributed in five groups. Prevalence values between groups were compared by χ² test. All statistical significance was justified at P < 0.05. Student’s t-test for unpaired values was used to determine the level of significance. Different between means were considered significant if P < 0.05. 

Results

Figure 1 shows the presence of serum autoantibodies against exorbital lacrimal gland acinar cell membranes in all the studied groups. The optical density values for both sera from pSS (group I) and sSS (group II) patients were similar. Sera from non-SS dry eye patients (group III) or for RA patients (group IV) show optical density values that do not differ from those of normal control sera (group V). These results point to the presence of IgG directed against lacrimal gland membrane acinar cells in both pSS and sSS patients. When myocardial cell membranes (lacking M₃ mAChR) were used as coating antigen in the ELISA procedure, sera from groups I and II gave negative results (Table 1) , pointing to the specificity of the reaction against the M₃ mAChR subtype. 

The presence of circulating autoantibodies against M₃ mAChR in the different groups was demonstrated by ELISA using a 25-mer peptide as coating antigen. The optical density values for each of the 110 subjects studied are shown in the scatterogram (Fig. 2) . The immune reactivity of sera from pSS dry eye patients (group I) was similar to that of sSS dry eye patients (group II). Furthermore, the immune reactivity of sera from pSS and sSS patients was significantly greater than that of sera from non-SS dry eye patients (group III;[ P < 0.0005]). The median optical density of sera from groups IV (non-SS non dry eye patients with RA) and V (control normal subjects) was not different from that of group III. The optical density of sera from group I and II was always higher than three SDs from those of sera of normal individuals (group V; Table 1 ). 

Figure 3 shows a concentration-dependent increase in optical density by serum, total IgG, and the corresponding affinity-purified anti-M₃ peptide IgG from group I. The left panel shows an immune reactivity of different dilution of sera; the right panel shows the concentration-dependent increase in optical density of total IgG and the corresponding affinity-purified anti-M₃ peptide IgG. The maximal increment induced by anti-M₃ peptide IgG was higher than that observed with the corresponding total IgG. The immunologic specificity of the ELISA assay was assessed by the ability of the M₃ synthetic peptide (10-fold concentration) to inhibit the reaction when sera or immunoglobulins were preincubated with the synthetic peptide for 30 minutes at 37°C and then added together in the microtiter plates (Fig. 3 , right and left panels). To determine the specificity of M₃ synthetic peptide, we also used M₂ synthetic peptide and obtained negative results (Table 1) . 

The frequency of antimembrane lacrimal acinar cells and anti-M₃ peptide mAChR autoantibodies in groups I and II were significantly higher than that observed on other groups (Table 2) ; which implies strong association between the existence of serum, antimembrane lacrimal acinar cell, and anti-M₃ peptide mAChRs autoantibodies, in SS dry eye patients. Moreover, the comparison of results of different immunoassays produced a high correlation between them in group I. Thus, in group I anti-Ro(SS-A), anti-La(SS-B) and ANA were the most strongly associated with the presence of antipeptide M₃ mAChR autoantibodies and antimembrane lacrimal acinar cells autoantibodies (P < 0.00001). As expected, in group II the anti-RF autoantibodies and anti-ANA autoantibodies correlated with both antibodies against lacrimal acinar cell membranes and anti-M₃ synthetic peptide (P < 0.00001). On the contrary, no association between anti-RF autoantibodies or anti-ANA autoantibodies with antimembrane lacrimal acinar cell and anti-M₃ peptide mAChR autoantibodies was observed in group IV. It is important to note that no significant differences in patient ages among the different groups were demonstrated (data not shown). Only women were selected for this study. 

The ocular surface evaluation in subjects from all the studied groups is shown in Table 3 . Results agree with previously reports and do not differentiate non-SS from SS dry eye. 

It is well known that the activation of NOS occurs by signal transduction on parasympathetic stimulation. To evaluate if the endogenous nitric oxide signaling system could be activated by autoantibodies from pSS or sSS patients, the affinity-purified antipeptide IgG from these groups was used to measure the NOS activity in exorbital lacrimal glands. As shown in Table 4 , total IgG from groups I and II increased NOS activity, achieving similar values to that obtained with carbachol (1 × 10⁻⁷ M). Moreover, the affinity-purified anti-M₃ peptide autoantibodies were able to mimick the action of the corresponding total IgG. These effects could be neutralized after preincubation of immunoglobulins with the M₃ synthetic peptide. Also, the specific M₃ antagonistic drug 4-DAMP blunted the reaction. The autoantibody effects on NOS activity could be blocked by a reversible NOS inhibitor l-NMMA and reversed by l-arginine (data not shown), which points to the specificity of the reaction. 

Discussion

Dry eye with tear deficiency is now classified into two categories: associated with SS and not associated with SS. ²⁵ Both non-SS and SS dry eye involve the same symptoms, in which the most frequent complaint is ocular fatigue. ²⁶ In this article we did not observe differences in ocular surface findings between patients from groups I, II, and III. However, in general, SS dry eye is more severe than non-SS dry eye, showing greater squamous metaplasia of the conjunctiva than that observed in non-SS dry eye. It also has been reported that there is a loss of the reflex tearing in response to nasal stimulation in SS rather than non-SS dry eye, ²⁷ whereas the reflex response to conjuntival stimulation is reduced in both groups of patients (see Table 3 , Schirmer test). 

The pathogenesis of ocular surface alterations differs between the two forms of the disease. Although SS is an autoimmune disorder characterized by lymphocytic infiltration and destruction of exocrine glands, ²⁸ in non-SS dry eye, lacrimal gland biopsy shows normal morphology and no lymphocytic infiltration. ^{29 30} Also, the gland produces tears in response to strong stimuli. ²⁷  

In this regard and in this article, the differences in serologic findings including the high frequency of anti-Ro(SS-A), anti-La(SS-B), ANA, and RF, which had been recognized in patients from group I but not in those from group III, confirm the autoimmune mechanism implicated in the pathophysiology of pSS dry eyes. But, serologic markers, in particular anti-Ro(SS-A) and anti-La(SS-B), vary greatly according to the origin of the patients’ sera. ¹⁵ On the other hand, these autoantibodies are associated with other connective tissue diseases. ¹⁵ The presence of anti-Ro/La was a mandatory condition for group I, which is why there was a high prevalence of anti-Ro/La in this group. 

The most important feature of this article relating to the autoimmune nature of SS, is the presence of autoantibodies to lacrimal gland M₃ mAChRs that, acting as an “agonist-like agent,” resulted in a primary, organ-specific dysfunction. Possibly, in pSS and sSS, direct M₃ mAChR antibody-mediated tissue damage might occur through nitric oxide generation and accumulation, with an adverse effect on the lacrimal glands. Immunologic generation of nitric oxide could have cytotoxic effects on the cell, through the production of free radicals. ³¹ This could have a special pathologic role, particularly in SS, where the increased release of inflammatory mediators could induce uncommonly high levels of nitric oxide. ¹⁹ Although the vasodilator role of nitric oxide in the secretory process is recognized, as well as its release in normal saliva after parasympathetic stimulation ^{32 33 34} ; nitric oxide accumulation does not appear to guarantee normal glandular function, as can be deduced from the observation that pSS patients have higher levels of nitrites in saliva, ¹⁹ but its pathologic role is still unclear. 

We have already reported autoantibodies against rat salivary and lacrimal glands M₃ mAChR, which trigger parasympathetic, receptor-mediated biological effects. ^{16 17} Here we have demonstrated that they are able to recognize a synthetic peptide corresponding in amino acid sequence to the second extracellular loop of the human M₃ mAChR. The distribution of the amino acid sequence between rat and human M₃ synthetic peptide has a great homology (84%). Moreover, the fact that an isolated fraction from pSS or sSS IgG enriched in anti-M₃ peptide antibodies could reproduce the effects of the corresponding whole immunoglobulins, strongly suggests a prominent role for anti-M₃ peptide antibody for the mAChR-mediated effects of total SS IgG. In addition, the synthetic peptide selectively suppressed the biological effects of SS anti-M₃ peptide autoantibody and the corresponding total IgG. This supports the view that the second extracellular loop is not only the main immunogenic region of the receptor ³⁵ but can be considered essential for the biological action of these autoantibodies. 

We also demonstrated in this article an association between the existence of circulating anti-M₃ peptide mAChR autoantibodies and the presence of ocular symptoms, surface alterations, and a selected number of antibodies that commonly are detected in SS, making these autoantibodies a valuable marker for dry eye associated with both pSS and sSS. Tsubota et al. ³⁶ have shown a good correlation between lacrimal function, serum interleukin-2 receptor, ANA, and RF in SS dry patients. 

Although evidence for desensitization of mAChR by antibodies has been reported previously, ³⁷ it is still unclear whether antibodies that interact with the second extracellular loop of mAChR are involved in the pathogenesis of lacrimal gland dysfunction. Cholinergic M₃ receptor stimulation is related to secretory function including release of proteins, electrolytes, and water. ^{38 39}  

It is possible that the chronic interaction of the autoantibodies with mAChR of the lacrimal gland, behaving as a muscarinic cholinergic agonist, could lead by accumulation of nitric oxide cell dysfunction or tissue damage. It is likely that, in addition to nitric oxide release, several different mechanisms may participate, including desensitization and/or downregulation of the receptor. This process could lead to a progressive blockade of mAChR an induce dry eyes, a classical sign of pSS and sSS. 

 Supported by a grant from CONICET and UBACYT.

 Submitted for publication February 14, 2000; revised May 24 and August 3, 2000; accepted August 30, 2000.

 Commercial relationships policy: P.

 

Figure 1.

 

Immunoreactivity of antimembrane lacrimal gland acinar cells antibodies of sera from different groups: 20 pSS dry eye patients (•), 17 sSS dry eye patients associated with RA (○), 24 postmenopausal dry eye patients without SS (□), 14 RA patients with neither SS nor dry eye (▪), and 35 normal control subjects (< ). Sera titers are expressed as the reciprocal of 1:2 serum dilutions from 1:10 to 1:2560 with optical density (OD) at 405 nm. Values are means ± SD of number of patients described above.

Table 1.

 

ELISA Assay on Different Coating Antigens

ELISA Antigens	Optical Density (405 nm)
		Group I	Group II	Group V
Lacrimal gland membranes	1.07 ± 0.04*	0.96 ± 0.03*	0.16 ± 0.01
Cardiac membranes	0.13 ± 0.01	0.12 ± 0.02	0.12 ± 0.02
M3 synthetic peptide	0.93 ± 0.03, †	0.88 ± 0.03, †	0.04 ± 0.01
M2 synthetic peptide	0.03 ± 0.01	0.04 ± 0.01	0.03 ± 0.01

 Values are means ± SD of 20 patients from group I, 17 from group II, and 35 from group V. Sera 1/20 dilution were assay sensitized microplates with 50 μg/ml lacrimal gland acinar cell membranes or cardiac membranes and 20 μg/ml M₃ or M₂ synthetic peptides.

*   P < 0.001 vs. group V.

†   P < 0.001 vs. group V.

Figure 2.

 

Scatterogram showing immunoreactivity of circulating IgG antibodies against the second extracellular loop of the M₃ mAChR tested by ELISA. The individual optical density (OD) value for each serum sample (1/100 dilution) from 20 pSS dry eye patients (group I), 17 sSS dry eye patients (group II), 26 non-SS postmenopausal dry eye patients (group III), 14 non-SS, non dry eye patients with RA (group IV), and 35 normal control subjects (Group V). Dotted line, cutoff value 0.272 (mean OD ± 2 SD for group V); black lines, median OD value. Prevalence values of anti-M₃ mAChRs antibodies in groups I and II were compared by the χ² test: group I (χ² = 20.69) and group II (χ² = 23.08) vs. group III, which showed significant independence. Group III versus group V (χ² = 0.15) and group I versus group II (χ ² = 0.45) did not show significant independence. All statistical significance was justified at P < 0.05.

Figure 3.

 

Immunoreactivity of anti-mAChR antibodies of SS sera from group I directed against second extracellular loop M₃ peptide tested by ELISA. Left: effect of pSS sera titers alone (•) or incubated in the presence of 10 μg M₃ peptide (○) (expressed as the reciprocal of 1:2 serum dilutions from 1:40 to 1:2560) on optical density (OD) at 405 nm. Microtiter wells were coated with 2 μg peptide, and enzyme immunoassay was carried out as described in Methods. Values are means ± SD of 20 pSS patients from group I. Right: effect of increased concentrations of total IgG pSS alone (○) or the corresponding affinity-purified anti-M₃ peptide IgG alone (•). Curves of total pSS IgG (▪) or anti-M₃ peptide IgG (□) incubated in the presence of 10 μg M₃ peptide are also shown. Values are ± SD of 10 pSS IgG and 10 affinity-purified IgG.

Table 2.

 

Serological Tests of Subjects from Different Groups

Serological Tests	Groups
		I	II	III	IV	V
Anti-Ro (SS/A)	20/20	2/17	0/24	0/14	0/35
	(100)	(11.8)	(0)	(0)	(0)
Anti-La (SS/B)	18/20	2/17	0/24	0/14	0/35
	(90)	(11.8)	(0)	(0)	(0)
Anti-RF abs	10/20	16/17	1/24	12/14	1/35
	(50)	(94.1)	(4.2)	(85.7)	(2.9)
ANA	20/20	15/17	2/24	6/14	1/35
	(100)	(88.2)	(8.3)	(42.9)	(2.9)
Antimembrane lacrimal acinar cells	17/20	12/17	2/24	1/14	2/35
	(85)	(70.6)	(8.3)	(7.1)	(5.7)
Antipeptide M3 mAChR	18/20	14/17	2/24	1/14	2/35
	(90)	(82.4)	(8.3)	(7.1)	(5.7)

 Distribution of the serological tests performed on different groups (I–V). The results were expressed as the number of positive per total with percentages in parentheses.

Table 3.

 

Ocular Surface Evaluation in Subjects from Different Groups

Groups	Schirmer Test	Break-up Time (sec)	Rose Bengal Staining	Impression Cytology
I	≤5 mm	≤10	≥3/9	GC: absent ECM: positive
II	≤5 mm	≤10	≥3/9	GC: absent ECM: positive
III	≤5 mm	≤10	≥3/9	GC: diminished ECM: negative
IV	≥5 mm	≥10	≤3/9	GC: normal ECM: negative
V	≥5 mm	≥10	≤3/9	GC: normal ECM: negative

 Ocular surface evaluation was performed on different groups (I–V) as described in Methods. GC, goblet cells; ECM, epithelial cells with metaplasia.

Table 4.

 

Activation of NOS Activity by Anti-M₃ mAChR Autoantibodies in Exorbital Lacrimal Glands

Additions	NOS Activity (pmol/g)
Basal	640 ± 38
T pSS IgG	1410 ± 78*
T sSS IgG	1390 ± 75*
T pSS IgG+ 4-DAMP	740 ± 35*
Carbachol	1520 ± 80*
Carbachol+ 4-DAMP	710 ± 42
Anti-pM3 pSS IgG	1320 ± 82*
Anti-pM3 pSS IgG+ 4-DAMP	710 ± 37
Anti-pM3 pSS IgG+ pM3	680 ± 36
Anti-pM3 pSS IgG+ l-NMMA	692 ± 41
T Normal IgG	638 ± 40

 Values are means ± SEM of five patients from groups I and II. Experiments were performed in duplicate. Total IgGs (T IgG) from pSS, sSS patients, and normal subjects were assayed at 1 × 10⁻⁶ M and carbachol at 1 × 10⁻⁷ M. The peptide (pM₃), 4-DAMP, and l-NMMA were used at 5 × 10⁻⁶ M. Anti-pM₃ IgG means the fraction reached on anti-M₃ peptide autoantibodies, isolated from pSS IgG of group I were used at 1 × 10⁻⁷ M.

*  Significantly different from basal with P < 0.05.

The authors thank Elvita Vannucchi for technical assistance.