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Cholinoreceptor Autoantibodies in Sjögren Syndrome

¹ Pharmacology Unit, School of Dentistry, University of Buenos Aires, M.T. de Alvear 2142-4° "B", 1122 AAH Buenos Aires, Argentina;
² Rheumatology Department, School of Medicine, Universidad Pontificia de Medellín, Colombia; and
³ Argentine National Research Council (CONICET), Buenos Aires, Argentina

^* corresponding author, enri{at}farmaco.odon.uba.ar

	ABSTRACT

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Previous studies have demonstrated that antibodies against cholinoreceptors of exocrine glands correlate with dry mouth in persons with primary Sjögren syndrome (pSS). The aim of the present investigation was to establish if serum IgG antibodies (pSS IgG) were able to interact with cholinoreceptors in rat submandibular gland-dependent stimulation of cyclooxygenase 2 (COX-2) mRNA expression and PGE₂ production. Our findings indicated that pSS IgG-stimulating M₃, M₄, and M₁ cholinoreceptors exerted an increase in COX-2 mRNA without affecting COX-1 mRNA expression and increased PGE₂ production. Inhibitors of phospholipase A₂, COX- s, L-type calcium channel currents, and Ca²⁺-ATPase from sarcoplasmic reticulum prevented the pSS IgG effect on PGE₂ production. An ionophore of calcium mimicked pSS IgG action, suggesting a crucial role of calcium homeostasis in the cholinoreceptor-stimulated increase in PGE₂ production. Moreover, the amounts of PGE₂ in saliva and in sera from persons with pSS were significantly higher than in pre- or post-menopausal women. These findings illustrate the importance of autoantibodies to cholinoreceptors in the generation of chronic inflammation of target tissues in SS.

KEY WORDS: PGE₂ • submandibular gland • Sjögren syndrome • autoantibodies • cholinoceptor antibodies

	INTRODUCTION

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Primary Sjögren syndrome (pSS) is a systemic autoimmune disease of unknown etiology. Many autoantibodies have been reported to be frequently present in pSS (Marczinovits et al., 2005). In particular, antibodies to the antigens SS-A/Ro and SS-B/La are commonly used in the diagnosis of the disease (Moutsopoulos and Talal, 1987). The presence of subtypes M₁- (Perez Leiros et al., 1999), M₃- (Bacman et al., 1996, 2001), and M₄- (Reina et al., 2005) specific autoantibodies in a majority of persons with pSS (83%–90%) (Kovacs et al., 2005) is an important advance toward an understanding of the pathogenesis of pSS, not only in terms of impaired glandular function, but also because of peripheral parasympathetic dysfunction in these individuals (Reina et al., 2004).

pSS-derived serum IgG samples binding to the glandular cholinoreceptors act as a partial agonist, and have been reported not only to activate the receptor, but also to impair the response to the authentic agonist (Berra et al., 2002), suggesting a defect in post-receptor signaling (Dawson et al., 2006). The most direct mechanism for preventing target organs from carrying out their biological function is that of early agonistic-promoting activation of cholinoreceptors, initiated by autoantibodies (Li et al., 2004), which bind to and persistently activate cholinoceptors (Waterman et al., 2000). Subsequently, the agonist activity displayed by these autoantibodies may induce cholinoceptor desensitization (Cha et al., 2006), internalization, and/or intracellular degradation, leading to a progressive decrease of expression and activity of these receptors (Li et al., 2004).

Xerostomia and keratoconjunctivitis sicca result from immune lymphocytic infiltration of the salivary (Ferguson, 1999) and lacrimal (Tsubota et al., 1999) glands. The infiltrating cells interfere with glandular function by cell-mediated glandular destruction and production of autoantibodies that interfere with cholinoceptors (Fox, 2005). Dental caries, resulting from the loss of salivary flow, may be associated with periodontal disease (Ravald and List, 1998).

Prostaglandins (PGs) are among the most relevant local mediators that participate in the modulation of acinar cell functions under basal conditions (Yuan et al., 2000). During inflammation or in early stages of autoimmune diseases, PGs are released in large amounts. In particular, overt production of PGs has been shown to occur in neuroinflammatory diseases (Pasinetti, 2001).

We hypothesized the presence of an exocrine-gland-specific antigen-antibody system in persons with pSS, and investigated whether the autoantibodies can induce cholinergic-PGE₂ production with overexpression of COX-2 mRNA in submandibular glands. This immuno-inflammatory influence could result in a functional disturbance, thereby triggering the clinical signs of pSS in the submandibular gland, maintaining the chronic inflammatory state of SS.

	MATERIALS & METHODS

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Participants and Serological Testing
Women (aged 35–55 yrs) free of treatment for 6 mos and with 7 to 15 yrs since diagnosis were selected from the metropolitan area of Buenos Aires (Argentina) and Medellín (Colombia). They were divided into three groups: group I, 15 women with primary Sjögren syndrome (pSS) and with dry mouth; group II, 16 post-menopausal women with dry mouth and without SS; or group III, 18 healthy, pre-menopausal women, mean age 45 ± 10 yrs, without any systemic diseases (control group). The diagnosis of SS was based on 4 or more of the criteria from Vitali et al.(1993). Biopsy results, degree of xerostomia and keratoconjunctivitis sicca, and serological tests of the different groups are presented in the Table. All participants gave their informed consent according to an approved protocol satisfying the Ethics Committee requirement of Buenos Aires University. The studies were conducted according to the tenets of the Declaration of Helsinki.

Peptides
A 22-mer peptide QYLVGERTVLAGQCYIQFLSQP (GeneBank P11229), a 22-mer peptide QYFVGKRTVPPGECFIQFLSEP (GeneBank P20309), and a 17-mer peptide TVYIIKGYW PLGAVVCD (GeneBank P08173), corresponding to the amino acid sequence of the second extracellular loop of the human muscarinic acetylcholine receptors (mAChRs) M₁, M₃, and M₄, respectively, were synthesized as previously reported (Berra et al., 2002).

Prostaglandin E₂ (PGE₂) Assay
PGE2 assays were performed in serum and saliva from groups I, II, and III and in rat submandibular glands exposed to serum or IgG from groups I, II, and III. Rat submandibular gland slices (10 mg) were incubated for 60 min in 0.50 mL assay buffer. Different concentrations of serum or IgG were added 30 min before the end of the incubation period, and blockers were added 30 min before the addition of serum or IgG. Tissues were then homogenized in a 1.5-mL polypropylene microcentrifuge tube. Serum (2.5 µL) or saliva (20 µL) was added directly to the microplate. Thereafter, all procedures were those indicated in the protocol of the Prostaglandin E₂ Biotrak Enzyme Immuno Assay (ELISA) System (Amersham Biosciences, Piscataway, NJ, USA). The PGE₂ results were expressed as nanograms per milliliter (ng/mL).

mRNA Isolation and cDNA Synthesis
Total RNA was extracted from rat submandibular gland slices by homogenization with the use of the guanidinium isothiocyanate method (Chomczynski and Sacchi, 1987). As previously described (Sterin-Borda et al., 2003), a 20-µL reaction mixture contained 2 ng of mRNA, 20 units of RNase inhibitor, 1 mM dNTPs, and 50 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). First-strand cDNA was synthesized at 37°C for 60 min.

Quantitative PCR Procedures
Cyclooxygenase (COX) isoform (COX-1, COX-2) mRNA levels were determined by a method that involves simultaneous co-amplification of both the target cDNA and a reference template (MIMIC) with a single set of primers. MIMICs for COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were constructed by means of a PCR MIMIC construction kit (Clontech Laboratories, Palo Alto, CA, USA). Each PCR MIMIC consisted of a heterologous DNA fragment with 5'- and 3'-end sequences that were recognized by a pair of gene-specific primers. Sizes of PCR MIMIC were distinct from those of native targets. PCR oligonucleotide primer sets for G3PDH, COX-1, and COX-2 were designed to amplify 452-, 160-, and 242-bp products, respectively. The following G3PDH primers were used: (5'–3') ACCAC AgTCCA TgCCAT CAC and TCCAC CACCC TgTTg CTgTA. Oligonucleotide primers for COX-1 were (5'–3') TAAgT ACCAg TgCTg gATgg and AgATC gTCgA gAAgA gCATCA; for COX-2, they were (5'–3') TCCAA TCgCT gTACA AgCAg and TCCCC AAAGA TAgCA TCTgg. Aliquots were taken from pooled first-strand cDNA from the same group and constituted one sample for PCR. A series of 10-fold dilutions of known concentrations of the MIMIC was added to PCR amplification reactions containing the first-strand cDNA. PCR MIMIC amplification was performed in 100 µL of a solution containing 1.5 mM MgCl₂, 0.4 µM primer, dNTPs, 2.5 U Taq DNA polymerase, and 0.056 µM Taq Start antibody (Clontech Laboratories). After initial denaturation at 94°C for 2 min, the cycle conditions were 30 sec of denaturation at 94°C, 30 sec of annealing at 60°C, and 45 sec for enzymatic primer extension at 72°C for 45 cycles for COX isoforms. The internal control was the mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH). PCR amplification was performed with initial denaturation at 94°C for 2 min, followed by 30 cycles of amplification. Each cycle consisted of 35 sec at 94°C, 35 sec at 58°C, and 45 sec at 72°C. Samples were incubated for an additional 8 min at 72°C before completion. PCR products were subjected to electrophoresis on ethidium-bromide-stained gels. Band intensity was quantified by densitometry with NIH Image software. Levels of mRNA were calculated from the point of equal density of the sample and MIMIC PCR products (Sterin-Borda et al., 2003). Cyclooxygenase isoform mRNA levels were normalized with the levels of G3PDH mRNA present in each sample, which served to control for variations in RNA purification and cDNA synthesis. Relative mRNA expressions of COX-1 and COX-2 were compared with those from the respective healthy individuals and persons with pSS, reported as a percentage of the healthy individuals.

Drugs
Pilocarpine, tropicamide, aspirin, verapamil, thansigargin, and pirenzepine were obtained from the Sigma Chemical Company (St. Louis, MO, USA). AF-DX 116 and 4-DAMP were kindly provided by Boehringer Ingelheim Pharmaceuticals Inc. (Ingelheim, Germany). 4-(4-octadecylphenyl)-4-oxobutenoic acid (OBAA) was provided by Tocris Cookson Inc. (Ellisville, MO, USA). Rofecoxib was provided by Merck (Darmstadt, Germany). Stock solutions were freshly prepared in the corresponding buffers. The drugs were diluted in the bath to achieve the final concentrations stated in the text.

Statistical Analysis
We used Student’s t test for unpaired values to determine the levels of significance. When multiple comparisons were necessary, after analysis of variance, the Student-Newman-Keuls test was applied. Differences between means were considered significant if P < 0.05.

	RESULTS

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Sera and the corresponding IgG from persons with pSS triggered PGE₂ production in a concentration-dependent manner in isolated rat submandibular glands (Figs. 1A, 1B). In contrast, the IgG from persons without pSS (groups II and III) had no effect. The selective M₁ (pirenzepine), M₃ (4-DAMP), and M₄ (tropicamide) cholinoreceptor antagonists decreased the production of PGE₂, shifting the dose-response curves of pSS IgG to the right (Fig. 1C). Moreover, M₁, M₃, and M₄ synthetic peptides inhibited pSS IgG-induced PGE2 production (Fig. 1D).

View larger version (19K): [in this window] [in a new window]   Figure 1. Actions of serum and IgG from pSS patients on PGE2 generation. (Upper panel) Effects of increasing concentrations of serum (A) and pSS IgG (B) on PGE2 production on an isolated rat submandibular gland. Values represent the mean ± SEM of 10 different individual serum or IgG samples from each group, performed in triplicate. *p < 0.001 vs. groups II and III. (Lower panel) Concentration-response curves of pSS IgG on PGE2 production on an isolated rat submandibular gland in the absence or in the presence of muscarinic acetylcholine receptor antagonists (C), and in the presence of M1, M3, and M4 synthetic peptides (D). The blocker agents were used at 1 x 10–6 M, and the peptides at 1 x 10–5 M. Values represent the mean ± SEM of 7 different individual serum or corresponding IgG samples from group I, performed in duplicate. *p < 0.01 vs. pirenzepine, 4-DAMP, and tropicamide. **p < 0.001 vs. M1 peptide, M3 peptide, and M4 peptide. [Group designations as in the Table: Group I, 15 women with primary Sjögren syndrome and with dry mouth; Group II, 16 post-menopausal women with dry mouth and without Sjögren syndrome; and Group III, 18 healthy, pre-menopausal women without any systemic disease (control group).]

View larger version (33K): [in this window] [in a new window]   Figure 2. Influence of different inhibitors on pSS IgG and RT-PCR analysis. (A) Concentration-response curves of pSS IgG in the absence or in the presence of 5 x 10–6 M OBAA or 1 x 10–4 M aspirin or 4 x 10–8 M rofecoxib. Results are expressed as mean ± SEM of 6 individual pSS IgG samples performed in duplicate. *p < 0.001 vs. pSS IgG alone; **p < 0.005 vs. pSS IgG alone. (B) Effect of pSS IgG on PGE2 production by a rat submandibular gland. Glands were treated with pSS IgG alone or in the presence of calcium-blocking agents (verapamil, 5 x 10–5 M; thansigargin, 1 x 10–6 M) or with a calcium ionophore agent (A 23187, 1 x 10–6 M) alone. Values are mean ± SEM of 6 individual pSS IgG samples performed in duplicate. *p < 0.005 vs. pSS IgG. (C) RT-PCR products of COX-1 and COX-2 obtained from rat submandibular gland alone (basal) or in the presence of 1 x 10–7 M pSS IgG. (D) The densitometry analysis of 7 individual IgG sample tests performed in duplicate. Values are mean ± SEM. Note that the COX-2 band in the presence of pSS IgG increased significantly (p < 0.001) compared with the basal COX-2 band.

View larger version (11K): [in this window] [in a new window]   Figure 3. PGE2 production on serum and saliva from different groups: 15 women with pSS (group I), 16 post-menopausal women with dry mouth and without SS (group II), and 18 healthy pre-menopausal women (group III). Solid lines: mean values ± SEM. p < 0.001 between group I and groups II and III.

	DISCUSSION

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We analyzed a possible role for pSS IgG in glandular inflammation through its capacity to trigger PGE₂ (a pro- inflammatory substance) production in submandibular glands. Results showed that pSS IgG interacting with glandular M₁, M₃, and M₄ cholinoreceptor subtypes to trigger PGE₂ production was prevented by specific antagonist subtypes and by M₁, M₃, and M₄ cholinoreceptor synthetic peptides. This mechanism was mediated, at least in part, by an increase in intracellular calcium concentrations, since it was inhibited by calcium blocker agents and was mimicked by a calcium ionophore. Thus, the mobilization of intracellular calcium is a key step in the activation of PLA₂ and COXs, which leads to stimulation of PGE₂ biosynthesis by pSS IgG. Thereby, pSS IgG raises intracellular calcium concentrations by increasing inositol tri-phosphate and L-type calcium channel current (Leiros et al. , 1997), and activates PLA₂ and COX-s, increasing PGE₂ production. Calcium ionophore-increased PGE₂ production by activation of PLA₂ and COX-s was reported in CHO cells (Lin et al., 1992).

The production of PGE₂ induced by pSS IgG appeared to be the result of the overexpression of pro-inflammatory COX-2 mRNA gene expression, with no modification of constitutive COX-1 mRNA levels. In fact, pSS IgG altered the rate of transcription of COX-2-mRNA in response to cholinoreceptor-activation at the cell membrane. The transcription factor was rapidly induced following receptor activation. Thus, during 1 hr of cholinoreceptor activation by pSS IgG, the expression of COX-2 was induced, which may play an important role in PGE₂ production triggered by the autoantibody. Both COX-1 and COX-2 mRNA are present in submandibular glands. However, the band for COX-2 expression appeared markedly increased by the action of pSS IgG, while the band for COX-1 did not change. Also, we showed, by a pharmacological approach, that rofecoxib (COX-2 specific inhibitor) was more effective than aspirin (COX-1 preferential inhibitor) in blocking the increment of PGE₂ production by pSS IgG. In contrast, an up-regulation of COX-1 expression was demonstrated in salivary glands from persons with SS, and the COX-1 expression correlated with glandular cell infiltration (Tominaga et al., 2000). Thus, COX-2-expressing glandular cells might depend on the effects of autoantibodies, while COX-1-expressing cells are due to the presence of glandular monocyte/macrophages infiltration.

On the basis of this evidence, it may be argued that pSS IgG increases PGE₂ production by the relative contributions of COX-1 and COX-2. This suggests that pSS IgG-evoked rapid PGE₂ production, caused primarily by COX-1, and, later, PGE₂ production are maintained by the induction of COX-2 mRNA, as occurs in other tissues (Tegeder et al., 2001). Chronic activation of COX-2 could then maintain the inflammatory processes on the submandibular glands in SS.

The ability of serum-derived pSS IgG to increase PGE₂ production was parallelled by increases in PGE₂ levels in both sera and saliva from the study participants. The rise in PGE₂ was synergistic with other mediators (Willams and Peck, 1977; Anderson et al., 1996; Bley et al. , 1998). PGE₂ has long been associated with inflammatory responses and is a key regulator of periodontal tissue destruction by directly or indirectly enhancing the expression and activation of matrix metalloproteinases and bone resorption (Graves et al., 2000). Moreover, the rise in PGE₂ levels within the crevicular fluid can serve as a risk factor for rates of attachment loss and bone resorption in periodontal disease (Offenbacher et al., 1993).

We hypothesized that cholinergic agonistic autoantibody activities might promote the development of inflammatory processes in submandibular glands, increasing PGE₂ production in SS. Therefore, PGE₂, together with anticholinergic pSS IgG, may serve as an important early marker of glandular inflammatory active processes in persons with pSS.

	ACKNOWLEDGMENTS

 
We thank Mrs. Elvita Vannucchi and Fabiana Solari for their technical assistance. This investigation was supported by the University of Buenos Aires (UBACyT, O 014) and by the Argentine National Research Council (CONICET, PIP 5680).

Received July 17, 2006; Last revision April 23, 2007; Accepted May 9, 2007

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Reina, S. Articles by Borda, E. Search for Related Content PubMed PubMed Citation Articles by Reina, S. Articles by Borda, E.