Atorvastatin inhibits the inflammatory response caused by anti-M(3) peptide IgG in patients with primary Sjögren's syndrome.

Experimental and clinical investigations have revealed that statins can down-regulate acute and chronic inflammatory processes. Whether statins express anti-inflammatory activities in the salivary glands in patients with primary Sjögren's syndrome (pSS) is not known. The in vitro and in vivo effect of atorvastatin on rat submandibular gland treated with anti-M(3) peptide IgG purified from SS patients was studied. The anti-inflammatory effects of atorvastatin were assessed by measuring the levels of IL-1ß, PGE(2) and MMP-3 by ELISA. Atorvastatin inhibited the increase in the production of IL-1ß, PGE(2) and MMP-3 in submandibular glands treated with anti-M(3) peptide IgG. A positive correlation between IL-1ß production with accumulation of PGE(2) and MMP-3 was observed. Rats pre-treated orally with atorvastatin (30 mg kg(-1)) or vehicle (phosphate-buffered solution) once a day for three consecutive days impaired the increment in the production of IL-1ß, PGE(2) and MMP-3 in the submandibular gland in the presence of anti-M(3) peptide IgG. In conclusion, the anti-inflammatory effects of atorvastatin are dependent upon inhibition of production of a pro-inflammatory cytokine (IL-1ß) and pro-inflammatory mediators such as PGE(2) and MMP-3. These data suggest that atorvastatin may constitute an anti-inflammatory effect in SS.

Cholinergic Autoantibodies from Primary Sjögren's Syndrome Inhibit Mucin Production via Phospholipase C and Cyclooxygenase-2 In the Rat Submandibular Gland.

BACKGROUND: Patients with primary Sjögren's syndrome (pSS) produce functional IgG against cholinoreceptor of exocrine glands modifying their activity. The aim of the present work was to demonstrate pSS IgG antibodies (pSS IgG) interacting with M(3) muscarinic acetylcholine receptors (mAChR) of rats submandibular glands that alter mucin release and production via phospholipase C (PLC) and cyclooxigenase-2 (COX-2) pathways. METHODS: Mucin release and production of prostaglandin E2 (PGE2), and total inositol phosphates (InsP) were measured in rat submandibular gland in the presence of pSS IgG auto antibodies. RESULTS: The auto antibodies interacting with M3 mAChR decreased mucin release and production through stimulation of PLC and COX-2. This stimulation leads to an incremental increase in InsP production and in PGE2 generation, inducing signalling through the prostaglandin membrane receptors subtype 2 (EP2). Moreover, the decrease in mucin production had negative correlation with PGE(2) generation and InsP accumulation. CONCLUSION: IgG in patients with pSS could play an important role in the pathoetiology of dry mouth, decreasing the salivary mucin through the production of proinflammatory substances and leading to the reduction in the protection of the oral tissues.

Anti-M(3) muscarinic cholinergic autoantibodies from patients with primary Sjögren's syndrome trigger production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E(2) (PGE(2)) from the submandibular glands.

BACKGROUND: We demonstrated that serum immunoglobulin G (IgG) from patients with primary Sjögren's syndrome (pSS), interacting with the second extracellular loop of human glandular M(3) muscarinic acetylcholine receptors (M(3) mAChR), trigger the production of matrix metalloproteinase-3 (MMP-3) and prostaglandin E(2) (PGE(2)). METHODS: Enzyme-linked immunosorbent assays (ELISAs) were performed in the presence of M(3) mAChR synthetic peptide as antigen to detect in serum the autoantibodies. Further, MMP-3 and PGE(2) production were determined in the presence of anti-M(3) mAChR autoantibodies. RESULTS: An association was observed between serum and anti-M(3) mAChR autoantibodies and serum levels of MMP-3 and PGE(2) in pSS patients. Thus, we established that serum anti-M(3) mAChR autoantibodies, MMP-3 and PGE(2) may be considered to be early markers of pSS associated with inflammation. Affinity-purified anti-M(3) mAChR peptide IgG from pSS patients, whilst stimulating salivary-gland M(3) mAChR, causes an increase in the level of MMP-3 and PGE(2) as a result of the activation of phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2) (but not COX-1). CONCLUSIONS: These results provide a novel insight into the role that cholinoceptor antibodies play in the development of glandular inflammation. This is the first report showing that an antibody interacting with glandular mAChR can induce the production of pro-inflammatory mediators (MMP-3/PGE(2)).

Cholinergic autoantibodies from primary Sjögren's syndrome modulate submandibular gland Na+/K+-ATPase activity via prostaglandin E2 and cyclic AMP.

We demonstrate that patients with primary Sjögren's syndrome (pSS) produce functional IgG autoantibodies that interact with the glandular M(3) muscarinic acetylcholine receptors (mAChRs). These autoantibodies act as a partial muscarinic agonist, increasing prostaglandin E(2) (PGE(2)) and cyclic AMP production through modifying Na(+)/K(+)-ATPase activity, but also interfere with the secretory effect of the parasympathetic neurotransmitter. The IgG from patients with pSS has two effects on the submandibular gland. On the one hand, it may act as an inducer of the proinflammatory molecule (PGE(2)) that, in turn, inhibits Na(+)/K(+)-ATPase activity. On the other hand, it plays a role in the pathogenesis of dry mouth, abolishing the Na(+)/K(+)-ATPase inhibition and the net K(+) efflux stimulation of the salivary gland in response to the authentic agonist pilocarpine, decreasing salivary fluid production.

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells.

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4-DAMP) and pirenzepine, indicating that M(1) and M(3) mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase-3 and JNK activation. The intracellular pathway leading to pilocarpine-induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U-73122, W-7, verapamil, BAPTA and BAPTA-AM blocked pilocarpine effects. L-NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process.

Cholinoceptor modulation on nitric oxide regulates prostaglandin E(2) and metalloproteinase-3 production in experimentally induced inflammation of rat dental pulp.

The purpose of this study was to investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of inducible nitric oxide synthase (iNOS) activity, prostaglandin E(2) (PGE(2)), and metalloproteinase-3 (MMP-3) in experimentally induced inflammation of rat incisors dental pulp. Inflammation was induced by application of bacterial lipopolysaccharide (LPS) to the pulp. Extirpated pulp tissue samples were incubated in saline solution until the various experiments were performed. Saline-treated pulp and healthy pulp were used as controls. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. PGE(2) and MMP-3 production were evaluated by an enzyme-linked immunosorbent assay (ELISA) and cyclooxygenase (cox-1 and cox-2) messenger RNA levels were measured using a reverse-transcriptase polymerase chain reaction by coamplification of target complementary DNA with a single set of primers. The application of LPS to the pulp increased NOS activity, PGE(2), and MMP-3 production associated with iNOS overactivity. Moreover, PGE(2) and MMP-3 production were the result of cox-2 expression. Pilocarpine (5 x 10(-11) mol/L to 5 x 10(-9) mol/L), acting on mAChRs, triggered a negative effect on NOS activity, PGE(2), and MMP-3 production. In control pulp, no action of pilocarpine was observed. Pulpitis changed mAChR conformation, increasing its coupling efficiency to transducing molecules that in turn activate iNOS. The capacity of pilocarpine to prevent iNOS activity, PGE(2), and MMP-3 by acting on mAChR mutation induced by pulpitis might be useful therapeutically as a local treatment.