Background/Purpose:

Sjögren’s syndrome (SS) is a chronic autoimmune inflammatory disease characterized by bilateral hypofunction of lacrimal and salivary glands leading to keratoconjuctivitis and xerostomia. The hallmark of SS is presence of antibodies against Ro (SS-A) and La (SS-B). Recent studies showed the presence of antibodies against M3 muscarinic acetylcholine receptor (M3R), a receptor found in endocrine and exocrine glands including salivary and lacrimal glands. M3R is a seven transmembrane GPCR having 3 extracellular and 3 intracellular domains/loops, responsible for smooth muscle contraction and glandular secretion. We hypothesized that antibodies derived from salivary gland against extracellular domains of M3R are functional and are responsible for decrease in salivary and lacrimal flow observed in Sjögren’s syndrome.

Methods:

Production of monoclonal antibodies (MAbs): Patients meeting the American College of Rheumatology (ACR) classification criteria for primary Sjögren’s syndrome (pSS) were evaluated at  OMRF Sjögren’s Research Clinic (OSRC). Control patients do not meet the criteria (DNMCs). A single cell suspension was prepared from labial salivary gland tissue obtained following biopsy. Antibody secreting cells (ASCs) having CD3- CD4- CD8- CD19+ CD27^high CD38^high IgG+ were sorted by fluorescence-activated single cell sorting. Recombinant MAbs were produced from ASCs by sequencing the V, D, J and CDR of the heavy and light chains amplified by RT-PCR and nested PCR, cloned into an expression vector, transfected to human cell line, expressed and purified. MAbs derived from pSS and DNMCs were 51 and 18 respectively.

M3R experiments: Peptides encoding the 2^nd (a.a. 213-228) and 3^rd(a.a. 514-527) ECL of M3R were employed in an ELISA to detect antibodies against these domains. FRET agonist/antagonist functional assays for MAbs using chimeric CHO-K1 NFAT bla cells, engineered to express human M3R along with beta lactamase reporter gene under control of NFAT response element, were carried out. CHO-K1 cells were incubated with 100 μg/ml mAb following incubation with either assay media or with carbachol at EC80 followed by substrate and fluorescence detection.

Results:

MAbs from pSS patients were highly reactive to 2^nd as well as 3^rd ECL as compared to DNMCs. We found significantly high O.D. values (> 4) in MAbs derived from pSS as compared to DNMCs. In MAbs from pSS, 9/51 were positive for 2^nd ECL and 5/51 were positive for 3^rd ECL. Where as in DNMCs none of the MAbs were positive for 2^nd ECL however two of the MAbs were found positive for 3^rdECL (cut-off 2 S.D. above DNMCs average O.D.). From the functional assays we have tested eight MAbs so far and found significant inhibition ranging from 14-66 % of the total stimulated response. None of these antibodies were found to be stimulatory.

Conclusion:

Anti-M3R antibodies have become a center of interest in rheumatology because of its possible pathogenesis and detection in SS as well as borderline SS patients. This data suggests inhibitory role of anti-M3R antibodies in producing SS symptoms. Second extracellular loop could play an antigenic role for anti-M3R antibodies. Our further studies will test whether passive transfer of MAbs into mice could induce Sjögren’s like disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-anti-muscarinic-receptor-3-monoclonal-antibodies-derived-from-salivary-gland-in-patients-of-sjogrens-syndrome/