Roles of sodium bicarbonate cotransporters in salivary glands and autoantibodies in Sjogren's syndrome

Abstract

Primary Sjögrens syndrome is a systemic autoimmune disease that impairs the structural integrity and function of the salivary and lachrymal glands, leading to dry mouth and eyes. According to previous studies, the disease is multifactorial, showing many extraglandular features such as arthritis, myalgia, pulmonary disease, vasculitis, lymphoma, renal tubular acidosis, poor dentition, leukopenia, and bicarbonate (HCO3-) secretion. This study aims to investigate Sjögrens syndrome-associated leukopenia and the physiological mechanisms of salivary sodium bicarbonate cotransporters (NBCs) associated with HCO3- secretion. Sjögrens syndrome patients have low salivary HCO3- concentration and buffer capacity in comparison to healthy patients. Salivary HCO3- buffers H+ ions produced from oral bacterial fermentation, so decreased salivary HCO3- secretion accelerates tooth decay and further compromises poor dentition. However, only a few studies have reported the roles and regulatory mechanisms of HCO3- secretion and intracellular pH regulation in salivary gland epithelial cells (SGECs). Therefore, chapter I mainly investigates intracellular pH (pHi) regulation and regulatory mechanisms of two different types of NBCs—electroneutral (NBCn1) and electrogenic NBC (NBCe1)—in human submandibular glands (hSMGs) and HSG cells (isolated from human submandibular gland intercalated duct cells). Intracellular pH and the pHi recovery rate from cell acidification induced by an NH4Cl pulse were recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. I determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2 (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo [3,4-d]pyrimidine), an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. In chapter II, a possible mechanism of Sjögrens syndrome-associated leukopenia is suggested and examined. Patients with primary Sjögrens syndrome have been shown to have serum autoantibodies that react with muscarinic acetylcholine type 3 receptors (M3R). Interestingly, leukopenia has been reported to be significantly more common in primary Sjögrens syndrome patients who have anti-M3R-autoantibodies in their sera. However, the pathophysiological relationship between leukopenia and Sjögrens syndrome is yet to be established. I hypothesized that anti-M3R autoantibodies would have effects on M3R and MHC I expression in T cells, which may lead to the death of T cells. Purified IgG antibodies were isolated from the serum of healthy individuals and primary Sjögrens syndrome patients. Jurkat cell line was used to represent T lymphocytes. In situ immunofluorescence confocal microscopy was used to confirm the binding reactivity of primary Sjögrens syndrome IgG antibodies to M3R. Co-immunoprecipitation and immunofluorescence results suggested a direct interaction between M3R and MHC I. Co-internalization of M3R and MHC I was observed when Jurkat cells were exposed to the primary Sjögrens syndrome IgG, but this co-internalization was prevented by the presence of exogenous interferon gamma (IFN-γ). Primary Sjögrens syndrome IgG itself did not affect the viability of Jurkat cells, but Jurkat cells exposed to primary Sjögrens syndrome IgG underwent significant cell death when co-cultured with primary Natural Killer cells. Taken together, I suggest that both NBCn1 and NBCe1-B play a role in pHi regulation in hSMG acinar and duct cells. NBCe1-B is phosphorylated by Src tyrosine kinase and translocates to the plasma membrane, whereas NBCn1 is not regulated by Src kinase. Further, anti-M3R autoantibodies in primary Sjögrens syndrome induced downregulation of plasma membrane M3R and MHC I molecules in leukocytes, followed by NK cell-mediated cell death. These physiological and immunological approaches may help to understand the pathophysiology of Sjögrens syndrome-associated diseases or phenotypes.