P2Y2R activation mediates cytokine release leading to salivary gland dysfunction

The G protein-coupled P2Y₂ receptor (P2Y₂R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of Sjögren's Syndrome (SS). Pro-inflammatory cytokines associated with SS can be produced by infiltrating lymphocytes or from salivary epithelium. Objectives: To 1) analyze the ability of the P2Y₂R activation to release pro-inflammatory cytokines in salivary epithelium, and 2) determine if cytokines alter transepithelial resistance (TER), short circuit current (Isc) and cell morphology. Methods: P2Y₂R-mediated activation of cytokine release in the polarized parotid cell line (Par-C10) was measured by ELISA. TER was measured with an epithelial volt-ohmeter. (Isc) was quantitated with an Ussing chamber. Agonist-induced increases of intracellular calcium concentration [Ca²⁺]_i was measured by using Fura-2 fluorescence technique. Electron and confocal microscopy were used to study changes in cell morphology. Results: P2Y₂R activation released TNFα from Par-C10 salivary cell monolayers. The SS-associated pro-inflammatory cytokines TNFα and IFNγ decreased TER, (Isc), altered cell morphology and caused loss of epithelial cell polarity. However, agonist induced increases in [Ca²⁺]_i remained unaltered. Conclusion: P2Y₂R mediated TNFα release in the Par-C10 cell monolayers indicating that salivary epithelium is a source of cytokines and P2Y₂Rs may play an important role in inflammation associated with salivary gland diseases, such as SS. Additionally, TNFα and IFNγ impaired the integrity of the Par-C10 monolayers. However, the lack of effect on [Ca²⁺]_iindicates that cytokine treatment did not affect cell signaling. Thus, the changes in dysfunction and integrity of the polarized monolayer could be attributed to TNFα and IFNγ effects on tight junctions. Supported by NIH-NIDCR grants R01DE017591-01, R01DE07389-19, K08DE017633-01 and the Sjögren's Syndrome Foundation Research Grant.