Extracellular Nucleotides and Interleukin-8 Production by ARPE Cells: Potential Role of Danger Signals in Blood-Retinal Barrier Activation

PURPOSE. RPE cell activation is an important feature of autoimmune uveitis. This investigation focused on whether extracellular nucleotides could contribute to this activation, and the effects of ATP gamma S, UTP, and UDP on the production of IL-8 by RPE cells was studied in relation to their expression of functional P2Y receptors. METHODS. ARPE-19 cells were cultured with ATP gamma S, UTP, UDP, and TNF. IL-8 gene transcription and protein production were measured by semiquantitative RT-PCR and ELISA. Western blot analysis and RT-PCR were used to investigate ERK 1/2 activation and P2Y expression. Changes in intracellular calcium and cAMP concentration were analyzed by spectrofluorometry and radioimmunoassay. RESULTS. Stimulation of ARPE-19 cells with ATP gamma S, UTP, and UDP induced IL- 8 gene transcription and protein secretion. TNF alpha induction of IL-8 secretion was also increased by ATP gamma S, UTP, and UDP. Nucleotide induction of IL-8 production was blocked by PD98059, and all nucleotides stimulated ERK 1/2 phosphorylation. P2Y(2) and P2Y(6) mRNAs were detected in ARPE-19 cells. All tested nucleotides induced a pulse of intracellular calcium. CONCLUSIONS. ATP gamma S, UTP, and UDP stimulate both basal and TNF alpha-induced IL-8 secretion in RPE cells through an ERK 1/2- dependent pathway. The results suggest that those effects are mediated by P2Y(2) and P2Y(6) receptors. ( Invest Ophthalmol Vis Sci. 2009; 50: 1241-1246) DOI:10.1167/iovs.08-1902