P2Y6 mediates HNP-induced IL-8 production in human lung epithelial cells. A549 cells grown in complete DMEM were transfected with 2.5 μM P2Y6 sense or antisense oligonucleotides for 4 hours. After washing, cells were incubated in complete medium overnight. (A) Total RNA was extracted, and RT-PCR of P2Y6 mRNA was performed. (B) Total P2Y6 protein level was determined from cell lysates by Western blotting (WB). After transfection with the oligonucleotides overnight, A549 cells (C) or SAEC cells (D) were incubated in serum-free DMEM containing the purified HNP for 8 hours. Three control groups were included to test the specificity of the antisense oligonucleotides on IL-8 release after stimulation with commercial synthetic HNP-1 (E), TNF-α (F), or UDP (G) in A549 cells. IL-8 levels in cell-culture supernatants were measured (n = 3). *P < .05; sense compared with antisense.
