PAR‐2 involvement in activation of human airway epithelial cells

To investigate if Alternaria proteases participate in the direct activation of airway epithelial cells, we are exploring a possible role for protease activated receptor type 2 (PAR‐2). We stimulated 16HBE140‐ cells with 7.5 μg/ml Alternaria culture filtrate (Alt), 100μM PAR‐2 agonist, media alone or Alt depleted of proteases by heat treatment (70°C 30min; heated Alt) for 3, 6 and 12 hours. After stimulations, supernatants were collected, frozen and batch assayed for cytokines (ENA78, FGFbasic, VEGF, IL1α, IL1ra, GMCSF, GCSF, IL6, IL8, TNF‐α and RANTES) by Luminex. At the 3 hour time, both Alt and PAR‐2 agonist increased production of GMCSF, VEGF, IL6, TNF‐α and IL8. At 6 hours, constitutive ENA‐78 production was inhibited by Alt but was increased by PAR‐2 agonist. At both 6 and 12 hr, GMCSF, GCSF,VEGF, IL6 and TNF‐α were increased by both stimulants. Stimulation with heated Alt significantly reduced GCSF, IL1α, IL‐6, IL‐8, TNF‐α. The reduction in these inflammatory cytokines by heated Alt suggests that the presence of proteases is necessary for their production by Alt. The similarities demonstrated for GCSF, IL1α, IL‐6, IL‐8 and TNF‐α production by Alt and PAR‐2 agonist and their reduction by heated Alt support the involvement of PAR‐2 in Alt activation of airway epithelial cells, whereas IL1ra, GMCSF and FGFbasic production and the inhibition of ENA‐78 and RANTES may occur via other pathways. Funded in part by Grants HL56177, AI42268, AI61811