Exposure to allergenic extracts induces both IL-8-transcription and IL-8 secretion. (A) Time-dependent IL-8 transcription after exposure to allergenic extracts. IL-8 mRNA expression was evaluated by RT-PCR and PCR. Total RNA of A549 cells exposed to Der f-HDMA (25 μg/ml) and GCA (12.5 μg/ml) was isolated at different time points. PCR-amplified IL-8 cDNA bands were quantified by densitometry and standardized with β-actin levels of intensity. Experiments were performed twice. Representative experiments are shown. (B) HDM, GCA and ACA induce IL-8 release in A549 cells in a concentration-dependent fashion. Following 24 hours of exposure to indicated concentrations of HDMA, GCA, and ACA. ELISA assessed IL-8 concentration in cell culture supernatants. The experiment was performed twice and mean values and standard deviations are shown, *, p < 0.05; * *, p < 0.005. (C) Allergens differ in their ability to induce detachment of A549 cells from monolayers. A549 cells were exposed to increasing concentrations (10–500 μg/ml) of Der f-HDMA, GCA or ACA. Cell detachment was assessed by methylene blue assay. For the assay, A549 cells were grown in 96-well plates (2.5 × 103 cells/well) and treated with various concentrations of allergen extracts. Following the 6-hour exposure period, the cells were fixed with formol saline, stained for 30 minutes with methylene blue and excess dye removed by several washings. The intracellular methylene blue was eluted with ethanol and the concentration of dye in each well quantified by spectrophotometry at a wavelength of 650 nm. The experiment was performed three times and a representative experiment is shown, *, p < 0.05.