Figure 2

Two week exposure to IL-4 or IL-13 increases the expression of the antimicrobial peptides hCAP18/LL-37 and hBD-2. PBEC from various donors were cultured at an ALI for two weeks in the presence of various concentrations of IL-4 or IL-13. (A) Quantitative real time PCR of LL-37 and hBD-2. Data were normalized for expression against two reference genes (β-actin and GAPDH). Data are represented as mean ± SEM of fold increase compared to medium alone for five different donors. * p < 0.05 Wilcoxon signed ranks test. (B) Densitometry results (fold increase compared to medium alone) from the dot blot analysis of hCAP18/LL-37 expression in apical washes of ALI cultures using cells from two donors. (C) Western Blot of LL-37 in cultures treated with medium alone (-), IL-4 (20 ng/ml), or IL-13 (20 ng/ml). From left to right: basal medium concentrated using Oasis cartridges, apical washes, apical washes concentrated using cartridges, cell lysates, cell lysates treated with DNaseI obtained from one culture, and a nasal secretion sample and 0.5 ng of synthetic LL-37 as controls. Similar results were obtained in basal medium from another donor using another antibody specific for hCAP18/LL-37. (D) ELISA for hBD-2 on apical washes and basal medium; mean ± SEM of n = 3 and n = 4 experiments, respectively. (E) Two ALI cultures that were differentiated in the absence or presence of IL-4 or IL-13 for two weeks were subsequently stimulated with 10⁸ CFU heat-inactivated P. aeruginosa PAO1 or with a combination of TNF-α and IL-1β (both at 20 ng/ml) for 24 h. Quantitative real time PCR of hCAP18/LL-37. Data were normalized for expression against two reference genes (β-actin and GAPDH). Two separate experiments are shown; data are represented as fold increase compared to medium alone.