Polarized Cytokine Secretion Figure 5. Effect of p38 MAPK, ERK1/2 MAPK, and NF-kB inhibitors on apical IL-6 and IL-8 release by 16HBE14o- cells. Cells were treated with inhibitor alone or co-incubated with poly-L-arginine (10 mM) in the presence of different concentrations of the inhibitors (mM) for 3 hours. IL-6 (A, B, and E) and IL-8 (C, D, and F) levels were measured with ELISA. Levels of IL-6 and IL-8 were corrected with vehicle control alone and normalized to 10 mM poly-L-arginine. Each column represents the mean 6 S.E. (n = 4). Statistically significant inhibitory effects compared with poly-L- arginine-treated control are marked with an asterisk (*p,0.05, Student’s t-test). doi:10.1371/journal.pone.0012091.g005 the apical application of 10 mM forskolin in control or treated epithelia (10 ng/ml IL-6 for 24 hours), respectively. Apical application of forskolin elicited an increase in ISC with no increase in [Ca2+] . Incubation with 10 ng/ml IL-6 had no i significant effect on the maximal ISC response (control: 49.962.7; treated: 44.564.3 mAcm22, n = 6). Similar results were obtained when the cells were incubated with IL-6 for a longer period of time (48 and 72 hours, data not shown). Using the same protocol, cells were incubated with IL-8 (10 ng/ ml) for 24–72 hours, and then stimulated with either apical application of UTP or forskolin. Essentially similar effects on ISC and [Ca2+]i were observed (data not shown). In general, chronic exposure of epithelia to IL-6 and IL-8 had no apparent effect on Ca2+- or cAMP-dependent Cl2 secretion across 16HBE14o- epithelia, despite a slight decrease in nucleotide-evoked [Ca2+]i increase. Discussion The airway epithelium participates in inflammation in a number of ways. The surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells [3]. Our results confirm that exposing bronchial epithelia to poly-L-arginine mimics the damage caused by MBP in airway inflammation. The decrease in TER may be due to an increase in paracellular permeability as seen in rabbit nasal epithelium [32]. Our antibody array data indicate that challenging the bronchial epithelia with poly-L-arginine resulted in increased levels of multiple proinflammatory proteins including IL-6, IL-6sR, IL-8, TNFa, IL-1a, IL-12p40, and Regulated on Activation Normal T Cell Expressed and Secreted (RANTES). In this study, we focused on the polarized secretion of IL-6 and IL-8 because these two PLoS ONE | www.plosone.org 7 August 2010 | Volume 5 | Issue 8 | e12091