Polarized Cytokine Secretion for 30 min. The nuclei (A, C, and E) were stained with Hoechst 33342, and NF-kB (B, D, and F) was stained with anti-NF-kB and Alexa Fluor 488 goat anti-rabbit secondary antibody. (magnification 620) (G) Immunofluorescence of NF-kB in the nuclear region and cytoplasmic region was quantified using NIH ImageJ software. Nucleus:cytoplasm ratios of NF-kB staining were calculated. Each column represents the mean 6 S.E. (n = 3). (*p,0.05 compared with vehicle control, Student’s t-test). doi:10.1371/journal.pone.0012091.g007 involved in polarized secretion are not fully clear. However, our finding may account for the attraction of neutrophils into the airway lumen following airway inflammatory responses. Together with a decrease in TER and hence barrier function of the epithelial layer upon poly-L-arginine exposure, the polarized secretion of potent chemoattractant would greatly facilitate the migration of immune cells to the lumen of the airway. As a consequence, the polarized secretion of IL-8 may lead to neutrophil recruitment and accumulation, as well as enhancement of IL-8 secretion by neutrophils [44], leading to a positive feedback cycle and progression of airway inflammation. In addition to the apical secretion of cytokines, basolaterally secreted cytokines from epithelia may play another role in supporting the inflammatory response by attracting immune cells from the underlying cellular matrix towards the subepithelial space. Moreover, polarized secretion of TNFa and RANTES by the airway epithelia was observed, which may further aggravate the inflammation and recruit more immune cells, respectively. In airway epithelial cells, the involvement of p38 MAPK activity in the production of IL-6 [45,46] and IL-8 [28] has been reported previously. Our experiments demonstrate that the p38 MAPK inhibitor, SB203580, inhibited poly-L-arginine-induced IL-6 and Figure 8. Simultaneous measurement of ISC and [Ca2+]i in 16HBE14o- epithelia treated with IL-6. Representative recordings of simultaneous measurements of ISC (upper trace) and [Ca2+]i (lower trace) in response to apical (ap) application of UTP (A and B) or forskolin (Fors; C and D) in epithelia treated with 1% BSA (A and C) or 10 ng/ml IL-6 (B and D) for 24 hours. 16HBE14o- cells were initially perfused bilaterally with normal K-H solution. The apical K-H solution was then changed to a K-H solution with reduced Cl2 concentration (not shown). Cells were then treated with apical application of 100 mM amiloride (Ami) 10 min prior to the apical application of 100 mM UTP or 10 mM forskolin (n = 6–8). doi:10.1371/journal.pone.0012091.g008 PLoS ONE | www.plosone.org 10 August 2010 | Volume 5 | Issue 8 | e12091