Polarized Cytokine Secretion Figure 6. Poly-L-arginine-induced ERK1/2 MAPK and p38 MAPK activation. 16HBE14o- cells were grown on 6-well plates for 9–10 days until confluent and were serum starved for 16 hours prior to treatment. Cells were then treated with 10 mM poly-L-arginine or vehicle control for the indicated periods of time. Total cell lysates were analyzed by SDS-PAGE and immunoblotted with antibodies specific for (A) phospho-p38 MAPK and p38 MAPK or (B) phospho-ERK1/2 MAPK and ERK1/2 MAPK. Quantification of the western blot is shown in (C–E). The band intensity of the phospho- MAPK is divided by that of the total MAPK. The phosphoMAPK:total MAPK ratio of the vehicle control was arbitrarily set to 1. IL-1 b (10 ng/ml) was employed as a positive control, and 0.1% BSA was the vehicle control. Data shown are representative of three independent experiments. (*p,0.05 compared with vehicle control, Student’s t-test). doi:10.1371/journal.pone.0012091.g006 cytokines were increased more significantly than the others. More importantly, these two cytokines are implicated in the initiation and perpetuation of local airway inflammatory responses. To measure the polarized secretion of IL-6 and IL-8 quantitatively, 16HBE14o- monolayers were grown on a perme- able support so that we could collect both apical and basolateral media for analysis. The results strongly indicate that there is a time- and concentration-dependent secretion of IL-6 and IL-8 by the epithelia into the apical compartment compared to the basolateral compartment. Polarized secretion of cytokines, includ- ing IL-6 and IL-8, has been observed in other epithelia. Preferential apical secretion was shown in lung (Calu-3 and A549) [33,34], mesothelial [35,36], and female reproductive tract epithelia [37]. In contrast, other epithelia, such as renal [38,39] and colonic [40] epithelia show a preferential basolateral secretion, whereas in retinal pigment epithelium, controversial results were obtained [41,42]. The direction of polarized secretion therefore appears to be cell-type-specific and may be related to the pathophysiology during inflammation and specific innate immune protection in different tissues. The polarized secretion of IL-6 and IL-8 (i.e., in an apical-to- basal gradient) may create a chemotactic gradient and induce transepithelial neutrophil migration in a manner analogous to the influx of neutrophils into the airway lumen. Our results strongly suggest that there is apically directed IL-6 and IL-8 secretion in polarized 16HBE14o- epithelia, and that the degree of polarized secretion is always greater in the apical compartment, regardless of whether the stimulus is applied from the apical or basolateral side of the membrane. In fact, the majority of eosinophil degranulation occurs in the lumen of the airway [43]. The exact mechanisms PLoS ONE | www.plosone.org 8 August 2010 | Volume 5 | Issue 8 | e12091