Polarized Cytokine Secretion Figure 3. Time course effect of poly-L-arginine on the release of IL-6 and IL-8 into apical (ap) and basolateral (bl) compartments of cultured 16HBE14o- cells. 16HBE14o- cells were exposed to 50 mM poly-L-arginine for various periods of time. Cell culture supernatants in both apical (A and C) and basolateral (B and D) compartments were collected at 1, 2, 3, and 6 hours with time-matched untreated epithelia as control. Each column represents the mean 6 S.E. (n = 4–9). Statistical significance between the apical or basolateral compartment of the same treatment period is indicated by an asterisk (*p,0.05, Student’s t-test). doi:10.1371/journal.pone.0012091.g003 expression was detected when the cells were stimulated with 10 mM poly-L-arginine (Fig. 4C and 4D). This cationic polypeptide had a more potent effect on IL-8 than IL-6 mRNA expression (.500-fold vs. 50-fold). Apart from measuring polarized secretion of IL-6 and IL-8 by 16HBE14o- epithelia, poly-L-arginine – induced TNFa and RANTES secretion into apical and basolateral compartments were also quantified by ELISA. Under basal conditions, there was minimal increase in TNFa secretion in both apical and basolateral compartments while the RANTES level (27.766.6 pg/ml) was increased in the apical but not basolateral compartment. When the epithelia were treated with 50 mM poly-L-arginine for 6 hours, there was a significantly higher concentration of TNFa release into the apical compartment (16.163.2 pg/ml) than into the basolat- eral compartment (2.961.0 pg/ml, n = 3, p,0.05). Similarly, the epithelia secreted much higher level of RANTES into apical (101.7617.9 pg/ml) than the basolateral (24.661.3 pg/ml, n = 3, p,0.05) compartment. Polarized secretion of IL-6 and IL-8 is regulated by p38 MAPK-, ERK1/2 MAPK-, and NF-kB-dependent pathways We next examined the effect of various MAPK and NF-kB inhibitors on poly-L-arginine–induced IL-6 and IL-8 release from 16HBE14o- epithelia because it has been shown that MAPKs (ERK1/2, p38) and NF-kB are involved in IL-6 [27] and IL-8 [28] release from epithelial cells. To evaluate the involvement of these signaling pathways in inducing IL-6 and IL-8 release, selective pharmacological inhibitors were used. For poly-L-arginine– induced apical IL-6 secretion, the p38 MAPK inhibitor, SB203580, suppressed IL-6 production significantly at concentra- tions of 30 and 50 mM (Fig. 5A). In contrast, the ERK1/2 inhibitor, PD98059, did not show any concentration-dependent inhibition of IL-6 secretion (Fig. 5B), with the exception of an effect at 10 mM PD98059. A similar inhibitory profile was also obtained for apical IL-8 secretion (Fig. 5C and 5D). SB203580 (10 and 50 mM) significantly inhibited IL-8 production, whereas PD98059 (10–50 mM) produced a similar inhibitory effect. The NF-kB inhibitor, BAY 11-7085 (10 mM), also significantly blocked IL-6 (Fig. 5E) and IL-8 (Fig. 5F) production. The effect of these inhibitors on basolateral IL-6 and IL-8 release was erratic, and no clear inhibitory effects were observed (data not shown), possibly due to the small and variable basolateral release of IL-6 and IL-8 compared to the apical release. Taken together, our data suggest that the apical secretion of IL-6 and IL-8 is mediated through p38 MAPK- and NF-kB-dependent pathways. However, the ERK1/2 pathway may also be involved in IL-8 secretion. Poly-L-arginine induces p38 and ERK1/2 phosphorylation and NF-kB translocation To verify that p38 and ERK1/2 MAPK are implicated in IL-6 and IL-8 release when the cells are challenged with poly-L- arginine, the ratio of phosphorylated to total p38 and ERK1/2 PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12091