Polarized Cytokine Secretion Figure 4. Polarized secretion of IL-6 and IL-8 into the apical compartment. 16HBE14o- cells were exposed to 1–50 mM poly-L-arginine, and cell culture supernatants were collected to measure IL-6 (A) or IL-8 (B) after 3 hours, with untreated epithelia as control. Each column represents the mean 6 S.E. (n = 3–9). Statistical significance between the apical and basolateral compartments of the same treatment concentration is indicated by an asterisk (* p,0.05, Student’s t-test). mRNA expression of IL-6 (C) and IL-8 (D) was analyzed with qRT-PCR. Epithelia were treated with poly-L- arginine for 3 or 6 hours. Relative expression of IL-6 and IL-8 was normalized to GAPDH and is shown as fold changes relative to untreated controls. Each column represents the mean 6 S.E. (n = 3). doi:10.1371/journal.pone.0012091.g004 MAPK was measured. Poly-L-arginine activated p38 MAPK in a time-dependent manner, with a maximum activation after stimulation for 30 min (Figs. 6A and 6C). Activation of the ERK1/2 pathway was also confirmed by western blotting, which showed that phosphorylated ERK1/2 MAPK was elevated in poly-L-arginine-treated cells (Fig. 6B). Both phosphorylated p42 and p44 were increased after treating the cells with poly-L-arginine for 10–30 min (Fig. 6D and 6E). IL-1b was used as a positive control because it activates p38 [29] and ERK1/2 [30] in 16HBE14o- cells (Fig. 6A and 6B). Immunofluorescent staining demonstrated translocation of NF- kB from the cytoplasm to the nucleus and confirmed NF-kB activation upon treatment of the epithelia with poly-L-arginine (Fig. 7A-F). A summary of these data is shown in Fig. 7G. When the cells were treated with poly-L-arginine (1 mM) for 30 min, the nucleus:cytoplasm ratio increased from 0.8660.07 to 1.1560.03, similar to the increase observed in cells (ratio = 1.1560.005) treated with an inflammatory cocktail consisting of IL-1b, TNFa, and IFNc [31]. In the presence of BAY 11-7085 (10 mM), NF-kB translocation was completely abolished (0.8260.05, n = 3). IL-6 and IL-8 do not affect Ca2+- or 39-59-cyclic adenosine monophosphate (cAMP)-dependent Cl2 secretion in 16HBE14o- epithelia Our previous study demonstrated that Cl2 secretion across the epithelia is mediated through both Ca2+- and cAMP-dependent pathways in 16HBE14o- cells [17]. Because certain cytokines may alter Ca2+- or cAMP-dependent Cl2 secretion [13,14], the effect of IL-6 and IL-8 on transepithelial Cl2 secretion and Ca2+ signaling in 16HBE14o- epithelia was monitored using a simultaneous measurement technique. Cells were first incubated with vehicle control (1% bovine serum albumin, BSA) or 10 ng/ml IL-6 for 24, 48, or 72 hours. The epithelia were then stimulated with apical application of the Ca2+- mobilizing agonist, UTP (100 mM), which activates P2Y4 receptors expressed in 16HBE14o- cells [17]. Fig. 8A (control) and 8B (IL-6– treated for 24 hours) show representative recordings of simulta- neous measurements of ISC and [Ca2+]i in response to UTP. Apical application of UTP elicited an increase in ISC, with a concomitant increase in [Ca2+]i. Incubation with 10 ng/ml IL-6 did not significantly affect the time course of the maximal ISC increase (control: 32.162.6; treated: 33.662.3 mAcm-2, n = 4–6). Similar results were obtained when epithelia were incubated with IL-6 for 48 (control: 38.362.0; treated: 35.062.3 mAcm22, n = 6, n.s.) or 72 hours (control: 25.865.2; treated: 25.164.2 mAcm22, n = 6, n.s.). Interestingly, incubation with 10 ng/ml IL-6 caused a small, albeit significant decrease in the UTP-induced Fura-2 ratio when the cells were treated with IL-6 for 24 (control: 0.4060.03; treated: 0.3160.03, n = 4–6, p,0.05) or 48 hours (control: 0.4160.02; treated: 0.2860.02 (n = 6, p,0.05), but not when cells were incubated for 72 hours (control: 0.4460.03; treated: 0.3360.06, n = 6, n.s.). In another series of experiments, a similar approach was used to examine the effect of IL-6 on cAMP-dependent Cl2 secretion. The epithelia were stimulated with the adenylate cyclase activator, forskolin (10 mM), to elevate the intracellular cAMP level. Fig. 8C and 8D show representative recordings of simultaneous measurements of ISC and [Ca2+]i in response to PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12091