alpha [TNFa], interferon-gamma [INF-c], and IL-1b) for 30 min. Measurement of translocation of NF-kB from the cytoplasm to the nucleus was performed using a Cellomics NF-kB Activation KitTM (Thermo Fisher Scientific, Pittsburgh, PA) according to the manufacturer’s validated protocol as described by others [23,24]. The nucleus was immunostained with Hoechst 33342, and NF-kB was detected with an NF-kB primary antibody and Alexa Fluor 488–conjugated goat anti-rabbit IgG secondary antibody. Micro- scopic images were captured with the MetaFluor Imaging System (v.7.5, Molecular Devices, Downingtown, PA) equipped with a cooled CCD camera (Quantix, Photometrics, Tucson, AZ). Image analysis was performed using NIH ImageJ software (v.3.91, http://rsbweb.nih.gov/ij/) as described by Noursadeghi M. et al. [25]. NF-kB nuclear translocation is represented as an increase in the nucleus:cytoplasm ratio. Simultaneous measurements of [Ca2+]i and ISC Agonist-induced calcium signals and anion secretion were measured simultaneously in polarized epithelium as described [17–19]. Positive ISC currents are displayed as upward deflections of the traces and were defined as those carried by anions moving from the basolateral to apical compartments. The effects of IL-6 and IL-8 on agonist-evoked ISC changes were tested by pre- incubating the epithelia with the cytokine for 1–3 days prior to addition of the agonist. Materials and solutions The bicarbonate-buffered K-H solution contained (in mM) NaCl, 117; NaHCO3, 25; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; and D-glucose, 11; pH 7.4 when bubbled with 5% CO2/95% O2. The low Cl2 solution (10 mM) was prepared by isosmotically replacing NaCl, KCl, CaCl2, and MgCl2 with Na- gluconate, K-gluconate, Ca-gluconate, and Mg-sulphate, respec- tively. The membrane permeant acetoxymethylester (AM) forms of Fura-2 and pluronic F127 were obtained from Molecular Probes (Eugene, OR). Uridine 59-triphosphate (UTP), forskolin, and poly-L-arginine were obtained from Sigma-Aldrich. The human cytokine antibody array kit was from RayBiotech Inc. (Norcross, GA). IL-6 and IL-8 ELISA kits were obtained from eBioscience (San Diego, CA), and recombinant human IL-6 and IL-8 were from PeproTech Inc. (Rocky Hill, NJ). All other general laboratory reagents were obtained from Sigma-Aldrich, and all cell culture reagents were obtained from Invitrogen. Data analysis Pooled data are presented as means 6 standard errors (S.E.), and values of n refer to the number of experiments in each group. Experimentally-induced changes (D) in the Fura-2 fluorescence ratio and ISC were quantified by measuring each parameter at the peak of a response and subtracting the equivalent values measured immediately prior to stimulation. Statistical comparisons between original data before normalization were performed using Student’s t-test and analysis of variance, where appropriate, with a p,0.05 considered significant. Results Effect of poly-L-arginine on TER TER is an electrical parameter that represents the resistance to ion flow through both paracellular and transcellular pathways. In this study, the effect of poly-L-arginine on TER of cultured 16HBE14o- cells was measured. Fig. 1 shows the time course of the effect of poly-L-arginine on cultured 16HBE14o- cells. The cultured epithelia were incubated with 10 mM poly-L-arginine for Polarized Cytokine Secretion Figure 1. TER measurement during poly-L-arginine exposure. TER was measured at 0, 1, 2, 3, 4, and 5 hours after cells were exposed to poly-L-arginine (50 mM, ap). Each TER measurement was normalized to the TER value of the control epithelia without poly-L-arginine treatment. The data represent the mean 6 S.E. (* p,0.05, n = 4, one- way ANOVA with Scheffe post-hoc test). doi:10.1371/journal.pone.0012091.g001 1–5 hours. Each TER measurement was normalized to the initial value without poly-L-arginine treatment. The TER decreased in a time-dependent manner and reached its minimum at 4 hours. Treating the epithelia for 2–5 hours produced a significant reduction in TER. The data therefore confirmed the damaging effect of poly-L-arginine as a surrogate for MBP on TER of the airway epithelium as seen in eosinophilic inflammation [26]. Poly-L-arginine triggered secretion of cytokines and chemokines by 16HBE14o- cells To further understand the effect of poly-L-arginine in stimulating bronchial epithelial cell cytokine and chemokine secretion, we first examined the cytokine profile in bronchial epithelial cells using the membrane-based RayBioH Human Inflammation Antibody Array III [21,22]. Fig. 2A and 2B show the control and poly-L-arginine-treated patterns of antibody array images, respectively. The mean optical density of each spot was normalized to the positive control and compared between control and treated epithelia (Fig. 2C). After exposure of the cultured epithelial cells to 10 mM poly-L-arginine for 3 hours, the intensities of two inflammation-related factors (IL-8 and RANTES) increased more than 2-fold, and two other factors (IL-6 and IL-6 soluble receptor [IL-6sR]) increased 5-fold as compared to the control (Fig. 2C). Moreover, the intensities of TNF-a, IL-1a, and IL-12 p40 also increased significantly. Apically directed secretion of IL-6, IL-8, TNFa and RANTES by polarized 16HBE14o- epithelia Because epithelia mainly secreted IL-6 and IL-8 into the supernatant in response to poly-L-arginine treatment, we next determined the concentrations of IL-6 and IL-8 secreted by the epithelia. To quantify the polarized secretion of IL-6 and IL-8, supernatant samples obtained from apical or basolateral compart- ments were analyzed by ELISA. Fig. 3A and 3B show the time course of the effect of poly-L-arginine on IL-6 release into apical and basolateral compartments, respectively. Exposure to 50 mM apical poly-L-arginine stimulated a time-dependent increase in PLoS ONE | www.plosone.org 3 August 2010 | Volume 5 | Issue 8 | e12091