epithelial function [11,12]. Certain inflammatory cytokines alter the fluid and electrolyte transport by the airway epithelium [13,14]. Therefore, asthma can be considered a disease of the bronchial epithelium, which may contribute to the pathophysiol- ogy of airway inflammation [15]. Polarized secretion of proinflammatory cytokines is important for establishing a specific microenvironment for airway inflam- mation. However, little is known about the polarized secretion of proinflammatory cytokines and its underlying signaling pathway in human bronchial epithelia damaged by cationic proteins. The aims of this study were to 1) investigate the inflammatory cytokine profile of human bronchial epithelia 16HBE14o- cells in response to a challenge with the cationic polypeptide poly-L-arginine; 2) study the polarized secretion of IL-6 and IL-8; 3) characterize the role of MAPK and NF-kB signaling pathways in the regulation of IL-6 and IL-8 secretion; and 4) examine the effect of IL-6 and IL-8 on transepithelial chloride (Cl2) secretion. Methods Cell Culture All experiments were performed using the immortalized cell line 16HBE14o-, which was derived from bronchial surface epithelial cells [16]. Cells were maintained in Minimum Essential Medium as described previously [17]. For cytokine quantification, cells were seeded onto 24-well Transwell-Clear filter inserts (Costar, Cambridge, MA) with a 0.4-mm pore diameter. For transepithelial resistance (TER) measurement and simultaneous measurements of intracellular calcium concentration ([Ca2+]i) and short-circuit current (ISC), cells were seeded onto Transwell-col filter mem- branes as previously described [18,19]. Cells reached confluence 2 after 10 days with a resistance greater than 150 V?cm . For quantitative real-time PCR (qRT-PCR) and other experiments, cells were grown on 6-well culture plates. RNA extraction and Real-time PCR Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions as described previously [17]. After treatment with DNase (Turbo DNA-free Kit, Ambion, Austin, TX), RNA was reverse tran- scribed to cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real- time PCR was performed with LightCycler 480 SYBR Green I Master Mix (Roche, Indianapolis, IN) on an iCycler thermal cycler (Bio-Rad Laboratories, CA). Primer sequences were as follows: IL-6 forward primer: 59-GCACTGGCAGAAAACA- ACCT-39, reverse primer: 59-TCAAACTCCAAAAGACCAG- TGA-39; IL-8 forward primer: 59-CCAACACAGAAATTATTG- TAAAGC-39, reverse primer: 59-TGAATTCTCAGCCCTCTT- CAA-39, GAPDH: forward primer: 59-TGCACCACCAAC- TGCTTAGC-39, reverse primer: 59-GGCATGGACTGTGGT- CATGAG-39. Relative expression of IL-6 and IL-8 was normalized to GAPDH and determined with the Pfaffl method [20]. A standard curve of Ct versus concentration was plotted to obtain the efficiency of each PCR reaction. This efficiency (E) can be obtained by the formula 10–(1/slope), with an acceptable range 1.8–2.2. Each PCR run included a no-template control and a sample without reverse transcriptase. All measurements were performed in duplicate. Polarized Cytokine Secretion solution. A transepithelial potential difference (p.d.) of 1 mV was applied periodically, and the resultant change in current was used to calculate the TER using Ohm’s law. After an equilibrium period of 30 min, the cultured epithelia were incubated with 10 mM poly-L-arginine for 1–5 hours with untreated epithelia as control. All TER values were determined after background subtraction (contributed by the filter and bath solution) and multiplied by the surface area of the filter. Measurement of cytokine secretion by antibody array and enzyme-linked immunosorbent assay (ELISA) 16HBE14o- cells were incubated with 10 mM poly-L-arginine for 3 hours, and cells incubated with fresh non-serum-containing medium were used for time-matched controls. Cell culture supernatants were collected and centrifuged at 30006g for 10 min at 4uC. The supernatants were then aliquotted and stored at 280uC until further use. The cytokine profile of the cell culture supernatants was analyzed with a RayBioH Human Inflammatory Antibody Array III kit according to the manufacturer’s instruc- tions. The membrane in this kit can simultaneously detect 40 different inflammation-related factors, including cytokines, che- mokines, soluble cytokine receptors, and growth factors [21,22]. The membranes were detected using chemiluminescence (Amer- sham Pharmacia Biotech, UK) for 1 min at room temperature. The membranes were then exposed to Fuji Film (Fuji, Japan) for 2 min. The signal intensity of individual spots was quantified with the FluorChemTM 8000 imaging system (Alpha Innotech Corp.) A positive control signal on each membrane was used to normalize the signal intensities of individual spots being compared on different membranes. Quantification of IL-6, IL-8, TNFa and RANTES secretion was done with enzyme-linked immunosorbent assay (ELISA). 16HBE14o- cells were exposed to different concentrations of poly- L-arginine, with or without pharmacological inhibitors, for various periods of times. Supernatant samples from the apical and basolateral compartments were collected, and IL-6, IL-8, TNFa (eBioscience, San Diego, CA) and RANTES (R&D Systems, Minneapolis, MN) secretion was assessed with commercially available ELISA kits, according to the manufacturer’s protocol. All conditions were measured in duplicate. Western Blot Western blotting was performed as described previously [17]. In brief, cells grown in a 6-well plate were lysed in RIPA buffer (1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl) supplemented with protease inhibitor cocktail. The protein samples were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore Corporation, Billerica, MA) and immunoblotted with specific primary antibody. All blots were developed by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ). The following primary antibodies were used: anti-phospho-p38 MAPK (Cell Signaling Technology, Beverly, MA; 1:500), anti-p38 MAPK (Cell Signaling Technology; 1:500), anti-phospho-p44/42 MAPK (ERK 1/2) (Cell Signaling Technology; 1:500), anti-p44/42 MAPK (ERK 1/2) (Cell Signaling Technology; 1:500), anti-b- tubulin (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500), and anti-GAPDH (Sigma, St. Louis, MO; 1:5,000). Measurement of transepithelial resistance (TER) Confluent 16HBE14o- epithelia were used to measure TER as described previously [19]. The monolayers were mounted in an Ussing chamber and bathed in normal Krebs-Henseleit (K–H) NF-kB translocation assay Cells were grown in black 96-well glass-bottomed plates (Costar) and stimulated with either vehicle control, 1 mM poly-L-arginine, or an inflammatory cocktail (10 ng/ml each tumor necrosis factor- PLoS ONE | www.plosone.org 2 August 2010 | Volume 5 | Issue 8 | e12091