IL-8 release. In addition, the involvement of p38 MAPK was confirmed by western blotting. Poly-L-arginine activated p38 MAPK phosphorylation, with a maximal activation after 30 min. In 16HBE14o- cells, IL-8 release may also be regulated by the ERK1/2 pathway [47,48]. Although the western blot data confirm a marked increase in p42- and p44–ERK phosphorylation following a poly-L-arginine challenge, the selective inhibitor, PD 98059, only produced a modest inhibitory effect on IL-8 secretion. In contrast, most concentrations of the inhibitor tested did not significantly suppress IL-6 release. Therefore, it appears that although IL-8, but not IL-6 release, may be regulated by an ERK1/2-dependent pathway, the p38 MAPK pathway may be more important in regulating polarized secretion of IL-6 and IL-8. Our study therefore provides a mechanistic explanation of how synthetic polycations induce inflammatory cell migration in the lungs of animal models [49]. Aside from the basal release of IL-6 and IL-8, poly-L-arginine- induced cytokine release into the apical compartment was readily detected 1 hour (IL-8) and 3 hours (IL-6) after stimulation. Our data indicate that within 30 min, the p38 MAPK, ERK1/2 MAPK, and NF-kB pathways were all activated. This relatively short period may indicate that the release originates from an intracellular presynthesized protein. In addition, our results indicate that the cationic polypeptide stimulates IL-6 and IL-8 in a transcription–dependent manner. Earlier studies have shown that cationic proteins stimulate IL-8 mRNA expression in eosinophils [50] and neutrophils [44]. Our results demonstrate a profound upregulation in both IL-6 and IL-8 gene transcription after 3 hours of poly-L-arginine stimulation. The effect was more profound when the cells were treated with poly-L-arginine for 6 hours. The increase in mRNA expression may be due to activation of NF-kB, which is considered to be a crucial inflammatory nuclear transcription factor and a central mediator of inflammatory responses [51]. Previous studies have suggested that both IL-6 and IL-8 secretion in airway epithelia may be regulated in an ERK/NF-kB- [48,52] or p38 MAPK/NF-kB- [28,46] dependent manner. Immunofluorescence data from the NF-kB activation assay demonstrates translocation from the cytoplasm to the nucleus after 30 min of poly-L-arginine exposure, similar to that observed with an inflammatory cocktail. In addition, blockade of NF-kB activation by its selective inhibitor, BAY 11-7085, inhibited both IL-6 and IL-8 protein release (,40– 50%), suggesting that the transcription factor NF-kB plays a role in regulating the expression of these two proinflammatory genes. However, transcriptional regulation may not be the only mechanism involved in enhancing cytokine release. In neutrophils, in addition to a transcriptional effect, the cationic protein may also regulate post-transcriptional events by prolonging the stability of mRNA [44]. In fact, p38 MAPK has been implicated in regulating both the post-transcriptional and translational mechanisms required for IL-8 protein expression in intestinal epithelia [53]. Our western blot data and the effect of the selective p38 MAPK inhibitor on IL-6 and IL-8 release strongly suggest that p38 References 1. Boucher RC (2002) An overview of the pathogenesis of cystic fibrosis lung disease. Adv Drug Deliv Rev 54: 1359–1371. 2. Randell SH, Boucher RC (2006) Effective mucus clearance is essential for respiratory health. Am J Respir Cell Mol Biol 35: 20–28. 3. Jeffery PK (2000) Pathological spectrum of airway inflammation. In: Page CF, Banner KH, Spina D, eds. Cellular Mechanisms in Airways Inflammation. Basel - Boston - Berlin: Birkhauser Verlag. pp 1–52. 4. Jeffery PK (2004) Remodeling and inflammation of bronchi in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1: 176–183. 5. Reed CE (1994) The importance of eosinophils in the immunology of asthma and allergic disease. Ann Allergy 72: 376–380. Polarized Cytokine Secretion MAPK activation was involved in the secretion process. Because 10 mM BAY 11-7085 did not completely abolish IL-6 and IL-8 secretion but did abolish NF-kB translocation, we cannot exclude the possibility that part of the effect of poly-L-arginine is independent of NF-kB activation and may involve other post- transcriptional mechanisms as seen in intestinal epithelial cells via activation of p38 MAPK [53]. Taken together, our data indicate that whereas NF-kB regulates IL-6 and IL-8 mRNA transcription in airway epithelia, whether the p38 MAPK pathway acts downstream of, and perhaps parallel to, this process awaits further investigation. Recent studies have suggested that certain inflammatory cytokines affect transepithelial ion transport. In 16HBE14o- cells, both IL-9 and IL-13 augment UTP-induced Cl2 secretion via an increased expression of hCLCA1, a Ca2+-activated Cl2 channel [54]. Therefore, certain cytokines change the balance between fluid absorption and secretion to favor the hydration of the airway surface and, consequently, mucus clearance [14]. However, our study does not support a role for IL-6 or IL-8 in modulating ion transport activities in 16HBE14o- epithelia. Incubating the epithelia for up to 72 hours with IL-6 or IL-8 did not affect Ca2+- or cAMP-dependent Cl2 secretion across the epithelia when stimulated with UTP or forskolin, respectively. Interestingly, a transient decrease in the UTP-mediated [Ca2+]i increase was observed in epithelia treated with IL-6 or IL-8 for 24 to 48 hours. It has been shown that in human airway epithelia, hyperin- flammatory responses lead to a larger bradykinin-mediated [Ca2+]i increase, which is due to the expanded endoplasmic reticulum/ Ca2+ store [55]. It appears that IL-6 or IL-8 may not be involved in mediating the inflammation-induced Ca2+ store expansion in airway epithelia. In summary, the cationic polypeptide, poly-L-arginine, induces a time- and concentration-dependent polarized IL-6 and IL-8 secretion towards the apical side of bronchial epithelial cells, effects that are mediated via p38-, ERK1/2-, and NF-kB- dependent pathways. This phenomenon may contribute to the pathophysiology of asthmatic inflammation and the development other inflammatory lung diseases. Further studies on the polarized expression of these cytokines by bronchial epithelia and the detailed underlying signaling mechanism(s) may lead to identifi- cation of important therapeutic targets of airway inflammation. Acknowledgments We would like to thank Dr. D. C. Gruenert (Burlington, VT, USA) for generously providing us with 16HBE14o- cells. In addition, we would like to thank Wallace C. Y. Yip for his excellent technical support. Author Contributions Conceived and designed the experiments: WhK. Performed the experi- ments: AWmC JFtL JScW YF. Analyzed the data: AWmC JFtL YF NLsT WhK. Contributed reagents/materials/analysis tools: NLsT WhK. Wrote the paper: WhK. 6. Adler KB, Fischer BM, Wright DT, Cohn LA, Becker S (1994) Interactions between respiratory epithelial cells and cytokines: relationships to lung inflammation. Ann N Y Acad Sci 725: 128–145. 7. Coyle AJ, Gutierrez-Ramos JC (2000) Eosinophils in asthma. In: Page CF, Banner KH, Spina D, eds. Cellular Mechanisms in Airways Inflammation. Basel - Boston - Berlin: Birkhauser Verlag. pp 147–158. 8. Homma T, Bates JH, Irvin CG (2005) Airway hyperresponsiveness induced by cationic proteins in vivo: site of action. Am J Physiol Lung Cell Mol Physiol 289: L413–L418. 9. Uchida DA, Ackerman SJ, Coyle AJ, Larsen GL, Weller PF, et al. (1993) The effect of human eosinophil granule major basic protein on airway responsiveness PLoS ONE | www.plosone.org 11 August 2010 | Volume 5 | Issue 8 | e12091