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• W2151966368 abstract "Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1α, 1औ, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor α). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B andMUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5Bexpression through the ERK signaling pathway. Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1α, 1औ, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor α). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B andMUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5Bexpression through the ERK signaling pathway. chronic obstructive pulmonary disease tracheobronchial epithelial cells extracellular signal-regulated kinase Janus kinase mitogen-activated protein phosphatidylinositol enzyme-linked immunosorbent assay nucleotides signal transducer and activator of transcription fluorescein isothiocyanate interleukin tumor necrosis factor Chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD),1and cystic fibrosis are all characterized by inflammation of the airways and mucus hypersecretion (1Murray J.F. Textbook of Respiratory Medicine. 3rd Ed. Saunders, Philadelphia2000: 443-538Google Scholar). The mucus hypersecretion by itself might increase morbidity and mortality in these conditions by obstructing the airways and impairing gas exchange (2Jeffery P.K. Am. J. Respir. Crit. Care Med. 2001; 164: S28-S38Crossref PubMed Scopus (773) Google Scholar). One of the main components of mucus secretion is the mucin protein. Mucins are a family of large glycoproteins that have a molecular mass of several thousand kilodaltons, and they are a major determinant of the viscoelasticity of mucus secretion (3King M. Fed. Proc. 1980; 39: 3080-3085PubMed Google Scholar). There are currently 19 identified mucin genes highly expressed in tissues, such as lung, nose, salivary glands, GI tract, and uterus (4Voynow J.A. Paediatr. Respir. Rev. 2002; 3: 98-103Crossref PubMed Scopus (46) Google Scholar, 5Kaliner M. Shelhamer J.H. Borson B. Nadel J. Patow C. Marom Z. Am. Rev. Respirat. Dis. 1986; 134: 612-621PubMed Google Scholar). In the lung, synthesis and secretion of mucins are restricted largely to the airway with little to no expression in alveolar airspaces (1Murray J.F. Textbook of Respiratory Medicine. 3rd Ed. Saunders, Philadelphia2000: 443-538Google Scholar). Although at least eight mucin genes (MUC 1, 2, 4, 5AC, 5B, 7, 8, 13) have been found to be expressed in adult human lung (4Voynow J.A. Paediatr. Respir. Rev. 2002; 3: 98-103Crossref PubMed Scopus (46) Google Scholar), MUC5AC andMUC5B appear to be the predominant genes expressed, and their glycoprotein products are the most abundant in mucus secretions (4Voynow J.A. Paediatr. Respir. Rev. 2002; 3: 98-103Crossref PubMed Scopus (46) Google Scholar, 6Hovenberg H.W. Davies J.R. Herrmann A. Lindén C.J. Carlstedt I. Glycoconjugate J. 1996; 13: 839-847Crossref PubMed Scopus (212) Google Scholar, 7Sheehan J.K. Howard M. Richardson P.S. Longwill T. Thornton D.J. Biochem. J. 1999; 338: 507-513Crossref PubMed Scopus (93) Google Scholar, 8Hovenberg H.W. Davies J.R. Carlstedt I. Biochem. J. 1996; 318: 319-324Crossref PubMed Scopus (248) Google Scholar, 9Thornton D.J. Howard M. Khan N. Sheehan J.K. J. Biol. Chem. 1997; 272: 9561-9566Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). MUC5AC appears to be produced mainly in the airway epithelium by goblet cells (8Hovenberg H.W. Davies J.R. Carlstedt I. Biochem. J. 1996; 318: 319-324Crossref PubMed Scopus (248) Google Scholar, 10Chen Y. Zhao Y.H. Di Y.P. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar), while MUC5B is mostly produced in the underlying submucosal glands (10Chen Y. Zhao Y.H. Di Y.P. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar). In contrast to this normal distribution pattern, we previously showed thatMUC5B could be expressed by surface airway epithelial cells in addition to the expression by submucosal gland cells in airway tissue sections obtained from COPD and asthma (10Chen Y. Zhao Y.H. Di Y.P. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar), whileMUC5AC expression was still restricted at the surface epithelial cells in these tissue sections. These results suggest that changes in MUC gene expression, especially MUC5B, are associated with airway diseases. The source of increased mucus production in diseases such as asthma and COPD is due at least partially to an increased number of goblet cells in the airway epithelium (11Jeffery P.K. Chest. 2000; 117: 251S-260SAbstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12Aikawa T. Shimura S. Sasaki H. Ebina M. Takishima T. Chest. 1992; 101: 916-921Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 13Carroll N. Carello S. Cooke C. James A. Eur. Respir. J. 1996; 9: 709-715Crossref PubMed Scopus (188) Google Scholar). Studies have linked goblet cell metaplasia to the increase of mucin gene expression in airway epithelial cells (10Chen Y. Zhao Y.H. Di Y.P. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar,14Zuhdi Alimam M. Piazza F.M. Selby D.M. Letwin N. Huang L. Rose M.C. Am. J. Respir. Cell Mol. Biol. 2000; 22: 253-260Crossref PubMed Scopus (214) Google Scholar). In recent years, inflammatory cytokines have been linked to increased mucus production by their effects on mucin gene expression in the airway epithelium (15Cohn L.A. Adler K.B. Exp. Lung Res. 1992; 18: 299-322Crossref PubMed Scopus (25) Google Scholar). For example, tumor necrosis factor (TNF) α has been shown to induce expression of MUC2 (16Levine S.J. Larivee P. Logun C. Angus C.W. Ognibene F.P. Shelhamer J.H. Am. J. Respir. Cell Mol. Biol. 1995; 12: 196-204Crossref PubMed Scopus (204) Google Scholar) andMUC5AC (17Borchers M.T. Carty M.P. Leikauf G.D. Am. J. Physiol. 1999; 276: L549-L555PubMed Google Scholar) in NCI-H292 cells. More recently, IL-1औ has also been shown to induce MUC2 and MUC5ACexpression in NCI-H292 cells (18Koo J.S. Kim Y.D. Jetten A.M. Belloni P. Nettesheim P. Exp. Lung Res. 2002; 28: 315-332Crossref PubMed Scopus (61) Google Scholar, 19Kim Y.D. Kwon E.J. Park D.W. Song S.Y. Yoon S.K. Baek S.H. Mol. Pharmacol. 2002; 62: 1112-1118Crossref PubMed Scopus (91) Google Scholar). From studies of asthma, evidence suggests that Th2 cytokines IL-4, 9, and 13 can also affect mucin gene expression. Transgenic overexpression of IL-4 in murine lungs causes mucous cell metaplasia and an induction of MUC5AC expression in the airway epithelium (20Temann U.A. Prasad B. Gallup M.W. Basbaum C. Ho S.B. Flavell R.A. Rankin J.A. Am. J. Respir. Cell Mol. Biol. 1997; 16: 471-478Crossref PubMed Scopus (239) Google Scholar). However, direct treatment of IL-4 on airway epithelial cell culture has produced conflicting results. One group reported a decrease in MUC5AC expression (21Jayawickreme S.P. Gray T. Nettesheim P. Eling T. Am. J. Physiol. 1999; 276: L596-L603PubMed Google Scholar), another reported no change (22Rose M.C. Piazza F.M. Chen Y.A. Alimam M.Z. Bautista M.V. Letwin N. Rajput B. J. Aerosol. Med. 2000; 13: 245-261Crossref PubMed Scopus (66) Google Scholar), and a third reported an increase ofMUC2 expression by IL-4 (23Dabbagh K. Takeyama K. Lee H.M. Ueki I.F. Lausier J.A. Nadel J.A. J. Immunol. 1999; 162: 6233-6237PubMed Google Scholar). It was later suggested that IL-13 was also important for the development of asthmatic phenotypes such as airway hyperreactivity, eosinophilic infiltration, and mucous cell metaplasia (24Grünig G. Warnock M. Wakil A.E. Venkayya R. Brombacher F. Rennick D.M. Sheppard D. Mohrs M. Donaldson D.D. Locksley R.M. Corry D.B. Science. 1998; 282: 2261-2263Crossref PubMed Scopus (1729) Google Scholar, 25Wills-Karp M. Luyimbazi J. Xu X. Schofield B. Neben T.Y. Karp C.L. Donaldson D.D. Science. 1998; 282: 2258-2261Crossref PubMed Scopus (2388) Google Scholar).In vivo models using transgenic mice (26Zhu Z. Homer R.J. Wang Z. Chen Q. Geba G.P. Wang J. Zhang Y. Elias J.A. J. Clin. Invest. 1999; 103: 779-788Crossref PubMed Scopus (1491) Google Scholar) and intranasal (14Zuhdi Alimam M. Piazza F.M. Selby D.M. Letwin N. Huang L. Rose M.C. Am. J. Respir. Cell Mol. Biol. 2000; 22: 253-260Crossref PubMed Scopus (214) Google Scholar) or intratracheal (27Singer M. Lefort J. Vargaftig B.B. Am. J. Respir. Cell Mol. Biol. 2002; 26: 74-84Crossref PubMed Scopus (49) Google Scholar) injections of IL-13 consistently showed increased goblet cells in the airways of mice. However, one limitation of these in vivo experiments was their inability to determine the exact mechanism of how the cytokine affects mucin gene expression. Does IL-13 interact directly with receptors on the airway epithelium to induce mucin gene expression, or are its effects mediated through inflammatory cell recruitment or the induction of local mediator release from surrounding cells such as fibroblasts or smooth muscle cells? For instance, significant infiltration of eosinophils and neutrophils within 4–8 h after the instillation of IL-13 was observed (28Shim J.J. Dabbagh K. Ueki I.F. Dao-Pick T. Burgel P.R. Takeyama K. Tam D.C. Nadel J.A. Am. J. Physiol. Lung Cell Mol. Physiol. 2001; 280: L134-L140Crossref PubMed Google Scholar). Since products from both neutrophils and eosinophils can induce mucin gene expression (29Voynow J.A. Young L.R. Wang Y. Horger T. Rose M.C. Fischer B.M. Am. J. Physiol. 1999; 276: L835-L843PubMed Google Scholar, 30Fischer B.M. Voynow J.A. Am. J. Respir. Cell Mol. Biol. 2002; 26: 447-452Crossref PubMed Scopus (179) Google Scholar, 31Burgel P.R. Lazarus S.C. Tam D.C. Ueki I.F. Atabai K. Birch M. Nadel J.A. J. Immunol. 2001; 167: 5948-5954Crossref PubMed Scopus (123) Google Scholar), one cannot determine convincingly whether it is IL-13 or the inflammatory cells and their products that are responsible for the mucous cell metaplasia. In airway epithelial cell cultures, IL-13 has been shown to enhance mucous cell differentiation in human nasal (32Laoukili J. Perret E. Willems T. Minty A. Parthoens E. Houcine O. Coste A. Jorissen M. Marano F. Caput D. Tournier F. J. Clin. Invest. 2001; 108: 1817-1824Crossref PubMed Scopus (202) Google Scholar) and pig tracheal (33Kondo M. Tamaoki J. Takeyama K. Nakata J. Nagai A. Am. J. Respir. Cell Mol. Biol. 2002; 27: 536-541Crossref PubMed Scopus (121) Google Scholar) epithelial cells. However, in these studies, the requirement of IL-13 treatment for 10–14 days is difficult to understand. In another recent study, IL-13 was also shown to inhibit MUC5AC gene expression in nasal epithelial cells (34Kim C.H. Song K.S. Koo J.S. Kim H.U. Cho J.Y. Kim H.J. Yoon J.H. Acta Otolaryngol. 2002; 122: 638-643Crossref PubMed Scopus (23) Google Scholar) and had no effect in NCI-H292 cells (22Rose M.C. Piazza F.M. Chen Y.A. Alimam M.Z. Bautista M.V. Letwin N. Rajput B. J. Aerosol. Med. 2000; 13: 245-261Crossref PubMed Scopus (66) Google Scholar). IL-9 has also been shown to have the ability to stimulate mucous cell hyperplasia in vivo (35Temann U.A. Geba G.P. Rankin J.A. Flavell R.A. J. Exp. Med. 1998; 188: 1307-1320Crossref PubMed Scopus (407) Google Scholar) as well as mucin gene expressionin vitro (36Longphre M. Li D. Gallup M. Drori E. Ordoñez C.L. Redman T. Wenzel S. Bice D.E. Fahy J.V. Basbaum C. J. Clin. Investig. 1999; 104: 1375-1382Crossref PubMed Scopus (246) Google Scholar, 37Louahed J. Toda M. Jen J. Hamid Q. Renauld J.C. Levitt R.C. Nicolaides N.C. Am. J. Respirat. Cell Mol. Biol. 2000; 22: 649-656Crossref PubMed Scopus (239) Google Scholar). However, gene knockout mice of IL-9 showed that IL-9 was necessary for mucous cell hyperplasia in a granuloma model of disease (38Townsend J.M. Fallon G.P. Matthews J.D. Smith P. Jolin E.H. McKenzie N.A. Immunity. 2000; 13: 573-583Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) but not in an allergic asthma model (39McMillan S.J. Bishop B. Townsend M.J. McKenzie A.N. Lloyd C.M. J. Exp. Med. 2002; 195: 51-57Crossref PubMed Scopus (127) Google Scholar). In cell cultures, stimulation of MUC5AC by IL-9 could be seen (36Longphre M. Li D. Gallup M. Drori E. Ordoñez C.L. Redman T. Wenzel S. Bice D.E. Fahy J.V. Basbaum C. J. Clin. Investig. 1999; 104: 1375-1382Crossref PubMed Scopus (246) Google Scholar, 37Louahed J. Toda M. Jen J. Hamid Q. Renauld J.C. Levitt R.C. Nicolaides N.C. Am. J. Respirat. Cell Mol. Biol. 2000; 22: 649-656Crossref PubMed Scopus (239) Google Scholar) but the cells used were undifferentiated TBE cells or cancerous cell lines. To further define the roles of cytokines in mucin gene expression, we used well differentiated primary cultures of human tracheobronchial epithelial (TBE) cells (40Wu R. Zhao Y.H. Chang M.M. European Respir. J. 1997; 10: 2398-2403Crossref PubMed Scopus (93) Google Scholar, 41Wu R. Schiff L.J. In Vitro Models of Respiratory Epithelium. CRC Press, Boca Raton, FL1986: 1-26Google Scholar) to determine which cytokines can stimulate MUC5AC and MUC5Bexpression. Our study has demonstrated that only IL-6 and IL-17, not Th2 cytokines, can directly stimulate mucin gene expression in these primary human TBE cells. A similar observation was extended to primary TBE cells derived from monkey and mouse. Human tracheobronchial tissues were obtained from the University of California at Davis Medical Center (Sacramento, CA) by patient consent. The University Human Subjects Review Committee approved all procedures involved in tissue procurement. In this study, tissues were collected only from patients without diagnosed lung-related disease. Monkey tissues were obtained from the California Regional Primate Research Center at the University of California, Davis, CA. Transgenic mice were generated by the in-house transgenic animal facility. Primary cultures derived from these airway tissues have been established before (42Chen Y. Zhao Y.H. Wu R. Am. J. Respir. Crit. Care Med. 2001; 164: 1059-1066Crossref PubMed Scopus (60) Google Scholar). Normally, tracheobronchial epithelial (TBE) cells were plated on a collagen gel substratum-coated Transwell™ (Corning Costar, Corning, NY) chamber (25 mm) at 1–2 × 104 cells/cm2, in a Ham's F12/Dulbecco's modified Eagle's medium (DMEM) (1:1) supplemented with insulin (5 ॖg/ml), transferrin (5 ॖg/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 ॖm), cholera toxin (10 ng/ml), bovine hypothalamus extract (15 ॖg/ml), bovine serum albumin (0.5 mg/ml), and all-trans-retinoic acid (30 nm). These primary TBE cultures, after a week in an immersed cultured condition, were transferred to an air-liquid interface (biphasic) culture condition. Under biphasic culture conditions, a mucociliary epithelium with the formation of cilia and mucus-secreting granules was observed (40Wu R. Zhao Y.H. Chang M.M. European Respir. J. 1997; 10: 2398-2403Crossref PubMed Scopus (93) Google Scholar). Cytokines, IL-1α, 1औ, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and TNFα were purchased from R&D systems Inc. (Minneapolis, MN). They were dissolved in phosphate-based saline with 17 bovine serum albumin and added directly to the primary TBE cultures at concentrations of 10 and 50 ng/ml. For additional dosage study (as indicated under “Results”), cytokine concentration was gradually increased up to 200 ng/ml. For the IL-6 neutralizing antibody study (R&D systems Inc. Minneapolis, MN), the antibody was added to culture at 0.05, 0.1, and 0.2 ॖg/ml at the time of IL-17 treatment and its continuous presence was maintained until the time of harvest. For the inhibitor study, AG490, PD98059, U0126, and wortmannin were purchased fromCalbiochem-Novabiochem Corporation (San Diego, CA), and they were dissolved in Me2SO. The dose for each of these selected inhibitors was AG490 (5 ॖm), U0126 (1 ॖm), PD98059 (25 ॖm), and wortmannin (10 ॖm). Each dose was determined to be optimal in the initial literature search and the following experimental trials. RNA was isolated from the cultures by a single step phenol/chloroform extraction (43Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63169) Google Scholar). For Northern blot hybridization, an equal amount of total RNA (20 ॖg/lane) was subjected to electrophoresis on a 1.07 agarose gel in the presence of 2.2 mm formaldehyde and transblotted onto Nytran membranes as described above (44Chen Y. Zhao Y.H. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 409-417Crossref PubMed Scopus (73) Google Scholar). For human and monkey cells, single-stranded antisense oligonucleotides corresponding to the tandem repeat unit of human MUC5B andMUC5AC, 5′-TGTGGTCAGCTTTGTGAGGATCCAGGTCGTCCCCGGAGTGGAGGAGGG-3′ (423–376 nt, GenBankTM: U63836) and 5′-AGGGGCAGAAGTTGTGCTGGTTGTGGGAGCAGAGGTTGTGCTGGTTGT-3′ (582–535 nt, GenBankTM: Z34277), respectively, were end-labeled with [γ-32P]ATP by T4 polynucleotide kinase. For mouseMuc5b gene detection, the clone corresponding to the 3′-end sequence of mouse Muc5b (42Chen Y. Zhao Y.H. Wu R. Am. J. Respir. Crit. Care Med. 2001; 164: 1059-1066Crossref PubMed Scopus (60) Google Scholar) was labeled with [α-32P]dCTP by a ready-to-goTM random labeling kit (Amersham Biosciences). All blots were exposed overnight to a phosphor screen and read by the STORMTM system (Molecular Dynamics, Sunnyvale, CA). The relative abundance ofMUC5B/MUC5AC message in Northern blots was normalized with the 18 S ribosomal RNA (rRNA) band. PCR approach was carried out to examine MUC5AC andMUC5B gene expression. cDNAs were generated from the RNA mentioned above by oligo(dT) primer. MUC5AC andMUC5B gene-specific primers were designed according to sequences retrieved from GenBankTM. Specifically, forMUC5AC, forward primer (5′-ACCCAGATCTGCAACACACACT-3′) and reverse primer (5′- GAGCGAGTACATGGAAGAGCTG-3′) were designed based on MUC5AC sequence (AJ001403). For MUC5B, forward primer (5′-ACATGTGTACCTGCCTCTCTGG-3′) and reverse primer (5′-TCTGCTGAGTACTTGGACGCTC-3′) were designed based onMUC5B sequence (Y09788). Each PCR reaction contained 10 ॖm primers for a total volume of 50 ॖl of PCR reaction solution. The initial denaturing step was 94 °C for 2 min, and the last elongation step was 72 °C for 7 min. These PCR reactions were all carried out the same way: denaturing at 94 °C for 30 s, annealing at 55 °C for 45 s and extension (or polymerizing) at 72 °C for 1 min per cycle. Cycle number was determined by experiment with different dilutions of the cDNA samples to avoid saturation. Ultimately 25 cycles were chosen for the PCR. औ-actin band was used as an internal control. The PCR products were separated by electrophoresis on a 1.27 agarose gel and visualized by ethidium bromide post-staining. A chimeric construct containing the proximal 4169 bp of the human MUC5B promoter region (10Chen Y. Zhao Y.H. Di Y.P. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar), and a luciferase reporter gene was prepared using pGL-3 vector. Transgenic mice were generated using B6 mice from Targeted Genomics Laboratory (University of California, Davis). The transgenic positive mice were determined by Southern blot and RT-PCR. The expression profiles of the human MUC5B promoter-driven luciferase were consistent with mouse Muc5b gene expression in the mouse tissues. Two different founder mice were used for this study. For the luciferase assay, cell extracts were prepared from mouse TBE cultures and incubated with Luclite plusTM (Packard Instrument, Meriden, CT) according to the manufacturer's protocol. The relative luciferase activity was expressed with the total protein concentration after normalization. The results were averaged from triplicate dishes of two separate cultures derived from two different founder mice. A human IL-6 QuantikineTMELISA kit (R&D systems Inc. Minneapolis, MN) was used to measure the secreted IL-6 concentrations in both the apical and basal media of biphasic cultured cells following the manufacturer's instructions. Anti-human IL-6R antibody (R&D systems Inc., Minneapolis, MN) was used to characterize the expression of IL-6 receptors in these primary TBE cultures and various human tracheal tissue sections. The staining was carried out by using FITC-conjugated anti-mouse secondary antibody (Vector Laboratories Inc. Burlingame, CA) and Vectashield mounting medium with propidium iodide (1.5 ॖg/ml), following the manufacturer's instructions. The staining pictures were captured by a digital camera attached to a Zeiss fluorescent microscope. Data are expressed as mean ± S.D. The number of repeats are described under “Results” and in the figure legends. Group differences were calculated by analysis of variance. When p < 0.01, the difference was considered significant. We treated primary tracheobronchial epithelial cells grown in an air liquid interface with a panel of cytokines (IL-1α, 1औ, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and TNFα) for 24 h. As shown in Fig.1, our cytokine panel at 10 ng/ml showed that only IL-6 and IL-17 could induce a dramatic increase in expression of MUC5AC and MUC5B. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate either MUC5AC orMUC5B. Treatment with a higher dose of 50 ng/ml yielded identical results (data not shown). MUC5B/5ACmRNA signals from three independent primary TBE cultures from different donors were normalized to 18 S rRNA signals and quantified. A 3–4-fold stimulation of both MUC5B and MUC5ACmessages was consistently observed after an overnight (16 h) treatment by IL-6 and IL-17 (Fig. 1, C and D). The effects of IL-6 and IL-17 on mucin gene expression were dose-dependent (Fig. 2,A and B). Concentrations as low as 2 ng/ml of IL-6 or 10 ng/ml of IL-17 elicited a significant stimulation ofMUC5B gene expression after 16 h of treatment on human primary TBE cultures. To ensure that our failure to find any induction by IL-4, IL-9, or IL-13 was not due to the generally lower sensitivity of northern blots (45McKenzie R.W. Jumblatt J.E. Jumblatt M.M. Invest. Ophthalmol. Vis. Sci. 2000; 41: 703-708PubMed Google Scholar), we repeated the experiments for them as well as IL-6 and IL-17 with reverse transcription polymerase chain reaction (RT-PCR). In addition, we used concentrations of up to 200 ng/ml to rule out that an inadequate cytokine concentration was a factor. As shown in Fig. 2B, even at 200 ng/ml, no effects onMUC5AC and MUC5B were detected in the IL-4, IL-9, or IL-13 treatments. In contrast, IL-6 showed a dose-dependent mucin-inducing activity at all concentrations up to 200 ng/ml, while IL-17 induced mucin genes at lower doses (10 and 50 ng/ml) but not at higher doses(100 and 200 ng/ml). The reason why lower levels of MUC5AC andMUC5B were seen at the higher concentrations of IL-17 is unclear. But it was apparently not due to toxicity because we routinely checked cell viability by the trypan blue dye exclusion test and found no evidence that higher levels of IL-17 were harmful to the cultures. For IL-4, IL-9, and IL-13, it was unlikely that a low level induction of MUC5AC or MUC5B occurred that could not be detected with the more sensitive RT-PCR (45McKenzie R.W. Jumblatt J.E. Jumblatt M.M. Invest. Ophthalmol. Vis. Sci. 2000; 41: 703-708PubMed Google Scholar). IL-2, 3, 5, 7, 8, and 18 appeared to have inhibited the expression of MUC5ACand MUC5B. These effects were not related to the cytotoxicity of the cytokines since the viability of the cells (greater than 957) was routinely checked by the trypan blue dye exclusion test with consistently positive results.Figure 2Dose- and time-dependent elevation ofMUC5B gene expression by cytokines. A, dose-dependent elevation of MUC5B message by IL-6 and IL-17, examined by Northern blot. IL-6 or IL-17 of various concentrations, as indicated, was used to treat primary human TBE cells overnight (16 h), as described in the legend to Fig. 1.B, IL-4, 6, 9, 13, 17 of various concentrations, as indicated, were used to treat primary human TBE cells overnight (16 h), as described in the legend to Fig. 1. Dose-dependent elevation of MUC5AC and MUC5B messages by IL-6 and IL-17 examined by RT-PCR. IL-17 had no induction at higher doses (100 ng/ml and 200 ng/ml). Notably, no inductions of MUC5Bmessage were seen in cultures treated with higher levels (up to 200 ng/ml) of IL-4, 9, and 13. C, time-dependent elevation of MUC5B message in monkey TBE cells after IL-6 (10 ng/ml) and IL-17 (10 ng/ml) treatments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A similar stimulation of MUC gene expression by these two cytokines was also seen in primary TBE cultures derived from monkey and mouse tissues (Figs. 2C and 4A, respectively). However, both animal cultures required a longer treatment time than human cells in order to see the stimulation by Northern blot analysis, which was probably related to the fact that human TBE cell culture has many mucous cells (goblet cell) while primary monkey and mouse TBE cell cultures have very few. 2R. Wu, Y. Chen, and Y. Zhao, unpublished observations. The significance of the stimulation by these two cytokines was further supported by the dramatic increase of the Alcian blue-PAS stained cell population in primary human TBE cultures (arrows in Fig.3, A–C), suggesting the elevation of mucous cell phenotypes in these cultures after IL-6 and IL-17 treatment. It is noteworthy to point out that throughout these studies, Th2 cytokine treatments had no stimulatory effect in these cultures (data not shown). In order to further clairfy the nature of mucin gene regulation, we looked into its transcriptional regulation. Because of the difficulty in transfecting well-differentiated primary human TBE cells, we examined the effects of IL-6 and IL-17 on primary cultures of mouse TBE cells derived from transgenic mice carrying multiple copies of aMUC5B promoter-luciferase reporter gene construct (10Chen Y. Zhao Y.H. Di Y.P. Wu R. Am. J. Respir. Cell Mol. Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar). These cells essentially acted as “stable-transfected” cells. We grew mouse TBE cells from two independent transgenic lines in culture and treated each of these cultures with 10 ng/ml of IL-6 and IL-17. As shown in Fig. 4B, the background luciferase activity was very low in these TBE cultures, which was consistent with the observation that there are not many mucous cells in mouse TBE cell culture. One day after treatment with IL-6 and IL-17, there was a significant (1.6-fold) increase of the luciferase activity. This increase continued to 9- and 8-fold at day 3, and it reached 223- and 96-fold by day 7 after IL-6 and IL-17 treatments, respectively. These increases were also consistent with the Northern blot analysis of mouse Muc5b message in these cultures (Fig. 4A)." @default.
• W2151966368 created "2016-06-24" @default.
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• W2151966368 date "2003-05-01" @default.
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• W2151966368 title "Stimulation of Airway Mucin Gene Expression by Interleukin (IL)-17 through IL-6 Paracrine/Autocrine Loop" @default.
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• W2151966368 doi "https://doi.org/10.1074/jbc.m210429200" @default.
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