title: Regulation of antiprotease and antimicrobial protein secretion by airway submucosal gland serous cells.
abstract: Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.
authors.affiliation: Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California 94305-2130, USA. nsjoo@psych.stanford.edu
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Joo
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Lee
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Winges
authors.forename: Arjun
authors.initials: A
authors.lastname: Rustagi
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Wine
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005576:Colforsin / Q000378:metabolism / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism*; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry*; D020545:Respiratory Mucosa / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine; D013997:Time Factors; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000737:chemistry
chemical_list: D000893:Anti-Inflammatory Agents; C505032:CFTR protein, human; D015221:Potassium Channels; D011480:Protease Inhibitors; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D012964:Sodium; D009113:Muramidase; D012697:Serine Endopeptidases; D005905:Glyburide