title: IL-13 induced increases in nitrite levels are primarily driven by increases in inducible nitric oxide synthase as compared with effects on arginases in human primary bronchial epithelial cells.
abstract: BACKGROUND
  Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T-helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells.
  
  
  OBJECTIVE
  We hypothesized that primary human airway epithelial cells in air-liquid interface (ALI) culture would increase iNOS expression and activity in response to IL-13, while decreasing arginase expression.
  
  
  METHODS
  iNOS and arginase mRNA (real-time PCR) and protein expression (Western blot and immunofluorescence) as well as iNOS activity (nitrite levels) were measured in ALI epithelial cells cultured from bronchial brushings of normal and asthmatic subjects following IL-13 stimulation.
  
  
  RESULTS
  IL-13 up-regulated iNOS mRNA primarily at a transcriptional level in epithelial cells. iNOS protein and activity also increased, arginase1 protein expression decreased while arginase 2 expression did not change. The changes in iNOS protein correlated strongly with changes in nitrites, and inclusion of arginase (1 or 2) did not substantially change the relationship. Interestingly, iNOS mRNA and protein were not correlated.
  
  
  CONCLUSIONS
  These results contrast with many previous results to confirm that Th2 stimuli enhance iNOS expression and activity. While arginase 1 protein decreases in response to IL-13, neither arginase appears to substantially impact nitrite levels in this system.
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA. chibanak@upmc.edu
authors.forename: K
authors.initials: K
authors.lastname: Chibana
authors.forename: J B
authors.initials: JB
authors.lastname: Trudeau
authors.forename: A T
authors.initials: AT
authors.lastname: Mustovich
authors.forename: A T
authors.initials: AT
authors.lastname: Mustovitch
authors.forename: H
authors.initials: H
authors.lastname: Hu
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: S
authors.initials: S
authors.lastname: Balzar
authors.forename: H W
authors.initials: HW
authors.lastname: Chu
authors.forename: S E
authors.initials: SE
authors.lastname: Wenzel
mesh_terms: D000328:Adult; D001119:Arginase / Q000235:genetics / Q000378:metabolism*; D001249:Asthma / Q000201:enzymology / Q000473:pathology; D015153:Blotting, Western; D001980:Bronchi / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D001999:Bronchoscopy; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D018793:Interleukin-13 / Q000494:pharmacology*; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D018709:Statistics, Nonparametric
chemical_list: D018793:Interleukin-13; D009573:Nitrites; D012333:RNA, Messenger; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D001119:Arginase