Increased nitric oxide production in nasal epithelial cells from allergic patients--RT-PCR analysis and direct imaging by a fluorescence indicator: DAF-2 DA.
BACKGROUND
Nitric oxide (NO) is believed to participate in the regulation of airway clearance and non-specific cellular immunity. Recent studies have suggested that airway epithelial cells of allergic and non-allergic individuals may differ in their ability to produce this molecule.


OBJECTIVE
The aim of this study was to detect the difference in NO production in human nasal epithelial cells between normal subjects and patients with perennial allergic rhinitis (AR), and to assess the relationship between the expression of nitric oxide synthase (NOS) isoforms and the severity of the disease.


METHODS
Nasal epithelial cells were obtained from the inferior turbinate. The expression of mRNAs encoding constitutive endothelial NOS (eNOS) and inducible NOS (iNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). Direct NO production in living cells was visualized and quantified by a fluorescent indicator, DAF-2 DA.


RESULTS
RT-PCR analysis demonstrated that AR patients with a RAST score of 5 or 6 showed significant increases in the levels of iNOS mRNA and slight reductions in those of eNOS mRNA. Patients with a RAST score of 2-4 also revealed the same tendency however, the difference was not significant. DAF-2 DA imaging demonstrated that epithelial cells, especially the ciliated cells, produced a larger amount of NO than non-epithelial inflammatory cells. Preincubation with L-NAME resulted in an approximate 40% decrease in both groups.


CONCLUSIONS
These results directly indicate that nasal epithelial cells of AR patients overall produce higher levels of NO through the concomitant expression of different NOS isoforms. Continuous NO production by the epithelial cells in normal subjects further support the hypothesis that NO derived from epithelium may play dual roles in the regulation of nasal airway clearance and in the host defense. In addition, the use of DAF-2 DA provides a reliable method to visualize and quantify the direct NO production of living cells.
Takeno
S
S
Department of Otolaryngology, Hiroshima University School of Medicine, Hiroshima, Japan. Sachio.Takeno@ma2.sekyou.ne.jp
Osada
R
R
Furukido
K
K
Chen
J H
JH
Yajin
K
K
D000199:Actins / Q000096:biosynthesis; D000328:Adult; D001120:Arginine / Q000008:administration & dosage; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D019793:Fluorescein; D005456:Fluorescent Dyes; D006416:Hematoxylin; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D009297:Nasal Mucosa / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000096:biosynthesis; D011852:Radioallergosorbent Test; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012221:Rhinitis, Allergic, Perennial / Q000175:diagnosis / Q000378:metabolism*; D012235:Rhodamines; D013194:Staining and Labeling
C414689:4,5-diaminofluorescein diacetate; D000199:Actins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012333:RNA, Messenger; D012235:Rhodamines; D009569:Nitric Oxide; C009434:tetramethylrhodamine isothiocyanate; D001120:Arginine; D019793:Fluorescein; D006416:Hematoxylin