Human bronchial fibroblasts express the 5-lipoxygenase pathway.
BACKGROUND
Fibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators. Fibroblast activity is influenced by members of the leukotriene family of bronchoconstrictor and inflammatory mediators, but it is not known whether human bronchial fibroblasts can synthesize leukotrienes.


METHODS
The expression of leukotriene biosynthetic enzymes and receptors was investigated in primary fibroblasts from the bronchi of normal and asthmatic adult subjects using RT-PCR, Western blotting, immunocytochemistry and flow cytometry.


RESULTS
These techniques revealed that human bronchial fibroblasts from both subject groups constitutively express 5-lipoxygenase, its activating protein FLAP, the terminal enzymes leukotriene A4 hydrolase and leukotriene C4 synthase, and receptors for leukotriene B4 (BLT1) and cysteinyl-leukotrienes (CysLT1). Human bronchial fibroblasts generated immunoreactive leukotriene B4 and cysteinyl-leukotrienes spontaneously and in increased amounts after calcium-dependent activation. Flow cytometry showed that human bronchial fibroblasts transformed to a myofibroblast-like phenotype by culture with transforming growth factor-beta1 expressed 320-400% more immunofluorescence for leukotriene C4 synthase and CysLT1 receptors, with 60-80% reductions in leukotriene A4 hydrolase and BLT1 receptors.


CONCLUSIONS
These results indicate that human bronchial fibroblasts may not only respond to exogenous leukotrienes but also generate leukotrienes implicated in narrowing, inflammation and remodeling of the asthmatic airway.
James
Anna J
AJ
Division of Infection, Inflammation and Repair (IIR), University of Southampton, School of Medicine, Southampton General Hospital, Southampton, SO16 6YD, UK. annajuliajames@hotmail.com
Penrose
John F
JF
Cazaly
Angelica M
AM
Holgate
Stephen T
ST
Sampson
Anthony P
AP
D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007975:Leukotriene B4 / Q000096:biosynthesis; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018077:Receptors, Leukotriene / Q000235:genetics / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000235:genetics / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000096:biosynthesis; D053773:Transforming Growth Factor beta1
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; C548593:Alox5ap protein, mouse; D002352:Carrier Proteins; D007476:Ionophores; C513240:LTB4R protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D018077:Receptors, Leukotriene; D018102:Receptors, Leukotriene B4; D018048:Receptors, Purinergic P2; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D007975:Leukotriene B4; D000001:Calcimycin; D001094:Arachidonate 5-Lipoxygenase; D004851:Epoxide Hydrolases; C083169:leukotriene D4 receptor; C043134:leukotriene A4 hydrolase