In situ amplification of 5-lipoxygenase and 5-lipoxygenase-activating protein in allergic airway inflammation and inhibition by leukotriene blockade.
Leukotrienes are important mediators of the eosinophilic influx and mucus hypersecretion in the lungs in a murine model of asthma. We used in situ PCR in this model of human asthma to detect lung mRNA for 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP), key proteins necessary for leukotriene synthesis. Lung tissue was obtained on day 28 from mice treated with i.p. (days 0 and 14) and intranasal (days 14, 25, 26, and 27) OVA or saline. After fixation, the tissue sections underwent protease- and RNase-free DNase digestion, before in situ RT-PCR using target-specific cDNA amplification. 5-LO and FLAP-specific mRNA was visualized by a digoxigenin detection system, and positive cells were analyzed by morphometry. 5-LO and FLAP-specific mRNA and protein were associated primarily with eosinophils and alveolar macrophages in the airways and pulmonary blood vessels in OVA-sensitized/challenged mice. 5-LO and FLAP protein expression increased on a per-cell basis in alveolar macrophages of OVA-treated mice compared with saline controls. Pulmonary blood vessel endothelial cells were also positive for 5-LO, FLAP mRNA, and protein. 5-LO inhibition significantly decreased 5-LO and FLAP-specific mRNA and protein expression in the lung inflammatory cells and endothelial cells. These studies demonstrate a marked increase in key 5-LO pathway proteins in the allergic lung inflammatory response and an important immunomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.
Chu
S J
SJ
Departments of Medicine and Pathology, University of Washington, Seattle, WA 98195, USA.
Tang
L O
LO
Watney
E
E
Chi
E Y
EY
Henderson
W R
WR
D058927:5-Lipoxygenase-Activating Proteins; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis / Q000235:genetics*; D001249:Asthma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D007274:Injections, Intraperitoneal; D020024:Leukotriene Antagonists; D016859:Lipoxygenase Inhibitors; D008168:Lung / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology*; D016676:Macrophages, Alveolar / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D009928:Organ Specificity / Q000235:genetics; D010047:Ovalbumin / Q000008:administration & dosage / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; C548593:Alox5ap protein, mouse; D002352:Carrier Proteins; D020024:Leukotriene Antagonists; D016859:Lipoxygenase Inhibitors; D008565:Membrane Proteins; D012333:RNA, Messenger; D010047:Ovalbumin; D001094:Arachidonate 5-Lipoxygenase