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 h ypersecretion 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 leukotrien e 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. Af ter 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 syste m, and positive cells were analyzed by morphometry, 5-LO and FLAP-specific mRNA and protein were associated primarily dth eosinophils and alveolar mac rophages in the airways and pulmonary blood vessels in OVA-sensitized/chall enged 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-sp ecific mRNA and protein expression in the lung inflammatory cells and endot helial cells. These studies demonstrate a marked increase in key 5-LO pathw ay proteins in the allergic lung inflammatory response and an important imm unomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.