EVIDENCE FOR MAJOR BASIC-PROTEIN IMMUNOREACTIVITY AND INTERLEUKIN-5 GENE ACTIVATION DURING THE LATE-PHASE RESPONSE IN EXPLANTED AIRWAYS

Current evidence suggests that the events subsequent to antigen challe nge in allergic asthmatics involve eosinophil activation and the synth esis of proinflammatory cytokines, in particular interleukin 5 (IL-5). However, little is known about how local inflammatory cell infiltrati on and activation are related to the changes in lung function followin g allergen exposure. We have developed a novel technique to investigat e the local inflammatory events during late-onset allergic bronchocons triction in lung explants from sensitized Brown-Norway (BN) rats. In t his study we tested the hypothesis that the in vitro late airway respo nse involves IL-5 gene activation and recruitment and activation of eo sinophils. Explants were prepared from excised lungs of BN rats (n = 9 ) sensitized 2 wk previously to ovalbumin (OVA). Lungs were inflated w ith liquid agarose solution (2% wt/vol, 48 ml/kg) following perfusion with cold Ca2+/Mg2+-free Hanks' solution, and refrigerated briefly to gel the agarose, and 0.5- to 1.0-mm slices were prepared and cultured overnight at 37 degrees C. Airways were identified and challenged by d irect application of OVA (20 mu g). Cryostat sections of explants were immunostained for major basic protein (MBP) and IL-5 mRNA was detecte d by a S-35-uridine triphosphate-labeled probe and in situ hybridizati on. Explants harvested immediately prior to challenge showed little ev idence of MBP and IL-5 mRNA expression. Explants harvested at 6 h whic h exhibited evidence of bronchoconstriction showed strong cell-associa ted immunostaining for MBP and high expression of IL-5 mRNA in the bro nchial mucosa. Colocalization studies performed in lung explants demon strating late-onset airway responses suggested that the majority of IL -5 mRNA expression was not found in MBP-positive cells. When compared with explants from sham-sensitized rats (n = 4), there was a significa nt increase in MBP-positive and IL-5 mRNA-positive cells per millimete r of basement membrane oi the airway. The presence of MBP immunoreacti vity and IL-5 gene expression was not observed in explants taken from sensitized BN rats which did not undergo late-onset airway responses, indicating an association between inflammatory cell activation and air way constriction. The increase in MBP-positive cells several hours aft er OVA suggests activation, local recruitment, and/or differentiation of eosinophils. This study provides direct evidence for a temporal ass ociation between IL-5 expression, eosinophil infiltration, and the lat e response in individual cultured airways.