EOSINOPHIL PENETRATION THROUGH CULTURED HUMAN AIRWAY EPITHELIAL-CELL LAYER

We investigated the mechanisms of eosinophil penetration and mannitol permeability through a multilayer of cultured human tracheal epithelia l cells. Wells of tissue culture plates were separated into the upper and the lower chambers by the cultured epithelial cell layer. Cr-51-la beled eosinophils or H-3-mannitol were put into the lower chamber. To stimulate the epithelial cells, platelet-activating factor (PAF) and/o r phorbol myristate acetate (PMA) were added to the upper chamber. Aft er 4 h of incubation, the eosinophil penetration rate was determined a s a percentage of the total count added to the lower chamber. PMA sign ificantly increased the eosinophil penetration rate in a dose-dependen t manner (4.0% at 10(-5) M), compared with control (0.67%), whereas PA F itself did not. Activation of eosinophils by the addition of PAF to the lower chamber produced a significant increase in the eosinophil pe netration (6.5% at 10(-6) M), which was inhibited by staurosporine. Fo r determining the mannitol permeability, PMA, PAF and/or supernatant f rom eosinophils were added to both upper and lower chambers and incuba ted for 30 min. PMA induced a significant increase in the mannitol per meability (175% of controls at 10(-5) M), whereas PAF itself did not a lter it. Supernatant from eosinophils activated by PAF (10(-6) M) sign ificantly increased the permeability (451% of controls), which was blo cked by staurosporine. Supernatants from AA861 (a 5-lipoxygenase inhib itor)-treated or phenidon (a phospholipase A(2) inhibitor)-treated eos inophils activated by PAF failed to alter the supernatant-induced incr eases in mannitol permeability. These findings suggest that substances released from activated eosinophils (not arachidonates) induce an inc rease in eosinophil penetration and/or airway permeability across epit helium, probably through the activation of protein kinase C in epithel ial cells.