Figure 7.

Allergen challenge of patients with asthma resulted in an eosinophil cPLA₂ and CoA-IT activation. Peripheral blood eosinophils were isolated from patients with asthma before and at 6 or 24 hours after allergen challenge. (A) cPLA₂ enzyme activity in the eosinophil cytosolic fraction was assayed in vitro by the hydrolysis of 2-arachidonoyl-glycerophosphatidylcholine. *P < 0.05 by unpaired t test versus nonasthmatic samples. (B) cPLA₂ was resolved by SDS-PAGE (50 μg/lane total protein) with Western blotting and the gel shift in cPLA₂ measured by densitometry. Individual examples shown in B include subject 1 before (lane 1) and after challenge (lane 2), and subject 2 before (lane 3) and after challenge (lane 4). (C) The mean proportion (± SEM) of gel-shifted (phosphorylated) protein was significantly higher both at baseline and after allergen challenge in the subjects with asthma compared with unstimulated control subjects without asthma. *P = 0.01 by unpaired t test of samples from subjects with asthma versus those from subjects without asthma. (D) Eosinophils from patients were assayed for CoA-IT activity at baseline, and at 6 or 24 hours after allergen challenge. Fold increase is shown normalized to the unstimulated baseline for each patient (P = 0.059 for difference between baseline versus 24 h, n = 3).