EFFECTS OF THEOPHYLLINE AND ROLIPRAM ON LEUKOTRIENE C-4 (LTC(4)) SYNTHESIS AND CHEMOTAXIS OF HUMAN EOSINOPHILS FROM NORMAL AND ATOPIC SUBJECTS

1 The effects of the non-selective phosphodiesterase (PDE) inhibitor t heophylline and the selective PDE4 inhibitor rolipram on leukotriene C -4 (LTC(4)) synthesis and chemotaxis of complement 5a (C5a)- and plate let-activating factor (PAF)-stimulated human eosinophils obtained from normal and atopic donors were investigated. 2 Eosinophils were purifi ed from peripheral venous blood of normal and atopic subjects by an im munomagnetic procedure to a purity >99%. Eosinophils were stimulated w ith PAF (0.1 mu M) or C5a (0.1 mu M) for 15 min and LTC(4) was measure d by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48-well microchambers (Boyden). 3 Under thes e conditions substantial amounts of LTC(4) (about 300-1000 pg per 10(6 ) cells) were only detectable in the presence of indomethacin (0.1-10 mu M). To explain this finding it was hypothesized that indomethacin r eversed the inhibition of LTC(4) synthesis by endogenously synthesized prostaglandins, in particular prostaglandin E(2) (PGE(2)). In fact, e osinophils release 23 pg PGE(2) per 10(6) cells following PAF stimulat ion; this PGE(2) synthesis was completely inhibited by indomethacin an d readdition of PGE(2) inhibited eosinophil LTC(4) synthesis (IC50=3 n M). The following experiments were performed in the presence of 10 mu M indomethacin. 4 Theophylline (IC(50)similar to 50 mu M) and rolipram (IC(50)similar to 0.03-0.2 mu M) suppressed PAF- and C5a-stimulated L TC(4) synthesis. This PDE inhibitor-induced suppression of LTC(4) gene ration is mediated by activation of protein kinase A, since it was rev ersed by the protein kinase A inhibitor Rp-8-Br-cyclic AMPS. In additi on, exogenous arachidonic acid concentration-dependently (0.3 mu M-3 m u M) reversed the inhibition of LTC(4) synthesis by the PDE inhibitors , indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The beta(2)-adrenoceptor agonist salbutamol inhib ited eosinophil LTC synthesis (IC50=0.08 mu M). The combination of sal butamol with theophylline (10 mu M) or rolipram (3 nM) appeared to be additive. 5 Theophylline (IC(50)similar to 40 mu M), rolipram (IC(50)s imilar to 0.02 mu M [C5a], similar to 0.6 mu M [PAF]) and PGE(2) (IC(5 0)similar to 3 mu M) inhibited C5a- and PAF-stimulated eosinophil chem otaxis. The combination of PGE(2) with theophylline resulted in an add itive effect. 6 Both C5a- and PAF-stimulated eosinophil chemotaxis and LTC(4) generation were significantly elevated in eosinophils from ato pic individuals compared to normal subjects. However, eosinophils from normal and atopic individuals were not different with respect to thei r total cyclic AMP-PDE and PDE4 isoenzyme activities as well as the po tencies of theophylline and rolipram to suppress LTC generation and ch emotaxis.