Effects of theophylline and rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of human eosinophils from normal and atopic subjects

1. The effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of complement 5a (C5a)-and platelet-activating factor (PAF)-stimulated human eosinophils obtained from normal and atopic donors were investigated. 2. Eosinophils were purified from peripheral venous blood of normal and atopic subjects by an immunomagnetic procedure to a purity > 99%. Eosinophils were stimulated with PAF (0.1 μm) or C5a (0.1 μm) for 15 min and LTC4 was measured by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48-well microchambers (Boyden). 3. Under these conditions substantial amounts of LTC4 (about 300–1000 pg per 106 cells) were only detectable in the presence of indomethacin (0.1–10 μm). To explain this finding it was hypothesized that indomethacin reversed the inhibition of LTC4 synthesis by endogenously synthesized prostaglandins, in particular prostaglandin E2 (PGE2). In fact, eosinophils release 23 pg PGE2 per 106 cells following PAF stimulation; this PGE2 synthesis was completely inhibited by indomethacin and readdition of PGE2 inhibited eosinophil LTC4 synthesis (IC50 = 3 nM). The following experiments were performed in the presence of 10 μm indomethacin. 4. Theophylline (IC50∼50 μm) and rolipram (IC50∼ 0.03-0.2 μm) suppressed PAF- and C5a-stimulated LTC4 synthesis. This PDE inhibitor-induced suppression of LTC4 generation is mediated by activation of protein kinase A, since it was reversed by the protein kinase A inhibitor Rp-8-Br-cyclic AMPS. In addition, exogenous arachidonic acid concentration-dependently (0.3 μm-3 μm) reversed the inhibition of LTC4 synthesis by the PDE inhibitors, indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The β-adrenoceptor agonist salbutamol inhibited eosinophil LTC4 synthesis (IC50 = 0.08 μm). The combination of salbutamol with theophylline (10 μm) or rolipram (3 nM) appeared to be additive. 5. Theophylline (IC50∼40 μm), rolipram (IC50∼0.02 μm [C5a], ∼0.6 μm [PAF]) and PGE2 (IC50∼3 nM) inhibited C5a- and PAF-stimulated eosinophil chemotaxis. The combination of PGE2 with theophylline resulted in an additive effect. 6. Both C5a- and PAF-stimulated eosinophil chemotaxis and LTC4 generation were significantly elevated in eosinophils from atopic individuals compared to normal subjects. However, eosinophils from normal and atopic individuals were not different with respect to their total cyclic AMP-PDE and PDE4 isoenzyme activities as well as the potencies of theophylline and rolipram to suppress LTC4 generation and chemotaxis.