Pharmacological comparison of LTB 4-induced NADPH oxidase activation in adherent and non-adherent guinea-pig eosinophils

1. Leukotriene B 4 (LTB 4) stimulation of guinea-pig peritoneal eosinophils, induced a biphasic activation of the NADPH oxidase composed of a rapid (<3 min) phase mediated by non-adherent cells and a sustained (3-120 min) phase mediated by CD11b/CD18 adherent eosinophils. Studies were undertaken to compare the intracellular mechanism that mediate these responses. 2. SB 203580 and PP1, inhibitors of p38 mitogen-activated protein (MAP) kinase and the src-family protein tyrosine kinases, respectively caused concentration-dependent attenuation of both the rapid (SB203580: pD 2= -6.31; PP1: pD 2= -5.50) and sustained (SB203580: pD 2= -6,50; PP1: pD 2= -5.73) phases. Similarly, the MAP kinase kinase-1 inhibitor, PD098059 produced partial inhibition of the both phases of superoxide generation. 3. The protein kinase C (PKC) inhibitors Ro-31 8220, GF 109203X and Gö 6976 attenuated the rapid NADPH oxidase response (pD 2S= -6.10, -6.72, -6.15 respectively) and, to a lesser extent, (pD 2S = -5.54, -6.02, -6.51 respectively) the sustained phase. 4. An inhibitor of phosphatidylinositol 3-kinase (PtdIns 3-kinase), wortmannin caused concentration dependent attenuation of the sustained (pD 2= -8.68) but not rapid phase of superoxide generation. In contrast, the syk kinase inhibitor, piceatannol abolished the rapid (pD 2= -6.43) but not sustained respiratory responses. 5. This study demonstrates that LTB 4-induced superoxide generation from adherent and non-adherent eosinophils is mediated via both common (p38 MAP kinase, MEK-1, PKC and the src kinases) and divergent intracellular pathways (syk kinases and PtdIns 3-kinase). This suggests the possibility of therapeutic intervention to selective attenuate activation of adherent tissue eosinophils.