Sphingosine blocks human polymorphonuclear leukocyte phagocytosis through inhibition of mitogen-activated protein kinase activation

In the present study, we investigated the mechanism by which sphingosine an d its analogues, dihydrosphingosine and phytosphingosine, inhibit polymorph onuclear leukocyte (PMN) phagocytosis of IgG-opsonized erythrocytes (EIgG) and inhibit ERK1 and ERK2 phosphorylation. We used antibodies that recogniz ed the phosphorylated forms of ERK1 (p44) and ERK2 (p42) (extracellular sig nal-regulated protein kinases 1 and 2). Sphingoid bases inhibited ERK1 and ERW activation and phagocytosis of EIgG in a concentration-dependent manner . Incubation with glycine, N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N -[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[(acetyloxy)methyl]ester (BAPTA,AM ), an intracellular chelator of calcium, failed to block either phagocytosi s or ERK1 and ERK2 phosphorylation, consistent with the absence of a role f or a calcium-dependent protein kinase C (PKC) in ERK1 and ERK2 phosphorylat ions. Western blotting demonstrated that sphingosine inhibited the transloc ation of Raf-1 and PKC delta from PMN cytosol to the plasma membrane during phagocytosis. These data are consistent with the interpretation that sphin gosine regulates ERK1 and ERK2 phosphorylation through inhibition of PKC de lta, and this in turn leads to inhibition of Raf-l translocation to the pla sma membrane, consistent with this interpretation, the sphingosine-mediated inhibition of phagocytosis, ERK2 activation, and PKC delta translocation t o the plasma membrane could be abrogated with a cell-permeable diacylglycer ol analog. The increase in the diacylglycerol mass correlated with the tran slocation of PKC delta and Raf-l to the plasma membrane by 3 minutes after the initiation of phagocytosis, Additionally, the diacylglycerol analog enh anced phagocytosis by initiating activation of PKC delta and its translocat ion to the plasma membrane. Because PMN generate sufficient levels of sphin gosine by 30 minutes during phagocytosis of EIgG to inhibit phagocytosis, i t appears that sphingosine can serve as an endogenous regulator of EIgG-med iated phagocytosis by downregulating ERK activation. (C) 1999 by The Americ an Society of Hematology.