Emb. Raeder et al., Sphingosine blocks human polymorphonuclear leukocyte phagocytosis through inhibition of mitogen-activated protein kinase activation, BLOOD, 93(2), 1999, pp. 686-693
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.