Tumor necrosis factor-alpha (TNF alpha)-induced maturation of dendritic cells (DC), with down-regulation of their endocytic ability, has been reported to be mediated by the accumulation of the lipid messenger ceramide...
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Tumor necrosis factor-alpha (TNF alpha)-induced maturation of dendritic cells (DC), with down-regulation of their endocytic ability, has been reported to be mediated by the accumulation of the lipid messenger ceramide. We have now studied the effects and mechanisms of action of NO on endocytosis, investigated with fluorescein isothiocyanate-labeled dextran using human monocyte-derived DC, both immature and after treatment with TNF alpha. Exposure of DC to NO, released by either bystander phagocytes or NO donors, reversed the inhibition of endocytosis induced by TNF alpha. The intracellular accumulation of ceramide induced by TNF alpha was also inhibited by NO. In addition, NO was found to exert an inhibitory effect downstream of the TNF alpha-triggered ceramide accumulation, because NO donors reversed the inhibition of endocytosis induced by the cell-permeant C-2-ceramide. These effects of NO were mimicked by the membrane-permeant cyclic GMP analogue, 8-Br cyclic GMP, and prevented by inhibition of the soluble guanylyl cyclase. At variance with rodents, the inducible isoform of the NO synthase was expressed neither in immature human DC nor after cell treatment with TNF alpha, interferon-gamma, and lipopolysaccharide, suggesting that regulation of these cells depends on exogenous NO, NO, working through cyclic GMP, might therefore prolong the ability of human DC to internalize antigens at the site of inflammation and thus modulate the initial steps leading to antigen-specific immune responses.
Sphingosine l-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low...
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Sphingosine l-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nm) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sph-inganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis;whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell Gi-coupled SPP rec
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