Abstract
Tumor necrosis factor-alpha (TNFalpha)-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 TNFalpha. Exposure of DC to NO, released by either bystander phagocytes or NO donors, reversed the inhibition of endocytosis induced by TNFalpha. The intracellular accumulation of ceramide induced by TNFalpha was also inhibited by NO. In addition, NO was found to exert an inhibitory effect downstream of the TNFalpha-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 TNFalpha, 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.
Highlights
NO is generated intracellularly by both constitutive NO synthases (NOSs), as in the case of B and T lymphocytes [4], or by the inducible isoform of the enzyme, expressed by macrophages after their activation with cytokines and bacterial products [5]
Our results show that NO, generated either by bystander phagocytes expressing iNOS or by NO donors, prevents in a cyclic GMP-dependent way the down-regulation of endocytosis induced in dendritic cells (DC) by exposure to TNF␣
DC were resuspended in cytokine-free medium and incubated with fluorescein isothiocyanate (FITC)-dextran, and the internalization of the latter was measured by flow cytometry
Summary
NO is generated intracellularly by both constitutive NO synthases (NOSs), as in the case of B and T lymphocytes [4], or by the inducible isoform of the enzyme (iNOS), expressed by macrophages after their activation with cytokines and bacterial products [5]. NO was found to exert an inhibitory effect downstream of the TNF␣-triggered ceramide accumulation, because NO donors reversed the inhibition of endocytosis induced by the cell-permeant C2-ceramide.
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