Dendritic cells (DCs) are specialized antigen-presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T cells. In this study, we analyzed the biological activity and intracellular signaling of adenosine by using reverse transcriptase-polymerase chain reaction assays to investigate mRNA expression of A(1), A(2a) and A(3) adenosine receptors in immature and mature human DCs. Functional experiments on adenosine stimulation showed chemotaxis, intracellular calcium transients, and actin polymerization, but no activation of adenylate cyclase in immature DCs. Experiments with receptor isotype-selective agonists and antagonists as well as pertussis toxin revealed that chemotaxis, calcium transients, and actin polymerization were mediated via G(i-) or G(0-)protein-coupled A(1) and A(3) receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down-regulation of A(1) and A(3) receptor mRNAs, although A(2a) receptor mRNA was still expressed. However, in LPS-differentiated DCs, adenosine and an A(2a) receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited interleukin 12 (IL-12) production. These effects could be completely prevented by pretreatment with A(2) receptor antagonist. These findings strongly suggest that adenosine has important but distinct biological effects in DCs activity as a chemotaxin for immature DCs and as a modulator of IL-12 production in mature DCs. These effects can be explained by differential expression of adenosine receptor subtypes.

Sphingosine 1-phosphate (S1P) is a potent extracellular lysolipid phosphoric acid mediator that is released after IgE-stimulation of mast cells. Here we investigated the biological activity and intracellular signaling of S1P on human dendritic cells (DC), which are specialized antigen presenting cells with the ability to migrate into peripheral tissues and lymph nodes, as well as control the activation of naive T cells. We show that immature and mature DC express the mRNA for different S1P receptors, such as endothelial differentiation gene (EDG)-1, EDG-3, EDG-5, and EDG-6. In immature DC, S1P stimulated pertussis toxin-sensitive Ca2+ increase actin-polymerization and chemotaxis. These responses were lost by DC matured with lipopolysaccharide. In maturing DC, however, S1P inhibited the secretion of tumor necrosis factor alpha and interleukin (IL)-12, whereas it enhanced secretion of IL-10. As a consequence, mature DC exposed to S1P showed a reduced and increased capacity to generate allogeneic Th1 and Th2 responses, respectively. In summary, our study implicates that S1P might regulate the trafficking of DC and ultimately favor Th2 lymphocyte-dominated immunity.

We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca(2+), membrane depolarization, and secretion of inflammatory cytokines. The ATP-activated Ca(2+) change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X(7), as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X(7) receptor ligand 2',3'-(4-benzoylbenzoyl)-ATP was a better agonist then ATP for Ca(2+) increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X(7) antagonist KN-62 inhibited both permeabilization and Ca(2+) changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y(1), P2Y(2), P2Y(5), P2Y(11) and P2X(1), P2X(4), P2X(7). Finally, stimulation of LPS-matured cells with ATP triggered release of IL-1 beta and TNF-alpha. Purinoceptors may provide a new avenue to modulation of dendritic cells function.

Dendritic cells (DCs) are considered the principal initiators of immune response because of their ability to migrate into peripheral tissues and lymphoid organs, process antigens, and activate naive T cells. There is evidence that extracellular nucleotides regulate certain functions of DCs via G-protein-coupled P2Y receptors (P2YR) and ion-channel-gated P2X receptors (P2XR). Here we investigated the chemotactic activity and analyzed the migration-associated intracellular signaling events such as actin reorganization and Ca ؉؉ transients induced by common P2R agonists such as adenosine 5-triphosphate (ATP) and 2-methylthioadenosine triphosphate, the P2YR agonists UTP and adenosine 5-diphosphate (ADP), or the P2XR agonists ␣␤-methylenadenosine-5-triphosphate and 2,3-(4-benzoyl)benzoyl-ATP. The common P2R agonists and the selective P2YR agonists turned out to be potent chemotactic stimuli for immature DCs, but not for mature DCs. In contrast, P2XR agonists had only marginal chemotactic activity in both DC types. Chemotaxis was paralleled by a rise in the intracellular Ca ؉؉ concentration and by actin polymerization. Studies with pertussis toxin implicated that intracellular signaling events such as actin polymerization, mobilization of intracellular Ca ؉؉ , and migration induced by nucleotides was mediated via G i/o protein-coupled P2YR. Moreover, functional studies revealed selective down-regulation of this G i/o protein-coupled chemotactic P2YR responsiveness during maturation, although immature and mature DCs expressed similar amounts of mRNA for the P2R subtypes (P2Y 2 R, P2Y 4 R, P2Y 5 R, P2Y 7 R, P2Y 11 R and P2X 1 R, P2X 4 R, P2X 7 R), and no major differences in respect to the mRNA expression of these receptors could be observed by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR). In summary, our data describe a differential chemotactic response of immature and mature DCs to nucleotides, and lend further support to the hypothesis that P2R are a novel class of immunomodulatory plasma membrane receptors suitable for pharmacological intervention. IntroductionDendritic cells (DCs) are antigen-presenting cells specialized to activate naive T lymphocytes and initiate primary immune responses. [1][2][3] They originate from hemopoietic stem cells and migrate into peripheral sites. Immature DCs reside in most unperturbed tissues, where they are adapted to capture antigens and alert for danger signals such as microorganisms, dying cells, and inflammatory cytokines. 4 Upon exposure to these factors, DCs undergo maturation, a process that involves acquisition of high levels of membrane major histocompatibility complex (MHC) and costimulatory molecules, and the production of a broad panel of cytokines. 1 As part of the maturation program, DCs acquire a propensity to migrate to secondary lymphoid organs for T-cell priming. 3 Adenosine 5Ј-triphosphate (ATP) is a well-known extracellular mediator in the nervous and cardiovascular systems. [5][6] Neurons, platelets, macrophages, T lymphocytes, ...