Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin–sensitive P2y receptors

Abstract

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, ...

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