Stefan Dichmann

Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses

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 α 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.

Expression and function of adenosine receptors in human dendritic cells

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 A1,A2a and A3 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 Gi‐or G0‐protein‐coupled A1 and A3 receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down‐regulation of A1 and A3 receptor mRNAs, although A2a receptor mRNA was still expressed. However, in LPS‐differentiated DCs, adenosine and an A2a receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited in‐terleukin 12 (IL‐12) production. These effects could be completely prevented by pretreatment with A2 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.

The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release

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 Ca2+, membrane depolarization, and secretion of inflammatory cytokines. The ATP‐activated Ca2+ 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 P2X7, as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X7 receptor ligand 2′,3′‐(4‐benzoylbenzoyl)‐ATP was a better agonist then ATP for Ca2+increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X7 antagonist KN‐62 inhibited both permeabilization and Ca2+ changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y1, P2Y2, P2Y5, P2Y11 and P2X1, P2X4, P2X7. Finally, stimulation of LPS‐matured cells with ATP triggered release of IL‐1 β and TNF‐a. Purinoceptors may provide a new avenue to modulation of dendritic cells function.—Ferrari, D., La Sala, A., Chiozzi, P., Morelli, A., Falzoni, S., Girolomoni, G., Idzko, M., Dichmann, S., Norgauer, J., Di Virgilio, F. The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release. FASEB J. 14, 2466–2476 (2000)

The Influence of Lysophosphatidic Acid on the Functions of Human Dendritic Cells

Lysophosphatidic acid (LPA) is a bioactive lipid mediator which is generated by secretory phospholipase A2. In this study, we studied the biological activity of LPA on human dendritic cells (DCs), which are specialized APCs characterized by their ability to migrate into target sites and secondary lymphoid organs to process Ags and activate naive T cells. We show that immature and mature DCs express the mRNA for different LPA receptors such as endothelial differentiation gene (EDG)-2, EDG-4, and EDG-7. In immature DCs, LPA stimulated pertussis toxin-sensitive Ca2+ increase, actin polymerization, and chemotaxis. During the maturation process, DCs lost their ability to respond toward LPA with Ca2+ transients, actin polymerization, and chemotaxis. However, LPA inhibited in a pertussis toxin-insensitive manner the secretion of IL-12 and TNFα as well as enhanced secretion of IL-10 from mature DCs. Moreover, LPA did not affect the endocytic or phagocytic capacities and the surface phenotype of DCs, although it increased the allostimulatory function of mature DC and inhibited their capacity to induce Th1 differentiation. In summary, our study implicates that LPA might regulate the trafficking, cytokine production, and T cell-activating functions of DCs.

P2 purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production

Extracellular nucleotides elicit multiple responses in eosinophils but no information on expression of purinergic receptors in these cells is available so far. In the present study we show that human eosinophils express the following P2Y and P2X subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2X1, P2X4, P2X7, whose stimulation results in intracellular Ca2+ increase and production of large amounts of reactive oxygen intermediates. These events are stimulated or inhibited, respectively, by P2 receptor agonists or antagonists.

Inflammation in stasis dermatitis upregulates MMP-1, MMP-2 and MMP-13 expression

Stasis dermatitis is a common disorder, which is a consequence of impaired venous drainage of the legs. It is characterized histologically by proliferation of small blood vessels in the papillary dermis. This neovascularization may lead occasionally to the formation of discrete papules due to inflammatory processes. In order to evaluate the role of matrix metalloproteinases (MMPs) in the acute phase of chronic venous insufficiency, we examined the production of MMP-1, -2, -13 and tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in lesional skin of stasis dermatitis. A total of 19 patients affected by stasis dermatitis were included in this experimental study. Polymerase chain reaction, western blot and immunohistochemical studies on tissue specimen were performed. In lesional skin of stasis dermatitis, there was elevated gene expression and immunoreactivity for MMP-1, -2 and -13 in comparison to healthy controls. In contrast, genexpression and immunoreactivity for TIMP-1 and -2 were diminished in stasis dermatitis in comparison with healthy controls. Overexpression and production of MMP-1, -2 and -13 without inhibitory effects could be the result of cytokine mediated induction. Matrix metalloproteinases (MMPs) may play an important role in the remodeling of lesional skin in stasis dermatitis.

Fractalkine induces chemotaxis and actin polymerization in human dendritic cells

Abstract.Objective and design: Dendritic cells (DCs) are considered as the principle initiators of immune responses by virtue of their ability to migrate into target sites, process antigens and activate naive T cells. Here, the chemotactic activity and intracellular signaling of fractalkine was analyzed and compared to well known chemotaxins.¶Methods: The mRNA-expression of G protein-coupled CX3CR1 was analyzed by RT-PCR. Chemotaxis was measured in 48-well Boyden chambers and actin polymerization by flow cytometry.¶Results: The mRNA-expression of CX3CR1 in immature and mature DCs was revealed. Fractalkine elicited actin polymerization and chemotaxis in a dose-dependent manner in DCs independent of their state of maturation.¶Conclusions: These results show that immature and mature DCs express mRNA for the CX3CR1 and that fractalkine induces chemotaxis and migration associated actin polymerization in immature as well as in mature DCs, contrasting with the action of other chemokines such as RANTES or MIP-3beta which act only on distinct maturation states of DCs.

Functional characterization of P2Y and P2X receptors in human eosinophils

Activation of purinoceptor by ATP induces in eosinophils various cell responses including calcium transients, actin polymerization, production of reactive oxygen metabolites, CD11b‐expression, and chemotaxis. Here, the effect of ion channel‐gated P2X and/or G protein‐coupled P2Y receptor agonists ATP, ATPγS, α,β‐meATP, 2‐MeSATP, BzATP, ADP, CTP, and UTP on the intracellular Ca2+‐mobilization, actin polymerization, production of reactive oxygen metabolites, CD11b expression and chemotaxis of human eosinophils were measured and the biological activity was analyzed. Although all tested nucleotides were able to induce all these cell responses, the biological activity of the analyzed nucleotides were distinct. Agonists of the G protein‐coupled P2Y receptors such as 2‐MeSATP, UTP, and ADP have a higher biological activity for production of reactive oxygen metabolites, actin polymerization and chemotaxis in comparison to the ion channel‐gated P2X agonists αβ‐meATP, BzATP, and CTP. In contrast, P2Y and P2X agonist showed similar potencies in respect to intracellular calcium transient and CD11b up‐regulation. This conclusion was further supported by experiments with receptor iso‐type antagonist KN62, EGTA or with the Gi protein‐inactivating pertussis toxin. These findings indicate participation of different purinorecptors in the regulation of cell responses in eosinophils.

Human dendritic cells are a physiological source of the chemotactic arachidonic acid metabolite 5-oxo-eicosatetraenoic acid.

Abstract:Objective: The arachidonic acid metabolite, 5-oxo-eicosatetraenoic acid (5-oxo-ETE), is a potent chemotaxin for neutrophils and eosinophils. The aim of this study was to identify physiological conditions and stimulators of 5-oxo-ETE synthesis, because no such conditions have yet been identified.¶Methods: Human neutrophils and monocyte-derived dendritic cells were prepared and 5-oxo-ETE synthesis analyzed using precolumn/reversed-phase HPLC under different conditions and with several physiological and unphysiological stimuli.¶Results: Incubation of neutrophils with 5-hydroxyeicosatetraenoic acid (5-HETE) resulted in the synthesis of about 3.4 nM 5-oxo-ETE per 106 cells in 1 ml under optimal conditions. The synthesis was enhanced about 8-fold with the unphysiological stimuli calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA). No significant effect was observed with different physiological activators. Under optimal conditions, human dendritic cells produced about 50 nM 5-oxo-ETE per 106 cells in 1 ml. The synthesis could be increased with PMA and A23187 by about 50%. Again, no effect could be observed with physiological agents for dendritic cells such as complement fragment C5a, platelet activating factor, N-formyl peptides and interleukin-5.¶Conclusions: These data identified dendritic cells as the only yet known physiological source of relevant amounts of 5-oxo-ETE. This suggests a regulatory function of dendritic cells in the induction of inflammatory neutrophil and eosinophil infiltration caused by 5-oxo-eicosatetraenoic acid.

Downregulation of platelet-activating factor responsiveness during maturation of human dendritic cells†

Dendritic cells (DCs) are specialized antigen‐presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T‐cells. Biological activities of platelet‐activating factor (PAF) and the cytokine macrophage inflammatory protein‐3β (MIP‐3β) as well as the mRNA expression of their receptors were characterized in human DCs during lipopolysaccharide (LPS)‐promoted maturation. Platelet‐activating factor induced calcium transients, migration‐associated actin polymerization response, and chemotaxis in immature human dendritic cells differentiated in vitro from monocytes with interleukin‐4 and granulocyte macrophage colony stimulating factor. In addition, RT‐PCR experiments indicated mRNA expression of the PAF receptor in these immature DCs. Cell studies and mRNA analyses further revealed that immature DCs neither respond to MIP‐3β nor express its specific receptor, CCR7. Induction of cell differentiation by LPS led to the loss of the mRNA expression of the PAF receptor, accompanied by decreasing intracellular calcium release, actin polymerization, and migration after stimulation with PAF. In contrast, LPS treatment induced increasing responsiveness toward MIP‐3β and mRNA expression of CCR7. Comparable data regarding mRNA expression of PAF receptor and PAF responsiveness were also obtained with another maturation protocol using TNFα instead of LPS. The direct comparison between the two different protocols showed a slower decrease of PAF responsiveness induced by TNFα than by LPS. These results show the loss of PAF responsiveness associated with downregulation of PAF receptor mRNA expression during LPS‐ and TNFα‐induced maturation in human DCs. Therefore, these findings point to a functional relevance of PAF in recruiting immature DCs, whereas MIP‐3β might regulate the migration of DCs at a later stage of maturation. J. Cell. Physiol. 185:394–400, 2000.