Yared Herouy

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 Serotoninergic Receptors of Human Dendritic Cells: Identification and Coupling to Cytokine Release

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is stored at peripheral sites in mast cells and released from this peripheral source upon IgE cross-linking. In this study, we investigated the expression of serotoninergic receptors (5-HTR), the signaling pathway, and biological activity of 5-HT on human dendritic cells (DC), showing that immature and mature DC expressed mRNA for different serotoninergic receptors. Thereby, the mRNA of 5-HTR1B, 5-HTR1E, 5-HTR2A, 5-HTR2B, one splicing variant of the 5-HTR3, 5-HTR4, and 5-HTR7 receptors were detected. Immature DC preferentially expressed mRNA for the heptahelical 5-HTR1B, 5-HTR1E, and 5-HTR2B receptors, while mature DC mostly expressed 5-HTR4 and 5-HTR7. The mRNA expression level of the ligand-gated cation channel 5-HTR3 and the heptahelical 5-HTR2A did not significantly change during maturation. Isotype-selective receptor agonists allowed us to show that 5-HT stimulated 5-HTR3-dependent Ca2+ influx in immature and mature DC. Moreover, we revealed that 5-HTR1 and 5-HTR2 receptor stimulation induced intracellular Ca2+ mobilization via Gi/o proteins in immature, but not mature, DC. Activation of 5-HTR4 and 5-HTR7 induced cAMP elevation in mature DC. Functional studies indicated that activation of 5-HTR4 and 5-HTR7 enhanced the release of the cytokines IL-1β and IL-8, while reducing the secretion of IL-12 and TNF-α in mature DC. In summary, our study shows that 5-HT stimulated, in a maturation-dependent manner, different signaling pathways in DC. These data point to a role for 5-HT in regulating the immune response at peripheral sites.

5-hydroxytryptamine modulates migration, cytokine and chemokine release and T-cell priming capacity of dendritic cells in vitro and in vivo.

Beside its well described role in the central and peripheral nervous system 5-hydroxytryptamine (5-HT), commonly known as serotonin, is also a potent immuno-modulator. Serotoninergic receptors (5-HTR) are expressed by a broad range of inflammatory cell types, including dendritic cells (DCs). In this study, we aimed to further characterize the immuno-biological properties of serotoninergic receptors on human monocyte-derived DCs. 5-HT was able to induce oriented migration in immature but not in LPS-matured DCs via activation of 5-HTR1 and 5-HTR2 receptor subtypes. Accordingly, 5-HT also increased migration of pulmonary DCs to draining lymph nodes in vivo. By binding to 5-HTR3, 5-HTR4 and 5-HTR7 receptors, 5-HT up-regulated production of the pro-inflammatory cytokine IL-6. Additionally, 5-HT influenced chemokine release by human monocyte-derived DCs: production of the potent Th1 chemoattractant IP-10/CXCL10 was inhibited in mature DCs, whereas CCL22/MDC secretion was up-regulated in both immature and mature DCs. Furthermore, DCs matured in the presence of 5-HT switched to a high IL-10 and low IL-12p70 secreting phenotype. Consistently, 5-HT favoured the outcome of a Th2 immune response both in vitro and in vivo. In summary, our study shows that 5-HT is a potent regulator of human dendritic cell function, and that targeting serotoninergic receptors might be a promising approach for the treatment of inflammatory disorders.

Elevated expression of extracellular matrix metalloproteinase inducer (CD147) and membrane-type matrix metalloproteinases in venous leg ulcers

Summary Background  Matrix metalloproteinases (MMPs) contribute to matrix remodelling in venous leg ulcers. Extracellular MMP inducer (EMMPRIN; CD147) has been reported to increase MMP expression, and membrane type 1 MMP (MT1‐MMP) has been implicated in the activation of MMPs.

EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation

UNLABELLED Upon coincubation with platelets, CD34(+) progenitor cells have the potential to differentiate into foam cells, and thereby may promote the progression of atherosclerosis. The exact mechanism of MMP-regulation during the cellular differentiation process to foam cells is still unclear. Thus, we investigated the role of EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) during foam cell formation originating from both monocytes/macrophages and CD34(+) progenitor cells. METHODS AND RESULTS Differentiation of CD34(+) progenitor to foam cells was analyzed in a coculture model of progenitor cells and platelets. While CD34(+) cells did not express EMMPRIN or MT1-MMP, mature foam cells strongly expressed EMMPRIN, which was associated with MT1-MMP expression as well as MMP-9. Gene silencing of EMMPRIN by siRNA during the cell differentiation process hindered not only the upregulation of MMPs (MT1-MMP, MMP-9), but also the secretion of the cytokine M-CSF. During the differentiation process CyPA was substantially released into the supernatant. The presence of the CyPA inhibitor NIM811 significantly reduced MMP-9 secretion during the differentiation process. Similar results were obtained using the classical pathway of foam cell formation by coincubating human macrophages with AcLDL. Additionally, the presence of soluble EMMPRIN ligands (CyPA, recombinant EMMPRIN) further enhanced MMP-9 secretion by mature foam cells. Consistently, CyPA and EMMPRIN were found in atherosclerotic plaques of ApoE-deficient mice by immunohistochemistry. CONCLUSION EMMPRIN is upregulated during the differentiation process from CD34(+) progenitor cells to foam cells, whereas its ligand, CyPA, is released. The CyPA/EMMPRIN activation pathway may play a relevant role in promoting the vulnerability of atherosclerotic plaques.

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.

Stimulation of P2 purinergic receptors induces the release of eosinophil cationic protein and interleukin-8 from human eosinophils

Extracellular nucleotides are the focus of increasing attention for their role as extracellular mediators since they are released into the extracellular environment in a regulated manner and/or as a consequence of cell damage. Here, we show that human eosinophils stimulated with different nucleotides release eosinophil cationic protein (ECP) and the chemokine interleukin 8 (IL‐8), and that release of these two proteins has a different nucleotide requirement. Release of ECP was triggered in a dose‐dependent manner by ATP, UTP and UDP, but not by 2′‐&3′‐o‐(4‐benzoyl‐benzoyl)adenosine 5′‐triphosphate (BzATP), ADP and α,β‐methylene adenosine 5′ triphosphate (α,β‐meATP). Release of IL‐8 was triggered by UDP, ATP, α,β‐meATP and BzATP, but not by UTP or ADP. Pretreatment with pertussis toxin abrogated nucleotide‐stimulated ECP but not IL‐8 release. Release of IL‐8 stimulated by BzATP was fully blocked by the P2X7 blocker KN‐62, while release triggered by ATP was only partially inhibited. IL‐8 secretion due to UDP was fully insensitive to KN‐62 inhibition. Priming of eosinophils with GM‐CSF increased IL‐8 secretion irrespectively of the nucleotide used as a stimulant. It is concluded that extracellular nucleotides trigger secretion of ECP by stimulating a receptor of the P2Y subfamily (possibly P2Y2), while, on the contrary, nucleotide‐stimulated secretion of IL‐8 can be due to activation of both P2Y (P2Y6) and P2X (P2X1 and P2X7) receptors.

Lysophosphatidic Acid Induces Chemotaxis, Oxygen Radical Production, CD11b Up-Regulation, Ca2+ Mobilization, and Actin Reorganization in Human Eosinophils via Pertussis Toxin-Sensitive G Proteins

Lysophosphatidic acid (LPA) is a bioactive lipid mediator, which is generated by secretory type II phospholipase A2 and is thought to play a major role in the pathogenesis of atopic diseases. In this study, the biological activity of LPA on human eosinophils was characterized. We showed by reverse transcription and PCR that human eosinophils express the mRNA of the LPA receptors endothelial differentiation gene (EDG)-2 and EDG-7. Experiments revealed that LPA has chemotactic activity toward eosinophils, stimulates the production of reactive oxygen metabolites, and induces up-regulation of the integrin CD11b. Signal pathway measurements indicated Ca2+-mobilization from intracellular stores and transient actin polymerization upon stimulation with LPA. Cell responses elicited by LPA were inhibited by pertussis toxin indicating that in eosinophils the LPA receptor(s), presumably EDG-2 and/or EDG-7, are coupled to Gi/o proteins. Moreover, LPA-induced activation of eosinophils could be completely blocked by the EDG-2/EDG-7 antagonist diacylglycerol pyrophosphate. In addition, at optimal doses the changes induced by LPA were comparable to those obtained by the other well-characterized chemotaxins. These results indicate that LPA is a strong chemotaxin and activator of eosinophils. These findings point to a novel role of LPA in the pathogenesis of diseases with eosinophilic inflammation such as atopic diseases as chemotaxin as well as activator of proinflammatory effector functions.

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.