Michael Horckmans

Extracellular nucleotides regulate CCL20 release from human primary airway epithelial cells, monocytes and monocyte-derived dendritic cells.

Extracellular nucleotides regulate ion transport and mucociliary clearance in human airway epithelial cells (HAECs) via the activation of P2 receptors, especially P2Y2. Therefore, P2Y2 receptor agonists represent potential pharmacotherapeutic agents to treat cystic fibrosis (CF). Nucleotides also modulate inflammatory properties of immune cells like dendritic cells (DCs), which play an important role in mucosal immunity. Using DNA‐microarray experiments, quantitative RT‐PCR and cytokine measurements, we show here that UTP up‐regulated ∼2‐ to 3‐fold the antimicrobial chemokine CCL20 expression and release in primary HAECs cultured on permeable supports at an air–liquid interface (ALI). Both P2Y2 (ATPγS, UTP, INS365) and P2Y6 (UDP, INS48823) agonists increased CCL20 release. UTP‐induced CCL20 release was insensitive to NF‐κB pathway inhibitors but sensitive to inhibitors of ERK1/2 and p38/MAPK pathways. Furthermore, UTP had no effect on interleukin‐(IL)‐8 release and reduced the release of both CCL20 and IL‐8 induced by TNF‐α and LPS. Accordingly, UTP reduced the capacity of basolateral supernatants of HAECs treated with TNF‐α or LPS to induce the chemoattraction of both CD4+ T lymphocytes and neutrophils. In addition, we show that, in monocyte‐derived DCs, ATPγS, and UDP but not UTP/INS365‐stimulated CCL20 release. Likewise, UDP but not ATPγS was also able to increase CCL20 release from monocytes. Pharmacological experiments suggested an involvement of P2Y11 or P2Y6 receptors through NF‐κB, ERK1/2, and p38/MAPK pathways. Altogether, our data demonstrate that nucleotides may modulate chemokine release and leukocyte recruitment in inflamed airways by acting on both epithelial and immune cells. Our results could be relevant for further clinical investigations in CF. J. Cell. Physiol. 211: 716–727, 2007.

P2Y2 Receptor Regulates VCAM-1 Membrane and Soluble Forms and Eosinophil Accumulation during Lung Inflammation

ATP has been defined as a key mediator of asthma. In this study, we evaluated lung inflammation in mice deficient for the P2Y2 purinergic receptor. We observed that eosinophil accumulation, a distinctive feature of lung allergic inflammation, was defective in OVA-treated P2Y2-deficient mice compared with OVA-treated wild type animals. Interestingly, the upregulation of VCAM-1 was lower on lung endothelial cells of OVA-treated P2Y2−/− mice compared with OVA-treated wild type animals. Adhesion assays demonstrated that the action of UTP on leukocyte adhesion through the regulation of endothelial VCAM-1 was abolished in P2Y2-deficient lung endothelial cells. Additionally, the level of soluble VCAM-1, reported as an inducer of eosinophil chemotaxis, was strongly reduced in the bronchoalveolar lavage fluid (BALF) of P2Y2-deficient mice. In contrast, we observed comparable infiltration of macrophages and neutrophils in the BALF of LPS-aerosolized P2Y2+/+ and P2Y2−/− mice. This difference could be related to the much lower level of ATP in the BALF of LPS-treated mice compared with OVA-treated mice. Our data define P2Y2 as a regulator of membrane and soluble forms of VCAM-1 and eosinophil accumulation during lung inflammation.

Extracellular Nucleotides and Interleukin-8 Production by ARPE Cells: Potential Role of Danger Signals in Blood–Retinal Barrier Activation

PURPOSE RPE cell activation is an important feature of autoimmune uveitis. This investigation focused on whether extracellular nucleotides could contribute to this activation, and the effects of ATPgammaS, UTP, and UDP on the production of IL-8 by RPE cells was studied in relation to their expression of functional P2Y receptors. METHODS ARPE-19 cells were cultured with ATPgammaS, UTP, UDP, and TNF. IL-8 gene transcription and protein production were measured by semiquantitative RT-PCR and ELISA. Western blot analysis and RT-PCR were used to investigate ERK 1/2 activation and P2Y expression. Changes in intracellular calcium and cAMP concentration were analyzed by spectrofluorometry and radioimmunoassay. RESULTS Stimulation of ARPE-19 cells with ATPgammaS, UTP, and UDP induced IL-8 gene transcription and protein secretion. TNFalpha induction of IL-8 secretion was also increased by ATPgammaS, UTP, and UDP. Nucleotide induction of IL-8 production was blocked by PD98059, and all nucleotides stimulated ERK 1/2 phosphorylation. P2Y(2) and P2Y(6) mRNAs were detected in ARPE-19 cells. All tested nucleotides induced a pulse of intracellular calcium. CONCLUSIONS ATPgammaS, UTP, and UDP stimulate both basal and TNFalpha-induced IL-8 secretion in RPE cells through an ERK 1/2-dependent pathway. The results suggest that those effects are mediated by P2Y(2) and P2Y(6) receptors.

Extracellular adenine nucleotides inhibit the release of major monocyte recruiters by human monocyte-derived dendritic cells.

Extracellular ATP is known to affect the maturation of monocyte‐derived dendritic cells mainly by regulation of cytokines and costimulatory molecules. The present study describes the inhibition of MCP‐1 (CCL2) and MIP‐1α (CCL3) release by human monocyte‐derived dendritic cells in response to adenine nucleotides. Our pharmacological data support the involvement of P2Y11 and P2Y1 purinergic receptors in the downregulation of these major monocyte recruiters. Migration assays have demonstrated that supernatants of dendritic cells treated with adenine nucleotides or anti‐MCP‐1/MIP‐1α blocking antibodies display a strongly reduced capacity to attract monocytes and immature dendritic cells.

P2Y(4) nucleotide receptor: a novel actor in post-natal cardiac development.

Communication between endothelial cells and cardiomyocytes is critical for cardiac development and regeneration. However the mechanisms involved in these endothelial-cardiomyocyte interactions remain poorly understood. Nucleotides are released within the heart, especially under ischemia or pressure overload. The function of P2Y nucleotide receptors in cardiac development has never been investigated. Here we show that adult P2Y4-null mice display microcardia. P2Y4 nucleotide receptor is expressed in cardiac endothelial cells but not in cardiomyocytes. Loss of P2Y4 in cardiac endothelial cells strongly inhibits their growth, migration and PDGF-B secretion in response to UTP. Proliferation of microvessels and cardiomyocytes is reduced in P2Y4-null hearts early after birth, resulting in reduced heart growth. Our study uncovers mouse P2Y4 receptor as an essential regulator of cardiac endothelial cell function, and illustrates the involvement of endothelial-cardiomyocyte interactions in post-natal heart development. We also detected P2Y4 expression in human cardiac microvessels. P2Y4 receptor could constitute a therapeutic target to regulate cardiac remodelling and post-ischemic revascularisation.

Connection between cardiac vascular permeability, myocardial edema, and inflammation during sepsis: role of the α1AMP-activated protein kinase isoform.

Objective:As adenosine monophosphate (AMP)-activated protein kinase both controls cytoskeleton organization in endothelial cells and exerts anti-inflammatory effects, we here postulated that it could influence vascular permeability and inflammation, thereby counteracting cardiac wall edema during sepsis. Design:Controlled animal study. Settings:University research laboratory. Subjects:C57BL/6J, &agr;1AMPK–/–, and &agr;1AMPK+/+ mice. Intervention:Sepsis was triggered in vivo using a sublethal injection of lipopolysaccharide (O55B5, 10 mg/kg), inducing systolic left ventricular dysfunction. Left ventricular function, edema, vascular permeability, and inflammation were assessed in vivo in both wild-type mice (&agr;1AMPK+/+) and &agr;1AMP-activated protein kinase–deficient mice (&agr;1AMPK–/–). The 5-aminoimidazole-4-carboxamide riboside served to study the impact of AMP-activated protein kinase activation on vascular permeability in vivo. The integrity of endothelial cell monolayers was also examined in vitro after lipopolysaccharide challenge in the presence of aminoimidazole-4-carboxamide riboside and/or after &agr;1AMP-activated protein kinase silencing. Measurements and Main Results:&agr;1AMP-activated protein kinase deficiency dramatically impaired tolerance to lipopolysaccharide challenge. Indeed, &agr;1AMPK–/– exhibited heightened cardiac vascular permeability after lipopolysaccharide challenge compared with &agr;1AMPK+/+. Consequently, an increase in left ventricular mass corresponding to exaggerated wall edema occurred in &agr;1AMPK–/–, without any further decrease in systolic function. Mechanistically, the lipopolysaccharide-induced &agr;1AMPK–/– cardiac phenotype could not be attributed to major changes in the systemic inflammatory response but was due to an increased disruption of interendothelial tight junctions. Accordingly, AMP-activated protein kinase activation by aminoimidazole-4-carboxamide riboside counteracted lipopolysaccharide-induced hyperpermeability in wild-type mice in vivo as well as in endothelial cells in vitro. This effect was associated with a potent protection of zonula occludens-1 linear border pattern in endothelial cells. Conclusions:Our results demonstrate for the first time the involvement of a signaling pathway in the control of left ventricular wall edema during sepsis. AMP-activated protein kinase exerts a protective action through the preservation of interendothelial tight junctions. Interestingly, exaggerated left ventricular wall edema was not coupled with aggravated systolic dysfunction. However, it could contribute to diastolic dysfunction in patients with sepsis.

Gene deletion of P2Y4 receptor lowers exercise capacity and reduces myocardial hypertrophy with swimming exercise

Nucleotides released within the heart under pathological conditions can be involved in cardioprotection or cardiac fibrosis through the activation purinergic P2Y(2) and P2Y(6) receptors, respectively. We previously demonstrated that adult P2Y(4)-null mice display a microcardia phenotype related to a cardiac angiogenic defect. To evaluate the functional consequences of this defect, we performed here a combination of cardiac monitoring and exercise tests. We investigated the exercise capacity of P2Y(4) wild-type and P2Y(4)-null mice in forced swimming and running tests. Analysis of their stress, locomotion, and resignation was realized in open field, black and white box, and tail suspension experiments. Exercise-induced cardiac hypertrophy was evaluated after repeated and prolonged exercise in P2Y(4) wild-type and P2Y(4)-null hearts. We showed that P2Y(4)-null mice have a lower exercise capacity in both swimming and treadmill tests. This was not related to decreased motivation or increased stress, since open field, white and black box, and mouse tail suspension tests gave comparable results in P2Y(4) wild-type and P2Y(4)-null mice. Heart rate and blood pressure rose normally in P2Y(4)-null swimming mice equipped with a telemetric implant. On the contrary, we observed a delayed recovery of postexercise blood pressure after exercise in P2Y(4)-null mice. The heart rate increment in response to catecholamines was also similar in P2Y(4) wild-type and P2Y(4)-null implanted mice, which is consistent with a similar level of cardiac β-receptor expression. Interestingly, the heart of P2Y(4)-null mice displayed a reduced sympathetic innervation associated with a decreased norepinephrine level. We also demonstrated that exercise-induced cardiac hypertrophy was lower in P2Y(4)-null mice after repeated and prolonged exercise. This was associated with a lower increase in cardiomyocyte size and microvessel density. In conclusion, besides its role in cardiac development, P2Y(4) receptor could constitute an important regulator of acute and chronic response to exercise.

Gene Expression Profiling Defines ATP as a Key Regulator of Human Dendritic Cell Functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5′-O-(3-thiotriphosphate) (ATPγS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPγS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPγS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPγS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPγS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPγS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPγS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPγS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPγS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.

Loss of Mouse P2Y4 Nucleotide Receptor Protects against Myocardial Infarction through Endothelin-1 Downregulation

Nucleotides are released in the heart under pathological conditions, but little is known about their contribution to cardiac inflammation. The present study defines the P2Y4 nucleotide receptor, expressed on cardiac microvascular endothelial cells and involved in postnatal heart development, as an important regulator of the inflammatory response to cardiac ischemia. P2Y4-null mice displayed smaller infarcts in the left descending artery ligation model, as well as reduced neutrophil infiltration and fibrosis. Gene profiling identified inter alia endothelin-1 (ET-1) as one of the target genes of P2Y4 in ischemic heart. The reduced level of ET-1 was correlated with reduction of microvascular hyperpermeability, neutrophil infiltration, and endothelial adhesion molecule expression, and it could be explained by the decreased number of endothelial cells in P2Y4-null mice. Expression analysis of metalloproteinases and their tissue inhibitors in ischemic heart revealed reduced expression of matrix metalloproteinase (MMP)-9, reported to be potentially regulated by ET-1, and MMP-8, considered as neutrophil collagenase, as well as reduction of tissue inhibitor of MMP-1 and tissue inhibitor of MMP-4 in P2Y4-null mice. Reduction of cardiac permeability and neutrophil infiltration was also observed in P2Y4-null mice in LPS-induced inflammation model. Protection against infarction resulting from loss of P2Y4 brings new therapeutic perspectives for cardiac ischemia and remodeling.

Loss of Mouse P2Y6 Nucleotide Receptor Is Associated with Physiological Macrocardia and Amplified Pathological Cardiac Hypertrophy

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy.