C1P Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Preventing NF-κB Activation in Neutrophils.

Recently, ceramide-1-phosphate (C1P) has been shown to modulate acute inflammatory events. Acute lung injury (Arnalich et al. 2000. Infect. Immun. 68: 1942-1945) is characterized by rapid alveolar injury, lung inflammation, induced cytokine production, neutrophil accumulation, and vascular leakage leading to lung edema. The aim of this study was to investigate the role of C1P during LPS-induced acute lung injury in mice. To evaluate the effect of C1P, we used a prophylactic and therapeutic LPS-induced ALI model in C57BL/6 male mice. Our studies revealed that intrapulmonary application of C1P before (prophylactic) or 24 h after (therapeutic) LPS instillation decreased neutrophil trafficking to the lung, proinflammatory cytokine levels in bronchoalveolar lavage, and alveolar capillary leakage. Mechanistically, C1P inhibited the LPS-triggered NF-κB levels in lung tissue in vivo. In addition, ex vivo experiments revealed that C1P also attenuates LPS-induced NF-κB phosphorylation and IL-8 production in human neutrophils. These results indicate C1P playing a role in dampening LPS-induced acute lung inflammation and suggest that C1P could be a valuable candidate for treatment of ALI.

Extracellular ATP Induces Vascular Inflammation and Atherosclerosis via Purinergic Receptor Y2 in Mice.

OBJECTIVE: A solid body of evidence supports a role of extracellular ATP and its P2 receptors in innate and adaptive immunity. It promotes inflammation as a danger signal in various chronic inflammatory diseases. Thus, we hypothesize contribution of extracellular ATP and its receptor P2Y2 in vascular inflammation and atherosclerosis. APPROACH AND RESULTS: Extracellular ATP induced leukocyte rolling, adhesion, and migration in vivo as assessed by intravital microscopy and in sterile peritonitis. To test the role of extracellular ATP in atherosclerosis, ATP or saline as control was injected intraperitoneally 3× a week in low-density lipoprotein receptor(-/-) mice consuming high cholesterol diet. Atherosclerosis significantly increased after 16 weeks in ATP-treated mice (n=13; control group, 0.26 mm2; ATP group, 0.33 mm2; P=0.01). To gain into the role of ATP-receptor P2Y2 in ATP-induced leukocyte recruitment, ATP was administered systemically in P2Y2-deficient or P2Y2-competent mice. In P2Y2-deficient mice, the ATP-induced leukocyte adhesion was significantly reduced as assessed by intravital microscopy. P2Y2 expression in atherosclerosis was measured by real-time polymerase chain reaction and immunohistochemistry and demonstrates an increased expression mainly caused by influx of P2Y2-expressing macrophages. To investigate the functional role of P2Y2 in atherogenesis, P2Y2-deficient low-density lipoprotein receptor(-/-) mice consumed high cholesterol diet. After 16 weeks, P2Y2-deficient mice showed significantly reduced atherosclerotic lesions with decreased macrophages compared with P2Y2-competent mice (n=11; aortic arch: control group, 0.25 mm(2); P2Y2-deficient, 0.14 mm2; P=0.04). Mechanistically, atherosclerotic lesions from P2Y2-deficient mice expressed less vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 RNA. CONCLUSIONS: We show that extracellular ATP induces vascular inflammation and atherosclerosis via activation of P2Y2.

NTPDase1/CD39 and aberrant purinergic signalling in the pathogenesis of COPD.

Purinergic receptor activation via extracellular ATP is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Nucleoside triphosphate diphosphohydrolase-1/CD39 hydrolyses extracellular ATP and modulates P2 receptor signalling.We aimed to investigate the expression and function of CD39 in the pathogenesis of cigarette smoke-induced lung inflammation in patients and preclinical mouse models. CD39 expression and soluble ATPase activity were quantified in sputum and bronchoalveolar lavage fluid (BALF) cells in nonsmokers, smokers and COPD patients or mice with cigarette smoke-induced lung inflammation. In mice, pulmonary ATP and cytokine concentrations, inflammation and emphysema were analysed in the presence or absence of CD39.Following acute cigarette smoke exposure CD39 was upregulated in BALF cells in smokers with further increases in COPD patients. Acute cigarette smoke exposure induced CD39 upregulation in murine lungs and BALF cells, and ATP degradation was accelerated in airway fluids. CD39 inhibition and deficiency led to augmented lung inflammation; treatment with ATPase during cigarette smoke exposure prevented emphysema.Pulmonary CD39 expression and activity are increased in COPD. CD39 deficiency leads to enhanced emphysema in mice, while external administration of a functional CD39 analogue partially rescues the phenotype. The compensatory upregulation of pulmonary CD39 might serve as a protective mechanism in cigarette smoke-induced lung damage.

Ceramide-1-phosphate inhibits cigarette smoke-induced airway inflammation.

Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1 s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation.

Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.

The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1­deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1(-/-) mice. The velocity of rolling leukocytes was higher in Tph1(-/-) mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1(-/-) mice. Diminished rolling in Tph1(-/-) mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1(-/-) mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1(-/-) mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity.

Uridine supplementation exerts anti-inflammatory and anti-fibrotic effects in an animal model of pulmonary fibrosis.

RATIONALE: Pulmonary fibrosis is a progressive disease with only few treatment options available at the moment. Recently, the nucleoside uridine has been shown to exert anti-inflammatory effects in different animal models, e.g. in acute lung injury or bronchial asthma. METHOD: Therefore, we investigated the influence of uridine supplementation on inflammation and fibrosis in the classical bleomycin model. Male C57BL/6 mice received an intratracheal injection of bleomycin on day 0 and were treated intraperitoneally with uridine or vehicle. The degree of inflammation and fibrosis was assessed at different time points. RESULTS: Uridine administration resulted in attenuated inflammation, as demonstrated by reduced leukocytes and pro-inflammatory cytokines in the broncho-alveolar lavage (BAL) fluid. Furthermore, collagen deposition in the lung interstitium was also reduced by uridine supplementation. Similar results were obtained in a model in which animals received repeated intraperitoneal bleomycin injections. In addition uridine inhibited collagen and TGF-ß synthesis by primary lung fibroblasts, the release of pro-inflammatory cytokines by human lung epithelial cells, as well as the production of reactive oxygen species by human neutrophils. CONCLUSION: In summary, we were able to show that uridine has potent anti-inflammatory and anti-fibrotic properties. As uridine supplementation has been shown to be well tolerated and safe in humans, this might be a new therapeutic approach for the treatment of fibrotic lung diseases.

P2Y6 deficiency limits vascular inflammation and atherosclerosis in mice.

OBJECTIVE: Nucleotides such as ATP, ADP, UTP, and UDP serve as proinflammatory danger signals via purinergic receptors on their release to the extracellular space by activated or dying cells. UDP binds to the purinergic receptor Y6 (P2Y6) and propagates vascular inflammation by inducing the expression of chemokines such as monocyte chemoattractant protein 1, interleukin-8, or its mouse homologsCCL1 (chemokine [C-C motif] ligand 1)/keratinocyte chemokine, CXCL2 (chemokine [C-X-C motif] ligand 2)/macrophage inflammatory protein 2, and CXCL5 (chemokine [C-X-C motif] ligand 5)/LIX, and adhesion molecules such as vascular cell adhesion molecule 1 and intercellular cell adhesion molecule 1. Thus, P2Y6 contributes to leukocyte recruitment and inflammation in conditions such as allergic asthma or sepsis. Because atherosclerosis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall, we hypothesized a role of P2Y6 in atherogenesis. APPROACH AND RESULTS: Intraperitoneal stimulation of wild-type mice with UDP induced rolling and adhesion of leukocytes to the vessel wall as assessed by intravital microscopy. This effect was not present in P2Y6-deficient mice. Atherosclerotic aortas of low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks expressed significantly more transcripts and protein of P2Y6 than respective controls. Finally, P2Y6 (-/-)/low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y6 (+/+)/low-density lipoprotein receptor-deficient mice. Bone marrow transplantation identified a crucial role of P2Y6 on vascular resident cells, most likely endothelial cells, on leukocyte recruitment and atherogenesis. Atherosclerotic lesions of P2Y6-deficient mice contained fewer macrophages and fewer lipids as determined by immunohistochemistry. Mechanistically, RNA expression of vascular cell adhesion molecule 1 and interleukin-6 was decreased in these lesions and P2Y6-deficient macrophages took up less modified low-density lipoprotein cholesterol. CONCLUSIONS: We show for the first time that P2Y6 deficiency limits atherosclerosis and plaque inflammation in mice.

Production of serotonin by tryptophan hydroxylase 1 and release via platelets contribute to allergic airway inflammation.

RATIONALE: 5-Hydroxytryptamine (5-HT) is involved in the pathogenesis of allergic airway inflammation (AAI). It is unclear, however, how 5-HT contributes to AAI and whether this depends on tryptophan hydroxylase (TPH) 1, the critical enzyme for peripheral 5-HT synthesis. OBJECTIVES: To elucidate the role of TPH1 and the peripheral source of 5-HT in asthma pathogenesis. METHODS: TPH1-deficient and TPH1-inhibitor-treated animals were challenged in ovalbumin and house dust mite models of AAI. Experiments with bone marrow chimera, mast cell-deficient animals, platelets transfusion, and bone marrow dendritic cells (BMDC) driven model of AAI were performed. 5-HT levels were measured in bronchoalveolar lavage fluid or serum of animals with AAI and in human asthma. MEASUREMENTS AND MAIN RESULTS: 5-HT levels are increased in bronchoalveolar lavage fluid of mice and people with asthma after allergen provocation. TPH1 deficiency and TPH1 inhibition reduced all cardinal features of AAI. Administration of exogenous 5-HT restored AAI in TPH1-deficient mice. The pivotal role of 5-HT production by structural cells was corroborated by bone marrow chimera experiments. Experiments in mast cell-deficient mice revealed that mast cells are not a source of 5-HT, whereas transfusion of platelets from wild-type and TPH1-deficient mice revealed that only platelets containing 5-HT enhanced AAI. Lack of endogenous 5-HT in vitro and in vivo was associated with an impaired Th2-priming capacity of BMDC. CONCLUSIONS: In summary, TPH1 deficiency or inhibition reduces AAI. Platelet- and not mast cell-derived 5-HT is pivotal in AAI, and lack of 5-HT leads to an impaired Th2-priming capacity of BMDC. Thus, targeting TPH1 could offer novel therapeutic options for asthma.

Blocking P2Y2 purinergic receptor prevents the development of lipopolysaccharide-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality resulting from a direct or indirect injury of the lung. It is characterized by a rapid alveolar injury, lung inflammation with neutrophil accumulation, elevated permeability of the microvascular-barrier leading to an aggregation of protein-rich fluid in the lungs, followed by impaired oxygenation in the arteries and eventual respiratory failure. Very recently, we have shown an involvement of the Gq-coupled P2Y2 purinergic receptor (P2RY2) in allergic airway inflammation (AAI). In the current study, we aimed to elucidate the contribution of the P2RY2 in lipopolysaccharide (LPS)-induced ARDS mouse model. We found that the expression of P2ry2 in neutrophils, macrophages and lung tissue from animals with LPS-induced ARDS was strongly upregulated at mRNA level. In addition, ATP-neutralization by apyrase in vivo markedly attenuated inflammation and blocking of P2RY2 by non-selective antagonist suramin partially decreased inflammation. This was indicated by a reduction in the number of neutrophils, concentration of proinflammatory cytokines in the BALF, microvascular plasma leakage and reduced features of inflammation in histological analysis of the lung. P2RY2 blocking has also attenuated polymorphonuclear neutrophil (PMN) migration into the interstitium of the lungs in ARDS mouse model. Consistently, treatment of P2ry2 deficient mice with LPS lead to an amelioration of the inflammatory response showed by reduced number of neutrophils and concentrations of proinflammatory cytokines. In attempts to identify the cell type specific role of P2RY2, a series of experiments with conditional P2ry2 knockout animals were performed. We observed that P2ry2 expression in neutrophils, but not in the airway epithelial cells or CD4+ cells, was associated with the inflammatory features caused by ARDS. Altogether, our findings imply for the first time that increased endogenous ATP concentration via activation of P2RY2 is related to the pathogenesis of LPS-induced lung inflammation and may represent a potential therapeutic target for the treatment of ARDS and predictably assess new treatments in ARDS.

Purinergic receptor inhibition prevents the development of smoke-induced lung injury and emphysema.

Extracellular ATP acts as a "danger signal" and can induce inflammation by binding to purinergic receptors. Chronic obstructive pulmonary disease is one of the most common inflammatory diseases associated with cigarette smoke inhalation, but the underlying mechanisms are incompletely understood. In this study, we show that endogenous pulmonary ATP levels are increased in a mouse model of smoke-induced acute lung inflammation and emphysema. ATP neutralization or nonspecific P2R-blockade markedly reduced smoke-induced lung inflammation and emphysema. We detected an upregulation the purinergic receptors subtypes on neutrophils (e.g., P2Y2R), macrophages, and lung tissue from animals with smoke-induced lung inflammation. By using P2Y(2)R deficient ((-/-)) animals, we show that ATP induces the recruitment of blood neutrophils to the lungs via P2Y(2)R. Moreover, P2Y(2)R deficient animals had a reduced pulmonary inflammation following acute smoke-exposure. A series of experiments with P2Y(2)R(-/-) and wild type chimera animals revealed that P2Y(2)R expression on hematopoietic cell plays the pivotal role in the observed effect. We demonstrate, for the first time, that endogenous ATP contributes to smoke-induced lung inflammation and then development of emphysema via activation of the purinergic receptor subtypes, such as P2Y(2)R.

Purinergic receptor type 6 contributes to airway inflammation and remodeling in experimental allergic airway inflammation.

RATIONALE: Extracellular nucleotides have recently been identified as proinflammatory mediators involved in asthma pathogenesis by signaling via purinergic receptors, but the role of the purinergic receptor type 6 (P2Y6R) has not been previously investigated. OBJECTIVES: To investigate the role of P2Y6R in asthma pathogenesis. METHODS: Acute and chronic OVA model and also HDM model of allergic inflammation in C57Bl/6 mice treated with specific P2Y6R antagonist and P2Y6R(-/-) mice were evaluated for classical features of asthmatic inflammation. In addition, primary epithelial cell culture from human and epithelial cell lines from mouse and human were stimulated with P2Y6R agonist and treated with P2Y6R antagonist and assessed for IL-6, IL-8/CXCL8 and KC levels. Experiments with P2Y6R(-/-) and P2Y6R(+/+) chimera were performed to discriminate the role of P2Y6R activation in structural lung cells and in cells from hematopoietic system. MEASUREMENTS AND MAIN RESULTS: We observed that the intratracheal application of a P2Y6R antagonist (MRS2578) and P2Y6R deficiency inhibited cardinal features of asthma, such as bronchoalveolar lavage eosinophilia, airway remodeling, Th2 cytokine production, and bronchial hyperresponsiveness in the ovalbumin-alum model. MRS2578 was also effective in reducing airway inflammation in a model using house dust mite extracts to induce allergic lung inflammation. Experiments with bone marrow chimeras revealed the importance of the P2Y6R expression on lung structural cells in airway inflammation. In accordance with this finding, we found a strong up-regulation of P2Y6 expression on airway epithelial cells of animals with experimental asthma. Concerning the underlying mechanism, we observed that MRS2578 inhibited the release of IL-6 and IL-8/KC by lung epithelial cells in vivo, whereas intrapulmonary application of the P2Y6R agonist uridine-5'-diphosphate increased the bronchoalveolar levels of IL-6 and KC. In addition, selective activation of P2Y6 receptors induced the release of IL-6 and KC/IL-8 by murine and human lung epithelial cells in vitro. CONCLUSIONS: P2Y6R expression on airway epithelial cells is up-regulated during acute and chronic allergic airway inflammation, and selective blocking of P2Y6R or P2Y6R deficiency on the structural cells reduces cardinal features of experimental asthma. Thus, blocking pulmonary P2Y6R might be a target for the treatment of allergic airway inflammation.

A potential role for P2X7R in allergic airway inflammation in mice and humans.

P2X7R deficiency is associated with a less severe outcome in acute and chronic inflammatory disorders. Recently, we demonstrated that extracellular adenosine triphosphate is involved in the pathogenesis of asthma by modulating the function of dendritic cells (DCs). However, the role of the purinergic receptor subtype P2X7 is unknown. To elucidate the role of P2X7R in allergic airway inflammation (AAI) in vitro and in vivo, P2X7R expression was measured in lung tissue and immune cells of mice or in humans with allergic asthma. By using a specific P2X7R-antagonist and P2X7R-deficient animals, the role of this receptor in acute and chronic experimental asthma was explored. P2X7R was found to be up-regulated during acute and chronic asthmatic airway inflammation in mice and humans. In vivo experiments revealed the functional relevance of this finding because selective P2X7R inhibition or P2X7R deficiency was associated with reduced features of acute and chronic asthma in the ovalbumin-alum or HDM model of AAI. Experiments with bone marrow chimeras emphasized that P2X7R expression on hematopoietic cells is responsible for the proasthmatic effects of P2X7R signaling. In the DC-driven model of AAI, P2X7R-deficient DCs showed a reduced capacity to induce Th2 immunity in vivo. Up-regulation of P2X7R on BAL macrophages and blood eosinophils could be observed in patients with chronic asthma. Our data suggest that targeting P2X7R on hematopoietic cells (e.g., DCs or eosinophils) might be a new therapeutic option for the treatment of asthma.

P2X7 receptor signaling in the pathogenesis of smoke-induced lung inflammation and emphysema.

Extracellular ATP is up-regulated in the airways of patients with chronic obstructive pulmonary disease, and may contribute to the pathogenesis of the disease. However, the precise mechanisms are poorly understood. Our objective was to investigate the functional role of the ATP receptor P2X(7) in the pathogenesis of cigarette smoke (CS)-induced lung inflammation and emphysema in vivo. Expression of the P2X(7) receptor (P2X(7)R) was measured in lung tissue und immune cells of mice with CS-induced lung inflammation. In a series of experiments using P2X(7) antagonists and genetically engineered mice, the functional role of the P2X(7)R in CS-induced lung inflammation was explored. CS-induced inflammation was associated with an up-regulation of the P2X(7)R on blood and airway neutrophils, alveolar macrophages, and in whole lung tissue. Selective intrapulmonary inhibition of the P2X(7)R reduced CS-induced lung inflammation and prevented the development of emphysema. Accordingly, P2X(7)R knockout mice showed a reduced pulmonary inflammation after acute CS exposure. Experiments with P2X(7)R chimera animals revealed that immune cell P2X(7)R expression plays an important role in CS-induced lung inflammation and emphysema. Extracellular ATP contributes to the development of CS-induced lung inflammation and emphysema via activation of the P2X(7)R. Inhibition of this receptor may be a new therapeutic target for the treatment of chronic obstructive pulmonary disease.

Extracellular adenosine triphosphate and chronic obstructive pulmonary disease.

RATIONALE: Extracellular ATP promotes inflammation, but its role in chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES: To analyze the expression of ATP and its functional consequences in never-smokers, asymptomatic smokers, and patients with COPD. METHODS: ATP was quantified in bronchoalveolar lavage fluid (BALF) of never-smokers, asymptomatic smokers, and patients with COPD of different severity. The expression of specific ATP (purinergic) receptors was measured in airway macrophages and blood neutrophils from control subjects and patients with COPD. The release of mediators by macrophages and neutrophils and neutrophil chemotaxis was assessed after ATP stimulation. MEASUREMENTS AND MAIN RESULTS: Chronic smokers had elevated ATP concentrations in BALF compared with never-smokers. Acute smoke exposure led to a further increase in endobronchial ATP concentrations. Highest ATP concentrations in BALF were present in smokers and ex-smokers with COPD. In patients with COPD, BALF ATP concentrations correlated negatively with lung function and positively with BALF neutrophil counts. ATP induced a stronger chemotaxis and a stronger elastase release in blood neutrophils from patients with COPD, as compared with control subjects. In addition, airway macrophages from patients with COPD responded with an increased secretion of proinflammatory and tissue-degrading mediators after ATP stimulation. These findings were accompanied by an up-regulation of specific purinergic receptors in blood neutrophils and airway macrophages of patients with COPD. CONCLUSIONS: COPD is characterized by a strong and persistent up-regulation of extracellular ATP in the airways. Extracellular ATP appears to contribute to the pathogenesis of COPD by promoting inflammation and tissue degradation.

P2Y2R Signaling Is Involved in the Onset of Glomerulonephritis.

Endogenously released adenosine-5'-triphosphate (ATP) is a key regulator of physiological function and inflammatory responses in the kidney. Genetic or pharmacological inhibition of purinergic receptors has been linked to attenuation of inflammatory disorders and hence constitutes promising new avenues for halting and reverting inflammatory renal diseases. However, the involvement of purinergic receptors in glomerulonephritis (GN) has only been incompletely mapped. Here, we demonstrate that induction of GN in an experimental antibody-mediated GN model results in a significant increase of urinary ATP-levels and an upregulation of P2Y2R expression in resident kidney cells as well as infiltrating leukocytes pointing toward a possible role of the ATP/P2Y2R-axis in glomerular disease initiation. In agreement, decreasing extracellular ATP-levels or inhibition of P2R during induction of antibody-mediated GN leads to a reduction in all cardinal features of GN such as proteinuria, glomerulosclerosis, and renal failure. The specific involvement of P2Y2R could be further substantiated by demonstrating the protective effect of the lack of P2Y2R in antibody-mediated GN. To systematically differentiate between the function of P2Y2R on resident renal cells versus infiltrating leukocytes, we performed bone marrow-chimera experiments revealing that P2Y2R on hematopoietic cells is the main driver of the ATP/P2Y2R-mediated disease progression in antibody-mediated GN. Thus, these data unravel an important pro-inflammatory role for P2Y2R in the pathogenesis of GN.

Extracellular ATP is a danger signal activating P2X7 receptor in a LPS mediated inflammation (ARDS/ALI).

Acute respiratory distress syndrome (ARDS) is a life-threating lung condition resulting from a direct and indirect injury to the lungs [1, 2]. Pathophysiologically it is characterized by an acute alveolar damage, an increased permeability of the microvascular-barrier, leading to protein-rich pulmonary edema and subsequent impairment of arterial oxygenation and respiratory failure [1]. This study examined the role of extracellular ATP in recruiting inflammatory cells to the lung after induction of acute lung injury with lipopolysaccharide (LPS). However, the precise mechanism is poorly understood. Our objective was to investigate the functional role of the P2X7 receptor in the pathogenesis of acute respiratory distress syndrome (ARDS/ acute lung injury (ALI)) in vitro and in vivo. We show that intratracheally applied LPS causes an acute accumulation of ATP in the BALF (bronchoalveolar lavage) and lungs of mice. Prophylactic and therapeutic inhibition of P2X7R signalling by a specific antagonist and knock-out experiments was able to ameliorate the inflammatory response demonstrated by reduced ATP-levels, number of neutrophils and concentration of pro-inflammatory cytokine levels in the BALF. Experiments with chimeric mice showed that P2X7R expression on immune cells was responsible for the observed effect. Consistently, the inflammatory response is diminished only by a cell-type specific knockdown of P2X7 receptor on non-stationary immune cells. Since the results of BALF from patients with acute ARDS or pneumonia simulated the in vivo data after LPS exposure, the P2X7 receptor may be a new therapeutic target for treatment in acute respiratory distress syndrome (ARDS/ALI).

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases.The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms.Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes.Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF.

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5'-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF.

P2rx4 deficiency in mice alleviates allergen-induced airway inflammation.

Compelling evidences point out a crucial role for extracellular nucleotides such as adenosine triphosphate (ATP) during inflammatory conditions. Once released into the extracellular space, ATP modulates migration, maturation and function of various inflammatory cells via activating of purinergic receptors of the P2Y- and P2X- family. P2RX4 is an ATP-guided ion channel expressed on structural cells such as alveolar epithelial and smooth muscle cells as well as inflammatory cells including macrophages, dendritic cells (DCs) and T cells. P2RX4 has been shown to interact with P2RX7 and promote NLRP3 inflammasome activation. Although P2RX7 has already been implicated in allergic asthma, the role of P2RX4 in airway inflammation has not been elucidated yet. Therefore, we used a selective pharmacological antagonist and genetic ablation to investigate the role of P2RX4 in an ovalbumin (OVA) driven model of allergen-induced airway inflammation (AAI). Both, P2RX4 antagonist 5-BDBD treatment and P2rx4 deficiency resulted in an alleviated broncho alveolar lavage fluid eosinophilia, peribronchial inflammation, Th2 cytokine production and bronchial hyperresponsiveness. Furthermore, P2rx4-deficient bone marrow derived DCs (BMDCs) showed a reduced IL-1ß production in response to ATP accompanied by a decreased P2rx7 expression and attenuated Th2 priming capacity compared to wild type (WT) BMDCs in vitro. Moreover, mice adoptively transferred with P2rx4-deficient BMDCs exhibit a diminished AAI in vivo. In conclusion our data suggests that P2RX4-signaling contributes to AAI pathogenesis by regulating DC mediated Th2 cell priming via modulating IL-1ß secretion and selective P2RX4-antagonists might be a new therapeutic option for allergic asthma.

Dendritic Cells Are Involved in the Effects of Exercise in a Model of Asthma.

INTRODUCTION: This study investigated the effects of aerobic exercise (AE) on both the maturation of dendritic cells (DC) and the activation of lymphocytes in a mouse model of chronic allergic airway inflammation. METHODS: C57BL/6 mice distributed into control, exercise, ovalbumin (OVA), and OVA + exercise groups were submitted to OVA sensitization and challenge. Treadmill training was performed for 4 wk, and mice were assessed for classical features of chronic allergic airway inflammation as well as dendritic cell activation and T-lymphocyte response. RESULTS: AE reduced OVA-induced eosinophilic inflammation as observed in bronchoalveolar lavage fluid (P < 0.001), airway walls (P < 0001), and also reduced collagen deposition (P < 0.001). AE also reduced bronchoalveolar lavage fluid cytokines (interleukin [IL]-4, P < 0.001; IL-5, P < 0.01; IL-6, P < 0.001; IL-13, P < 0.01; and tumor necrosis factor α, P < 0.01). Cells derived from mediastinal lymphnodes of AE animals that were restimulated with OVA produced less IL-4 (P < 0.01), IL-5 (P < 0.01), and IL-13 (P < 0.001). In addition, AE reduced both DC activation, as demonstrated by reduced release of IL-6 (P < 0.001), CXCL1/KC (P < 0.01), IL-12p70 (P < 0.01), and tumor necrosis factor α (P < 0.05) and DC maturation, as demonstrated by lower MCH-II expression (P < 0.001). CONCLUSION: AE attenuated dendritic cell and lymphocyte activation and maturation, which contributed to reduced airway inflammation and remodeling in the OVA model of chronic allergic airway inflammation.