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Abstract 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.

Abstract 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.

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-HTR(1) and 5-HTR(2) receptor subtypes. Accordingly, 5-HT also increased migration of pulmonary DCs to draining lymph nodes in vivo. By binding to 5-HTR(3), 5-HTR(4) and 5-HTR(7) 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.

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.

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 P2X7 in the pathogenesis of cigarette smoke (CS)–induced lung inflammation and emphysema in vivo. Expression of the P2X7 receptor (P2X7R) was measured in lung tissue und immune cells of mice with CS-induced lung inflammation. In a series of experiments using P2X7 antagonists and genetically engineered mice, the functional role of the P2X7R in CS-induced lung inflammation was explored. CS-induced inflammation was associated with an up-regulation of the P2X7R on blood and airway neutrophils, alveolar macrophages, and in whole lung tissue. Selective intrapulmonary inhibition of the P2X7R reduced CS-induced lung inflammation and prevented the development of emphysema. Accordingly, P2X7R knockout mice showed a reduced pulmonary inflammation after acute CS exposure. Experiments with P2X7R chimera animals revealed that immune cell P2X7R 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 P2X7R. Inhibition of this receptor may be a new therapeutic target for the treatment of chronic obstructive pulmonary disease.

Abstract 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.

The nucleotide adenosine-5'-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1) and A(2a) receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca(2+) concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1) receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a) receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+)CD45RA(+) T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

Background Hypertension is one of the key factors causing cardiovascular diseases which make up the most frequent cause of death in industrialised nations. However about 60% of hypertensive patients in Germany treated with antihypertensives do not reach the recommended target blood pressure. The involvement of patients in medical decision making fulfils not only an ethical imperative but, furthermore, has the potential of higher treatment success. One concept to enhance the active role of patients is shared decision making. Until now there exists little information on the effects of shared decision making trainings for general practitioners on patient participation and on lowering blood pressure in hypertensive patients. Methods/Design In a cluster-randomised controlled trial 1800 patients receiving antihypertensives will be screened with 24 h ambulatory blood pressure monitoring in their general practitioners’ practices. Only patients who have not reached their blood pressure target (approximately 1200) will remain in the study (T1 – T3). General practitioners of the intervention group will take part in a shared decision making-training after baseline assessment (T0). General practitioners of the control group will treat their patients as usual. Primary endpoints are change of systolic blood pressure and change of patients’ perceived participation. Secondary endpoints are changes of diastolic blood pressure, knowledge, medical adherence and cardiovascular risk. Data analysis will be performed with mixed effects models. Discussion The hypothesis underlying this study is that shared decision making, realised by a shared decision making training for general practitioners, activates patients, facilitates patients’ empowerment and contributes to a better hypertension control. This study is the first one that tests this hypothesis with a (cluster-) randomised trial and a large sample size. Trial registration WHO International Clinical Trials: http://apps.who.int/trialsearch/Trial.aspx?TrialID=DRKS00000125

Opioids are the drugs of choice for treating moderate-to-severe pain, especially for patients in the end stage of cancer or other advanced illnesses, and also in critical care or for the treatment of chronic pain. Side effects such as nausea, pruritus, dizziness and constipation have to be controlled in order to use these drugs to their full potential. Opioid-induced bowel syndrome and constipation caused by activation of μ-receptors in the gut can have such distressing effects that some patients prefer to forego adequate pain control. Methylnaltrexone is a μ-opioid receptor antagonist that, unlike naltrexone or naloxone, does not pass the blood-brain barrier, and therefore does not impair the centrally mediated analgesic effect of opioids. It is licensed for the treatment of opioid-induced constipation in palliative care in more than 50 countries. This article presents practically relevant pharmacological data, basic research results and evidence from clinical research about methylnaltrexone, and outlines potential future therapeutic options for this promising drug.

The nucleotide adenosine-5'-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A.sub.1 and A.sub.2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca.sup.2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A.sub.1 receptors. AMP strongly inhibited secretion of TNF-[alpha] and IL-12p70, while it enhanced production of IL-10 both via activation of A.sub.2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4.sup.+ CD45RA.sup.+ T cells inhibited IFN-[gamma] production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.