Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1ß release by mononuclear phagocytes.

Objective: The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in host defense against infections. High systemic IL-1ß levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1ß release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1ß release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Methods: Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1ß was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. Results: The inhibitory effect of nAChR agonists on the BzATP-induced IL-1ß release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1ß release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1ß release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. Conclusion: We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1ß release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats.

BACKGROUND: Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-alpha and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations. METHODS: AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA. RESULTS: In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-alpha production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12. CONCLUSION: NO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.

Impaired superoxide radical production by bronchoalveolar lavage cells from NO(2)-exposed rats.

Production of superoxide radicals is a central property of professional phagocytes used to combat invading microorganisms. Even though the number of macrophages and neutrophils is often increased in the lungs of patients with chronic lung diseases, these patients frequently suffer from bacterially induced exacerbations. To understand the underlying mechanisms, we investigated the production of superoxide radicals by bronchoalveolar lavage (BAL) cells in a rat NO(2) exposure model (10 ppm NO(2) for 1, 3, or 20 days). We showed that cells from NO(2)-exposed animals display a significantly impaired superoxide radical release after zymosan stimulation. The use of specific inhibitors (antimycin or diphenyleneiodonium [DPI]) revealed that the major enzyme systems, NADPH oxidase and complex III of the respiratory chain, are affected. In addition, we investigated gene expression and enzyme activities of antioxidant enzymes. mRNA expression was significantly enhanced for glutathione peroxidase (GPx)-3 and CuZn-superoxide dismutase (SOD) in BAL cells from animals exposed 3 and 20 days, and GPx and SOD enzyme activities were increased in BAL cells from rats exposed 20 days. In conclusion, concomitant occurrence of reduced production and increased scavenging of superoxide radicals resulted in the drastically impaired release of these radicals from BAL cells of NO(2)-exposed rats.

IL-24 is expressed by rat and human macrophages.

Recently, a number of interleukin-10 (IL-10) homologues, among them IL-24 formerly known as melanocyte differentiation factor-7 (mda-7), has been described. Since IL-10 is released by macrophages and plays an important role in the resolution of inflammatory processes, we hypothesized that IL-24 might also be expressed in cells of the monocyte/macrophage lineage. We analyzed IL-24 expression on the mRNA and protein level in stimulated rat and human macrophages. In rat alveolar macrophages and NR8383 cells, IL-24 mRNA induction was observed following stimulation with LPS and IL-4 whereas TNF-alpha failed. Intracellular IL-24 protein was detected in unstimulated and IL-4 stimulated NR8383 cells. Also human blood monocytes showed a strong up-regulation of IL-24 mRNA following preparation which was enhanced by LPS and lowered by IL-10. Furthermore, infection of human monocytes with influenza A virus A/PR/8 caused an induction of IL-24 mRNA expression. In conclusion, our data show that IL-24 expression is induced in stimulated and infected rat and human macrophages, however, more insights into the functions of IL-24 are necessary to define its physiological relevance.

[Deliberative participation of citizens in the prioritisation debate: what is their contribution?]. / Deliberative Bürgerbeteiligung in der Priorisierungsdebatte: Welchen Beitrag können Bürger leisten?

The German debate on prioritisation in medicine is getting well under way. This development has raised the question of which substantial and procedural criteria should be used to guide fair and legitimate prioritisation. It seems to be obvious that in a pluralist, democratic society citizens should be involved in such a discussion. But which is the adequate method, and what is the potential of citizen participation? In this paper we compare the results of a regional citizens' conference on prioritisation in medicine with various European reports on principles and criteria for prioritisation, and thereby aim to identify the conference members'contributions to the German debate on prioritisation criteria. The results of this exemplary deliberative event can provide hints towards the general potential of discursive participation. (As supplied by publisher).

[Stakeholder participation in priority setting - a consideration of the normative status of quantitative and qualitative methods]. / Stakeholderpartizipation und Priorisierung - eine Betrachtung des normativen Status quantitativer und qualitativer Methoden.

Priority setting in medicine is generally regarded as an appropriate means for preparing just allocation of medical resources. By involving the general public or affected stakeholders in priority setting, advocates hope to legitimise this process and increase the acceptability of future decisions on resource allocation. Here, we differentiate between two ideal-typical methods of stakeholder involvement: 1) qualitative and 2) quantitative ones. We argue that the level of information of participants is important to the quality of the outcome of participatory events. Qualitative methods aim at fostering deliberative discussions among well-informed stakeholders. By contrast, quantitative methods usually do not have the capacity to ensure or, at least, control the level of information that participants use to guide their decisions. Hence, we conclude that in the context of priority setting qualitative and especially deliberative methods are preferable to quantitative approaches.

Shift toward an alternatively activated macrophage response in lungs of NO2-exposed rats.

Inflammatory mechanisms are thought to play an important role in the pathogenesis of acute and chronic obstructive pulmonary diseases. In a rat inhalation model using continuous exposure to 10 ppm nitrogen dioxide for 1, 3, and 20 d, we investigated the inflammatory response with particular focus on the activation state of alveolar macrophages. Whereas the number of inflammatory cells and total protein concentration were increased in the bronchoalveolar lavage (BAL), the amount of the proinflammatory cytokine tumor necrosis factor-alpha was markedly reduced with increasing exposure time. In contrast, interleukin (IL)-10 and IL-6 were found at elevated levels and intracellular amounts of suppressor of cytokine signaling-3 protein increased in BAL cells. Upon in vitro lipopolysaccharide stimulation, BAL cells revealed reduced capability to produce the proinflammatory mediators tumor necrosis factor-alpha, IL-1 beta, and nitric oxide, but showed markedly increased transcription and protein release for IL-10. In addition, elevated levels of IL-6, scavenger receptor B, and suppressor of cytokine signaling-3 mRNA were detected in BAL cells from exposed animals. Analyses of highly purified alveolar macrophages indicated that changes in the activation state of these cells were responsible for the observed effects. In conclusion, a priming toward development of the alternatively activated macrophage phenotype occurred in the lungs of rats following nitrogen dioxide inhalation.