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.
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.
The cytotoxic necrotizing factors CNF1 and CNF2 produced by pathogenic Escherichia coli strains and CNF Y of Yersinia pseudotuberculosis constitutively activate small GTPases of the Rho family. They deamidate a glutamine (Gln63 in RhoA), which is crucial for GTP hydrolysis. CNF1 and CNF Y exhibit 61% identity on the amino acid level, with equal distribution over the whole molecule. Although the two toxins are homologous in the receptor binding domain, we show that they bind to different cellular receptors. CNF Y does not enter Caco-2 and CHO-K1 cells, which are responsive to CNF1. In contrast, HeLa, Hep-2, and HEK 293 cells do respond to both toxins. Competition studies with catalytically inactive mutants of the toxins revealed that binding of CNF1 has no influence on the uptake of CNF Y into HeLa cells. In contrast, uptake of CNF1 is retarded after preincubation of HeLa cells with the catalytically inactive mutant of CNF Y , suggesting that the toxin receptors overlap. Moreover, we compared the pathways of the toxins from receptor binding into the cytosol and showed that both toxins are taken up independent of the presence of clathrin or lipid rafts and are released into the cytosol from acidified endosomes.Cytotoxic necrotizing factor 1 (CNF1) is an AB-type toxin expressed by pathogenic Escherichia coli strains which cause urinary tract infections and neonatal meningitis. CNF1 is a 115-kDa single-chain molecule comprising an N-terminal receptor binding domain and a C-terminal catalytic domain, which contains deamidase activity (for a review, see reference 9). These two domains are separated by a putative translocation domain, which contains two hydrophobic helices involved in membrane translocation (16). CNF1 deamidates small GTPbinding proteins of the Rho family at glutamine 63/61, leading to constitutive activation of the small GTPases by blocking their intrinsic and GTPase activating protein-stimulated GTP hydrolysis (5, 17).CNF1 has been shown to enter cells by receptor-mediated endocytosis independent of clathrin and independent of sphingolipid-cholesterol-rich membrane microdomains (lipid rafts), including caveolae (4). Based on interaction studies, using a yeast two-hybrid system, it has been suggested that cell binding of the toxin is mediated by the laminin receptor precursor p37, a subunit of the mature nonintegrin 67-kDa laminin receptor (3, 11). Moreover, it was shown that CNF1 contributes to E. coli K1 invasion of human brain microvascular endothelial cells via the 67-kDa laminin receptor. Following internalization, CNF1 is delivered to late endosomes by microtubule-dependent transport. From there, it is released into the cytosol in an acidic pH-dependent manner. Specific acidic residues located in the hydrophilic loop, connecting the two hydrophobic helices of the translocation domain, appear to be involved in the translocation process (16).More recently, a cytotoxic necrotizing factor (CNF) produced by Yersinia pseudotuberculosis (CNF Y ) was discovered, which catalyzes the same deamidation reac...
Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schägger and von Jagow 1991); (Schägger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN-and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.
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