1a). Open in a separate window Figure 1 Immature monocyte derived dendritic cells express the C5aR around the protein level (a) and mRNA level (b). polymerization was increased when C5aR was up-regulated by PGE2. Activation of DC with C5a resulted in interleukin-10 production which was significantly increased after C5aR up-regulation with TNF- and PGE2. Therefore, up-regulation of the C5aR on human DC alters their chemotactic and immunologic response to C5a. Introduction The C5a-receptor mediates proinflammatory and immunomodulatory effects of the anaphylatoxin C5a and its natural catabolite C5adesArg.1 It belongs to the superfamily of G-protein coupled receptors with seven hydrophobic domains and a serpentine transmembrane structure2,3 and is expressed on cells of myeloid origin such as granulocytes and monocytes/macrophages.4,5 Its ligand, C5a, is generated by cleavage of Tilorone dihydrochloride the fifth component of complement (C5) upon activation of the classical or the alternative pathway of the complement system. It has several proinflammatory effects which may lead to changes in blood flow and impairment of vascular integrity associated with oedema.6 These effects are exerted by deliberation of enzymes, activation of effector mechanisms such as production of reactive oxygen species and rapid changes in expression of membrane molecules.7C10 By inducing the synthesis of inflammatory mediators such as interleukin-1 (IL-1), IL-6, IL-8 and tumour necrosis factor- (TNF-) and suppressing the production of IL-12 in monocytes, C5a Tilorone dihydrochloride modulates the course of inflammation.11C15 Recently, C5a has been shown to play an important role in allergy by initiating T-cell recruitment in a murine model of contact hypersensitivity.16,17 Furthermore, in humans the gene locus of C5a has been shown to play a relevant role in the susceptibility to allergic bronchial hyperreactivity.18 Therefore, C5a appears to have a significant role in allergic reactions; however, its exact immunological role has not been elucidated yet. In particular the target cells of C5a are not known. Myeloid dendritic cells (DC) are a possible target for C5aR signalling in allergic reactions, because (1) they Tilorone dihydrochloride are present in allergic inflammation;19C22 (2) they express C5aR; and (3) they show a chemotactic migration towards a C5a gradient.23C26 Myeloid dendritic cells are crucial in initiating the primary immune response.27 They reside in an immature state in many non-lymphoid tissues which are under high pathogen exposure like skin or airways mucosa as sentinels of the immune system.28,29 After receiving activation signals such as bacterial products, cytokines like TNF- and IL-1, or cognate signals like CD40 ligation, they migrate to the draining lymph nodes where they encounter naive T cells. After antigen capture and during their migration DC convert from an antigen-capturing to an antigen-presenting mode, which is usually termed maturation. They up-regulate major histocompatibility complex (MHC) class II and costimulatory molecules such as CD80, CD86 and CD40. During Rabbit Polyclonal to CRHR2 this process, the anaphylatoxin C5a might influence immune or chemotactic functions of DC. Therefore, we investigated the regulation of C5aR expression on human DC and immunomodulatory effects of C5a activation with regard to receptor expression.24,25 We demonstrate that this C5a receptor is differentially expressed on DC depending on their mode of maturation. DC matured in the absence of prostaglandin E2 (PGE2) down-regulated the C5aR, whereas PGE2 up-regulated the receptor. This up-regulation was associated with an increased response of DC to C5a activation with regard to F-actin polymerization and IL-10 production. Materials and methods Preparation and fluorescence-activated cell sorting (FACS) analysis of monocyte-derived dendritic cellsPeripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation of heparinized leucocyte-enriched Tilorone dihydrochloride buffy-coats and allowed to adhere in cell culture flasks in humidified atmosphere at 37 and 5% CO2. After 2 hr, non-adherent cells were removed by five vigorous washes with phosphate-buffered saline Tilorone dihydrochloride (PBS). The remaining adherent cells were cultured in Iscove’s altered Dulbecco’s medium (IMDM) supplemented with 4% heat-inactivated human serum, 250 U/ml IL-4 (R&D Systems, Wiesbaden, Germany), and 1000 U/ml granulocyteCmacrophage colony-stimulating factro (GM-CSF; Novartis Pharma, Nrnberg, Germany). The cultures were fed with new medium and cytokines on day 3 of culture. Non-adherent cells, thereafter called immature dendritic cells (DC), were harvested at day 7. Before initiation of further experiments, cells were analysed by double colour circulation cytometry for.