The role of eicosanoids in dendritic cell biology

Dendritic cells (DC) which are potent antigen-presenting cells of the immune system can differentiate from human monocytes in the presence of GM-CSF and IL-4. In this culture system, TGF-ß promotes the development of dendritic Langerhans cells and pro-inflammatory cytokines such as TNF-alpha and IL-1 induce the terminal maturation of potent immunostimulatory DC. Eicosanoids are a family of oxygenated derivatives of arachidonic acid (AA) which include lipoxygenase products (leukotrienes and lipoxins) and cyclooxygenase products (prostaglandins and thromboxanes). Intriguingly, GM-CSF, IL-4 and TGF-ß induce a strong bias towards the lipoxygenase arm of the AA metabolisms, whereas the maturation-inducing cytokines have been shown to stimulate the cyclooxygenase arm. In the proposed project, the importance of endogenous and exogenous eicosanoids for DC development and function will be examined. Reverse transcription PCR (RT-PCR) analysis will be used to reveal potentially relevant pathways of AA metabolism in DC. DC will then be tested for their capacity to elaborate distinct eicosanoids using enzyme- immunoassays (EIA), radioimmunoassays (RIA) as well as high performance liquid chromatography (HPLC). Enzyme inhibitors, antisense oligonucleotides as well as receptor antagonists will be used to investigate the importance of individual enzymes and their products for DC development and function. The capacity of DC to respond to exogenous eicosanoids which they cannot produce will also be studied. Elements of prostaglandin E2 (PGE2)-induced signaling pathways (cAMP, protein kinase A, phosphodiesterases) will be investigated in DC. The effects of DC matured in the presence of PGE2 on the T helper (Th) cell bias will also be studied. We will test the hypothesis that DC activated with TNF-alpha plus PGE2 support an extreme Th1 bias which favours the activation of Th2 type regulatory T-cells. This would explain the contradictory results in the literature. These studies will also include the characterization of antigen-specific T-cell clones derived from patients who have been treated with DC pulsed with antigen and activated with TNF-alpha plus PGE2.