Studies on 15-lipoxygenase in dendritic cells and leukotriene receptors in Hodkin lymphoma

Sammanfattning: Classical Hodgkin Lymphoma (cHL) is histologically characterized by a minority of malignant cells, the so-called Hodgkin-Reed Sternberg (H-RS) cells, surrounded by inflammatory cells such as T lymphocytes, eosinophils, macrophages and mast cells. It is generally believed that various molecules released by the H-RS cells are of major importance in the pathophysiology of cHL. In paper I, we report a link between inflammatory cell-derived arachidonic acid metabolites, the cysteinyl leukotrienes (CysLT), to tumor cell growth and function in this disease entity. Two HL cell lines, L 1236 and KMH2, were shown to express functional CysLT, receptors, responding with a robust calcium signal upon challenge with LTD4. Incubation of L1236 cells with LTD4 not only stimulated DNA synthesis but also the transcription and protein release of tumor necrosis factor-á, interleukin-6 and -8. Importantly, all these LTD4-induced effects were blocked by the CysLT1, receptor-specific antagonist zafirlukast. Immunohistochemical studies of classical HL biopsies and microarray analysis of laser captured cells revealed that the CysLT1, receptor is expressed also by the Hodgkin Reed-Stemberg cells ex vivo. Since these cells are surrounded by CysLT-producing eosinophils, macrophages and mast cells, our results suggest the CysLTs as novel mediators in the pathogenesis of classical HL by contributing to the growth and the cytokine features of this tumor. Lipoxygenases are highly regulated enzymes that catalyze the introduction of molecular oxygen in polyunsaturated fatty acids. The lipoxygenases are classified with respect to their positional selectivity of the substrate arachidonic acid. 15-lipoxygenase- 1 (15-LO-1), an enzyme implicated in several pathophysiological conditions, possesses the ability to oxygenate free fatty acids and fatty acids bound to membrane phospholipids. The regulation of the enzymatic activity of membrane associated 15- LO-1 is poorly understood. In paper II we demonstrate that calcium ionophore stimulates the translocation of 15-LO-1 to the plasma membrane in human dendritic cells. Furthermore, as shown in a protein-lipid overlay assay, 15-LO-1 was shown to interact with several phosphoinositides. In the presence of calcium, addition of phosphatidylinositol-4.5-bisphosphate (PI(4.5)P2) or PI(3.4)P2 to the vesicles containing arachidonic acid led to a significantly increased formation of 15- hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) compared to vesicles without phosphoinositides. Kinetic studies further revealed that vesicles containing PI(4.5)P2) or PI(3.4)P2 lowered the Km value (substrate concentration required for maximum enzymatic velocity) for 15-LO-1, whereas Vmax (the maximum enzymatic velocity) was unaffected. These results suggest that 15-LO-1 activity also might be regulated by the phospholipid constitution of membranes.