Studies on the activation of cytosolic phospholipase A2 and 15-lipoxygenase-1 in hematopoietic cells

Sammanfattning: Arachidonic acid is a polyunsaturated fatty acid that has four double bonds and belongs to the omega-6 family of fatty acids. Although it can be formed in humans from one essential fatty acid (linoleic acid), most of the arachidonic acid in the body comes with the intake of food. In mammalian cells, arachidonic acid is found esterified in cellular membranes. Phospholipases release arachidonic acid upon cellular stimulation. There are several different enzyme families of mammalian lipases, but only cytosolic phospholipase A2 (cPLA2-alpha) has been shown to preferentially release arachidonic acid. Depending on the cell and stimuli, arachidonic acid can be further metabolized into biologically active fatty acids, including prostaglandins, thromboxan A2, leukotrienes and eoxins. The first part of this thesis investigates the arachidonic acid release induced by cPLA2-alpha in human platelets after stimulation with polychlorinated biphenyls (PCBs). The release of arachidonic acid by PCBs was shown to be cPLA2-alpha dependent, since it was completely blocked by the cPLA2-alpha inhibitors AACOCF3 or pyrrolidine-1. Two anti-estrogens, nafoxidin and tamoxifen - but not 17?-estradiol - inhibited PCB-induced arachidonic acid release. Platelets incubated with PCBs did not aggregate, even though a robust release of arachidonic acid was observed. A unique feature of 15-LO-1 is that it translocates to internal cellular membranes and oxygenates free fatty acids, as well as fatty acids esterified into lipids. 15-LO-1 is expressed in lung epithelial cells, eosinophils, reticulocytes, mast cells and interleukin- 4 (IL-4) stimulated monocytes and dendritic cells. In the second part, the 15-lipoxygenase type 1 (15-LO-1) was investigated in different hematopoietic cells. The enzyme translocated to the plasma membrane in IL-4 stimulated human dendritic cells upon calcium stimulation. In addition, 15-LO-1 bound to certain phospholipids, particularly phosphatidylinositols in a lipid-overlay assay. A vesicle assay model was set up, and kinetic assays were performed. The Vmax was shown to be unchanged, but the apparent Km of 15-LO-1 towards arachidonic acid was significantly lower in the presence of PI(4.5)P2 or PI(3.4)P2 in the vesicles. The expression of 15-LO-1 was investigated in biopsies from Hodgkin lymphoma (HL) tumors. 15-LO-1 was found in Hodgkin Reed-Sternberg cells, which constitutes only 1-2% of all tumor cells and is believed to orchestrate the infiltration of eosinophils, mast cells, neutrophils and T-cells into the HL tumor. The HL cell lineL1236 also expressed 15-LO-1 constitutively and produced the inflammatory eoxins. In addition, the IL-13 induction of 15-LO-1 was investigated in the non-Hodgkinlymphoma cell line Karpas-1106P, which is originating from a primary mediastinal Bcell lymphoma (PMBCL). Upon IL-13 stimulation, this cell line acquired several proinflammatory features that made the PMBCL cells more like the HL cell line L1236, demonstrating the biological similarities between the two diseases. In summary, the results presented in this thesis demonstrate that 15-LO-1 and cPLA2-alpha have important functions in the arachidonic acid cascade. The discovery of 15-LO-1 in the HL disease and the human cell lines supports the pro-inflammatory role of the enzyme and establishes a cellular model system that can help to further elucidate the biological role of human 15-LO-1.