Studies on arachidonic acid metabolism in normal and malignant hematopoietic cells

Sammanfattning: This thesis deals with the metabolism of arachidonic acid via the 5-lipoxygenase- and the 15- lipoxygenase-1 pathways in normal and malignant hematopoietic cells. The first part of the thesis (papers I & II) describes the expression of genes involved in arachidonic acid metabolism and the capacity to generate leukotriene (LT) B4 via the 5-lipoxygenase pathway in blood tumor cells from patients with precursor B-ALL (acute lymphoblastic leukemia) and AML (acute myeloid leukemia). In addition, peripheral blood CD34+ pluripotent stem cells from three patients with non-myeloid malignancy were analyzed. Based on immunophenotyping, both precursor BALL clones and AML clones were shown to represent clones at various stages of maturation. In total, eight patients with precursor B-ALL, sixteen patients with AML and CD34+ cells from three patients with non-myeloid malignancies were included. RT-PCR analysis of the precursor B-ALL clones demonstrated that four of the investigated clones (the three most immature clones in addition to one clone with a more mature phenotype) expressed the gene coding for cytosolic phospholipase A2 (cPLA2), but not the gene coding for 5-lipoxygenase. In contrast, the remaining four clones expressed 5-lipoxygenase but not cPLA2. The capacity of the precursor B-ALL clones to express the 5-lipoxygenase protein and to produce LTB4 upon stimulation, correlated with the expression of the 5-lipoxygenase mRNA. The CD34+ stem cells expressed the cPLA2 protein, but not the 5-lipoxygenase protein. The enzyme cPLA2 was also abundantly expressed in all sixteen AML clones and the activity of cPLA2 was high in certain clones. On the contrary, 5-lipoxygenase, although expressed in all sixteen AML clones, seemed to possess low activity in general, since only the more mature clones had the ability to produce LTB4 upon cell stimulation. Taken together, these results indicate that the capacity to produce leukotrienes via the 5-lipoxygenase pathway is under development during early hematopoiesis, and the capacity to generate leukotrienes is gained upon maturation. The second part of the thesis (papers III, IV & V) deals with metabolism of arachidonic acid via the 15-lipoxygenase-1 pathway in human eosinophils, mast cells, the Hodgkin lymphoma and nasal polyps. Human eosinophils contain abundant amounts of 15-lipoxygenase-1. Also the Hodgkin lymphoma cell line L1236 was found to possess large amounts of this enzyme. Incubation of L1236 cells or eosinophils, isolated from human whole blood, with arachidonic acid led to formation of a product with an UV absorbance maximum at 282 nm and shorter retention time than LTC4 in reversephase HPLC. Analysis with positive-ion electrospray tandem MS identified the metabolite as 14,15- LTC4. This metabolite could be metabolized into 14,15-LTD4 and 14,15-LTE4. To avoid confusion with 5-lipoxygenase derived LTs and to emphasize the original finding of these metabolites in eosinophils, we suggested that the 14,15-LTs should be denoted eoxins (EXs), and hence 14,15-LTC4, 14,15-LTD4 and 14,15-LTE4 are now referred to as EXC4, EXD4 and EXE4, respectively. Cord-blood derived mast cells and surgically removed nasal polyps from allergic subjects were also found to produce EXC4. The EXC4 synthase activity was characterized in eosinophils and in L1236 cells. The enzyme LTC4 synthase catalyzed the conversion of EXA4 to EXC4 in eosinophils whereas the soluble glutathione transferase GSTM1b-1b catalyzed the corresponding reaction in L1236 cells. We also provided data showing that the EXs act as proinflammatory agents with profound effects on vascular permeability leading to plasma leakage, a hallmark of inflammation, in an in vitro model using confluent human endothelial cells. In addition, following receptor-mediated activation by LTC4, PGD2 or IL-5, eosinophils were shown to produce EXC4, and this finding supports a physiologically relevant role for EXs.