Identification of arachidonic acid metabolites formed through the 15-lipoxygenase-1 pathway

Abstract: The polyunsaturated fatty acid arachidonic acid is the precursor of many biologically active lipid mediators. This thesis is focused on the arachidonic acid metabolites formed through the 15-lipoxygenase-1 (15-LO-1) pathway. The formation and the biological effects of mediators formed through cyclooxygenases and 5-lipoxygenase pathways are well characterized. The 15-LO-1 is less studied but several lines of evidence suggest a role for 15-LO-1 in asthma and other inflammatory diseases. In this thesis, eosinophils, mast cells, airway epithelial cells and the Hodgkin L1236 cell line were found to express 15-LO-1. In mast cells and airway epithelial cells, IL-4 stimulation increased the expression of 15-LO-1. Stimulation of eosinophils with pro-inflammatory agents and osmotic activation of mast cells by mannitol resulted in activation of the 15-LO-1 pathway. Bacterial infection as well as mechanical injury of the epithelial cells in the respiratory tract are well-known triggers of asthma attacks. These stimuli also increased the formation of 15-HETE in primary airway epithelial cells. Increased amounts of 15-LO-1 were found in bronchial biopsies from asthmatic patients compared to healthy volunteers. 15-LO-1 was also positively stained in Hodgkin lymphoma biopsies localized in the Hodgkin- Reed Sternberg cells, indicating a therapeutic or diagnostic relevance for 15-LO-1 in Hodgkin lymphoma. Analysis with LC-MS/MS identified the novel metabolite 14,15-LTC4. This metabolite was given the name eoxin C4 (EXC4) since eosinophils were a rich source of this metabolite. In addition, mast cells, airway epithelial cells, nasal polyps and L1236 cells could also produce EXC4. This metabolite was converted to EXD4 and EXE4 in eosinophils and L1236 cells. The cysteinyl-eoxins were shown to be 100 times more potent than histamine and almost as potent as cysteinyl-leukotrienes to increase the transendothelial permeability. Increased permeability and vascular leakage is a hallmark of inflammation, which is an important feature of asthma. Both LTC4 synthase and certain soluble glutathione S-transferases were found to catalyze the conjugation of glutathione with EXA4 leading to the formation of EXC4. The animal ortholog of 15-LO-1, the 12/15-LO, was also found to generate EXC4, indicating that studies on animals can be predictive of the function of 15-LO-1 in human. Besides the cysteinyl-eoxins, the Hodgkin L1236 cell line also converted arachidonic acid into the 14,15- hepoxilin (Hx) A3 11(S) and 14,15-HxB3 13(R) as well as 14,15-HxA3-C and 14,15-HxA3-D. The 14,15-HxA3 11(S) and 14,15-HxB3 13(R) were also identified in eosinophils, dendritic cells and nasal polyps. The Hodgkin lymphoma tumor only consists of a minority of malignant cells and the main part is inflammatory cells, such as eosinophils and mast cells. A potential role of 15-LO-1 could be to facilitate the inflammatory features of this disease. In essence, this thesis demonstrates that the 15-LO-1 pathway can convert arachidonic acid to many different metabolites with potential pro-inflammatory effects.