Biosynthesis and biological role of leukotrienes in human lymphocytes

Abstract: Leukotrienes (LT) are biologically active metabolites of arachidonic acid. The key enzyme in leukotriene biosynthesis is 5-lipoxygenase (5-LO). Leukotriene B4 exerts its biological effect mainly via BLT1, the high affinity receptor for LTB4. Leukotriene B4 is a potent proinflammatory and chemotactic mediator. Myeloid cells are the main LT producing cells in humans and the role of LTB4 in these immune cells is well established. In contrast, the role of LTs in lymphocytes is not fully understood. B lymphocytes express 5-LO and BLT1, and activated T cells express BLT1. In this thesis the biosynthesis and biological function of LTs in human lymphocytes were studied. The role of 5-LO and LTB4 in chronic B lymphocytic leukemia cells (B-CLL) was investigated. Treatment of B-CLL cells with CD40-ligand resulted in cellular activation as measured by increased expression of cell surface markers (CD23, CD54, CD150) and thymidine incorporation. This activation could be inhibited by LT biosynthesis inhibitors. Addition of exogenous LTB4 counteracted the inhibitory action of the inhibitors on B-CLL cells. To elucidate the expression of 5-LO in various subtypes of normal B cells, B cells from tonsils were isolated. Using Western blot, immunohistochemistry and RT-PCR, it was shown that mantle zone B cells and memory B cells expressed high amounts of 5-LO. In contrast, germinal center B cells and plasma cells contained low or undetectable amounts of 5-LO, respectively. Further studies in tumor biopsies from mantle cell lymphoma (MCL) patients demonstrated high expression of 5-LO. MCL cell lines also expressed 5-LO. Studies on the chemical properties of 5-LO protein in MCL cell lines (Granta519, JEKO1, Rec1) showed that the enzyme was phosphorylated on serine 523. In contrast, native 5-LO in neutrophils was not phosphorylated on serine 523. Phosphorylated 5-LO was purified from Rec1 cells using an ATP-agarose column, and the purified enzyme could be dephosphorylated with alkaline phosphatase. The MCL cell lines constitutivelyexpressed phosphorylated 5-LO and this phosphorylation could be induced by activation of protein kinase A. Western blot analysis of biopsies and peripheral blood from patients suffering from MCL or B-CLL demonstrated that these cells also expressed pSer523-5- LO. We have studied the role of LTB4 in cellular immune responses. Due to the lack of EBV specific cellular memory, cord blood mononuclear cells (CBMC) are well suited tostudy immunological interactions during primary EBV infections. Polysaccharide K (PSK) and thioredoxin 80 (Trx80) are immunostimulating compounds and have been demonstrated to activate T and NK cells which inhibit the proliferation of EBV infected B cells. We have found that LTB4 activated T cells which in turn inhibited the proliferation of EBV infected B cells. We have also shown that LTB4 alone is as good as PSK and Trx80 to inhibit proliferation of EBV infected B cells. In conclusion, these studies demonstrate the expression of 5-LO in various subtypes of B cells and for the first time we demonstrate a chemical difference between 5-LO in B cells (express pSer523-5-LO) and neutrophils. Furthermore, the importance of LTB4 in activation of B-CLL cells and in cellular immune responses to EBV infections is also shown.