Biosynthesis and biological role of leukotrienes in B lymphocytes

Abstract

Leukotrienes (LT) are potent lipid mediators, synthesized from arachidonic acid (AA) upon cell activation. The essential enzyme for leukotriene biosynthesis is 5-lipoxygenase (5-LO), converting AA, through a two step process to LTA4. The activity of 5-LO is dependent on 5-lipoxygenase activating protein (FLAP). Two other enzymes can further metabolize LTA4. Leukotriene A4 hydrolase converts LTA4 to LTB4, a potent pro-inflammatory and chemotactic compound. Leukotriene B4 exerts its biological action through two receptors known as BLT1 and BLT2. The second enzyme able to convert LTA4 is LTC4 synthase, which converts LTA4 to LTC4.

Leukotriene C4 is the first metabolite in the group of cysteinyl containing Us (cys-LT), where LTD4 and LTE4 are the other two. Cysteinyl Us; are generally believed to be involved in asthmatic and allergic diseases and exert their biological actions through three receptors, CysLT1, CysLT2 and the newly GPR1 7. The BLT1 expression, the activity of 5-LO and the inhibitory action of leukotriene biosynthesis inhibitors were investigated in chronic B lymphocytic leukemia cells (B-CLL). Chronic B lymphocytic leukemia cells produce LTB4 in Similar amounts as neutrophils when stimulated with azodicarboxylic acid his(dimethylamide) (diamide), AA and calcium ionophore (A23187). The mechanism of diamide is not known, but it is believed to change the cellular redox, status of the cell. FACS analysis revealed the expression of BLT1 in all B-CLL clones investigated.

Cultivation of B-CLL cells with CD40-ligand (CD40L) expressing cells for 96 hours induced DNA synthesis and antigen expression of CD23, CD54 and CD150. MK-886 (a specific FLAP inhibitor) and BWA4C (a specific 5-LO inhibitor) markedly reduced the DNA synthesis and the expression of antigens. Exogenously LTB4 reversed the effects of inhibitors. These results demonstrate the intrinsic and exogenous role of LTB4 in B-CLL cells.

To better understand the expression of enzymes and receptors of the leukotriene biosynthesis cascade in B lymphocyte biology, subsets of B cells were purified and analyzed. Immunohistochemical analysis of tonsils revealed a high expression of 5-LO in mantle zone B cells. Western blot analysis was in agreement with the immunohistochemical findings. In addition, the western blot result also indicated that memory B cells expressed similar amounts of 5-LO as mantle zone B cells. RT-PCR demonstrated the expression of 5-LO, FLAP and LTA4 hydrolase in memory B cells and mantle zone B cells. Mantle zone B cells and memory B cells also expressed the CysLT1 transcript. The transcripts of LTC4 synthase, CysLT2 and BLT2 were not detected and a very low amount of BLT1 was observed. Mantle cell lymphoma (MCL) cell lines and cyclin D1 positive B cell prolymphocytic leukemia (B-PLL) (which is considered to be leukemic form of MCL) were all able to produce LTB4 Upon stimulation with diamide, AA and A23187. Cysteinyl leukotrienes induced a robust calcium response which was in agreement with the PCR data. The calcium response could be abrogated with a selective CysLT1 antagonist. Immunohistochemical staining of MCL tumor biopsies stained positive for 5-LO.

In conclusion, the present thesis demonstrates the potential involvement of leukotrienes in B cell malignancies and that 5-LO in normal B cells is primarily expressed in mantle zone and memory B cells.