REACTIVE OXYGEN SPECIES AND NOT LIPOXYGENASE PRODUCTS ARE REQUIRED FOR MOUSE B-LYMPHOCYTE ACTIVATION AND DIFFERENTIATION

A potential role for lipoxygenase (LO) products and reactive oxygen sp ecies (ROS) in mouse B-lymphocyte activation and differentiation was i nvestigated. Previously published investigations with the nonspecific 5-LO (EC 1.13.11.34) and 12-LO (EC 1.13.11.31) inhibitors such as nord ihydroguaiaretic acid (NDGA) and 6,7-dihydroxycoumarin (Esculetin), ar e misleading in that they suggest lymphocyte LO activity is required f or activation and differentiation of these cells. In initial support o f this concept, we report that NDGA and Esculetin completely inhibited B-lymphocyte activation mediated by either membrane immunoglobulin (m ig), or the lipopolysaccharide (LPS) receptor. NDGA and Esculetin comp letely inhibited cell enlargement and proliferation, exhibiting half m aximal inhibitory concentrations (IC50S) Of approximately 1 x 10(-6) M . In contrast, the highly specific 5-LO inhibitors BAY X 1005, MK-886 and Wy 50,295 did not inhibit cell enlargement or proliferation. Moreo ver, 5,8,11-eicosatriynoic acid (ETI) which inhibits 5- and 12-LO, and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) which inhibits all known LO s did not affect B-lymphocyte proliferation. Interestingly, NDGA and E sculetin are antioxidants, unlike BAY X 1005, MK-886, Wy 50,295, ETI a nd ETYA. Our hypothesis was that the antioxidant activities of NDGA an d Esculetin were responsible for inhibiting B-lymphocyte activation an d proliferation and we speculated that ROS and not LO activity was req uired for both processes. Additional antioxidants such as butylated hy droxy toluene, o-phenanthroline, thiourea, and alpha-tocopherol (vitam in E), also inhibited B-lymphocyte proliferation induced by either the LPS or mig receptors. These agents exhibited IC50S of 1 x 10(-8) M, 5 x 10(-10) M, 6 x 10(-3) M and 5 x 10(-5) M, respectively. When restin g B-lymphocytes were treated with a source of ROS (1 x 10(-5) M H2O2), cells enlarged in a temperature-sensitive manner, which is similar to LPS-induced enlargement. Both NDGA and Esculetin completely inhibited H2O2-induced enlargement. These results further indicate that ROS are required for B-lymphocyte activation and proliferation. Similar resul ts were obtained for B-lymphocyte differentiation. NDGA and Esculetin completley inhibited the development of plasma cells and displayed IC5 0S of 5 x 10(-6) M. Conversely, BAY X 1005, MK-886, Wy 50,295, ETI, an d ETYA did not block the formation of plasma cells. Therefore, ROS are also crucial for differentiation into plasma cells. These experiments are the first to directly illustrate that intracellular ROS mediate B -lymphocyte activation, proliferation and differentiation and that LO products are not required for these processes. Moreover, this investig ation illustrates that NDGA and Esculetin are not specific LO inhibito rs and that their use cannot implicate a role for LO activity.