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 species (ROS) in mouse B-lymphocyte activation and differentiation was investigated. Previously published investigations with the nonspecific 5-LO (EC 1.13.11.34) and 12-LO (EC 1.13.11.31) inhibitors such as nordihydroguaiaretic acid (NDGA) and 6,7-dihydroxycoumarin (Esculetin), are misleading in that they suggest lymphocyte LO activity is required for activation and differentiation of these cells. In initial support of this concept, we report that NDGA and Esculetin completely inhibited B-lymphocyte activation mediated by either membrane immunoglobulin (mlg), or the lipopolysaccharide (LPS) receptor. NDGA and Esculetin completely inhibited cell enlargement and proliferation, exhibiting half maximal inhibitory concentrations (IC₅₀S) of approximately 1 × 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. Moreover, 5,8,11-eicosatriynoic acid (ETI) which inhibits 5- and 12-LO, and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) which inhibits all known LOs did not affect B-lymphocyte proliferation. Interestingly, NDGA and Esculetin are antioxidants, unlike BAY X 1005, MK-886, Wy 50,295, ETI and ETYA. Our hypothesis was that the antioxidant activities of NDGA and Esculetin were responsible for inhibiting B-lymphocyte activation and proliferation and we speculated that ROS and not LO activity was required for both processes. Additional antioxidants such as butylated hydroxy toluene, o-phenanthroline, thiourea, and α-tocopherol (vitamin E), also inhibited B-lymphocyte proliferation induced by either the LPS or mIg receptors. These agents exhibited IC₅₀s of 1 × 10⁻⁸ M, 5 × 10⁻¹⁰ M, 6 × 10⁻³ M and 5 × 10⁻⁵ M, respectively. When resting B-lymphocytes were treated with a source of ROS (1 × 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 results were obtained for B-lymphocyte differentiation. NDGA and Esculetin completely inhibited the development of plasma cells and displayed IC₅₀S of 5 × 10⁻⁶ M. Conversely, BAY X 1005, MK-886, Wy 50,295, ETI, and 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 investigation ilustrates that NDGA and Esculetin are not specific LO inhibitors and that their use cannot implicate a role for LO activity.