Reactive oxygen species released from granulocytes stimulate 5-lipoxygenase activity in a B-lymphocytic cell line.

B-lymphocytes express 5-lipoxygenase (5-LO) protein but cellular leukotriene production is suppressed by selenium-dependent peroxidases. Thus it was of interest to check whether reactive oxygen species (ROS) which are released under inflammatory conditions can stimulate B-lymphocyte 5-LO and counteract peroxidase-mediated suppression of cellular 5-LO activity. It was found that 5-LO in the Epstein-Barr virus-transformed B-lymphocytic cell line BL41-E95-A is activated by addition of hydrogen peroxide or xanthine/xanthine oxidase and after increasing the oxidative state of the cell by azodicarboxylic acid bis(dimethylamide). Generation of endogenous ROS from mitochondria by antimycin A also lead to a threefold upregulation of 5-LO activity in B-cells. There was almost no detectable endogenous superoxide formation in BL41-E95-A cells after stimulation with 4beta-phorbol 12-myristate 13-acetate. Co-incubation experiments with BL41-E95-A cells and granulocytes demonstrated that granulocyte-derived ROS can activate B-lymphocyte 5-LO. Addition of superoxide dismutase and/or catalase to the B-lymphocyte/granulocyte co-incubations and to B-lymphocyte homogenates revealed that the 5-LO activation is due to the superoxide-derived release of hydroperoxides or hydrogen peroxide from granulocytes. The data suggest that ROS formation plays an important role in the regulation of cellular 5-LO activity in B-lymphocytes. As leukotrienes affect B-cell functions like cell proliferation, activation and maturation, this finding provides a new link between the formation of ROS and the regulation of immune responses.

Nonredox 5-lipoxygenase inhibitors require glutathione peroxidase for efficient inhibition of 5-lipoxygenase activity.

Nonredox type 5-lipoxygenase (5-LO) inhibitors, such as ZM 230487, its methyl analogue ZD 2138, or the Merck compound L-739,010, suppress cellular leukotriene synthesis of ionophore stimulated granulocytes with IC50 values of about 50 nM. However, in cell homogenates or in preparations of purified enzyme, up to 150-fold higher concentrations are required for similar inhibition of 5-LO activity. This loss of 5-LO inhibition in cell homogenates was reversed by addition of glutathione or dithiothreitol, which increased the inhibitory potency of ZM 230487 or L-739,010 by about 100 to 150-fold so that 5-LO inhibition was comparable with that of intact cells. In the presence of thiols, addition of hydroperoxide [13(S)-HpODE], glutathione-peroxidase inhibition by iodacetate or selenium-deficiency lead to impaired 5-LO inhibition by ZM 230487 in cell homogenates. Moreover, addition of glutathione peroxidase was required for efficient inhibition of purified human 5-LO by ZM 230487. The data suggest that low hydroperoxide concentrations are important for efficient 5-LO inhibition by ZM 230487. The kinetic analysis revealed a noncompetitive inhibition of 5-LO by ZM 230487 at low hydroperoxide levels, whereas it acted as a competitive inhibitor with low affinity under nonreducing conditions in granulocyte homogenates. No such redox-dependent effects were observed with the 5-LO inhibitor BWA4C, the 5-LO activating protein-inhibitor MK-886 or the pentacyclic triterpene acetyl-11-keto-beta-boswellic acid. These data suggest that physiological conditions associated with oxidative stress and increased peroxide levels lead to impaired efficacy of nonredox type 5-LO inhibitors like ZM 230487 or L-739,010. This could explain the reported lack of activity of this class of 5-LO inhibitors in chronic inflammatory processes.