Assessment of leukotriene B4 synthesis in human lymphocytes by using high performance liquid chromatography and radioimmunoassay methods.

Leukotrienes (LT), mainly LTB4, have been shown recently to affect several functions of human lymphocytes in vitro, and they are regarded as putative modulators of the immune response. Although it is recognized that human neutrophils, eosinophils, monocyte-macrophages, and mast cells can generate LTs, the synthesis of 5-lipoxygenase products by lymphocytes is still the subject of a controversy. Human peripheral blood mononuclear leukocytes, nylon wool-purified lymphocytes, CD4+, CD4- T cells, large granular lymphocytes, and various fractions of pure lymphocyte preparations obtained by counter flow centrifugal elutriation were stimulated for 10 min to 24 hr with ionophore A23187, phytohemagglutinin, concanavalin A, or lipopolysaccharide with or without exogenous arachidonic acid (AA); supernatants were analyzed by reverse-phase high performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) methods for the presence of LTB4. Pure human lymphocyte preparations, which were shown to be free of monocytes, did not release any detectable amount of LTB4. Increasing percentage of contaminating monocytes was clearly paralleled by increasing amounts of LTB4. Murine thymocytes, interleukin 2-dependent CTLL2 cytotoxic lymphocytes, EL4 thymoma cells, and human Jurkatt cells were also found to be unable to generate detectable amounts of LTB4 after stimulation with ionophore A23187, phytohemagglutinin, phorbol myristate acetate, recombinant interleukin 1, or interleukin 2 with or without exogenous AA. The addition of increasing numbers of adherence-purified monocytes to Jurkatt cells was followed by increased synthesis of LTB4. In conclusion, the present study indicates that the synthesis of LTB4 by pure human lymphocyte preparations or some human and animal lymphoid cell lines is not detectable by combined HPLC-RIA methods in any of the conditions used.