Characterization of hydrogen peroxide-potentiating factor, a lymphokine that increases the capacity of human monocytes and monocyte-like cell lines to produce hydrogen peroxide.

Abstract

A study was made of a lymphokine produced by human T lymphocytes that mediates activation of human monocytes and monocyte-like cell lines, measured by increased production of H2O2. The lymphokine was produced either by stimulation of human nonadherent peripheral blood mononuclear cells with concanavalin A (Con A) or by stimulation of a human T cell line, HSB2, with Con A and phorbol myristic acetate (PMA). When incubated with freshly isolated peripheral blood monocytes for 48 to 72 hr, the H2O2-potentiating factor (HPPF) stimulated increased production of H2O2, measured in a PMA-triggered assay for H2O2 secretion. Because variations occurred in the response of normal blood donors to the HPPF, human monocyte-like cell lines were used as homogeneous and consistently responsive targets for the lymphokine to facilitate biochemical characterization studies of the factor. Two cell lines were studied: HL60, a human promyelocytic cell line, and U937, a human histiocytic cell line. When target cells of either type were incubated in the presence of the HPPF for 48 to 72 hr, they produced increased amounts of H2O2 in a dose-dependent fashion. H2O2 levels were assessed by means of a microassay that measures peroxide-mediated oxidation of phenol red after an oxidative burst triggered with PMA. By using this assay, HPPF was found to have an apparent m.w. of 54,000 and an isoelectric point of 5.5. The bouyant density was determined to be 1.307, indicating that HPPF is a protein. The utilization of cell lines for both the production and assay of HPPF should facilitate the purification of this lymphokine and the subsequent evaluation of its relationship to other lymphokines known to affect macrophage microbicidal and tumoricidal function.