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Partial characterization of lymphokines that change the surface charge density of human polymorphonuclear leukocytes
Abstract:
We have investigated the effects of stimulated human lymphocyte supernatants on the surface charge density of human polymorphonuclear leukocytes using analytical free-flow electrophoresis. Unfractionated ConA-induced human lymphocyte supernatants were found to decrease the electrophoretic mobility of PMN leukocytes by 10-16%. Optimal conditions for the production of this lymphokine as well as for the PMN leukocyte-lymphokine interaction are described. In order to obtain more information about the factor responsible for the effect, active and control supernatants were fractionated by Sephacryl S-200 column chromatography, Sephadex G-50, and Amicon membrane filtration. The fractions obtained were dialysed, concentrated, and tested for activity on PMN leukocytes in free-flow electrophoresis. Fractions obtained in parallel were tested for Leukocyte Inhibition Factor (LIF) activity using the Clausen assay. Decrease in electrophoretic mobility of human PMN leukocytes was found to be due to two distinct lymphokines with molecular weights of about 10,000-20,000 and 50,000-60,000 d, respectively. These fractions showed no activity in the Clausen assay. Fraction V (60,000-80,000 d) revealed strong activity in the Clausen assay, but had no effect on the surface charge density of human PMN leukocytes. The ConA-induced lymphokines that changed the surface charge density of PMN leukocytes retained their activity after heating to 56 degrees C for 30 min. They were not synthesized in the presence of puromycin, but mitomycin C treatment of the lymphocytes had no effect on the production of the lymphokines. Moreover, pretreatment of human PMN leukocytes with puromycin for 30 min blocked the reaction, indicating an active role of the PMN leukocytes following the reaction with the lymphokines. Finally these lymphokines preferentially influenced the surface charge density of human PMN leukocytes but not that of rat or guinea-pig peritoneal exudate cells.