Modulation of lymphocyte migration by human lymphokines. II. Purification of a lymphotactic factor (LCF).

Abstract

Delayed-type hypersensitivity reactions are characterized by mononuclear cell infiltration consisting of macrophages and lymphocytes, the majority of which are not specifically sensitized for the antigen eliciting the reaction. Although chemotactic and migration inhibitory factors for macrophages have been extensively characterized, little is known about similar human chemoattractants generated by lymphocytes for lymphocytes. A companion paper describes some of the biologic characteristics of a human mitogen-induced lymphokine that is specifically chemokinetic for nonadherent lymphocytes over other cell types. Further chemical characterization of lymphotactic activity derived from crude Con A-stimulated peripheral blood lymphocyte supernatants by Sephadex G-100 gel filtration chromatography revealed a peak that eluted in a region corresponding to a m.w. of 56,200. This activity was cationic, eluting from a QAE Sephadex anion-exchange column at 4 to 6 mS. Subsequent isoelectric focusing in sucrose revealed an isoelectric point of 9.0–9.1. Activity after sequential Sephadex G-100 and QAE chromatography and isoelectric focusing was subjected to electrophoresis in sodium dodecyl sulfate Polyacrylamide gels (SDS-PAGE). A single band of stainable protein was evident in both nonreduced and reduced gels, which corresponded to a m.w. of 14,000, and to a region from which lymphotactic activity could be detected in eluates from slices of unstained gels run in parallel. Subsequent Sephadex G-50 gel filtration chromatography demonstrated a m.w. of 14,400 of the lymphotactic activity after interaction with SDS. It is postulated that in vitro stimulation of peripheral blood lymphocytes by Con A results in the secretion of a cationic lymphocyte chemokinetic activity that is present in aggregated tetrametric form. Interaction with SDS is sufficient to reduce the factor to a monomeric form of 14,000 without significant loss of activity. The simultaneous generation of lymphocyte migration inhibitory activity (approximately 25,000 daltons) in size could provide a mechanism for immobilization of nonsensitized lymphocytes once attracted to sites of delayed-type hypersensitivity reactions.