Purification of a lymphoid cell line product with leukocyte inhibitory factor activity.

Abstract

Leukocyte inhibitory factor (LIF), a lymphokine that inhibits the random and directed migration of polymorphonuclear (PMN) leukocytes, was purified from a human non-T, non-B leukemia cell line (Reh). From 10 l of serum-free supernatant, 1.3 .mu.g of protein with LIF activity was obtained by the sequential use of affinity chromatography with concanavalin A-Sepharose, hydrophobic chromatography with hexylagarose and gel filtration chromatography. The specific activity of LIF recovered represented an 80,000-fold purification over that of the initial crude serum-free supernatants and the preparation at that point was .apprx. 80-90% pure. To both assess the purity of the preparation and provide a further purification step, Reh LIF activity recovered by the above procedures was subjected to isoelectric focusing. One major stainable protein band was identified; its isoelectric point was pH 5.4-5.5. Gels run in parallel for recovery of biologic activity revealed only 1 region (pH 5.4-5.5) with ability to inhibit PMN leukocyte migration. Iodination of Reh LIF resulted in a loss of biologic activity, but isoelectric focusing of this material revealed 1 major 125I-labeled band (pH 5.1) and several minor bands. The coincidence of biologic LIF activity with 1 stainable protein band as identified by isoelectric focusing implies that the final product may be homogeneous.