Partial purification of a bone-resorbing factor elaborated from human allogeneic cultures

Abstract

Human mixed leukocyte culture supernatants contain a factor(s) capable of effecting resorption of fetal rat bones in vitro. We have investigated procedures for the large-scale generation and partial purification of this bone-resorbing factor(s). Human mononuclear leukocytes obtained by leukapheresis from two healthy donors (average of 1 × 10 10 cells/donor) were cultured together (2.5 × 10 6 cells/ml) for 40 hr, the supernatants harvested and assayed for bone-resorbing activity by measurement of 45Ca release from fetal rat bones in vitro. These supernatants consistently induced significant bone resorption when compared to supernatants from a single donor's cells. Neither the release of this bone-resorbing factor nor its biologic activity were abrogated by addition of indomethacin. The bone-resorbing factor(s) eluted from Sephadex G-75 at an apparent molecular weight of 11,000 to 18,000 and appeared only in the breakthrough when fractionated on DEAE-cellulose (0.005 M phosphate, 0.02 M NaCl, pH 7.5). A concentrated eluate containing bone-resorbing activity after two cycle Sephadex G-75 gel filtration and DEAE-cellulose chromatography was applied to anionic polyacrylamide disc gels. A single peak of bone-resorbing activity was eluted, although this activity could not be attributed to a single stainable protein band. The results indicate that human mixed leukocyte cultures elaborate a low-molecular-weight boneresorbing factor(s) resembling the previously-described osteoclast-activating factor. Methods presented for the large-scale generation and partial purification of this activity should facilitate clarification of the role of this mediator in tissue destruction.