Conditioned media (CM) from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix contains a factor that induces macrophage-like maturation of HL-60 cells. This factor was purified from the CM of HL-60 cells grown on bone marrow stroma by ammonium sulfate precipitation, then sequential chromatography on DEAE, affi-gel blue affinity, gel exclusion, and wheat germ affinity columns, followed by C-4 reverse phase HPLC, and SDS-PAGE. The maturation promoting activity of the CM was identified in a single 31 kD protein. Amino acid sequence analysis of four internal tryptic peptides of this protein confirmed significant homology with amino acid residues 48-60, 138-147, 215-220, and 221236 of human cytoskeletal α-actinin. An immunoaffinity purified rabbit polyclonal anti- chicken α-actinin inhibited the activity of HL-60 conditioned media. A 27 kD amino-terminal fragment of α-actinin produced by thermolysin digestion of chicken gizzard α-actinin, but not intact α-actinin, had maturation promoting activity on several cell types, including blood monocytes, as measured by lysozyme secretion and tartrateresistant acid phosphatase staining. We conclude that an extracellular α-actinin fragment can promote monocyte/macrophage maturation. This represents the first example of a fragment of a cytoskeletal component, which may be released during tissue remodeling and repair, playing a role in phagocyte maturation.
Bibliographical noteFunding Information:
The authors would like to thank Dr. Kenneth Williams and Kathy Stone of the W.M. Keck Foundation Biotechnology Resource Laboratory for peptide sequencing. This work was supported by the Department of Veterans Affairs.