HUMAN INTERFERON-INDUCIBLE PROTEIN-10 - EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN DEMONSTRATE INHIBITION OF EARLY HUMAN HEMATOPOIETIC PROGENITORS

Human interferon-inducible protein 10 (IP-10), a member of the family of the small secreted proteins called intercrine cytokines or chemokin es, is secreted by interferon gamma-stimulated T cells,. monocytes, en dothelial cells, and keratinocytes. We have begun to explore the biolo gical properties of IP-10 by cloning and overexpression in baculovirus and in bacterial protein expression systems. A 9.9-kD protein was sec reted by infected insect cells, which on sodium dodecyl sulfate-polyac rilamide gel electrophoresis comigrated with keratinocyte IP-10 and wi th f(22-98), a bacterial recombinant fragment lacking the signal seque nce but containing all other residues of IP-10. All three reacted with antibodies recognizing residues 10-98 (alphaIP-10) and 77-98 of IP-10 (alpha22), demonstrating that it is secreted by keratinocytes and ins ect cells after removal of the signal sequence but without proteolysis of the COOH-terminal end. Purified rIP-10 suppresses in vitro colony formation by early human bone marrow progenitor cells which need r-ste el factor (rSLF) and rGM-CSF or rSLF and r-erythropoeitin (rEPO). The inhibition is dose dependent, is complete at concentrations greater-th an-or-equal-to 50 ng/ml, is prevented by preincubation of rIP-10 with alphaIP-10, but not by alpha22, and is seen with highly purified CD34 cells, suggesting direct effect of rIP-10 on the progenitors. Combina tion of rIP-10 and other chemokines at inactive concentrations inhibit ed colony formation in a synergistic manner. rIP-10 did not affect col ony formation in the absence of any growth factors or in the presence of rEPO or rGM-CSF but in absence of rSLF. The effects of IP-10 may be relevant to normal marrow function and might be harnessed to protect human hematopoietic progenitors from the cytotoxic effects of chemothe rapy.