HIGH-ACTIVITY SUPPRESSION OF MYELOID PROGENITOR PROLIFERATION BY CHIMERIC MUTANTS OF INTERLEUKIN-8 AND PLATELET FACTOR-4

The proliferation of human myeloid progenitor cells is negatively regu lated in the presence of certain members of the chemokine family of mo lecules. This includes interleukin 8 (IL 8) and platelet factor 4 (PF4 ), which in combination are able to synergize, resulting in cell suppr ession at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in. vitro colony formatio n assay for myeloid progenitor cells to assess domains of these protei ns that are required for activity. Mutation of either of the two DLQ m otifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were in capable of suppressing colony formation, A class of chimeric mutants c onsisting of domains of either PF4 and IL-8, Gro-alpha and PF4, or Gro -beta and PF4 were observed to inhibit myeloid cell proliferation at c oncentrations which were between 500 and 5000-fold lower than either t he IL 8 or PF4 wild-type proteins alone. These chimeric mutants posses sed activities that were comparable to or better than the activity obs erved when IL-8 and PF4 were added together in vitro. One of these hig hly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferatio n but also the cell cycling of myeloid progenitor cells following intr avenous injection of the mutant into mice. Examination of additional I L-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal ''ELR'' motif, resulted in the obs ervation that the highly active IL-8 mutant required both aspartic aci d at amino acid residue 4 and either glutamine or asparagine at residu e 6, Single mutations at either of these positions resulted in mutants with myelosuppressive activity equivalent to wild-type IL-8. Mutants such as IL-8M1 and IL-8M10 were observed to be significantly reduced i n their ability to activate isolated human neutrophils, suggesting tha t separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines.