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 regulated in the presence of certain members of the chemokine family of molecules. This includes interleukin 8 (IL-8) and platelet factor 4 (PF4), which in combination are able to synergize, resulting in cell suppression at very low concentrations of these molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells to assess domains of these proteins that are required for activity. Mutation of either of the two DLQ motifs within PF4 resulted in an inactive protein. Perturbations within the IL-8 dimer interface region also resulted in mutants that were incapable of suppressing colony formation. A class of chimeric mutants consisting 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 concentrations which were between 500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins alone. These chimeric mutants possessed activities that were comparable to or better than the activity observed when IL-8 and PF4 were added together in vitro. One of these highly active chimeric proteins was observed to be 1000-fold more active than either IL-8 or PF4 alone in suppressing not only the proliferation but also the cell cycling of myeloid progenitor cells following intravenous injection of the mutant into mice. Examination of additional IL-8-based mutants in the colony formation assay, which centered on the perturbation of the amino-terminal "ELR" motif, resulted in the observation that the highly active IL-8 mutant required both aspartic acid at amino acid residue 4 and either glutamine or asparagine at residue 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 in their ability to activate isolated human neutrophils, suggesting that separate mechanisms may exist by which myeloid progenitor cells and neutrophils are affected by chemokines.
Daly
T J
TJ
Repligen Corporation, Cambridge Massachusetts 02139, USA.
LaRosa
G J
GJ
Dolich
S
S
Maione
T E
TE
Cooper
S
S
Broxmeyer
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D018925:Chemokines / Q000235:genetics / Q000494:pharmacology; D006224:Cricetinae; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009154:Mutation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010978:Platelet Factor 4 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018123:Receptors, Interleukin / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship
D015703:Antigens, CD; D018925:Chemokines; D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D010978:Platelet Factor 4