SPECIFIC HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ANTAGONISTS

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a p leiotropic hemopoietic growth factor and activator of mature myeloid c ell function. We have previously shown that residue 21 in the first he lix of GM-CSF plays a critical role in both biological activity and hi gh-affinity receptor binding. We have now generated analogues of GM-CS F mutated at residue 21, expressed them in Escherichia coli, and exami ned them for binding, agonistic, and antagonistic activities. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K boun d to the GM-CSF receptor cy chain with a similar affinity to wild type GM-CSF and had lost high-affinity binding to the GM-CSF receptor cy-c hain-common P-chain complex. From these mutants, only the charge rever sal mutants E21R and E21K were completely devoid of agonistic activity . Significantly we found that E21R and E21K antagonized the proliferat ive effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as web as GM-CSF-mediated stimulation of neu trophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferati on or tumor necrosis Factor cy-mediated stimulation of neutrophil supe roxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-ty pe GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical signi ficance for the management of inflammatory diseases and certain leukem ias where GM-CSF plays a pathogenic role.