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Separation of functionally distinct human granulocyte-macrophage colony-
stimulating factors
NA Nicola, D Metcalf, GR Johnson and AW Burgess
Human placental conditioned medium (HPCM) contans colony-stimulating
factors (CSFs) required for the growth in vitro of neutrophilic
granulocyte-macrophage (GM) and eosinophilic (EO) progenitor cells from
human bone marrow. Fractionation of CSFs in HPCM was achieved by
manipulation of the elution conditions on a column of phenyl-Sepharose.
After equilibration of the phenyl-Sepharose column at high ionic strength
(1 M ammonium sulfate), all of the CSF bound; one species of GM-CSF (alpha)
and all of the elutable EO-CSF were eluted from the column simply by
reducing the salt concentration, whereas the second species of GM-CSF
(beta) was free of EO-CSF and was eluted only by increasing the
concentration of tehylene glycol in the elution buffer. The two GM-CSFs
were functionally distinct. GM-CSF alpha preferentially stimulated colony
formation by day 14 of culture, and there was a decreased proportion of
neutrophil colonies and increased proportion of macrophage colonies as the
strength of the stimulus was decreased; GM- CSF beta, on the other hand,
preferentially stimulated colony formation by day 7 of culture, and the
proportion of neutrophil colonies was high (average 80%) and independent of
the concentration of GM-CSF beta. GM- CSF alpha and GM-CSF beta were
indistinguishable on the basis of apparent molecular size on tel filtration
columns (molecular weight 30,000), charge properties on isoelectric
focusing beds (isoelectric point, 4.9), and were not related to each other
as a sialoglycoprotein is related to its asialo form. Adherent cell removal
of the target bone marrow cells (to remove colony-stimulating cells)
suggested that both GM-CSFs acted directly rather than by stimulating the
production of GM- CSF. Mixing and titration experiments indicated that the
differences in functional specificities of the two GM-CSFs (and the lack of
EO-CSF associated with GM-CSF beta) were not due to the presence of
specific inhibitory molecules or lower absolute levels of CSF in one
fraction relative to the other. These two species of GM-CSF should be
useful in separately enumerating subpopulations of different GM-progenitor
cells inhuman hemopoietic disorders.
Volume 54,
Issue 3,
pp. 614-627,
09/01/1979
Copyright © 1979 by The American Society of Hematology
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