Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the in vitro proliferation and differentiation of granulocytic and macrophage cells. This regulator is now known to act at other levels of hemopoietic regulation. The heterogeneity of GM-CSFs is not only related to the tissue of origin and the in vitro production method, but also to functional subclasses of the molecule that have distinct biologic specificities. Most adult mouse organs produce GM-CSF (mol wt 23,000), but a macrophage (M)-CSF has been detected in fetal conditioned medium (CM) and isolated from L-cell CM. Murine endotoxin serum appears to contain a M-CSF, GM-CSF, and G-CSF, the last of which cofractionates with a differentiation factor active on leukemic cells. Human GM-CSFs, G-CSF, and EO-CSFs active on human cells have been detected in a variety of CM, but as yet none have been purified. Again, there are subclasses of progenitor cells that respond to particular forms of human active CSFs. GM-CSF isolated from mouse lung CM stimulates multipotential progenitor cells, the initial proliferatin of progenitors in the erythroid, eosinophil, and megakaryocyte series, as well as mature cells in the GM series. While GM-CSF is also able to stimulate the differentiation of myeloid leukemic cells, other factors appear to be more potent in this respect. Information on the regulation of GM-CSF production, on the modulators of its action on specific target cells, and on its role in vivo will be required before the physiologic function of this molecule can be properly assessed.

Granulocyte-macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte-macrophage stimulus, GM-CSF, or by the predominantly macrophage stimulus, M-CSF. After 24 hours, paired daughter cells of granulocyte-macrophage colony-forming cells (GM-CFC) were separated by micromanipulation and one cultured in GM-CSF, the other in M-CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and day 3 clones were cross-transferred to GM-CSF or M-CSF recipient cultures, irreversible commitment was more obvious. Most M-CSF-initiated clones exhibited irreversible commitment to macrophage formation in GM-CSF cultures and a high proportion of GM-CSF-initiated clones continued to produce granulocyte progeny after transfer to M-CSF. The results indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC respectively to granulocyte or macrophage production. While for women GM-CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions. Calculations from the data indicated that two-thirds of GM-CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM-CSF and M-CSF.

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The Epidermal Growth Factor (EGF) receptor appears to require a fully active tyrosine kinase domain to transmit mitogenic signals. However, waved-2 mice carrying a mutation in the alpha-helix C of their EGF-R, which abolishes tyrosine kinase activity, only display a mild phenotype and are fully viable. This suggests that the mutant EGF-R signals through heterodimerization with endogenous, kinase active members of the EGF-R family such as ErbB-2 or ErbB-4. We have examined the biochemistry of EGF-Rs carrying mutations in the alpha-helix C of the human EGF-R (V741G and Y740F), in the ATP binding site (K721R) and at the C-terminus (CT957), by expression in BaF/3 cells which are devoid of EGF-R family members. The in vitro kinase activity of the alpha-helix C EGF-R mutants was severely impaired as a result of reduced phosphotransfer activity without appreciable changes in the affinity for either ATP or peptide substrate. Surprisingly, EGF stimulation of cells carrying the different mutant or wild type EGF-Rs resulted in tyrosine phosphorylation of EGF-R proteins; this phosphorylation was abolished in crude plasma membrane preparations, and appears to be due to activation of a membrane-associated or a cytosolic kinase. Receptor-mediated internalization of EGF was profoundly suppressed in the V741G, K721R and CT957 receptor mutant, and high affinity EGF binding was undetectable in the V741G and K721R receptors. We conclude that specific residues in the C-helix of the EGF-R kinase are essential for full kinase activity; mutations in this region do not affect ATP binding, but impair the receptors' phosphotransfer ability. High affinity binding of EGF is not dependent on tyrosine kinase activity or sequences in the C-terminus.

Medium conditioned by human placental tissue was found to stimulate granulocytic and monocytic colony formation by human marrow cells in semisolid agar cultures. The colony-stimulating activity of unfractionated conditioned medium was equivalent to the activity of standard peripheral blood underlayers. Placentas were a reliable source of active material, and one placenta provided enough material to stimulate 5,000–10,000 cultures of normal or leukemic cells. The colony- stimulating factor in human placental conditioned medium (CSFHPCM) was concentrated and purified 1800-fold using ammonium sulfate precipitation, calcium phosphate gel absorption, DEAE-cellulose batch absorption, gel filtration on Sephadex G-150, and polyarcylamide gel gel electrophoresis. The active factor behaved on gel filtration as a macromolecule with an apparent molecular weight of 30,000 daltons. The active factor in placental conditioned medium was not dependent on the presence of adherent marrow cells with endogenous colony-stimulating activity.

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.

Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli. The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties. The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence. The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation. This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium. Human GM-CSF had no activity on murine bone marrow or murine leukemic cells. There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF. Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells. The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl- phenylalanine (FMLP)-induced superoxide production and lysozyme secretion. Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay.

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