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Year XVI -Issue 06 - 2000
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 CURRICULUM |
ABSTRACT |
BIBLIOGRAFIA |
Donald Metcalf
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Historical Background Prior to 1965, there was no knowledge of how the basal production of granulocytes and macrophages was controlled. Cell levels and production clearly changed during bacterial infections but how this might happen also was not understood. The introduction in the mid-1960s of clonal culture techniques allowing precursors of granulocytes and macrophages to generate colonies of maturing progeny (1,2) not only provided a versatile method for quantifying such cellular events but also a bioassay system allowing the search for specific regulators of granulocyte and macrophage production. It was to require twenty years of sustained effort before the fruits of this work were ready for clinical application. There were several reasons for this long development period. First, notions of how granulocyte and macrophage production might be controlled were initially based on the one known hematopoietic regulator, erythropoietin, which appeared to be the single regulator required to control red cell formation. This proved to be a misleading model, leading to a decade or more of confusion before it became obvious that multiple regulators existed that interacted to control granulocyte and macrophage formation. Second, the initial goal of purifying these regulators, which proved to be glycoproteins, was not technically feasible in the late 1960s and for much of the 1970s. These regulators have extremely high specific activity, meaning that their concentrations in the serum and tissues are extremely low. The up to one million-fold purification required to purify these regulators was not achievable until the introduction of high-performance liquid chromatography in the late 1970s. Third, the amounts able to be purified indicated that native regulators could never be produced by biochemical fractionation in the quantities likely to be needed for in vivo work, either in animals or patients. This required application of the then relatively new technology of gene cloning to isolate the genes encoding these regulators, then to develop satisfactory methods for mass-producing recombinant versions of the regulators with equivalent biological properties to those of the native glycoproteins. Progress through each one of these stages required working at the edge of achievable technology and, when viewed in this light, the twenty-year development period becomes more understandable and a very creditable achievement by the groups involved. After commencement of clinical use of the regulators of granulocytes and macrophages, an unexpected further phase of experimental work became necessary - the generation of mice lacking specific regulator genes. This was needed because of the obvious complexity of the control systems and the necessity to establish the exact role being played by each regulator in normal health and disease. The most recent phase of research on these regulators has involved characterization of their membrane receptors, the signaling pathways initiated when a regulator binds to its receptor and the nuclear transcription factors and other interactive molecules that modulate and determine the various types of cellular response actually occurring following regulator stimulation. The Colony Stimulating Factors The data from clonal cultures showed that, although mature granulocytes and monocyte-macrophages differ radically in morphology and many functional properties, they are in fact closely related cell lineages, most showing common bipotential lineage-committed progenitor ancestors (3). These are the cells able to form colonies of granulocytes and/or macrophages in vitro, with the committed progenitor cells themselves being the progeny of multipotential stem cells. As analyzed in clonal culture, the proliferation of these progenitor cells primarily depends on stimulation by four glycoprotein colony stimulating factors - GM-CSF, G-CSF, M-CSF and Multi-CSF (IL-3) (Figure1)(4). These were the original four regulators identified and purified because of their capacity to stimulate colony formation hence the name for these regulators. Subsequent work has shown that two other regulators, stem cell factor and IL-6, also have stimulating actions on granulocyte proliferation in vitro (5,6). If the cellular proliferation by which stem cells generate granulocyte-macrophage progenitor cells is also considered, then all six of these regulators can have important actions on this process as can thrombopoietin, IL-11, IL-12 Flk-ligand and LIF. It is a feature of this cell system that most stem and progenitor cells seem to simultaneously coexpress receptors for multiple regulators, with little evidence that subsets exist that respond exclusively only to a single regulator. The
prototypic and most active regulators of granulocyte and macrophage
formation - the CSFs - are all glycoproteins with core polypeptide molecular
weights ranging from 14 to 63 kD (4). The carbohydrate component of
each is not needed for biological activity in vitro and appears to serve
merely to extend the half-life of the molecules in vivo - a phenomenon
much more evident for the prototypic regulator, erythropoietin. |
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Donald Metcalf |
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Human G-CSF (long-chain 4-helix bundle) |
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LEGENDA G-CSF = granulocyte colonystimulating factor GM-CSF = granulocyte-macrophage colony stimulating factor M-CSF = macrophage colony stimulating factor Multi-CSF = multi-potential colony stimulating factor |
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