Hematopoietic Growth Factors Nicos A. Nicola* The Walter and Eliza Hall Institute of Medical Research and Cooperative Research Centre for Cellular Growth Factors, PO Royal Melbourne Hospital, Parkville, Victoria 3050, Australia * corresponding author tel: 61-3-9345-2526, fax: 61-3-9345-2616, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.02009.
THE HEMATOPOIETIC CELL SYSTEM The mature hematopoietic cell compartment consists of a variety of cells responsible for oxygen transport (red blood cells), hemostasis (platelets derived from megakaryocytes), innate immunity against infections (granulocytes, monocytes/macrophages, and mast cells), and acquired immunity (T and B lymphocytes). Monocytes are also the precursors of antigenpresenting dendritic cells and of the bone-remodeling osteoclasts. Bone marrow transplantation studies have revealed that all of these cells are derived from a common ancestral hematopoietic stem cell, relatively small numbers of which are maintained throughout adult life by a process of self-renewal (Harrison et al., 1993). During development, stem cells originate from embryonic mesoderm. Primitive hematopoiesis originates extraembryonically in the blood islands of the yolk sac while definitive or adult hematopoiesis originates in the embryonic aorta±gonadal mesonephros (AGM) region. During development the major site of hematopoiesis in the embryo moves first to the fetal liver, then the spleen, and finally the bone marrow (Dzierzak, 1999). T lymphocyte maturation occurs in the thymus while B lymphocyte maturation occurs in the germinal centers of the spleen. Several biological assays have been devised to define various stages of hematopoietic cell differentiation and have given rise to terminology that may be confusing to the uninitiated. Stem cells were first defined by their capacity after transplantation to generate hematopoietic colonies in the spleens of
irradiated mice, and were called colony-forming units±spleen (CFU-S) (Till and McCulloch, 1961). Alternate assays that detect the same or similar cells measure the capacity to repopulate all hematopoietic lineages in otherwise lethally irradiated mice (longterm repopulating cells); the ability to initiate longterm cultures of bone marrow cells in vitro (long-term culture-initiating cells, LTC-IC) (Eaves et al., 1991); or the ability to generate colonies in vitro that contain self-renewing primitive blast cells (blast colonyforming cells or B-CFC) (Nakahata and Ogawa, 1982) or form very large colonies (high proliferative potential colony-forming cell or HPP-CFC) (McNiece et al., 1990). The development of bone marrow colony-forming assays in semisolid cultures in vitro (Metcalf and Nicola, 1995) led to the colony-forming cell (CFC) or colony-forming unit (CFU) terminologies for lineagecommitted progenitor cells. The types of cells found in these colonies were indicated by a prefix; thus granulocyte CFC (G-CFC), macrophage CFC (M-CFC), eosinophilic CFC (Eo-CFC), megakaryocyte CFC (Meg-CFC), granulocyte±macrophage CFC (GM-CFC), etc. Erythroid colonies were observed to come in two categories: large multicentric colonies (burst-forming units-erythroid or BFU-E) or smaller clusters (colony-forming units-erythroid or CFU-E).
HISTORY OF DISCOVERY OF THE HEMATOPOIETIC GROWTH FACTORS The first hematopoietic growth factor to be described was erythropoietin, which was postulated to be a
850 Nicos A. Nicola humoral hormone released when the red blood cell mass fell below normal levels. Although erythropoietin was not purified until 1977, the postulate was ultimately proved correct. Hypoxia sensors primarily in the kidney activate transcription factors that increase the production of eythropoietin. The released eythropoietin travels through the bloodstream to the bone marrow where it acts on erythroid progenitor cells to increase the rate of production of red blood cells. The increased oxygen tension in the tissues then turns off the transcription of the erythropoietin gene (Fisher, 1997). A similar argument was used to postulate the existence of thrombopoietin that would regulate the production of platelets. However, the purification of thrombopoietin proved even more elusive than that of erythropoietin and was not accomplished until 1993, when several groups, acting on a clue that the mpl receptor might be the receptor for a thrombopoietic factor, used it to purify and clone thrombopoietin. Unlike erythropoietin, thrombopoietin is produced constitutively by the liver, kidneys, and other organs but it is degraded by platelets so that the circulating levels reflect the platelet mass (Wendling and Vainchenker, 1998). The major regulators of granulocyte and macrophage production were discovered by accident using in vitro agar cultures. In attempts to stimulate leukemic cell growth in vitro, bone marrow cells were plated in the agar underlayer. The leukemic cells did not grow but colonies of granulocytes and macrophages did form in the bone marrow feeder layer and it was postulated that the bone marrow cells elaborated a granulocyte and macrophage colony-stimulating factor (GM-CSF). Over time it became apparent that tissues produced several different molecular species with differential specificity for the formation of granulocytes and macrophages. GM-CSF and macrophage CSF (M-CSF) were both purified in 1977 and granulocyte CSF (G-CSF) was purified in 1984, based on its capacity to induce terminal differentiation of the leukemic cell line WEHI-3B, as well as to stimulate the formation of pure granulocytic colonies. Because they were originally discovered and purified based on in vitro assays it was not possible to determine the in vivo roles of the CSFs until the respective cDNAs and genes were cloned in the mid1980s. It then became apparent that G-CSF was the major regulator of neutrophilic granulocyte production and that M-CSF was a major regulator of the macrophage and osteoclast lineages. Surprisingly, despite its potent colony-stimulating activity in vitro, GM-CSF had little effect on the production of granulocytes and macrophages in vivo. Instead, it was essential for the correct functioning of alveolar
macrophages and was important in regulating macrophage hyperinflammatory responses and dendritic cell function (Metcalf and Nicola, 1995). During the analysis of CSFs produced by different tissues, it was noted that lectin-stimulated spleen cells produced CSFs that stimulated multipotential colonies containing granulocytes, macrophages, eosinophils, mast cells, megakaryocytes, and erythroid cells. This CSF was called multi-CSF but it was first purified as a factor that stimulated the growth of a factordependent cell line (FDC-P1) in vitro and called interleukin 3 (IL-3) (Ihle et al., 1982). IL-3 remains an enigma. It can rarely be detected in vivo and, despite its potent colony-stimulating activity in vitro, its presence or absence in vivo has little effect on blood cell levels or their activities (Metcalf and Nicola, 1995). A study of eosinophil production in vitro demonstrated the requirement for an eosinophil differentiation factor while studies of B cell differentiation resulted in the discovery of B cell growth factor II (BCGF-II). Subsequent cloning revealed that these were the same molecule, which was called interleukin 5 (IL-5). IL-5 has no activity on B cells in humans and its major role in the mouse is the production of eosinophils in response to helminthic infections (Karlen et al., 1998). The discovery of stem cell factor (SCF) was based on its synergistic stimulation of large macrophage colonies (with M-CSF) or on two natural mutant strains of mice with severe anemia. Steel (Sl) mice contain stem cells which are functional when transplanted to other mice, suggesting that the mice lack an essential blood cell growth factor. White spotted mice (W), on the other hand, have defective stem cells but can be cured by bone marrow transplantation (including by Sl bone marrow), suggesting that they have an intact microenvironment. Because of their similar phenotype it was postulated that W mice might contain a defective stem cell receptor and Sl mice might contain a defective ligand for the same receptor. This proved to be the case with the W locus encoding the kit tyrosine kinase receptor and the Sl locus encoding its ligand, stem cell factor (Bernstein et al., 1990; Broudy, 1997). The Flt-3/Flk2 receptor was cloned as an orphan tyrosine kinase receptor. It was then used to purify and clone the Flt-3 ligand which was shown initially to stimulate, in a synergistic fashion, the growth of myeloid cells in vitro. Ultimately it was also shown both in vitro and in vivo to stimulate the production and function of a class of antigen-presenting dendritic cells (Lyman, 1998). A number of other cytokines and growth factors originally discovered using different assays have
Hematopoietic Growth Factors subsequently been shown to influence the growth or function of hematopoietic cells. However, since they are covered in different sections of this book, they will not be discussed further here.
SUBCLASSIFICATIONS OF HEMATOPOIETIC GROWTH FACTORS Although there was no reason initially to believe that the hematopoietic growth factors would be related to each other (and this was certainly not obvious from their amino acid sequences), more recent structural data and their usage of receptors indicate that they do constitute recognizable families (Nicola, 1994). All of the above cytokines have a similar subunit size and are known or postulated to take up the structure of an antiparallel four helical bundle. Some are monomers (erythropoietin, thrombopoietin, G-CSF, GM-CSF, IL-3) while others are disulfide-linked or noncovalent dimers (M-CSF, SCF, Flt-3 ligand, IL-5). Most bind to highly homologous type I cytokine receptors which are homodimers (erythropoietin, thrombopoietin, G-CSF) or heterodimers which share a common subunit (GM-CSF, IL-3, and IL-5). The others bind to related homodimeric tyrosine kinase receptors and can themselves be cell surfaceassociated transmembrane proteins (M-CSF, SCF, Flt-3 ligand). All of the hematopoietic cytokines have multiple activities on their target cells, including the prevention of apoptosis, stimulation of cell proliferation, induction of differentiation, and functional activation of the mature cells. Gene deletion studies in mice have revealed that some of these growth factors are relatively specific in their actions on end cells and are the major physiological regulators of the steady-state levels of granulocytes, eosinophils, erythroid cells, and platelets (G-CSF, IL-5, erythropoietin, and thrombopoietin, respectively). However, despite their specificity in terms of end cell production, at least two of these cytokines (thrombopoietin and GCSF) also have important effects on the maintenance of stem cell numbers and/or the mobilization of stem and progenitor cells out of the bone marrow into the blood. GM-CSF, IL-3, and IL-5 also play important roles in host defense and are each produced by a variety of cells, including activated T cells after antigenic stimulation. These roles include the activation of macrophages, granulocytes, and dendritic cells (GMCSF), mast cells (IL-3), and eosinophils (IL-5). Their major physiological role may be to be produced
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locally in response to particular microorganisms or their products and to act on resident cells mediating innate immunity. To some extent, the tyrosine kinase ligands (M-CSF, SCF, and Flt-3 ligand) may also play an important role in host defense (activating macrophages, mast cells, and dendritic cells, respectively). However, all three also have synergistic effects on very primitive hematopoietic cells in the bone marrow. Recently, hematopoietic stem cells capable of long-term reconstitution of the entire hematopoietic system have been purified and their growth in vitro has been shown to require the action of multiple growth factors acting synergistically. SCF and Flt-3 ligand have little colony-stimulating activity when acting alone but act synergistically with other CSFs to form giant colonies, often with self-renewing capacity and with multiple cell lineages expressed (Haylock et al., 1997). M-CSF also generates giant macrophage colonies with high proliferative potential when combined with other stimulii such as IL-1, SCF, and Flt-3 ligand. These cytokines also have significant effects outside of the traditional hematopoietic system. M-CSF acts on bone-destroying osteoclasts and on placental trophoblasts during pregnancy. SCF acts on melanocytes and on primordial germ cells and Flt-3 ligand acts on dendritic cells.
CLINICAL APPLICATIONS OF THE HEMATOPOIETIC GROWTH FACTORS As might have been expected, the cytokines with the most specific effects on blood cell development were the first to find widespread clinical use. Erythropoietin has found widespread use in cases of erythropoietin insufficiency such as renal disease, anemia associated with cancer, and anemia associated with infection or inflammation (Fisher, 1997). Because of its specificity, there are few side-effects with erythropoietin other than hypertension associated with excess red blood cell numbers. Similarly, G-CSF has found extensive use in neutrophil recovery after cancer chemotherapy and/or bone marrow transplantation and has proved useful in the treatment of cyclic neutropenia (Welte et al., 1996). Perhaps surprisingly, high elevations of neutrophil levels in response to G-CSF were not associated with severe toxicity. The normal route of discovery of cytokine functions was also reversed in G-CSF clinical trials with the unexpected discovery that GCSF could dramatically elevate circulating stem and progenitor cell levels: this was later confirmed in
852 Nicos A. Nicola animal studies. This latter activity has seen a major usage of G-CSF to allow for peripheral blood stem cell transplants as a replacement for bone marrow transplantation. As mentioned above, thrombopoietin is also highly specific, at least in its actions on end-stage cells. However, here there is a lesson in the difficulty of bringing promising drug candidates to clinical utility. Initial clinical trials of a modified thrombopoietin resulted in the production of antibodies that recognized endogenous thrombopoietin and therefore had the exact opposite of the desired effect ± the induction of a thrombocytopenic state. Additionally, it appears that the action of thrombopoietin in stimulating platelet production after cancer chemotherapy is somewhat delayed so that only a modest change in the rate of platelet recovery is seen. Of the related cytokines, GM-CSF, IL-3, and IL-5 (each of which uses the common chain in its receptor complexes), only GM-CSF has found current clinical utility. Like G-CSF, it has been used to accelerate neutrophil recovery after cancer chemotherapy and to mobilize hematopoietic stem cells into the blood. However, GM-CSF is somewhat less effective than G-CSF and has potentially more adverse effects, as also revealed by animal studies which demonstrated the macrophage-activating actions and consequent tissue damage mediated by excess levels of GM-CSF. Nevertheless, the same actions of GM-CSF as well as its actions on dendritic cell function may in the future see the use of this molecule in cancer immunotherapy and serious infections (Armitage, 1998). Initial clinical trials of IL-3 were discouraging because of its broad activity and adverse events associated with activation of mast cells (Royer and Arock, 1998). Similarly, the involvement of IL-5 in the activation of eosinophils in allergic reactions has seen clinical interest center on the development of IL-5 antagonists rather than agonists. Clinical trials of stem cell factor have shown that it too can accelerate hematopoietic recovery following cancer chemotherapy and can mobilize hematopoietic stem cells into the blood in a synergistic way with GCSF. Like IL-3, SCF also has the potential to activate mast cells and basophils and some adverse reactions have been reported, but it may find clinical utility for stem cell mobilization in cases where G-CSF alone is ineffective (Shpall, 1999). Flt-3 ligand has not yet undergone extensive clinical trials but its actions in animal models of synergistically stimulating early myeloid cells and mobilizing stem cells, dendritic cells, and natural killer cells suggest that it might be useful as an adjunct in cancer chemotherapy and in cancer immunotherapy and infection (Lyman, 1998). Surprisingly little has been reported on clinical trials
of M-CSF but it appears to offer little additional benefit over G-CSF in hematopoietic recovery after chemotherapy.
CONCLUSIONS AND OUTLOOK The hematopoietic growth factors have developed into useful clinical reagents or have the potential to do so when applied in the appropriate clinical setting. None of them are as specific in their actions as was originally thought, but some have been shown by genetic deletion experiments to be the major regulators of the production of specific hematopoietic cell types. To some extent, in vitro experiments have been misleading. The potent in vitro colony-stimulating factors GM-CSF and IL-3, for example, have turned out to be rather weak stimulators of granulocyte and macrophage production in vivo. Instead, they are probably more important as mediators of antibacterial and inflammatory responses and their effective clinical use may rely on the determination of the particular infectious agents against which they are effective. Other hematopoietic growth factors such as SCF and M-CSF have quite distinct actions on different hematopoietic and nonhematopoietic cells and their effective use may depend on the timing and site of administration of the growth factor. Despite the successful use of some of these cytokines in the clinic, we still know relatively little about their physiological sites of production and the inductive stimulii that regulate their production. Production of most of these cytokines is widespread and usually at low levels, so these issues have been difficult to resolve, but it is important to do so because it will reveal the physiological and pathological situations in which it is most appropriate to use each cytokine. It is important also to recognize that the body has evolved mechanisms to eliminate cytokines after they are no longer needed since they can sometimes exert considerable collateral damage to tissues if left unchecked. Such mechanisms include release of inhibitory soluble receptors, receptormediated destruction of the cytokine, and a range of intracellular feedback inhibitors of cytokine signaling. There is currently no corresponding sophistication in the delivery, elimination, and temporality of cytokine action in clinical medicine so often there are adverse events in their use. The biology of hematopoietic cells has taught us that they are rarely exposed to single cytokines in vivo. Rather, they respond to a continuously changing milieu of a mixture of stimulatory and inhibitory cytokines and this serves to prevent dramatic fluctuations in cell numbers or their biological responses.
Hematopoietic Growth Factors Indeed, multiple cytokines appear mandatory for the stimulation of the most primitive hematopoietic cells. Yet current clinical practice is to use large doses of single cytokines applied intermittently. It is hoped that this may change as our understanding of how cytokines are regulated physiologically improves. Finally, it has recently become possible to conceive of improvements in the cytokines themselves. Small molecule agonists of erythropoietin and G-CSF are being developed and these may be orally available (rather than requiring injection), may be easier to target, and may have better pharmacodynamics than the cytokines. Attempts are also being made to circumvent the entire cytokine/receptor system by developing agonists and antagonists of intracellular cytokine signaling pathways. Some of these could be more precise clinical tools than the cytokines because they could, on the one hand, target only one of the several actions induced by a cytokine or, on the other hand, target common pathways that are used by several cytokine systems. The rapid advances occurring in our understanding of hematopoietic cytokine action may make these possibilities a reality in the next few decades.
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