Hemopoietic Growth Factors: a Review George Morstyn and Antony W

(CANCER RESEARCH 48. 5624-5637, October 15. 1988] Review Hemopoietic Growth Factors: A Review George Morstyn and Antony W. Burgess1

Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Australia 3050

Abstract chemical properties of the hemopoietic growth factors have been studied in depth and the clinical indications for these Several hemopoietic growth factors have now been purified, cloned, factors can be predicted with some degree of confidence. For and produced in bacteria. Granulocyte colony stimulating factor and the first time we are confronted with biological reagents which granulocyte-macrophage colony stimulating factor are already being used should selectively "Stimulate the phagocytes" (Shaw, 1906) in clinical trials. Within 12 months two more hemopoietic growth factors, macrophage colony stimulating factor (also called colony stimulating ( 15). Our current knowledge suggests that these reagents should factor 1) and interleukin 3 (also called multi-colony stimulating factor) have immediate applications for patients with impaired phag will be used for patient treatment. This review discusses the recent ocyte function. advances in our knowledge of the molecular properties and biological specificities of these factors. It is now clear that these molecules are able Hemopoiesis to modulate selectively the activity of mature blood cells as well as Hemopoietic Cells. The mature cells in the hemopoietic sys stimulating the production of specific lineages of blood cells. The avail ability of recombinant hemopoietic growth factors purified from animal tem are lymphocytes, monocytes, erythrocytes, neutrophils, or yeast cell conditioned medium or bacteria has facilitated in vivo eosinophils, basophils, and platelets. Each of these cells has a experiments, as well as the clinical trials. Each of the growth factors has limited life span and only lymphocytes and monocytes are a unique spectrum of biological activities and it appears that the growth capable of cell proliferation. All of these cells are produced as factors will enhance the recovery and function of circulating white blood a result of the balance between self-renewal and commitment cells after cancer therapy or bone marrow transplantation. of hemopoietic stem cells. In special situations where there is increased loss of cells, the stem and progenitor cell compart ments respond in an appropriate way by increasing the levels Introduction of mature cells. These changes appear predominantly to be achieved by alterations in the rate of production of mature cells With the present enthusiasm for the use of biological re and by the shifting of cells from the progenitor compartments sponse modifiers for cancer therapy, one could be forgiven for to the differentiating compartments. There appears to be little thinking that the activation of blood cells is a new paradigm in control over the rate of cell destruction during normal hemo- molecular medicine. However, Elias Metchnikoff ( 1) and Al- poiesis. moth Wright (2) eloquently noted the same possibilities many There is clear evidence (Fig. 1) that the organization of the years ago, but our incomplete knowledge of the production, hemopoietic system is hierarchical, with multipotential stem activation and biology of leukocytes has delayed the effective cells leading to mature nonproliferating cells (3). The nomen testing of this hypothesis for almost a century. During the last clature for progenitor cells is based on the mature cells (3) 5 years there has been a major change in our understanding of which they produce. Thus, the progenitor cells are designated the molecules which control both the production and activation as colony forming unit erythroid, BFU-e,2 GM-CFC, eosinophil of leukocytes (3-10). It is already evident that some of these colony forming cell, and megakaryocyte colony forming cell. molecules are likely to have important roles in the development All of the progenitor cells (except perhaps the earliest stem of new therapies for cancer patients (11), for the treatment of patients with life-threatening infections, for wound healing, and cells) can be detected by in vitro culture and their proliferation is totally dependent on specific growth factors (16). The evi perhaps even for the prophylactic boosting of our cellular dence for distinct subsets of progenitor cells is that they can be defense systems in aged or immunocompromised patients. partially separated from each other by velocity sedimentation, In this review we will discuss the structure, biology, and likely isopyknic centrifugation, and on the basis of surface markers therapeutic indications of some of the hemopoietic growth by flow cytometry (17-22). factors. Since many properties of both murine and human It is not clear whether the direction of differentiation of the hemopoietic growth factors have been tabulated in a number of recent reviews (8, 10, 12-14), we will concentrate on the more stem cells is determined stochastically or whether growth reg ulators influence it. The studies of Koike et al. (23) and Metcalf recent publications, attempting to highlight the properties of and Burgess (24) suggest that the direction of differentiation is the molecules which may have relevance as therapeutic agents. influenced by the presence and concentration of specific he We will address some of the broader concepts indicated by this mopoietic growth factors. work as well as summarize some of the recent progress which Progenitor cells recently identified by in vitro techniques are has been made on the genetics, synthesis, and use of the the high proliferative potential colony forming cells and can be hemopoietic growth factors. detected by stimulation in vitro with M-CSF (also called CSF- Compared to other biological response modifiers, some of which have already been tested in patients, the biological and 2The abbreviations used are: BFU-e, burst forming unit erythroid; GM-CFC, granulocyte-macrophage colony forming cell; M-CSF, monocyte colony stimulat Received 12/30/87; revised 6/14/88; accepted 7/6/88. ing factor; CSF, colony stimulating factor; multi-CSF, multi-colony stimulating The costs of publication of this article were defrayed in part by the payment factor; IL-1. -2, -3, and -4, interleukins 1, 2, 3, and 4, G-CSF, granulocytic colony of page charges. This article must therefore be hereby marked advertisement in stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; accordance with 18 U.S.C. Section 1734 solely to indicate this fact. GM, granulocyte-macrophage; cDNA, complementary DNA; PWM-SCM, ' To whom requests for reprints should be addressed, at Ludwig Institute for pokeweed mitogen stimulated spleen cell conditioned medium. HGF. hemopoietic Cancer Research, P. O. Royal Melbourne Hospital, Melbourne, Victoria 3050, growth factor; AML, acute myeloid leukemia; rG-CSF, recombinant granulocyte Australia. colony stimulating factor. 5624

1) and hemopoietin 1 (25-29). Nakahata et al. (30) have sug ml, unless a hemopoietic growth factor is present, all of the gested that there is also another group of multipotential, self- cells die. However, if one of the hemopoietic growth factors is renewing cells which appear to enter randomly into cycle from present, the early myeloid cells continue to proliferate and Go and which can then be stimulated to produce blast cell differentiate, producing a colony of mature granulocytes and/ colonies by multi-CSF (IL-3). Perhaps hemopoietin 1 is re or macrophages. When human marrow is used, the colonies sponsible for the initial stimulation of these cells from G0 to require 7 to 14 days to develop between 50 and 5000 mature GÃ¬(31-33). Recently it has been shown that IL-4 acts on these cells and the number and type of differentiated cells depend on early cells (34). the concentration and species of the hemopoietic growth factor. Subpopulations of Granulocyte Progenitor Cells. There ap Two names were given to the first hemopoietic growth factor: pears to be a hierarchy of human granulocyte-macrophage macrophage-granulocyte inducer (40) and CSF (39). These progenitor cells with two separate growth factors stimulating names proved to be inadequate as another growth factor for different subsets of the GM-CFC. These have been operation murine marrow cells was also discovered (41). This molecule ally called Day 14 GM-CFC and Day 7 GM-CFC. There is stimulated the production of macrophage colonies in preference evidence that some of the Day 14 GM-CFC are precursors of to granulocyte colonies. Since this second hemopoietic growth Day 7 GM-CFC (35). Morstyn et al. (36) were able to partially factor is also a colony stimulating factor, confusion between purify these two subpopulations using lectin binding and cell the two molecules was inevitable. Two sets of nomenclature sorting. The Day 14 population was enriched for myeloblasts were used to distinguish between these two molecules: GM- and the Day 7 population for promyelocytes and myelocytes. CSF by one group (42) and CSF-2 by other groups (43); the Begley et al. (37), using a monoclonal antibody to mature cells, other molecule was renamed CSF-1 or M-CSF. Most com have confirmed that myelocytes and promyelocytes are a dis monly, GM-CSF and CSF-1 are used to distinguish between tinct subset of progenitor cells. The distinction between the these two molecules (44). Day 7 and Day 14 granulocyte progenitor cells is important A third hemopoietic growth factor specific for granulocytic because they are preferentially stimulated, by G-CSF (originally cells was discovered in the conditioned medium from human called CSF0) and GM-CFC (formerly called CSFa) (36). A placenta (45) and in the serum of mice treated with endotoxin further complication is that Francis et al. (38) have shown that (46). This molecule is widely known as G-CSF (47). as cells differentiate in the GM pathway they become more The fourth hemopoietic growth factor is also capable of sensitive to conditioned media containing CSFs, i.e., to GM- stimulating the production of granulocytes and macrophages. CSF and G-CSF. This molecule was identified by several laboratories working on the different properties of the conditioned medium at lectin Hemopoietic Growth Factors stimulated murine spleen cells (48) and the conditioned medium from a "myeloid" cell line WEHI3B(D~) (49, 50). Initial obser Biological Specificity. Since the discovery of the method for vations suggested that this conditioned medium induced I - growing blood cells in culture (39, 40) four "granulocyte-mac rophage" CSFs have been isolated. The first hemopoietic lymphocyte differentiation; therefore the component responsi ble for this effect was named interleukin 3 (51). Subsequent growth factor was detected because of its ability to stimulate investigations have shown that the apparent effects on T-lym- the production of granulocytes and macrophages from murine phocytes were actually associated with the production of neu- spleen and marrow cells. The same assay system has been used trophils and macrophages. However, it was known that this to detect the other hemopoietic growth factors (16). When marrow cells are dispersed in agar at approximately 10s cells/ conditioned medium stimulated the production of eosinophils, megakaryocytes, and erythroid cells as well as granulocytes and macrophages. While Chromatographie investigations indicated that pokeweed mitogen stimulated spleen cell conditioned me dium contained a GM-CSF, the purification data were also

Stem cells compatible with the existence of another molecule capable of stimulating the production of all mature hemopoietic cells (48). Thus the molecule was also called multi-CSF. Indeed, it was Proliferation subsequently shown that the conditioned medium from WEHI3B(D~) cells did not contain GM-CSF and that the hemopoietic growth factor activity was all associated with multi-CSF (IL-3) (50, 52). Murine multi-CSF (IL-3) was the first hemopoietic growth factor to be molecularly cloned (53) Progenitor cells and the human equivalent of multi-CSF has been cloned re / \ Proliferation cently (54). Its range of biological activities has not been ex plored in detail; however, the cDNA for gibbon IL-3 has also been cloned (54) and this molecule supports the growth of erythroid and myeloid precursors in human marrow cells. Maturation and Thus, the production of granulocytes and macrophages from Activation the progenitor cells of mouse marrow can be stimulated by any one of four distinct molecules, multi-CSF (IL-3), GM-CSF, M- CSF (CSF-1), and G-CSF. There are human equivalents of all four molecules and three of these, multi-CSF, GM-CSF, and G-CSF, have been shown to stimulate the proliferation of Mature cells leukemic progenitor cells (54-56). Other terms have been used Fig. 1. Schema for the organization of the hemopoietic system. Colony stim ulating factors stimulate not only the proliferation and differentiation of cells in to describe mixtures (or impure forms) of these hemopoietic the myeloid lineages but also the activation of the mature cells. growth factors, e.g., CSFa, CSF/3, pluripoietin, pluripoietin a, 5625

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. HEMOPOIETIC GROWTH FACTORS and burst promoting activity (8). Usually the properties of these sequence. From a full length cDNA it has been possible to conditioned media can be adequately accounted for by the deduce the complete amino acid sequence of pro-GM-CSF (71) properties of one of the four CSFs. Other molecules are able to and a putative prepro-GM-CSF (77). The hypothetical prepro- stimulate (or modify) the lineage specific proliferation and/or GM-CSF would have a structure similar to that of a transmem differentiation of marrow progenitor cells, e.g., interleukin 1 brane molecule; however, in situations where this molecule (57), hemopoietin 1 (25), eosinophil differentiation factor (58- would be expected to be synthesized, only the secreted form of 60), erythropoietin (61); interleukin 2 (62, 63), interleukin 4 the protein has been detected. Initial amino acid sequencing on (64) and interleukin 6. We have not attempted to include murine GM-CSF from lung conditioned medium yielded NH2- descriptions of the biology or chemistry of these molecules in terminal X-Thr-Val-Glu-Arg-Pro-Trp-Lys-His ... (78); this review. i.e., the NH2 terminus commenced six residues after the ex GM-CSF is known to stimulate eosinophil production (45, pected cleavage site (71 ), between Ser29 and Alaao. The Ser-Ala 65) and the initial divisions of progenitor cells in the erythroid cleavage site is used for GM-CSF derived murine T-lympho- and megakaryocyte lineages (32, 66). In the mouse high con cytes stimulated by concanavalin A. It is clear that limited NH2- centrations of G-CSF are also able to stimulate macrophage terminal truncations can be tolerated without influencing the production; however, only the initial proliferation of the other biological activity. Since E. coli synthesized human and murine hemopoietic progenitors is effected by G-CSF. M-CSF predom GM-CSFs are fully biologically active in vitro we know that the inantly produces macrophages, but at high concentrations some carbohydrate moieties are not necessary for either receptor granulocytes are produced (67). M-CSF does not seem to effect binding or activation. Studies on the serum half-life of bacte- even the initial divisions of the progenitor cells in the other rially synthesized murine GM-CSF indicate that the serum hemopoietic lineages. half-life (6-10 min) is similar for the native molecule. Another class of hemopoietic cell "response modulators" Analysis of the structure of the GM-CSF mRNA has revealed needs to be mentioned, i.e., those molecules that appear to a segment of nucleotide sequence in the 3'-untranslated section influence hemopoiesis either indirectly or in the presence of of the mRNA which is important for controlling the breakdown one of the CSFs. Molecules such as IL-1 stimulate monocytes of the mRNA (79). The GM-CSF gene is inducible and mRNA to produce hemopoietic growth factors (68). Although IL-1 turnover is rapid. It is as though short bursts of synthesis rather may not act directly on myelopoiesis, activation of the synthesis than chronic production are used to control GM-CSF levels. It of M-CSF and GM-CSF will stimulate the production of gran may be possible to manipulate the 3'-untranslated section of ulocytes and macrophages. Indeed, depending on the number, the cDNA to produce a GM-CSF mRNA with a longer half- distribution, and state of activation of the tissue monocytes (or life. This would lead to increased mRNA levels and presumably macrophages) molecules such as IL-1 could initiate a cascade a more efficient animal cell production system for GM-CSF to which leads to high concentrations of the hemopoietic growth be developed. factors and consequently the large scale activation, accumula There has been considerable interest in the range of biological tion, and production of leukocytes. activities associated with the different hemopoietic growth fac At least two other mechanisms of amplifying the effects of tors (80, 81). The availability of large amounts of recombinant the hemopoietic growth factors have been proposed. The overall GM-CSF is allowing the biological specificity to be explored in production of mature cells depends on the number of available more detail both in vitro and in vivo. Purified murine GM-CSF progenitor cells. By increasing the production of progenitor stimulates a single cell to proliferate and differentiate to pro cells, the production of mature cells can be accelerated. For duce neutrophils, eosinophils, granulocytes, or macrophages example, 'hemopoietin 1 stimulates the production of macro (65, 82). The low frequency of eosinophil progenitor cells phage progenitors (25) and in the presence of both hemopoietin initially masked the detection of the eosinophils produced by 1 and M-CSF, monocyte production can be increased consid murine GM-CSF. Although it had not been purified, it was erably. Other molecules appear to act synergistically with he suggested that human GM-CSF could also stimulate eosinophil mopoietic growth factors, stimulating the rate of proliferation progenitors (83). The purification and cloning of sufficient of progenitor cells in response to multi-CSF; e.g., interleukin 4 quantities of both murine (32, 66) and human GM-CSF con stimulates the rate of multi-CSF dependent mast cell prolifer firmed that these three cell lineages are all stimulated by GM- ation (64). CSF (75, 80, 84). Granulocyte-Macrophage Colony Stimulating Factor. The The stimulation of isolated hemopoietic progenitor cells in molecule known as GM-CSF has been purified to homogeneity dicates that GM-CSF can initiate proliferation in both the from a human cell line (69) and murine lung conditioned erythroid and megakaryocytic lineages, but other molecules are medium (70). To some extent the native GM-CSF has been necessary to complete the production of mature cells in these used to define the range of biological activities of GM-CSF. lineages (32). The discovery of murine multi-CSF established However, the purified proteins also provided amino acid se the concept that a single molecule could stimulate the produc quence information which led to the identification and sequenc tion of all hemopoietic cells (48). Although earlier studies did ing of cDNAs corresponding to both the human and murine not detect megakaryocyte production in response to GM-CSF, GM-CSFs. It has been possible to express recombinant GM- it is now apparent that a proportion of megakaryocyte progen CSF for both species in COS cells (55, 71), yeast (72, 73), and itors can be stimulated with either murine (66) or human (84, Escherichia coli (74, 75). Indeed biologically active, recombi 85) GM-CSF. nant GM-CSFs have been purified from each of these sources The search for an equivalent molecule produced by human and these have been used to define some of the activities of this cells initially led to the detection of mixtures of molecules molecule in vivo (11, 76). which were thought to be pluripoietins and multilineage growth The availability of purified GM-CSF, whether natural or factors. In most cases these activities have proved to be mixtures recombinant, will allow progress toward an understanding of of GM-CSF and G-CSF. its structure and function. At present our knowledge of the The biological specificity of human GM-CSF has proved to structure of GM-CSF is essentially limited to the amino acid be more difficult to determine than the corresponding murine 5626

molecule. One major reason for this problem is the ability of molecules. There is a particular exception to this situation; human bone marrow cells to produce growth factors which when mice are given injections of endotoxin, several CSFs can interact in the colony stimulating assays. Even small numbers be detected in the blood. Two groups initiated studies designed of contaminating monocytes, T-cells, endothelial cells, or to characterize the molecule(s) in endotoxin serum which was strdiiial cells are likely to produce significant levels of molecules responsible for inducing leukemic cell differentiation (46, 94). which can synergize with and modulate the activity of human Both groups discovered that most of the colony stimulating GM-CSF (86-88). Thus, even a population of BFU-e which is activity in endotoxin serum could be neutralized by antisera 50% pure may contain cells which will respond to GM-CSF to raised against partially purified M-CSF (CSF-1) prepared from produce molecules able to effect BFU-e proliferation. It is L-cell conditioned medium (95). However, the ability of endo difficult to ascertain whether a proportion of BFU-e is able to toxin serum to induce WEHI3B(D*) cells was not diminished respond directly to GM-CSF or whether the GM-CSF "in by this antiserum. Lotem et al. (94) did not detect any effect of duces" the production of another molecule which acts on some the partially purified endotoxin serum differentiation factor or all of the BFU-e. (macrophage-granulocyte inducer 2) on normal cells. This It should be emphasized that this "biological" specificity has group has developed a novel hypothesis concerning the link generally been determined by in vitro responses. Indeed, the between proliferation and differentiation, they propose that analytical efforts to determine which cells proliferate in direct hemopoietic regulatory molecules either induce differentiation response to GM-CSF rather than those which proliferate as a or stimulate proliferation (96). In contrast Burgess and Metcalf result of indirect stimulation need to be considered in the (46) found that the endotoxin serum differentiation factor for appropriate context. When GM-CSF is released in vivo, its full WEHI3B(D+) cells was associated with some colony stimulat range of effects will be manifest (i.e., direct and indirect), and ing activity. All of the colonies stimulated by this CSF were proliferative or differentiative responses can be expected to composed of granulocytes. Thus, they suggested that a G-CSF occur in a wide range of cell lineages. was responsible for the differentiation of the WEHI3B(D+) Although the discovery of GM-CSF occurred because of its cells. Although other differentiation factors may exist (97), it in vitro proliferative effects on hemopoietic progenitor cells in is likely that the ability of endotoxin serum to induce leukemic the marrow and spleen, even in the first reports, it was recog cell differentiation is due to the G-CSF. This molecule was nized that this proliferation was associated with differentiation. purified from mouse lung conditioned medium (47) and the During recent years there has been an increasing awareness of purified molecule stimulates the proliferation of normal gran the action of GM-CSF on mature neutrophils and macrophages. ulocytic progenitor cells as well as the differentiation of It is now apparent that in the presence of initiator molecules, WEHI3B(D+) cells. GM-CSF is capable of activating both neutrophils and eosino- Molecular cloning and receptor competition studies have phils (65, 75, 80). made it clear that many conditioned media are likely to contain Granulocyte-Colony Stimulating Factor. The conditioned me mixtures of GM-CSF, G-CSF, and M-CSF. A particular ex dia from mouse 1.-cells and human urine stimulate almost ample is the identity of pluripoietin with G-CSF and pluripoie- exclusively macrophage progenitors (89), whereas mouse lung tin a with GM-CSF (98). Although GM-CSF and G-CSF are conditioned medium stimulates both granulocyte and macro becoming generally accepted terms, it is important to note that phage progenitors (66) and mitogen-stimulated spleen cells the cellular specificity of these two hemopoietic growth factors produce growth "factors" which produce granulocytes, macro is considerably broader than their names imply. Both GM-CSF phages, erythroid, eosinophil, and megakaryocyte precursors and G-CSF are capable of stimulating a limited number of (90). The CSFs in each of these preparations are heterogeneous divisions early in the erythroid, megakaryocyte, and eosinophil and proved to be difficult to purify, but there were some lineages (99). However, it is currently a matter of controversy indications that several distinctly different molecules could whether either molecule can stimulate the production of mature stimulate granulocyte (91, 92) and macrophage progenitors. cells in the erythroid and megakaryocyte lineages (100). In vitro Two potent sources of GM-CSF for human bone marrow cells, and in vivo both GM-CSF and G-CSF can stimulate cell pro human leukocytes stimulated by phytohemagglutinin and hu duction in other hemopoietic lineages. However, these effects man placenta! conditioned medium, appear to contain several may be indirect (i.e., by inducing the production of other growth CSFs. Phytohemagglutinin-stimulated human leukocytes were factors or modulators) (11, 85). This phenomenon is still being separated into a component capable of stimulating murine investigated in detail. macrophage progenitors and the "GM-CSF" which stimulated Human G-CSF has been synthesized by recombinant DNA human progenitor cells (93). Using phenyl-Sepharose chroma- methods using bacteria and appears to be active on its relevant tography, human placenta! conditioned medium was separated target cells (101, 102). Two mRNAs have been detected for into two distinct colony stimulating factors, a and ÃŸ(45):CSFo human G-CSF; one of these mRNAs encodes 3 extra residues stimulated granulocyte, macrophage and eosinophil progeni (101). It is likely that the larger molecule arises from a splicing tors; whereas CSFÃŸstimulated only granulocyte and macro artifact inasmuch as the shorter G-CSF is considerably more phage progenitors (45). When used at low concentrations the active in the bioassay (102). There appear to be no detectable CSFa preparation selectively stimulated macrophage progeni difference between the natural G-CSF (56) and the bacterially tors, whereas at low concentrations CSFÃŸpreferentially stim synthesized G-CSF in vivo. Thus the carbohydrate moiety does ulated granulocyte progenitors. The human CSFa activity had not appear to influence the interaction between G-CSF and its properties similar to those of murine GM-CSF. At low concen target cell. Apart from erythropoietin, there is no definitive trations the human CSFÃŸactivity mimicked the biological information which gives us a clue to the function of the carbo activity of the granulocytic specific murine G-CSF. hydrate moiety attached to the hemopoietic growth factors. Most hemopoietic growth factor preparations are produced The homology between human and murine G-CSF is approx from tissues or cells cultured in vitro. Indeed, the concentration imately 75% [cf., the homology between the murine and human of these molecules in fresh tissue specimens and body fluids is GM-CSF which is 56% (103)] and there is almost full biological extremely low and it is difficult to detect and characterize these cross-reactivity between the different species of G-CSF (104). 5627

At present there is insufficient structural information to indi CSF and other hemopoietic growth factors in vitro (23, 25, cate which parts of the G-CSF molecule are required for its 110). Many of these interactions should be manifest in vivo and biological activity. There is a single free sulfhydryl near the even small concentrations of Multi-CSF or G-CSF could be NH2 terminus (101) but this does not seem to be important for expected to synergize with the effects of M-CSF on both pro the activity of the molecule. Peptide analogues may help to genitor cells and mature cells. define the binding domain by allowing the epitope for neutral Multi-Colony Stimulating Factor (Multi-CSF, IL-3). The dis izing antibodies to be defined. Once location of the binding site covery of GM-CSF and M-CSF encouraged a search for other can be identified, analogues incorporating these residues can be molecules responsible for stem cell proliferation and production prepared to study the linkage between binding and mitogenesis. of eosinophils, megakaryocytes, and erythroid cells. Numerous Macrophage-Colony Stimulating Factor. There is a high mo sources of GM-CSF and M-CSF were discovered, but the other lecular weight hemopoietic growth factor, M-CSF [also called colony types were difficult to produce in vitro. It was known CSF-1 by the group which achieved the first purification of this that the conditioned medium from mitogen stimulated mono- molecule (89)], which has a propensity to stimulate the prolif nuclear blood cells, together with erythropoietin, was able to eration of macrophage progenitors. M-CSF has been detected stimulate granulocyte, macrophage, and erythroid colonies in a wide range of human and murine tissues where it is present (111). Parker and Metcalf (112) produced PWM-SCM which as a glycosylated homodimer (105). M-CSF has a variable supported the proliferation of progenitors for natural killer amount of carbohydrate; human urinary M-CSF has an appar cells, eosinophils, megakaryocytes, and erythroid cells. The only ent molecular weight between 45,000 and 60,000 (43) and the other source of growth factors for these cells was the condi molecular weight of the murine M-CSF homodimer varies from tioned medium from a murine myelomonocytic leukemia 60,000 to 80,000 (43). M-CSF is active only in the dimer form; WEHI3B(D~) (49). Since GM-CSF was a distinct molecular when it is dissociated by urea-mercaptoethanol or acidic ace- entity, it was initially assumed that PWM-SCM and WEHI3B(D~) conditioned medium also contained distinct eo- tonitrile the monomer is no longer active (67, 105). M-CSF has been available only in microgram amounts and sinophil CSF, megakaryocyte CSF, and erythroid CSFs. How the characterization of the important structural features has ever, attempts to separate these molecules from PWM-SCM been quite difficult. However, in 1985 human M-CSF was invariably yielded two classes of CSF: GM-CSF; and another molecularly cloned (106). Interestingly, the open reading frame biologically active pool which contained a molecule(s) capable for the M-CSF cDNA predicts a polypeptide 224 amino acids of stimulating all of the other progenitors (48, 90). Murine long (equivalent to a molecular weight of 26,000). There is a multi-CSF (IL-3) was the first of the hemopoietic growth factors putative leader sequence of 32 amino acids in front of the NH2- to be molecularly cloned (53, 113). Factor dependent mast cell terminal sequence determined from the purified human urinary clones (114, 115, 116) were used to assay products secreted by M-CSF. Since the size of the M-CSF polypeptide chain appears Xenopus oocytes previously treated with injections of subfrac- tions of WEHI3B(D") mRNA. The enriched mRNA was used to be 14,500, some posttranslational processing of the polypep tide must occur. to prepare a cDNA library and the appropriate cDNA pools Attempts to isolate an equivalent cDNA for murine M-CSF from the library screened in the oocyte assay using hybrid have proved to be even more difficult. However, one clone has selection and elution. The availability of a full length cDNA been isolated (107) and there is considerable homology at the and NH2-terminal sequences for multi-CSF indicated that con level of amino acid sequence. siderable heterogeneity could occur at the NH2 terminus (pre It is important to consider the range of biological activities sumably due to differential processing). Despite some limited of M-CSFs on mouse and human cells. Human urinary M-CSF homology at the signal peptide cleavage site (117), no other does not stimulate human colony formation directly (108), but significant homologies have been detected between the pre it is able to stimulate mouse marrow colony formation. Human dicted amino acid sequences for murine multi-CSF, GM-CSF, urinary M-CSF stimulates granulocytic clusters to form in G-CSF, or M-CSF peptide chains. unfractionated human marrow. This occurs by the stimulation Attempts to locate the human equivalent of multi-CSF using of monocytes to produce G-CSF. Thus, the human urinary M- the murine multi-CSF cDNA were not successful. There is no CSF seems to function as a stimulus for the final stages of overlap in biological activity between murine and human IL-3. granulocytic development in humans and a stimulator of the Even the rat multi-CSF (118) amino acid sequence is signifi early monocytic progenitor cells in mouse marrow. Murine M- cantly different from murine multi-CSF. CSF stimulates murine marrow cells to form macrophages, but Further analysis of the murine GM-CSF and multi-CSF does not stimulate human marrow cells. Even though murine cDNA clones indicated that as well as the secreted forms, the M-CSF is predominantly a stimulator of murine macrophage longer clones could encode pre-CSF molecules with a number and monocyte progenitors, there is still some stimulation of of charged residues at the NH2 terminus. This NH2-terminal neutrophil formation. Indeed, in serum-free cultures of individ arrangement of these longer molecules would generate a ''trans- ual hemopoietic progenitor cells, murine M-CSF appears to be membrane-like'' domain allowing the GM-CSF or multi-CSF an excellent stimulus for neutrophil-macrophage progenitors to anchor in the plasma membrane. At present neither the (23). M-CSF is also a powerful stimulus for mononuclear mRNA nor the protein corresponding to the transmembrane phagocytes (41) and murine monocytes require the presence of form of these two molecules has been detected. either GM-CSF or M-CSF for their survival (109). Interactions between Hemopoietic Growth Factors. The HGFs Both human and murine M-CSF can induce proliferation of can act synergistically; i.e., combinations of two or more of the murine mononuclear phagocytes; however, although human HGFs can induce proliferation and differentiation more effec mononuclear phagocytes can be functionally activated by M- tively than the individual growth factors. Until each of the CSF, as yet human monocytes have not responded mitogeni- HGFs had been purified it was difficult to study these synergis cally to any agent. tic effects definitively and even now it is important to note that It will be interesting to study the biological effects of M-CSF there are many other potent modulators of hemopoiesis in vitro, in vivo. There are strongly synergistic interactions between M- e.g., fetal calf serum (23, 119), hemopoietin 1 (25), and IL-4 5628

(64). The molecular basis for these synergistic effects is poorly but there will be a different potency for each growth factor at understood. The situation will be even more complex in vivo each phase. For example multi-CSF and GM-CSF appear to where the high density of cells in the marrow and spleen can be be two of the preferred stimuli for expanding the early prolif expected to produce significant concentrations of other modu eration steps. Although both are also capable of inducing dif lating agents (120-124). ferentiation and activating end cell function, they may mediate It is simple to show the interactions between the different these effects through the transmodulation pathways via the M - growth factors. When murine marrow cells are stimulated by CSF and G-CSF receptors. M-CSF (CSF-1) in medium containing fetal calf serum most of In contrast, while G-CSF may influence the early proliferative the colonies contain only monocytes/macrophages. Using con events in hemopoiesis, this regulator appears to be a more centrations of M-CSF which maximally stimulate marrow col potent activator of neutrophil function than multi-CSF (133). ony formation, 95% of the colonies are composed of monocytes. In order to manipulate the hemopoietic system we will need to However, when M-CSF is mixed with GM-CSF many granu- learn how to use these regulators (or their antagonists) to locytic and mixed granulocyte/macrophage colonies develop stimulate (or inhibit) the different phases of hemopoiesis. The (110). Indeed, even when the GM-CSF is used at low concen linkages between these three aspects of hemopoiesis are re tration, as long as there is maximal stimulation by M-CSF, quired to maintain the number of mature blood cells. In the granulocytic colonies will form. Thus, as long as there is a murine system several myeloid cell lines have been isolated strong proliferative stimulus, even a weak differentiation signal which are still dependent on growth factor but appear to be can influence the pattern of hemopoietic differentiation. blocked at different stages of differentiation. When this block In serum free cultures a combination of multi-CSF and M- occurs before the acquisition of G-CSF or M-CSF receptor, the CSF increased the size (i.e., number of cells) of both granulocyte cells will not differentiate, even in the presence of these regu and macrophage colonies (23). Furthermore, the number of lators. However, in other cell lines such as WEHI3B(D+), GM- GM-progenitor cells responding to the combination of multi- CSF and G-CSF receptors coexist but do not appear to be CSF and M-CSF is greater than the sum of the number of linked (127), i.e., occupancy of the GM-CSF receptor fails to colonies stimulated by the individual factors. transmodulate the G-CSF receptor. Thus, even though GM- Hemopoietin 1 is a molecule which appears to stimulate the CSF stimulates the proliferation rate of WEHI3B(D+) it is not initial proliferation of multipotential hemopoietic cells and a potent inducer of differentiation. These cells can still differ early macrophage progenitors (25), but it is not able to induce entiate if they are treated directly with G-CSF or actinomycin complete maturation. Mature cells are produced only in the D. A similar situation may occur in many primary leukemias presence of the other hemopoietic regulators. There also ap and it is imperative that we learn more about the biochemistry pears to be a synergistic interaction between hemopoietin 1 and of receptor transmodulation in normal and neoplastic cells. At multi-CSF (23). In the presence of both hemopoietin 1 and present our knowledge of the receptors for the hemopoietic multi-CSF the number of colonies formed by multipotential growth factors limits our ability to understand the biochemical progenitors increases almost 30-fold and many of the colonies interactions which mediate the receptor signal (72, 134-136). still contain multipotential progenitors. The molecular basis of this synergism has not been studied in detail, although in other cellular systems (fibroblast and Effects of Colony Stimulating Factors on Mature Cells epithelial) synergistic interactions have been reported to occur Studies with radiolabeled (iodinated) stimulating factors have among epidermal growth factor, platelet derived growth factor confirmed that mature as well as immature cells in the granu (125), and human transforming growth factor ÃŸ(126).There is locyte and macrophage cell series have receptors for colony some evidence that the synergistic interactions are modulated stimulating factors (133). G-CSF receptors are lineage re at the level of the cell surface receptor via an hierarchical stricted to the neutrophil cell series and M-CSF receptors to network (127). Thus it has been proposed that the activated the monocyte cell series. The mature cells have more G-CSF multi-CSF and GM-CSF receptors transmodulate the M-CSF and M-CSF receptors than the immature cells. GM-CSF and and G-CSF receptors and that it is these interactions which are multi-CSF receptors are present on neutrophils, monocytes, responsible for the production of granulocytes and macrophages and eosinophils and the mature cells have less receptors than in the presence of Multi- or GM-CSF. By extrapolation, the the early cells (133). generation of eosinophils, megakaryocytes, and erythroid cells The presence of receptors for the HGFs on mature cells is by multi-CSF and eosinophils by GM-CSF might be expected consistent with the observations that the colony stimulating to be mediated by the transmodulation of the receptors for factors have many effects on mature cells as well as on progen eosinophil (59, 128, 129) megakaryocyte (130, 131), or eryth itor cells. The activities of GM-CSF on mature cells are listed roid (132) growth regulatory proteins. Receptor transmodula in Table 1. tion could be a vital link between proliferation and differentia The effects of purified native and bacterially synthesized tion. Normal progenitor cells stimulated by multi-CSF would human GM-CSF on mature neutrophils and on eosinophils are be expected to proliferate and those which acquire G-CSF or similar (75, 141). Superoxide production, phagocytosis, lyso- M-CSF receptors would then be induced to differentiate (even zyme secretion, and antibody dependent cell mediated cytotox- in the absence of G-CSF or M-CSF). Multi-CSF and GM-CSF might be capable of producing granulocytes and macrophages Table I Effects of GM-CSF on neutrophils and macrophages at sites not normally endowed with sufficient concentrations of Function Ref. M-CSF or G-CSF. Chemotaxis 98 The interrelationship among three phases of hemopoietic cell Inhibition of locomotion 69 production (i.e.. proliferation, differentiation, and activation) Adherence phagocytosis 137,138 Survival in vitro 139 needs to be considered when analyzing leukemic cell popula Superoxide generation 140,141 tions. Most of the hemopoietic growth factors are probably Antibody dependent cell cytotoxicity 80,129,140,142,143 capable of influencing these different phases of hemopoiesis, Parasite killing 144,145 5629

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. HEMOPOIETIC GROWTH FACTORS Â¡cityby"primed" neutrophils are all stimulated by natural and antibodies which recognized the GM-CSF; these antibodies, bacterially synthesized GM-CSF. The concentrations of bacte- however, did not interfere with hemopoiesis. A major problem rially synthesized GM-CSF required to stimulate these func with this study was that the human GM-CSF was only partially tions were in the range of 1 to 10 pM. This is similar to the purified from COS cell conditioned medium. Nevertheless as potency of the COS cell derived human GM-CSFs (141). suming that the findings are reproducible with pure GM-CSF These studies suggest that nonglycosylated GM-CSF is as one can speculate that GM-CSF may be useful in conditions effective as glycosylated GM-CSF when stimulation of mature such as acquired immunodeficiency syndrome in which there is cell function is required. It is clear that low levels of neutrophils a neutropenia perhaps secondary to a deficiency of lymphokines (<1000/mm3), but particularly levels below 500/mm,3 are as produced by lymphocytes. sociated with an increased rate of infections (146). The mortal Multi-Colony Stimulating Factor. Kindler et al. (153) have ity of febrile neutropenia with current treatment is low. Pizzo reported the effects of multi-CSF in mice. Similar results have et al. (147) reported 22 deaths in 550 episodes of fever. The been obtained by Metcalf et al. (154) and Lord et al. (155). morbidity of febrile neutropenic episodes can be significant and Kindler et al. (153) used doses of 0.03-0.1 Mg/24 h for 7 days. even with current antibiotics the time to defervescence of the The total number of progenitor cells in normal mice was fever is prolonged (mean, 4 days; range, 1 to 35 days) (147). It increased 2-fold and, in particular, there was a rise in the is not clear whether during infections the low numbers of erythroid and myeloid progenitor cells in the spleen. If mice neutrophils are maximally activated or whether additional GM- were irradiated prior to therapy there was a 10-fold increase in CSF might cause further activation and additional antibacterial progenitor cell levels compared to control animals. Multi-CSF effect. There are also reports that patients being treated with injected into the peritoneal cavity produced increased numbers chemotherapy may exhibit diminished neutrophil function of monocytes (154) and these monocytes appeared to function (148). This functional inhibition may be overcome by GM-CSF. as more effective phagocytes. The human equivalent of murine This possibility is under investigation. Another question relates IL-3 has been cloned recently but its activity in vivo is not yet to the functional status of neutrophils in the elderly and whether known. GM-CSF might prove a useful activator. Granulocyte Colony Stimulating Factor. One group (156) has There is an important distinction between GM-CSF and G- reported on the in vivo activity of purified rG-CSF. The rG- CSF; GM-CSF activates both mature neutrophils and eosino- CSF was administered s.c. Following a single injection there phils (140, 149), whereas G-CSF activates only neutrophils. M- was a rise in neutrophil counts within 2 h. After 15 days of CSF activates mature monocytes and, in particular, causes the treatment there was an 8-fold rise in neutrophil levels. When production of interferon (150) and plasminogen activator (151, rG-CSF was withdrawn the blood neutrophil levels returned to 152). These actions of M-CSF may have clinical applications normal within 24 h (Fig. 2). Repeated s.c. injections of 2.5 fig/ in the treatment of infections. mouse/day resulted in sustained elevated neutrophil counts for more than 3 weeks. Daily single injections of 10 Mgof rG-CSF In Vivo Effects of CSFs for 14 days resulted in a 20-fold increase in neutrophil levels Cranulocyte-Macrophage Colony Stimulating Factor. Until recently insufficient amounts of GM-CSF were available for 106, definitive in vivo studies. The availability of all of the CSFs in gram quantities is now allowing the pharmacology and physi ology of each to be studied in detail. Further, many laboratories are testing multiple combinations of the CSFs with other mod ulators such as the interferons and interleukins. 10= Murine Studies. Metcalf et al. (76) have studied the effects of red blood cells bacterially synthesized murine GM-CSF in vivo. The doses used were in the range 6-200 ng/animal. Since the serum half-life E of GM-CSF is short (<5 min), multiple (up to 6) injections 104 lymphocytes were used. These injections resulted in elevated peripheral blood

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. HEMOPOIETIC GROWTH FACTORS over the basal level. In the mouse the effects were restricted to represent the later stages of some cases of myeloid leukemia. the myeloid system and there were no consistent changes in the However, their direct relevance to spontaneously arising leu levels of lymphocytes, monocytes, platelets, or RBC. In the kemias is yet to be established. mouse a transient effect on lymphocytes was apparent. G-CSF Morstyn et al. (165) demonstrated that normal promyelocytes also increased the number of CFU-spleen in the spleen. It is and myelocytes were highly responsive to partially purified likely that this is an indirect effect, perhaps representing re human G-CSF. Recently, human G-CSF has been shown to be cruitment, since G-CSF has no in vitro effect on multipotential very similar to murine G-CSF and I25l-Iabeled murine G-CSF progenitor cells. can be used to study human G-CSF receptors on leukemic cells When purified G-CSF was given after an alkylating drug such (104); 14 patients with myeloid leukemia had near normal G- as cyclophosphamide, the neutropenia produced by this agent CSF receptor levels. The data for human GM receptors (166) was completely abrogated (156). Administration of G-CSF can also demonstrate that leukemic myeloid cells have near normal preempt the death of mice which have been infected with a receptor levels. In many ways this evidence suggests that these lethal dose of Gram negative microorganisms (Pseudomonas) leukemias are not driven by autocrine stimulation. (157). Pseudomonas is a common cause of infections and deaths Leukemogenesis. Murine marrow cells can be forced to adopt in cancer patients being treated with cytotoxic chemotherapy. the leukemic phenotype in several different ways. Continuous cell lines have been derived from apparently normal long-term marrow culture. These cell lines are invariably dependent on Hemopoietic Growth Factors and Leukemia multi-CSF (115, 167, 168). However, cell lines dependent on The acute myeloid leukemias are subdivided into 7 categories G-CSF (169) and M-CSF (170) have also been obtained. by the French-American-British classification system (158). These cell lines which are absolutely dependent on the he- Each leukemia is characterized according to the degree of mopoietic growth factor for proliferation are blocked at an early proliferation of the leukemic clone in the marrow. The variants stage of differentiation but are not leukemogenic in syngeneic of myeloid leukemia are further distinguished by the morpho recipients. They can be made independent of CSF by sponta logical and biochemical properties of the leukemic cells (158). neous mutation (168), by transformation with the Abelson A feature of all cases of acute myeloid leukemia is that there murine leukemia virus (171, 172), or by infection with a recom appears to be a block to the normal maturation of blast cells. binant retrovirus that encodes and expresses GM-CSF (164). This block may be associated with a disturbance in the biochem The molecular lesions leading to leukemia in the case of inser ical pathways associated with receptor cross-modulation (126) tion of the GM-CSF gene and the spontaneous mutations are or with the control of expression of the genes encoding differ associated with the autocrine secretion of GM-CSF or multi- entiation factors (96). Therapy of acute myeloid leukemias CSF. The \-abl gene appears to circumvent the requirements usually leads to the eradication of the leukemic clone and the for GM-CSF and probably does this by producing a tyrosine recovery of normal hemopoiesis (159), although in some cases kinase which mimics the intracellular events normally triggered by the GM-CSF-GM-CSF receptor complex. Dunn3 has re normal differentiation and maturation of the leukemic cells are achieved (159). cently found that after the transfection of factor dependent cells Are the CSFs involved in the development or maintenance with a retrovirus which encodes and expresses the polyoma of the myeloid leukemias? Would it be possible by manipulating middle T-antigen one class of cells becomes leukemic but re the levels of one or more of the CSFs to improve the therapy mains dependent on GM-CSF for growth in vitro. These cells of leukemia patients? It will be important to determine the appear to resemble human leukemic cells. basis of the suppression of normal hemopoiesis by the leukemic Acute Myelogenous Leukemia. It has been shown recently cells and the levels of each of the CSFs in the tissues and serum that the genes for M-CSF, GM-CSF, multi-CSF, interleukin 4, of patients with AML. Further, an understanding of the de interleukin 5, and M-CSF receptor are all clustered on chro pendence of the proliferation and differentiation of AML cells mosome 5 (173, 174). There is a clinical syndrome called the on the normal CSFs and other biological response modifiers 5q- syndrome that involves a deletion of the long arm of should help define the conditions to create for attacking the chromosome 5 (174). It is intriguing to speculate that the leukemic cells with cytotoxic drugs. disorder in these patients is due to activation of a hemopoietic Both the autocrine and autonomous models for leukemic cell growth factor or receptor gene. However, this has not yet been population predict factor independent myeloid leukemias. investigated. However, in vitro studies suggest that at the time of diagnosis Promyelocytic leukemia has a 17/15 translocation and the most myeloid leukemias require exogenous CSF to proliferate G-CSF gene is on chromosome 17. The abnormality in this (160). There are a few leukemias which do not require CSF situation may be the absence of a differentiation stimulus (G- (161). In some cases the leukemic cells may produce CSF (162). CSF) leading to a maturation block at the level of the promye- It has also been found that the dose-response curves for CSF locyte (175). stimulation of normal and leukemic cells are similar (38, 163). The number of G-CSF receptors on normal and leukemic cells Production and Storage of GM-CSF is similar at the equivalent stages of development. Some cases of AML, particularly during the later phases of the disease, Most organs can produce the granulocyte-macrophage colony may not be dependent on an exogenous source of CSF but this stimulating factors. The organs that have been shown to pro has not been extensively tested. However, it should be noted duce GM-CSF using bioassays (176) include salivary gland, that many leukemias in blast crisis do not proliferate in vitro, lung, thymus, kidney, spleen, lymph node, pancreas, brain, even in the presence of the CSFs. Even those that do not grow in vitro obviously proliferate in vivo, suggesting there are he- heart, small intestine, testis, skeletal muscle, liver, and skin. Nicola et al. (177) have demonstrated that GM-CSF synthe mopoietic growth factors still to be identified. It is not clear sized by different organs had very similar biochemical proper- which growth factor is stimulating these leukemias in vivo. It may be that the leukemia models developed by Lang et al. (164) 3A. Dunn, personal communication. 5631

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. HEMOPOIETIC GROWTH FACTORS ties. These studies indicate either that GM-CSF is made by Table 2 Relationship between CSF levels and clinical states cells present in all tissues such as monocytes, endothelial cells stateCyclicClinical levelCSF CSF (177, 178), or fibroblasts or that cells of many different types neutropeniaAplastic precederiselevels cycle and make GM-CSF. This has not been clarified directly as yet. inWBCElevated anemiaCongenital urineDiminishedCSF in Endotoxin and tumor necrosis factor appear to stimulate CSF neutropeniaEndotoxin levelElevatedserum CSF release (87, 179). The presence of GM-CSF in serum is still a administrationInfectionInfectious urine"GM-CSF-Elevatedserum and 187188. matter of some contention. Undoubtedly serum contains he- CSFLow serum and urine 189190191192193194195-198 mopoietic growth factors, but whether GM-CSF, multi-CSF, mononucleosisAcute CSFElevatedlevels of G-CSF, M-CSF, or IL-1 is responsible for the biological re neutropeniaChronic levelsCSFG-CSF withoutinfectionChemotherapyneutropenia levelsnormalElevated sponses has not been studied with the appropriate reagents. A recent study (180) has demonstrated GM-CSF production lym-phomaTherapyfor levelsElevatedCSF by endothelial cells, smooth muscle, and fibroblasts and that anti-neutrophilantibodyHumanwith levelsElevatedCSF these can be stimulated to produce CSF by tumor necrosis factor. Broudy et al. (180) also showed that protein synthesis associatedwithcancers CSF levelsRef.183184185186, was required. Koury and Pragnell (181) demonstrated CSF neutrophiliaSerum production by fibroblasts. The in vivo importance of these observations is unclear. The kinetics of release in vivo and the Table 3 Potential clinical applications of G-CSF and GM-CSF failure of cycloheximide to reduce accumulation of CSFs in 1. Mitigation of chemotherapy induced neutropenia. 2. Improvement of host defense. endotoxin serum suggest that at least one of the CSFs exists in 3. Management of infections. a stored form (e.g., in membrane bound vesicles, in granules, 4. Recovery following marrow transplantation. or even on the surface of cells). The initial release of these 5. Expansion of progenitor cells prior to transplantation. 6. Marrow failure. growth factors is probably connected with their ability to acti 7. Improvement of granulocytc function. vate mature cells rather than to increase cell production. 8. Anticancer effects. Another approach to identifying the tissues and cell types that produce GM-CSF is to identify the cells and tissues where distribution of each of the CSFs in various diseases. Studies of GM-CSF mRNA is present. Recently, Chan et al. (182) used CSF levels in normal and disease states are now under way and full length GM-CSF cDNA as a probe and screened, by dot an enzyme-linked immunosorbent assay is available for moni blot hybridization analysis, 64 tissue samples including liver, toring exogenous bacterially synthesized GM-CSF adminis colon, esophagus, rectum, stomach, breast, lung, skin, and tered to patients (199). This assay also detects native GM-CSF various solid tumors and leukemias. There was no constitutive in serum. production of detectable levels of GM-CSF mRNA in any sample studied. Since smooth muscle, endothelial cells, and Early Clinical Trials with G-CSF fibroblasts would have been present in these tissues and other techniques suggest they synthesize GM-CSF the approach may The potential clinical applications of the CSFs are listed in have been too insensitive. However, it is also possible that the Table 3. One possible application of the CSFs is to prevent or GM-CSF mRNA only appears 8-12 hours after the tissues have reverse the neutropenia that follows cytotoxic chemotherapy been stimulated. Chan et al. (182) did confirm that mRNA for and thus reduce the incidence or duration of infections. Morstyn GM-CSF is present in activated T-lymphocytes and were able et al. (200) have reported a Phase I/II study of G-CSF in to identify the region of the GM-CSF gene involved in regulat patients receiving i.v. melphalan. The patients received G-CSF ing the transcription of the GM-CSF mRNA. for 5 days prior to chemotherapy to assess the effect of G-CSF. The major findings were that G-CSF caused an immediate Clinical Disorders and Abnormal GM-CSF Levels transient fall in circulating neutrophils followed by a rise which was dependent on the dose of G-CSF administered. Circulating There is both direct and indirect evidence that CSFs can levels of neutrophils over 70,000/mm3 were achieved. The control hemopoiesis in vivo. This evidence includes a correlation neutrophils exhibited granulation and Dohle bodies. The major between urine CSF levels and WBC levels (183), a demonstra change in the marrow was an increase in the proportion of tion that tumors that produce CSF are associated with elevated promyelocytes to over 30%. These changes would appear to WBC and that the in vivo administration of purified CSFs reflect initial margination or sequestration of neutrophils fol produces high WBC. Table 2 contains a list of clinical condi lowed by release and new synthesis of neutrophils after 4-5 tions that are associated with altered levels of CSF. In each days. Following melphalan administration the period of neutro case CSF was measured by the marrow bioassay. All of these penia was reduced from 8 days to 0 days in patients who had studies need to be reassessed because the bioassay does not not received prior cytotoxic therapy. Patients responded less distinguish among multi-CSF, GM-CSF, or mixtures of M- well to G-CSF if they had been extensively pretreated with CSF and G-CSF. Also some studies (184, 188) on the distri chemotherapy or radiotherapy. Bronchud et al. (201) have bution of human CSFs were undertaken with murine cells as reported in a study of 12 patients a reduction in the neutropenia targets. The latter assays presumably detected human G-CSF caused by combination chemotherapy in small cell lung cancer, but not human GM-CSF since only human G-CSF acts on and Gabrilove et al. (202) have reported a reduction in neutro murine targets. It is also possible that M-CSF which acts penia in patients receiving combination chemotherapy for blad indirectly to stimulate G-CSF release may have been detected der cancer. These studies involved the administration of G-CSF in some of these studies. In cases such as those associated with during one cycle of chemotherapy. Studies are now required of infections where levels of endotoxin may have been high, indi G-CSF administration for longer periods of time and random rect effects of endotoxin in the bioassay rather than GM-CSF ized studies are required to confirm that G-CSF causes not only levels may also interfere with the assay. More direct assays a reduction in the period of neutropenia but also protection (e.g., a radioimmunoassay) are required to characterize the against bacterial and fungal infections. The pharmacokinetics 5632

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1988 American Association for Cancer Research. HEMOPOIETIC GROWTH FACTORS of G-CSF following i.v. administration reveals at least one hemopoietic series. However, Ruff et al. (207) have reported phase of elimination of about 110 min. Morstyn et al. (200) that at high concentrations GM-CSF inhibits the proliferation also reported a brief first phase lasting for 3 to 8 minutes by of small cell lung cancer cells. It suggests either that malignant bioassay. The best route of administration of G-CSF has not cells may express GM-CSF receptor aberrantly or that these yet been defined; however, we have given 10-day continuous tumors are closely related to hemopoietic cells. Baldwin et al. s.c. infusion with good effect and no local reactions. G-CSF is (218) have reported G- and GM-CSF receptors on small cell well tolerated although bone pain has been reported (200). A lung cancers. We do not know what effect the CSFs will have maximum tolerated dose has not been identified and studies on such tumors or whether we will lose the selective protection have stopped because it was considered that WBC levels above of the marrow. 75,000/mm3 were undesirable. Potential Side Effects of CSF Therapy. The direct effects of the colony stimulating factors will be confined to cells that Early Clinical Studies with GM-CSF contain CSF receptors; however, the stimulated cells may be induced to secrete other biological response modifiers which Three forms of GM-CSF are in clinical trials: mammalian could influence the action of these molecules. These could derived material (Sandoz); material produced by yeast (Immu- include IL-1 and tumor necrosis factor. Accumulation of gran- nex); and bacterial!) synthesized material (Schering Plough). ulocytes and/or monocytes in the lungs, liver, pericardium, and Like G-CSF, GM-CSF causes elevations in WBC levels. At low peritoneum is possible. Such infiltrates have been observed in doses the predominant rise is in neutrophils; however, at higher preclinical studies (76). doses a rise in monocytes and eosinophils is also seen. The Another potential side effect is the development of antibodies immediate effect of GM-CSF is a transient fall in the WBC to CSF. Such antibodies are theoretically more likely to develop level (203) which occurs after both i.v. and s.c. administration in response to glycosylated than to nonglycosylated CSF; how and which is followed by a rise. The best route of administration ever, even nonglycosylated CSF may prove antigenic due to of GM-CSF has not been identified. GM-CSF has been shown minor differences in the three dimensional structure or its state to reduce the period of neutropenia after autologous marrow of aggregation. transplantation from 16 to 10 days (204). It has been shown to Antibodies to hGM-CSF were detected in some of the mon cause elevations in the WBC levels in patients with acquired keys treated with the COS cell derived human GM-CSF (11). immunodeficiency syndrome. CebÃ³n et al. (199) have studied However, in this situation there was a species difference between the pharmacokinetics of GM-CSF and found that after a single the GM-CSF and the animals and the carbohydrate moiety s.c. dose of GM-CSF of 10 Mg/kg the serum level of GM-CSF would be quite different. The antibodies were not neutralizing is sustained above 1 ng/ml for more than 12 h. There would and appeared to be of little pathological significance. therefore appear to be no advantage to continuous i.v. infusions Role of Combinations of Hemopoietic Growth Factors. Many of GM-CSF. This is particularly important because GM-CSF combinations of biological response modifiers will be used in is a chemoattractant and might cause leukostasis and throm an attempt to modify hemopoiesis and phagocyte function and bosis at the catheter tip. The s.c. route of administration, it is a little too early to predict the best combinations of however, has been associated with local rashes in some patients. hemopoietic growth factors, modulators, and cytotoxic drugs. Vadhan-Raj et al. (205) have reported a beneficial effect in A considerable effort will be required to define the in vivo effects patients with myelodysplasia. However, Mertelsmann (206) of the hemopoietic growth factors so that appropriate baseline reported at a recent meeting that in some cases where the data are available from which their use can be optimized. The proportion of blasts in the marrow is greater than 20% GM- actions of GM-CSF and G-CSF are synergistic (209, 210). CSF causes a worsening of the leukemia. An apparent difference However, the testing of the combinations of these growth between the early clinical studies using G-CSF (200-202) and factors should be delayed until we have adequate baseline data GM-CSF (199, 203-205) is that no maximum tolerated dose on the individual molecules. of G-CSF has been identified, whereas there is clearly a maxi The hemopoietic growth factors have been characterized in mum tolerated dose of GM-CSF. The dose limiting toxicity of detail and we have a considerable knowledge of their biology in GM-CSF may reflect direct effects or indirect effects perhaps vitro. The exquisite specificity of these growth factors makes as a consequence of monocyte activation and release of other them potentially powerful modulators of blood cell production cytokines. The toxicities of GM-CSF include bone pain, fever, and function. At present we can only hope that this potential edema, rashes, and serositis, including pericarditis. can be combined with new or current therapies to help trans plant or cancer patients. The results of the initial animal studies Future Directions have been so encouraging that it is tempting to suggest that the Anticancer Effects of GM-CSF. GM-CSF can function as a hemopoietic growth factors will lead to entirely new approaches macrophage activating factor and consequently can promote in oncology. Now that neutrophil levels can be restored more the ability of macrophages to recognize and destroy human quickly, it is essential to develop similar strategies for the melanoma cells. GM-CSF also activates antibody dependent elevation of platelets. However, it is also clear that the control cell cytotoxicity (140). Administered systemically, the CSFs are of hemopoiesis is complex and depends on the interaction of unlikely to have anticancer effects. However, there are clinical many factors. We do not fully understand this network and situations, e.g., ovarian cancer, in which the malignancy is much work remains before we can ensure that the hemopoietic confined to the peritoneal cavity for a significant period of time. growth factors can be used for the maximum benefit of the In this situation the i.p. infusion of GM-CSF and tumor di patient. rected antibodies may be able to stimulate an anticancer effect via the activation of antibody mediated monocyte cytotoxicity. Acknowledgments Effects of Colony Stimulating Factors on Nonhemopoietic Cells. Studies on G-CSF receptors (133) and GM-CSF recep The authors wish to thank Jean Kingett for her care and patience in tors (134) indicate that both are restricted to cells of the the preparation of this manuscript. We are also grateful to Henrike 5633"Â»

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George Morstyn and Antony W. Burgess

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