Biological Activities of Human Granulocyte- Macrophage Colony-Stimulating Factor

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1 Concise Review International Journal of Cell Cloning 6: (1988) Biological Activities of Human Granulocyte- Macrophage Colony-Stimulating Factor Steven C. Clark Genetics Institute, Cambridge, Massachusetts, USA Key Words. GM-CSF Hematopoietic growth factors Interleukin 3 Regulation of hematopoiesis Abstract. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as an important regulation for hematopoietic cell development and function. Within the myeloid lineages, GM-CSF serves as a growth and developmental factor for intermediatestage progenitors between early, interleukin 3-responsive and late granulocyte colonystimulating factor-responsive precursors. GM-CSF also serves as an activator of circulating effector cells. The ability of GM-CSF to induce monocyte expression of tumor necrosis factor, interleukin 1 and other factors, further ties this hormone into a network of cytokines that interact to regulate many hematologic and immunologic responses. The availability of large quantities of recombinant GM-CSF now provides the opportunity and challenge not only for unraveling the mechanisms regulating hematopoiesis, but also for developing new therapies for enhancement of host defense against infection that were not previously possible. Introduction In both mouse and man, families of hematopoietic growth factors have been identified that serve to control the growth and development in culture of the various lineages of hematopoietic cells [I, 21. The members of this family in each species include granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF) and erythropoietin (Epo), factors that are relatively selective in supporting proliferation of neutrophilic granulocyte, macrophage and erythroid progenitors, respectively. In addition, granulocyte-macrophage Correspondence: Steven C. Clark, Genetics Institute, 87 Cambridgehrk Drive, Cambridge, MA 02140, USA. Received August 18, 1988; accepted for publication August 18, /88/$2.00/0 QAlphaMed Press

2 Clark 366 colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3, also known as multilineage-csf) are members of the family that display broader spectra of activities reflecting their abilities to interact with earlier, more multipotent progenitors than the lineage-restricted factors. As might be expected, this rather simple picture has become considerably more complex as investigations from different fields have converged. For example, cytokines thought to be important in B cell development have proved to exert a variety of effects with many different target cell populations [3-51. This has led to the recognition that there is a rather large, complex network of interacting cytokines that plays a major role in regulating the production, development and activation of cells within the immune and hematopoietic systems, as well as in integrating the responses of these systems with those of other systems in the organism. The classical hematopoietic growth factors are now known to constitute only a small subset of the cytokine network; other important members are the interleukins (IL-1 through 1L-7), the tumor necrosis factors (TNFs) and the interferons [6]. Although many of these cytokines were originally thought to be relatively specific for selected target cells, it has become increasingly clear that most of them interact with a variety of cell types, often exerting different effects with different target cells. Initial studies with recombinant cytokines focused on the biological properties of the individual cytokines with specific target cells. However, in vivo we are almost certain that individual cells are never exposed to a single cytokine, but rather a cocktail of factors representing a combination of different members of the cytokine network. The resulting signal presented to the cell may not merely represent the sum of the effects provided by the isolated cytokines. Thus, a clear understanding of the individual factors requires analysis of the biological properties of each factor in isolation and in combination with other members of the cytokine network. While GM-CSF has thus far not proved to have as broad a range of responsive target cells as some of the other cytokines (such as IL-1 or IL-6), it too is clearly an interactive member of the network and must be dealt with in that context. GM-CSF was originally identified as a growth factor capable of supporting the clonal proliferation of normal granulocyte-macrophage progenitors (CFU-gm) in culture [I, 21. From analysis of the colony types supported by natural GM-CSF, MetcaZfer al. [7] recognized that GM-CSF (also known as CSF-a) additionally supported proliferation of eosinophil progenitors, a property not shared with G-CSF. Lusis andkbej7er [8], beginning with conditioned medium from an HTLV II-transformed T cell line demonstrated that the myeloid leukemic cells, KG-I, would form colonies in response to GM-CSF, a property that we exploited to molecularly clone a cdna encoding the human factor [9]. Subsequent analysis of the recombinant GM-CSF has confirmed many of the findings of investigators

3 Biological Activities of Human GM-CSF 367 who analyzed the properties of the natural molecule [7, However, the detailed analysis of the recombinant protein has also greatly expanded our knowledge of GM-CSF as an effector cell activator, as a growth factor for megakaryocytic and erythroid progenitors and as a potential participant, either directly or indirectly, in regulating T cell and B cell function. These studies have underscored the importance of GM-CSF as a member of the cytokine network with much broader effects than orignally recognized. Clearly, all of these effects must be considered in planning clinical applications of GM-CSF. Here I will review what we have learned about the structure and function of GM-CSF and how it interacts with other members of the cytokine network. Structure of GM-CSF Both the natural and mammalian cell-derived GM-CSF are extremely heterogeneous glycoproteins with an apparent range of molecular masses between 14,500 and 35,000 [14]. This extreme size heterogeneity is largely due to a variation in the type and extent of addition of complex carbohydrate to the polypeptide chain at two sites for addition of N-linked carbohydrate (Fig. 1). Elimination of either or both of these sites results in a substantial reduction in the range of species of GM-CSF expressed in mammalian cells [u]. Glycosylation of the polypeptide on two serine residues near the amino terminus generates the remainder of the heterogeneity [El. The mature polypeptide itself is a discrete 127 amino acid polymer held in a bilobed configuration by two disulfide bridges (Fig. 1). Although biologic activity is lost upon reduction, the fully denatured and reduced polypeptide is readily renatured, yielding fully active and uniformly refolded GM-CSF, indicating that the native configuration is very stable. Because of this feature, GM-CSF is readily produced in bacteria. The remarkable size heterogeneity of GM-CSF raises the possibility that different species of the hormone might mediate different subsets of its biologic activities. Thus far, however, comparisons of non-glycosylated GM-CSF expressed in E. coli. with the different species derived from mammalian cells have revealed quantitative, but not qualitative, differences in biologic activity [14]. Interestingly, the heavily glycosylated forms of GM-CSF are significantly less active in vitro in supporting cell proliferation and effector cell function than is the non-glycosylated E. co1i.-derived molecule. This decrease in activity with increase in size also correlates with a decrease in the affinity of GM-CSF for its receptor [16]. Thus, the large amount of carbohydrate, which in the largest forms of GM-CSF accounts for more than 50% of the mass of the molecule, appears to interfere with the interaction of GM-CSF and its receptor.

4 QI oa Fig. 1. Predicted structure of GM-CSF. The amino acid sequence, in single-letter code, was originally determined from the nucleotide sequence of the cdna. This sequence as well as the disulfide structure and sites of carbohydrate modification were confirmed by analysis of complete amino acid sequences of all possible tryptic fragments of the recombinant protein. The different secondary structural elements were predicted from Chou-Fasman analysis of the sequence.

5 Biological Activities of Human GM-CSF 369 If the carbohydrate on GM-CSF actually interferes with the activity of the molecule in culture then what purpose could this modification serve? One clue has come from plasma clearance studies in rats [15]. In these studies, the disappearance of radiolabeled GM-CSF from plasma displayed biphasic kinetics. The initial rapid phase, which is usually termed the alpha phase (a phase), represents distribution of the factor into all accessible extra-cellular compartments in the animal while the longer beta phase (p phase) represents true organ-mediated plasma clearance. Analysis of the kinetics of clearance of fully glycosylated (both N-linked sugar sites occupied), partially glycosylated (only one N-linked site occupied) and non-glycosylated GM-CSF revealed that all three forms of the molecule are cleared (p phase) in the kidney with an apparent half-life of about 15 minutes. Interestingly, however, each molecule displayed substantially different kinetics of distribution (a phase). Little if any of the fully glycosylated GM-CSF left circulation during the a phase and most of the GM-CSF was cleared in the kidneys during the p phase. In contrast, more than 70% of the IV-administered nonglycosylated GM-CSF left the circulation during the initial distribution phase lasting 3-5 minutes. The singly modified GM-CSF was intermediate between the two extremes. These findings at least raise the possibility that the different forms of GM-CSF might distribute differently in the body and, therefore, might function in vivo differently by being more or less effective at reaching different populations of responsive cells. For example, high molecular weight GM-CSF secreted at a distant site of infection might be more effective than the smaller forms in passing through the circulation to reach the marrow to signal the production and release of more neutrophils. Although this possibility is still highly speculative it identifies an interesting and important avenue to be explored before we fully understand the function of GM-CSF in vivo. Growth Factor Activity of GM-CSF Many investigators have characterized in detail the cell lineages in colonies supported by GM-CSF in cultures of normal human hematopoietic progenitor cells [7, 10-13, 17-19]. Although different conditions favor different lineages, and there is frequently considerable donor to donor variation, a reasonably consistent picture has emerged (Table I). In various serum-containing cultures, GM-CSF will effectively support the formation of a variety of colony types containing macrophages, neutrophils and eosinophils. In the presence of erythropoietin, hemoglobinized erythroid colonies as well as mixed colonies appear which frequently contain megakaryocytes. In the presence of M-CSF subliminal concentrations of GM-CSF greatly enhance macrophage colony formation [19], In cultures specifically optimized for megakaxyocyte growth, GM-CSF does serve as a growth factor

6 Clark 370 able I. Activities of human GM-CSF Effect Target cell 1. Growth factor neutrophil progenitors eosinophil progenitors monocyte progenitors erythroid progenitors (in combination with Epo) megakaryocyte progenitors T cells (in combination with IL-2) acute and chronic myelogenous leukemias 2. Survival factor hematopoietic progenitors neutrophils eosinophils monocyteslmacrophages 3. Activating factor neutrophils eosinophils monocyteslmacrophages 4. Cytokine-inducing factor monocyteslmacrophages (induction of TNF and L-1) for megakaryocyte progenitors [13, 181. Thus, GM-CSF clearly interacts with a much broader range of progenitors than originally thought. However, in comparison with IL-3, GM-CSF is less effective in supporting primitive blast cell, erythroid and megakaryocyte colony formation, while in comparision with G-CSF, GM-CSF is less effective in supporting neutrophil colony formation. These observations suggest that within many of the lineages, GM-CSF interacts with committed progenitors after they have been exposed to an early-acting factor such as IL-3, but before they become dependent on late-acting factors such as G-CSF, M-CSF or Epo. The recent development of serum-free culture systems for human hematopoietic cells has provided further confirmation bf this model [20]. Sonada et af. found that in serum-free cultures, none of the individual CSFs will support colony formation, although a combination of factors will in the same cultures. Analysis of all of the combinations of CSFs in the system revealed that within most lineages, colony formation is dependent on the presence of both early- (IL-3 or GM-CSF) and late-acting (G-CSF, Epo) factors to yield colonies. These studies demonstrated that GM-CSF alone will not support colony formation unless serum components (CSFs?) or other CSFs are present in the culture providing further evidence for the requirement for multiple factors in blood cell development. In addition to serving as a growth factor for normal hematopoietic cells, many primary myeloid leukemias and leukemic cell lines are GM-CSF-dependent (or at least GM-CSF-responsive) for growth in culture [8, Analysis of many

7 Biological Activities of Human GM-CSF 371 different patient samples has revealed that almost every leukemia exhibits a different response to GM-CSF and to the other hematopoietic growth factors. In some cases, the leukemic cells proliferate while maintaining their ability to self-renew. In other cases, GM-CSF-supported proliferation occurs along with cellular differentiation resulting in a rapid decline in self-renewal capacity. The propensity to retain or lose self-renewal capacity can also be influenced by combination with other cytokines including IL-1, IL-6, G-CSF, IL-3 and M-CSF. These studies have already begun to provide new insights into the mechanisms of growth regulation of leukemic cells and, perhaps, by extension into the properties of the normal progenitors from which the leukemia was derived. In addition to the myeloid leukemias, several T cell-acute lymphocytic leukemias have been described which have proven to be GM-CSF-responsive [23]. Kupper et al. using the IL-Zdependent T cell line, HT2, as a target purified a novel, keratinocytederived T cell growth factor which proved to be GM-CSF [25]. Such observations led Santoli and colleagues to test GM-CSF and IL-3 for effects on normal human T lymphocytes [26]. Although neither IL-3 nor GM-CSF alone were able to support the long-term proliferation of peripheral human T cells, either factor substantially potentiated the IL-Zdependent proliferation of either resting or mitogen-activated T cells. In such experiments with mixed cell populations, it is always difficult to distinguish direct and indirect effects. Nevertheless, cultures of purified T cells survived and grew longer in the presence of IL-2 and either GM-CSF or IL-3 than in the presence of any of the single factors alone, yielding cultures consisting of greater than 98% CD3' cells after three to four weeks. These results have provided strong evidence that both GM-CSF and IL-3, like IL-4, IL-5 and IL-6, interact with both lymphoid and myeloid cells and suggest the possibility that, in addition to their myeloid growth-promoting functions, they may serve some role, like IL-2, in regulating the immune response. Again it should be remembered that these effects with normal cells were observed only in combination with a second cytokine, IL-2, providing yet another example of cytokine combinations yielding results different from those expected from the isolated factors. GM-CSF Activation of Effector Cell Function Like the other CSFs, GM-CSF has proved to be a potent activator of mature effector cell function [12]. This was recognized by Weisbart et al. who purified a cytokine, NIF-T, that inhibited neutrophil migration in vitro that proved to be identical to GM-CSF Analysis of the effects of GM-CSF revealed that this cytokine primes the neutrophils for enhanced oxidative metabolism causing a pronounced release of superoxide anions in response to second signals such as

8 Clark 372 f-met-leu-phe and leukotriene B4. GM-CSF promotes neutrophil survival in vitro and enhances other neutrophil functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of opsonized yeast [l2]. Similar effects have been observed with GM-CSF-treated eosinophils: the cytokine-treated cells are more effective in ADCC killing of Schistosome larvae and have increased capacity to synthesize and secrete leukotriene C4 in response to calcium ionophore [28]. Interestingly, the lineage-restricted G-CSF, which supports neutrophil progenitor proliferation, displays similar activities with neutrophils, but not eosinophils, while the multilineage factor IL-3 displays the reverse target cell specificity [29]. Mononuclear phagocytes are also highly responsive to GM-CSF. Monocytes cultured in the presence of GM-CSF have enhanced ability to kill tumor target cells [30, 311. The GM-CSF-augmented tumoricidal capacity appears to be mediated by enhancement of macrophage TNF transcription [31]. Actual release of TNF requires a second signal such as that provided by bacterial LPS, phorbol ester or, possibly, tumor cell contact. Similarly, normal monocytes stimulated with GM-CSF have increased capacity to produce both IL-la and IL-l@ [32]. The stimulation of production of these cytokines in culture might be expected to lead to an enhanced primary antibody response: indeed, GM-CSF has been shown to enhance the function of antigen-presenting cells which is at least partly mediated by IL-1 production [33]. The ability of GM-CSF to interact with monocytes and either directly or indirectly with T and B cells, thereby resulting in increased cytokine production (IL-1, TNF, IL-6, etc.), further illustrates the complexity of the cytokine network and the importance of GM-CSF within that network. GM-CSF Stimulation of Hematopoiesis In Vivo As predicted from all of the in vitro studies, GM-CSF has proven to be a potent stimulator of hematopoiesis both in humans and non-human primates. Donahue et al, initially showed that administraiion of GM-CSF to macaques by continuous IV or subcutaneous infusion elicited a rapid and dramatic leukocytosis in dose-dependent fashion [34]. With the higher doses tested (10-20 pg/kg/day), total white counts were 5 to 10 fold above normal (normal in the macaque is about 8,000 cells/pl) within 48 to 72 hours from the onset of infusion. Analysis of differential cell counts revealed that the increase in circulating blood cells was due to increased numbers of neutrophils, eosinophils, monocytes and lymphocytes. The leukocytosis resolved quickly upon termination of the administration; normal values were typically observed within 3-4 days following the infusion. In one experiment, an animal was administered GM-CSF for one month during which time the white count was maintained at 40,000 or higher without signifi-

9 Biological Activities of Human GM-CSF 373 cant side effects. Muyer et ul. extended these studies to show that effector cells isolated from the treated animals were functional and displayed characteristics similar to cells treated in vitro with the cytokine [35]. These studies led to the first clinical trials with GM-CSF in cytopenic AIDS patients who demonstrated hematologic response to the factor much as predicted from the animal studies [36]. Other recent investigations have documented the effects of GM-CSF on hematopoietic reconstitution following irradiation either with or without a bone marrow transplant [ These studies, both in monkeys and in people, have provided some evidence that GM-CSF can accelerate the return of peripheral granulocytes by several days over untreated controls and possibly decrease the risk of infection. Similar encouraging results have been obtained in patients with myelodysplastic syndrome [41]. All of these studies have shown that GM-CSF has considerable potential as a stimulator of the hematopoietic system in patients. Because GM-CSF appears to act within the hematopoietic system at an intermediate stage with a target cell population that is on average at a later stage of development than the targets of IL-3, Donuhue et af. tested the possibility that IL-3 and GM-CSF might reveal synergistic interactions in vivo [42]. To test this prediction, an animal was pretreated with IL-3 for seven days (a treatment which has only a modest effect on the level of circulating peripheral blood cells) followed by a low dose of GM-CSF. This low dose of GM-CSF elicited a rapid and dramatic leukocytosis that was equivalent to that achieved only with much higher doses of GM-CSF by itself. This observation supports the model that IL-3 elicited an expansion in a population of GM-CSF-responsive progenitors, thereby priming the hematopoietic system to become hyper-responsive to GM-CSF, The experiment further suggests that administration of combinations of cytokines in vivo may well have much more dramatic effects that achieved with either of the individual cytokines and that such combinations may be designed to directly attack particular clinical problems. Conclusions Progress in understanding the actions of GM-CSF as well as of the other CSFs has been rapid indeed. In the early 1980 s, the various factors were only poorly characterized activities in samples of medium conditioned by various cell lines. Now, however, gram quantities of each of the recombinant CSFs have been prepared and many clinical as well as animal investigations are underway. Significant progress has already been made in designing protocols that should ameliorate the myelosuppression induced by radiation or chemotherapy, thereby demonstrating tremendous potential for helping cancer patients. And these studies

10 Clark 374 have only begun. The next few years should witness an explosion in the numbers of patients treated with the recombinant CSFs. The availability of the recombinant GM-CSF has also greatly facilitated our analysis of the actions of this hormone in vitro. GM-CSF by itself serves as a growth and differentiation factor for intermediate stages of development in the neutrophilic, monocytic, eosinophilic, megakaryocytic and erythroid lineages. However, GM-CSF alone is insufficient (at least in serum-free culture) for supporting the terminal stages of development in each of these lineages as revealed in the requirement for secondary late-acting factors such as G-CSF, M-CSF and Epo. Because GM-CSF acts on intermediatestage progenitors, combinations with either early-acting (IL-3) or late-acting myeloid growth factors often yield non-additive responses. The ability of GM-CSF to enhance the IL-2-dependent proliferation of T cells extends the growth-promoting capacity of GM-CSF to include at least some lymphoid cells as well. The role of GM-CSF as an effector cell activator greatly extends the potential for this cytokine as a regulator in different systems. In addition to enhancing the function of the effector cells in host defense, GM-CSF also enhances production of other physiologic regulators by granulocytes, monocytes and macrophages. The mononuclear phagocyte appears to be one of the key regulatory cells in the cytokine network. Stimulation of these cells to produce IL-1 and TNF should initiate a cascade of production of many cytokines, including GM-CSF, by fibroblasts, endothelial cells and lymphocytes. Mechanisms that are still not understood must come into play to prevent infinite cellular activation with resulting cytokine production that would have catastrophic consequences. Future analysis of both positive and negative regulation of cytokine gene expression will be required to analyze these mechanisms. Finally, the realization that GM-CSF functions within a much broader cytokine network that coordinates many different physiologic responses has focused attention on combinations of growth factors. Within the network, one cytokine frequently elicits a cascade of multiple cytokines (GM-CSF being a prominent one) and responsive target cells are usually not exposed to individual cytokines but more likely a complex mixture of factors. These combinations often elicit very different responses than achieved with the individual components of the mixtures. These observations still leave open considerable room for analysis of the effects of GM-CSF in combination with other cytokines both in vitro and in vivo. Although GM-CSF by itself has shown tremendous potential in the clinic, in the long run that potential may be greatly expanded through careful combination with other cytokines.

11 Biological Activities of Human GM-CSF 375 Acknowledgments I wish to thank R. &en, R. Hm'ck and M. Recny for performing the detailed structural analysis of GM-CSF and many helpful discussions; all of the members of the Hematopoiesis Program at Genetics Institute for their contributions to the work and f? Holding for preparation of the manuscript. References Metcalf D. The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood 1986;67: Clark SC, Kamen R. The human hematopoietic colony-stimulating factors. Science 1987,236: Peschel C, Paul WE, O'Hara J, Green I. Effects of B cell stimulatory factor-l/ interleukin-4 on hematopoietic progenitor cells. Blood 1987;70: Sanderson CJ, O'Garrao A, Warren DJ, Klaus GGB. Eosinophil differentiation factor also has B-cell growth factor activity: proposed name interleukin 4. Proc Natl Acad Sci USA 1986;83: Wong GG, Witek-Giannotti JS, Temple PA, et al. Stimulation of murine hemopoietic colony formation by human IL-6. J Immunol 1987;140: Wong G, Clark S. Multiple actions of interleukin 6 within a cytokine network. Immunology Today 1988;9: Metcalf D, Begley CG, Johnson GR, et al. Biologic properties in vitro of recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1986; Lusis A, Koeffler HP. Action of granulocyte-macrophage colony-stimulating factors: studies using a human leukemic cell line. Proc Natl Acad Sci USA Wong GG, Witek JS, Temple PA, et al. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 1985;228: Tomonaga M, Golde DW, Gasson JC. Biosynthetic (recombinant) human granulocytemacrophage colony-stimulating factor: effect on normal bone marrow and leukemic cell lines. Blood 1986; Sieff C, Emerson SG, Donahue RE, et al. Human recombinant granulocytemacrophage colony-stimulating factor: a multilineage hematopoietin. Science 1985;230: Lopez AF, Williamson DJ, Gamble JR, et al. Recombinant human granulocytemacrophage colony-stimulating factor stimulates in vitro mature human neutrophil and esoinophil function, surface receptor expression, and survival. J Clin Invest 1986;78: Mazur EM, Cohen JL, Wong GG, Clark SC. Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells. Exp Hematol 1987;15: Wong GG, Witek JS, Temple PA, et al. Molecular cloning of human and gibbon T-cell-derived GM-CSF cdnas and purification of the natural and recombinant human proteins. Cancer Cells 1985;3:

12 Clark Donahue RE, Wang EA, Kaufman RJ, et al. Effects of N-linked carbohydrate on the in vivo properties of human GM-CSF. Cold Spring Harbor Symposia on Quantitative Biology. New York: Cold Spring Harbor, 1986;LI: Gasson J, Golde D. Personal communication. 17 Leary AG, Yang YC, Clark SC, Gasson JC, Golde DW, Ogawa M. Recombinant gibbon interleukin 3 supports formation of human mulitlineage colonies and blast cell colonies in culture: comparison with recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1987;70: Messner HA, Ymasaki K, Jamal N, et al. Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci USA 1987;84: Caracciolo D, Shirsat N, Wong GG, Lange B, Clark SC, &era G. Recombinant human macrophage colony-stimulating factor (M-CSF) requires subliminal concentrations of granulocyte/macrophage (GM)-CSF for optimal stimulation of human macrophage colony formation in vitro. J Exp Med 1987;166: Sieff CA, Niemeyer CM, Nathan DG, et al. Stimulation of human hematopoietic colony formation by recombinant gibbon multi-colony-stimulating factor or interleukin 3. J Clin Invest 1987;80: Sonoda Y, Yang YC, Wong GG, Clark SC, Ogawa M. Analysis in serum-free culture of the targets of recombinant human hemopoietic growth factors: interleukin 3 and granulaytelmacrop~e-colony-stimulating factor are specific for early developmental stages. Proc Natl had Sci USA 1988;85: Griffin JD, Lowenberg B. Clonogenic cells in acute myeloblastic leukemia. Blood 1986;68: Santoli D, Yang YC, Clark SC, Kreider BL, Caracciolo D, Rovera G. Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-la, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM- CSFdependent leukemic cell lines. J Immunol 1987;139: Hoang T, Nara N, Wong G, Clark S, Minden MD, McCulloch EA. Effects of recombinant GM-CSF on the blast cells of acute myeloblastic leukemia. Blood 1986;68: Kupper T, Flood P, Coleman LD, Horowitz M. Growth of an interleukin 2/interleukin 4dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). J Immunol 1987;138: Santoli D, Clark SC, Kreider BL, Mash PA, Rovera G. Amplification of IL-2driven T cell proliferation by recombinant human IL-3 and granulocyte-macrophage colonystimulating factor. J Immunol 1988;141: Weisbart RH, Golde DW, Clark SC, Wong GG, Gasson JC. Human granulocytemacrophage colony-stimulating factor is a neutrophil activator. Nature 1985;314: Silverstein DS, Owen WF, Gasson JC, et al. Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocytemacrophage colony-stimulating factor. J Immunol 1986;l37: Lopez AF, To LB, Yang YC, et al. Stimulation of proliferation, differentiation, and function of human cells by primate interleukin 3. Proc Natl Acad Sci USA 198x84:

13 Biological Activities of Human GM-CSF Grabstein KH, Urdal DL, fishinski RJ, et al. Induction of macrophage tumoricidal activity by granulocyte-macrophage colony -stimulating factor. Science 1986; Cannistra SA, Rambaldi A, Spriggs DR, Herrmann F, Kufe D, Griffin JD. Human granulocyte-macrophage colony-stimulating factor induces expression of the tumor necrosis factor gene by the U937 cell line and by normal monocytes. J Clin Invest 1987;79: Sisson SD, Dinarello CA. Personal communication. Momssey PJ, Bressler L, Park LS, Alpert A, Gillis S. Granulocyte-macrophage colonystimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells. J Immunol 1987;139:11l Donahue RE, Wang EA, Stone DK, et al. Stimulation of haematopoiesis in primates by continuous infusion of recombinant human GM-CSF. Nature 1986;321: Mayer P, Lam C, Obengus H, Liehl E, Besemer J. Recombinant human GM-CSF induces leukocytosis and activates peripheral blood poly morphonuclear neutrophils in nonhuman primates. Blood : Groopman JE, Mitsuyasu RT, DeLeo MJ, Oette DH, Golde DH. Effect of recombinant human granulocyte-macrophage colony-stimulating factor on myelopoiesis in the acquired immunodeficiency syndrome. N Engl J Med 1987;317: Nienhuis AW, Donahue RE, Karlsson S, et al. Recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) shortens the period of neutropenia after autologous bone marrow transplantation in a primate model. J Clin Invest : Monroy RL, Skelly RR, MacVittie TJ, et al. The effect of recombinant GM-CSF on the recovery of monkeys transplanted with autologous bone marrow. Blood : Monroy RL, Skelly RR, Taylor P, Dubois A, Donahue RE, MacVittie TJ. Recovery from severe hematopoietic suppression using recombinant human granulocytemacrophage colony-stimulating factor. Exp Hematol 1988;16: Brandt SJ, Peters WP, Atwater SK, et al. Effect of recombinant human granulocytemacrophage colony-stimulating factor on reconstitution after high dose chemotherapy and autologous bone marrow transplantation. N Engl J Med 1988;318: Vadhan-Raj S, bating M, LeMaistre A, et al. Effects of recombinant human granulocyte-macrophage colony -stimulating factor in patients with myelodysplastic syndromes. N Engl J Med : Donahue RE, Seehra J, Metzger M, et al. Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates. Science (in press).