International Journal of Cell Cloning 8:374-390 Suppl 1 (1990)

Disease States Induced by Hemopoietic Growth Factor Excess: Their Implications in Medicine Donald Metcalf The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

Key Words. Colony-stimulating factor Leukemia inhibitory factor damage * Pathological changes Excess hemopoiesis

Tissue

Abstract. Sustained excess levels of hemopoietic regulators can induce a variety of disease states in mice in addition to the anticipated hyperplasiaof the responding hemopoietic lineages. In all the models examined so far, there is a complicating problem that at least some responding cells are also producing the excess regulator concerned. The development of the various disease states may therefore not necessarily be the simple consequence of overstimulation by excess regulator levels. The various disease states develop rapidly in a high proportion of animals and should serve as useful models for a variety of disease states in man.

Introduction The use of various in vitro culture systems for hemopoietic cells has allowed the identification of a group of glycoproteins able to control the proliferation, differentiationand functional activity of hemopoietic cells. In the case of the paired granulocyte-macrophagelineage, five glycoproteins have been identified as proliferative stimuli: granulocyte-macrophagecolony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), multi-potential colony-stimulating factor (Multi-CSF or IL-3) and interleukin 6 (IL-6) [l]. Receptors for a sixth molecule, leukemia inhibitory factor (LIF), exist on monocyte-macrophages [2], and while not appearing to act as a CSF, LIF may have other actions on monocyte-macrophages in the above categories. The cloning of cDNAs for each tactor and the demonstration that recombinant factors have equivalent biological activity to the native molecules have allowed studies on the in vivo actions of these regulators [3]. In general, each appears Correspondence: Professor D. Metcalf, The Walter and E l k Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, 3050 Victoria, Australia. Received September 18, 1989; accepted for publication September 18, 1989. 0737-1454/90/$2.00/00AlphaMed Press

Metcalf

375

to produce observable effects in vivo in agreement with the expectationsof in vitro studies. Extensive clinical trials have now been initiated using recombinant GMCSF and G-CSF with early trials on Multi-CSF and M-CSF [4]. These have shown in a variety of patients that GM-CSF and G-CSF can stimulate elevations of granulocyte and/or monocyte and eosinophil levels in the peripheral blood, and the CSFs should therefore prove to be of value in enhancing resistance to a variety of infections. Based on these developments,the time seemed appropriate to explore the consequences of excess stimulation of granulocytes, macrophages and allied populations by these same regulators using several model systems to achieve sustained high levels of each factor. Such studies can be used to address a variety of questions, such as: 1) Can such models predict patterns of adverse responses likely to be encountered clinically in patients injected with the factor? 2) Does chronic excess stimulationof one lineage lead to severe stem cell depletion or major depletions in other lineages? 3) Does chronic excess stimulation lead to leukemic transformation? 4) Does chronic excess stimulation lead to tissue injury because of the ability of these agents to stimulate mature cells to produce excessive levels of factors that are toxic? 5 ) Do induced tissue lesions provide possible clues to the mechanisms underlying a variety of chronic disease states of previously unknown pathogenesis? This discussion will concentrate mainly on the last two questions and consider the nature and possible implications of various disease states arising in animals with excess levels of the CSFs or LIE

General Considerations Before consideringthe results from the various model systemsused, the known end-cell actions of the various molecules need to be summarized, since induced products of these cells are the most likely agents responsible for any tissue lesions developingin the presence of excess levels of these molecules. The cell type most likely to generate toxic products is the monocyte-macrophage. Studies have shown that GM-CSF or M-CSF can stimulate these cells to produce interleukin 1 (IL-1) [5], tumor necrosis factor (TNF) [6], prostaglandin E [7], interferon [8], plasminogen activator [9, 101 and other regulatory molecules such as G-CSF and M-CSF [ll, 121. Fewer receptors exist on monocytes and macrophages for G-CSF or Multi-CSF [l3,141 and these molecules might therefore be less likely to have comparable actions. In principle, GM-CSF should be able to stimulateeosinophils to release toxic products, but studies so far have only documented an enhanced production of leukotriene [U]. Similarly, GM-CSF, G-CSF and Multi-CSF (the latter in the mouse only since human neutrophils lack Multi-CSF receptors) are able to stimulate the functional activity of mature neutrophils, e.g., stimulating leukotriene [16] and

Metcalf

376

superoxide production [17] and enhanced antibody-mediated cytotoxicity [18]. Multi-CSF could stimulate mast cells to release mediators of inflammation and histamine as agents likely to elicit tissue damage [19]. A recent report stated that GM-CSF and G-CSF can directly stimulate the chemotaxis and proliferation of endothelial cells [20], but it remains speculative whether such actions in vivo would result in tissue damage. While defined products of viable end cells are potentially able to cause tissue damage, there is an important additional source of damaging products from such cells. Although the CSFs can enhance the survival of mature cells in vitro [21], whether this also occurs in vivo is not known. Regardless of whether such an action occurs in vivo, most mature granulocytes, monocytes and eosinophils are likely to have a short lifespan and many will break down in vivo. If the total number of such cells is increased markedly by CSF-stimulated production, greater numbers of cells need to be destroyed. If the elimination system becomes overloaded or breakdown is occurring in unusual tissue sites, it is likely that a wide variety of rather non-specific proteolytic enzymes and other potentially damaging products of degradingcells will be released and possibly damage adjacent cells. In general terms, therefore, actions of the CSFs might be predicted as being able to cause tissue damage which might be particularly evident at sites of macrophage accumulation or might be systemic if high circulating levels of an agent, like TNF, were generated. The magnitude of such damage should correlate with the intensity of stimulation by the factors, and thus be most readily demonstrable in situations where chronically elevated levels of factors have been generated.

Tissue Lesions Induced by Excess GM-CSF Levels Information on the toxic consequences of excess GM-CSF levels has come from studies on GM-CSF transgenic mice and from irradiated mice repopulated by hemopoietic cells in which dysregulated GM-CSF cDNA has been inserted using a retroviral vector. The disease states observed in the two systems have many similarities, suggesting that the likely initiating cause of the diseases is either excess circulating and local levels of GM-CSF or the abnormal behavior of hemopoietic cells when a growth factor to which they are responsive is being produced. Two independent lines of GM-CSF transgenic (C57BL x SJL) F2mice were developed in which insertion of the transgene with regulation by a Moloney virus LTR promoter had occurred in one line in an autosomal chromosome (male line) and in the other on an X chromosome (female line) [22]. Observations have now been completed on 5,000 such transgenic mice. Both transgenic lines exhibit a drastically shortened lifespan (mean survival time 145 days for male-line mice and 95 days for female-line mice). In both lines, the levels of serum GM-CSF are equally elevated approximately 80-to 100-fold (2,000-4,000U/ml) [22, 231. The lines differ in that GM-CSF is not detectable in the urine of female-line mice or in female mice of the male line, but is present in concentrations often exceed-

Diseases from Growth Factor Excess

377

ing serum levels in the urine of all males of the male line [23]. The basis for this difference has not been clarified completely. Female kidneys clear injected GMCSF less efficiently than do male kidneys, and the transgenic bladder of male mice produces more GM-CSF in organ culture than the bladder from female-line mice [23], but these differences alone seem unsatisfactory to account wholly for the dramatic differencesobserved. No specific lesions have been observed in the kidneys of either transgenic line other than a general atrophy associated with the cachectic state exhibited by many of these transgenic mice. Litter size is normal in transgenic matings, and transgenic mice appear in good health for the first two months of life with the notable exception that the eyes are opaque from the time the eyelids open [22]. This is an invariable lesion of transgenic mice of both lines and is based upon macrophage infiltration in the anterior and posterior eye chambers with progressive destruction of the photoreceptor layer of the retina, leading to retinal detachment and breakdown. The overall volume of the eyes and the size of the eye sockets are also usually abnormally small. No obvious organ abnormalities are evident in apparently healthy two-monthold transgenic mice with the striking exception of the presence of massive numbers of cells (mainly macrophages) in the peritoneal and pleural cavities [22,24]. These levels are significantly higher in male-line mice (mean 100 x lo6cells in the peritoneal cavity versus a normal number of 2 X lo6to 4 X lo6)than in femaleline mice (mean 50 x lo6cells) and comparable cell numbers are present in the pleural cavity. The origin of these massive cell numbers is unclear. Little mitotic activity is evident in local cell populations; white blood cell levels are normal as are total hemopoietic and progenitor cell numbers in the marrow and usually the spleen. A point of some importance is that despite continuous exposure to high levels of GM-CSF, the GM-CSF receptors on relevant cells are not downregulated and are present in a normal pattern and number on differentiating marrow and spleen populations. Indeed, the enlarged transgenic macrophages of the peritoneal and pleural cavities express significantly higher numbers of receptors (2-4 fold) than corresponding normal macrophages in these locations. Therefore, these cells are equipped to respond to stimulation by GM-CSF. Thus far, the only systematic study undertaken on these transgenic mice on a GM-CSF-stimulated macrophage product of potential toxicity has been an analysis of IL-1 levels. IL-1 levels were shown to be elevated in peritoneal and pleural cavity fluids and often in the circulation (with serum levels up to 100 U/ml) [25]. In general, IL-1levels in the two cavities paralleled macrophage cell numbers and the concentration of GM-CSF in these locations. The disease states developing in the two transgenic lines exhibit characteristic differences. Most female-line mice develop a cachectic state with fine muscle tremors and terminally, hind leg paresis or paralysis. At autopsy, all organs are atrophic and one-third of the mice exhibit a blackened small bowel based on congestion, with microscopic hemorrhages into the gut cavity [24]-a lesion typical

Metcalf

378

of a local action of TNF, particularly in the presence of endotoxin. The most prominent tissue lesion in more than 90% of female-linemice is the large focal infiltrate in skeletal muscle tissue associated with local destruction of muscle cells and an overall reduction in muscle cell size [22,24] (Fig. 1). The focal infiltrates initially are composed mainly of macrophages, but in advanced lesions eosinophils, granulocytes and lymphocytes are also present. The lymph nodes are usually atrophic, and most often the spleen is atrophic with a marked reduction in lymphoid follicles and sclerotic thickening of the spleen capsule and trabeculae. All other organs show a general reduction in the size of parenchymal cells, paralleling the cachechtic state of the animals. Many male-line transgenic mice develop a similar wasting syndrome, but about one-third become moribund without cachexia and, in these the spleen is enlarged and contains an excess population of hemopoietic cells, particularly erythroid cells. Inflammatory foci are present in the skeletal muscle of 50%of male-line mice but are usually smaller in size than in female-line mice [24]. Unique to male-line mice is the development in 50-70%of the mice of multiple fibrotic nodules in the pleural and particularly the peritoneal cavity, most typically seen as small nodules on the abdominal surface of the diaphragm [22, 241. The second model achieving elevated serum GM-CSF levels involved the insertion, using the Zen retroviral vector of GM-CSF cDNA under the control of the strong MPSV LTR promoter [26]. Within two weeks, irradiated recipients of these marrow cells exhibited a mean level of 3 x lo5U of GM-CSF/ml in the serum, and within three weeks most mice became seriously ill with wasting and hind leg paralysis similar to that seen in transgenic mice. These mice exhibited focal muscle infiltrations in the heart and skeletal muscle similar to those seen in transgenic mice but, in addition, massive areas of proliferating granulocytic and macrophagic populations in the lung and liver. This was associated with extreme thymus atrophy. Some ocular lesions were noted, resembling those in transgenic GM-CSF mice. The interpretation of the tissue lesions developing in both GM-CSF models is that the lesions are likely to be the consequence of GM-CSF stimulation of macrophages to produce tissue-damaging products, including TNF, fibroblast growth factor (FGF), IL-1and y-interferon. In both models, a possible complication arises because the macrophages themselves are producing GM-CSF, and this intracellular source of CSF might lead to deranged macrophage function. The different lesions in the two transgenic lines are paralleled by morphological and functional differences between their macrophages, suggesting that the insertional site of the transgene has also significantly altered macrophage function [24]. Because of these complications, the lesions cannot be assumed to represent the simple consequences of overstimulation of otherwise normal cells by highcirculating GM-CSF levels. However, the development of pericarditis in patients on high GM-CSF dosage [27] may be based on lesions similar to the pericarditis, myocardial infiltration and pericardial nodules noted in the models.

Diseases from Growth Factor Excess

379

Fig. 1. Skeletal muscle from a GM-CSFtransgenic mouse showing part of a focal infiltration of macrophages and other cells with destruction of adjacent muscle cells.

Are there any disease states in which lesions resembling those of these transgenic mice occur and which therefore might be based on similar mechanisms? The childhood disease malignant histiocytosis exhibits tissue lesions identical to those in transgenic GM-CSF mice, and this disease warrants careful analysis on this basis. Similarly, a variety of chronic inflammatory states, such as polymyositis, could have a similar pathogenesis and such diseases also warrant analysis for evidence either of high-circulating GM-CSF levels or of an acquired capacity of the cells in the lesions to produce GM-CSF. No vascular lesions have been seen so far in mice with elevated GM-CSF levels, but local production of CSF by or acting on sub-endothelial macrophages is possibly of relevance in the development of atherosclerosis [28]. The predisposition for inflammatory foci to be localized in the eye and skeletal muscle is curious, but both are sites normally of macrophage foci during ontogeny. For example, a normal three-week-old mouse exhibits skeletal muscle tissue that contains focal aggregates of cells similar to those seen in transgenic lesions. These aggregates presumably are involved in organ remodeling, but are not associated with overt muscle damage and do not produce excess levels of TNF or IL-1(Lang R4,personal communication). Similarly, macrophage-like cells are normally present in the developing eye. It may be that in situations with excess GM-CSF levels such foci become persistent, rather than transient, and with the hyperactivity of the macrophages elicit progressive tissue damage. This might ex-

Metcalf

380

plain the lesion distribution in transgenic mice where the transgene might be expressed early in life, but would not account for the development of comparable lesions in adult transplanted mice. An alternative is to postulate that these locations persist in adult life as potential sites of macrophage localization.

Tissue Lesions Resulting from Excess G-CSF Lesions It might have been predicted that by stimulating polymorphs G-CSF would elicit a variety of tissue damage comparable, for example, with that seen in the acute respiratory syndrome. However, in patients G-CSF has exhibited no doselimiting toxicity [29]. Similarly, irradiated mice transplanted with G-CSF-producing hemopoietic cells develop massive elevations of serum G-CSF (up to 1 x lo6 U/ml) and of circulating granulocytes (up to 1 X lo5 U/ml). However, this excess production and possible functional stimulation of polymorphs elicits virtually no tissue damage [30]. The mice remain in good health, exhibit no major deficiency in hemopoietic cell production in other lineages, and exhibit no extramedullary hemopoiesis and no histological abnormalities in non-hemopoietic tissue other than a not very remarkable excess number of granulocytes in the lung.

Tissue Lesions from Excess Multi-CSF Lesions The anticipated novel aspect of excess stimulation by Multi-CSF was an increase in mast cell populations, possibly resulting in tissue damage, since MultiCSF is the only CSF stimulating the proliferation of mast cells 111. Irradiated mice injected with Multi-CSF-producing hemopoietic cells developed a lethal syndrome [31,32], but with death occurring significantly later than in GM-CSF mice (mean survival time 80 days). A unique aspect of these mice was a pronounced tendency to scratch, in some cases resulting in loss of pinnae and multiple skin lesions containing mast cells, usually with ulceration and infection probably as a consequenceof scratching [32]. The mice showed large increases in monocytes, macrophages, eosinophils, neutrophils and nucleated-erythroidcells in the spleen, blood and peritoneal cavity with gross spleen enlargement. Massive accumulations of mast cells were present in the spleen (the major site of mast cell accumulation following the injection of Multi-CSF), and this was accompanied by a curious fibrotic reaction surrounding the spleen capsule and containing many mast cells. The liver was extensively infiltrated by hemopoietic cells as were the lung alveoli, where the dominant infiltrating population was neutrophils. The development of inflammatory foci in heart and skeletal muscle tissue was highly variable with some mice showing no lesions. Where lesions were present they contained macrophages, neutrophils, eosinophils and occasional mast cells and megakaryocytes.

Diseases from Growth Factor Excess

381

Lesions from Excess Erythropoietin (Epo) Levels Transgenic Epo mice [33] developed a moderate polycythemia with extensive extramedullary hemopoiesis. A more extreme model has been generated in this laboratory by retroviral insertion of the Epo cDNA into hemopoietic cells and repopulation of irradiated recipients by such cells. Levels of serum Epo reached up to 10 U/ml, and the mice developed hematocrits reaching 90%.The mean survival time of the animals was 70 days with death occurring from vascular accidents (villevul J-L and Merculf D,unpublished data). The tissues exhibited remarkably few histological abnormalities other than uniform engorgement and expansion of all vessels. These animals developed grossly enlarged spleens containing a high percentage of nucleated erythroid cells, but showed minimal extramedullary hemopoiesis in the liver and none in other locations.

Tissue Lesions from Excess LIF Levels The LIF was purified and cloned on the basis of its capacity to induce differentiation in M1 leukemic cells [34-361. LIF has yet to be convincingly demonstrated to be a major regulator of hemopoietic cells, although it is a proliferative stimulus for one continuous hemopoietic cell line (DA1) [37], has proliferative effects on a m-transformedmurine erythroid cell line (Cory S4, Muekawa Tand MercuFD, unpublished data) and has been reported to have some action on human blast progenitor cells [38]. Furthermore, there are LIF receptors on monocytes and macrophages [2], and potentially, LIF could be a functional stimulus for these cells. Many of the diverse actions of LIF may represent direct effects since LIF receptors are present on a wide variety of cells, including osteoblasts, liver cells, embryonic stem cells, adrenal cortex cells and placental cells. LIF has a correspondingly wide series of reported actions. LIF is the hepatocyte-stimulating factor III inducing release by liver cells of acute phase proteins [39], can release calcium from bone tissue [40], can modify the action of autonomic nerves, is a lipoprotein lipase inhibitor [41]-the differentiationinhibitory factor @IA) preventing spontaneous differentiation in commitment in normal embryonic stem cells [42,43] and HILDA [37]-although the eosinophil-stimulating activity claimed for HILDA has not been observed with recombinant LIF, and no receptors for LIF are present on eosinophils. The effects of chronic elevation of LIF levels have been determined by transplanting FDC-PI cells into syngeneic DEW2 mice, a procedure leading to engraftment of the marrow,spleen and lymph nodes by a 510% population of FDC-P1 cells. Serum levels of up to 1,OOO U LIF/ml versus undetectable levels in normal mice were achieved in grafted animals by employing FDC-P1 cells in which LIF cDNA was inserted (using the Zen retrovirus which contained the MPSV LTR promoter) and were selected to be high LIF producers (producing up to 1 U LIF/ cell/day) [MI.

Metcalf

3 82

Fig. 2. Marrow cavity from a mouse engrafted with FDC-PI cells producing LIE Note abnormal formation of new bone trabeculae, the presence of excess numbers of osteoblasts and the depletion of pre-existing hemopoietic tissue.

Mice bearingLIF-producingFDC-P1 cells (LIF/FD mice) developed a mpidly fatal syndrome (mean survival time in unirradiated recipients 50 days and in irradiated recipients 22 days). The syndrome was characterizedby progressive weight loss with a curious state of imtability and hypermobility. At post-mortem, all subcutaneous and abdominal fat was absent, the bones were filled by osteoblasts and newly formed osteoid tissue (Fig. 2) with compensatory splenomegaly and extramedullary hemopoiesis in the spleen and liver [44,45]. Other lesions seen in virtually all mice were foci of calcification in the heart, skeletal muscle and sometimes liver, liver necrosis and fibrosis, loss of the inner adrenal cortex, thymus atrophy, pancreatic edema and necrosis, often accompanied by mononuclear cell infiltration, failure of spermatogenesisand defective formation of ovarian corpora lutea. Initial studies using injections of 1 X lo5U LIF three times daily have duplicated some of these changes. The interpretation of the pathogenesis of such a variety of tissue lesions is complex, since both direct and indirect actions of LIF might be involved. The major affected cell types-osteoblasts, liver cells, pancreas cells and inner adrenal cortex cells-are known to exhibit LIF receptors, and actions on these tissues could be direct responses to LIF. The lipoprotein lipase inhibitory action of LIF could be responsible for the curious loss of body fat in these mice, while their irritable behavior might be a consequence of the action of LIF on autonomic nerve signal-

Diseases from Growth Factor Excess

383

'hble I. Murine models of excess factor levels Regulator

Model

GM-CSF

Transgenic-Male line -Female line Irradiated, repopulated Irradiated. repopulated Irradiated, repopulated Irradiated, repopulated Transgenic Irradiated, repopulated FDC-PI repopulated

G-CSF Multi-CSF (IL-3) Epo LIF

Mean factor Mean survival levels in time (days) serum (U/ml) 2,000-4,ooO 2,000-4,ooO 300,000 105-107

1o,o0O-2o,o00 30 m U 0.2-50 50-1,ooO

145 95

200

Reference

22, 24 22, 24 26 30

-

31

< 35 > 365

32 33

70 20-50

44.45

ing. Other changes, such as thymus atrophy, may be secondary to body weight loss and/or adrenal changes. The ability of LIF to induce the production of other biologically active molecules is in obvious need of exploration.

Discussion This approach to the abnormal biology of hemopoietic regulators is obviously only in its initial phases. All of the models used so far (Table I) have a common

problem complicating the interpretation of the results, namely the unknown consequences of hctor production within the responding end cells. Even if this complexity proves to be inconsequential, there remains the difficulty in identifLing candidate mediator molecules elicited by regulator stimulation and in distinguishing their effects from possible direct actions of the regulators themselves on nonhemopoietic cells. This latter problem is obvious with LIF, but will become an equal problem with the broadly acting IL-6 and remains as a potential problem even with regulators such as GM-CSF and G-CSF. The characterization of the actual mechanisms operating in a single lesion, such as the focal inflammatory foci with associated muscle damage in mice with excess GM-CSF levels, will require a major research program and may remain partially unresolved until all possible candidate mediator molecules have been identified and monitored. At the practical level, it can be concluded that excess levels of some regulators seem to induce remarkably little tissue pathology. The best current examples of such regulators are G-CSF and Epo.Conversely, GM-CSF has emerged as a consistent inducer of tissue damage, both in the form of inflammatory lesions and, in a generalized form, as cachexia. While the models used are so complex that there is no necessary reason why this ranking of toxicity should also be seen with

Metcalf

384

injected regulators in vivo, it is nonetheless interesting that GM-CSF has emerged clinically as being more prone in high doses to induce serious adverse responses than Epo, G-CSF and possibly Multi-CSF. The present type of experimental approach has a more positive aspect than merely demonstrating toxic effects of excess regulator levels. The regular development of characteristic lesions raises the possibility that important pathogenic mechanisms have been uncovered for some naturally occurring disease states. Inflammatory foci and nodules as seen with GM-CSF resemble lesions found in the number of disease states ranging from polymyositis and rheumatoid arthritis to malignant histiocytosis. This suggests the value of exploring the possibility that cells in such lesions are producing abnormal levels of CSF or, less likely, that circulating levels of GM-CSF are elevated. A similar line of reasoning can be applied to other types of lesions. For example, is there evidence of abnormal transcription or production of LIF in osteosclerosis, pancreatitis, ovarian dysfunction, etc.? At the clinical level such an approach will be slow because of difficulty in obtaining tissue specimens.However the experimental lesions are readily reproducible and provide valuable models for exploring disease states for which no previous models existed. Thus, regardless of whether these diseases are usually initiated by excess regulator levels, the lesions can still be induced by this maneuvre to generate animal models for useful studies. In one respect the studies on excess factor levels were clearly negative. Hyperstimulation of cell proliferation in otherwise normal hemopoietic cells produces massive hyperplasia, but not leukemic transformation. This information is relevant and useful at the clinical level. More important, animals of this type can be used to explore the action of proto-oncogene products, e.g., of myc, myb or abl to establish what combinations result in leukemic transformation.

References Metcalf D. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature 1989;339:27-30. Hilton DJ, Nicola NA, Metcalf D. Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells. Proc Natl Acad Sci USA 1988;85: 591-5975. Metcalf D. The molecular control of blood cells. Boston: Harvard University Press, 1988:l-165. Morstyn G,Lieschke G,Sheridan W, et al. Clinical experience with recombinant human G-CSF and GM-CSF. Semin Hematol 1989;26:9-U. Moore RN, Oppenheim JJ, Farrar JJ, Carter CS, Waheed A, Shadduck RK. Production of lymphocyte-activating factor (interleukin 1) by macrophages activated with colony-stimulating factors. J Immunol 1980;125:1302-l305. Cannistra SA, Rambaldi A, Sprigs DR, Hermann F, Kufe D, Griffin JD. Human granulocyte-macrophagecolony-stimulating factor induces expression of the tumor necrosis factor gene by the U937 cell line and by normal human monocytes. J Clin Invest 1987:79:1720-1728.

Diseases from Growth Factor Excess

385

7 Kurland JI, Pelus LM. Ralph P, Bockman RS, Moore MAS. Induction of prostaglandin E synthesis in normal and neoplastic macrophages: role for colony-stimulating factor(s) distinct from effects on myeloid progenitor cell proliferation. P m Natl Acad Sci USA 1979;76:2326-2330. 8 Warren K, Ralph P. Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor and colony stimulating activity. J Immunol 1986;1372281-2285. 9 Lin H-S, Gordon S. Secretion of plasminogen activator by bone marrow-derived mononuclear phagocytes and its enhancement of colony-stimulating factor. J Exp Med 1979;150:231-245. 10 Hamilton JA, Stanley ER, Burgess AW, Shadduck RK.Stimulation of macrophage plasminogen activator activity by colony-stimulating factors. J Cell Physiol 1980; 103:435-445. 11 Metcalf D, Nicola NA. Synthesis by mouse peritoneal cells of G-CSF, the differentiation inducer for myeloid leukemia cells: stimulation by endotoxin, M-CSF and MultiCSF. Leuk Res 1985;9:35-50. 12 Ishizaka Y,Motoyoshi K, Hatake K, Saito M, Takaku F, Miura Y.Mode of action of human urinary colony-stimulating factor. Exp Hematol 1986;14:1-8. 13 Nicola NA, Metcalf D. Binding of 12sI-labeledgranulocytecolony-stimulatingfactor to n o d murine hemopoietic cells. J Cell Physiol 1985;124:313-321. 14 Nicola NA, Metcalf D. Binding of iodinated multipotentialcolony-stimulatingfactor (interleukin-3) to murine bone marrow cells. J Cell Physiol 1986;U8:180-186. 15 Silberstein SD,Owen WF, Gasson JC, et al. Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor. J Immunol 1986;1373290-3294. 16 Dahinden CA,Zingg J. Maly F,de Weck AL. Leukotriene production in human neutrophils primed by recombinant human granulocytelmacrophagecolony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals. J Exp Med 1988;167:1281-1295. 17 Weisbart RH, Golde DW, Clarke SC, Wong GG,Gasson JC. Human granulocytemacrophage colony-stimulating factor is a neutrophil activator. Nature 1985;314: 361-363. 18 Lopez AF, Williams DJ, Gamble JR, et al. Recombinant human granulocytemacrophage colony stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression and survival. J Clin Invest 1986;78:l220-1228. 19 Seldin DC, Austin W.Mast cell subclasses and their growth dependence in mice, rats and humans. Lymphokines l988;15:313-340. 20 Bussolino F, Wang JM, Defilippi P, et al. Granulocyte-and granulocyte-macrophage colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature 1989;337:471-473. 21 Begley CG, Lopez AF, Metcalf D, Nicola NA, Warren CJ, Sanderson CJ. Purified colony stimulating factors enhance the survival of human neutrophils and eosinophils in vitro: a rapid and sensitive microassay for colony stimulating factors. Blood 1986; 68:162-166. 22 Lang RA, Metcalf D, Cuthbertson RA, et al. Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF)develop accumulations of macrophages, blindness and a fatal syndrome of tissue damage. Cell 1987;51:675-686. 23 Metcalf D. Mechanisms contributing to the sex difference in levels of granulocytemacrophage colony stimulating factor in the urine of GM-CSF transgenic mice. Exp Hematol 1988;16:794-800.

Metcalf

386

24 Metcalf D, Moore JG. Divergent disease patterns in granulocyte-macrophagecolonystimulating factor transgenic mice associated with different transgene insertion sites. Proc Natl Acad Sci USA 1988;85:7767-7771. 25 Gearing N, Metcalf D, Moore JG, Nicola NA. Elevated levels of GM-CSF and IL-1 in the serum, peritoneal and pleural cavities of GM-CSF transgenic mice. Immunology 1989;67:216-220. 26 Johnson GR, Gonda TJ, Metcalf D, Hariharan IK, Cory SA. A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte-macrophage colony-stimulating factor. EMBO J l989;8:441-448. 27 Lieschke GJ, Maher D, Cebon J, et al. Effects of bacterially-synthesizedrecombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy. Annals Int Med 1989;110:357-364. 28 Ross R. The pathogenesis of atherosclerosis-an update. N Engl J Med 1986;314: 488-500. 29 Morstyn G,Campbell L, Souza LM, et al. Effect of granulocyte colony stimulating factor on neutropenia induced by cytotoxic chemotherapy. Lancet 1988;i:667-672. 30 Chang JM, Metcalf D, Gonda TJ, Johnson GR. Long-term exposure to retrovirallyexpressed G-CSF induces a non-neoplastic granulocytic and progenitor cell hyperplasia without tissue damage in mice. J Clin Invest 1989 (in press). 31 Wong PMC, Chung S-W, Dunbar GF, Bodine DM, Ruscetti S, Nienhuis AW. Retrovirus-mediatedtransfer and expression of the interleukin-3gene in mouse hematopoietic cells results in a myeloproliferativedisorder. Mol Cell Biol 1989;9:798-808. 32 Chang JM, Metcalf D, Lang RA, Gonda TJ, Johnson GR. Non-neoplastic hemopoietic myeloproliferative syndrome induced by dysregulated Multi-CSF (IL-3) expression. Blood 1989;73~1487-1497. 33 Semenza GL, Traystman MD, Gearhart JD, Antonarakis SE. Polycythemia in transgenic mice expressing the human erythropoietin gene. Proc Natl Acad Sci USA 1989;86:2301-2305. 34 Tomida M, Yamamoto-Yamaguchi Y, Hozumi M. Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J Biol Chem 1984;259:10978-10982. 35 Hilton DJ, Nicola NA, Metcalf D. Purification of a murine leukemia inhibitory factor from Krebs ascites cells. Anal Biochem 1988;173:359-367. 36 Gearing DP, Gough NM, King JA, et al. Molecular cloning and expression of cDNA encoding a murine myeloid leukemia inhibitory factor (LIF). EMBO J 19876: 3995-4002. 37 Moreau J-F, Donaldson DD, Bennett F, Witek-Giannotti J, Clark SC, Wong GG.Leukemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells. Nature 1988;336:690-692. 38 Leary AG, Wong GG,Clark SC, Ogawa M. Leukemia inhibitory factor (LIF)/differentiation inducing activity (DIA) is a synergistic factor for human hemopoietic stem cells. Exp Hematol 1989;17:524a. 39 Baumann H, Wong GG.Hepatocyte-stimulatingfactor 111shares structural and functional identify with leukemia-inhibitory factor. J Immunol 1989;143:1163-1167. 40 Abe E, Tanaka H,Ishimi Y, et al. Differentiation-inducingfactor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption. Proc Natl Acad Sci USA 1986;83:5958-5962. 41 Mori M, Yamaguchi K, Abe K. Purification of a lipoprotein lipase-inhibitingprotein produced by a melanoma cell line associated with cancer cachexia. Biochem Biophys Res Commun 1989:160:1085-1092.

Diseases from Growth Factor Excess

387

42 Williams RL, Hilton DJ, Pease S, et al. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-687. 43 Smith AG, Heath JK, Donaldson DD, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988;336:688-690. 44 Metcalf D,Gearing DP. A fatal syndrome in mice engrafted with cells producing high levels of the leukemia inhibitory factor (LIF). Proc Natl Acad Sci USA 1989;86: 5948-5952. 45 Metcalf D, Gearing DP. A myelosclerotic syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor (LIF). Leukemia 1989 (in press).

Discussion O’ReiUy: In the mice with excess LIF levels, the thymus atrophy and, in particular, the testis lesions raised the possibility of severe fluoride-zinc deficiency or cadmium poisoning. DNA and RNA polymerase are both metalloenzymes requiring zinc. I was wondering whether you are investigating these possibilities? Metcalf: Such metalloenzymes may be abnormal, but so far we have only been measuring calcium levels. These are certainly elevated. O’ReiUy: In addition to the changes in body fat and also the parametastatic calcification, you also noted that these are rather high-strung mice. What do you see in the brain?

Metcalf: Histologically, the brain appears normal, but pharmacologically,the brain may be far from normal. There will be an interesting study published later this year, which is not mine, and which I can’t, in politeness, talk about in detail. The work may however explain why the mice are sojumpy. We have recently started to inject LIF into mice. The reason this work was delayed is because LIF has a half-life of 8 minutes in vivo, and we needed to know what target tissues to pay attention to before starting a laborious course of injections. If you inject LIF into mice, they lose weight very abruptly in the first three days, and this occurs also in C3H/HE.I mice. The latter mice are normally docile, but they too become jumpy after LIF injections.

B m e y e r : The hmrdependent cells, with inserted cDNAs, produce high levels of growth factors and are a very interesting system. The question is, how relevant are these models? I’m not aware of any disease states in humans where you have such tremendous excesses of growth factors, and while it may be a great way to study what happens when you have totally unregulated growth factor production, I wander if such studies are not going to give us a false impression of what the potential of these molecules really is in a human. Metcalf: Yes, I accept this criticism. However, we had a very difficult problem on our hands. All our earlier work with the CSFs had been progressive. We became very familiar with what the molecules did, as we painfully tried to purify them. When recombinant CSFs became available for in vivo use, it was no big surprise what was observed in vivo. We k n e w what tissues to look at. With LIF we developed recombinant, purified material based on an in vitro assay using leukemic cells without having the faintest idea of what its normal function was. My argument was, and still is, that one of the quickest ways to get a general idea of the function of a molecule is to generate a mouse with excess levels of the factor; then, sit back and see what happens. When you follow such experiments with the injection of reasonable doses of the factor, you know which tissues to monitor. In the case

Metcalf

388

of LIF, we established by this method that the parameters to monitor were, for example, serum calcium levels, liver damage, the release of acute-phase proteins and changes in erythrocyte sedimentation rates. There wouldn’t have been any reason at the beginning to choose such parameters. Of course, it’s an abnormal model, and in retrospect, we did exactly what we needed to do without realizing it, which is to have a local population of cells in the bone marrow producing an agent which was going to stimulate bone formation. I suspect that we may not be able to reproduce these bone changes by injecting LIF, because although you can get LIF into the bone marrow, it will not be in very high concentrations. You might say: “Well, okay, that’s fine, but there is no disease state where that’s going on.” I would agree-I don’t thinkthere is such disease state, although the situation in osteosclerosisand myelofibrcsis obviously needs to be checked carefully. Even if there are no naturally occurring diseases exactly like the model, a new way to induce osteosclerosis has been developed, and this should be a useful model for studying this disease.

Broxmeyer: I was very intrigued by the fact that when you put the factor-dependent cells in, they didn’t completely take over the hemopoietic tissues of the animal. You said that there were only about 10% of such cells in the bone marrow and spleen. What is holding them from taking over the whole tissue? Metcalf: The detailed biology of mice injected with FDC-P1 cells has been published elsewhere. Eventually, these mice do develop leukemia, derived from the cells injected. But for some curious reason, the natural build up of the population is restricted by normal regulatory mechanisms, and there is a period of about two months when a population of stable size is present.

Greenberg: Don, one of the surprising things you demonstrated was an increase in mast cells infiltrating organs in animals carrying cells with the G-CSF insert. Metcalf: No, mast cell accumulation is only seen in mice engrafted with Multi-CSF (IL-3)producing cells. The response is simply an exaaeration,of what you see when you inject IL-3. It is certainly dramatic to see large numbers of mast cells in sections of the bone marrow which, in a normal mouse, never contains mast cells. These mice also develop a very curious fibrosis around their spleen, which is full of mast cells. Shadduck: We speak a lot about secondary cytokine production. It would seem to me that these would be ideal models in which to look for secondary cytokine production, because of the very high levels of the primary factors. Have you in fact measured the levels of the various CSFs in the sera or urine of these animals?

Metcalf: Yes, I have, and my silence on the matter was significant. For example, GMCSF in vitro will stimulate macrophages to make G-CSF. One therefore might expect to observe elevated G-CSF levels in GM-CSF transgenic mice. However, no G-CSF is detectable in the serum. On the other hand, I did describe elevated IL-1 levels in such mice, and the interesting situation then is that you now have an animal with elevated levels of GM-CSF plus IL-1. From the short-term studies in irradiated mice, this situation might be expected to produce an excess number of progenitor cells and excess hemopoiesis. However, GM-CSF transgenic mice have normal progenitor cells levels. It may be therefore that the acute models are a little bit misleading. In general, we haven’t found other factors to be elevated by bioassay in any of these models. Here, one needs to be a bit careful. It may be that in a lesion there is a local production of other factors, so assays on serum may underestimate what is going on.

Diseases from Growth Factor Excess

389

O’Reilly: I would like to ask your thoughts on the possible basis for the difference in the presence of extramedullary collections of inflammatory cells in GM-CSF versus G-CSF mice with transfected hemopoietic cells. Does this have any implications for different leukemias that may or may not develop foci of leukemic cells within extramedullary sites, such as muscle?

Metcalf: I don’t know. The tw CSF models are completely different. You never see muscle lesions in mice with excess G-CSF levels. How do the macrophages get into the muscle and why in focal aggregates? A normal three-week-old mouse has identical focal accumulations of macrophages in muscle tissue to the ones I showed you in GM-CSF transgenic mice, but with one important difference-there is no muscle damage. When you examine those focal macrophage lesions in normal developing muscle, there is no excess production of IL-1 or TNF. The point is that in normal development, foci of macrophages develop in these locations. Macrophages are also normally present during development in the eye. One possibility is, therefore, that in transgenic mice the cells arrive as part of the normal developmentalsequence, but do not leave as they should normally. Then, because of excess production of the GM-CSF, they induce progressive, damaging foci of inflammatory cells. This type of explanation will not, however, explain how you can generate similar lesions in an adult animal by irradimting the animal and injecting GM-CSF-producing hemopoietic cells. So, it may be necessary to postulate an intrinsic ability of macrophages to home to certain regions in muscle and other tissue, regardless of the retroviral insert. O’Reilly: However, if you look at the macrophages in muscle lesions, they do have the integrated vector?

Metcalf: Yes, they are expressing the GM-CSF insert. The lesions are the only place in which excess GM-CSF production is demonstrated by in situ hybridization. DiPersio: I think you may have answered my question already, but if you were to inject a mouse and achieve comparable levels of GM-CSF as you have in the transgenics, would you see a neutrophilia response to the growth factor? And, if so, why is it that in the transgenics you do not? Do they have a reduced responsiveness to GM-CSF, and why is this? Is it because the cells responding are actually those cells that are making GM-CSF?

Metcalf: I have to say that in all the studies we did in injecting GM-CSF into mice, we really never saw a convincing elevation in peripheral blood neutrophil counts. I was surprised, therefore, when later studies in primates and man did show that GM-CSF could elevate blood neutrophil levels. The fact is that GM-CSF is a poor stimulus in the mouse for elevating blood neutrophil levels. So, the fact that GM-CSF transgenic mice had absolutely normal white cell levels did not differ from our previous experience. Of course, if you increase the GM-CSF concentration a further lOO-fold, as in the experiment using injected hemopoietic cells, you do get very high neutrophil rises. Are the GM-CSF transgenic macrophages unresponsiveto GM-CSF? No, they have normal or excess numbers of receptors and can be stimulated by GM-CSF in vitro to exhibit increased functional activity. Gupta: My question concerns the gene constructs. I‘m wondering whether the constructs were made with the homologous promoter and control sequencesor with non-homologous.

MetcaV: No, in all the models, what was used were cDNAs. and a viral LTR promoter w s placed in front. Initially, we used the Moloney virus LTR because it was the best then available. In later experiments, we used the more powerful myeloproliferative sarcoma virus LTR.

Metcalf

390

Gupta: The reason I asked the question is that I am raising the possibility that you might then have expression in abnormal cell types. Could this have any implications for the biological responses you are seeing?

Metcalf Yes, this may well have happened except perhaps in the models where the insert w a s put into hemopoietic cells. In the latter models, the other tissues of the animal are normal and have no cells that are expressing the cDNA insert, because the construct does not result in the production of infectious virus. Seiler: You have shown us how selective factor excess contributes to shortening of life span and to other diseases. One would like also to know what effects would result from the selective deletion of a particular factor. How would this affect the life span of an animal? What would happen to a mouse lacking LIF? Metcalf: Well, now that LIF exists, it is possible to do such gene-deletion experiments, because LIF is needed to maintain normal embryonic stem cells in a totipotential state. With the use of LIF, genes can be deleted selectively from cells which can then be used to generate chimeric mice and eventually transgenic mice. Experiments of the type you suggested are now being attempted. O’Reilly: You dropped the other bomb, and that was that you see this extraordinary difference in both pathology and secondary cytokine generation in GM-CSF transgenic based on the chromosomal location of the insert. My first question is, are the integrations basically random or are there selective integrations sites? Secondly, how do you interpret the differences seen? Is there, for example, a regulator gene that can downregulate secondary cytokine generation? Metcalf The integration site is not random in the sense that once inserted, all progeny cells have the same integration site. O’Reilly: But, if you locate many different founder mice, what is observed?

Metcalf: The amount of effort involved in getting one or two sublines is so great that few studies of this nature have been attempted in any transgenic system. The difference between our two GM-CSF transgenic lines seems to be based on differences in the morphology and function of the transgenic macrophages. The simplest explanation is that the insertion site of the transgene somehow results in an altered function of the macrophage bearing the insert. Possibly, adjacent genes are modified, but the differences seen will need much further analysis before a possible molecular mechanism can be proposed.