Growth Factors, 1992, Vol. 7, pp. 169-173 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
Mini-Review
Leukemia Inhibitory Factor-A Polyfunctional Regulator
Puzzling
DONALD METCALF The Walter arid Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
KEYWORDS: differentiation, cell proliferation, stem cells, platelets, neuronal differentiation
INTRODUCTION
Professor Donald Metcalf
The leukemia inhibitory factor (LIF) is a heavily glycosylated single chain polypeptide of Mr 58,000 that is the prototype example of an emerging group of puzzling polyfunctional regulatory molecules, other members of which are interleukins-6 and -11. These regulators exhibit the polyfunctionality of some classical hormones but differ in that they do not have a single organ origin. They differ also from typical hemopoietic regulators, such as the colony stimulating factors, that have a similar multi-tissue origin but a relatively restricted range of responding target cells. The polyfunctionality of LIF led to its discovery and rediscovery by workers in quite separate disciplines: as a molecule able to induce differentiation in and suppression of the proliferation of clonogenic cells of a mouse myeloid leukemic cell line, M1 (Tomida et al., 1984; Hilton et al., 1988);as a factor preventing differentiation commitment in normal embryonic stem cells (differentiation inhibitory activity (DIA)) (Williams et al., 1988; Smith et al., 1988); as a factor able to switch signalling of autonomic nerves from an adrenergic to cholinergic mode (Yamamori et al., 1989); as an hepatocyte stimulating factor able to stimulate the production of acute phase proteins (hepatocyte stimulating factor 3) (Baumann and Wong, 1989); and as an inhibitor of lipoprotein lipase, blocking lipid transport to adipocytes (Mori et al., 1989).
HEMOPOIETIC ACTIVITIES OF LIF IN VITRO AND IN VIVO When acting on murine myeloid leukemic M1 cells, LIF suppresses the capacity of the clonogenic cells for self-renewal, leading to suppression Address correspondence to: Prof. D. Metcalf, at above address, Tel: 61-3-345-2555.Fax: 61-3-347-0852.
of colony formation (Metcalf et al., 1988). It also induces maturation of the cells into the macrophage lineage as documented by morphological changes, expression of Fc receptors and phagocytic activity or the production of lysozyme (Tomida et al., 1984; Lotem et al., 1989). When used in combination with G-CSF, GM-CSF or IL6, enhanced suppression of the human myeloid leukemic cell lines HL60 and U937 is observable
I69
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
I70
METCALF
(Maekawa and Metcalf, 1989; Maekawa et al., 1990). Because a number of hemopoietic regulators such as G-CSF, IL-6, GM-CSF and M-CSF are able to induce differentiation in certain myeloid leukemia cell lines, it was initially expected that LIF would prove to be another such hemopoietic regulator. Receptors for LIF were demonstrated by autoradiography on immature and mature macrophages, megakaryocytes and a subset of small lymphocyte-like cells in bone marrow, spleen and thymus (Hilton et al., 1988a, 1991). However, in semisolid cultures of normal marrow cells LIF failed to reveal a capacity, when acting alone, to stimulate the clonal proliferation of cells in any hemopoietic lineage (Metcalf et al., 1988). Furthermore, although mature macrophages express relatively high numbers of LIF receptors, LIF was not able to influence their phagocytic activity, production of other cytokines or the production of lysozyme or superoxide. A human-derived factor, termed HILDA and later shown to be LIF, can stimulate the proliferation of one murine continuous cell line, the DAl line (Moreau et al., 1988). However, the eosinoPhil-stimulating activity originally described for HILDA has not been verified by studies using purified LIF and no receptors for LIF are present on eosinophils. LIF is able, when acting with IL-3, to potentiate the proliferation of certain murine myc-transformed erythroleukemic cell lines (Cory et al., 1991) and megakaryocyte colony formation by normal murine progenitor cells (Metcalf et al., 1991). There have also been reports that LIF may have an action on hemopoietic stem cells as determined either by an acceleration of their ability to initiate clonal proliferation i n uifro (Leary et al., 1990) or by facilitation of retroviral insertion of genetic material into such cells (Fletcher et al., 1990). In view of the relatively weak actions of LIF when potentiating IL-3-stimulated megakaryocyte colony formation in ziitro, it was somewhat surprising that, when injected into normal mice, LIF was able to induce rises in platelet levels (Metcalf et al., 1990) that were comparable with the elevations induced by several other agents with more prominent megakaryocyte stimulating activity in vitro-Multi-CSF, IL-6 and IL-11. In this LIF-induced response in vim there was a
sequential rise in megakaryocyte progenitors, then mature megakaryocytes before platelet levels began to rise after 5-7 days of injections; LIF had no detectable capacity to cause acute platelet release from existing megakaryocytes. The platelets induced by LIF have normal morphology and functional, activity but platelets from mice with elevated LIF levels exhibit increased aggregability probably due to the elevated levels of fibrinogen circulating in this state (Waring, P., unpublished data). Assays for LIF in human serum using a receptor competition assay have failed to detect elevated LIF levels either in states of thrombocytopenia or thrombocythemia (Waring, P., personal communication) so there is no evidence at present that circulating LIF plays an important role either in maintaining normal platelet levels or in increasing platelet production in disease states.
NON-HEMOPOIETIC ACTIONS OF LIF Insight into the pleiotropic actions of LIF was gained by an analysis of tissue changes occurring in mice with chronically elevated levels of LIF. These were achieved by engrafting mice with cells of the hemopoietic line FDC-P1 which were actively producing LIF following retroviral insertion of LIF cDNA linked to a strong retroviral promoter (Metcalf and Gearing, 1989,1990). These mice developed a syndrome leading to death within two to three months that was characterized by a hyper-excitable state, complete loss of subcutaneous and abdominal fat, thymus atrophy, hypercalcemia, calcium deposits in heart, skeletal muscle and liver, cirrhosis, destruction of pancreatic acini, loss of the inner third of the adrenal cortex, failure of corpora lutea formation and complete suppression of spermatogenesis. More dramatic was the development of massive numbers of osteoblasts in the marrow cavity, leading to osteosclerosis and a displacement of hemopoietic populations to the spleen and liver. Injections of high doses of LIF for two weeks into adult mice were able to reproduce the excitable state, loss of body fat and thymus atrophy. Such mice exhibited stimulation of megakaryocyte and platelet formation and acute phase protein responses but they did not develop any of
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
BIOLOGY OF LIF
the other organ pathology of mice with chronically elevated LIF levels (Metcalf et al., 1990). I n vitro studies have shown that LIF can release calcium from adult bone tissue by a novel mechanism initiated by stimulation of osteoblasts (Abe et al., 1986; Allen et al., 1990). The action of LIF in simultaneously elevating new bone formation, serum calcium levels and tissue deposits of calcium is unusual and suggests a quite complex action on calcium metabolism. In zdro, LIF has been reported to stimulate the proliferation of osteoblasts (Reid et al., 1990), an action that could explain in part the massive accumulation of osteoblasts with increased mitotic activity in mice with chronically elevated LIF levels. The excitable state and erection of hair at the nape of the neck might in part be due to hypercalcemia but is likely to be based on the ability of LIF to directly influence autonomic nerve signalling, including the production of vasoactive peptides. In this context it is of interest that injections of LIF induced pylorospasm and stomach dilatation in C3H/HeJ mice, although not in DBA mice (Metcalf et al., 1990). The kinetics of LIF stimulation of hepatocytes to produce acutephase proteins are similar to those noted for IL-6 but the pattern of protein response differs slightly, suggesting that each agent operates independently on hepatocytes. The loss of body fat induced by LIF seems likely to be based on the presence of LIF receptors on adipocytes and the ability of LIF to inhibit lipoprotein lipase presumably necessary for lipid transport to, and accumulation in, adipocytes. The cellular basis for the gonadal and pancreatic changes seen in mice with excess LIF levels is so far uncharacterized.
NON-HEMOPOIETIC ACTIONS OF LIF IN EMBRYONIC LIFE LIF exhibits the unusual feature of probably having quite different functions in embryonic versus adult life. LIF can prevent differentiation coinmitment i n pluripotential embryonic stem cclls (Williams et al., 1988; Smith et al., 1988) which implies that LIF is likely to be of major importance in early events following fertilization, hlastocyst development and implantation. Although LIF is incapable of crossing the fully-
171
developed placenta to the fetus (Hilton et al., 1991), active transcription of LIF has been noted in the metrial gland in the mouse uterine wall adjacent to the blastocyst implantation site (Croy et al., 1991) and this may be a local source of LIF during this early development period. Transcription of LIF has also been noted in blastocyst cells and in embryonic stem cells (Brown, M., unpublished data), so the developing embryo may itself provide much of the LIF needed in this period. Little is known yet about the possible actions of LIF at later stages in embryonic development with the exception of the central nervous system. In ziitro studies using day 9 undifferentiated murine neural crest cells have shown LIF to be a potent agent in promoting survival and sensory neuronal differentiation in such cells (Murphy et al., 1991). Thus LIF can exhibit diametrically opposite actions on differentiation commitment: (a) on embryonic stem cells where differentiation commitment is inhibited, and (b) on myeloid leukemic stem cells and embryonic neural crest cells where differentiation is induced by LIF action. Differentiation commitment has the nature of an irreversible all-or-none event occurring during the G1 to S transit period. The ability of LIF to both promote and prevent this process according to the nature of the responding cell suggests the possible activation by LIF of a particular set of nuclear transcription factors whose action is entirely dependent on the context in which activation occurs.
LIF RECEPTORS Both high- and low-affinity receptors have been noted on various cells (Hilton and Nicola, 1992). The cloned LIF receptor cDNA encodes a receptor with low-affinity (Gearing et al., 1991) and, in other cell systems, receptors of high-affinity may be generated either by dimer formation or by combination with one or more separate subunit polypeptides which themselves may or may not be able to bind the ligand. It is possible that some of pleiotropic actions of LIF may be based on the interaction of LIF with receptors of high- or lowaffinity or even on the association in different cells of differing B subunits with the low-affinity LIFa receptor chain. A soluble LIF receptor has recently been puri-
I72
METCALF
fied from serum and is present in high levels (2,uglml) in mouse serum (Layton, M. and Nicola, N.A., personal communication). It is not yet known whether combination of LIF with soluble receptors results in biological inactivation of LIF or, whether as in the case of IL-6 receptors, the LIF-soluble receptor complex is itself capable of stimulating cells expressing a p subunit.
appears usually to be produced and to function locally in various tissues, an arrangement that would minimize unwanted actions of this polyfunctional regulator. Nevertheless it remains puzzling what purpose is achieved by use of a regulator with potent actions on such a wide range of apparently unrelated tissues.
REFERENCES
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
LIF AND DISEASE DEVELOPMENT Many of the actions of LIF are shared by other regulators. Similarly, although LIF levels are elevated in many local inflammatory lesions (Waring, P., personal communication) so also are proinflammatory agents like IL-6 and IL-1 and the macrophage-active M-CSF and GM-CSF. The special role played by LIF in situations where there are multiple regulator perturbations can probably only be determined by deletion experiments using either anti-LIF antibodies or gene knockout transgenic mice. The latter could be difficult to generate if LIF production in the blastocyte is crucial for early embryo development. Many of the disease states seen in mice with excess LIF levels such as pancreatitis, osteoslcerosis, gonadal failure and thrombocytopenia have human counterparts that are of unknown pathogenesis. However, assays on serum LIF levels in humans with these disease states have failed to detect elevated LIF levels, which might argue against LIF being involved in the development of these diseases. It needs to be commented however that the biology of LIF production seems to involve usually local production in multiple sites with no release of detectable LIF to the circulation. In an arrangement like this, possible links between LIF and the development of various local diseases will need investigation of local LIF production in the affected tissue either by immunoassay of mature protein or by analysis for LIF transcription.
SUMMARY LIF seems likely to have important functions in the early developing embryo and in adult life can influence platelet formation, osteoblast and neuronal function, calcium and lipid metabolism and the production of acute-phase proteins. LIF
Abe, E., Tanaka, H., Ishimi, Y. et al. (1986) Differentiationinducing factor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption. Proc. Natl. Acad. Sci. U S A 83, 5958-5962. Allan, E. H., Hilton, D. J., Brown, M. A., et al. (1990) Osteoblasts display receptors for and responses to leukemia inhibitory factor. I . Cell. Physiol. 145,110-119. Baumann, H. and Wong, G. G. (1989) Hepatocyte-stimulating factor I11 shares structural and functional identity with leukemia-inhibitory factor. 1. Immunol. 143, 1163-1 167. Cory, S., Maekawa, T., McNeall, J. and Metcalf, D. (1991) Murine erythroid cell lines derived with c-myc retroviruses respond to leukemia-inhibitory factor, erythropoietin and interleukin 3. Cell Growth and Differelltiation 2, 165-172. Croy, B. A,, Guilbert, L. J., Brown, M. A., et al. (1991) Characterization of cytokine production by the metrial gland and granulated metrial gland cells. 1. Reproduc. lmmunol. 19, 149-1 66. Fletcher, F. A,, Williams, D. F., Miliszewski, C., Anderson, D., Rives, A. and Belmont, J. W. (1990) Murine leukemia inhibitory factor enhances retroviral-vector infection efficiency of hematopoietic progenitors. Blood 76, 1098-1 103. Gearing, D. P., Thut, C. J., Van den BOS, T., et al. (1991) Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. E M B O I . 10,2839-2848. Hilton, D. J. and Nicola, N. A. (1992) Kinetic analysis of the binding of leukemia inhibitory factor to receptors o n cells, membranes and in detergent solution. /. Bid. Chem. (in press). Hilton, D. J., Nicola, N. A. and Metcalf, D. (1988) Purification of a murine leukemia inhibitory factor from Krebs ascites cells. Ann/. Biocliern. 173, 359-367. Hilton, D. J., Nicola, N. A. and Metcalf, D. (1988a) Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells. Proc. Nntl. Acad. Sci. U S A 85, 5971-5975. Hilton, D. J., Nicola, N. A. and Metcalf, D. (1991) Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells. /. Cell. Physrol. 146,207-215. Leary, A. G., Wong, G. G., Clark, S. C., Smith, A. G. and Ogawa, M. (1990) Leukemia inhibitory factor differentiation-inhibitory activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells. Blood 75,1960-1964. Lotem, I., Shabo, Y. and Sachs, L. (1989) Clonal variation in susceptibility to differentiation by different protein inducers in the myeloid leukemia cell line, M1. Leukemia 3, 804-807. Maekawa, T. and Metcalf, D. (1989) Clonal suppression of HL60 and U937 cells by recombinant leukemia inhibitory factor in combination with GM-CSF or G-CSF. Leukeniin 3, 270-276. Maekawa, T., Metcalf, D. and Gearing, D. P. (1990) Enhanced suppression of human myeloid leukemic cell lines by com-
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
BIOLOGY OF LIF binations of IL-6, LIF, GM-CSF and C-CSF. I t ~ t 1. . Cnfrrcr 45, 353-358. Metcalf, D. and Gearing, D. P. (1989) A fatal syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor. Proc. Nntl. Acad. Sci. USA 86, 5948-5952. Metcalf, D. and Gearing, D. P. (1990) A myelosclerotic syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor (LIF). Leirkrruia 3, 847-852. Metcalf, D., Hilton, D. J. and Nicola, N. A. (1988) Clonal analysis of the action of the murine inhibitory factor on leukemic and normal murine hemopoietic cells. Lrirkiwrin 2, 216-221. Metcalf, D., Hilton, D. and Nicola, N. A. (1991) Leukemia inhibitory factor can potentiate murine megakaryorvtc production iii z~itro.Blood 77, 2150-2153. Metcalf, D., Nicola, N. A. and Gearing, D. P. (1990) Effects of injected leukemia inhibitory factor (LIF) on heniopoietic and other tissues in mice. B h J d 76, 50-56. Moreau, J,-F.,Donaldson, D. D., Bennett, F., Witek-Gianotti, J. A,, Clark, 5. C. and Wong, G. G. (1988) Leukemia inhihitory factor is identical to the myeloid growth factor human interleukin for DA cells. Nnfirrc 336,690-692. Mori, M., Yamaguchi, K. and Abe, K. (1989) Purification of a lipoprotein lipase-inhibiting protein produced by a mela-
173
noma cell line associated with cancer cachexia. Biochrnr. Bioyhys. Res. Cornfnirri. 160, 1085-1092. Murphy, M., Reid, K., Hilton, D. J . and Bartlett, P. F. (1991) Generation of sensory neurones is stimulated by leukemia inhibitory factor. Proc. Natl. A d . Sci. USA 88, 3498-3501. Reid, I. R., Lowe, C., Cornish, J . . et al. (1990) Leukemia inhibitory factor: a novel bone-active cytokine. E~irlocririolugy126, 1416-1420. Smith, A. G., Heath, J. K., Donaldson, D. D., et al. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. N n f i t r e 336, 688-690. Tomida, M., Yamamoto-Yamaguchi, Y. and Hozumi, M. (1984) Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. 1. Biol. Clicrrr. 259, 10978-10982. Williams, R. L., Hilton, D. J., Pease, S., et al. (1988) Myeloid leukemia inhibitory factor (LIF) maintains the developmental potential of embryonic stem cells. Nafirrc, 336,684-687. Yamamori, T., Fukada, K., Aebersold, R., Korsching, S., Fann, M.-J. and Patterson, P. H. (1989) The cholinergic neuronal differentiation factor from heart cells is identical to leu kemia inhibitory factor. Sriiwctl 246, 1412-1416.
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
Mini-Review
Leukemia Inhibitory Factor-A Polyfunctional Regulator
Puzzling
DONALD METCALF The Walter arid Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
KEYWORDS: differentiation, cell proliferation, stem cells, platelets, neuronal differentiation
INTRODUCTION
Professor Donald Metcalf
The leukemia inhibitory factor (LIF) is a heavily glycosylated single chain polypeptide of Mr 58,000 that is the prototype example of an emerging group of puzzling polyfunctional regulatory molecules, other members of which are interleukins-6 and -11. These regulators exhibit the polyfunctionality of some classical hormones but differ in that they do not have a single organ origin. They differ also from typical hemopoietic regulators, such as the colony stimulating factors, that have a similar multi-tissue origin but a relatively restricted range of responding target cells. The polyfunctionality of LIF led to its discovery and rediscovery by workers in quite separate disciplines: as a molecule able to induce differentiation in and suppression of the proliferation of clonogenic cells of a mouse myeloid leukemic cell line, M1 (Tomida et al., 1984; Hilton et al., 1988);as a factor preventing differentiation commitment in normal embryonic stem cells (differentiation inhibitory activity (DIA)) (Williams et al., 1988; Smith et al., 1988); as a factor able to switch signalling of autonomic nerves from an adrenergic to cholinergic mode (Yamamori et al., 1989); as an hepatocyte stimulating factor able to stimulate the production of acute phase proteins (hepatocyte stimulating factor 3) (Baumann and Wong, 1989); and as an inhibitor of lipoprotein lipase, blocking lipid transport to adipocytes (Mori et al., 1989).
HEMOPOIETIC ACTIVITIES OF LIF IN VITRO AND IN VIVO When acting on murine myeloid leukemic M1 cells, LIF suppresses the capacity of the clonogenic cells for self-renewal, leading to suppression Address correspondence to: Prof. D. Metcalf, at above address, Tel: 61-3-345-2555.Fax: 61-3-347-0852.
of colony formation (Metcalf et al., 1988). It also induces maturation of the cells into the macrophage lineage as documented by morphological changes, expression of Fc receptors and phagocytic activity or the production of lysozyme (Tomida et al., 1984; Lotem et al., 1989). When used in combination with G-CSF, GM-CSF or IL6, enhanced suppression of the human myeloid leukemic cell lines HL60 and U937 is observable
I69
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
I70
METCALF
(Maekawa and Metcalf, 1989; Maekawa et al., 1990). Because a number of hemopoietic regulators such as G-CSF, IL-6, GM-CSF and M-CSF are able to induce differentiation in certain myeloid leukemia cell lines, it was initially expected that LIF would prove to be another such hemopoietic regulator. Receptors for LIF were demonstrated by autoradiography on immature and mature macrophages, megakaryocytes and a subset of small lymphocyte-like cells in bone marrow, spleen and thymus (Hilton et al., 1988a, 1991). However, in semisolid cultures of normal marrow cells LIF failed to reveal a capacity, when acting alone, to stimulate the clonal proliferation of cells in any hemopoietic lineage (Metcalf et al., 1988). Furthermore, although mature macrophages express relatively high numbers of LIF receptors, LIF was not able to influence their phagocytic activity, production of other cytokines or the production of lysozyme or superoxide. A human-derived factor, termed HILDA and later shown to be LIF, can stimulate the proliferation of one murine continuous cell line, the DAl line (Moreau et al., 1988). However, the eosinoPhil-stimulating activity originally described for HILDA has not been verified by studies using purified LIF and no receptors for LIF are present on eosinophils. LIF is able, when acting with IL-3, to potentiate the proliferation of certain murine myc-transformed erythroleukemic cell lines (Cory et al., 1991) and megakaryocyte colony formation by normal murine progenitor cells (Metcalf et al., 1991). There have also been reports that LIF may have an action on hemopoietic stem cells as determined either by an acceleration of their ability to initiate clonal proliferation i n uifro (Leary et al., 1990) or by facilitation of retroviral insertion of genetic material into such cells (Fletcher et al., 1990). In view of the relatively weak actions of LIF when potentiating IL-3-stimulated megakaryocyte colony formation in ziitro, it was somewhat surprising that, when injected into normal mice, LIF was able to induce rises in platelet levels (Metcalf et al., 1990) that were comparable with the elevations induced by several other agents with more prominent megakaryocyte stimulating activity in vitro-Multi-CSF, IL-6 and IL-11. In this LIF-induced response in vim there was a
sequential rise in megakaryocyte progenitors, then mature megakaryocytes before platelet levels began to rise after 5-7 days of injections; LIF had no detectable capacity to cause acute platelet release from existing megakaryocytes. The platelets induced by LIF have normal morphology and functional, activity but platelets from mice with elevated LIF levels exhibit increased aggregability probably due to the elevated levels of fibrinogen circulating in this state (Waring, P., unpublished data). Assays for LIF in human serum using a receptor competition assay have failed to detect elevated LIF levels either in states of thrombocytopenia or thrombocythemia (Waring, P., personal communication) so there is no evidence at present that circulating LIF plays an important role either in maintaining normal platelet levels or in increasing platelet production in disease states.
NON-HEMOPOIETIC ACTIONS OF LIF Insight into the pleiotropic actions of LIF was gained by an analysis of tissue changes occurring in mice with chronically elevated levels of LIF. These were achieved by engrafting mice with cells of the hemopoietic line FDC-P1 which were actively producing LIF following retroviral insertion of LIF cDNA linked to a strong retroviral promoter (Metcalf and Gearing, 1989,1990). These mice developed a syndrome leading to death within two to three months that was characterized by a hyper-excitable state, complete loss of subcutaneous and abdominal fat, thymus atrophy, hypercalcemia, calcium deposits in heart, skeletal muscle and liver, cirrhosis, destruction of pancreatic acini, loss of the inner third of the adrenal cortex, failure of corpora lutea formation and complete suppression of spermatogenesis. More dramatic was the development of massive numbers of osteoblasts in the marrow cavity, leading to osteosclerosis and a displacement of hemopoietic populations to the spleen and liver. Injections of high doses of LIF for two weeks into adult mice were able to reproduce the excitable state, loss of body fat and thymus atrophy. Such mice exhibited stimulation of megakaryocyte and platelet formation and acute phase protein responses but they did not develop any of
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
BIOLOGY OF LIF
the other organ pathology of mice with chronically elevated LIF levels (Metcalf et al., 1990). I n vitro studies have shown that LIF can release calcium from adult bone tissue by a novel mechanism initiated by stimulation of osteoblasts (Abe et al., 1986; Allen et al., 1990). The action of LIF in simultaneously elevating new bone formation, serum calcium levels and tissue deposits of calcium is unusual and suggests a quite complex action on calcium metabolism. In zdro, LIF has been reported to stimulate the proliferation of osteoblasts (Reid et al., 1990), an action that could explain in part the massive accumulation of osteoblasts with increased mitotic activity in mice with chronically elevated LIF levels. The excitable state and erection of hair at the nape of the neck might in part be due to hypercalcemia but is likely to be based on the ability of LIF to directly influence autonomic nerve signalling, including the production of vasoactive peptides. In this context it is of interest that injections of LIF induced pylorospasm and stomach dilatation in C3H/HeJ mice, although not in DBA mice (Metcalf et al., 1990). The kinetics of LIF stimulation of hepatocytes to produce acutephase proteins are similar to those noted for IL-6 but the pattern of protein response differs slightly, suggesting that each agent operates independently on hepatocytes. The loss of body fat induced by LIF seems likely to be based on the presence of LIF receptors on adipocytes and the ability of LIF to inhibit lipoprotein lipase presumably necessary for lipid transport to, and accumulation in, adipocytes. The cellular basis for the gonadal and pancreatic changes seen in mice with excess LIF levels is so far uncharacterized.
NON-HEMOPOIETIC ACTIONS OF LIF IN EMBRYONIC LIFE LIF exhibits the unusual feature of probably having quite different functions in embryonic versus adult life. LIF can prevent differentiation coinmitment i n pluripotential embryonic stem cclls (Williams et al., 1988; Smith et al., 1988) which implies that LIF is likely to be of major importance in early events following fertilization, hlastocyst development and implantation. Although LIF is incapable of crossing the fully-
171
developed placenta to the fetus (Hilton et al., 1991), active transcription of LIF has been noted in the metrial gland in the mouse uterine wall adjacent to the blastocyst implantation site (Croy et al., 1991) and this may be a local source of LIF during this early development period. Transcription of LIF has also been noted in blastocyst cells and in embryonic stem cells (Brown, M., unpublished data), so the developing embryo may itself provide much of the LIF needed in this period. Little is known yet about the possible actions of LIF at later stages in embryonic development with the exception of the central nervous system. In ziitro studies using day 9 undifferentiated murine neural crest cells have shown LIF to be a potent agent in promoting survival and sensory neuronal differentiation in such cells (Murphy et al., 1991). Thus LIF can exhibit diametrically opposite actions on differentiation commitment: (a) on embryonic stem cells where differentiation commitment is inhibited, and (b) on myeloid leukemic stem cells and embryonic neural crest cells where differentiation is induced by LIF action. Differentiation commitment has the nature of an irreversible all-or-none event occurring during the G1 to S transit period. The ability of LIF to both promote and prevent this process according to the nature of the responding cell suggests the possible activation by LIF of a particular set of nuclear transcription factors whose action is entirely dependent on the context in which activation occurs.
LIF RECEPTORS Both high- and low-affinity receptors have been noted on various cells (Hilton and Nicola, 1992). The cloned LIF receptor cDNA encodes a receptor with low-affinity (Gearing et al., 1991) and, in other cell systems, receptors of high-affinity may be generated either by dimer formation or by combination with one or more separate subunit polypeptides which themselves may or may not be able to bind the ligand. It is possible that some of pleiotropic actions of LIF may be based on the interaction of LIF with receptors of high- or lowaffinity or even on the association in different cells of differing B subunits with the low-affinity LIFa receptor chain. A soluble LIF receptor has recently been puri-
I72
METCALF
fied from serum and is present in high levels (2,uglml) in mouse serum (Layton, M. and Nicola, N.A., personal communication). It is not yet known whether combination of LIF with soluble receptors results in biological inactivation of LIF or, whether as in the case of IL-6 receptors, the LIF-soluble receptor complex is itself capable of stimulating cells expressing a p subunit.
appears usually to be produced and to function locally in various tissues, an arrangement that would minimize unwanted actions of this polyfunctional regulator. Nevertheless it remains puzzling what purpose is achieved by use of a regulator with potent actions on such a wide range of apparently unrelated tissues.
REFERENCES
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
LIF AND DISEASE DEVELOPMENT Many of the actions of LIF are shared by other regulators. Similarly, although LIF levels are elevated in many local inflammatory lesions (Waring, P., personal communication) so also are proinflammatory agents like IL-6 and IL-1 and the macrophage-active M-CSF and GM-CSF. The special role played by LIF in situations where there are multiple regulator perturbations can probably only be determined by deletion experiments using either anti-LIF antibodies or gene knockout transgenic mice. The latter could be difficult to generate if LIF production in the blastocyte is crucial for early embryo development. Many of the disease states seen in mice with excess LIF levels such as pancreatitis, osteoslcerosis, gonadal failure and thrombocytopenia have human counterparts that are of unknown pathogenesis. However, assays on serum LIF levels in humans with these disease states have failed to detect elevated LIF levels, which might argue against LIF being involved in the development of these diseases. It needs to be commented however that the biology of LIF production seems to involve usually local production in multiple sites with no release of detectable LIF to the circulation. In an arrangement like this, possible links between LIF and the development of various local diseases will need investigation of local LIF production in the affected tissue either by immunoassay of mature protein or by analysis for LIF transcription.
SUMMARY LIF seems likely to have important functions in the early developing embryo and in adult life can influence platelet formation, osteoblast and neuronal function, calcium and lipid metabolism and the production of acute-phase proteins. LIF
Abe, E., Tanaka, H., Ishimi, Y. et al. (1986) Differentiationinducing factor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption. Proc. Natl. Acad. Sci. U S A 83, 5958-5962. Allan, E. H., Hilton, D. J., Brown, M. A., et al. (1990) Osteoblasts display receptors for and responses to leukemia inhibitory factor. I . Cell. Physiol. 145,110-119. Baumann, H. and Wong, G. G. (1989) Hepatocyte-stimulating factor I11 shares structural and functional identity with leukemia-inhibitory factor. 1. Immunol. 143, 1163-1 167. Cory, S., Maekawa, T., McNeall, J. and Metcalf, D. (1991) Murine erythroid cell lines derived with c-myc retroviruses respond to leukemia-inhibitory factor, erythropoietin and interleukin 3. Cell Growth and Differelltiation 2, 165-172. Croy, B. A,, Guilbert, L. J., Brown, M. A., et al. (1991) Characterization of cytokine production by the metrial gland and granulated metrial gland cells. 1. Reproduc. lmmunol. 19, 149-1 66. Fletcher, F. A,, Williams, D. F., Miliszewski, C., Anderson, D., Rives, A. and Belmont, J. W. (1990) Murine leukemia inhibitory factor enhances retroviral-vector infection efficiency of hematopoietic progenitors. Blood 76, 1098-1 103. Gearing, D. P., Thut, C. J., Van den BOS, T., et al. (1991) Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. E M B O I . 10,2839-2848. Hilton, D. J. and Nicola, N. A. (1992) Kinetic analysis of the binding of leukemia inhibitory factor to receptors o n cells, membranes and in detergent solution. /. Bid. Chem. (in press). Hilton, D. J., Nicola, N. A. and Metcalf, D. (1988) Purification of a murine leukemia inhibitory factor from Krebs ascites cells. Ann/. Biocliern. 173, 359-367. Hilton, D. J., Nicola, N. A. and Metcalf, D. (1988a) Specific binding of murine leukemia inhibitory factor to normal and leukemic monocytic cells. Proc. Nntl. Acad. Sci. U S A 85, 5971-5975. Hilton, D. J., Nicola, N. A. and Metcalf, D. (1991) Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells. /. Cell. Physrol. 146,207-215. Leary, A. G., Wong, G. G., Clark, S. C., Smith, A. G. and Ogawa, M. (1990) Leukemia inhibitory factor differentiation-inhibitory activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells. Blood 75,1960-1964. Lotem, I., Shabo, Y. and Sachs, L. (1989) Clonal variation in susceptibility to differentiation by different protein inducers in the myeloid leukemia cell line, M1. Leukemia 3, 804-807. Maekawa, T. and Metcalf, D. (1989) Clonal suppression of HL60 and U937 cells by recombinant leukemia inhibitory factor in combination with GM-CSF or G-CSF. Leukeniin 3, 270-276. Maekawa, T., Metcalf, D. and Gearing, D. P. (1990) Enhanced suppression of human myeloid leukemic cell lines by com-
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/07/15 For personal use only.
BIOLOGY OF LIF binations of IL-6, LIF, GM-CSF and C-CSF. I t ~ t 1. . Cnfrrcr 45, 353-358. Metcalf, D. and Gearing, D. P. (1989) A fatal syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor. Proc. Nntl. Acad. Sci. USA 86, 5948-5952. Metcalf, D. and Gearing, D. P. (1990) A myelosclerotic syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor (LIF). Leirkrruia 3, 847-852. Metcalf, D., Hilton, D. J. and Nicola, N. A. (1988) Clonal analysis of the action of the murine inhibitory factor on leukemic and normal murine hemopoietic cells. Lrirkiwrin 2, 216-221. Metcalf, D., Hilton, D. and Nicola, N. A. (1991) Leukemia inhibitory factor can potentiate murine megakaryorvtc production iii z~itro.Blood 77, 2150-2153. Metcalf, D., Nicola, N. A. and Gearing, D. P. (1990) Effects of injected leukemia inhibitory factor (LIF) on heniopoietic and other tissues in mice. B h J d 76, 50-56. Moreau, J,-F.,Donaldson, D. D., Bennett, F., Witek-Gianotti, J. A,, Clark, 5. C. and Wong, G. G. (1988) Leukemia inhihitory factor is identical to the myeloid growth factor human interleukin for DA cells. Nnfirrc 336,690-692. Mori, M., Yamaguchi, K. and Abe, K. (1989) Purification of a lipoprotein lipase-inhibiting protein produced by a mela-
173
noma cell line associated with cancer cachexia. Biochrnr. Bioyhys. Res. Cornfnirri. 160, 1085-1092. Murphy, M., Reid, K., Hilton, D. J . and Bartlett, P. F. (1991) Generation of sensory neurones is stimulated by leukemia inhibitory factor. Proc. Natl. A d . Sci. USA 88, 3498-3501. Reid, I. R., Lowe, C., Cornish, J . . et al. (1990) Leukemia inhibitory factor: a novel bone-active cytokine. E~irlocririolugy126, 1416-1420. Smith, A. G., Heath, J. K., Donaldson, D. D., et al. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. N n f i t r e 336, 688-690. Tomida, M., Yamamoto-Yamaguchi, Y. and Hozumi, M. (1984) Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. 1. Biol. Clicrrr. 259, 10978-10982. Williams, R. L., Hilton, D. J., Pease, S., et al. (1988) Myeloid leukemia inhibitory factor (LIF) maintains the developmental potential of embryonic stem cells. Nafirrc, 336,684-687. Yamamori, T., Fukada, K., Aebersold, R., Korsching, S., Fann, M.-J. and Patterson, P. H. (1989) The cholinergic neuronal differentiation factor from heart cells is identical to leu kemia inhibitory factor. Sriiwctl 246, 1412-1416.
