0163-769X/91/1203-0208$03.00/0 Endocrine Reviews Copyright © 1991 by The Endocrine Society

Vol. 12, No. 3 Printed in U.S.A.

LIF: Not Just a Leukemia Inhibitory Factor RAZELLE KURZROCK, ZEEV ESTROV, MEIR WETZLER, JORDAN U. GUTTERMAN, AND MOSHE TALPAZ Department of Clinical Immunology and Biological Therapy, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

I. Introduction

A. Nomenclature

L

EUKEMIA inhibitory factor (LIF) is a pleiotropic cytokine with the ability to influence the function and/or development of seemingly unrelated body elements (Table 1): blood cells, embryonal cells, hepatocytes, neurons, adipose tissues, and bone. These diverse properties account for the bewildering array of names under which this factor has been published (Table 2). Although the most common designation is leukemia inhibitory factor, this molecule can promote both suppression and stimulation of proliferation and/or differentiation depending on the leukemic cell line examined. In addition, LIF may serve as part of the body's energy storage communication network. In this regard, some of the abnormalities that manifest in mammalian hosts with chronic illness include increased serum acute phase proteins and a wasting diathesis accompanied by hypertriglyceridemia; the latter is caused by suppression of lipoprotein lipase, the key enzyme of triglyceride metabolism. It is therefore of interest that LIF induces cachexia in mice, inhibits lipoprotein lipase, and stimulates hepatocyte release of acute phase proteins. In a different arena, the potent effects of LIF on embryonal stem cells suggest a critical role for this molecule in the earliest stages of embryogenesis. Finally, LIF also promotes remodeling of bone and functions as a neuronal differentiation factor. It remains unclear how one molecule can affect distinct organ systems without simultaneous side effects on its other target tissues, although recent data demonstrating that LIF exists both as a diffusible form and a form incorporated into the extracellular matrix (18) suggest that immobilization may serve to restrict its spheres of influence. In this review, we will discuss the multifaceted aspects of LIF functions and implications for understanding the cytokine network.

The diverse nomenclature of LIF reflects the panoply of properties that led researchers in different fields to independent discovery of this protein (Table 2). This explains the distinct and contradictory designations such as differentiation-inducing factor, differentiation-retarding factor, hepatocyte- stimulating factor III, cholinergic neuronal differentiation factor, and melanoma-derived lipoprotein lipase inhibitor I. The most common term for this molecule in current use is, however, LIF. B. Cellular sources Cellular sources for LIF are listed in Table 3. They include certain alloreactive T cell clones, many tumor cell lines, osteoblasts, cultured keratinocytes, and thymic epithelium. In the mouse system, LIF is synthesized in extraembryonic tissue and may therefore regulate embryonic tissues during development (24). Further, embryonic stem cells themselves may express small amounts of LIF, and expression is increased with differentiation induction (25). Recently, we have found that LIF messenger RNA is constitutively expressed by longterm cultures of bone marrow adherent cells, a system felt to be the in vitro analog of the stroma of the bone marrow microenvironment (26). Unstimulated monocytes and lymphocytes do not express LIF, whereas cultured skin fibroblasts and stimulated monocytes do (26, 31); it is therefore the fibroblast-like component of stromal cultures that are probably the LIF producers. Phytohemagglutin-stimulated T lymphocytes also produce LIF (26). C. Molecular biology and biochemistry The reported mol wts of LIF range from 38 to 67K. Much of this heterogeneity can be ascribed to variable glycosylation as removal of the carbohydrate moiety reduces the mol wt to 25K (34, 35). Recombinant forms of LIF that display a different pattern of glycosylation

* Research conducted, in part, by the Clayton Foundation for Research. Dr. Gutterman is a Senior Clayton Foundation Investigator. Address requests for reprints to: Razelle Kurzrock, M.D., Department CIBT, Box 41, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030.

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LEUKEMIA INHIBITORY FACTOR

(yeast-derived) or no glycosylation (Escherichia coliderived) are still active (1, 35). Both murine and human LIF have been cloned (1, 3537), and the complete nucleotide sequence for their genes is 8.7 and 7.6 kilobase pairs, respectively (38). Both genes comprise three exons, two introns, and an unusually long 3'-untranslated region (3.2 kilobase pairs) (38). The LIF transcript is 4.2 kilobases in length. The murine and human clones that have been isolated predict a sequence with 179 residues for the mature protein (1, 2, 35, 36) and a 79% amino acid sequence identity between the two species. This primary form of LIF is a biologically active, "diffusible" glycoprotein. In addition to the molecule described above, Rathjen and co-workers (18) have recently shown that an alternate "immobilized" form of LIF exists; the latter is incorporated into the extracellular matrix. These different forms are a consequence of the expression of alternative transcripts that diverge throughout the first exon and use distinct promoters. Diffusible and immobilized LIF variants are therefore encoded by mRNAs that are differentially spliced at the exon 1/exon 2 boundary. A transcript termed D (diffusible), encodes the diffusible form; a transcript termed M (matrix-associated) is a result of splicing a novel 5' exon to exons 2 and 3 of the LIF trancription unit, and encodes the matrix-associated form. It should be noted that the two transcripts comigrate on agarose gels and can therefore be distinguished by ribonuclease protection analysis but not by Northern blots. The differential localization of the two forms of LIF can be explained by the modular organization of the

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TABLE 2. LIF nomenclature

Name

Abbreviation

References

Leukemia inhibitory factor Differentiation factor Differentiation inhibitory activity Differentiation inducing factor Differentiation retarding factor Human IL for DA cells Hepatocyte stimulating factor III Melanoma-derived lipoprotein lipase inhibitor I 9. Cholinergic neuronal differentiation factor

LIF D-factor DIA DIF DRF HILDA HSFIII MLPLI

1,10 11-13 14 12 14, 15 2, 16, 17 8 7

1. 2. 3. 4. 5. 6. 7. 8.

gene. Exons 2 and 3 produce the core hydrophobic secretory sequences and biologically active protein, while extracellular localization is specified by the first exon. Incorporation of mature, functional LIF into the extracellular matrix is therefore directed by changes in the amino terminal of the primary translation product. D. Chromosomal localization LIF has been localized to chromosome 22ql2 in man (39) and 11A1-A2 in the mouse (40). The 22ql2 region in man is of interest because the majority of Ewings sarcomas have a translocation involving this cytogenetic band [(t(ll;22)(q24;ql2)]. However, analyses using somatic cell hybrids and pulsed field gel electrophoresis suggest that the LIF gene is distal to the breakpoint and not close enough to be involved (41). E. Biological properties

TABLE 1. Biological properties of LIF Hematopoiesis In vitro, induces differentiation and inhibits proliferation, or stimulates proliferation without differentiation depending on the cells analyzed (1, 2) In vivo, increases erythroid and megakaryocytic elements while decreasing lymphocytes in mice (3)

LIF, like many other members of the cytokine family, affects diverse body tissues. In the case of LIF, actions on embryonal cells, hematopoietic elements, liver cells, nervous system development, and bone growth have been documented (Table 1). II. Embryogenesis

Embryonal cells Inhibits in vitro differentiation of totipotent mouse embryonal (ES) stem cells, without affecting proliferation (4, 5) Bone Stimulates bone remodeling in vitro (6) Lipid metabolism Inhibits lipoprotein lipase in vitro and may therefore mediate, in part, cachexia (7) Hepatic Stimulates acute phase protein synthesis in hepatocytes (8) Nervous system Directs choice of neurotransmitter phenotype made by sympathetic neurons in vitro (9)

Extraembryonic tissues lie in close proximity to the embryo and secrete factors that influence the development of the latter element. In this regard, Conquet and Brulet (24) have demonstrated that LIF gene transcription is discernible in the extraembryonic tissue of 7.5day, and in the placenta of 9.5-, 10.5-, and 12.5-day (midgestation) postcoitum embryos. LIF transcripts were also detected at the preimplantation mouse blastocyst stage (3.5 days post coitum) (24). The expression of LIF in blastocysts before the appearance of hematopoiesis suggests that this cytokine may regulate embryonal stem cells or the development of trophoblasts (42), and its continued presence in midgestation further implicates this molecule in embryogenesis. A study by Rathjen and

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KURZROCK ETAL.

210 TABLE 3. Cellular sources of LIF

Mouse Krebs II cells (19, 20) Mitogen-treated splenocytes (12) Ehrlich ascites cells (11) L929 leukemia cells (21) Neutrophils and macrophages of peritoneal infiltrates (22) T cell lines (1) Osteoblast cell line MC3T3E1 (23) Extraembryonic tissue (24) Embryonic stem (ES) cells [differentiation by retinoic acid or 3methoxybenzamide increases LIF levels (25)] Mouse embryonic fibroblasts after induction by IL-la, TGF/3, and bFGF (25) Human Cultures of bone marrow stromal cells (LIF mRNA is expressed constitutively, and expression can be increased by IL-la, IL-1/3, TNFa, and TGF0) (26) Thymic epithelial cells (27) T cell clones (alloreactive) from lymphocytes rejecting kidney allografts (2,10, 17) Cultured keratinocytes (28) Tumor cell lines including 5637 (bladder carcinoma), SVK14 (SV40 transformed keratinocyte), NCI-H23 (lung adenocarcinoma), HLFa (epidermoid carcinoma), SW948 and HRT18 (colon adenocarcinoma), MIA PACA (pancreatic carcinoma), C32 (amelanotic melanoma) HBL 100 (SV-40-transformed mammary epithelium), MDA (breast adenocarcinoma) (29), SEKI (melanoma) (7), COLO-16 (squamous carcinoma) (8), THP-1 (monocytic leukemia) (30) Phytohemagglutinin stimulated T cells (26) Activated monocytes (31) Other Buffalo rat liver cells (14) Rat Yoshida sarcoma cells (32) Rat heart cells (9) Rat osteoblasts (retinoic acid, TNFa, and, in some experiments, TGFjS increases LIF levels) (33)

co-workers (25) on relative expression of LIF D and M transcripts in egg cylinder-stage mouse embryos showed that neither transcript was detectable in the 6.5 or 7.5 day decidua nor in the 7.5 day embryonic regions, and only very low levels of the M transcript were discerned in the 6.5 day embryonic region. However, both D and M transcripts were found at relatively high levels in the extraembryonic region, with the D form approximately 5-fold more abundant than the M form. Expression of both transcripts was greater at 6.5 days than at 7.5 days, with the M transcript barely detectable at the latter timepoint. These data establish both temporal and tissue-specific regulation of LIF D and M transcripts. The activity of LIF has also been studied with the use of in vitro model systems. Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, have been exploited for several years to investigate differentiation and development during embryogenesis. Although these cells demonstrate many phenotypic characteristics of

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early embryos, they are cytogenetically abnormal, display only limited differentiation potential, and can induce tumors when introduced into embryos. In contrast, embryonal stem (ES) cells, the pluripotent cell lines derived from preimplantation embryos, develop normally and can contribute to all somatic and germ lineages. It has been known for several years that EC and ES cells require the presence of a feeder layer to prevent their differentiation in vitro (43). The feeder factor maintains the embryonal cells (even after multiple passages) in the totipotent state without affecting proliferation (44; reviewed in Ref. 45); it was initially designated differentiation-inhibitory activity or differentiation-retarding factor (14, 15) and is now known to be identical to LIF (4, 5). The above data suggest that LIF functions as a developmental regulatory factor in vivo. How can a pleiotropic molecule that evokes protean responses in different cell types orchestrate complex developmental processes? Some of the answers to this question may lie in the work of Rathjan and co-workers (25); they showed that relative production of the diffusible and matrix-associated, immobilized forms of LIF are developmentally programmed and can be modulated by other bioregulatory molecules. To expand, undifferentiated ES cells express LIF transcript M, albeit at low levels. The possibility of an autocrine process during development of the inner cell mass/primitive ectoderm is thus implied. The level of expression after differentiation is dependent on the particular developmental program. Differentiation induction by exposure to retinoic acid or 3-methoxybenzamide results in a significant increase in steady state levels of both LIF transcripts, whereas only a modest induction occurs in cells that differentiate because of the withdrawal of LIF. These investigators also studied the retinoic acid-induced differentiation of three EC lines. These cells mature into various embryonic cell types whose phenotype varies with the parental cell line; the morphologically distinct progeny exhibit different patterns of LIF expression. Such disparities in relative expression of the two forms of LIF suggest that developmental regulation of the two promoters and/or processing of the two primary transcripts is autonomous. It is therefore conceivable that the diffusible and matrix-associated forms of LIF are produced in different embryonal compartments. Further, the demonstration of enhanced LIF expression in differentiating ES cells suggests a feedback mechanism for stem cell renewal accompanying differentiation (25). The salient characteristic of this model is that early activation of LIF expression by differentiated progeny results in the inhibition of subsequent differentiation and stimulation of self-renewal. One wonders whether such a phenomenon also participates in the control of the differentiation/self renewal program of

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other systems in which LIF has been implicated, i.e. hematopoiesis. In addition to being developmentally regulated, the expression of LIF is modulated by other bioregulatory molecules including transforming growth factor (TGF)/3, interleukin-1 (IL-1), bovine fibroblast growth factor (bFGF), and steroids. TGF/3 and FGF induce LIF expression in embryonic fibroblasts within 30 and 60 min, respectively (25). The rapid kinetics of this effect and the fact that it proceeds in the presence of protein synthesis inhibition indicate that LIF is a primary response gene and its induction is not mediated via other gene products. Analysis of relative expression of the two transcripts reveals that, in response to some factors (bFGF and IL-1 a), both transcripts increase, whereas, in response to other factors (TGF/3), the increase in LIF D transcript is disproportionately high. In addition, dexamethasone inhibits induction of LIF by IL-la but not by bFGF. The latter result establishes the existence of negative regulators for LIF, a phenomenon that may play a crucial role in restricting physiological expression of LIF. Combinations of a variety of specific inducers and suppressors of LIF coupled with independent developmental programming of the two LIF promoters may therefore contribute to precise regulation of the activity of the two forms of this molecule. Finally, differentiation-based activation of LIF expression ensures stem cell renewal, and hence protects the stem cell pool from exhaustion.

III. Hematopoiesis A. In vitro LIF has a myriad of hematologic effects, and specific effects vary from cell line to cell line (1-3,10, 11,16-22, 34, 46-58) (Table 4). Additionally, some of the findings of different investigators remain to be reconciled, and the data concerning LIF's effects on normal human hematopoietic progenitors are still fraught with uncertainties. LIF was originally characterized by virtue of its ability to induce differentiation of the murine myeloid leukemia cell line Ml (1, 20, 36, 46), a property which it shares with MGI-2 (now known as IL-6) (47). LIF and IL-6 activity have been compared on Ml cells and a second murine myelomonocytic cell line WEHI 3B D+. Despite LIF's potent effects as an inducer of macrophage differentiation of Ml cells (48), it has no effect on WEHI 3B D+ cells, and the latter have no LIF receptors (10, 48). In contrast, IL-6 actively differentiates both Ml and WEHI 3B D+ cells (47, 49). The action of LIF on Ml cells is very rapid, with a decrease in clonogenic cells evident within 24 h of LIF exposure, and significant differentiation induced within 48 h (48). Addition of Mcolony stimulating factor (CSF) to Ml cultures contain-

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TABLE 4. Hematopoietic effects of LIF In vitro Murine Induces macrophage and granulocyte differentiation and inhibits proliferation of Ml murine myeloid leukemia cells (1) Stimulates proliferation without differentiation of murine IL-3 dependent DA-la leukemia cells (2)

In vivo Blood | platelets Bone marrow [ lymphocytes f erythroid cells | megakaryocytic cells (3)

Enhances IL-3 induced stimulation of megakaryocyte colony formation (58) Human Enhances IL-3 dependent colony formation of very primitive blast colony forming cells (53) and of megakaryocytes (58) In combination with G-CSF, GM-CSF, or IL-6 suppresses clonogenicity of U937 and HL-60 myeloid leukemia cells (51, 52)

ing LIF partially abrogates the reduction in colony number and size that is a consequence of LIF exposure, suggesting that the effects of LIF on Ml cells may be due, in part, to their transformation to CSF-dependent macrophages (48). Examination of LIF's actions on human leukemia cell lines has shown variable results. Wang and colleagues (50) demonstrated that, in two of three acute myelogenous leukemia lines, LIF increased the doubling time of the clonogenic population, possibly by prolonging the stem cell cycle. Additionally, LIF, in combination with G-CSF, GM-CSF, or IL-6, suppresses clonogenicity of U937 and HL-60 myeloid leukemia cells (51, 52). In contrast, LIF also stimulates proliferation of the murine myeloid IL-3-dependent DA-1A leukemia cell line (16, 17). In regard to the effects of LIF on normal progenitors, Metcalf and colleagues (48) found that LIF has no observable colony-stimulating activity in the murine system. Prior exposure of normal progenitor cells to LIF did, however, reduce their survival and/or their subsequent ability to proliferate after stimulation by CSFs (48). Even so, it should be noted that LIF is identical by cloning to a factor originally called HILDA, and activities attributed to HILDA include erythroid burst-promoting activity on human hemopoietic progenitors and the ability to be a chemoattractant and activator of mouse and human eosinophils (16, 17). Because LIF receptors have not been found on eosinophils (10), it has been suggested that these activities could be the result of contamination

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with other molecules. On the other hand, certain investigators have also discerned LIF effects on hematopoietic progenitors. In particular, Leary and colleagues (53) felt that this cytokine was as effective as IL-6 or G-CSF in the enhancement of IL-3-dependent colony formation of very primitive human blast colony-forming cells. LIF alone, however, had no effect on CD34-positive human bone marrow cells. Additionally, Verfaillie and McGlave (54) noted that in serum-containing media (but not in serum-free cultures), LIF stimulated the growth of CFUMIX and CFU-EO in a dose-dependent fashion, and promoted increased CFU-MIX and BFU-E colony size. Depletion of accessory cells had no deleterious effect on these activities. A similar increase was induced by the addition of LIF to CD34+ cells stimulated with IL-3 combined with IL-6. Finally, Metcalf and colleagues (58) have shown that while LIF alone has no effect on murine megakaryocyte colonies, the megakaryocyte colony formation stimulated by IL-3 (multipotential CSF) could be enhanced by the addition of LIF. Recently, we have found that LIF mRNA is constitutively expressed by long-term cultures of bone marrow adherent layers, a population of cells believed to reflect the stroma of the bone marrow microenvironment (26). A role for LIF in modulating hematopoietic processes may therefore be implied, as our stromal cell cultures constitutively express only LIF, TGF/3, and M-CSF, whereas other hemopoietic regulatory cytokine transcripts [IL-la, IL-10, IL-6, GM-CSF, G-CSF, and tumor necrosis factor-a (TNFa)] are only produced after exposure to specific induction stimuli (55). Further, stromal cultures derived from some patients with chronic myelogenous leukemia produce increased amounts of LIF mRNA (26). Increased LIF expression correlates with advanced disease in these patients and is accompanied by the appearance of uninduced expression of IL-1/3 in both the primary malignant cells and in the stromal cells (56). The latter event may serve to drive the disease in an autocrine or paracrine fashion. Since our data also indicate that stromal cell LIF expression can be significantly augmented by IL-lft IL-la, TNF-a, and TGF-0 (26), it may be that enhanced LIF expression in advanced chronic myelogenous leukemia is the corollary of increased IL-1/3 production. B. In vivo Mice engrafted with cells producing high levels of LIF develop a fatal syndrome that includes a neutrophilic leukocytosis and splenic enlargement with excessive hematopoietic cells in the spleen (6, 57). Marrow cellularity is reduced with selective survival of cells derived from the granulocytic lineage. Some of the bone marrow changes are probably caused by myelosclerosis, as in-

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creased bone marrow osteoblasts is a cardinal feature of the syndrome (57). When purified recombinant murine LIF is injected into mice (3), many of the characteristics of the engraftment model fail to be reproduced. At high doses (2 ng, three times daily) LIF administration is followed by toxic effects such as behavior changes, loss of body fat, and thymus atrophy. Dose-related rises are observed in blood platelets, erythrocytes, sedimentation rate, and serum calcium-albumin ratios. The latter changes are seen at dose schedules with no toxic effects. LIF also induces a decrease in bone marrow lymphocytes and elevation of erythroid populations. Additionally, a rise in megakaryocytes in the bone marrow and in the spleen are noted. IV. Bone Remodeling Several cytokines have been implicated in bone remodeling. The term "osteoclast activating factor" was originally applied to the bone-resorbing activity accompanying certain hematological malignancies. Osteoclast activating factor activity is now known to be mediated by a family of cytokines that includes IL-1 and TNFa and TNFjfl. Recently, LIF has also been added as a member of this class of substances (12). In vitro, both catabolic and anabolic effects on bone are displayed by LIF (12, 33, 57, 59-62). Moreover, mice engrafted with cells producing high levels of LIF exhibit pronounced bone resorption, calcium deposition in myocardium and skeletal muscle, and the accumulation of osteoblasts in the bone marrow accompanied by excess new bone formation (6, 57). The catabolic effects of LIF are prostaglandin-dependent; the anabolic effects are not. These activities may therefore be mediated through distinct pathways. Alternatively, LIF stimulation of osteoclasts and bone resorption may require the presence of osteoblasts as has been demonstrated for IL-1 and TNF (63, 64). Support for the latter premise is also derived from the observation that osteoblasts, but not multinucleated osteoclasts, display LIF receptors (33). Specific LIF actions on osteoblasts have been documented. In UMR106 cells (rat osteogenic sarcoma cells with osteoblast-like features), LIF increases plasminogen activator inhibitor (33, 59). The subsequent reduction in plasmin generation could be expected to reduce neutral protease activity and contribute to a net positive effect on bone formation. This type of effect is similar to that displayed by TGF/3. In MC3T3E1 cells (murine osteoblast-like line), LIF increases the level of osteopontin (a molecule that plays a role in cell attachment and proliferation), inhibits proliferation and DNA synthesis, and suppresses alkaline phosphatase activity (a differentiation marker) (60). Yet, in a different cell line (RCT-1, a preosteoblastic rat calvaria line), LIF potentiates the

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increase in alkaline phosphatase produced by retinoic acid (61). The reason for the discrepancy in LIF action on these two cell lines is not immediately apparent, but it should be remembered that the lines are derived from different species and perhaps represent distinct stages of osteoblastic maturation. Reduction of the level of type I procollagen mRNA occurs in both MC3T3E1 and RCT1 cells exposed to LIF. Evidence for the bone resorption activity of LIF has been accrued by assessment of in vitro release of calcium from prelabeled mouse fetal calvaria (12, 62). Bone resorption is associated with an increase in the number of osteoclasts, and the osteolytic effect is inhibited by indomethacin suggesting that it is mediated via local production of prostaglandin. As mentioned earlier, LIF shares many of its prostaglandin-dependent, bone resorption activities with other cytokines such as IL-1 (a and 0), TNF {a and 0), and TGF0. Integration of in vitro observations have indicated that an osteoblast-like cell is probably the primary target of LIF action (59), and bone resorption could therefore be associated with LIF stimulation of osteoblast prostaglandin production. It is also known that LIF transcripts and protein are produced by primary osteoblastic populations and by osteoblast-like cell lines. Further, in these cells, LIF levels can be increased in response to retinoic acid, TNFa, and, in some experiments, TGF0 (33). Since TNFa and TGF0 both affect bone metabolism, these observations identify a potentially interesting cytokine interaction reminiscent of that described above for the embryo and for the bone marrow microenvironment (25, 26). Overall, several lines of evidence converge to implicate LIF as an important paracrine or autocrine modulator of bone remodeling.

V. Hepatic Functions A combination of IL-6, IL-1 (or TNF), and glucocorticoids is minimally required to stimulate the synthesis of most major acute phase plasma proteins in the liver. Cultured keratinocytes and their malignant counterparts, squamous carcinoma cell lines, release additional hepatocyte stimulating factors (HSF) (8, 28). HSFIII is now known to be identical to LIF (8). Interestingly, LIF induces the same set of acute phase plasma proteins in the liver as IL-6. IL-6 is structurally distinct from LIF, and both are bound to a unique receptor. The mechanism for the similarity in their modulatory activity on hepatic cells is not known but could involve common regulatory sequences or receptor subunit similarities. However, since the overall hepatic cell response to LIF and IL-6 also shows some differences, cytokine-specific elements in the regulatory mechanism must exist.

213 VI. Lipid Metabolism

Mammals suffering from chronic infection or malignancy manifest several common clinical and biochemical abnormalities. These abnormalities include a catabolic state with weight loss accompanied by hypertriglyceridemia. The hypertriglyceridemia is associated with elevation of very low-density lipoproteins. The high levels of very low-density lipoproteins result from a clearing defect caused by suppression of the key enzyme of triglyceride metabolism, lipoprotein lipase (65). Inhibition of lipoprotein lipase activity may reduce the intake of fatty acids by adipocytes, resulting in lipid catabolism in adipose tissue and loss of fat. These derangements can be mediated by TNF, IL-1, or interferon-7 (66-68). Recently, investigators have demonstrated that LIF can also inhibit lipoprotein lipase (7). It is, therefore, possible that the cachexia exhibited by mice with high circulating levels of LIF may be explained by the ability of this molecule to suppress adipogenic processes (6).

VII. Nervous System Development In the nervous system, phenotypic decisions can be controlled by factors in the local environment termed neuronal differentiation factors. Rat heart cell conditioned medium produces a protein, cholinergic neuronal differentiation factor, which acts on postmitotic rat sympathetic neurons to specifically induce the expression of acetylcholine synthesis and cholinergic function while suppressing catecholamine synthesis and noradrenergic function (9). It is designated a differentiation factor because it controls phenotypic choices in these neurons without affecting their survival or growth. It has now been established that cholinergic neuronal differentiation factor is identical to LIF (9). Recently, Murphy and colleagues (69) have also shown that LIF stimulates the generation of sensory neurons in cultures of mouse neural crest. They also demonstrate that LIF supports the generation and/or maturation of sensory neurons in cultures of cells from embryonic dorsal root ganglia and, in cultures of postnatal dorsal root ganglia, which contain mature sensory neurons, LIF acts directly to promote neuronal survival. These results suggest that LIF may be a critical part of the process that regulates the development of peripheral neurons from their precursors in the embryonic neural crest. LIF also affects the regulation of the neuropeptide substance P (SP) in sympathetic neurons. In particular, it increases SP in both pure neuronal cell cultures and in cultures containing a mixture of neuronal and nonneuronal cells (70). Interestingly, IL-10 can also increase neuropeptide SP expression; however, unlike LIF, IL-10 has this effect only in dissociated cultures of ganglion neuronal and nonneuronal cells and not in pure neuronal

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214

cultures. The cell type necessary for the IL-1/3 action is probably the ganglion Schwann cell. Importantly, there is significant additional evidence for the importance of hemopoietic cytokines in neuronal development. IL-1 is found in the brain and receptors for IL-1, -2, and -4 are found on neural cell lines (71-74; reviewed in Ref. 9). IL-1, IL-2, IL-6, and 7-interferon all can influence nervous system cell growth or differentiation (75-78). The opposite is also true: nerve growth factor can promote hemopoietic cell differentiation and growth (79, 80). In fact, there are many parallels in the questions of lineage decisions in the neural crest and hematopoietic systems (81). A. Receptors Cells showing LIF receptors include: rat osteoblasts (33); murine myeloid leukemia Ml cell lines (10, 11); normal murine monocyte macrophages (10); murine embryonal stem cells (ES and EC lines) (4); immature and mature murine megakaryocytes (58); and fetal and adult hepatocytes (82). Receptors are not found on murine neutrophils, mast cells, eosinophils, erythrocytes, or lymphocytes (10). Multinucleated osteoclasts also do not express receptors (33), even though it has been suggested that these cells are derived from the monocyte/macrophage lineage (83). Several distinct LIF-responsive cells have similar numbers of receptors. UMR106-06 rat (osteoblast-like) osteogenic sarcoma cells and RCT-1 preosteoblastic rat calvaria cells each have 300 LIF receptors per cell; their apparent dissociation constant (KD) is 60 and 20 pM, respectively (33, 61). Ml myeloid leukemia cells and ES embryonal cells display 100-500 high-affinity receptors per cell (apparent KD, 100-200 pM) (4, 5,10; reviewed in Ref. 34), and 20% receptor occupancy is needed for biological effects on these cell lines. Thus, the disparate effects of LIF on Ml cells (induction of differentiation) and ES cells (inhibition of differentiation) involve an unclarified molecular basis. A recent study by Hilton et al (82) showed that both fetal and adult parenchymal hepatocytes display a higher number of receptors than monocytes. Specifically, the number of receptors per positive cell ranged from 150 for bone marrow monocytes to 2,000 for adult hepatocytes. In each case, however, binding was of high affinity, with an apparent KD of 34100 pM. VIII. Summary Increasingly it seems that many cytokines are pleiotropic, and individual molecules may have critical roles in several different organ systems. LIF exemplifies this phenomenon: it influences embryogenesis, bone and lipid metabolism, and hematopoietic and nervous system

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function. Many of LIF's effects are reminiscent of those of IL-1, TNF, and TGF-,9. Further, even within a single system, LIF can display totally different effects, i.e. induction of differentiation of one leukemic cell line vs. stimulation of proliferation of another. The corollary to these observations is that there appears to be many parallels in developmental systems. For instance, in the case of neuronal "lineage commitment," the events that relate to migration of neural crest cells along various pathways and their ultimate arrest in different locales demonstrate sufficient analogies to hematopoietic lineage commitment phenomena that, in a provocative review, Anderson coined the term "neuropoiesis" (81). This type of analogy becomes even more intriguing when one realizes that some of the same molecules are regulating neuronal and hematopoietic "lineage" proliferation and differentiation. In this respect, several interleukins in addition to LIF are important in neuronal development, and nerve growth factor turns out to also be a hematopoietic regulatory molecule. Similar parallels are enacted in other organ systems as well. The mediation of identical effects by distinct cytokines bound to unique receptors could conceivably be explained by receptor transmodulation or by overlapping signaling sequences. It is nevertheless also unclear how a single cytokine attached to a single receptor can accomplish varied and opposing effects, although divergent intracellular signaling mechanisms could account for some of these phenomena. Yet another enigma relates to how cells from one system can be properly influenced by a pleiotropic molecule such as LIF without significant "cross-effects" on other potentially responsive systems. Cytokine production that is restricted to certain developmental stages, or very localized distribution and spheres of influence within a microenvironment, could be explanatory. The findings of Rathjan and colleagues (18), i.e. that LIF exists as both a diffusible molecule and as a molecule incorporated into the extracellular matrix, is of special interest in relation to the above questions. Indeed, the distinctions between the roles of diffusible and immobilized signaling molecules could be crucial to the multiplicity of LIF's actions (18). Diffusible regulatory factors allow communication between spatially separated cells. Cellular responsiveness to such factors is dictated by the presence of appropriate receptors and postreceptor machinery. In contrast, immobilization of a factor in the extracellular matrix confines its signal to restricted sites. Furthermore, in embryonic cells, differential expression of the two LIF transcripts appears to be developmentally regulated and to be modulated by other cytokines such as IL-la, TGF/?, and bFGF, a phenomenon that may be responsible for fine-tuning control of LIF actions in different physiological situations. It is possible that similar control over localized

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production of different forms of LIF or analogous stem cell regulators by other specialized microenvironments such as the bone marrow also occurs. Such a concept is strengthened by findings that cultured bone marrow stromal cells produce LIF and that production in this system is also increased by cytokines that include IL-1 and TGF0 as well as TNFa (26). Similarly, TNF« and TGF/? can also increase LIF levels in osteoblasts (33). Overall, the concept which emerges with LIF as a paradigm is that, in many cases, the body does not produce a cytokine for an isolated purpose, but rather exploits individual molecules to perform a multiplicity of distinct functions.

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American Institute for Cancer Research Conference on "Exercise, Calories, Fat & Cancer" September 4-5, 1991 Ritz Carlton Hotel, Pentagon City, Virginia (Metropolitan Washington, D. C.) Current research will be presented by leading scientists on the role of exercise, calories, and fat in the prevention and treatment of cancer. Platform and poster presentations made by AICR grant recipients, invited participants, and guests will provide a forum to increase understanding of the influences, mechanisms and practical applications of nutritional research on the cancer process, [9 CME hours for RDs and DTs by the ADA and 8.5 hours of AMA CME Category I credit by Georgetown University School of Medicine have been approved.] For information contact Rita Taliferro, Conference Management Division, Associate Consultants, Inc., 1726 M Street, NW, Suite 400, Washington, D. C. 20036. Phone: (202)737-8062.

7th European Workshop on Molecular and Cellular Endocrinology of the Testis Castle Elmau, Bavarian Alps, Germany, May 5-10, 1992 Plenary Sessions: Endocrine and paracrine regulation of testicular function/Inhibin, activin and growth factors/ Gonadotropin receptors/Androgen receptor and testosterone metabolism/Spatial arrangement of germ cells/Genetic control of spermatogenesis/Sperm maturation in the epididymis Invited Speakers: E. Y. Adashi (USA), A. O. Brinkman (NL), B. A. Cooke (UK), T. G. Cooper (D), B. Jegou (F), L. Martini (I), J. Mather (USA), M. van Noort (NL), W. Schulze (D), P. Vogt (D), D. Wolgemuth (USA), P. Wong (Hongkong) Miniposters: Ample time is allocated for the discussion of submitted miniposters and there will be extensive Meet-the-expert-sessions. Deadline for submission of miniposters and registration: January 4, 1992. Instructions for miniposters and registration forms are available from: Prof. Dr. E, Nieschlag, Institute of Reproductive Medicine of the University, Steinfurter Str. 107, D-4400 Minister, Germany, Phone: 49-251-836097, Fax: 49-251-836093.

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