Leukemia inhibitory factor: Recent advances and implications in biotechnology

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with several functions in health and disease ranging from inflammation to cancer. LIF is also a potential target and/or therapeutic agent for diseases such as multiple sclerosis, stroke and even psychological disorders, where the function of LIF as a neurotrophic factor has only recently been explored. In recent years, a limited number of LIF clinical trials have been completed, which partially explains the shortage of effective applications as a therapeutic agent. With the increasing interest from biotechnology companies producing recombinant LIF, this status quo will certainly change, and the potential impact of LIF in terms of disease diagnosis, treatment and management will be realized.     

Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre-implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF-deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre-implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley-Liss, Inc.     

Immunohistochemical localization of leukemia inhibitory factor, interleukins 1 and 6 at the primary implantation site in the rhesus monkey

Blastocyst implantation and placentation involve localized inflammatory type of responses at and around the site of nidation. In the present study, the likely involvement of inflammatory cytokines, namely, leukemia inhibitory factor (LIF), interleukins 1 alpha and 1 beta (IL-1alpha and IL-1beta) and IL-6 at the primary implantation site of the rhesus monkey was examined immunocytochemically during lacunar (n=6) and villous (n=8) stages of gestation. Trophoblast cells and extraembryonic mesenchymal cells were immunopositive for LIF and IL-1alpha. The distribution of IL-1beta and IL-6 in trophoblast cells was low in lacunar stage samples, however, a higher degree of immunopositivity for IL-6 was observed in villous stage samples. Decidual cells were immunopositive for all the cytokines studied. In lacunar stage samples, plaque cells adjacent to implanted nidus were immunopositive for all the cytokines examined, and the degree of their immunoprecipitation increased, except that of IL-1beta, during the villous stage. Luminal and glandular epithelial cells were immunopositive for LIF, IL-1alpha, IL-1beta and IL-6 in lacunar and in villous stage samples. LIF immunopositivity was detected in endothelial cells of blood vessels within and below chorionic plate and cytotrophoblast shell, while vascular smooth muscle cells were positive for all the cytokines studied. The temporo-spatial characteristics of LIF, IL-1alpha, IL-1beta and IL-6 protein expressions in primary implantation sites of the rhesus monkey suggest that these pro-inflammatory cytokines play specific roles in regulating trophoblast cell proliferation, differentiation, invasion and associated maternal tissue remodelling during early gestation.     

Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro.

The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytes maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL-11 (10 ng/ml), or IL-6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum-free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin-3 (IL-3), LIF, IL-6, and IL-11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-11 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3+IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-11, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and IL-11 promote murine and human Mk maturation in vitro, although the effect of IL-11 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on Mk ploidy, IL-11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Leukemia inhibitory factor protects against liver steatosis in nonalcoholic fatty liver disease patients and obese mice

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide. However, the molecular mechanisms that promote dysregulation of hepatic triglyceride metabolism and lead to NAFLD are poorly understood, and effective treatments are limited. Leukemia inhibitory factor (LIF) is a member of the interleukin-6 cytokine family and has been shown to regulate a variety of physiological processes, although its role in hepatic triglyceride metabolism remains unknown. In the present study, we measured circulating LIF levels by ELISA in 214 patients with biopsy-diagnosed NAFLD as well as 314 normal control patients. We further investigated the potential role and mechanism of LIF on hepatic lipid metabolism in obese mice. We found that circulating LIF levels correlated with the severity of liver steatosis. Patients with ballooning, fibrosis, lobular inflammation, and abnormally elevated liver injury markers alanine transaminase and aspartate aminotransferase also had higher levels of serum LIF than control patients. Furthermore, animal studies showed that white adipose tissue–derived LIF could ameliorate liver steatosis through activation of hepatic LIF receptor signaling pathways. Together, our results suggested that targeting LIF-LIF receptor signaling might be a promising strategy for treating NAFLD.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

白细胞介素-11研究和开发进展

白细胞介素 １１是美国ＦＤＡ在１９９７年批准的第一个促血小板生成的细胞因子药品。本文就促血小板生成类细胞因子的Ｒ＆Ｄ、ＩＬ １１基因与蛋白结构、ＩＬ １１生物学活性、药学研究以及临床应用作一综述。     

Differential expression of genes linked to the leukemia inhibitor factor signaling pathway during the estrus cycle and early pregnancy in the porcine endometrium

The objective of this study was to determine the expression profiles of leukemia inhibitory factor (LIF) and its receptor (LIFR), interleukin 6 receptor (IL6R), tumor protein p53 (TP53) and B-cell CLL/lymphoma 2 (BCL2) in the porcine endometrium on selected days of the estrous cycle and pregnancy. Time- and reproductive status (estrous cycle/pregnancy)-specific patterns of expression were identified for all investigated genes. The most pronounced changes were seen on Days 12 and 14 of pregnancy when maternal recognition of pregnancy and implantation, respectively, occurs in pigs.     

Leukemia inhibitory factor coordinates the down-regulation of the visual cycle in the retina and retinal-pigmented epithelium

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.     

Distribution of cholinergic neuronal differentiation factor/leukemia inhibitory factor binding sites in the developing and adult rat nervous system in vivo

Cholinergic neuronal differentiation factor/leukemia inhibitory factor (CDF/LIF) is a multi-functional cytokine that affects neurons as well as many other cell types. Toward elucidating its neural functions in vivo, we previously investigated the distribution of CDF/LIF binding sites with iodinated native CDF/LIF in embryonic to postnatal day 0 (P0) rats. In the present study, we have extended our examination to postnatal ages and find that specific CDF/LIF binding sites are present at defined developmental stages in additional brain regions not previously exhibiting binding by P0. High levels of binding are detected in all P7 sensory and autonomic ganglia examined, but only in restricted postnatal central nervous system structures. Cranial motor and mesencephalic trigeminal neurons maintain high levels throughout, while binding to spinal motor neurons, which decreases to low levels at P0, reappears by P14 and increases with age. Most other structures, which show detectable binding by P0, exhibit higher levels at postnatal ages, including the red, deep, ventral cochlear, trapezoid, superior olivary, vestibular, ventral tegmental, and ventral posterior thalamic nuclei as well as the glomerular layer of the olfactory bulb. High levels are also detected in several structures for the first time after P0, including the cerebellar cortex (molecular and Purkinje cell layers), lateral reticular nucleus of the medulla and reticular formation, as well as the reticulotegmental, medial geniculate, solitary (rostral, dorsomedial, and commissural regions), medial septal, lateral mammillary, and lateral habenular nuclei. These results not only identify regions of potential CDF/LIF-responsive neurons and glia throughout development but suggest new CDF/LIF roles in the nervous system. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 163–192, 1997.     

Leukemia inhibitory factor, leukemia inhibitory factor receptor, and glycoprotein 130 in rhesus monkey uterus during menstrual cycle and early pregnancy

This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.     

Expression of a bioactive recombinant human interleukin-11 in chicken HD11 cell line

To direct the synthesis and secretion of recombinant human interleukin-11 (rhIL-11) in chicken HD11 cells, a plasmid targeting the c-lysozyme gene has been constructed which contains the mature cytokine cDNA in frame with the lysozyme leader sequence. The upregulation of rhIL-11 mediated by LPS proves the knock-in of hIL-11 cDNA in the lysozyme gene. The bioactivity of the expressed protein is demonstrated and quantified with the hIL-11 dependent 7TD1 and B9 cell lines. The electrophoretic mobility, receptor binding properties and growth promoting effect of the chicken-derived cytokine are identical to those of a rhIL-11 expressed in Escherichia coli. These results describe the secretion of a biologically active rhIL-11 expressed by an avian cellular machinery.     

Expression of the fusion protein recombinant human granulocyte-macrophage colony stimulating factor and leukemia inhibitory factor in a baculovirus vector system

A fusion gene coding human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) cDNAs was inserted into the transfer vector pSXIVVI⁺ X3 with the control of Syn and XIV promoters. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). The fusion protein recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) could be synthesized in cells infected with recombinant virus at a level of about 23% of their total cellular protein. Activity analysis of the fusion protein in infected cells revealed that it exhibited the dual activities of GM-CSF and LIF. Western blot analysis of the expressed fusion protein in infected larvae showed that the virus-mediated fusion protein, with a molecular weight of ∼35 kDa, is confirmed with immunoreactivity. Received 02 December 1998/ Accepted in revised form 07 May 1999     

Combined action of stem cell factor,leukemia inhibitory factor,and cAMP on in vitro proliferation of mouse primordial germ cells

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre-formed cell feeder layers. The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 μM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5-bromo-2′-deoxyuridin (BrdU) incorporation. These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro. © 1993 Wiley-Liss, Inc.     

Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor

Leukemia inhibitory factor (LIF) can regulate the survival and differentiation of certain neurons and glial cells in culture. To determine the role of this cytokine in the central nervous system in vivo, we examined the brains of young and adult mice in which the LIF gene was disrupted. Immunohistochemical staining of neurons for choline acetyltransferase, tyrosine hydroxylase, serotonin, parvalbumin, calbindin, neuropeptide Y, vasoactive intestinal polypeptide, and calcitonin gene-related peptide revealed no significant differences between null mutant and wild-type (WT) brains. In contrast, analysis of glial phenotypes demonstrated striking deficits in the LIF-knockout brain. Staining with several anti-glial fibrillary acidic protein (GFAP) antibodies showed that the number of GFAP-positive cells in various regions of the hippocampus in the female mutant is much lower than in the WT. The null male hippocampus also displays a significant, though less marked deficit. The number of astrocytes in the mutant hippocampus, as determined by S-100 staining, is not, however, significantly different from WT. In addition, quantification of immunohistochemical staining of female, but not male, mutants reveals a significant deficit in myelin basic protein content in three brain regions, suggesting alterations in oligodendrocytes as well. Thus, while overall brain histology appears normal, the absence of LIF in vivo leads to specific, sexually dimorphic alterations in glial phenotype. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 509–524, 1998