anti-LIF Antibody Research Articles

Impact of anti leukemia inhibitory factor antibody on immune related gene expression in breast cancer Balb/c mouse model

Leukemia inhibitory factor (LIF) is involved in the progression of different cancers. In this study, we investigated the effect of anti-LIF antibodies on immune-related gene expression in the Balb/c mouse model of breast cancer. To immunize mice against LIF, recombinant LIF with Freund adjuvant was injected into the test group, whereas the control group received phosphate-buffered saline with adjuvant. Tumor induction (4T1 cell line) was performed by increasing the antibody titer. The expression of immune-related genes was evaluated by real-time PCR. The anti-LIF titer was significantly increased in the immunized group. The expression of genes related to the differentiation of T helper (Th)-1, Th-2, and Th-17 cells was significantly higher in the immunized group than in the control group. In addition, anti-LIF did not have a significant effect on the expression of genes related to the differentiation of regulatory T cells, and immune checkpoint-associated genes. Additionally, the test group had higher survival and lower tumor development rates. The results demonstrated that the anti-LIF antibody may potentially play a role in the differentiation of immune cells or immune responses. However, further studies utilizing advanced techniques are necessary to validate its function.

Antagonist anti-LIF antibody derived from naive human scFv phage library inhibited tumor growth in mice

BackgroundLeukemia inhibitory factor (LIF) is a multifunctional member of the IL-6 cytokine family that activates downstream signaling pathways by binding to the heterodimer consisting of LIFR and gp130 on the cell surface. Previous research has shown that LIF is highly expressed in various tumor tissues (e.g. pancreatic cancer, breast cancer, prostate cancer, and colorectal cancer) and promotes cancer cell proliferation, migration, invasion, and differentiation. Moreover, the overexpression of LIF correlates with poor clinicopathological characteristics. Therefore, we hypothesized that LIF could be a promising target for the treatment of cancer. In this work, we developed the antagonist antibody 1G11 against LIF and investigated its anti-tumor mechanism and its therapeutic efficacy in mouse models.ResultsA series of single-chain variable fragments (scFvs) targeting LIF were screened from a naive human scFv phage library. These scFvs were reconstructed in complete IgG form and produced by the mammalian transient expression system. Among the antibodies, 1G11 exhibited the excellent binding activity to human, cynomolgus monkey and mouse LIF. Functional analysis demonstrated 1G11 could block LIF binding to LIFR and inhibit the intracellular STAT3 phosphorylation signal. Interestingly, 1G11 did not block LIF binding to gp130, another LIF receptor that is involved in forming the receptor complex together with LIFR. In vivo, intraperitoneal administration of 1G11 inhibited tumor growth in CT26 and MC38 models of colorectal cancer. IHC analysis demonstrated that p-STAT3 and Ki67 were decreased in tumor tissue, while c-caspase 3 was increased. Furthermore, 1G11 treatment improves CD3+, CD4 + and CD8 + T cell infiltration in tumor tissue.ConclusionsWe developed antagonist antibodies targeting LIF/LIFR signaling pathway from a naive human scFv phage library. Antagonist anti-LIF antibody exerts antitumor effects by specifically reducing p-STAT3. Further studies revealed that anti-LIF antibody 1G11 increased immune cell infiltration in tumor tissues.

Optimizing the Expression and Solubilization of an E. coli-Produced Leukemia Inhibitory Factor for Anti-LIF Antibody Production and Use Thereof for Contraception in Mice

Leukemia inhibitory factor (LIF) is an essential cytokine for blastocyst implantation. This study evaluated the effect of LIF inhibition on the blockage of embryo implantation. A truncated mouse LIF (tmLIF) was designed and expressed in E. coli. The protein expression was optimized using different culture media and inducers. To block pregnancy, the mice were immunized by the purified protein via maternal injection of the protein or in utero injection of the anti-LIF serum. The expression of implantation-relevant genes was quantified in the uterine tissue. The results showed that the protein was expressed in aggregated form in E. coli. The highest yield of protein was produced in the M9 medium. The insoluble protein was completely dissociated by SDS and 2-ME combination, but not by urea. The maternal immunization reduced the number of offspring, but not significantly. Instead, in utero injection of the anti-LIF serum prevented the blastocyst implantation. Gene expression analyses showed decrease of Jam2, Msx1and HB-EGF genes and increase of Muc1 gene as the result of intrauterine administration of theanti-LIF serums. In conclusion, SDS-mediated solubilization of inclusion bodies was compatible with in vivo studies. The intrauterine administration of anti-LIF serum could prevent mouse pregnancy. This indicates that in utero application of LIF antibodies might be used as a contraceptive.

Induced Pluripotent Stem Cell-Derived Conditioned Medium Promotes Endogenous Leukemia Inhibitory Factor to Attenuate Endotoxin-Induced Acute Lung Injury.

The conditioned medium of induced pluripotent stem cells (iPSC-CM) can attenuate neutrophil recruitment and endothelial leakage of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Therefore, we investigated the mechanisms by which iPSC-CM regulate the interaction between neutrophils and the endothelium in ALI. Murine iPSCs (miPSCs) were delivered intravenously to male C57BL/6 mice (8–12 weeks old) 4 h after intratracheal LPS injection. A miPSC-derived conditioned medium (miPSC-CM) was delivered intravenously to mice after intratracheal LPS injection. DMSO-induced HL-60 cells (D-HL-60, neutrophil-like cells) and human umbilical vein endothelial cells (HUVECs) were used as in vitro models to assess the interaction of neutrophils and endothelial cells. miPSC-CM diminished the histopathological changes in the lungs and the neutrophil count in bronchoalveolar lavage fluids of ALI mice. miPSC-CM attenuated the expression of adhesion molecules in the lungs of ALI mice. Human iPSC conditioned medium (hiPSC-CM) reduced the expression of adhesion molecules in a HUVEC and D-HL-60 co-culture after LPS stimulation, which decreased the transendothelial migration (TEM) of D-HL-60. A human angiogenesis factors protein array revealed that leukemia inhibitory factor (LIF) was not detected in the absence of D-HL-60 and hiPSC-CM groups. hiPSC-CM significantly promoted the production of endogenous LIF in in vitro models. Administration of an anti-LIF antibody not only reversed the effect of iPSC-CM in ALI mice, but also blocked the effect of iPSC-CM on neutrophils TEM in in vitro models. However, a controlled IgG had no such effect. Our study demonstrated that iPSC-CM promoted endogenous LIF to inhibit neutrophils TEM and attenuate the severity of sepsis-induced ALI.

Abstract CT014: Initial results from the Phase I study of MSC-1, a humanized anti-LIF monoclonal antibody, in patients with advanced solid tumors

Abstract Background: Leukemia Inhibitory Factor (LIF) is a pleiotropic cytokine involved in many physiological and pathological processes. LIF is highly expressed in a subset of tumors across multiple solid tumor types and has been shown to correlate with poor prognosis. LIF is hypothesized to contribute to tumor growth and progression by acting on multiple aspects of cancer biology, including immunosuppression of the tumor microenvironment (TME), and regulation of cancer initiating cells (CICs), which are thought to underpin tumor growth, metastasis, and resistance to therapy. MSC-1 is a first-in-class humanized IgG1 monoclonal antibody that potently and selectively inhibits LIF. Blocking LIF with MSC-1 decreased tumor growth in multiple mouse tumor models, drove reprogramming of the TME through effects on immunosuppressive macrophages, and generated durable regressions when combined with anti-PD1. These findings form the basis of a robust therapeutic hypothesis, that MSC-1 treatment may lead to clinical activity in multiple cancer indications. Methods: The Phase 1 study of MSC-1 is enrolling patients with advanced relapsed/refractory solid tumors. The study employs an accelerated 3+3 escalation design to explore safety, PK, LIF peripheral target engagement, immuno-regulatory activity, and preliminary anti-tumor activity of MSC-1. Patients receive treatment with MSC-1 intravenously once every three-weeks until confirmed disease progression or intolerable toxicity. At the top three dose levels, the cohorts will be expanded to further assess safety, PK/target engagement, and to preliminarily assess MOA biomarkers in paired pre- and on treatment tumor tissue from patients. The Dose and Expanded Escalation will enroll patients without regard to their pretreatment LIF tumor levels. Results: As of January 28, 2019, dosing has occurred in 14 patients in the 5 preplanned Dose Escalation cohorts (225mg-1500mg) as well as in 15 patients in the expanded cohorts at 750mg and 1125mg doses for additional safety, PK/PD and biomarker analysis, including analysis of pretreatment and on treatment tumor biopsies in a subset. There have been no DLTs observed at any dose, and analysis of data to select a RP2D for Dose Expansion is ongoing. Citation Format: Alison Schram, Anna Spreafico, Marc Oliva, Irene Brana, Elena Garralda, Nehal Lakhani, Daniel Von Hoff, Erkut Borazanci, Naimish Pandya, Kimberly Hoffman, Robin Hallett, Patricia Giblin, Judit Anido, Adrianne Kelly, Robert Wasserman, Joan Seoane, Lillian Siu, David M. Hyman, Josep Tabernero. Initial results from the Phase I study of MSC-1, a humanized anti-LIF monoclonal antibody, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT014.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

STAT3 signaling maintains homeostasis through a barrier function and cell survival in corneal endothelial cells

The cornea protects the eye from inflammation, which is one of the leading causes of blindness. Severe inflammation in the anterior chamber disrupts the barrier function of corneal endothelial cells (CECs) leading to severe visual loss. However, the mechanism by which such inflammation affects CEC function and survival is unknown. Activation of STAT3 signaling regulates various intracellular responses through inflammation and generally mediates expression of the barrier function marker zonula occludens-1 (ZO-1). In this study, we investigated the relationship between the corneal endothelial barrier function and activation of STAT3 signaling through a variety of cytokines in human CECs. Phosphorylated STAT3 (pSTAT3) was expressed in human and mouse CECs. Inhibition of pSTAT3 remarkably decreased the expression of the ZO-1 protein, reduced the trans-endothelial electric resistance, and induced cell apoptosis. The expression level of ZO-1 mRNA was correlated with that of STAT3 mRNA in the human corneal endothelium. pSTAT3 was increased with the addition of LIF, IL-6, and IFN-γ. LIF expressed in CECs suppressed pSTAT3 activation as observed experimentally using an anti-LIF antibody. Promoter regions of ZO-1 and SOCS3 were directly regulated by transcriptional activation of STAT3. These findings suggest that regulation of the STAT3 pathway is essential for corneal endothelial homeostasis via barrier function and may protect from various inflammatory factors.

Leukemia inhibitory factor functions in parallel with interleukin-6 to promote ovarian cancer growth.

Ovarian carcinoma-associated mesenchymal stem cells (CA-MSC) produce not only high levels of IL6 but also the related cytokine leukemia inhibitory factor (LIF). Interleukin 6 (IL6) mediated activation of STAT3 is implicated as a critical therapeutic target for cancer therapy. Less is known about the role of LIF, which can similarly activate STAT3, in ovarian cancer. We therefore sought to evaluate the tumorigenic effects of CA-MSC paracrine LIF signaling and the redundancy of IL6 and LIF in activating ovarian cancer STAT3 mediated cancer growth. As expected, we found that both IL6 and LIF induce STAT3 phosphorylation in tumor cells. In addition, both IL6 and LIF increased the percentage of ALDH+ ovarian cancer stem-like cells (CSC). Supporting redundancy of function by the two cytokines, CA-MSC induced STAT3 phosphorylation and increased cancer cell ‘stemness’. This effect was not inhibited by LIF or IL6 blocking antibodies alone, but was prevented by dual IL6/LIF blockade or JAK2 inhibition. Similarly, small hairpin RNA (shRNA)-mediated reduction of IL6 or LIF in CA-MSC partially decreased but could not completely abrograte the ability of CA-MSC to induce STAT3 phosphorylation and stemness. Importantly, the in vivo pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with the JAK2 inhibitor ruxolitinib to a much greater extent than treatment with anti-IL6 or anti-LIF antibody alone. Ruxolitinib treatment also improves survival in the immunocompetent ovarian cancer mouse model system with ID8 tumor cells plus MSC. Ruxolitinib-treated tumors in both the immunocompromised and immunocompetent animal models demonstrate decreased phospho-STAT3, indicating on-target activity. In conclusion, CA-MSC activate ovarian cancer cell STAT3 signaling via IL6 and LIF and increase tumorigenesis cancer stemness. This functional redundancy suggests that therapeutic targeting of a single cytokine may be less effective than strategies such as dual inhibitor therapy or targeting shared downstream factors of the JAK/STAT pathway.

Abstract LB-B34: LIF as a novel cancer immunotherapy target: modulating the tumor microenvironment with MSC-1, a humanized anti-LIF monoclonal antibody

Abstract Leukemia Inhibitory Factor (LIF) is a pleiotropic cytokine involved in many physiological and pathological processes. LIF is highly expressed in a subset of tumors across multiple tumor types and has been shown to correlate with poor prognosis. LIF is hypothesized to contribute to tumor growth and progression by acting on multiple aspects of cancer biology, including immunosuppression of the tumor microenvironment and is a key regulator of cancer initiating cells (CICs). MSC-1, a first-in-class, humanized monoclonal antibody (IgG1), is a potent and selective inhibitor of LIF. MSC-1 leads to STAT3 inhibition by disrupting LIF signaling through the LIF receptor (LIFR). Blocking LIF with MSC-1 decreased tumor growth in multiple mouse tumor models and drove reprogramming of the tumor microenvironment through modulation of immunosuppressive macrophages and of several immune cell types. These findings form the basis of a robust therapeutic hypothesis, whereby MSC-1 treatment may lead to clinical activity in multiple cancer indications. Clinical testing is planned to initiate in the end of 2017 and trials will incorporate target engagement and PD biomarkers as well as efficacy endpoints. Citation Format: Jeanne Magram, Naimish Pandya, Kimberly Hoffman, Robin Hallett, Patricia Giblin, Angus Sinclair, Judit Anido, Isabel Huber Ruano, Ada Sala, Monica Pascual, Vanessa Chiganças, Robert Wasserman, Joan Seoane. LIF as a novel cancer immunotherapy target: modulating the tumor microenvironment with MSC-1, a humanized anti-LIF monoclonal antibody [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B34.

Abstract TMEM-030: INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT

Abstract PURPOSE: We have previously isolated mesenchymal stem cells from the microenvironment of tumors from ovarian cancer patients (cancer-associated MSC, CA-MSC). We found that CA-MSC promote tumorigenesis in vitro and in vivo by increasing cancer stem cells (CSC) in the epithelial tumor cells. Microarray analysis demonstrates the upregulation of the cytokines IL6 and LIF in CA-MSC versus control MSC. The purpose of this work is to characterize the role of cytokines interleukin-6 (IL6) and leukemia inhibitory factor (LIF) secreted by CA-MSC in ovarian cancer tumorigenesis. EXPERIMENTAL PROCEDURES: We have isolated and established cultures of CA-MSC. Healthy donor adipose-derived MSC were used as controls (Invitrogen). Ovarian cancer cells were treated with MSC-conditioned media or recombinant IL6 and/or LIF (EBioscience). Cytokine function was inhibited with IL6 blocking antibody, LIF blocking antibody, both antibodies or the JAK2 inhibitor ruxolitinib for dual blockade. Phosphorylated STAT3 and total STAT3 immunoblotting was performed by standard methods. Sphere assays were performed using low-adherence plates and X-Vivo20 serum free media (Lonza). Flow cytometry was carried out using the Aldefluor assay for aldehyde dehydrogenase activity (Stem Cell Technologies). For in vivo studies, NSG mice were injected with OVCAR3 ovarian cancer cells without or with CA-MSC, and then either mock treated or treated with IL6 blocking antibody (tocilizumab, Genentech), LIF blocking antibody or ruxolitinib. Tumor size was monitored both during treatment and at sacrifice, and tumors were analyzed. RESULTS: Treatment of ovarian cancer cells with either recombinant IL6 or LIF alone leads to STAT3 phosphorylation, suggesting functional redundancy in stimulation of this pathway. Neither LIF nor IL6 blocking antibodies alone can prevent upregulation of phospho-STAT3, but combined therapy with both LIF and IL6 blocking antibodies prevents STAT3 phosphorylation. The JAK2 inhibitor ruxolitinib inhibits cytokine-mediated upregulation of phospho-STAT3. Either LIF or IL6 treatment results in an increase the percentage of cancer stem cells in ovarian cancer cell lines, as measured using both sphere assays and aldehyde dehydrogenase activity. Ruxolitinib blocks both LIF and IL6 mediated increase in cancer stem cells. In vivo, the pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with ruxolitinib but not by treatment with anti-IL6 antibody alone or anti-LIF antibody alone. Ruxolitinib treated tumors demonstrate decreased phospho-STAT3, indicating on-target activity. CONCLUSIONS: IL6 and LIF are secreted by CA-MSC in the ovarian cancer tumor microenvironment. Both cytokines upregulate phospho-STAT3 and increase the percentage of cancer stem cells in epithelial tumor cells. Functional inhibition of these effects cannot be achieved in vitro or in vivo with blockade of either cytokine individually; dual blockade with compounds such as the JAK2 inhibitor ruxolitinib is necessary. Thus the shared downstream signaling molecules of the JAK/STAT pathway may be preferred targets for novel anticancer therapeutics for ovarian cancer. Citation Format: Karen McLean, Lijun Tan and Ronald J. Buckanovich. INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-030.

Research on the expression of integrin \u03b23 and leukaemia inhibitory factor in the decidua of women with cesarean scar pregnancy

BackgroundCesarean scar pregnancy (CSP) is a late serious complication of cesarean section. There has been an increase in the incidence of CSP worldwide in recent years. It’s a life-threatening condition because of the high risk of uncontrolled hemorrhage and uterine rupture. The mechanism of CSP is still unclear. The endometrial receptivity might be different in the cesarean scar between CSP and normal pregnancies. Endometrial expression of integrin β3 and LIF positively correlates with endometrial receptivity and embryo implantation. The purpose of the study is to explore the mechanism of CSP.MethodsThe EnVision two-step immunohistochemical staining technique was used to detect the expression of integrin β3 and LIF in the decidua of women with CSP (20 cases) and normal pregnancies (20 cases). The distribution and staining intensity of integrin β3 and LIF in the two groups were observed. Observation of the staining were done using microscope within five randomly selected high-power fields (HPF, 10 × 40). All data analyses were conducted with SPSS 17.0 and the statistical significance was set at P <0.05.ResultsThe decidua in the different parts of both two groups that stained with the anti-integrin β3 and anti-LIF antibody: most of the integrin β3 and LIF positive cells were located in glandular epithelium. The expression intensity of integrin β3 in the cesarean scar in CSP group was significant higher than the uterine cavity in CSP group and the cesarean scar in normal pregnancy group. It’s similar with the uterine cavity in normal pregnancy group. The expression intensity of LIF in the cesarean scar in CSP group was significant higher than the uterine cavity in CSP group and the cesarean scar in normal pregnancy group. It’s significant lower than the uterine cavity in normal pregnancy group.ConclusionsThe decidual integrin β3 and LIF might play an important role in the mechanism of CSP. The increase expression of integrin β3 and LIF in the cesarean scar decidua might be associated with embryo implantation in cesarean scar. The occurrence of CSP might be related to the changes of endometrial receptivity in local cesarean scar.

Analysis of the Effect of Leukemia Inhibitory Factor on Follicular Growth in Cultured Murine Ovarian Tissue.

Leukemia inhibitory factor (LIF) is expressed in the ovary and controls follicular growth. LIF has been reported to accelerate the primordial to primary follicle transition, the growth of cultured preantral follicles, and the maturation of oocytes. Previous reports on factors that regulate follicular growth have largely employed cultured follicles. However, there are several types of follicles and somatic cells in the ovary that are likely to interact with one another to regulate follicular growth. Therefore, a novel approach is essential for understanding the function of factors that regulate follicular growth in the ovary. In this study, we evaluated the function of LIF using cultured ovarian tissue. Ovarian tissue slices were cultured in the presence or absence of recombinant LIF and neutralizing anti-LIF antibody to enable continuous monitoring of follicular growth within the context of the ovary as well as analysis of the process of follicular growth. The results revealed that LIF inhibited the growth of primary, secondary, and antral follicles. Furthermore, we verified the inhibitory function of LIF using the neutralizing antibody, which accelerated follicular growth. These results suggest that LIF is likely to coordinate follicular growth in the ovary. The culture and analysis methods employed in this study are thus effective for clarifying the tissue-level functions of factors that regulate follicular growth within the ovary.

Embryo Implantation is Blocked by Intraperitoneal Injection with Anti-LIF Antibody in Mice

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fibroblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1), NANOG, SOX2, and NODAL, but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specific JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals.

Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss

Autoimmune injury to oligodendrocytes evokes an endogenous response in the central nervous system, which initially limits the acute injury to oligodendrocytes and myelin, and subsequently promotes remyelination. The key molecular and cellular events responsible for this beneficial outcome are incompletely understood. In this article, we utilize murine autoimmune encephalomyelitis (EAE) to focus on the effect of endogenously produced leukemia inhibitory factor (LIF) upon mature oligodendrocyte survival after demyelinating injury. We show that the mRNA for LIF is markedly upregulated in the spinal cord in the context of acute inflammatory demyelination. After clinical disease onset, administration of neutralizing anti-LIF antibodies over a four day period significantly worsens disease severity in two different murine EAE models. We also show that administration of neutralizing antibodies results in reduced activation of the cognate LIF receptor components in the spinal cord. Histologically, anti-LIF antibody administration increases the extent of acute demyelination (P < 0.01) and doubles the oligodendrocyte loss already induced by EAE (P < 0.05), without altering the extent of inflammatory infiltration into the spinal cord. Although acute EAE induces a rapid, three-fold increase in the proliferation of NG2 positive oligodendrocyte progenitors (P < 0.001), this response is not diminished by antagonism of endogenous LIF. We conclude that endogenous LIF is induced in response to autoimmune demyelination in the spinal cord and protects mature oligodendrocytes from demyelinating injury and cell death, thereby resulting in attenuation of clinical disease severity.

296 LEUKEMIA INHIBITORY FACTOR INFLUENCES SHEEP OOCYTE PARTHENOGENETIC DEVELOPMENT DURING THE TRANSITION FROM GERMINAL VESICLE TO EARLY PRONUCLEAR STAGE

Leukemia inhibitory factor (LIF) is an indispensable cytokine for female fertility. The influence of LIF on embryo development and particularly implantation has been recently confirmed; however, the effect of this cytokine on the oocyte has not been studied. The presence of LIF in human follicular fluid implies its possible role in the acquisition of oocyte competence. Furthermore, the up-regulation of LIF by steroid hormones in sheep makes entirely feasible the hypothesis that ovulatory estradiol peak plays a role in the preparation of female gamete for fertilization. With this in mind, we studied the effect of LIF during in vitro development of sheep oocytes mimicking the physiological expression of LIF induced by the ovulatory peak of estradiol in mice. GV stage oocytes matured and chemically activated in the presence of LIF and anti-LIF antibody were cultured to the blastocyst stage in our standard media. To eliminate the effect of the putative presence of LIF in heat inactivated fetal calf serum used for oocyte maturation, aliquots of LIF were treated at 56°C for 30 min and added to the maturation medium. The proportion of embryos that reached the blastocyst stage in vitro was significantly higher (P &lt; 0.001) for oocytes matured and activated with LIF (36/93; 39%) than for the group incubated with antibody against LIF (6/68; 9%). The significant effect of anti-LIF antibody (P &lt; 0.001) was also observed when compared with blastocysts developed from the control group of oocytes matured without LIF addition (31/106; 29%). Although the beneficial influence of LIF treatment on embryo development demonstrated with those preliminary data was not confirmed statistically, due to low number of oocytes involved, the proportion of embryos reaching the blastocyst stage in vitro was about 10% higher for those incubated with LIF than for either those cultured without the cytokine or those, matured in the presence of heat-treated LIF (15/55; 27%); however, the rate of blastocyst development appeared very similar to that of the control group. This study revealed for the first time a role of LIF in determining oocyte competence. Further investigation to determine how LIF achieves its effects on the oocyte are ongoing in our laboratory. This work was supported by FIRB RBNE01HPMX, COFIN 2002074357, COFIN 2003073943 002, and British Council 2004.

GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygous knockout mice

Glial cell line-derived neurotrophic factor (GDNF) can induce neuron-like differentiation of mouse pheochromocytoma (MPC) cell lines derived from mice with a heterozygous knockout mutation of nf1, the murine counterpart of the human gene mutated in neurofibromatosis type 1 (NF1). Here, we show that GDNF-induced differentiation in the MPC 862L cell line is mediated by the MEK/extracellular signal-regulated kinase (ERK) pathway. Neurite outgrowth, increased expression of growth-associated protein 43, and decreased incorporation of bromodeoxyuridine (BrdU) were induced by treatment with GDNF, H-RasV12, or a constitutively active MEK2. GDNF also induces leukemia inhibitory factor (LIF) via the MEK/ERK pathway, and LIF itself can elicit these differentiative changes via a cell-extrinsic autocrine/paracrine pathway. Treatment with anti-LIF neutralizing antibody depleted the differentiative activity of the conditioned medium from cells stimulated for MEK/ERK signaling, while recombinant LIF could induce differentiation in MPC cells, indicating that LIF is the sole factor with differentiative activity. LIF could activate MEK1/2 and STAT3, but LIF-induced differentiation was blocked only by the MEK1/2-specific inhibitor U0126, indicating that the MEK/ERK pathway is necessary for LIF action in MPC cells. Our findings suggest that LIF may be utilized for signaling mediated by GDNF and may be important in the pathobiology of neuroendocrine tumors.

Leukemia inhibitory factor is a mediator of Escherichia coli lipopolysaccharide-induced acute thymic atrophy.

Septic shock in animals and humans is associated with thymic atrophy and generalized lymphocyte apoptosis that impairs T cell responses. Injection of animals with E. coli lipopolysaccharide (LPS) mimics bacterial sepsis-induced thymic atrophy. Leukemia inhibitory factor (LIF) induces pituitary ACTH release in vivo, and LIF administration to mice induces acute thymic atrophy. We have investigated the role of LIF in LPS-induced thymic atrophy. LPS significantly elevated serum LIF within 1 h and induced thymic LIF mRNA within 6 h. Moreover, pretreatment of mice with anti-LIF antibody significantly reduced LPS-induced thymic atrophy. Using adrenalectomized mice and 17-day fetal thymic organ cultures treated with the steroidogenic enzyme inhibitor metyrapone, we demonstrated that LIF induced both systemic and intrathymic corticosteroid production. These data demonstrate systemic and intrathymic pathways of E. coli LPS-induced acute thymic atrophy mediated by LIF and corticosteroids.

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.

Identification of leukemia inhibitory factor as a potent mast cell growth-enhancing factor produced by mouse keratinocyte cell line, KCMH-1.

Inoculation of KCMH-1 cells, a keratinocyte-derived cell line established from a chemically induced skin tumor, into the skin of mice results in accumulation of mast cells around the resulting tumors. The conditioned medium of KCMH-1 cells enhances the growth of mast cells in vitro when they are cultured in the presence of NIH/3T3 fibroblasts, suggesting an important role for keratinocytes in mast cell hyperplasia in the skin. The aim of this study was to identify this mast cell growth-enhancing factor (MCGEF) by screening a KCMH-1 cDNA library. We first established a polyclonal antibody raised against the partially purified factor obtained from KCMH-1-conditioned medium which neutralized the MCGEF activity in KCMH-1-conditioned medium. Expression cloning of 1 x 10(6) cDNAs from the KCMH-1 cDNA library led to 16 cDNAs. One of these cloned cDNAs was found to be leukemia inhibitory factor (LIF). Both LIF produced by COS cells and the recombinant protein obtained commercially showed MCGEF activity when added to mast cell/fibroblast cocultures. MCGEF activity in KCMH-1-conditioned medium was completely neutralized by an anti-LIF monoclonal antibody. These results suggest that MCGEF produced by KCMH-1 cells is identical to LIF.