Leukemia Inhibitory Factor Receptor Research Articles

Leukaemia inhibitory factor receptor and gp130 in the human Fallopian tube and endometrium before and after mifepristone treatment and in the human preimplantation embryo

Leukaemia inhibitory factor (LIF) is a cytokine, which is associated with reproductive processes such as embryo development and implantation. The objectives of this study were to detect the presence of LIF receptor (LIFR) and glycoprotein 130 (gp 130) in the human Fallopian tube, endometrium and preimplantation embryo and to study the effect of mifepristone on the expression of LIFR and gp130 in the Fallopian tube. Twenty-two healthy fertile women received a single dose of 200 mg mifepristone or placebo immediately after ovulation (LH + 2). Biopsies were obtained from the Fallopian tubes during laparoscopic sterilization once between days LH + 4 and LH + 6 and from endometrium once between days LH + 6 and LH + 8. Preimplantation embryos were received from couples undergoing in vitro fertilization treatment. Immunohistochemistry was used to detect the presence of LIFR and gp130 in the Fallopian tube, endometrium and preimplantation embryo. Real-time PCR was used to study LIFR and gp130 expression in the Fallopian tube and endometrium. LIFR and gp130 were localized in the Fallopian tube, preimplantation embryo and endometrium. LIFR was more abundant in the Fallopian tube than in the endometrium. In the blastocyst, the staining of gp130 was mainly localized in the inner cell mass, whereas LIFR was expressed in all cells. The presence of LIFR and gp130 in the Fallopian tube and preimplantation embryo indicates a role for LIF in communication between the embryo and the Fallopian tube. Mifepristone did not affect the expression of LIFR and gp130 in the Fallopian tube, nor in the endometrium suggesting that progesterone might not be directly involved in the regulation of LIFR or gp130.

The role of SOCS3 in modulating leukaemia inhibitory factor signalling during murine placental development

Cytokines are an integral part of the adaptive and innate immune responses. The signalling pathways triggered by receptor engagement translate exposure to cytokine into a coordinated biological response. To contain these responses, the initiation, duration and magnitude of the signal is controlled at multiple levels. Suppressor of cytokine signalling (SOCS) proteins act in a negative feedback loop to inhibit signal transduction. Mice with a deletion of SOCS3 die at midgestion due to placental insufficiency. SOCS3-null placentae have increased numbers of mature trophoblast giant cells, disruption of the labyrinthine layer and a decrease in the spongiotrophoblast layer. Genetic crosses have revealed that the phenotype is due to dysregulation of signalling downstream of the leukaemia inhibitory factor (LIF) receptor alpha (LIFRα) and that the ligand responsible for this, LIF, is produced by embryonic tissues and acts in a paracrine fashion. These observations highlight the role of LIF as an extrinsic factor regulating trophoblast differentiation in vivo. The creation of mice with conditional deletion of SOCS3 in different tissues has also uncovered critical roles for SOCS3 in the regulation of IL-6, G-CSF and leptin signalling.

The Role of STAT-3 and LIF in regulating IgM secretion in B-1 cells: CD25+ B-1 cells and their characteristics (86.12)

Abstract B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major shortfall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we identify a novel subset of B-1 cells that express the high affinity IL-2 receptor (IL-2Ra/CD25) and preferentially have the activated form of the signal transducer and activator of transcription-3 (STAT3). We show that upon inhibition of pSTAT3, Ig secretion is elevated preferentially in the CD25+ subset. Consistent with the induction of Ig secretion, we detected increases in the expression of key regulators of the plasmacytic program, B lymphocyte inducer of maturation program 1 (BLIMP-1) and X-box binding protein 1 (XBP-1). In contrast to the CD25- subset of B-1, we also found that the CD25+ subset differentially expresses a number of genes including the STAT3 target SOCS3, the cytokine receptor GP130 and the leukemia inhibitory factor receptor (LIF-R). We demonstrate that treatment of B-1 cells with LIF induces Ig secretion preferentially in the CD25+ subset. Together, these data identify STAT3 and LIF as possible regulators of Ig secretion in B-1 cells. Furthermore, these results implicate a role for cytokines in regulating B-1 Ig secretion.

In VitroEvaluation of Leukemia Inhibitory Factor Receptor Antagonists as Candidate Therapeutics for Inflammatory Arthritis

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) are found in appreciable concentrations in synovial fluid from patients with rheumatoid arthritis (RA) but not osteoarthritis. Accordingly, both are potential therapeutic targets in inflammatory diseases of the joints. Several LIF antagonists have been developed. They have the capacity to inhibit the biologic activities of not only LIF but also other interleukin-6 (IL-6) subfamily cytokines, including OSM. Both LIF and OSM share the same receptor, which is part of a cytokine receptor super family in which the glycoprotein 130 (gp130) subunit is a common constituent. The aim of this study was to evaluate the antagonistic potentials of two LIF mutants, LIF05 and MH35-BD. Both are mutant forms of human LIF with reduced affinity for gp130 and greater LIF receptor (LIFR) binding affinity. The results, using Ba/F3 cell proliferation assay, acute-phase protein (haptoglobin) induction analysis in HepG2 human hepatoma cells, a porcine cartilage glycosaminoglycan release assessment for proteoglycan degradation, and a collagen release assay, show that these antagonists inhibit relevant LIF, OSM, and other IL-6 subfamily cytokines in vitro albeit with differential potencies and have, therefore, therapeutic potential for treatment of RA and perhaps other diseases.

Leukemia Inhibitory Factor: A Newly Identified Metastatic Factor in Rhabdomyosarcomas

Rhabdomyosarcoma frequently infiltrates bone marrow and this process involves the stromal-derived factor-1 (SDF-1)-CXCR4 axis. Because leukemia inhibitory factor (LIF), like SDF-1, is secreted by bone marrow stroma and directs the regeneration of skeletal muscles, we examined whether the LIF-LIF receptor (LIF-R) axis affects the biology of rhabdomyosarcoma cells. We found that in rhabdomyosarcoma cells, LIF stimulates the following: (a) phosphorylation of mitogen-activated protein kinase p42/44, AKT, and signal transducers and activators of transcription 3, (b) adhesion and chemotaxis, and (c) increased resistance to cytostatics. To compare the biological effects of LIF versus SDF-1, we examined the RH30 cell line, which is highly responsive to both ligands, and found that the chemotaxis of these cells is significantly reduced when the inhibitors of both receptors (T140 for CXCR4 and gp190 blocking antibody for LIF-R) are added simultaneously. Subsequently, by using repetitive chemotaxis to LIF or SDF-1, we selected from the RH30 line subpopulations of cells that respond to LIF but not SDF-1 (RH30-L) or to SDF-1 but not LIF (RH30-S). We found that (a) RH30-L cells seed better to the bone marrow, liver, and lymph nodes of immunodeficient mice than RH30-S cells and (b) mice inoculated i.m. with the RH30-L cells had more rhabdomyosarcoma cells in the bone marrow and lung after 6 weeks. Thus, we present the first evidence that the LIF-LIF-R axis may direct rhabdomyosarcoma metastasis. Further, because we showed that the in vivo metastasis of RH30 cells is inhibited by small interfering RNA against LIF-R, molecular targeting of this axis could become a new strategy to control the metastasis of rhabdomyosarcoma.

Leukemia Inhibitory Factor Induces the Chemomigration of Immortalized Gonadotropin-Releasing Hormone Neurons through the Independent Activation of the Janus Kinase/Signal Transducer and Activator of Transcription 3, Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase 1/2, and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 superfamily. LIF acts through a cell-surface receptor complex formed by two subunits, the specific LIF receptor beta (LIFRbeta) and the glycoprotein 130. Little is known about LIF involvement in modulating the neuroendocrine circuitry governing the reproductive function and, specifically, the development of GnRH-secreting neurons. In the present study, we evaluated the effect of LIF on the in vitro migration of GN11 cells, a model of immature and migratory GnRH neurons, and the signaling pathways involved in this process. GN11 cells expressed both LIFRbeta and glycoprotein 130 subunits. Exposure of GN11 cells to 100 ng/ml LIF resulted in activation of the Janus kinases (Jaks)/signal transducer and activator of transcription 3, MAPK/ERK1/2, and phosphatidylinositol 3-kinase/protein kinase B/Akt pathways. The selective inhibition of Jaks, MAPK kinase, and phosphatidylinositol 3-kinase indicated that these signaling pathways were activated independently by LIF and that Jak2 is not the main kinase involved in LIF signaling. Exposure of GN11 cells to LIF for 3 h induced a concentration-dependent chemotactic response, with a plateau at 100 ng/ml LIF. LIF was also found to induce chemokinesis of GN11 cells. Furthermore, LIF-promoted GN11 migration was the result of the partial and independent contribution of all the three signaling pathways activated by LIF. The present data, together with the observation that LIF and LIFRbeta are expressed prenatally in the mouse nasal compartment, would suggest that LIF might participate in the migration of GnRH neurons.

WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived CD133 + cells in vitro

Cord blood-derived CD133 + cells have a degree of non-hematopoietic potential and express transcripts of pluripotency markers including Oct-4, Sox-2, Rex-1, and leukemia inhibitory factor (LIF) receptor, as well as markers of progenitor cells, such as HoxB4, brachyury, and nestin. Having shown by transcriptome analysis that the mouse embryonic fibroblast (MEF) cells routinely used to maintain pluripotent embryonic stem cells express transcripts of the WNT/BMP families of signaling factors, we have assessed the effects on proliferation and differentiation of CD133 + cells of medium conditioned (CM) by MEF, by NIH3T3, and by NIH3T3 cells stably expressing WNT1, WNT3a, WNT4, WNT5a, and WNT11. Cultivation of CD133 + cells in MEF-CM led to a significant increase in cell number after 7 days of culture, while WNT-1, WNT3a-, and WNT11-CM increased the cell number significantly by 14 days of culture. During this period, WNT3a-CM increased the proportion of nestin-expressing cells and increased the ratio of blast-like cells to macrophages, suggesting that these signaling molecules contribute to the maintenance of an undifferentiated, blast-like phenotype. The number of cells expressing the endothelial-related marker CD31 + was significantly increased following culture in WNT5a- and WNT11-CM, whereas the number of cells positive for von Willebrand (vW) factor was maintained during 14 days of culture only in the presence of WNT4-CM. In addition, WNT5a-CM led to increased β-catenin mRNA levels and the presence of β-catenin protein in the cytoplasm and nucleus, consistent with the activation of the WNT signaling pathway. We conclude that in vitro conditioning of CD133 + cells by media containing specific WNT signaling factors influences the non-hematopoietic potential of CD133 + cells and dynamically alters the expression of the neural stem/progenitor cell marker nestin and the endothelial-related cell surface markers CD31 and vW factor.

Differential stimulation‐induced receptor localization in lipid rafts for interleukin‐6 family cytokines signaling through the gp130/leukemia inhibitory factor receptor complex

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are cytokines which signal through receptor complexes that include the receptor subunits glycoprotein 130 (gp130) and the LIF receptor (LIFR), but CNTF also requires the non-signal transducing CNTF receptor (CNTFR) for binding. We show here that in IMR-32 neuronal cells endogenously expressing the receptor subunits for LIF and CNTF, CNTFR, but not gp130 or LIFR, is found in detergent-resistant lipid rafts. In addition, stimulation of these cells with CNTF resulted in a rapid translocation of a portion of gp130 and LIFR into detergent-resistant lipid rafts while an equivalent stimulation with LIF did not. Disruption of lipid rafts by cholesterol depletion of cell membranes blocked the CNTF-induced translocation of LIFR and gp130. Interestingly, while cholesterol-depletion did not inhibit signal transducer and activator of transcription 3 phosphorylation by either CNTF or LIF stimulation, it strongly inhibited both CNTF- and LIF-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 and Akt. LIF and CNTF generally appear to have redundant effects in cells responsive to both cytokines. Intriguingly, the data presented here suggest a possible mechanism whereby CNTF or other cytokines that signal through CNTFR could generate signals distinct from those elicited by cytokines such as LIF which utilize a LIFR/gp130 heterodimer, via association with or exclusion from lipid rafts.

A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.

PPARγ2 Regulates a Molecular Signature of Marrow Mesenchymal Stem Cells

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. Bone marrow mesenchymal stromal/stem cells (MSC) contain a compartment that provides progeny with bone forming osteoblasts and fat laden adipocytes as well as fibroblasts, chondrocytes, and muscle cells. In addition, marrow MSC provide an environment for support of hematopoiesis, including the development of bone resorbing osteoclasts. The PPAR2 nuclear receptor is an adipocyte-specific transcription factor that controls marrow MSC lineage allocation toward adipocytes and osteoblasts. Increased expression of PPAR2 with aging correlates with changes in the MSC status in respect to both their intrinsic differentiation potential and production of signaling molecules that contribute to the formation of a specific marrow micro-environment. Here, we investigated the effect of PPAR2 on MSC molecular signature in respect to the expression of gene markers associated exclusively with stem cell phenotype, as well as genes involved in the formation of a stem cell supporting marrow environment. We found that PPAR2 is a powerful modulator of stem cell-related gene expression. In general, PPAR2 affects the expression of genes specific for the maintenance of stem cell phenotype, including LIF, LIF receptor, Kit ligand, SDF-1, Rex-1/Zfp42, and Oct-4. Moreover, the antidiabetic PPAR agonist TZD rosiglitazone specifically affects the expression of “stemness” genes, including ABCG2, Egfr, and CD44. Our data indicate that aging and anti-diabetic TZD therapy may affect mesenchymal stem cell phenotype through modulation of PPAR2 activity. These observations may have important therapeutic consequences and indicate a need for more detailed studies of PPAR2 role in stem cell biology.

An In Vitro and In Vivo Evidence That Downregulation of Leukemia Inhibitory Factor (LIF) Receptor (LIF-R) Decreases the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Rhabdomyosarcoma (RMS) and skeletal muscle-derived tumors frequently infiltrate bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1-CXCR4 receptor (Blood 2002; 100:2597) and hepatocyte growth factor (HGF)-c-Met receptor (Cancer Res. 2003;63:7926) play an important role in RMS metastasis to BM. Leukemia inhibitory factor (LIF) is a well known factor that plays an important role in skeletal muscle development/regeneration and similarly as SDF-1 and HGF is secreted by BM stroma. This prompted us to examine whether the LIF-LIF receptor (LIF-R) axis affects the biology/metastasis of RMS cells. We employed in our studies, human established RMS cell lines, as well as RMS samples isolated from patients and noticed that LIF-R was expressed not only on established human RMS cell lines (7/7) but more importantly, it was also detectable in patient samples (23/23). We also found that in RMS cells LIF stimulatesphosphorylation of MAPKp42/44, AKT and STAT3,chemotaxis and adhesion andincreases resistance to cytostatics (e.g., etoposide).These LIF-mediated effects were inhibited after downregulating the LIF-R by siRNA. To learn more on the biological significance of the LIF-LIF-R axis in vivo we employed two models. First, human RMS cells (RH-30) were exposed or not exposed to LIF-R siRNA and subsequently injected into SCID™-Beige immunodeficient mice. To estimate the number of RMS cells that seed to BM and liver in these animals, we isolated DNA and using real- time RT-PCR, amplified human a-satellite sequences and murine b-actin. The number of human cells present in murine organs was subsequently calculated from a standard curve derived from mixing varying numbers of human cells with a constant number of murine cells. We noticed that downregulation of LIF-R by siRNA significantly decreased the number of human RMS cells in murine BM and liver (x4 and x2 respectively). In a second model, the RH30 cell line was selected by repetitive chemotaxis for cells that are highly responsive to LIF (RH-30 L) and subsequently the cells from parental RH-30 cell line and RH-30 L cells were injected intramuscularly. Six weeks after tumour inoculation, we detected more metastasis in bone marrow and lungs in mice injected with RH-30L cells as compared to parental RH-30 clone (x6 and x3 respectively). In conclusion, we present evidence for the first time that the inhibition of LIF-LIF-R axis may decrease the invasive potential of human RMS both in vitro and in vivo. Hence, molecular targeting of LIF-LIF-R axis could possibly become a more effective new strategy to control the progression and metastasis of RMS.

Leukemia Inhibitory Factor (LIF): Marrow-Derived Human Mesenchymal Stem Cell (huMSC) Secretion and Potential Impact on Umbilical Cord Blood (UCB) CD133+ Hematopoietic Stem Cells (HSC) Differentiation.

In vitro expansion of UCB using cytokines has been pursued to overcome the limited stem cell content of a single UCB graft. We have previously demonstrated that a feeder layer of huMSC inhibits UCB HSC differentiation during short-term cytokine-driven expansion in vitro. The protein LIF has been shown to inhibit the differentiation of neurosphere stem cells during 3 week culture over a monolayer of murine stromal cells (C. Shih, et al., 2001). We sought to investigate the hypothesis that LIF secreted by bone marrow (BM) derived huMSCs is involved in inhibition of UCB HSC differentiation during short-term cytokine-driven expansion in vitro. BM derived huMSCs (third passage) were cultured at 2x10^6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72 hours and analyzed for LIF secretion levels by ELISA (Quantikine). LIF secreted by huMSC was noted at all four time points, with peak secretion at 48 h (mean 52.1±3.3 pg/ml) (n=3). UCB was obtained according to institutional guidelines after normal full-term deliveries, collected into bags with citrate dextrose (Allegiance), and MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi) and surface stained for LIF receptor (LIF-R). Surface expression of LIF-R on gated CD133+ cells was 2.61%. Total LIF-R expression in isolated CD133+ cells was further confirmed by Western blot (n=3) using anti-LIF-R antibody (Chemicon). Isolated UCB CD133+ were plated in 24 well plates at 3.3x10^3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B (Gibco). UCB CD133+ were culture-expanded for 96h with or without the addition of recombinant human LIF (Chemicon) (10ng/ml) in a combination of cytokines including: IL-3 (20 ng/mL), IL-6 (20 ng/mL), Flt-3L (100 ng/mL) (R&D), SCF (100 ng/mL), G-CSF (20 ng/mL), and EPO (0.1 U/mL) (Amgen Inc.). At 0, 48, and 96 h cell counts were obtained and flow analysis was performed including surface staining for: CD133, CD34, CD38, HLA-DR, CD33, CD71, and CD41B (Becton Dickinson). At 48 hours, higher cell counts in cultures without LIF were noted 6.3x10^4 ± 0.9/ml, compared with cultures with LIF 4.4x10^4 ± 0.8/ml. However, at 96 h cell counts equalized when comparing cultures with or without LIF at 7.510x10^4 ± 0.9/ml, and 7.8x10^4±0.9/ml respectively (n=6). Surface expression of differentiation markers on gated CD133+ cells did not differ when comparing cultures (n=3). In summary, we observed LIF secretion by MSC peaks at 48 h at a concentration 3 logs lower than that previously used to inhibit stem cell differentiation (10 ng/ml). Although LIF-R is expressed on CD133+ HSC, no difference in cell expansion nor surface phenotype of UCB 133+ cells was observed at early time points (96h) during expansion in cytokines with or without the addition of LIF. Taken together, huMSC mediated inhibition of cytokine-driven UCB HSC differentiation is not attributable to LIF secretion alone and may require direct cell contact between UCB CD133 HSC and huMSC. Studies are ongoing to determine whether LIF may augment huMSC-based UCB CD133 expansion.

Oncostatin M (OSM) Cytostasis of Breast Tumor Cells: Characterization of an OSM Receptor β–Specific Kernel

The interleukin-6 cytokine oncostatin M (OSM) induces potent growth-inhibitory and morphogenic responses in several different tumor cell types, highlighting the importance of OSM signaling mechanisms as targets for therapeutic intervention. The specific molecular pathways involved are not well understood, as OSM can signal through two separate heterodimeric receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) alpha and gp130/OSM receptor beta (OSMRbeta). In this investigation, we used a LIFR antagonist to help resolve signaling responses and identify patterns of gene expression elicited by the different receptor complexes. OSM-induced biological effects on breast tumor-derived cell lines were specifically mediated through the gp130/OSMRbeta complex. Each cytokine tested exhibited differential signaling capability and manifested both shared and unique patterns of gene activation, emphasizing compositional differences in activator protein-1 transcription factor activity and expression. In particular, OSM strongly activated the c-Jun NH(2)-terminal kinase (JNK) serine/threonine kinase and downstream components, including activating transcription factor (ATF)/cyclic AMP-responsive element binding protein family member, ATF3. JNK/stress-activated protein kinase kinase inhibition abrogated cell morphogenesis induced by OSM, indicating an important role for this pathway in OSM specificity. These findings identify a core signaling/transcriptional mechanism specific to the OSMRbeta in breast tumor cells.

S-SHIP Associates with Receptor Complexes Essential for Pluripotent Stem Cell Growth and Survival

Embryonic stem (ES) cells are pluripotent cells that have the ability to either self-renew or differentiate into any cell type found in the mammalian body. The signaling pathways required for self-renewal of these cells are yet to be defined. Previously we identified a stem cell-specific isoform of the protein SH2 domain-containing 5'-inositol phosphatase (SHIP) that we call s-SHIP, which is expressed in both pluripotent ES cells and adult tissue-specific multipotent cells, such as hematopoietic stem cells (HSCs). s-SHIP lacks an SH2 domain but contains a 5'-inositol phosphatase domain and several protein-protein interaction domains that potentially enable its participation in many different signaling pathways. Here we show that s-SHIP associates with gp130, which forms a heterodimeric complex with the leukemia inhibitory factor receptor (LIFR). Signaling through LIFR and other receptors that heterodimerize with gp130 is critical for growth and survival of ES cells and HSCs. Our findings provide biochemical evidence that s-SHIP participates in signaling pathways important for the maintenance of pluripotent stem cell populations.

Altered neuronal responses and regulation of neurotrophic proteins in the medial septum following fimbria‐fornix transection in CNTF‐ and leukaemia inhibitory factor‐deficient mice

Degeneration of axotomized GABAergic septohippocampal neurones has been shown to be enhanced in ciliary neurotrophic factor (CNTF)-deficient mice following fimbria-fornix transection (FFT), indicating a neuroprotective function of endogenous CNTF. Paradoxically, however, the cholinergic population of septohippocampal neurones was more resistant to axotomy in these mutants. As leukaemia inhibitory factor (LIF) has been identified as a potential neuroprotective factor for the cholinergic medial septum (MS) neurones, FFT-induced responses were compared in CNTF(-/-), LIF(-/-) and CNTF/LIF double knockout mice. In CNTF(-/-) mice, FFT-induced cholinergic degeneration was confirmed to be attenuated as compared with wildtype mice. The expression of both LIF and LIF receptor beta was increased in the MS providing a possible explanation for the enhanced neuronal resistance to FFT in these animals. However, ablation of the LIF gene also produced paradoxical effects; following FFT in LIF(-/-) mice no loss of GABAergic or cholinergic MS neurones was detectable during the first postlesional week, suggesting that other efficient neuroprotective mechanisms are activated in these animals. In fact, enhanced activation of astrocytes, a source of neurotrophic proteins, was indicated by increased up-regulation of glial fibrillary acidic protein and vimentin expression. In addition, mRNA levels for neurotrophin signalling components (e.g. nerve growth factor, p75(NTR)) were differentially regulated. The positive effect on axotomized cholinergic neurones seen in CNTF(-/-) and LIF(-/-) mice as well as the increased up-regulation of astrogliose markers was abolished in CNTF/LIF double knockout animals. Our results indicate that endogenous CNTF and LIF are involved in the regulation of neuronal survival following central nervous system lesion and are integrated into a network of neurotrophic signals that mutually influence their expression and function.

Optimization of a flow cytometry‐based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow

To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation. Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45low D7-FIB+ LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases. In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45low D7-FIB+ LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, 15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45low D7-FIB+ LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45low D7-FIB+ LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively). This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease.

Expression of leukemia inhibitory factor and its receptors is increased during differentiation of human embryonic stem cells

To investigate gene expression profiles during the early spontaneous differentiation of human embryonic stem cells (hESCs), with particular emphasis on leukemia inhibitory factor (LIF)-induced pathways and the ultrastructural surface morphology of the undifferentiated and spontaneously differentiated hESCs. Prospective experimental study. University laboratory. Four hESC cell lines. The effect of LIF on receptor expression level was studied in cultures. Gene expression in the hESC line HS237 was analyzed using microarrays. Real-time reverse-transcription polymerase chain reaction was used to validate the microarray results in four hESC lines (HS181, HS235, HS237, HS293). Immunohistochemistry was used to assay LIF, LIF receptor, and gp130 protein expression. Cell surface morphology was studied using scanning electron microscopy. The expression of LIF, LIF receptor, and gp130 messenger RNA and protein was increased in spontaneously differentiated HS237 cells compared with undifferentiated cells, with high expression of an inhibitor of LIF-mediated signaling, suppressor of cytokine signaling-1, in undifferentiated hESCs. Genes, those expressed specifically and those shared in undifferentiated hESCs, differentiated cells, and in fibroblasts, were identified. Supplementation with LIF did not affect the LIF receptor expression. The expression of LIF and its receptors is low in undifferentiated hESCs but increases during differentiation. Added LIF does not prevent spontaneous differentiation. Suppressor of cytokine signaling-1 may prevent LIF signaling in hESCs.

Continued Administration of Ciliary Neurotrophic Factor Protects Mice from Inflammatory Pathology in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. We show that daily administration (days 8 to 24) of murine ciliary neurotrophic factor (CNTF), a neurotrophic factor that has been described as a survival and differentiation factor for neurons and oligodendrocytes, significantly ameliorates the clinical course of a mouse model of multiple sclerosis. In the acute phase of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein peptide 35-55, treatment with CNTF did not change the peripheral immune response but did reduce the number of perivascular infiltrates and T cells and the level of diffuse microglial activation in spinal cord. Blood brain barrier permeability was significantly reduced in CNTF-treated animals. Beneficial effects of CNTF did not persist after it was withdrawn. After cessation of CNTF treatment, inflammation and symptoms returned to control levels. However, slight but significantly higher numbers of oligodendrocytes, NG2-positive cells, axons, and neurons were observed in mice that had been treated with high concentrations of CNTF. Our results show that CNTF inhibits inflammation in the spinal cord, resulting in amelioration of the clinical course of experimental autoimmune encephalomyelitis during time of treatment.

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c‐Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.

Regulation of pre–B cell colony‐enhancing factor by STAT‐3–dependent interleukin‐6 trans‐signaling: Implications in the pathogenesis of rheumatoid arthritis

To determine whether interleukin-6 (IL-6) trans-signaling directs the expression of pre-B cell colony-enhancing factor (PBEF) in vitro and in vivo. Complementary DNA from rheumatoid arthritis (RA) synovial fibroblasts treated with IL-6 and soluble IL-6 receptor (sIL-6R) was used to probe a cytokine microarray. PBEF regulation by the IL-6-related cytokines, IL-6, sIL-6R, oncostatin M (OSM), IL-11, and leukemia inhibitory factor (LIF) was determined by reverse transcription-polymerase chain reaction analysis. IL-6-mediated STAT-3 regulation of PBEF was determined using a cell-permeable STAT-3 inhibitor peptide. Antigen-induced arthritis (AIA) was induced in wild-type (IL-6(+/+)) and IL-6-deficient (IL-6(-/-)) mice. PBEF and STAT were detected by immunohistochemistry, immunoblotting, and electrophoretic mobility shift assay. Synovial levels of PBEF were quantified by enzyme immunoassay. IL-6 trans-signaling regulated PBEF in a STAT-3-dependent manner. In addition, PBEF was regulated by the IL-6-related cytokine OSM, but not IL-11 or LIF. Flow cytometric analysis of the IL-6-related cognate receptors suggested that OSM regulates PBEF via its OSM receptor beta and not its LIF receptor. The involvement of PBEF in arthritis progression was confirmed in vivo, where induction of AIA resulted in a 4-fold increase in the synovial expression of PBEF. In contrast, little or no change was observed in IL-6(-/-) mice, in which the inflammatory infiltrate was markedly reduced and synovial STAT-1/3 activity was also impaired. Analysis of human RA synovial tissue confirmed that PBEF immunolocalized in apical synovial membrane cells, endothelial cells, adipocytes, and lymphoid aggregates. Synovial fluid levels of PBEF were significantly higher in RA patients than in osteoarthritis patients. Experiments presented herein demonstrate that PBEF is regulated via IL-6 trans-signaling and the IL-6-related cytokine OSM. PBEF is also actively expressed during arthritis. Although these data confirm an involvement of PBEF in disease progression, the consequence of its action remains to be determined.