An antagonist for the leukemia inhibitory factor receptor inhibits leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M

The leukemia inhibitory factor receptor (LIF-R) is activated not only by LIF, but also by cardiotrophin-1, ciliary neurotrophic factor with its receptor, and oncostatin M (OSM). Each of these cytokines induces the hetero-oligomerization of LIF-R with gp130, a signal-transducing subunit shared with interleukin-6 and interleukin-11. The introduction of mutations into human LIF that reduced the affinity for gp130 while retaining affinity for LIF-R has generated antagonists for LIF. In the current study, a LIF antagonist that was free of detectable agonistic activity was tested for antagonism against the family of LIF-R ligands. On cells that express LIF-R and gp130, all LIF-R ligands were antagonized. On cells that also express OSM receptor, OSM was not antagonized, demonstrating that the antagonist is specific for LIF-R. Ligand-triggered tyrosine phosphorylation of both LIF-R and gp130 was blocked by the antagonist. The antagonist is therefore likely to work by preventing receptor oligomerization.     

Reconstitution of the response to leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in hepatoma cells

Ciliary neurotrophic factor (CNTF) has been described as a neuro-active cytokine that shares functional similarities with the leukemia inhibitory factor (LIF). We demonstrate here that, like LIF, CNTF stimulates expression of acute phase plasma proteins in rat H-35 hepatoma cells. Transfection of the LIF receptor into Hep3B hepatoma cells reconstituted LIF and oncostatin M regulation of acute phase plasma protein genes. Co-expression of the LIF receptor and the CNTF receptor, but not expression of either subunit alone, generated CNTF responsiveness in Hep3B cells, suggesting cooperativity of these receptor subunits. Evidence is presented for direct interaction of the LIF receptor with the intracellular signal transduction machinery.     

Modulation of integrin alpha-v-beta-1 expression on human tumor cells by leukemia inhibitory factor (LIF) and oncostatin M (OSM)

Corticostatins/defensins are a family of cationic peptides recently isolated from phagocytotic cells of the myeloid lineage. Natural killer (NK) cells are spontaneously cytotoxic large granular lymphocytes that are involved in immunosurveillance against cancer and infections. Their activity is modulated by hormones of the hypothalamic-pituitary-adrenal axis. We wished to determine whether two human corticostatins/defensins, HP-1 and HP-4, are able to change in vitro the spontaneous NK activity of human peripheral blood mononuclear cells (PBMC) and the responses either to the stimulatory cytokines immune interferon (IFN-gamma) or interleukin 2 (IL-2) and to the inhibitory hormone cortisol. NK cell activity was measured in a 4-h direct cytotoxicity assay with K562 cells as a target. HP-1 and HP-4 (10 (-8) -10 (-9) M) significantly inhibited the spontaneous and cytokine-inducible NK activity, and increased the cortisol-dependent inhibition. Radioimmunoassay of HPLC purified fractions obtained from sonicated NK cells showed HP-1 in the two cell preparations examined. We also evaluated the effects of HP-1 and HP-4 (10 (-8) M -10(-9) M) upo IFN-gamma and interleukin 6 (IL-6) production by PBMC stimulated with phytohemagglutinin (PHA) or concanavalin A (ConA). IFN-gamma was titrated with the biological assay using WISH cells as indicators and vescicular stomatitis virus (VSV) as the challenge virus. IL-6 was measured using an enzyme amplified sensitivity immunoassay. Both HP-1 and HP-4 significantly reduced cytokine production. Our data indicate that corticostatins/defensins are novel modulators of lymphocyte functions in vitro. Their immunodepressing properties could add complexity and plasticity to hypothalamic-pituitary-adrenal-cytokine circuits in vivo.     

Effects of leukemia inhibitory factor and oncostatin M on bone mineral formed in in vitro rat bone-marrow stromal cell culture: physicochemical aspects

Leukemia inhibitory factor (LIF) and oncostatin M (OSM), two pleiotropic cytokines involved in bone remodeling, have both anabolic and catabolic activities. This study analyzed the effects of LIF and OSM on the physicochemical characteristics of mineral phases formed in a rat bone-marrow stromal cell culture model. Stromal cells were cultured for three weeks in the presence of 10(-8) M dexamethasone, 50 microgram/mL ascorbic acid and 10 mM Na beta-glycerophosphate with or without 10 ng/ml LIF or OSM. Subsequently, the physicochemical characteristics of the mineralization nodules formed were analyzed by energy dispersive X ray microanalysis (EDX) and Fourier transform-infrared (FT-IR) and FT-Raman spectroscopy. EDX and FT-IR spectroscopy revealed the influence of LIF and OSM on the physicochemical characteristics of mineral phases. FT-Raman spectroscopy showed modifications of the main vibrational modes of the organic matrix. These alterations induced by growth factors could help define new strategies for the prevention and treatment of skeletal disorders.     

Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells

Alpha-1 proteinase inhibitor (A1-Pi) is the main serine proteinase inhibitor found in human plasma and is a potent elastase inhibitor in various tissues, including lung. A1-Pi is expressed and induced in liver during inflammatory responses but can also be produced by epithelial cells. Since hepatocyte A1-Pi production is stimulated by interleukin-6 (IL-6) and other gp130-cytokines, such as leukemia inhibitory factor (LIF) and oncostatin M (OM), we investigated the role of these cytokines in regulating A1-Pi in lung epithelial cells. We show that OM, a monocyte and T cell product, can specifically and potently induce A1-Pi production in lung-derived A549 alveolar (epithelial) cells, as well as in liver-derived HepG2 cells. Both A1-Pi protein (as detected by ELISA and Western blots) and mRNA levels were enhanced 20-fold to 30-fold in A549 cells. OM was also able to stimulate the expression of tissue inhibitor of metalloproteinase-1 in these cells. Interestingly, other members of the IL-6 family (IL-6 and LIF) had little or no effect on A549 cells, and proinflammatory cytokines, such as IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) also had no stimulatory effect on A1-Pi synthesis in A549 cells. Costimulation with IL-1 beta resulted in a decrease in A1-Pi production from OM-stimulated A549 cells. However, IL-6 production was synergistically enhanced. OM was also able to stimulate A1-Pi production from a bronchial epithelial primary cell line, whereas an intestinal epithelial cell line HT29 responded to IL-6 but not OM. These results suggest that lung levels A1-Pi could be derived not only from liver and inflammatory cells but also from epithelial cells, which can be upregulated on stimulation by OM. This may have implications for regulation of local activity of human neutrophil elastase (HNE) in such diseases as emphysema and cystic fibrosis.     

Upmodulation of alpha v beta 1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: correlation with increased cell adhesion on fibronectin

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane alpha v beta 1 1.5-2-fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. alpha v beta 1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against alpha v or beta 1 integrin subunits. Similar results were obtained after TNF-alpha treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.     

Blocking signaling through the Gp130 receptor chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth and sensitizes the tumor cells to etoposide and cisplatin-mediated cytotoxicity

BACKGROUND: The mechanisms of drug resistance associated with advanced, hormone-independent prostate carcinoma are poorly understood. The human prostate carcinoma PC-3 cell line, derived from a metastatic tumor and lacking androgen receptors, represents a useful model to investigate drug resistance. METHODS: The effects of oncostatin M (OM), antiinterleukin-6 (IL-6) treatment, or interference with the gp130-mediated signaling on etoposide- or cisplatin-mediated cytotoxicity were investigated. RESULTS: Both endogenous and exogenous IL-6 and exogenous OM up-regulated cell growth and enhanced resistance of PC-3 tumor cells to both etoposide and cisplatin. The influence of IL-6 is controlled by treating PC-3 tumor cells with anti-IL-6 neutralizing antibody and, more efficiently, by a mutated IL-6, Sant7. Sant7 has a high affinity binding to the IL-6 receptor-alpha (IL-6Ralpha) subunit, but does not bind to the signaling subunit gp130; therefore, it behaves as a receptor antagonist. Both IL-6- and OM-mediated effects are inhibited by the treatment of PC-3 with an antisense oligodeoxynucleotide against gp130, the protein kinase inhibitor genistein (GNS), or the monoterpene perillic acid (PA), a posttranslational inhibitor of p21ras isoprenylation. CONCLUSIONS: These results demonstrate the protective roles in drug sensitivity of IL-6 and OM through signaling of the common chain gp130 and, most likely, a downstream ras-dependent pathway in PC-3 tumor cells. These findings suggest the potential clinical application of anticytokine therapy or interference with gp130 signaling in the treatment of drug resistant prostate carcinoma.     

Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.     

Interleukin-4 but not interleukin-10 inhibits the production of leukemia inhibitory factor by rheumatoid synovium and synoviocytes

The expression of the proinflammatory cytokine leukemia inhibitory factor (LIF) has been reported in the cartilage and synovium of rheumatoid arthritis (RA) patients. Here, we show that high levels of LIF were constitutively produced by cultures of synovium pieces. Low levels of LIF were produced spontaneously by isolated synoviocytes, but interleukin (IL)-1 beta caused a fourfold enhancement of this secretion. The anti-inflammatory cytokine IL-4 reduced the production of LIF by synovium pieces by 75%, as observed earlier with IL-6, IL-1 beta and tumor necrosis factor (TNF)-alpha. IL-4 had a direct effect since it inhibited LIF production by unstimulated and IL-1 beta- or TNF-alpha-stimulated synoviocytes. Conversely, IL-4 enhanced the production of IL-6, which shares with LIF biological activities and receptor components. The inhibitory effect of IL-4 was dose dependent and was reversed using a blocking anti-IL-4 receptor antibody. Similar inhibitory action of IL-4 on LIF production was observed on synovium pieces from patients with osteoarthritis and on normal synoviocytes. IL-10, another anti-inflammatory cytokine acting on monocytes, had no effect on LIF production by either synovium pieces or isolated synoviocytes. Thus, the production of LIF by synovium tissue was inhibited by IL-4 through both a direct effect on synoviocytes and an indirect effect by inhibition of the production of LIF-inducing cytokines.     

Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling

trkB encodes a receptor tyrosine kinase activated by three neurotrophins--brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4/5. In vivo, three isoforms of the receptor are generated by differential splicing--gp145trkB or the full-length trkB receptor, and trkB.T1 and trkB.T2, two cytoplasmically truncated receptors that lack kinases, but contain unique C termini. Although the truncated receptors appear to be precisely regulated during nervous system development and regeneration, their role in neurotrophin signaling has not been directly tested. In this paper, we studied the signaling properties and interactions of gp145trkB, trkB.T1, and trkB.T2 by expressing the receptors in a Xenopus oocyte microinjection assay. We found that oocytes expressing gp145trkB, but not trkB.T1 or trkB.T2, were capable of eliciting 45Ca efflux responses (a phospholipase C-gamma-mediated mechanism) after stimulation by BDNF. When trkB.T1 and trkB.T2 were coexpressed with gp145trkB, they acted as dominant negative receptors, inhibiting the BDNF signal by forming nonfunctional heterodimers with the full-length receptors. An ATP-binding mutant of gp145trkB had similar dominant inhibitory effects. Our data suggest that naturally occurring truncated trkB receptors function as inhibitory modulators of neurotrophin responsiveness. Furthermore, the homodimerization of gp145trkB appears to be an essential step in activation of the BDNF signaling cascade.     

Elevated amniotic fluid levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 in intra-amniotic infection

OBJECTIVE: The study's objective was to determine and correlate amniotic fluid levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 in patients with and without intra-amniotic infection. STUDY DESIGN: Amniocentesis was performed on 41 pregnant women with preterm contractions, labor, or premature rupture of membranes. Intra-amniotic infection was defined as the presence of a positive amniotic fluid culture result. Amniotic fluid tests for Gram stain, glucose, leukocyte counts, creatinine level, pH, and specific gravity were performed. Amniotic fluid levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 were measured by an enzyme-linked immunoassay. Unlike in previous reports, cytokines were normalized by amniotic fluid creatinine levels. RESULTS: Fifteen patients had intra-amniotic infection and 26 did not. Amniotic fluid median levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 were significantly higher in pregnant women with intra-amniotic infection than in those without intra-amniotic infection (leukemia inhibitory factor median 3912 pg/mg creatinine, range 0.0-199314, vs 56 pg/mg creatinine, range 0. 0-12148, P =.01; interleukin 6 median 2005 ng/mg creatinine, range 27-4071, vs 990 ng/mg creatinine, range 7.5-3409, P =.005; interleukin 8: median 4933 ng/mg creatinine, range 0.0-55058, vs 61 ng/mg creatinine, range 0.0-2399, P =.005). Amniotic fluid levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 were positively correlated. CONCLUSIONS: The data indicate that leukemia inhibitory factor plays an important role in the pathogenesis of intra-amniotic infection. In addition, significant elevations of and correlations among amniotic fluid levels of leukemia inhibitory factor, interleukin 6, and interleukin 8 suggest that measurements of these cytokines in amniotic fluid may be of diagnostic and prognostic importance.     

Expression of interleukin-6 and interleukin-6 receptors in human granulosa lutein cells

Prolonged isolated thrombocytopenia, defined as recovery of other cell counts with continuous dependence on platelet transfusions for greater than 90 days after hematopoietic stem cell transplantation (HSCT), develops in approximately 5% of patients who undergo HSCT. Although the clinical conditions associated with prolonged isolated thrombocytopenia have been studied, a systematic review of bone marrow biopsies has not been performed and the pathophysiologic basis has not been defined. We reviewed all HSCT at one center from 1990 to 1995 (n = 454) and found 12 cases that met criteria for prolonged isolated thrombocytopenia (incidence = 12/454 or 3%). Bone marrow core biopsies from 12 patients with prolonged isolated thrombocytopenia were reviewed to determine cellularity, numbers of megakaryocytes, the presence of atypical forms, and clusters of megakaryocytes. These marrow megakaryocyte counts were compared to age and disease matched controls, and 11 normal donors. Patients (aged 1-56 years, mean 32 years) who underwent HSCT (four sibling HLA-identical, five autologous bone marrow, three autologous peripheral stem cell) with prolonged isolated thrombocytopenia had a statistically significant lower absolute megakaryocyte count in bone marrow biopsies performed before transplantation and more than 30 days after transplantation compared to control patients (aged 4 months to 50 years, mean 31 years) who underwent HSCT (four sibling HLA-identical, four autologous bone marrow, four autologous peripheral stem cell) for similar conditions. No apparent differences were seen in size of megakaryocytes, nuclear-cytoplasmic ratios, or clustering of megakaryocytes. Overall marrow cellularities were similar in the three groups. These findings suggest that decreased differentiation of megakaryocytes from stem cells, rather than ineffective platelet production or peripheral destruction of platelets, causes prolonged isolated thrombocytopenia in HSCT patients. Low megakaryocyte counts prior to HSCT may be a useful prognostic indicator, as this feature was associated with the development of prolonged isolated thrombocytopenia.     

Spillover-mediated transmission at inhibitory synapses promoted by high affinity alpha6 subunit GABA(A) receptors and glomerular geometry

Divergence and convergence of synaptic connections make a crucial contribution to the information processing capacity of the brain. Until recently, it was thought that transmitter released at a synapse affected only a specific postsynaptic cell. We show here that spillover of inhibitory transmitter at the Golgi to granule cell synapse produces significant cross-talk to non-postsynaptic cells, which is promoted both by the anatomical specialization of this glomerular synapse and by the presence of the high affinity alpha6 subunit-containing GABA(A) receptor in granule cells. Cross-talk is manifested as a novel slow rising and decaying small amplitude inhibitory postsynaptic current (IPSC) that can also contribute a long-lasting component to more typical IPSCs, which is prolonged by inhibition of the neuronal GABA transporter GAT-1. Because of the long duration of IPSCs generated by spillover, the total charge carried is three times that of IPSCs generated by directly connected terminals. GABA spillover within the mossy fiber glomerulus may play an important role in regulating the number of granule cells active in the cerebellar cortex, a regulation that is suggested by theoretical models to optimize cerebellar information processing.     

Macrophage colony-stimulating factor augments beta-amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells

In Alzheimer's disease (AD), a chronic cerebral inflammatory state is thought to lead to neuronal injury. Microglia, intrinsic cerebral immune effector cells, are likely to be key in the pathophysiology of this inflammatory state. We showed that macrophage colony-stimulating factor, a microglial activator found at increased levels in the central nervous system in AD, dramatically augments beta-amyloid peptide (betaAP)-induced microglial production of interleukin-1, interleukin-6, and nitric oxide. In contrast, granulocyte macrophage colony-stimulating factor, another hematopoietic cytokine found in the AD brain, did not augment betaAP-induced microglial secretory activity. These results indicate that increased macrophage colony-stimulating factor levels in AD could magnify betaAP-induced microglial inflammatory cytokine and nitric oxide production, which in turn could intensify the cerebral inflammatory state by activating astrocytes and additional microglia, as well as directly injuring neurons.     

Leukemia inhibitory factor and oncostatin M influence the mineral phases formed in a murine heterotopic calcification model: a Fourier transform-infrared microspectroscopic study

The study of bone mineralization processes is of considerable interest in understanding bone diseases and developing new therapies for skeletal disorders, particularly since bone homeostasis requires numerous cell types and a large cytokine network. Cell culture models of mineralization have often been used to study the cellular mechanisms of mineralization, but few data have been reported concerning the influence of extracellular matrix components and cytokines on the physicochemical properties of mineral. The purpose of this study was to analyze the effects of two cytokines, leukemia inhibitory factor (LIF) and oncostatin M (OSM), involved in bone metabolism on the physicochemical properties of bone mineral formed in a murine in vivo mineralization model. Murine bone marrow cells implanted under the kidney capsule in the presence or absence of cytokines led to heterotopic ossicle formation. A scanning electron microscopic microprobe revealed that heterotopic calcification had a lower (approximately 20%) Ca/P ratio after cytokine treatment as compared with the control without cytokine. Transmission electron microscopic analysis of cytokine-treated ossicles showed numerous areas with low mineral density, whereas electron diffraction pattern revealed an apatitic phase. These areas were not observed in the absence of cytokine. Moreover, Fourier transform-infrared microspectroscopy showed at the molecular level that the presence of either cytokine induced many microscopic areas in which short-range order organization, such as incorporation of carbonate and crystallinity/maturity of ossicle mineral, were modified. LIF and OSM influenced mineral phase formation in the present model and may thus be key protagonists in bone mineral development and skeletal diseases.