Leukemia Inhibitory Factor Induction Research Articles

Nerve growth factor inhibits sympathetic neurons' response to an injury cytokine.

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Transcriptional regulation of the human `leukemia inhibitory factor' gene: modulation by glucocorticoids and estradiol

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite its possible importance as a therapeutic target, very little is known about the regulation of human LIF. In particular, its regulation at the promoter level has not been studied so far, and was, therefore, the focus of the present work. After showing that Jurkat T lymphoma cells can be induced to express endogenous LIF mRNA, we used this cell line as a model to study the regulation of the human LIF promoter in transient transfection assays. For this purpose, a 666 bp fragment of the human LIF 5′-flanking region, amplified from genomic DNA by nested polymerase chain reaction (PCR), was used for the construction of a luciferase reporter plasmid (hLIF666-Luc). In unstimulated Jurkat cells, the human LIF promoter showed low constitutive activity. The promoter was induced upon stimulation with phorbol ester (TPA). Combined stimulation with TPA and the calcium ionophore ionomycin resulted in strong synergistic induction of luciferase activity from the LIF promoter. Transfection experiments with deletion constructs (hLIF274-Luc and hLIF82-Luc) located the region required for this induction to a 192 bp portion of the promoter, which carries two putative c-ets binding sites. We then investigated the effect of glucocorticoids and estradiol by cotransfecting the respective receptors. Both hormones strongly inhibited the stimulation of the LIF promoter by TPA and ionomycin. Since LIF is implicated in the pathogenesis of inflammatory and degenerative conditions, such as rheumatoid arthritis and osteoporosis, the finding that therapeutic agents employed in the treatment of such conditions, i.e. glucocorticoids and estrogens, can modulate the induction of LIF at the transcriptional level, is of particular interest.

Changes in Gene Expression in Adult Sympathetic Neurons after Axonal Injury

Publisher Summary This chapter summarizes the advances in the knowledge of the phenotype of axotomized peripheral neurons, concentrating on the phenotypic changes that occur in axotomized sympathetic neurons and the signals that trigger these changes. The decrease in tyrosine hydroxylase (TH) activity in axotomized sympathetic neurons is accompanied by a decrease in TH mRNA. Changes can also be found in the mRNA levels of other proteins involved in synaptic transmission. Decreases in transmitter synthesis also occur in axotomized sensory and motor neurons. In addition to changes in neurotransmitter-neuromodulator levels in peripheral neurons after axotomy, changes have been found in their complement of receptors for these signaling molecules. Axotomy of peripheral neurons also produces increases in expression of certain proteins known to be involved in regeneration, such as tubulin and growth-associated protein (GAP)-43. In addition, several neuropeptides, not normally expressed by these neurons, are induced in response to axotomy. Some of these peptides might promote neuronal survival and/or fiber outgrowth under conditions in which the neurons are deprived of their target-derived trophic factors. Two events involved in triggering these phenotypic changes following axotomy have been identified: the induction and release of leukemia inhibitory factor (LIF) in nonneuronal cells within the superior cervical ganglion (SCG) and/or at the site of nerve transection and the reduction in levels of nerve growth factor (NGF) in the SCG as a consequence of the disconnection of the neurons from their target tissues. Other evidence confirms that dramatic changes occur in the nonneuronal population of the SCG after axotomy. Evidence for an endogenous LIF-inducing factor has also been reported in the chapter. In addition to LIF induction, a reduction in NGF levels in the SCG also plays an important role in triggering these changes.

Leukemia inhibitory factor mediates cytokine-induced suppression of myenteric neurotransmitter release from rat intestine

BACKGROUND & AIMS: Exposure of rat longitudinal muscle myenteric plexus to the proinflammatory cytokine interleukin (IL) 1 beta mimics the effects of nematode infection on enteric nerve function through a hitherto unidentified protein intermediate. It is postulated that leukemia inhibitory factor (LIF), the downstream intermediate of several IL-1-induced neuroimmune interactions, mediates IL-1 beta- induced suppression of acetylcholine release from rat jejunum. METHODS: Preparations were preloaded with [3H]choline, and [3H]-acetylcholine release was induced by either electrical field stimulation or by 50 mmol/L KCl. Cytokines and anti-LIF antibodies were added to the preincubation media or to the superfusate before stimulation. RESULTS: Human recombinant LIF had no immediate effects, but preincubation with the cytokine induced a concentration-dependent (2-100 ng/mL) and reversible suppression of acetylcholine release from rat longitudinal muscle myenteric plexus. The effects of human recombinant LIF on acetylcholine release were reversed by anti-human recombinant LIF- neutralizing antibody. Human recombinant IL-1 beta (10 ng/mL) induced a similar suppression of acetylcholine release, and the addition of anti- rat LIF antibody abolished the effects of exogenous IL-1 on acetylcholine release. CONCLUSIONS: IL-1 beta suppresses neurotransmitter release from rat myenteric plexus via the induction of LIF as a downstream intermediate. (Gastroenterology 1996 Sep;111(3):674-81)

Activation of acidic sphingomyelinase and protein kinase C zeta is required for IL-1 induction of LIF mRNA in a Schwann cell line.

Axotomy of sympathetic superior cervical ganglia (SCG) causes Schwann cells to induce mRNA encoding leukemia inhibitory factor (LIF), a neuropoietic cytokine that has been shown to promote sympathetic neuron survival and peptide gene regulation. LIF mRNA is virtually undetectable in uninjured SCG, but is induced by the inflammatory cytokine interleukin-1 (IL-1). The SC1 Schwann cell line was used to study this regulatory mechanism. LIF mRNA increased five-to-tenfold in SC1 cells when IL-1 receptors were stimulated with IL-1. The action of IL-1 is thought to be mediated by the type I IL-1 receptor (IL-1RI), which has been suggested to stimulate a ceramide-dependent protein kinase pathway, much like tumor necrosis factor-alpha. However, stimulation of the ceramide-dependent protein kinase pathways in SC1 cells with either 2-acetylceramide or sphingomyelinase treatment does not induce LIF mRNA accumulation, but 2-acetylceramide addition induces cyclooxygenase-2 mRNA in parallel experiments. Inhibition of phosphotidylcholine-phospholipase C activity, endosomal acidification, or activity of atypical protein kinase C reduce LIF induction by IL-1. These results are consistent with IL-1 regulation of LIF mRNA through stimulation of the endosomal, acidic sphingomyelinase pathway, leading to ceramide activation of protein kinase C zeta. Utilization of this branch of the ceramide signaling pathway may be cell type specific or may be specific for the LIF mRNA response.

Hypothalamic and pituitary leukemia inhibitory factor gene expression in vivo: a novel endotoxin-inducible neuro-endocrine interface

We have recently shown expression of leukemia inhibitory factor (LIF) in human fetal pituitary tissue and its in vitro induction of POMC transcription. We now use qualitative and semiquantitative RT-PCR to demonstrate that LIF and LIF-receptor (LIF-R) are constitutively expressed in the normal mouse hypothalamus and pituitary. Hypothalamic and pituitary LIF and LIF-R are significantly induced (up to 6- and 4-fold, respectively) in vivo in response to lipopolysaccharide endotoxin (LPS) administered to B6D2F1 and C57BL/6 mice. In contrast to the nearly exclusive expression of matrix-associated LIF messenger RNA (mRNA) in control hypothalamus and pituitary, both diffusible and matrix-associated LIF mRNA alternate transcripts are induced by LPS. Furthermore, the time course of peripheral ACTH-response to LPS peaks at 60 min, whereas hypothalamic LIF mRNA increase occurs at 30 min and pituitary LIF induction occurs at 60 min. These results show that mLIF is a novel LPS-inducible proinflammatory neuroendocrine cytokine and the alternatively spliced diffusible LIF may play a paracrine role in activating pituitary ACTH secretion in synergy with hypothalamic CRH, implying a mechanism for central nervous system cytokine responses to immune signals.

Leukemia inhibitory factor (LIF) stimulates proopiomelanocortin (POMC) expression in a corticotroph cell line. Role of STAT pathway.

We recently described the expression of leukemia inhibitory factor (LIF) in human fetal and murine corticotrophs. LIF and the related cytokine oncostatin M induced basal, and corticotropin-releasing hormone (CRH) induced proopiomelanocortin (POMC) mRNA and ACTH secretion in AtT20 cells. LIF signaling and regulation of POMC gene transcription were therefore tested. Dexamethasone inhibited both basal- and LIF-induced ACTH secretion (P<0.05) and LIF induction of ACTH was also attenuated by immuneutralization of either the LIF receptor (35%, P<0.05) or the gp130 affinity converter (41%, P<0.05). These antisera also attenuated basal ACTH secretion in the absence of added ligand (P<0.05). To examine intrapituitary LIF signaling, phosphorylation of post-receptor substrates was measured. 1 nM LIF rapidly induced tyrosyl phosphorylation of STAT 1 and STAT 3 proteins, as well as tyrosyl phosphorylation of a 115-kD protein, coimmunoprecipitated with STAT 1. The transfected rat POMC promoter -706/+64, fused to the luciferase reporter gene, was induced by LIF, which exerted strong (18-fold) synergy with CRH. Deletion of the major CRH responsive region in POMC (-323/-166) abolished CRH induction of transcription and severely limited LIF synergy. Although 8 bromo cAMP or forskolin modestly enhanced POMC transcription (2.8-fold), LIF markedly potentiated (7.4-fold) these cAMP activators. These results demonstrate that corticotroph LIF action is receptor mediated and involves activation of STAT signaling pathways. LIF potently synergizes with both CRH and cAMP induction of POMC transcription. This novel intrapituitary signaling mechanism may mediate a neuroimmune pituitary interface.

Signals Triggering the Induction of Leukemia Inhibitory Factor in Sympathetic Superior Cervical Ganglia and Their Nerve Trunks after Axonal Injury

Leukemia inhibitory factor (LIF) plays an important role in regulating neuropeptide expression in sympathetic and sensory neurons after axonal transection. By 2 h after axotomy, LIF mRNA increased in nonneuronal cells in sympathetic ganglia and peripheral nerve. In addition, within 1 h of explanting sympathetic ganglia or segments of sympathetic nerve trunks, a protein factor(s) that was able to induce LIF mRNA both in sympathetic cultures and in intact gangliain vivowas released. This factor(s) appeared to be present in sympathetic ganglia and their nerve trunks under normal conditions and to be activated and/or released after axonal injury. Since the factor(s) has a molecular weight(s) greater than 66 kDa, and no other proteins of such high molecular weight have been previously identified with LIF-inducing activity, it appears to be a novel inducer of LIF.

Human Cytomegalovirus Increases Constitutive Production of Interleukin-6 and Leukemia Inhibitory Factor by Bone Marrow Stromal Cells

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony-stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL-6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS-stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.

Human cytomegalovirus increases constitutive production of interleukin- 6 and leukemia inhibitory factor by bone marrow stromal cells

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony- stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL- 6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS- stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.

Cytokine-induced expression of leukemia inhibitory factor in renal mesangial cells

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine, which shares many characteristics with interleukin-6 (IL-6). Recent observations indicate a role for LIF in inflammatory processes. To examine the potential involvement of LIF in the regulation of mesangial cell behavior, we studied LIF expression in early primary cultures of rat and human mesangial cells, as well as the response of mesangial cells to exogenous LIF. Growing or growth-arrested rat mesangial cells constitutively expressed very low levels of LIF mRNA, barely detectable by Northern blot analysis. Strong induction of LIF mRNA expression was caused by cytokines, such as interleukin-1 beta (5 ng/ml), tumor necrosis factor alpha (100 ng/ml) and PDGF (100 ng/ml), as well as LPS (200 ng/ml). The induction was transient with a peak after three to five hours. Dexamethasone (0.1 microM) almost completely inhibited the induction of LIF. Weak induction of LIF mRNA was observed after stimulation with basic fibroblast growth factor, endothelin and transforming growth factor beta. In combination with IL-1 beta, TGF beta showed synergistic effects on LIF induction. LIF itself or IL-6 had no effect on LIF mRNA expression. A similar induction pattern was observed for the expression of IL-6 mRNA. LIF protein was detected by specific ELISA in the supernatants of human mesangial cells stimulated by LPS or IL-1 beta. In addition, we found that mesangial cells not only express LIF but they are also target cells for LIF. Recombinant LIF effectively induced transient expression of the immediate early genes, c-fos, jun-B and Egr-1 in rat mesangial cells, with a maximum at 30 to 60 minutes. LIF was not mitogenic for mesangial cells. Our findings indicate that glomerular mesangial cells produce and react to LIF. As a cytokine with autocrine potential, LIF may play a physiological and/or pathophysiological role in the glomerulus, the exact nature and relevance of which remain to be explored.

Cytokine-cytokine synergy and protein kinase C in the regulation of lung fibroblast leukemia inhibitory factor

Studies were undertaken to characterize the cytokines and cytokine-cytokine interactions that stimulate human lung fibroblast leukemia inhibitory factor (LIF) production and the mechanisms of these regulatory effects were investigated. Unstimulated fibroblasts did not produce significant amounts of LIF, whereas recombinant interleukin-1 alpha (rIL-1 alpha), transforming growth factor-beta (TGF-beta), and recombinant tumor necrosis factor (rTNF) were dose-dependent stimulators of LIF production. TGF-beta and rIL-1 alpha also interacted in a synergistic fashion to further increase LIF elaboration. Under all conditions alterations in LIF production were associated with comparable alterations in LIF mRNA accumulation. The kinetics of mRNA induction, however, differed with rIL-1-induced LIF mRNA being readily detected after 2 h, TGF-beta 1 induction peaking after 16-24 h, and the induction caused by rIL-1 alpha plus TGF-beta 1 being most prominent after 2-4 h and decreasing with additional incubation. Protein synthesis was not required for LIF induction. In addition, even though A23187 was an effective stimulator of LIF production, the calmodulin antagonists W-7 and trifluoperazone dichoride (TFP) did not significantly alter the LIF-stimulatory effects of IL-1 and TGF-beta. PKC did appear to play an important role in this induction, however, since LIF was induced by PMA and cytokine induction of LIF production was markedly diminished by chronic phorbol ester preincubation, staurosporine, and H-7, but not by HA1004. These studies demonstrate that 1) rIL-1, TGF-beta, TNF, agents that increase intracellular calcium and agents that activate PKC, stimulate lung fibroblast LIF production; 2) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast LIF production; and 3) rIL-1 and TGF-beta stimulate lung fibroblast LIF production via a pretranslational activation pathway that is largely PKC-dependent and protein synthesis-, cyclic nucleotide-, and calmodulin-independent. Cytokine-stimulated LIF production may play an important role in homeostasis and repair in the human lung.

Interleukin-1 induces substance P in sympathetic ganglia through the induction of leukemia inhibitory factor (LIF)

It has become increasingly clear that immune cytokines perform growth and differentiation functions in the nervous system similar to those performed in the immune system. In previous studies we have shown that interleukin-1 beta (IL-1 beta) raises substance P (SP) and the mRNA coding for its preprotachykinin precursor in cultured sympathetic superior cervical ganglia (SCG) (Jonakait and Schotland, 1990; Hart et al., 1991a). The action of IL-1 is blocked both by depolarization of the ganglia and by glucocorticoid hormones (Hart et al., 1991a). In the present report, we have found that IL-1 does not act directly upon neurons to raise SP, but rather induces the production of a soluble intermediate molecule that raises both SP and the cholinergic-specific enzyme ChAT. Its induction by IL-1 is blocked by the synthetic glucocorticoid hormone dexamethasone; its action is compromised under depolarizing conditions. Because medium conditioned by IL-1 (IL-1CM) is functionally similar to leukemia inhibitory factor (LIF), we sought to determine whether this molecule might be an active constituent of IL-1CM. Immunoprecipitation with an antiserum directed against LIF eliminated large proportions of SP-inducing activity from IL-1CM. In addition, steady-state levels of mRNA coding for LIF are increased by IL-1 treatment of SCG. These data suggest that LIF, induced by IL-1, may ultimately be responsible for the IL-1 induction of SP.

Induction of leukemia inhibitory factor in human synovial fibroblasts by IL-1 and tumor necrosis factor-alpha.

The cytokines, IL-1 alpha and TNF-alpha, induced a dose-dependent production of leukemia inhibitory factor (LIF) in cultured human synovial fibroblast-like cells, as measured by a radioreceptor competition assay. Significant levels of LIF were first detected in cell supernatants between 2 and 4 h after cytokine addition. Increases were also observed in LIF mRNA steady-state expression. Evidence is presented for down-regulation of the IL-1-induced LIF activity by an endogenous cyclo-oxygenase product(s); the glucocorticoid, dexamethasone, lowered the IL-1-induced LIF activity and mRNA expression. A synergistic effect was noted between the actions of IL-1 alpha and TNF-alpha, and between IL-1 and transforming growth factor-beta. IFN-gamma could not induce LIF formation in the synovial cells but inhibited the stimulatory effect of IL-1. These results suggest that cytokine-stimulated synovial fibroblasts may be a major source of intraarticular LIF production in the joints of patients with inflammatory arthritis. Synoviocyte-derived LIF may activate monocyte/macrophages in the lesions and may contribute to the bone changes and certain systemic manifestations in patients with inflammatory joint disease.