Activation of acidic sphingomyelinase and protein kinase C 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-α. 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 Cζ. Utilization of this branch of the ceramide signaling pathway may be cell type specific or may be specific for the LIF mRNA response. © 1996 Wiley-Liss, Inc.     

Interleukin-1 involvement in the induction of leukemia inhibitory factor mRNA expression following axotomy of sympathetic ganglia

Axotomy of superior cervical (sympathetic) ganglia (SCG) results in increased neuropeptide gene expression. In vitro, neuropeptide gene expression is similarly increased by exposure to the inflammatory cytokine interleukin-1 (IL-1). The effect of IL-1 in vitro has been shown to be mediated by leukemia inhibitory factor (LIF). Since IL-1 regulates neuropeptide expression via LIF in vitro, we asked whether axotomy in vivo produces an increase in LIF mRNA, and whether that increase is regulated by IL-1 activity. Within 6 h following axotomy, ganglionic LIF mRNA is substantially elevated. Moreover, axotomy produces a rapid and transient increase in intraganglionic IL-1β mRNA, followed rapidly by an increase in ICAM-1 mRNA, thereby suggesting a local source of IL-1 activity. Pretreatment with the anti-inflammatory agent dexamethasone (DEX) reduces the increases of both IL-1β and LIF mRNAs following axotomy. mRNA encoding the specific signal-transducing Type I IL-1 receptor is present in unlesioned SCG in vivo, and increases following axotomy. Local application of IL-1β in vivo induces LIF mRNA even in uninjured ganglia, though not to the extent seen with axotomy. DEX treatment blocks this IL-1β-mediated increase in LIF mRNA. Therefore, DEX blocks the induction of LIF mRNA by inhibiting both the production of IL-1 and its action on LIF gene expression. Axotomy of a homozygous IL-1 receptor type I gene knockout mouse leads to a delayed and/or diminished induction of LIF mRNA in SCG, but does not prevent LIF mRNA expression. We conclude that while IL-1 is likely to be involved in the cascade of gene expression that follows axotomy, it alone is not sufficient to mediate the full induction of LIF mRNA by axotomy.     

Modulation of schwann cell phenotype by TGF-β1: Inhibition of P0 mRNA expression and downregulation of the low affinity NGF receptor

The phenotype of a fully differentiated, mature Schwann cell is appar-ently largely determined by Schwann cell-axon interactions. In vitro, elevation of intra-cellular cAMP levels in Schwann cells induces a phenotype which resembles that of a mature, i.e., axon-related, Schwann cell. Therefore, an important role for cAMP as a second messenger of axon-Schwann cell interactions in vivo is assumed. However, the effects of cAMP on Schwann cells are not restricted to induction of features of a mature phenotype. For example, elevation of intracellular cAMP levels results of also in a markedly increased synthesis of nerve growth factor (NGF) mRNA, which is barely detectable in intact sciatic nerves of adult animals. Furthermore, since cAMP induces myelin gene expression in cultured Schwann cells, additional regulatory mechanisms have to be postulated for the induction and maintenance of a mature non-myelinating Schwann cell phenotype. Here we show that a soluble protein “growth factor” can partially induce a non-myelinating mature Schwann cell phenotype in vitro. Treatment with transforming growth factor β1 (TGF-β1) results in a marked and rapid downregulation of the low affinty NGF receptor (NGFR) on cultured Schwann cells without induction of PO gene expression. In contrast, in agreement with previous studies, an increase in PO mRNA levels and a reduction in NGFR mRNA after cAMP elevation is much slower when compared with the effect of TGF-β1, suggesting the involvement of different intracellular mechanisms. Consistent with this hypothesis, we did not observe an induction of mRNA coding for TGR-β isoforms after cAMP elevation in cultured Schwann cells which constitutively synthesize TGF-β1 mRNA. © 1993 Wiley-Liss, Inc.     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

Complementary anti-inflammatory actions of the β₂-adrenoceptor agonist clenbuterol and the glucocorticoid dexamethasone in rat brain

Systemic administration of the β(2)-adrenoceptor agonist clenbuterol induces expression of IL-1β and its negative regulators, interleukin-1 receptor antagonist (IL-1ra) and the interleukin-1 type II decoy receptor (IL-1RII) in rat brain. Clenbuterol also increases central expression of the broad spectrum anti-inflammatory cytokine interleukin-10 (IL-10) and its downstream signalling molecule, suppressor of cytokine signalling-3 (SOCS-3). Here we examine the impact of combined treatment with clenbuterol (0.5mg/kg) and the glucocorticoid dexamethasone (1mg/kg) on mRNA expression of IL-1β and the IL-1β-inducible gene iNOS, on IκBα mRNA expression and NFκB activation, and on mRNA expression of the anti-inflammatory molecules IL-1ra, IL-1RII, IL-10 and SOCS-3 in rat cortex, striatum and hippocampus. Dexamethasone inhibited induction of IL-1β and iNOS mRNA expression by clenbuterol in all three brain regions, without altering its ability to induce IL-1ra mRNA expression. In the case of IL-1RII, dexamethasone further augmented clenbuterol-induced IL-1RII mRNA expression in hippocampus and striatum. These data highlight a mechanistic dissociation between the ability of β(2)-adrenoceptor activation to induce expression of IL-1β, and its negative regulators IL-1ra and IL-1RII in the brain. Treatment with either dexamethasone or clenbuterol alone independently induced IκBα mRNA expression, and elicited a concomitant decrease in the DNA binding of NFκB in all three brain regions. In the hippocampus and striatum dexamethasone treatment did not influence the ability of clenbuterol to induce IL-10 mRNA expression. In contrast in the cortex, induction of IL-10 and SOCS-3 mRNA expression by clenbuterol administered in combination with dexamethasone was less than induced by clenbuterol alone. Overall these data indicate that combined treatment with dexamethasone and the β(2)-adrenoceptor agonist clenbuterol elicit complementary anti-inflammatory actions in the CNS. Specifically, dexamethasone inhibits expression of pro-inflammatory cytokines, whereas clenbuterol has the added benefit of promoting expression of anti-inflammatory molecules including IL-1ra, IL-1RII, IL-10 and SOCS-3.     

Microglial Activation Results in Inhibition of TGF-β-Regulated Gene Expression

Chronic inflammation mediated by persistent microglial activation is associated with the pathogenesis of neurodegenerative diseases. The mechanisms underlying chronic microglial activation are poorly understood. We have previously shown that anti-inflammatory TGF-β signaling is inhibited in LPS-treated microglia. In this study, we assessed whether different disease-related microglial activators could downregulate TGF-β induction of gene expression. We examined the effects of amyloid β (Aβ) (1–42)- or heat-killed Listeria monocytogenes (HKLM) on the TGF-β-regulated gene expression in primary rat microglia. We found that Aβ (1–42) oligomers and HKLM, in addition to LPS, suppressed TGF-β-mediated induction of gene expression in part through reducing expression of TβR1 mRNA encoding the TGF-β receptor 1 in primary microglia. Aβ (1–42) and LPS also prevented induction of TGF-β-induced genes in primary microglia. Additionally, Aβ (1–42) rescued primary microglia from TGF-β-mediated cell death without increasing cell proliferation. Blockage of NFκB signaling, but not the ERK or IRF3 pathways, inhibited Aβ (1–42)- and LPS-mediated reduction of TβR1 mRNA. Finally, LPS and Aβ (1–42) induced transient upregulation of mRNAs encoding SnoN and Bambi, inhibitors of TGF-β signaling. Our data indicate that one mechanism through which activators may prolong microglial stimulation is through direct inhibition of anti-inflammatory signaling. A more detailed understanding of the interaction between inflammatory and anti-inflammatory pathways may reveal potential targets for ameliorating chronic inflammation and hence speed the development of therapeutics to address neurodegenerative diseases.     

Lymphocyte regulation of neuropeptide gene expression after neuronal injury

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are induced strongly in neurons after several types of injury, and exhibit neuroprotective actions in vitro and in vivo. It is thought that changes in expression of neuropeptides and other molecules in injured neurons are mediated by new factors produced in Schwann and immune cells at the injury site, a loss of target-derived factors, or a combination of mediators. To begin to determine the role of the inflammatory mediators, we investigated axotomy-induced changes in VIP and PACAP gene expression in the facial motor nucleus in severe combined immunodeficient (SCID) mice, and in mice with targeted mutations in specific cytokine genes. In normal mice, VIP and PACAP mRNA was induced strongly in facial motor neurons 4 days after axotomy. The increase in PACAP mRNA was blocked selectively in SCID mice, indicating that mechanisms responsible for VIP and PACAP gene induction are not identical. The loss of PACAP gene expression in SCID mice after axotomy was fully reversed by an infusion of normal splenocytes, suggesting that PACAP mRNA induction requires inflammatory mediators. PACAP and VIP mRNA inductions, however, were maintained in mice lacking leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), and in mice lacking both receptors for tumor necrosis factor alpha (TNFalpha). The data suggest that an inflammatory response, most likely involving T lymphocytes, is necessary for the axotomy-induced increase in PACAP but not in VIP. LIF, IL-6, and TNFalpha, however, are not required for this response to injury.     

Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve

The transforming growth factors beta (TGF-β), a family of regulatory polypeptides, are involved in numerous vital processes including inflammation and wound healing. Since repair of a peripheral nerve lesion includes a series of well-defined steps of cellular actions possibly controlled by TGF-βs, and since TGF-β mRNA and immunoreactivity have been found in the normal peripheral nerve, we have examined TGF-β mRNA regulation and protein expression in the lesioned peripheral nerve. Sciatic nerves of adult rats were either crushed (allowing axonal regenration) or transected (to prevent axonal regeneration and to induce Wallerian degeneration in the distal stump). After intervals of 6 hours, 2 and 6 days post-lesion, the rats were sacrificed and each nerve was cut into four segments, two proximal and two distal to the lesion site. TGF-β 1-3 mRNA were determined for each segment. We demonstrate that TGF-ß1 mRNA levels are higher than those of TGF-ß3; the amplitude of mRNA regulation depends on time, type of lesion and localization relative to the lesion site. TGF-ß2 mRNA could not be detected. For TGF-ß1-3 immunocytochemistry, animals were sacrificed 12, 24, 48, 72 hours and 7 and 14 days after surgery. TGF-β immunoreactivity (IR) was observed for all isoforms in lesioned and unlesioned nerves. In the segment directly adjacent to the lesion at its proximal side, an increase of TGF-β-IR became apparent as soon as 12 hours after surgery; it remained elevated during the whole period observed in both models. In the segment adjoining the distal side of the lesion, an increase of TGF-β-IR was observed after 48 hours, which was still present after 14 days. At day 7 after crush or transection, an increase of TGF-β-IR was detected in the most distal segments, which reached its highest levels at the end of our observation period. Our results suggest that the presence of axonal contact might induce an enhancement of TGF-β expression by Schwann cells in the distal stump of a lesioned and regenerating peripheral nerve. Since we demonstrate an increase of TGF-β mRNA and protein expression also in the distal stump of transected nerves where Schwann cells are not able to contact sprouting axons from the proximal part, other regulatory pathways must exist. The acquisition of a “reactive” Schwann cell phenotype after peripheral nerve lesion might involve an upregulation of TGF-β expression. © 1994 Wiley-Liss, Inc.     

Dibutyryl cyclic AMP and inflammatory cytokines mediate C3 expression in schwann cells

Schwann cells (SchC), the myelinating glia of the peripheral nervous system, are immunocompetent cells and secrete a variety of immune and inflammatory mediators. In this report, we show that rat SchC in vitro express both C3 mRNA and protein in response to dibutyryl cyclic AMP (dbcAMP) and the cytokines IFN-γ, TNF-α, and IL-1β. SchC in culture constitutively expressed low levels of C3 which were significantly upregulated upon stimulation with 1mM dbcAMP by 24 hours, and persisted up to 120 hours. This response was minimally enhanced by costimulation with 100 U/ml IFN-γ, whereas costimulation with 100 U/ml IFN-γ together with 150–450 ng/ml TNF-α induced a greatly increased C3 response. TNF-α alone did not induce C3 expression in SchC. Cycloheximide inhibited this dbcAMP-dependent delayed C3 production, thus implying an intermediary signal in the induction pathway requiring protein synthesis. Treatment with 0.1–10 ng/ml IL-1β for 0–72 hours induced C3 mRNA and protein in a dose-dependent manner. C3 mRNA was detectable at 1 hour and mRNA and protein peaked by 6–12 hours on stimulation with 10 ng/ml IL-1β, or at 48 hours with 1.0 ng/ml IL-1β. Furthermore, IL-1β mRNA was detected at 6 hours in dbcAMP-treated SchC, preceding the dbcAMP-induced C3 expression by 18 hours. Induction of C3 mRNA and protein by dbcAMP at 24 hours was inhibited >85% by a neutralizing anti-IL-1β antibody and 76% with an IL-1 receptor antagonist. This suggests that dbcAMP-induced synthesis of IL-1β mediates the C3 production by SchC in an autocrine/paracrine fashion by binding to a functional IL-1 receptor expressed on the surface of SchC. Endoneurial IL-1 and C3 production by SchC may therefore contribute to the inflammatory events associated with peripheral nerve demyelination. GLIA 20:308–321, 1997. © 1997 Wiley-Liss, Inc.     

Central Nervous System IL-1β System and Neuropeptide Y mRNAs During IL-1β-induced Anorexia in Rats

Interleukin-1β (IL-1β) induces anorexia and neuropeptide Y (NPY) increases feeding by direct action in the central nervous system (CNS). IL-1β, depending on the dose, attenuates or blocks NPY-induced feeding. This suggests that IL-1β-NPY interactions may be involved in IL-1β-induced anorexia. Here, RNase protection assays were used to investigate the effects of the chronic intracerebroventricular (ICV) administration of IL-1β (at a dose that yields estimated pathophysiological concentrations in the cerebrospinal fluid) on mRNA levels of IL-1β system components and NPY in the cerebellum, parietofrontal cortex, hippocampus, hypothalamus, and midbrain. The results show that the chronic ICV administration of IL-1β (8.0 ng/24 h for 72 h) differentially induced IL-1β system components across brain regions in anorectic rats. IL-1β mRNA and IL-1 receptor antagonist (IL-1Ra) mRNA were induced similarly, exhibiting highest and lowest expression levels in the hypothalamus and hippocampus, respectively. IL-1 receptor type I (IL-1RI) mRNA and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA were also induced in the hypothalamus and cerebellum. NPY mRNA expression showed a small, but significant decrease in the hypothalamus. Heat-inactivated IL-1β (8.0 ng/24 h for 72 h) had no effect on the behavioral or molecular profiles. The results suggest that endogenous upregulation of IL-1β contributes to IL-1β-induced anorexia, and that modification of NPY mechanisms also may be involved.     

Effect of transforming growth factor-β1 and -β2 on Schwann cell proliferation on neurites

Mechanisms regulating Schwann cell proliferation during development are unclear. Schwann cell division is known to be driven by an unidentified mitogen present on the surface of axons, but it is not known whether other molecules play a role in regulating this proliferation. Transforming growth factor-beta (TGF-β) which is found in the developing peripheral nervous system (PNS) and is mitogenic for neuron-free Schwann cells in vitro could be involved. We have investigated the effects of TGF-β 1, TGF-β 2 and antibodies to TGF-β and TGF-β 2 on axon driven Schwann cell proliferation. Rat embryonic dorsal root ganglion neurons (DRG) neurons and Schwann cells from the sciatic nerve were isolated, purified and recombined in vitro. Confirming earlier reports by others, we observed that TGF-β 1 and TGF-β 2 added to the culture medium stimulated the proliferation of Schwann cells in the absence of neurons. However, when added to neuron-Schwann cell co-cultures, TGFβ caused a variable response ranging from no effect to moderate inhibition of Schwann cell proliferation in different experiments. A stimulation of Schwann cell proliferation by TGFβ was never observed in neuron-Schwann cell co-cultures. Antibodies to TGF-β and TGF-β 2 did not influence axon driven Schwann cell proliferation. To further determine the role of TGF-β in Schwann cell proliferation and myelination, we studied Schwann cell proliferation in cultures from mice in which the TGF-β 1 gene was delected by homologous recombination. Neuron-Schwann cell cultures from wild-type, heterozygous and homozygous mice were used. No differences were observed in either Schwann cell proliferation or myelination between cultures obtained from homozygous mutants and their heterozygous and wild-type controls. These findings suggest that TGF-β does not function as a part of the mitogenic mechanism presented by neurons to Schwann cells, but that the presence of active TGFβ in the cellular environment might regulate the degree of proliferation induced by neuronal contact. Copy 1995 Wiley-Liss, Inc.