Oxidant-induced atrogin-1 and transforming growth factor-beta1 precede alcohol-related myopathy in rats

Alcohol-related chronic myopathy is characterized by severe biochemical and structural changes to skeletal muscle. Our goals were to: (1) identify early regulatory elements that precede the overt manifestation of plantaris atrophy; and (2) circumvent these derangements by supplementing alcohol-fed rats with the glutathione precursor, procysteine. After 6 weeks of daily ingestion, before the development of overt atrophy of the plantaris muscle, alcohol increased several markers of oxidative stress and increased gene expressions of atrogin-1 and transforming growth factor-beta1 (TGF-beta1) by approximately 60- and approximately 65-fold, respectively, which were attenuated by procysteine supplementation. Interestingly, after 28 weeks of alcohol ingestion, when overt plantaris atrophy had developed, atrogin-1 and TGF-beta1 gene expression had returned to baseline levels. Together, these findings suggest that alcohol-induced, redox-sensitive alterations drive pro-atrophy signaling pathways that precede muscle atrophy. Therefore, targeted anti-oxidant treatments such as procysteine supplementation may benefit individuals with chronic alcohol abuse, particularly if given prior to the development of clinically significant myopathy.     

Isoform-specific role of transforming growth factor-beta2 in the regulation of proliferation and differentiation of murine adrenal chromaffin cells in vivo

Chromaffin cells, the neuroendocrine cells of the adrenal medulla, play an important role in molecular, cellular, and developmental neurobiology. Unlike the closely related sympathetic neurons, chromaffin cells are able to proliferate throughout their whole life span. Proliferation of chromaffin cells in vivo is thought to be regulated by the interaction of neurogenic and hormonal signals. Previous studies have shown that chromaffin cells synthesize and release transforming growth factor-betas (TGF-betas). In the present study, effects of TGF-betas on proliferation and differentiation of chromaffin cells in mouse adrenal chromaffin cells were investigated in a genetic mouse model. We observed a significant increase in the total number of tyrosine hydroxylase-positive (TH(+)) cells in Tgfbeta2(-/-) knockout mouse embryos at embryonic day (E) 18.5 compared with wild-type animals (Tgfbeta2(+/+)), but no changes in the number of TH(+) cells were observed in Tgfbeta3(-/-) mouse mutants. At E15.5, but not at E18.5, there was a marked increase in the number of proliferative cell nuclear antigen-positive chromaffin cells in Tgfbeta2(-/-) knockout embryos compared with the wild-type group. On the other hand, there was a clear decrease in the ratio of total number of phenylethanolamine-N-methyltransferase-positive cells to the total TH(+) in Tgfbeta2(-/-) mice embryos at E18.5 compared with wild-type animals. This is the first documentation of the physiological significance of the TGF-beta2, an isoform that has been suggested to play a role in the regulation of chromaffin cells proliferation and differentiation based on in vitro experiments.     

HIV-1 non-specifically stimulates production of transforming growth factor-β1 transfer in primary astrocytes

HIV-1 expression in monocytes/macrophages can be controlled by transforming growth factor-beta l (TGF-β1). TGF-β1 is present in astrocytes surrounding HIV-1-infected monocyte/macrophages in brain tissue from patients with AIDS but not from seronegative, normal individuals. We sought to determine whether or not production of TGF-β1 can be directly stimulated by HIV-1 in astrocytes. Astrocytes from neonatal rat cortex grown in primary culture were exposed to HIV-1 virions for 24 h. One day later, TGF-β1 was measured in culture supernatants by a biological assay. HIV-1 caused 1.7-2.1-fold increase in extracellular concentration of TGF-β1. TGF-β1 production also was stimulated by recombinant HIV-1 proteins gp120, p66 and p24. Gpl20 labeled with fluorescein was visualized inside astrocytes and its stimulatory effect was not blocked by antibodies against rat CD4. The effect was not specific to HIV-1 and its proteins, because non-opsonized Latex particles and leucine methyl ester (LME) (known to be phagocytosed and endocytosed, respectively, by astrocytes) also stimulated TGF-β1 production. The effect was inhibited by two inhibitors of the phago/endocytotic pathway, chloroquine and leupeptin. These results may be relevant to the neuropathogenesis of HIV-1 infection.     

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: A role for transforming growth factor β

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1β (IL-1β) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1β expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1β was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B₄-isolectin and an antibody against GFAP. Production of IL-1β and NO was determined by measurement of IL-1β and nitrite concentrations in cell lysates and the culture medium, respectively. TGFβ, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGFβ. The potential effect of TGFβ was studied by using immunoneutralizing antibodies against TGFβ1 and TGFβ2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGFβ antibodies (3 μg/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1β was not affected. The antibodies against TGFβ1 and TGFβ2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGFβ1 and TGFβ2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGFβ on NO production by microglial cells (possibly) by activation of TGFβ or by increasing the sensitivity of microglial cells for TGFβ. GLIA 19:190–198, 1997. © 1997 Wiley-Liss, Inc.     

Immunofluorescence and biochemical studies of the type VI collagen expression by human glioblastoma cells in vitro

Abstract

The human glioblastoma cell line U-87 MG was found to express a 140 kD polypeptide which was recognized on immunoblot analysis by a monoclonal antibody to type VI collagen. This polypeptide was digestible by a highly purified bacterial collagenase. After treatment of U-87 MG cells by pepsin, the protein profile revealed the two major pepsin-resistant fragments identical in Mr to those of collagen VI extracted from human placenta. The respective peptide maps from V8 protease one-dimensional gels of these two fragments were identical to those obtained with human collagen VI. Immunofluorescent staining by antibodies to type VI collagen was observed in the extracellular matrix. Moreover; U-87 MG cells were found to be positive for A2B5, a cell surface marker specific for 0-2A type glial precursor cells. These data indicate that the human glioblastoma cell line U-87 MG exhibits the properties of glial precursor cells and expresses collagen type VI in vitro. This cell line therefore may prove valuable for comparative investigations of the regulation of type VI collagen synthesis, and may be useful as a model to study the function and pathological importance of type VI collagen in human brain tumours, both in vitro and in vivo. [Neurol Res 1994; 16: 370-375]     

Inhibition of glial scarring in the injured rat brain by a recombinant human monoclonal antibody to transforming growth factor-beta2.

The transforming growth factor-betas (TGF-betas) are potent fibrogenic factors implicated in numerous central nervous system (CNS) pathologies in which fibrosis and neural dysfunction are causally associated. In this study, we aim to limit the fibrogenic process in a model of CNS scarring using a recombinant human monoclonal antibody, derived from phage display libraries and specific to the active form of the TGF-beta2 isoform. The implicit inference of the work was that, as such antibodies are potential pharmacological agents for the treatment of human CNS fibrotic diseases, validation of efficacy in a mammalian animal model is a first step towards this end. Treatment of cerebral wounds with the anti-TGF-beta2 antibody led to a marked attenuation of all aspects of CNS scarring, including matrix deposition, formation of an accessory glial-limiting membrane, inflammation and angiogenesis. For example, in the wound, levels of: (i) the connective tissue components fibronectin, laminin and chondroitin sulphate proteoglycan; and (ii) wound-responsive cells including astrocytes and macrophages/microglia, were markedly reduced. Our findings suggest that such synthetic anti-fibrotic TGF-beta antibodies are potentially applicable to a number of human CNS fibrotic diseases to arrest the deposition of excessive extracellular matrix components, and maintain and/or restore functional integrity.     

Genetic variation in the transforming growth factor β1 gene in multiple sclerosis

Transforming growth factor β1 (TGFβ1) is a Th2 cytokine encoded on chromosome 19q13, a region possibly linked to multiple sclerosis (MS). TGFβ1 exerts favorable effects on experimental allergic encephalomyelitis. We performed a comprehensive search for genetic variants in this gene in 122 population-based sporadic cases of MS. We detected six variants, including three missense variants. We tested for association of the variants with susceptibility and course of MS and for linkage and transmission disequilibrium in a family series consisting of 395 samples in 59 pedigrees. Genetic variation in TGFB1 does not appear to contribute in a major way to susceptibility to MS.     

Secretory products of central nervous system glial cells induce Schwann cell proliferation and protect from cytokine-mediated death

There continues to be interest in Schwann cells (SC) as a possible source of myelinating cells for transplantation into the central nervous system (CNS) of patients with multiple sclerosis (MS) and spinal cord injury. It has been suggested that CNS glial cells interfere with SC migration, survival, maturation, and clinically significant remyelination in the CNS. To investigate the effects of CNS glial cells on SC, we examined the effects of serum-free supernatants obtained from rat mixed CNS glial cultures on rat neonatal SC cultures. Supernatants from 1-, 3-, and 5-day CNS glial cultures induced proliferation of SC assayed at 5 days in vitro but did not induce SC differentiation as measured by induction of surface expression of galactolipids (GalL). High concentrations of cAMP simulate many of the effects of axolemma on SC; CNS glial cell supernatants did not inhibit cAMP induction of SC differentiation. CNS glial cell supernatants had no apparent effect on SC viability at 48 hr as measured by trypan blue exclusion. We have previously demonstrated that incubation of SC with transforming growth factor-beta1 (TGF-beta1) + tumor necrosis factor-alpha (TNF-alpha) induces SC death via apoptosis. We now show that CNS glial supernatants inhibits TGF-beta1/TNF-alpha-induced SC death. Our data show that soluble products of CNS glial cells do not induce or inhibit SC differentiation or increase cell death but have the potential to increase proliferation of SC and their resistance to cytokine-mediated death, and thus may affect the outcome of SC transplantation into the CNS.     

Involvement of prostaglandin E2 released from leptomeningeal cells in increased expression of transforming growth factor-beta in glial cells and cortical neurons during systemic inflammation

The leptomeninges play a central role in the antiinflammatory response through the glia-neuron interaction during systemic inflammation. In the present study, we examined the possible production of two potent antiinflammatory mediators, prostaglandin E(2) (PGE(2)) and transforming growth factor-beta1 (TGF-beta1) by leptomeningeal cells during systemic inflammation. After immunization with the complete Freund's adjuvant (CFA), cyclooxygenase (COX)-2 and membrane-bound PGE synthase-1 (mPGES-1) were induced in the leptomeninges. Primary cultured leptomeningeal cells secreted PGE(2) after treatment with lipopolysaccharide (LPS) or proinflammatory cytokines. The LPS-induced release of PGE(2) was depressed by a selective COX-2 inhibitor, NS-398. On the other hand, TGF-beta1 and TGF-beta receptor II (TGF-betaRII) both markedly increased in the leptomeninges and the parenchymal cells after the CFA injection. Double-staining immunohistochemistry demonstrated TGF-beta1 to be induced in both glial cells and cortical neurons, whereas TGF-betaRII was induced only in cortical neurons. Furthermore, the conditioned medium prepared from the leptomeningeal cells after LPS stimulation was able to induce an increased expression of TGF-beta1 and TGF-betaRII in the primary cultured glial cells and cortical neurons. This increased expression was suppressed by NS-398. PGE(2) was found to increase directly the production of TGF-beta1 and TGF-betaRII in the primary cultured cells. These observations strongly suggest that PGE(2), which is biosynthesized by the leptomeninges, mainly regulates the production of TGF-beta1 by glial cells and cortical neuron, thus playing a protective role in the cortical neurons during systemic inflammation. Furthermore, TGF-beta1 may also exert a protective effect directly on the cortical neurons.     

Interleukin-1beta but not tumor necrosis factor-alpha potentiates neuronal damage by quinolinic acid: protection by an adenosine A2A receptor antagonist

Quinolinic acid is an agonist at glutamate receptors sensitive to N-methyl-D-aspartate (NMDA). It has been implicated in neural dysfunction associated with infections, trauma, and ischemia, although its neurotoxic potency is relatively low. This study was designed to examine the effects of a combination of quinolinic acid and the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Compounds were administered to the hippocampus of anesthetized male rats, animals being allowed to recover for 7 days before histological analysis of the hippocampus for neuronal damage estimated by counting of intact, healthy neurons. A low dose of quinolinic acid or IL-1beta produced no damage by itself, but the two together induced a significant loss of pyramidal neurons in the hippocampus. Higher doses produced almost total loss of pyramidal cells. Intrahippocampal TNF-alpha produced no effect alone but significantly reduced the neuronal loss produced by quinolinic acid. The adenosine A(2A) receptor antagonist ZM241385 reduced neuronal loss produced by the combinations of quinolinic acid and IL-1beta. The results suggest that simultaneous quinolinic acid and IL-1beta, both being induced by cerebral infection or injury, are synergistic in the production of neuronal damage and could together contribute substantially to traumatic, infective, or ischemic cerebral damage. Antagonism of adenosine A(2A) receptors protects neurons against the combination of quinolinic acid and IL-1beta.     

ATP binding cassette transporter ABC1 is required for the release of interleukin-1beta by P2X7-stimulated and lipopolysaccharide-primed mouse Schwann cells

Schwann cells are best known as myelinating glial cells of the peripheral nervous system, but they also participate actively in the sphere of immunity by producing pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). In a previous study, we demonstrated that posttranslational processing of IL-1beta by immune-challenged Schwann cells required the P2X7 receptor. Remarkably, the release of IL-1beta was not associated with cell death, indicating the involvement of an active mechanism. ATP binding cassette (ABC) transporters are known to transport leaderless secretory proteins, such as IL-1beta; therefore, we investigated whether such transporters were at work in Schwann cells. Mouse Schwann cells expressed ABC1 transporter mRNA and displayed the functional protein. Glybenclamide and diisothiocyanato-stilbene-disulfonic acid (DIDS), two blockers of chloride fluxes that drive the export activity of ABC1 transporters, inhibited IL-1beta release without altering its intracellular processing. Enhancing chloride efflux potentiated the release of IL-1beta, while decreasing it led to a strong reduction in its release. Because the stimulation of the P2X7 receptor also activates a chloride conductance, we investigated the possibility of a sole anionic pathway mobilized by the P2X7 receptor and ABC1. Glybenclamide and DIDS had no significant effects on the P2X7-activated chloride current suggesting therefore the existence of two different pathways. In summary, ABC1 transporters are required for the release of IL-1beta by mouse Schwann cells. Being associated together with chloride conductance, P2X7 receptors and ABC1 transporters delineate a subtle and complex regulation of IL-1beta production in mammalian Schwann cells. Furthermore, ABC1 transporters could be a target of therapeutic interest for regulating IL-1beta activity in neuroinflammation disorders.     

Transforming growth factor-beta2 modulates synaptic efficacy and plasticity and induces phosphorylation of CREB in hippocampal neurons

Transforming growth factor-betas (TGF-betas) are widely expressed and play roles as multifunctional growth factors and regulators of key events in development, disease, and repair. However, it is not known whether TGF-betas affect the plasticity of hippocampal neurons. As a first step to address this issue, we examined whether TGF-beta2 modulated the electrophysiological and biochemical properties of cultured hippocampal neurons. We found that prolonged 24 h treatment with TGF-beta2 induced facilitation of evoked postsynaptic currents (ePSCs). This facilitation was associated with a decrease in short-term synaptic depression of ePSCs and increases in both the amplitude and frequency of spontaneous miniature postsynaptic currents (mPSCs). The long-term changes of ePSCs and mPSCs may be associated with cAMP response element-binding protein (CREB), which has been previously implicated in long-term potentiation. Immunofluorescence techniques and Western blot analysis both revealed that TGF-beta2 enhanced the phosphorylation of CREB. Together, these results suggest that TGF-beta2 may play a role in the cascade of events underlying long-term synaptic facilitation in hippocampus, and that CREB may be an important mediator of these effects.     

Interferon lambda inhibits herpes simplex virus type I infection of human astrocytes and neurons

Herpes simplex virus Type I (HSV-1) is a neurotropic virus that is capable of infecting not only neurons, but also microglia and astrocytes and can establish latent infection in the central nervous system (CNS). We investigated whether IFN lambda (IFN-λ), a newly identified member of IFN family, has the ability to inhibit HSV-1 infection of primary human astrocytes and neurons. Both astrocytes and neurons were found to be highly susceptible to HSV-1 infection. However, upon IFN-λ treatment, HSV-1 replication in both astrocytes and neurons was significantly suppressed, which was evidenced by the reduced expression of HSV-1 DNA and proteins. This IFN-λ-mediated action on HSV-1 could be partially neutralized by antibody to IFN-λ receptor. Investigation of the mechanisms showed that IFN-λ treatment of astrocytes and neurons resulted in the upregulation of endogenous IFN-α/β and several IFN-stimulated genes (ISGs). To block IFN-α/β receptor by a specific antibody could compromise the IFN-λ actions on HSV-1 inhibition and ISG induction. In addition, IFN-λ treatment induced the expression of IFN regulatory factors (IRFs) in astrocytes and neurons. Furthermore, IFN-λ treatment of astrocytes and neurons resulted in the suppression of suppressor of cytokine signaling 1 (SOCS-1), a key negative regulator of IFN pathway. These data suggest that IFN-λ possesses the anti-HSV-1 function by promoting Type I IFN-mediated innate antiviral immune response in the CNS cells.     

Contribution of the interleukin-1beta gene polymorphism in multiple system atrophy.

We studied genetic polymorphisms in the promoter region at position -511 of the interleukin (IL) -1beta gene (IL-1B-511) and at position -889 of the IL-1alpha gene (IL-1A-889), in 111 Japanese patients with multiple system atrophy (MSA) and 160 controls. The distribution of IL-1B-511 was significantly different between MSA patients and controls, because of the under-representation of patients with homozygotes for allele 2 (IL-1B-511*2), a high producer of IL-1beta. The frequency of IL-1A-889*2, a high secretor of IL-1alpha, was also decreased in MSA patients. Our findings suggest that abnormal cytokine expression may be implicated in the pathogenesis of MSA.     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

Alterations in matrix metalloproteinase-9 levels and tissue inhibitor of matrix metalloproteinases-1 expression in a transforming growth factor-beta transgenic model of hydrocephalus

The development of spontaneous hydrocephalus in mouse models resulting from the overexpression of transforming growth factor-beta (TGFbeta-1) has been previously described, although the mechanism by which this occurs remains obscure. It has been previously demonstrated that increased expression of TGFbeta has consequences for the levels of matrix metalloproteinases (MMPs) and their specific inhibitors (tissue inhibitors of MMPs, or TIMPs). These remodeling proteins play an important role in extracellular matrix (ECM) maintenance through degradation and deposition of ECM components. The present study investigated the relationship between elevated levels of TGFbeta-1, the ECM modulators TIMP-1 and MMP-9, and development of hydrocephalus in the neonatal mouse. In newborn pups, TIMP-1 mRNA levels were equal between animals expressing the TGFbeta-1 transgene and littermates without the transgene. However, immunohistochemistry of littermate pups shows that the distribution of TIMP-1 was changed from homogeneous with large punctate concentrations of signal to uniform, dense staining in hydrocephalic animals carrying the TGFbeta-1 transgene. The mRNA levels of MMP-9 were decreased in the transgenic animals, as were the activity levels MMP-9. These results suggest that the remodeling protein MMP-9 and its specific inhibitor, TIMP-1, may contribute to the spontaneous development of hydrocephalus in this transgenic model by altering the ECM environment.