Transcriptional regulation of chemokine gene expression in astrocytes.
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Animals↗
Biomedical subjects
R M Ransohoff
Publications and source records attributed to R M Ransohoff.
Characterization of beta-R1, a gene that is selectively induced by interferon beta (IFN-beta) compared with IFN-alpha.
We report preliminary characterization of a gene designated beta-R1, which is selectively expressed in response to interferon beta (IFN-beta) compared with IFN-alpha. In human astrocytoma cells, beta-R1 was induced to an equivalent extent by 10 IU/mL IFN-beta or 2500 IU/mL IFN-alpha2. To address the mechanism of this differential response, we analyzed induction of the beta-R1 gene in fibrosarcoma cells and derivative mutant cells lacking components required for signaling by type I IFNs. beta-R1 was readily induced by IFN-beta in the parental 2fTGH cell line, but not by recombinant IFN-alpha2, IFN-alpha Con1, or a mixture of IFN-alpha subtypes. IFN-alpha8 induced beta-R1 weakly. beta-R1 was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-alpha and -beta receptor, also failed to express beta-R1. U1A cells are partially responsive to IFN-beta and IFN-alpha8 but lacked beta-R1 expression, indicating that TYK2 protein is essential for induction of this gene. Taken together, these results suggest that the expression of beta-R1 in response to type I IFN requires IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-beta compared with IFN-alpha.
Astrocytoma↗
Neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes.
Chemokines (pro-inflammatory chemoattractant cytokines) are expressed in pathological conditions of the central nervous system (CNS). Previous studies suggested that the CNS is relatively resistant to leukocyte diapedesis after chemokine injection, leaving their functional role unresolved. The CNS function of N51/KC, a neutrophil-selective chemokine, was addressed by expressing N51/KC under control of the myelin basic protein (MBP) promoter in transgenic (tg) mice (MBP-N51/KC mice). CNS-specific N51/KC expression produced remarkable neutrophil infiltration into perivascular, meningeal, and parenchymal sites, demonstrating that this chemokine exerts the multiple functions in vivo required to recruit leukocytes into the CNS. MBP-N5 1/KC mice represent an incisive model for the molecular dissection of neutrophil entry into the CNS. Unexpectedly, MBP-N51/KC mice developed a neurological syndrome of pronounced postural instability and rigidity at high frequency beginning at 40 days of age, well after peak chemokine expression. 68/182 mice in one tg fine were found dead before one year of age, with prominent neurological symptoms premortem in 26 (38%). Florid microglial activation and blood-brain barrier disruption without dysmyelination were the major neuropathological alterations. Late-onset neurological symptoms in MBP-N51/KC mice may indicate unanticipated consequences of CNS chemokine expression.
Animals↗
Stimulus-specific inhibition of intracellular adhesion molecule-1 gene expression by TGF-beta.
Astrocytes and microglia, the two major glial cells within the central nervous system, can function as immune effector cells upon activation. Intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein involved in extravasation into inflamed tissue and Ag-specific activation of T lymphocytes, can be induced in astrocytes and microglia by numerous stimuli. In this study, we investigated the role of TGF-beta, an immunosuppressive cytokine, in regulating ICAM-1 expression in glial cells. We previously demonstrated that TNF-alpha, IL-1 beta, IFN-gamma, or IFN-gamma plus LPS (IFN-gamma/LPS) can enhance ICAM-1 expression in astrocytes, while microglia express ICAM-1 only in response to IFN-gamma or IFN-gamma/LPS. TGF-beta alone has a minimal effect on constitutive ICAM-1 expression in either astrocytes or microglia, but inhibits, in a time-dependent manner, TNF-alpha- or IL-1 beta-induced ICAM-1 mRNA and protein expression in astrocytes. Interestingly, TGF-beta has no effect on IFN-gamma- or IFN-gamma/LPS-induced ICAM-1 expression in astrocytes or microglia. Inhibition of TNF-alpha- or IL-1 beta-induced ICAM-1 mRNA levels by TGF-beta in astrocytes was not due to degradation of ICAM-1 message, rather, inhibition was mediated at the transcriptional level. Similar results were observed in two human astroglioma cell lines, CRT and STT; TGF-beta inhibited TNF-alpha- and IL-1 beta-induced ICAM-1 expression, but IFN-gamma induction of ICAM-1 was unaffected. These results indicate that TGF-beta suppresses ICAM-1 expression in glial cells in a stimulus-specific manner.
Astrocytoma↗
Chemokine monocyte chemoattractant protein-1 is expressed by astrocytes after mechanical injury to the brain.
By 24 h after mechanical trauma to the cerebral cortex, astroglial reaction begins and injury sites are infiltrated by activated mononuclear phagocytes derived from blood-borne monocytes and endogenous microglia. There is little information about cellular interactions between astrocytes and leukocytes during this process. We previously showed that murine astrocytes produce chemokines including monocyte chemoattractant protein-1 (MCP-1) during experimental autoimmune encephalomyelitis. In this study, we asked whether astrocytes produce MCP-1 in the absence of immune mediated inflammation. To address this question, we analyzed the time course and cellular source of MCP-1 in mouse brain after penetrating mechanical injury, with particular focus on early time points before histologic detection of infiltrating mononuclear phagocytes. We observed sharply increased steady state levels of MCP-1 mRNA within 3 h after nitrocellulose membrane stab or implant injury to the adult mouse brain, and MCP-1 protein elevations were documented at 12 h postinjury. In situ hybridization combined with immunohistochemistry for the glial fibrillary acidic protein astrocyte marker showed that astrocytes were the cellular source of MCP-1 mRNA at these early time points after mechanical brain injury. Stab injury to the neonatal brain evoked neither MCP-1 expression nor astrogliosis. These results demonstrate that chemokine gene expression comprises one component of the astrocyte activation program. The data are consistent with a role for MCP-1 in the central nervous system inflammatory response to trauma.
Animals↗
Interferon beta induces interleukin-10 expression: relevance to multiple sclerosis.
Interferon-beta decreases the relapse rate, relapse severity, progression of neurological disability, and development of new brain lesions observed with brain magnetic resonance imaging in relapsing-remitting multiple sclerosis patients. The mechanism of action of this effect is presently unknown. This study was based on the hypothesis that immunoregulatory effects of interferon-beta may underlie its demonstrated clinical efficacy. The objective of the study was to determine the effect of interferon-beta-1a on the expression of interleukin-10, a cytokine that strongly inhibits cell-mediated immune responses. Interferon-beta-1a induced accumulation of interleukin-10 messenger RNA and protein secretion by cultured peripheral blood mononuclear cells. The observed in vitro effects were similar for healthy control subjects and multiple sclerosis patients. Intramuscular injections of interferon-beta-1a increased serum levels of interleukin-10 at 12 and 24 hours following the injection. Greater increases were induced with 12 x 10(6)-IU than 6 x 10(6)-IU injections. The effect of interferon-beta-1a was relatively specific for interleukin-10, as treatment with interferon-beta-1a did not result in accumulation of transforming growth factor-beta messenger RNA. Upregulation of interleukin-10 represents a possible mechanism of action of interferon-beta's therapeutic effect in relapsing-remitting multiple sclerosis, and has implications for therapy of other autoimmune diseases.
Antibodies, Monoclonal↗
Chemokines in immune-mediated inflammation of the central nervous system.
Inflammatory cell recruitment to the central nervous system (CNS) is a cardinal feature of physiological and pathological processes, including multiple sclerosis (MS). Despite recent progress, the soluble signals that attract inflammatory cells from the vascular compartment into the CNS parenchyma remain obscure. We favor the hypothesis that chemoattractant cytokines termed 'chemokines' are uniquely important for mediating leukocyte entry into CNS tissues during immune-mediated inflammation. Three lines of evidence supporting this hypothesis will be reviewed. The first regards expression of chemokines in animal models of immune-mediated CNS inflammation and in the human disease, multiple sclerosis. The second line of evidence involves interventional studies of chemokine blockade in such model disorders. The third line of evidence comprises function of chemokines in the CNS, as analysed in transgenic mice. Investigation of CNS chemokine function will enhance our understanding of leukocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.
Animals↗
In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis.
Reactive astrogliosis is a prominent pathological feature of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. It is characterized by hypertrophy of astrocytes with increased content of glial fibrillary acidic protein (GFAP.) Studies of reactive astrocytes in acute experimental autoimmune encephalomyelitis have been complicated by the observation that the diffuse increase in GFAP immunohistochemical staining at the onset of central nervous system inflammation does not parallel the gradual increase in GFAP content probably because tissue edema enhances GFAP immunostaining. To characterize changes in GFAP expression, we performed in situ hybridization at 3- to 7-day intervals during the course of acute murine experimental autoimmune encephalomyelitis. We found a biphasic course of GFAP expression: an early phase of astrocyte reaction surrounding perivascular inflammatory cuffs and submeningeal infiltrates at the onset of central nervous system inflammation and clinical disease and a later phase of increased GFAP mRNA expression in regions of demyelination during resolution of inflammation and clinical improvement. IP-10, a member of a family of proinflammatory chemoattractant cytokines called chemokines, was expressed by astrocytes in a similar distribution as those expressing increased GFAP mRNA in the early phase of inflammation but was no detected in astrocytes in the later phase of activation. These results indicate that location and function of reactive astrocytes may vary during the course of immune-mediated demyelination.
Acute Disease↗
Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the beta 2 integrin LFA-1 in multiple sclerosis lesions.
To identify potential molecular substrates for leukocyte trafficking and activation in multiple sclerosis (MS) brain, we determined the immunocytochemical distribution of the beta, integrin lymphocyte-function-associated antigen-1 (LFA-1) and its major ligands, intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 in MS tissue. Colocalization of these adhesion molecules with lineage-specific markers was analyzed by dual-labeling immunocytochemistry and confocal microscopy. ICAM-1 and ICAM-2 were detected on endothelial cells, and ICAM-3 immunoreactivity was restricted to infiltrating leukocytes. In control brain, 10% of glucose transporter-1 positive vessels contained ICAM-1 immunoreactivity on their luminal surface and 21% were ICAM-2-positive. A significant increase in ICAM-1-positive vessels was found in MS brains. This increase was greater in MS lesions (81% of vessels) than in nonlesion areas (37% of vessels). A significant increase in ICAM-1-positive vessels was found in encephalitis (55% of vessels) but not in Parkinson's (17% of vessels) brains. The percentage of vessels expressing ICAM-2 was not increased in MS, encephalitis, or Parkinson's brains. Both ICAM-3 and LFA-1 were detected on the vast majority of infiltrating lymphocytes and monocytes in and near MS lesions, and these cells were often closely apposed to each other. In addition, LFA-1 was detected on activated microglia located close to the edge of demyelinating lesions. ICAM-3-positive leukocytes were often closely apposed to LFA-1-positive microglia. These results suggest a role for ICAM-1, -2, and LFA-1 in the transendothelial migration of leukocytes into MS brain and a role for ICAM 3/LFA-1 interactions in the activation of lymphocytes, monocytes, and microglia in MS lesions.
Adolescent↗
Interferon (IFN) beta acts downstream of IFN-gamma-induced class II transactivator messenger RNA accumulation to block major histocompatibility complex class II gene expression and requires the 48-kD DNA-binding protein, ISGF3-gamma.
Interferon (IFN) gamma, a cardinal proinflammatory cytokine, induces expression of the gene products of the class II locus of the major histocompatibility complex (MHC), whereas IFN-alpha or -beta suppresses MHC class II expression. The mechanism of IFN-beta-mediated MHC class II inhibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-gamma-induced MHC class II transcription. We studied the status of IFN-gamma-induced CIITA messenger RNA (mRNA) accumulation and CIITA-driven transactivation in IFN-beta-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-beta treatment did not suppress IFN-gamma-induced accumulation of CIITA mRNA. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were actively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-beta. These results suggest that IFN-beta acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) gamma was essential for IFN-beta to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-gamma or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-beta-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters.
DNA-Binding Proteins↗
Cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) by astrocytes and astrocytoma cell lines.
Astrocytes in the central nervous system (CNS) associate intimately with vascular endothelial cells and are integral to the blood-brain barrier (BBB). Leukocyte transmigration across the BBB is a cardinal feature of CNS inflammation, as observed in experimental autoimmune encephalomyelitis, and very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1) interactions have recently been proposed as essential for this process. VCAM-1 expression by astrocytes was recently reported. We addressed the regulation of VCAM-1 expression by inflammatory cytokines in primary human astrocytes and two human astrocytoma cell lines. Astrocytoma cells up-regulated surface VCAM-1 expression in response to cytokines in the following order: IFN-gamma plus TNF-alpha > IFN-gamma plus IL-1 beta > TNF-alpha > IFN-gamma. Cytokine-activated astrocytoma cells expressed 7-domain VCAM-1, as indicated by accumulation of 3.2-kb VCAM-1 mRNA and immunoprecipitation of a 100-kDa protein with anti-VCAM-1 mAb. Lymphoblast adhesion to cytokine-activated astrocytoma cell monolayers was significantly blocked by mAb specific for VCAM-1 and VLA-4, indicating that astrocytoma cell VCAM-1 was functional. Astrocytoma cell expression of VCAM-1 could be a constituent of the astrocyte phenotype. To support this possibility, we demonstrated that cytokine-treated adult human and rat primary astrocytes expressed VCAM-1, and the rank order of cytokine potency for VCAM-1 induction in primary and neoplastic astrocytes was strikingly similar. This is the first documentation of VCAM-1 expression by adult human astrocytes. Expression of VCAM-1 by astrocytes at the BBB could play a role in mononuclear leukocyte entry into the CNS.
Astrocytes↗
The interferons: biological effects, mechanisms of action, and use in multiple sclerosis.
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Humans↗
Central nervous system chemokine mRNA accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis.
Central nervous system (CNS) expression of two chemokine mRNAs, encoding monocyte chemoattractant protein-1 (MCP-1) and IFN-gamma-inducible protein (IP-10), was previously shown to be closely related to the onset of clinical signs of murine experimental autoimmune encephalomyelitis (EAE). Chemokine mRNAs accumulated in a striking, transient burst within astrocytes, near inflammatory leukocyte infiltrates. It remained unclear if chemokines functioned to initiate leukocyte entry into CNS tissues, or to amplify the intrathecal inflammatory reaction. To address this issue, we determined the expression of chemokine mRNAs at the earliest evidence of CNS immune-mediated inflammation. For these experiments, mice were sacrificed in pairs at varying times after immunization. Only one member of each pair was symptomatic for EAE at the time of sacrifice. Symptom presence correlated well with histological inflammation at the time of sacrifice. RNA was prepared from two CNS sites, brain and spinal cord, and expression of chemokine mRNAs was analyzed by a sensitive and quantitative reverse transcriptase/polymerase chain reaction dot-blot hybridization assay. CNS expressions of MCP-1 and IP-10 gene were correlated tightly with histological inflammation; indeed, chemokine expression was never detected in the absence of leukocyte infiltrates. In situ hybridizations showed that astrocytes expressed chemokine transcripts. These findings provide new information about mechanisms controlling chemokine mRNA expression during immune-mediated inflammation in EAE and are consistent with a role for chemokines as amplifiers of CNS inflammatory reactions.
Animals↗
Biologic effects of interferons: relevance to multiple sclerosis.
Recombinant interferon beta has established efficacy for relapsing-remitting MS, but the mechanisms of action in the disease are not well understood. Interferons (IFNs) mediate biologic effects by receptor-mediated gene activation. Binding by IFNs to high-affinity surface receptors results in physophorylation and activation of two cytoplasmic tyrosine kinases. This leads to activation of latent transcription factors in cell cytoplasm that translocate to the nucleus, where activated transcriptional elements interact with the interferon-stimulated response element (ISRE), leading to transcription of the interferon-stimulated genes (ISGs). IFN's biologic effects are mediated by function of the ISGs. The biologic effects of IFNs have been classified as antiviral, antiproliferative and immunomodulatory; effects are complex, however, because there are two separate types of IFN, four separate varieties of type I IFN and approximately 30 known interferon-regulated genes. Known effects of IFN that may plausibly relate to therapy efficacy in MS are discussed.
Antineoplastic Agents↗
The immunology of multiple sclerosis: new intricacies and new insights.
New epidemiology and genetics data have supported the broad concept that multiple sclerosis represents an acquired autoimmunity, which is determined in part by inheritance. Furthermore, it is now widely believed that the pathogenesis of multiple sclerosis involves autoimmune reactivity that is directed against myelin proteins. Much of our understanding of myelin-directed autoimmunity has come from studies on experimental autoimmune encephalomyelitis, a T-cell-mediated, multiple sclerosis-like disease, which is provoked by immunizing animals with myelin proteins. During the past year, significant progress has been made in delineating immune reactivity to myelin antigens. Models that feature restricted epitope recognition and limited T-cell receptor gene utilization have been challenged (often by their original authors). Unifying new concepts include dynamic temporal diversification of the autoimmune response and clear-cut distinctions between the potential and engaged autoimmune repertoire. An abundance of new information about the biologic determinants of T-cell immunopathogenicity is leading rapidly to innovative therapeutic strategies.
Animals↗
Do chemokines mediate inflammatory cell invasion of the central nervous system parenchyma?
Inflammatory cell recruitment into the central nervous system (CNS) is a critical step in the response to diverse insults, including infection, trauma and infarction, as well as immune-mediated disorders such as multiple sclerosis (MS). Despite considerable advances in understanding immune surveillance and antigen recognition in the CNS, the signals resulting in parenchymal inflammation are incompletely understood. Members of a novel family of chemo-attractant cytokines, the chemokines, are made in the CNS and are emerging as likely mediators of inflammatory cell migration into the CNS.
Animals↗
Inhibition of interferon-gamma-induced major histocompatibility complex class II gene transcription by interferon-beta and type beta 1 transforming growth factor in human astrocytoma cells. Definition of cis-element.
To address mechanisms by which cytokines inhibit interferon-gamma (IFN gamma)-induced gene expression in astrocytic cells, we have been studying effects of type beta 1 transforming growth factor (TGF beta 1) and interferon-beta (IFN beta) on IFN gamma-induced expression of the well-characterized human major histocompatibility complex (MHC) class II gene DRA. This investigation was motivated by the observations that IFN gamma-induced expression of MHC class II antigen expression on astrocytic cells can be blocked in a tissue-specific fashion by several cytokines and neurotransmitters in tissue culture and that astrocyte expression of MHC class II is severely restricted in vivo. We previously showed that IFN beta inhibited IFN gamma-induced DRA expression at the transcriptional level. This inhibition was not global, since IFN gamma-induction of intercellular adhesion molecule-1 was not affected. Here, TGF beta 1-mediated inhibition of DRA is shown to exhibit similar characteristics. To address the mechanism of this inhibition, sequence requirements for IFN beta and TGF beta 1 to suppress IFN gamma-induced transcription of DRA were determined. A 135-base pair DRA sequence element containing the IFN gamma-responsive region and transcriptional start site was sufficient to direct IFN beta- or TGF beta 1-mediated suppression of a reporter gene. These experiments suggested that either IFN beta or TGF beta 1 could repress IFN gamma-induced DRA transcription directly, without requiring a cis-element extrinsic to the IFN gamma-inducible DRA sequences. Consistently similar effects of IFN beta and TGF beta 1 observed in these experiments prompted comparison of other gene regulatory effects of the two cytokines. TGF beta 1, unlike IFN beta, induced gene expression directed by upstream elements of the gene encoding plasminogen activator inhibitor type-1. IFN beta, unlike TGF beta 1, enhanced levels of 2'-5'-oligoadenylate synthetase activity. Additionally, direct assay of TGF beta 1 and monoclonal anti-TGF beta antibody blocking experiments were used to determine that cells treated with IFN beta did not produce increased amounts of active or latent TGF beta. These data argued that IFN beta and TGF beta 1 utilized distinct pathways to mediate the inhibition of IFN gamma-induced DRA transcription.
2',5'-Oligoadenylate Synthetase↗
Astrocyte expression of mRNA encoding cytokines IP-10 and JE/MCP-1 in experimental autoimmune encephalomyelitis.
Mononuclear leukocytes preferentially accumulate in the central nervous system (CNS) during the course of experimental autoimmune encephalomyelitis (EAE). To address factors that govern leukocyte trafficking in EAE, we monitored expression of mRNAs encoding IP-10 and JE/MCP-1, which are members of a family of chemoattractant cytokines. A transient burst of IP-10 and JE/MCP-1 mRNA accumulation in the CNS occurred, in close relation to the onset of histologic and clinical disease. In situ hybridizations showed, unexpectedly, that astrocytes were the major source of mRNAs encoding IP-10 and JE/MCP-1. These observations implicate astrocyte-derived cytokines as potential chemoattractants for inflammatory cells during EAE.
Animals↗