Differential selectivity of CIITA promoter activation by IFN-γ and IRF- 1 in astrocytes and macrophages: CIITA promoter activation is not affected by TNF-α

During demyelinating disease of the central nervous system (CNS), locally elevated cytokine levels may induce upregulation of MHC class II molecules on otherwise low expressing or negative cell types such as microglia and astrocytes, since IFN-γ has been shown to induce MHC class II expression on these cell types in vitro. While many transcription factors are involved with MHC class II expression, only the class II transactivator (CIITA) is tightly coordinated with IFN-γ-inducibility. Control of CIITA gene expression is complex, involving four distinct promoters, two of which (promoters III and IV) are IFN-γ-inducible in certain cell types. Here we demonstrate that IFN-γ treatment of rat astrocytes induces only CIITA promoter IV activity in contrast to the murine macrophage cell line RAW 264.7 that uses both IFN-γ-inducible promoters. In contrast to previously published reports, promoter IV activation is completely dependent upon an intact interferon regulatory factor-1 (IRF-1) but not STAT1 binding site using promoter constructs specifically mutated at these positions. Importantly, while TNF-α is able to synergize with IFN-γ to increase astrocyte MHC class II expression in vitro, we show that treatment of rat astrocytes with TNF-α has no effect on CIITA promoter activity. These data demonstrate that TNF-α augments MHC class II expression through a mechanism downstream or independent of CIITA induction

Mechanisms and specificity of lentivirus neurotoxicity

Lentiviruses such as Maedi-Visna Virus (MVV) in sheep and Human Immunodeficiency Virus (HIV) often cause a variety of neurological symptoms in later stages of infection. In sheep, MVV infection results in paralysis and ataxia, with post-mortem investigations revealing astrocytosis, demyelination and axonal damage. At present it is unclear how such neurodegeneration is mediated as MVV, like HIV, does not directly infect neurons. There is much debate as to the mechanisms involved in lentivirus related brain damage, centred around three main possibilities. Firstly, there may be a direct detrimental action of a viral fragment on neuronal cell types. Secondly, MVV infected cells may be releasing diffusible mediators such as cytokines which could cause neural damage or act on astrocytes and microglia causing them to produce factors to induce neurodegeneration. Thirdly, the prospect of an immune response occuring within the brain, involving infected macrophages or microglia, is highly probable.In order to investigate the possible mechanisms at work in lentivirus induced neurodegeneration, several sets of experiments were carried out. Firstly, a putative model of MVV encephalopathy was established in severe combined immunodeficient (,scid) mice. Scid mice lack T and B cells and are thus unable to mount any immune response. Blood derived macrophages were obtained from healthy sheep and infected with MVV in vitro. These were intracerebrally injected into scid mice, as were uninfected macrophages, in order to assess the occurrence of any neural cell damage. Cell preparations of peripheral blood mononuclear cells (PBMCs), uninfected, supplemented with virus, or obtained from productively MVV infected sheep, were also administered to scid mice in the same manner, to evaluate the role of immune cells in causing any neurodegeneration. The results show that the administration of MVV infected macrophages produces greater gliosis and astrocytosis in the brains of these mice as compared to those injected with control uninfected macrophages or vehicle. However, the injections of PBMCs did not cause any detectable differences in astrocytes and microglia between the groups. No neuronal loss or demyelination was evident with any of the injections. These results suggest that neural cell disruption in MVV encephalopathy may be due to the presence of MVV infected macrophages in the Central Nervous System (CNS) of infected sheep, acting indirectly through the release of diffusible mediators.To investigate further the underlying causes of these changes, the possible detrimental contribution of cytokines in this model was evaluated through the use of the reverse transcriptase-polymerase chain reaction (RT-PCR) of MVV infected and uninfected blood derived macrophages in vitro. The results show an increase in the levels of the pro-inflammatory cytokine interleukin-1 beta (TL-113) mRNA in MVV infected macrophages. Interpretation of this model therefore suggests that MVV exerts an indirect disruptive effect on neural cells via the release of pro-inflammatory cytokines from infected macrophages, causing microgliosis and astrocytosis which in turn lead to major disturbances of brain function.The possibility of a neurotoxic action of a viral product was also investigated. Peptides derived from the basic region of the transactivating protein Tat from both MVV and HIV have previously been shown to be lethal to neurons in vivo and in vitro, through a mechanism believed to involve Nitric Oxide (NO)-mediated glutamate neurotoxicity and cytokines. The actions of the MVV tat peptide when injected intracerebrally in rats are diminished one week post-operatively by the Nmethyl-D-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,ll-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine maleate (MK801) and the Nitric Oxide Synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Here, its short term effects were examined and its neurotoxic mechanisms probed through the use of in vivo stereotaxic injections and pharmacologic manipulations. The results show that the MVV and HIV tat peptides were very rapidly neurotoxic, causing neuronal cell death within 0.5 hours, and displayed a distinctive and unusual lesion profile. Changes in astrocytes and microglia were also observed. The acute effects of the MVV tat peptide were blocked by MK801, an NMDA receptor antagonist. However, the administration of L-NAME, a NOS inhibitor, alpha melanocyte stimulating hormone (aMSH), a TNFa inhibitor, or 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo- (F)-quinoxaline (NBQX), a non-NMDA glutamate receptor antagonist, did not reduce the volume of the lesion. This suggests that the MVV tat peptide acts at the NMDA receptor to cause cell death either directly or via an excitotoxic mechanism

The modulatory effect of cytokines on cell proliferation in C6 glioma cells.

by Liu Heng.Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.Includes bibliographical references (leaves 115-138).Acknowledgments --- p.IList of Abbreviations --- p.IIAbstract --- p.VChapter Chapter 1: --- IntroductionChapter 1.1 --- Cytokines in the Central Nervous System --- p.1Chapter 1.1.1 --- Basic Properties of Cytokines --- p.1Chapter 1.1.2 --- The General Characteristics of Glial Cells --- p.4Chapter 1.1.2.1 --- Astrocytes --- p.4Chapter 1.1.2.2 --- Oligodendrocytes --- p.6Chapter 1.1.2.3 --- Microglial --- p.7Chapter 1.1.3 --- The Effects of Cytokines on Neural Cells --- p.7Chapter 1.1.3.1 --- TNF-α and Neural Cells --- p.8Chapter 1.1.3.2 --- LIF and Neural Cells --- p.10Chapter 1.1.3.3 --- IL-1 and Neural Cells --- p.12Chapter 1.1.3.4 --- IL-6 and Neural Cells --- p.14Chapter 1.1.4 --- Immune Response in the Central Nervous System --- p.16Chapter 1.2 --- The C6 Glioma as a Model for the Study of Glial Cell Growth and Differentiation --- p.21Chapter 1.2.1 --- The Rat C6 Glioma Cells --- p.21Chapter 1.2.2 --- The Differentiation and Proliferation of C6 Glioma Cells --- p.23Chapter 1.3 --- Signal Transduction Pathways in Cytokine-stimulated Glial Cells --- p.28Chapter 1.3.1 --- Intracellular Signalling Pathways of Cytokines --- p.28Chapter 1.3.1.1 --- Protein Kinase C Pathway --- p.29Chapter 1.3.1.2 --- Tyrosine Kinase Pathway --- p.30Chapter 1.3.1.3 --- Cyclic Nucleotide Pathway --- p.32Chapter 1.3.1.4 --- Nitric Oxide Pathway --- p.33Chapter 1.3.2 --- Intracellular Signalling Pathways in Cytokine-stimulated C6 Glioma Cells --- p.34Chapter 1.4 --- The Aims of This Thesis Project --- p.37Chapter Chapter 2: --- Materials and Methods --- p.41Chapter 2.1 --- Rat C6 Glioma Cell Culture --- p.41Chapter 2.1.1 --- Preparation of Culture Media --- p.41Chapter 2.1.1.1 --- Complete Dulbecco's Modified Eagle Medium --- p.41Chapter 2.1.1.2 --- Complete Roswell Park Memorial Institute1640 Medium --- p.42Chapter 2.1.2 --- Maintenance of the C6 Cell Line --- p.42Chapter 2.1.3 --- Cell Preparation for Assays --- p.43Chapter 2.2 --- Determination of Cell Proliferation --- p.44Chapter 2.2.1 --- Determination of Cell Proliferation by [3H]-Thymidine Incorporation --- p.44Chapter 2.2.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.45Chapter 2.2.3 --- Data Analysis --- p.45Chapter 2.3 --- Effects of Cytokines and Lipopolysaccharide on C6 Cell Proliferation --- p.46Chapter 2.4 --- Effects of Protein Kinase C Activators and Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.47Chapter 2.5 --- Effects of cAMP or cGMP on Cytokine-induced C6 Cell Proliferation --- p.48Chapter 2.6 --- Effects of Tyrosine Kinase Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.48Chapter 2.7 --- Effects of Calcium Ion on Cytokine-induced C6 Cell Proliferation --- p.49Chapter 2.8 --- Effects of Nitric Oxide on Cytokine-induced C6 Cell Proliferation --- p.49Chapter 2.8.1 --- Effects of Sodium Nitroprusside and Nitric Oxide Synthase Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.49Chapter 2.8.2 --- Nitric Oxide Production Assay --- p.50Chapter 2.9 --- Effects of β-Adrenergic Receptor Agonist and Antagonist on Cytokine-induced C6 Cell Proliferation --- p.51Chapter 2.10 --- Morphological Studies on Cytokine-Treated C6 Glioma Cells --- p.51Chapter 2.10.1 --- Wright-Giesma Staining --- p.52Chapter 2.10.2 --- Glial Fibrillary Acidic Protein Staining --- p.52Chapter 2.10.3 --- Hematoxylin Staining --- p.53Chapter Chapter 3: --- Results --- p.55Chapter 3.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.55Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.56Chapter 3.1.2 --- The Time Course of Cytokine-induced C6 Cell Proliferation --- p.59Chapter 3.1.3 --- Effects of Lipopolysaccharide on C6 Cell Proliferation --- p.61Chapter 3.1.4 --- Effects of Cytokines on the Growth of C6 Cells --- p.64Chapter 3.2 --- Morphology and GFAP Expression in Cytokine-treated C6 Glioma Cells --- p.64Chapter 3.2.1 --- Effects of Cytokines on the Morphology of C6 Cells --- p.64Chapter 3.2.2 --- Effects of Cytokines on GFAP Expression in C6 Glioma Cells --- p.66Chapter 3.3 --- The Signalling Pathway of Cytokine-induced C6 Cell Proliferation --- p.69Chapter 3.3.1 --- The Involvement of Protein Kinase C in Cytokine-induced C6Cell Proliferation --- p.71Chapter 3.3.2 --- The Involvement of Tyrosine Kinase in the Cytokine- induced C6 Cell Proliferation --- p.81Chapter 3.3.3 --- The Involvement of Calcium Ions in Cytokine-induced C6 Cell Proliferation --- p.87Chapter 3.3.4 --- The Involvement of Cyclic Nucleotides in Cytokine- induced C6 Cell Proliferation --- p.92Chapter 3.3.5 --- The Involvement of Nitric Oxide in Cytokine-induced C6 Cell proliferation --- p.94Chapter 3.3.6 --- The Involvement of P-Adrenergic Receptor in Cytokine- induced C6 Cell Proliferation --- p.101Chapter Chapter 4: --- Discussion and Conclusions --- p.104References --- p.11

Distribution and regulation of proteins related to neuronal degeneration

Using in situ hybridisation, Tau 1 and Tau 2 gene expression has been studied in adult and neonatal rats in the basal forebrain and hippocampal regions. Tau 1 mRNA levels are significantly higher in the neonatal (gestation day 17) rats, with Tau 2 mRNA levels being significantly higher in the adult rats (Chapter 3). Using in situ hybridisation, Tau 1 and Tau 2 gene expression in the basal forebrain and hippocampal regions has been studied in adult rats in response to intraperitoneal administration of the following compounds: ondansetron (100ng/kg), MK801 (1mg/kg), GYKI52466 (30mg/kg), indomethacin (5mg/kg) and aniracetam (l0mg/kg). None of the compounds cause a significance change in either Taul or Tau 2 mRNA levels after a 24 hour period (Chapter 3). Glial cell cultures have been used to study the distribution and expression of NADPH diaphorase expression in response to exposure of the cells to cytokines, excitatory amino acid agonists and pro teases. The glial cultures have been characterised by positive staining to glial fibrillary acidic protein and NADPH diaphorase. Points 3-5 summarise the work carried out using these cultures (Chapter 4). Lipopolysaccharide (100mug/ml) significantly increases NADPH diaphorase staining. A maximum induction is observed after a 6 hour time period. Interleukin-1 (0.25ng/ml) also significantly increases NADPH diaphorase staining, and again maximum induction occurs after a 6 hour time period. The lipopolysaccharide-induced increase in NADPH diaphorase staining remains unaltered following combined exposure of the cells to lipopolysaccharide (100mug/ml ) and the calmodulin anatgonist W7 (400muM). The excitatory amino acid agonists glutamate (50muM), AMPA (50muM) and NMDA (50muM and 100muM) significantly increase NADPH diaphorase staining. This induction occurs after a 24 hour time period. The inhibitors APV (100muM) and CNQX (100muM) reduce the glutamate and AMPA-induced increase in NADPH diaphorase staining to near control values, again after a 24 hour time period. The glutamate-induced increase in NADPH diaphorase staining is reduced following combined exposure of the cells to glutamate (50muM) and the calmodulin antagonist W7 (400muM). Exposure of the cells to the proteases chymotrypsin (300ng/ml) and trypsin (500ng/ml) increase NADPH diaphorase staining. This induction occurs after a 24 hour time period. This increase is reduced to near control levels following combined exposure of the cells to the proteases and their respective inhibitors chymostatin (100muM) and trypsin inhibitor (l?/ml), again after a 24 hour time period. 6\ Basal forebrain primary cultures have been used to study the distribution and expression of cytoskeletal proteins and NADPH diaphorase staining following exposure of the cells to nitric oxide, excitatory amino acid agonists and cytokines. The cells have been characterised by positive immunostaining to choline acetyltransferase (ChaT), neuron specific enolase, neurofilament and microtubule-associated protein 2 (MAP2). Points 6-10 summarise the work carried out using these cultures (Chapter 5). (Abstract shortened by ProQuest.)