Inhibition of Interferon-γ Signaling in Oligodendroglia Delays Coronavirus Clearance without Altering Demyelination
2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.050496
ISSN1525-2191
AutoresJohn Mario González, Cornelia C. Bergmann, Chandran Ramakrishna, David R. Hinton, Roscoe Atkinson, Jason B. Hoskin, Wendy B. Macklin, Stephen A. Stohlman,
Tópico(s)interferon and immune responses
ResumoInfection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-γ (IFN-γ) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-γ receptor on oligodendroglia. Defective IFN-γ signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8+ T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-γ receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient. Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-γ (IFN-γ) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-γ receptor on oligodendroglia. Defective IFN-γ signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8+ T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-γ receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient. The central nervous system (CNS), which is composed of highly specialized cells with limited renewal capacity, is the target of many viral infections.1Tyler KL Gonzalez-Scarano F Viral diseases of the central nervous system.in: Nathanson N Viral pathogenesis. Lippincott-Raven, Philadelphia1996: 837-855Google Scholar, 2Griffin DE Immune responses to RNA-virus infections of the CNS.Nat Rev Immunol. 2003; 3: 493-502Crossref PubMed Scopus (198) Google Scholar Although the host immune response to viral CNS infections is often effective at limiting acute disease, it can also contribute to immunopathology.3Doherty PC Allan JE Lynch F Ceredig R Dissection of an inflammatory process induced by CD8+ T cells.Immunol Today. 1990; 11: 55-59Abstract Full Text PDF PubMed Scopus (141) Google Scholar The definition of immune effector mechanisms that control virus replication within specific subsets of CNS cells has been facilitated by analysis of models differing in cellular tropism. For example, viruses that preferentially infect and/or persist in neurons are generally controlled by noncytolytic effector strategies, including neutralizing antibodies (Ab) and interferon-γ (IFN-γ).2Griffin DE Immune responses to RNA-virus infections of the CNS.Nat Rev Immunol. 2003; 3: 493-502Crossref PubMed Scopus (198) Google Scholar By contrast, acute lymphocytic choriomeningitis virus infection, which results in inflammation of the choroid plexus and meninges, is controlled by CD8+ T cells that use perforin-dependent cytolysis.3Doherty PC Allan JE Lynch F Ceredig R Dissection of an inflammatory process induced by CD8+ T cells.Immunol Today. 1990; 11: 55-59Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 4Kagi D Vignaux F Ledermann B Burki K Depraetere V Nagata S Hengartner H Golstein P Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity.Science. 1994; 265: 528-530Crossref PubMed Scopus (1495) Google Scholar However, during chronic lymphocytic choriomeningitis virus infection in which virus persists predominantly in neurons, cytolytic CD8+ T-cell mechanisms are ineffective with recovery mediated by Ab and cytokines.5Oldstone MB Blount P Southern PJ Lampert PW Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system.Nature. 1986; 321: 239-243Crossref PubMed Scopus (208) Google Scholar, 6Planz O Ehl S Furrer E Horvath E Brundler MA Hengartner H Zinkernagel RM A critical role for neutralizing-antibody-producing B cells, CD4+ T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers.Proc Natl Acad Sci USA. 1997; 94: 6874-6879Crossref PubMed Scopus (190) Google Scholar The neurotropic coronavirus, mouse hepatitis virus (MHV) strain JHM (JHMV), induces an acute encephalomyelitis associated with myelin loss; however, an insufficient immune response results in viral persistence.7Marten NW Stohlman SA Bergmann CC MHV infection of the CNS: mechanisms of immune-mediated control.Viral Immunol. 2001; 14: 1-18Crossref PubMed Scopus (85) Google Scholar, 8Wang FI Hinton DR Gilmore W Trousdale MD Fleming JO Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.Lab Invest. 1992; 66: 744-754PubMed Google Scholar JHMV initially infects ependymal cells, but as infection progresses it becomes polytropic and infects astrocytes, microglia, and macrophages.8Wang FI Hinton DR Gilmore W Trousdale MD Fleming JO Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.Lab Invest. 1992; 66: 744-754PubMed Google Scholar As infection enters the white matter, oligodendroglia are primary targets of JHMV replication.8Wang FI Hinton DR Gilmore W Trousdale MD Fleming JO Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.Lab Invest. 1992; 66: 744-754PubMed Google Scholar A concerted immune response, involving cellular and humoral effector mechanisms, controls acute and chronic JHMV infection.7Marten NW Stohlman SA Bergmann CC MHV infection of the CNS: mechanisms of immune-mediated control.Viral Immunol. 2001; 14: 1-18Crossref PubMed Scopus (85) Google Scholar, 9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar, 10Bergmann CC Parra B Hinton DR Ramakrishna C Dowdell KC Stohlman SA Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells.J Virol. 2004; 78: 1739-1750Crossref PubMed Scopus (89) Google Scholar, 11Ramakrishna C Stohlman SA Atkinson RD Shlomchik MJ Bergmann CC Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells.J Immunol. 2002; 168: 1204-1211PubMed Google Scholar JHMV infection induces CNS recruitment of inflammatory cells comprising components of both the innate and adaptive immune responses. Virus-specific CD4+ and CD8+ T cells are both present within the inflamed CNS.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar However, CD8+ T cells are directly associated with the control of infectious virus.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar, 10Bergmann CC Parra B Hinton DR Ramakrishna C Dowdell KC Stohlman SA Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells.J Virol. 2004; 78: 1739-1750Crossref PubMed Scopus (89) Google Scholar By contrast CD4+ T cells appear to play an auxiliary role by promoting CD8+ T cell expansion and survival12Stohlman SA Bergmann CC Lin MT Cua DJ Hinton DR CTL effector function within the central nervous system requires CD4+ T cells.J Immunol. 1998; 160: 2896-2904PubMed Google Scholar and have been implicated in the physiopathology of myelin loss.13Wu GF Dandekar AA Pewe L Perlman S CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination.J Immunol. 2000; 165: 2278-2286PubMed Google Scholar, 14Liu MT Chen BP Oertel P Buchmeier MJ Armstrong D Hamilton TA Lane TE The T cell chemoattractant IFN-inducible protein 10 is essential in host defense against viral-induced neurologic disease.J Immunol. 2000; 165: 2327-2330PubMed Google Scholar JHMV replication in microglia, macrophages, and astrocytes is controlled via a perforin-dependent mechanism.10Bergmann CC Parra B Hinton DR Ramakrishna C Dowdell KC Stohlman SA Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells.J Virol. 2004; 78: 1739-1750Crossref PubMed Scopus (89) Google Scholar, 15Lin MT Stohlman SA Hinton DR Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis.J Virol. 1997; 71: 383-391PubMed Google Scholar By contrast, IFN-γ, but not perforin, is critical for the control of virus replication in oligodendroglia, the cells that synthesize and maintain CNS myelin.16Parra B Hinton DR Marten NW Bergmann CC Lin MT Yang CS Stohlman SA IFN-gamma is required for viral clearance from central nervous system oligodendroglia.J Immunol. 1999; 162: 1641-1647PubMed Google Scholar Whether a noncytolytic mechanism, ie, IFN-γ, directly controls JHMV replication in oligodendroglia and how signaling via this mediator impacts myelin loss are unknown. Virally induced demyelination has also been suggested to be the result of virus-induced oligodendroglial dysfunction or death.17Richardson-Burns SM Kleinschmidt-DeMasters BK DeBiasi RL Tyler KL Progressive multifocal leukoencephalopathy and apoptosis of infected oligodendrocytes in the central nervous system of patients with and without AIDS.Arch Neurol. 2002; 59: 1930-1936Crossref PubMed Scopus (55) Google Scholar, 18Barac-Latas V Suchanek G Breitschopf H Stuehler A Wege H Lassmann H Patterns of oligodendrocyte pathology in coronavirus-induced subacute demyelinating encephalomyelitis in the Lewis rat.Glia. 1997; 19: 1-12Crossref PubMed Scopus (52) Google Scholar, 19Schobesberger M Zurbriggen A Doherr MG Weissenbock H Vandevelde M Lassmann H Griot C Demyelination precedes oligodendrocyte loss in canine distemper virus-induced encephalitis.Acta Neuropathol (Berl). 2002; 103: 11-19Crossref PubMed Scopus (21) Google Scholar, 20Lampert PW Sims JK Kniazeff AJ Mechanism of demyelination in JHM virus encephalomyelitis. Electron microscopic studies.Acta Neuropathol (Berl). 1973; 24: 76-85Crossref PubMed Scopus (196) Google Scholar However, virus alone is insufficient to mediate myelin destruction following JHMV infection, and both T cells and macrophages are necessary for demyelination.10Bergmann CC Parra B Hinton DR Ramakrishna C Dowdell KC Stohlman SA Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells.J Virol. 2004; 78: 1739-1750Crossref PubMed Scopus (89) Google Scholar, 13Wu GF Dandekar AA Pewe L Perlman S CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination.J Immunol. 2000; 165: 2278-2286PubMed Google Scholar, 21Wang FI Stohlman SA Fleming JO Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated.J Neuroimmunol. 1990; 30: 31-41Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 22Wu GF Perlman S Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus.J Virol. 1999; 73: 8771-8780PubMed Google Scholar To determine the contribution of IFN-γ signaling in oligodendroglia to both immune-mediated control of JHMV replication and the subsequent myelin loss, pathogenesis of JHMV-infected transgenic (Tg) mice expressing a dominant-negative IFN-γ receptor (dnIFN-γR) specifically on oligodendroglia was compared to syngeneic wild-type (Wt) mice. The results demonstrate that IFN-γ signaling in oligodendroglia is required for virus control, eliminating potential secondary effects. Surprisingly, the high rate of infection did not increase oligodendroglial apoptosis or influence myelin loss, despite the presence of highly activated T cells capable of expressing anti-viral effector functions. These data thus indicate that, although CNS virus infection of glial cells associated with T-cell inflammation precipitates demyelination, extensive JHMV infection of oligodendroglia per se is not directly associated with cell death or myelin loss, even in the presence of activated T cells and macrophages. Homozygous Tg mice on the H-2b background expressing a dnIFN-γR1 under control of the proteolipid protein promoter, designated PLP-25/B6, were bred and maintained in the animal facilities of the University of Southern California. The transgene is expressed exclusively on oligodendroglia and severely impairs IFN-γ signaling.23Gonzalez JM Bergmann CC Fuss B Hinton DR Kangas C Macklin WB Stohlman SA Expression of a dominant negative IFN-gamma receptor on mouse oligodendrocytes.Glia. 2005; 51: 22-34Crossref PubMed Scopus (24) Google Scholar Syngeneic Wt C57BL/6 (H-2b) mice were purchased from the National Cancer Institute (Frederick, MD). Mice of both sexes were used at 6 to 7 weeks of age, and no gender-dependent differences were detected. All procedures were performed in compliance with protocols approved by the Keck School of Medicine Institutional Animal Care and Use Committee. Mice were infected with the neutralizing monoclonal antibody (mAb)-derived variant 2.2v-1 of the neurotropic JHMV strain of MHV.24Fleming JO Trousdale MD el-Zaatari FA Stohlman SA Weiner LP Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.J Virol. 1986; 58: 869-875PubMed Google Scholar Virus was propagated in the presence of mAb J.2.2, and titers were determined by plaque assay on monolayers of the delayed brain tumor murine astrocytoma as previously described.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar, 24Fleming JO Trousdale MD el-Zaatari FA Stohlman SA Weiner LP Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.J Virol. 1986; 58: 869-875PubMed Google Scholar Mice were injected in the left brain hemisphere with 30 μl containing 250 plaque forming units of JHMV in endotoxin-free Dulbecco's modified phosphate-buffered saline (PBS). Infected mice were scored daily for clinical signs as follows: 0) no clinical signs or healthy, 1) hunched back, 2) partial hind limbs paralysis, 3) complete hind limbs paralysis, and 4) moribund or death.24Fleming JO Trousdale MD el-Zaatari FA Stohlman SA Weiner LP Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.J Virol. 1986; 58: 869-875PubMed Google Scholar To determine CNS virus replication, brains were dissected in the midsagittal plane, and one-half brain was homogenized in RPMI 1640 medium containing 25 mmol/L HEPES, pH 7.2, using Tenbroeck homogenizers. Cells were recovered by centrifugation at 500 × g for 7 minutes, and supernatants were stored at −70°C until use. Virus titers were determined by plaque assay on delayed brain tumor monolayers as previously described.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar, 12Stohlman SA Bergmann CC Lin MT Cua DJ Hinton DR CTL effector function within the central nervous system requires CD4+ T cells.J Immunol. 1998; 160: 2896-2904PubMed Google Scholar CNS cells were obtained from homogenized brains and separated from myelin by differential centrifugation on Percoll gradients (Amersham Biosciences, Uppsala, Sweden) as previously described.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar Briefly, homogenates were suspended in RPMI containing 25 mmol/L HEPES, pH 7.2, and adjusted to 30% Percoll. Cells were isolated by centrifugation at 800 × g for 25 minutes at 4°C onto a 1-ml 70% Percoll cushion. Cells recovered from the 30/70% interface were washed and counted using trypan blue exclusion. Cells were examined for surface molecule expression by flow cytometry. Nonspecific binding was inhibited by incubation with mouse Fc block CD16/CD32 (Clone 2.4G2) and a mixture of normal goat, human, mouse, and rat serum for 10 minutes on ice. All mAbs were obtained from BD Pharmingen (San Diego, CA) unless otherwise noted. Cells were stained for 30 minutes at 4°C with mAb specific for CD4 (L3T4, clone GK1.5), CD8a (Ly-2, clone 53-6.7), CD19 (1D3), CD45 (Ly-5, clone 30-F11), and NK 1.1 (PK136). Macrophages were identified as F4/80+ (Serotec, Oxford, UK). Cells were washed with PBS containing 1% bovine serum albumin and fixed in 2% paraformaldehyde before analysis using a FACSCalibur and CellQuest Software (BD Biosciences, Mountain View, CA). Intact cells were included according to the side scatter versus forward scatter profiles. At least 1 × 104 cells were acquired in each sample. Virus-specific CD8+ T cells were detected with fluorescein isothiocyanate-labeled anti-CD8 mAb and phycoerythrin-labeled DbS510 tetramers.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar Antigen-specific intracellular IFN-γ production was determined by incubating 1 × 106 CNS-derived cells in 100 μl of RPMI 1640 supplemented with 10% fetal calf serum, 1 μmol/L S510 peptide, representing the H-2Db immunodominant JHMV-specific CD8+ T-cell epitope,9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.J Immunol. 1999; 163: 3379-3387PubMed Google Scholar, 25Castro RF Perlman S CD8+ T-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity.J Virol. 1995; 69: 8127-8131PubMed Google Scholar and 1 μg/ml monensin (BD Pharmingen) for 5 hours at 37°C. Peptide was omitted in control samples. Following surface staining, cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Pharmingen) and stained with a fluorescein isothiocyanate-labeled IFN-γ-specific mAb (clone XMG1.2) as recommended by the supplier (BD Pharmingen). Virus-specific Ab responses were determined by enzyme-linked immunosorbent assay as described previously.16Parra B Hinton DR Marten NW Bergmann CC Lin MT Yang CS Stohlman SA IFN-gamma is required for viral clearance from central nervous system oligodendroglia.J Immunol. 1999; 162: 1641-1647PubMed Google Scholar, 26Tschen SI Bergmann CC Ramakrishna C Morales S Atkinson R Stohlman SA Recruitment kinetics and composition of antibody-secreting cells within the central nervous system following viral encephalomyelitis.J Immunol. 2002; 168: 2922-2929PubMed Google Scholar Briefly, serial twofold serum dilutions were adsorbed onto JHMV-coated plates overnight at 4°C. Virus-specific Abs were detected with biotinylated anti-mouse IgG, anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA), anti-mouse IgG1 or anti-mouse IgG2a (Southern Biotech, Birmingham, AL), streptavidin/peroxidase (Sigma-Aldrich, St. Louis, MO), and 2,2-azino-di(3-ethylbenzthiazoline)sulfonic acid as substrate. Optical densities were determined at 405 nm, and the titers were calculated as reciprocal of the highest dilution that exceeded three standard deviations over mean negative control. Neutralization titers were determined as previously described.26Tschen SI Bergmann CC Ramakrishna C Morales S Atkinson R Stohlman SA Recruitment kinetics and composition of antibody-secreting cells within the central nervous system following viral encephalomyelitis.J Immunol. 2002; 168: 2922-2929PubMed Google Scholar Briefly, following heat inactivation (56°C for 30 minutes), serial twofold serum dilutions were incubated with 200 plaque forming units of JHMV in 96-well tissue culture plates for 90 minutes at 37°C. Delayed brain tumor cells (9 × 104 cell per well) were added before incubation at 37°C and 5% of CO2 for 48 hours.26Tschen SI Bergmann CC Ramakrishna C Morales S Atkinson R Stohlman SA Recruitment kinetics and composition of antibody-secreting cells within the central nervous system following viral encephalomyelitis.J Immunol. 2002; 168: 2922-2929PubMed Google Scholar Brains and spinal cords were removed and either fixed with Clark's solution (75% ethanol and 25% glacial acetic acid) and embedded in paraffin or snap frozen. Serial paraffin or frozen sections from tissues obtained at 5, 7, 10, 14, 16, 18, 21, and 30 days post infected (p.i.) were stained with either hematoxylin and eosin for inflammation or luxol fast blue for demyelination. The relative extent of myelin loss in serial longitudinal sections was determined by demarcating the demyelinated area in luxol fast blue-stained serial sections under a dissecting microscope. Sections were digitized with an Epson 2000 flatbed scanner at 720 dpi, and the percentage area of demyelination was calculated with Optimas image analysis software (Media Cybernectics, Silver Spring, MD) and an LCD drawing slate (Wacom Technology Corp., Vancouver, WA). Viral antigen in oligodendroglia was co-localized in frozen sections of spinal cord obtained from mice perfused with 3% paraformaldehyde in PBS. Oligodendroglia were identified with anti-adenomatus ployposis coli mAb (Ab-7 Oncogene Research Products, Cambridge, MA) and visualized with peroxidase a NovaRED substrate kit (Vector Laboratories, Burlingame, CA). Viral antigen was detected with mAb J.3.3, specific for the viral nucleocapsid protein,10Bergmann CC Parra B Hinton DR Ramakrishna C Dowdell KC Stohlman SA Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells.J Virol. 2004; 78: 1739-1750Crossref PubMed Scopus (89) Google Scholar, 15Lin MT Stohlman SA Hinton DR Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis.J Virol. 1997; 71: 383-391PubMed Google Scholar, 16Parra B Hinton DR Marten NW Bergmann CC Lin MT Yang CS Stohlman SA IFN-gamma is required for viral clearance from central nervous system oligodendroglia.J Immunol. 1999; 162: 1641-1647PubMed Google Scholar and visualized with the peroxidase SG substrate kit (Vector Laboratories). Axonal integrity was examined in paraffin spinal cord sections using anti-phosphoneurofilament mAb SMI-31 (Sternberger Monoclonals Inc., Lutherville, MD), followed by peroxidase Vectastain ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine (Sigma-Aldrich). Infiltrating cells were identified by immunoperoxidase staining of acetone-fixed frozen sections with F4/80 (Serotec), anti-CD8 (Ly-2), and anti-CD4 (L3T4) mAbs (BD Pharmingen). Primary mAbs were detected by immunoperoxidase staining with a biotinylated rabbit anti-rat Ab (Vector Laboratories) and a Vectastain ABC kit and aminoethyl carbazol as chromogen (Vector Laboratories). For identification of viral antigen, paraffin sections were stained with mAb J.3.3 followed by peroxidase Vectastain ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine (Sigma-Aldrich). Viral antigens were quantified manually in serial spinal cord sections and are reported as positive cells/mm2. Data were compared using Mann-Whitney U-test. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling using the ApopTag detection kit (Serological Corp., Norcross, GA). Briefly, serial frozen sections were fixed in 1% paraformaldehyde in PBS, pH 7.4, and postfixed in ethanol-acetic acid (2:1). Endogenous peroxidase activity was quenched with 3% H2O2 in PBS. Slides were incubated 1 hour at 37°C with terminal deoxy-nucleotidyl transferase-digoxigenin-deoxynucleoside triphosphate mixture, and an anti-digoxigenin peroxidase conjugated-Ab and Vector NovaRed as substrate (Vector Laboratories) were used to detect end-labeled DNA fragments. Slides were counterstained with hematoxylin. Numbers of apoptotic cells were quantified manually and reported as positive cells/mm2. Infected Tg and Wt mice were compared to determine how impaired oligodendroglia responses to INF-γ-influenced JHMV pathogenesis. No differences in clinical disease or mortality were detected comparing Wt and Tg mice up to day 30 p.i. (data not shown). Following infection clinical symptoms were initially apparent in both Tg and Wt mice by day 6 p.i. and peaked at days 10 to 11 p.i. Virus replication was examined to determine whether diminished IFN-γ signaling in oligodendroglia altered overall CNS virus replication or clearance. Consistent with initial JHMV replication in ependymal cells, astrocytes, and microglia,8Wang FI Hinton DR Gilmore W Trousdale MD Fleming JO Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination.Lab Invest. 1992; 66: 744-754PubMed Google Scholar similar levels of infectious virus were detected at days 5 and 7 p.i. (Figure 1) Although infectious virus within the CNS began to decline in both groups by day 10 p.i. (Figure 1), immune-mediated clearance was less efficient in Tg mice. Infectious virus was low, but detectable, in the CNS of ∼40% of Wt mice at day 14 p.i. and dropped to undetectable levels in all Wt mice by day 16 p.i. By contrast, all infected Tg mice harbored infectious virus in the CNS at day 14 p.i., and infectious virus was detected in five of six Tg mice at day 16 p.i. Although infectious virus was still recovered from 23% of Tg mice at day 21 p.i., complete CNS clearance was achieved at day 30 p.i. (Figure 1). These data suggest that the decreased ability of oligodendroglia to respond to IFN-γ had a dramatic effect on infectious virus clearance from the CNS, without affecting clinical disease or mortality. The anti-viral Ab response plays no role in controlling acute JHMV infection in Wt mice but is critical in maintaining CNS persistence.11Ramakrishna C Stohlman SA Atkinson RD Shlomchik MJ Bergmann CC Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells.J Immunol. 2002; 168: 1204-1211PubMed Google Scholar, 26Tschen SI Bergmann CC Ramakrishna C Morales S Atkinson R Stohlman SA Recruitment kinetics and composition of antibody-secreting cells within the central nervous system following viral encephalomyelitis.J Immunol. 2002; 168: 2922-2929PubMed Google Scholar, 27Lin MT Hinton DR Marten NW Bergmann CC Stohlman SA Antibody prevents virus reactivation within the central nervous system.J Immunol. 1999; 162: 7358-7368PubMed Google Scholar, 28Ramakrishna C Bergmann CC Atkinson R Stohlman SA Control of central nervous system viral persistence by neutralizing antibody.J Virol. 2003; 77: 4670-4678Crossref PubMed Scopus (54) Google Scholar Anti-viral Ab induction in Tg and Wt mice was compared to ensure that the delay in controlling infectious virus did not correlate with a defect in humoral immunity. No differences in kinetics, magnitude, or isotype of total anti-viral Ab or levels of serum virus neutralizing Ab were detected when comparing Tg and Wt mice (data not shown). CNS recruitment of inflammatory cells was analyzed to determine whether differences in numbers or phenotype correlated with delayed virus clearance in Tg mice. Total numbers of cells recovered from the CNS were similar in both groups, with maximal numbers present at day 10 p.i. (Figure 2). Consistent with the increased cellularity, bone marrow-derived (CD45hi) CNS-infiltrating cells were maximal at day 10 p.i. (Figure 2) and decreased by day 14 p.i. in both groups (Figure 2). Despite the continued presence of infectious virus in the CNS of Tg mice between days 14 and 21 p.i. (Figure 1), no differences in either yields or percentages of CD45hi cells were detected (Figure 2). In addition, CD45hi inflammatory cells remained within the CNS of both groups until day 30 p.i. (Figure 2), consistent with previous data.9Bergmann CC Altman JD Hinton D Stohlman SA Inverted immunodominance and impaired cytolyti
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