Role of Neuronal Interferon-γ in the Development of Myelopathy in Rats Infected with Human T-Cell Leukemia Virus Type 1
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.051225
ISSN1525-2191
AutoresYukiko Miyatake, Hitoshi Ikeda, Akihiro Ishizu, Tomohisa Baba, Toru Ichihashi, Akira Suzuki, Utano Tomaru, Masanori Kasahara, Takashi Yoshiki,
Tópico(s)Animal Disease Management and Epidemiology
ResumoHuman T-cell leukemia virus type 1 (HTLV-1) is the causative agent of not only adult T-cell leukemia but also HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Among the rat strains infected with HTLV-1, chronic progressive myelopathy, named HAM rat disease, occurs exclusively in WKAH rats. In the present study, we found that HTLV-1 infection induces interferon (IFN)-γ production in the spinal cords of HAM-resistant strains but not in those of WKAH rats. Neurons were the major cells that produced IFN-γ in HTLV-1-infected, HAM-resistant strains. Administration of IFN-γ suppressed expression of pX, the gene critically involved in the onset of HAM rat disease, in an HTLV-1-immortalized rat T-cell line, indicating that IFN-γ protects against the development of HAM rat disease. The inability of WKAH spinal cord neurons to produce IFN-γ after infection appeared to stem from defects in signaling through the interleukin (IL)-12 receptor. Specifically, WKAH-derived spinal cord cells were unable to up-regulate the IL-12 receptor β2 gene in response to IL-12 stimulation. We suggest that the failure of spinal cord neurons to produce IFN-γ through the IL-12 pathway is involved in the development of HAM rat disease. Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of not only adult T-cell leukemia but also HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Among the rat strains infected with HTLV-1, chronic progressive myelopathy, named HAM rat disease, occurs exclusively in WKAH rats. In the present study, we found that HTLV-1 infection induces interferon (IFN)-γ production in the spinal cords of HAM-resistant strains but not in those of WKAH rats. Neurons were the major cells that produced IFN-γ in HTLV-1-infected, HAM-resistant strains. Administration of IFN-γ suppressed expression of pX, the gene critically involved in the onset of HAM rat disease, in an HTLV-1-immortalized rat T-cell line, indicating that IFN-γ protects against the development of HAM rat disease. The inability of WKAH spinal cord neurons to produce IFN-γ after infection appeared to stem from defects in signaling through the interleukin (IL)-12 receptor. Specifically, WKAH-derived spinal cord cells were unable to up-regulate the IL-12 receptor β2 gene in response to IL-12 stimulation. We suggest that the failure of spinal cord neurons to produce IFN-γ through the IL-12 pathway is involved in the development of HAM rat disease. Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL)1Poiesz BJ Ruscetti FW Gazdar AF Bunn PA Minna JD Gallo RC Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma.Proc Natl Acad Sci USA. 1980; 77: 7415-7419Crossref PubMed Scopus (3953) Google Scholar, 2Yoshida M Miyoshi I Hinuma Y Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease.Proc Natl Acad Sci USA. 1982; 79: 2031-2035Crossref PubMed Scopus (1715) Google Scholar and so-called HTLV-1-associated diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP),3Gessain A Barin F Vernant JC Gout O Maurs L Calender A de Thé G Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis.Lancet. 1985; 2: 407-410Abstract PubMed Scopus (2419) Google Scholar, 4Osame M Usuku K Izumo S Ijichi N Amitani H Igata A Matsumoto M Tara M HTLV-I associated myelopathy, a new clinical entity.Lancet. 1986; 1: 1031-1032Abstract PubMed Scopus (1922) Google Scholar HTLV-1 uveitis (HU),5Mochizuki M Watanabe T Yamaguchi K Takatsuki K Yoshimura K Shirao M Nakashima S Mori S Araki S Miyata N HTLV-I uveitis: a distinct clinical entity caused by HTLV-I.Jpn J Cancer Res. 1992; 83: 236-239Crossref PubMed Scopus (274) Google Scholar HTLV-1-associated arthropathy (HAAP),6Nishioka K Maruyama I Sato K Kitajima I Nakajima Y Osame M Chronic inflammatory arthropathy associated with HTLV-I.Lancet. 1989; 1: 441Abstract PubMed Scopus (350) Google Scholar T-cell alveolitis,7Sugimoto M Nakashima H Watanabe S Uyama E Tanaka F Ando M Araki S Kawasaki S T-lymphocyte alveolitis in HTLV-I-associated myelopathy.Lancet. 1987; 2: 1220Abstract PubMed Scopus (211) Google Scholar Sjögren's syndrome,8Vernant JC Buisson G Magdeleine J De Thore J Jouannelle A Neisson-Vernant C Monplaisir N T-lymphocyte alveolitis, tropical spastic paresis, and Sjögren syndrome.Lancet. 1988; 1: 177Abstract PubMed Scopus (183) Google Scholar polymyositis,9Morgan OS Rodgers-Johnson P Mora C Char G HTLV-1 and polymyositis in Jamaica.Lancet. 1989; 2: 1184-1187Abstract PubMed Scopus (293) Google Scholar and infective dermatitis.10LaGrenade L Hanchard B Fletcher V Cranston B Blattner W Infective dermatitis of Jamaican children: a marker for HTLV-I infection.Lancet. 1990; 336: 1345-1347Abstract PubMed Scopus (365) Google Scholar Only a small proportion (<5%) of HTLV-1-infected individuals develop ATL or HTLV-1-associated diseases, whereas more than 95% of carriers remain asymptomatic for life.11Hollsberg P Hafler DA Seminars in medicine of the Beth Israel Hospital, Boston. Pathogenesis of diseases induced by human lymphotropic virus type I infection.N Engl J Med. 1993; 328: 1173-1182Crossref PubMed Scopus (293) Google Scholar Little is known about the factors that govern susceptibility to diseases caused by HTLV-1. We previously established a rat model of HAM/TSP in which chronic progressive myelopathy with paraparesis of lower limbs occurred in WKAH rats 15 to 22 months after HTLV-1 infection.12Ishiguro N Abe M Seto K Sakurai H Ikeda H Wakisaka A Togashi T Tateno M Yoshiki T A rat model of human T lymphocyte virus type I (HTLV-I) infection. 1. Humoral antibody response, provirus integration, and HTLV-I-associated myelopathy/tropical spastic paraparesis-like myelopathy in seronegative HTLV-I carrier rats.J Exp Med. 1992; 176: 981-989Crossref PubMed Scopus (119) Google Scholar Although the provirus was detected in the systemic organs of all HTLV-1-infected strains examined, myelopathy, hereafter referred to as HAM rat disease, occurred exclusively in WKAH rats. Histopathological alterations were limited to the thoracic spinal cord in HAM rat disease. The most crucial finding was apoptotic cell death of oligodendrocytes in the anterior and lateral funiculi of the upper thoracic cord, which became manifest 7 months after inoculation with HTLV-1.13Seto K Abe M Ohya O Itakura O Ishiguro N Ikeda H Wakisaka A Yoshiki T A rat model of HTLV-I infection: development of chronic progressive myeloneuropathy in seropositive WKAH rats and related apoptosis.Acta Neuropathol (Berl). 1995; 89: 483-490Crossref PubMed Scopus (29) Google Scholar Subsequently, demyelination occurred with infiltration of activated macrophages, and at the end stage of the disease, proliferation of astrocytes was observed in the affected region.14Ohya O Ikeda H Tomaru U Yamashita I Kasai T Morita K Wakisaka A Yoshiki T Human T-lymphocyte virus type I (HTLV-I)-induced myeloneuropathy in rats: oligodendrocytes undergo apoptosis in the presence of HTLV-I.APMIS. 2000; 108: 459-466Crossref PubMed Scopus (13) Google Scholar Interestingly, lymphocytic infiltration into the spinal cord, which is characteristic of human HAM/TSP,15Jacobson S Immunopathogenesis of human T cell lymphotropic virus type I-associated neurologic disease.J Infect Dis. 2002; 186: S187-S192Crossref PubMed Scopus (124) Google Scholar was absent throughout the disease process in the HAM rat model. Although the significance of this finding is not clear, lymphocytic infiltration in human HAM/TSP may represents a cellular response to tissue damage. The HTLV-1 provirus, which predominantly localizes in microglia and macrophages, becomes detectable in the spinal cord of both HAM-resistant and -susceptible rats 3 months after infection.16Kasai T Ikeda H Tomaru U Yamashita I Ohya O Morita K Wakisaka A Matsuoka E Moritoyo T Hashimoto K Higuchi I Izumo S Osame M Yoshiki T A rat model of human T lymphocyte virus type I (HTLV-I) infection: in situ detection of HTLV-I provirus DNA in microglia/macrophages in affected spinal cords of rats with HTLV-I-induced chronic progressive myeloneuropathy.Acta Neuropathol (Berl). 1999; 97: 107-112Crossref PubMed Scopus (26) Google Scholar Selective expression of the HTLV-1 pX gene peaks 7 months after infection, accompanied by an increase in tumor necrosis factor-α levels in the spinal cord and down-regulation of the anti-apoptotic bcl-2 gene in oligodendrocytes.17Tomaru U Ikeda H Ohya O Abe M Kasai T Yamasita I Morita K Wakisaka A Yoshiki T Human T lymphocyte virus type I-induced myeloneuropathy in rats: implication of local activation of the pX and tumor necrosis factor-alpha genes in pathogenesis.J Infect Dis. 1996; 174: 318-323Crossref PubMed Scopus (27) Google Scholar, 18Jiang X Ikeda H Tomaru U Morita K Tanaka Y Yoshiki T A rat model for human T lymphocyte virus type I-associated myeloneuropathy. Down-regulation of bcl-2 expression and increase in sensitivity to TNF-alpha of the spinal oligodendrocytes.J Neuroimmunol. 2000; 106: 105-113Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Thus, we reasoned that the most crucial molecular events occurred ∼7 months after HTLV-1 infection in our rat model. The proinflammatory cytokine interferon (IFN)-γ, secreted from activated T and NK cells, increases MHC class I and II expression on a wide variety of cells and then induces a Th1-type immune response. Until recently, IFN-γ had been considered a deleterious factor for central nervous system (CNS) disorders such as multiple sclerosis and experimental autoimmune encephalomyelitis.19Panitch HS Hirsch RL Haley AS Johnson KP Exacerbations of multiple sclerosis in patients treated with gamma interferon.Lancet. 1987; 1: 893-895Abstract PubMed Scopus (640) Google Scholar, 20Renno T Taupin V Bourbonniere L Verge G Tran E De Simone R Krakowski M Rodriguez M Peterson A Owens T Interferon-gamma in progression to chronic demyelination and neurological deficit following acute EAE.Mol Cell Neurosci. 1998; 12: 376-389Crossref PubMed Scopus (96) Google Scholar However, several lines of evidence indicate that, in some instances, IFN-γ exerts protective effects against CNS disorders. First, inactivation of the IFN-γ gene by gene knockout converts an otherwise experimental autoimmune encephalomyelitis-resistant mouse strain to experimental autoimmune encephalomyelitis-susceptible.21Krakowski M Owens T Interferon-gamma confers resistance to experimental allergic encephalomyelitis.Eur J Immunol. 1996; 26: 1641-1646Crossref PubMed Scopus (418) Google Scholar Second, a low level of IFN-γ expression in the CNS plays a protective role in cuprizone-induced demyelination.22Gao X Gillig TA Ye P D'Ercole AJ Matsushima GK Popko B Interferon-gamma protects against cuprizone-induced demyelination.Mol Cell Neurosci. 2000; 16: 338-349Crossref PubMed Scopus (71) Google Scholar Third, BALB/c mice treated with an anti-IFN-γ antibody become susceptible to measles virus encephalitis, and viral clearance from the CNS is impaired.23Finke D Brinckmann UG ter Meulen V Liebert UG Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.J Virol. 1995; 69: 5469-5474PubMed Google Scholar Fourth, treatment with IFN-γ results in inhibition of viral replication in primary cultured nerve cells infected with measles virus.24Patterson CE Lawrence DM Echols LA Rall GF Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent.J Virol. 2002; 76: 4497-4506Crossref PubMed Scopus (126) Google Scholar Fifth, IFN-γ protects neurons from apoptosis during destructive encephalitis induced by herpes simplex virus type 1.25Geiger KD Nash TC Sawyer S Krahl T Patstone G Reed JC Krajewski S Dalton D Buchmeier MJ Sarvetnick N Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death.Virology. 1997; 238: 189-197Crossref PubMed Scopus (105) Google Scholar In these reports,21Krakowski M Owens T Interferon-gamma confers resistance to experimental allergic encephalomyelitis.Eur J Immunol. 1996; 26: 1641-1646Crossref PubMed Scopus (418) Google Scholar, 22Gao X Gillig TA Ye P D'Ercole AJ Matsushima GK Popko B Interferon-gamma protects against cuprizone-induced demyelination.Mol Cell Neurosci. 2000; 16: 338-349Crossref PubMed Scopus (71) Google Scholar, 23Finke D Brinckmann UG ter Meulen V Liebert UG Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.J Virol. 1995; 69: 5469-5474PubMed Google Scholar, 24Patterson CE Lawrence DM Echols LA Rall GF Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent.J Virol. 2002; 76: 4497-4506Crossref PubMed Scopus (126) Google Scholar, 25Geiger KD Nash TC Sawyer S Krahl T Patstone G Reed JC Krajewski S Dalton D Buchmeier MJ Sarvetnick N Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death.Virology. 1997; 238: 189-197Crossref PubMed Scopus (105) Google Scholar the authors assumed that IFN-γ was derived from mononuclear cells, including T and NK cells, infiltrating into the CNS. We show here that IFN-γ levels in the spinal cord are significantly increased in HAM-resistant ACI and LEW rats 7 months after HTLV-1 infection, whereas no such increase occurs in HAM-susceptible WKAH rats. Infiltration of mononuclear cells was never seen in the CNS of HTLV-1-infected rats, indicating that IFN-γ was produced by resident cells of the CNS. By confocal laser-scanning microscopy, we identified IFN-γ-producing cells in the spinal cord of HAM-resistant rats as neurons. We suggest that IFN-γ produced by neurons in response to HTLV-1 infection has a protective role against the development of myelopathy. Inbred ACI, LEW, and WKAH rats were obtained from the Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine. HTLV-1 infection was achieved as described.12Ishiguro N Abe M Seto K Sakurai H Ikeda H Wakisaka A Togashi T Tateno M Yoshiki T A rat model of human T lymphocyte virus type I (HTLV-I) infection. 1. Humoral antibody response, provirus integration, and HTLV-I-associated myelopathy/tropical spastic paraparesis-like myelopathy in seronegative HTLV-I carrier rats.J Exp Med. 1992; 176: 981-989Crossref PubMed Scopus (119) Google Scholar Briefly, HTLV-1-immortalized MT-226Miyoshi I Kubonishi I Yoshimoto S Akagi T Ohtsuki Y Shiraishi Y Nagata K Hinuma Y Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells.Nature. 1981; 294: 770-771Crossref PubMed Scopus (1145) Google Scholar was injected into the peritoneal cavity of newborn rats (1 × 107 cells/rat). All HTLV-1-infected rats were maintained in the P3 room. At least three rats were used in each experiment. All rats used in this study were anesthetized with sodium pentobarbital and then intravascularly perfused with ice-cold saline. All animal experiments were done in accordance with the Guide for Care and Use of Laboratory Animals in Hokkaido University Graduate School of Medicine. After perfusion with ice-cold saline, the spinal cord, cerebrum, and spleen were harvested, flash-frozen in liquid nitrogen, and served as samples for mRNA extraction. Microglia- and neuron-rich populations were prepared from the spinal cord as follows: the harvested spinal cord was dissected and then incubated in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 0.25% collagenase (Worthington Biochemical Corp., Freehold, NJ) and 700 U DNase I (Takara, Otsu, Japan) for 30 minutes at 37°C. Microglia-rich populations were separated from the solution by Percoll-gradient centrifugation as described by Tomaru and colleagues17Tomaru U Ikeda H Ohya O Abe M Kasai T Yamasita I Morita K Wakisaka A Yoshiki T Human T lymphocyte virus type I-induced myeloneuropathy in rats: implication of local activation of the pX and tumor necrosis factor-alpha genes in pathogenesis.J Infect Dis. 1996; 174: 318-323Crossref PubMed Scopus (27) Google Scholar and Jiang and colleagues.18Jiang X Ikeda H Tomaru U Morita K Tanaka Y Yoshiki T A rat model for human T lymphocyte virus type I-associated myeloneuropathy. Down-regulation of bcl-2 expression and increase in sensitivity to TNF-alpha of the spinal oligodendrocytes.J Neuroimmunol. 2000; 106: 105-113Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar For separation of neuron-rich populations, myelin residues were removed from the solution by centrifugation at 6 × g for 1 minute, and then neurons in the supernatant were collected by centrifugation at 36 × g for 7 minutes. All samples were stored at −80°C until use. Total RNAs were extracted using Isogen (Nippon Gene, Tokyo, Japan) and purified using the RNeasy mini kit (Qiagen, Alameda, CA). The purified total RNAs were reverse-transcribed using the Super Script III first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). Quantitative real-time RT-PCR was done with the cDNAs, SYBR Green I dye (SYBR Green PCR Master Mix; Qiagen), and the primer set for IFN-γ (sense: 5′-GATCCA-GCACAAAGCTGTCA-3′, anti-sense: 5′-GACTCCTTT-TCCGCTTCCTT-3′), interferon regulatory factor 1 (IRF-1) (sense: 5′-TGAAGCTGCAACAGATGAGG-3′, anti-sense: 5′-AGCAAGTATCCCTTGCCATC-3′), IL-12p40 (sense: 5′-AGGTGCGTTCCTCGTAGAGA-3′, anti-sense: 5′-CC-ATTTGCTGCATGATGAAT-3′), IL-12 receptor β1 (IL-12Rβ1) (sense: 5′-AGGTGCAGATTTCCCGTTTA-3′, anti-sense: 5′-CAGCCCTGTTTAAGCCAATG-3′), IL-12 receptor β2 (IL-12Rβ2) (sense: 5′-TGCCACCAATCCACAAACTA-3′, anti-sense: 5′-CCTGCTTCCTAGCACCTTGT-3′), IL-23p19 (sense: 5′-CACCACTGGGAGACTCAACA-3′, anti-sense: 5′-AGGATCTTGGAACGGAGA-AGA-3′), IL-23 receptor (IL-23R) (sense: 5′-TTGATG-AATTGTGCCTCGTT-3′, anti-sense: 5′-GTCTGCGCTG-GGATAGTTTC-3′), IL-27 (sense: 5′-ACTCTGCTTCCT-CGCTACCA-3′, anti-sense: 5′-GGAGATCCAGCCTCA-TTGC-3′), IL-27 receptor (IL-27R, WSX-1) (sense: 5′-AGCCCAGGGATAAAGGTGAC-3′, anti-sense: 5′-AGA-CGGGTCCAGTTGAGCTT-3′), or GAPDH (sense: 5′-ATGGGAGTTGCTGTTGAAGTCA-3′, anti-sense: 5′-CC-GAGGGCCCACTAAAGG-3′). PCR was performed in a two-step reaction (95°C for 30 seconds, 60°C for 30 seconds) for 45 cycles after initial denaturation (95°C, 15 minutes), using the ABI Prism 7000 sequence detector system (Applied Biosystems, Foster City, CA). Relative expression of target genes was analyzed using the ΔΔCT-method.27Bloch G Toma DP Robinson GE Behavioral rhythmicity, age, division of labor and period expression in the honey bee brain.J Biol Rhythms. 2001; 16: 444-456Crossref PubMed Scopus (127) Google Scholar The amount of specific mRNA was quantified at the point where the system detected uptake in the exponential phase of PCR accumulation and normalized to GAPDH mRNA levels. ELISA for rat IFN-γ was performed using a kit (BioSource, Camarillo, CA). In brief, after perfusion with ice-cold saline, harvested spinal cords were homogenized with 1 ml of phosphate-buffered saline (PBS) containing 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml phenylmethyl sulfonyl fluoride. Duplicate samples (100 μl) of spinal cord homogenates were subjected to ELISA according to the manufacturer's instructions. The detection limit of the kit was 13 pg/ml. Saline-perfused spinal cords were obtained from rats 7 months after HTLV-1 infection and from age-matched control rats. Harvested spinal cords were dissected and digested as described above. Cell suspensions were centrifuged, and then the pellet was resuspended in 30% Percoll (Amersham Biosciences, Uppsala, Sweden) diluted with Hanks' balanced salt solution (Invitrogen, Carlsbad, CA). The cell suspension was laid gently on 80% Percoll solution. The gradient solution was centrifuged at 1800 × g for 40 minutes. Cells in the 30% Percoll layer were dissociated, washed, and then plated sparsely on poly-l-lysine-coated dishes in Dulbecco's modified Eagle's medium/Ham's F12 medium (Invitrogen) supplemented with 10% fetal calf serum and 50 ng/ml of nerve growth factor 2.5S (Invitrogen) at 37°C in an atmosphere of 5% CO2. Recombinant rat IFN-γ was purchased from PeproTech EC (London, UK). Recombinant mouse interleukin (IL)-12, previously shown to function in rats,28Pelidou SH Zou LP Deretzi G Nennesmo I Wei L Mix E Van Der Meide PH Zhu J Intranasal administration of recombinant mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats.Scand J Immunol. 2000; 51: 29-35Crossref PubMed Scopus (15) Google Scholar was purchased from R&D Systems (Minneapolis, MN). Cells cultured on poly-l-lysine/laminin-coated glasses for 5 days were fixed with 4% paraformaldehyde for 15 minutes. For intracellular staining, cells were treated with PBS containing 0.1% Triton-X and 0.05% bovine serum albumin for 4 minutes and then fixed with ice-cold 70% methanol for 4 minutes. Nonspecific binding was blocked with PBT (0.05% Tween-20/0.1% bovine serum albumin in PBS) containing 0.1% goat serum for 10 minutes. Primary antibodies used were mouse monoclonal anti-rat IFN-γ (DB1; PBL Biomedical Laboratories, Piscataway, NJ), mouse monoclonal anti-rat CD68 (ED-1; Serotec, Oxford, UK), rabbit polyclonal anti-neurofilament (NF) 150-kd molecule (AB1981; Chemicon International, Temecula, CA), and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (Dakocytomation, Glostrup, Denmark). For double staining, cells were labeled with DB1 and AB1981, DB1 and anti-GFAP, or ED-1 and AB1981 followed by labeling with Alexa Fluor 488-conjugated goat polyclonal antibody to mouse IgG and Alexa Fluor 568-conjugated goat polyclonal antibody to rabbit IgG. Confocal images were acquired with a laser-scanning microscope (MRC-1024; Bio-Rad Laboratories, Hercules, CA). LEW-S1,12Ishiguro N Abe M Seto K Sakurai H Ikeda H Wakisaka A Togashi T Tateno M Yoshiki T A rat model of human T lymphocyte virus type I (HTLV-I) infection. 1. Humoral antibody response, provirus integration, and HTLV-I-associated myelopathy/tropical spastic paraparesis-like myelopathy in seronegative HTLV-I carrier rats.J Exp Med. 1992; 176: 981-989Crossref PubMed Scopus (119) Google Scholar an HTLV-1-immortalized rat T-cell line, was incubated with 100 or 1000 U/ml recombinant rat IFN-γ for 3 hours, and the relative expression of the HTLV-1 pX gene to the structural gag gene was calculated using the quantitative real-time RT-PCR method. Primer sets used were 5′-ATCCCGTGGAGACTCCTCAA-3′ (sense) and 5′-CCAAACACGTAGACTGGGTATCC-3′ (anti-sense) for pX and 5′-CCAATGCAAACAAAGAATGC-3′ (sense) and 5′-AGCCCGCAACATATCTCCTA-3′ (anti-sense) for gag. Genomic DNA was extracted from the tails of ACI, LEW, and WKAH rats using the DNeasy tissue kit (Qiagen). The 5′-flanking region of the IL-12Rβ2 gene (1.8 kb) was amplified by nested PCR (outer primers, 5′-ACCACA-CCTCTTGCCATTTT-3′ and 5′-CGAATCGGAGTACACTGCTG-3′; inner primers, 5′-CCCAGAGGCACTTTAAG-CA-3′ and 5′-ACCGATGGACAATGGGTATC-3′). After gel electrophoresis, the PCR products were purified with Freeze ’N Squeeze DNA gel extraction spin columns (Bio-Rad Laboratories) and subjected to direct sequencing with the CEQ 2000XL DNA analysis system (Beckman-Coulter, Fullerton, CA). Sequences were aligned using the online ClustalW service () and potential binding sites of transcription factors were identified using the Transfec database (). Data were analyzed with either Student's t-test or repeated measures analysis of variance, appropriately. P values less than 0.05 were considered to be significant. In view of the fact that IFN-γ exerts protective effects against CNS disease,21Krakowski M Owens T Interferon-gamma confers resistance to experimental allergic encephalomyelitis.Eur J Immunol. 1996; 26: 1641-1646Crossref PubMed Scopus (418) Google Scholar, 22Gao X Gillig TA Ye P D'Ercole AJ Matsushima GK Popko B Interferon-gamma protects against cuprizone-induced demyelination.Mol Cell Neurosci. 2000; 16: 338-349Crossref PubMed Scopus (71) Google Scholar, 23Finke D Brinckmann UG ter Meulen V Liebert UG Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.J Virol. 1995; 69: 5469-5474PubMed Google Scholar, 24Patterson CE Lawrence DM Echols LA Rall GF Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent.J Virol. 2002; 76: 4497-4506Crossref PubMed Scopus (126) Google Scholar, 25Geiger KD Nash TC Sawyer S Krahl T Patstone G Reed JC Krajewski S Dalton D Buchmeier MJ Sarvetnick N Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death.Virology. 1997; 238: 189-197Crossref PubMed Scopus (105) Google Scholar we compared the expression levels of IFN-γ mRNA between HAM-susceptible and -resistant rats 7 months after inoculation with HTLV-1 (Figure 1). Expression of IFN-γ was quantified by real-time RT-PCR in the spinal cord, cerebrum, and spleen. We used tissue samples obtained 7 months after infection because our previous work indicated that critical molecular events leading to the development of HAM rat disease occurred at this time period.17Tomaru U Ikeda H Ohya O Abe M Kasai T Yamasita I Morita K Wakisaka A Yoshiki T Human T lymphocyte virus type I-induced myeloneuropathy in rats: implication of local activation of the pX and tumor necrosis factor-alpha genes in pathogenesis.J Infect Dis. 1996; 174: 318-323Crossref PubMed Scopus (27) Google Scholar, 18Jiang X Ikeda H Tomaru U Morita K Tanaka Y Yoshiki T A rat model for human T lymphocyte virus type I-associated myeloneuropathy. Down-regulation of bcl-2 expression and increase in sensitivity to TNF-alpha of the spinal oligodendrocytes.J Neuroimmunol. 2000; 106: 105-113Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 29Tomaru U Ikeda H Jiang X Ohya O Yoshiki T Provirus expansion and deregulation of apoptosis-related genes in the spinal cord of a rat model for human T-lymphocyte virus type I-associated myeloneuropathy.J Neurovirol. 2003; 9: 530-538Crossref PubMed Scopus (11) Google Scholar The expression of IFN-γ in the spinal cord, an organ affected in HAM rat disease, was significantly elevated in HAM-resistant ACI and LEW rats compared with age-matched, uninfected controls, whereas the expression levels of IFN-γ in the spinal cord of HAM-susceptible WKAH rats were almost the same as those in uninfected controls (Figure 1A, left). The expression of IFN-γ in the cerebrum, an organ never affected in HAM rat disease, was remarkably increased in infected rats regardless of whether they were HAM-resistant or -susceptible (Figure 1A, right). In spleen cells, the mRNA level of IFN-γ did not change by infection; however, even in the absence of infection, IFN-γ was expressed more abundantly than in the cerebrum of infected rats (data not shown). Increased expression of IFN-γ mRNA was not evident in the spinal cord of HAM-resistant rats 3 months after infection, when the provirus was barely detected (data not shown). To evaluate expression of IFN-γ at the protein level, spinal cords were harvested from infected and age-matched uninfected rats and the tissue extracts subjected to assay using an ELISA kit. Consistent with the results obtained at the mRNA level, the amount of IFN-γ proteins in the spinal cord was increased in HAM-resistant ACI and LEW rats but not in HAM-susceptible WKAH rats when measured 7 months after HTLV-1 infection (Figure 1B). We next examined expression of the IRF-1 gene. IRF-1 is known as a downstream molecule induced by IFN-γ.30Kroger A Koster M Schroeder K Hauser H Mueller PP Activities of IRF-1.J Interferon Cytokine Res. 2002; 22: 5-14Crossref PubMed Scopus (226) Google Scholar Like IFN-γ, expression of IRF-1 was significantly increased in the spinal cord of ACI and LEW rats 7 months after infection, whereas no such increase was seen in the spinal cord of WKAH rats (Figure 1C). Thus, collective evidence clearly indicated that IFN-γ was induced by HTLV-1 infection only in the spinal cords of HAM-resistant strains. IFN-γ was recently shown to have a negative regulatory role against HTLV-1 gene expression.31D'Onofrio C Franzese O Puglianiello A Peci E Lanzilli G Bonmassar E Antiviral activity of individual versus combined treatments with interferon alpha, beta and gamma on early infection with HTLV-I in vitro.Int J Immunopharmacol. 1992; 14: 1069-1079Crossref PubMed Scopus (19) Google Scholar To examine whether IFN-γ can suppress expression of the pX gene, previously shown to play a critical role in the onset of HAM rat disease,17Tomaru U Ikeda H Ohya O Abe M Kasai T Yamasita I Morita K Wakisaka A Yoshiki T Human T lymphocyte virus type I-induced myeloneuropathy in rats: implication of local activation of the pX and tumor necrosis factor-alpha genes in pathogenesis.J Infect Dis. 1996; 174: 318-323Crossref PubMed Scopus (27) Google Scholar, 18Jiang X Ikeda H Tomaru U Morita K Tanaka Y Yoshiki T A rat model for human T lymphocyte virus type I-associated myeloneuropathy. Down-regulation of bcl-2 expression and increase in sensitivity to TNF-alpha of the spinal oligodendrocytes.J Neuroimmunol. 2000; 106: 105-113Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar we treated the HTLV-1-immortalized rat T-cell line LEW-S112Ishiguro N Abe M Seto K Sakurai H Ikeda H Wakisaka A Togashi T Tateno M Yoshiki T A rat model of human T lymphocyte virus type I (HTLV-I) infection. 1. Humoral antibody response, provirus integration, and HTLV-I-associated myelopathy/tropical spastic paraparesis-like myelopathy in seronegative HTLV-I carrier rats.J Exp Med. 1992; 176: 981-989Crossref PubMed Scopus (119) Google Scholar with IFN-γ for 3 hours in vitro
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