Virus-Specific Antibody, in the Absence of T Cells, Mediates Demyelination in Mice Infected with a Neurotropic Coronavirus
2005; Elsevier BV; Volume: 166; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)62301-2
ISSN1525-2191
Autores Tópico(s)Multiple Sclerosis Research Studies
ResumoMice infected with mouse hepatitis virus strain JHM develop an inflammatory demyelinating disease in the central nervous system with many similarities to human multiple sclerosis. The mouse disease is primarily immune-mediated because demyelination is not detected in JHM-infected mice lacking T or B cells but does occur after transfer of JHM-specific T cells. Although less is known about the ability of antibodies to mediate demyelination, the presence of oligoclonally expanded B cells and high concentrations of antibodies (against self or infectious agents) in the central nervous system of many multiple sclerosis patients suggests that antibodies may also contribute to myelin destruction. Here, we show that anti-JHM antibodies, in the absence of T or B cells, caused demyelination in JHM-infected mice. Anti-JHM antibody was detected adjacent to areas of demyelination, consistent with a direct interaction between antibody and infected cells. Demyelination was reduced by 85 to 90% in infected RAG1−/− mice lacking normal expression of activating Fc receptors (FcRγ−/−) and by ∼76% when complement was depleted by treatment with cobra venom factor. These data demonstrate that JHM-specific antibodies are sufficient to cause demyelination and that myelin destruction in the presence of anti-virus antibodies results from a combination of complement- and Fc receptor-dependent mechanisms. Mice infected with mouse hepatitis virus strain JHM develop an inflammatory demyelinating disease in the central nervous system with many similarities to human multiple sclerosis. The mouse disease is primarily immune-mediated because demyelination is not detected in JHM-infected mice lacking T or B cells but does occur after transfer of JHM-specific T cells. Although less is known about the ability of antibodies to mediate demyelination, the presence of oligoclonally expanded B cells and high concentrations of antibodies (against self or infectious agents) in the central nervous system of many multiple sclerosis patients suggests that antibodies may also contribute to myelin destruction. Here, we show that anti-JHM antibodies, in the absence of T or B cells, caused demyelination in JHM-infected mice. Anti-JHM antibody was detected adjacent to areas of demyelination, consistent with a direct interaction between antibody and infected cells. Demyelination was reduced by 85 to 90% in infected RAG1−/− mice lacking normal expression of activating Fc receptors (FcRγ−/−) and by ∼76% when complement was depleted by treatment with cobra venom factor. These data demonstrate that JHM-specific antibodies are sufficient to cause demyelination and that myelin destruction in the presence of anti-virus antibodies results from a combination of complement- and Fc receptor-dependent mechanisms. The human disease multiple sclerosis (MS) is an immune-mediated, chronic inflammatory disease manifested clinically by neurological deficits and histologically by multiple foci of demyelination. T cells are detected in active demyelinating lesions and a critical role for these cells in demyelination has been clearly demonstrated in several animal models of demyelination, including rodents with experimental autoimmune encephalitis (EAE) and mice infected with coronaviruses or Theiler's murine encephalomyelitis virus.1Hemmer B Archelos JJ Hartung HP New concepts in the immunopathogenesis of multiple sclerosis.Nat Rev Neurosci. 2002; 3: 291-301Crossref PubMed Scopus (505) Google Scholar, 2Noseworthy JH Progress in determining the causes and treatment of multiple sclerosis.Nature. 1999; 399: A40-A47Crossref PubMed Scopus (244) Google Scholar, 3Steinman L Assessment of animal models for MS and demyelinating disease in the design of rational therapy.Neuron. 1999; 24: 511-514Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHM) develop acute and chronic demyelinating diseases. We and others4Wu 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, 5Wang F 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, 6Houtman JJ Fleming JO Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM.J Neurovirol. 1996; 2: 101-110Crossref PubMed Scopus (102) Google Scholar have shown that demyelination was not detected in JHM-infected mice lacking T and B cells [either mice with severe combined immunodeficiency or mice lacking functional recombination activating enzyme 1 (RAG1−/−)]. However, adoptive transfer of syngeneic splenocytes from JHM-immune mice resulted in rapid and reproducible demyelination.6Houtman JJ Fleming JO Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM.J Neurovirol. 1996; 2: 101-110Crossref PubMed Scopus (102) Google Scholar, 7Wu 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 Depletion of T cells abrogated demyelination showing that T cells were necessary and B or other splenic cells were not sufficient for demyelination to occur. Either CD8 or CD4 T cells, in the absence of the other subset, were able to mediate demyelination in this model.4Wu 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 In these experiments, T cells were transferred into RAG1−/− mice 4 days after they were immunized with JHM. The innate immune system was activated by JHM infection before T-cell transfer, as shown by up-regulated expression of several proinflammatory cytokines and chemokines, such as tumor necrosis factor-α, MIP-2, CCL7 (MCP-3), CCL4 (MIP-1β), CCL2 (MCP-1), CXCR10 (IP-10), and CCL5 (RANTES) in the central nervous system (CNS).8Haring JS Pewe LL Perlman S Bystander CD8 T cell-mediated demyelination after viral infection of the central nervous system.J Immunol. 2002; 169: 1550-1555PubMed Google Scholar This intense inflammatory milieu is likely critical for the rapid recruitment and activation of T cells to the CNS after adoptive transfer. Less is known about the role of humoral immune factors in MS, but several features suggest that B cells or antibodies are involved in myelin destruction.9Archelos JJ Storch MK Hartung HP The role of B cells and autoantibodies in multiple sclerosis.Ann Neurol. 2000; 47: 694-706Crossref PubMed Scopus (227) Google Scholar Oligoclonal expansion of B cells is observed in the cerebrospinal fluid of patients with MS. Also, high levels of immunoglobulin are detected in the cerebrospinal fluid.10Owens GP Ritchie AM Burgoon MP Williamson RA Corboy JR Gilden DH Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid.J Immunol. 2003; 171: 2725-2733PubMed Google Scholar Some of these cerebrospinal fluid-derived antibodies are directed against myelin proteins and pathogens such as Epstein-Barr virus11Levin LI Munger KL Rubertone MV Peck CA Lennette ET Spiegelman D Ascherio A Multiple sclerosis and Epstein-Barr virus.JAMA. 2003; 289: 1533-1536Crossref PubMed Scopus (127) Google Scholar and varicella-zoster virus.12Burgoon MP Hammack BN Owens GP Maybach AL Eikelenboom MJ Gilden DH Oligoclonal immunoglobulins in cerebrospinal fluid during varicella zoster virus (VZV) vasculopathy are directed against VZV.Ann Neurol. 2003; 54: 459-463Crossref PubMed Scopus (42) Google Scholar In addition, circulating antibodies against myelin proteins are detected in patients with MS13Raine CS Cannella B Hauser SL Genain CP Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: a case for antigen-specific antibody mediation.Ann Neurol. 1999; 46: 144-160Crossref PubMed Scopus (261) Google Scholar, 14Noseworthy JH Lucchinetti C Rodriguez M Weinshenker BG Multiple sclerosis.N Engl J Med. 2000; 343: 938-952Crossref PubMed Scopus (3098) Google Scholar and are a marker for the subsequent development of MS in patients with single episodes of a first neurological event.15Berger T Rubner P Schautzer F Egg R Ulmer H Mayringer I Dilitz E Deisenhammer F Reindl M Antimyelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event.N Engl J Med. 2003; 349: 139-145Crossref PubMed Scopus (556) Google Scholar Furthermore, depositions of IgG and complement have been detected at sites of active demyelination in these patients.16Lucchinetti C Bruck W Parisi J Scheithauer B Rodriguez M Lassmann H Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination.Ann Neurol. 2000; 47: 707-717Crossref PubMed Scopus (2757) Google Scholar Multiple studies using rodent models of EAE also indicate that antibodies may have an important role in the demyelinating process. In mice, rats, and marmosets, treatment with antibody directed against an epitope of myelin oligodendrocyte glycoprotein resulted in the rapid onset of demyelination.17Wekerle H Remembering MOG: autoantibody mediated demyelination in multiple sclerosis?.Nat Med. 1999; 5: 153-154Crossref PubMed Scopus (26) Google Scholar Antibody was detected at sites of myelin destruction.13Raine CS Cannella B Hauser SL Genain CP Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: a case for antigen-specific antibody mediation.Ann Neurol. 1999; 46: 144-160Crossref PubMed Scopus (261) Google Scholar, 18Genain CP Cannella B Hauser SL Raine CS Identification of autoantibodies associated with myelin damage in multiple sclerosis.Nat Med. 1999; 5: 170-175Crossref PubMed Scopus (786) Google Scholar The mechanism of antibody-mediated demyelination is not known with certainty. Several studies showed that complement depletion with cobra venom factor (CVF) resulted in delayed onset of EAE and a reduction in demyelination.19Linington C Morgan BP Scolding NJ Wilkins P Piddlesden S Compston DA The role of complement in the pathogenesis of experimental allergic encephalomyelitis.Brain. 1989; 112: 895-911Crossref PubMed Scopus (145) Google Scholar EAE has been reported to be ameliorated20Nataf S Carroll SL Wetsel RA Szalai AJ Barnum SR Attenuation of experimental autoimmune demyelination in complement-deficient mice.J Immunol. 2000; 165: 5867-5873PubMed Google Scholar or unaffected21Calida DM Constantinescu C Purev E Zhang GX Ventura ES Lavi E Rostami A Cutting edge: C3, a key component of complement activation, is not required for the development of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in mice.J Immunol. 2001; 166: 723-726PubMed Google Scholar in mice deficient in C3 expression. Other studies implicate a role for the terminal components of complement in demyelination, via formation of membrane attack complex (MAC).22Tran GT Hodgkinson SJ Carter N Killingsworth M Spicer ST Hall BM Attenuation of experimental allergic encephalomyelitis in complement component 6-deficient rats is associated with reduced complement C9 deposition, P-selectin expression, and cellular infiltrate in spinal cords.J Immunol. 2002; 168: 4293-4300PubMed Google Scholar, 23Mead RJ Singhrao SK Neal JW Lassmann H Morgan BP The membrane attack complex of complement causes severe demyelination associated with acute axonal injury.J Immunol. 2002; 168: 458-465PubMed Google Scholar MAC has multiple functions, including direct cell lysis and enhancement of phagocytosis. Fc receptors (FcR) that are involved in the interaction of antibodies with effector cells, including macrophages, have also been implicated in antibody-induced demyelination.24Abdul-Majid KB Stefferl A Bourquin C Lassmann H Linington C Olsson T Kleinau S Harris RA Fc receptors are critical for autoimmune inflammatory damage to the central nervous system in experimental autoimmune encephalomyelitis.Scand J Immunol. 2002; 55: 70-81Crossref PubMed Scopus (80) Google Scholar Mice deficient in expression of activating FcR (FcγRI and FcγRIII) develop less disease and demyelination whereas disease is exacerbated in mice lacking expression of the inhibitory FcγRII molecule. These studies suggest that antibody directed against a CNS antigen can mediate demyelination and that this process involves both signaling through the FcR and complement activation. However, these studies were always performed in animals with intact T- and B-cell compartments, making it difficult to determine the precise role of antibody in the demyelinating process. For example, C3a and C5a both increase expression of adhesion molecules and can serve as chemoattractants for macrophages and T cells.25Song WC Sarrias MR Lambris JD Complement and innate immunity.Immunopharmacology. 2000; 49: 187-198Crossref PubMed Scopus (113) Google Scholar C5a is also required for optimal T-cell activation because treatment of mice with a C5a antagonist results in a diminished CD8 T-cell response.26Kim AH Dimitriou ID Holland MC Mastellos D Mueller YM Altman JD Lambris JD Katsikis PD Complement C5a receptor is essential for the optimal generation of antiviral CD8+ T cell responses.J Immunol. 2004; 173: 2524-2529PubMed Google Scholar Therefore, administration of anti-myelin oligodendrocyte glycoprotein antibody may initiate the demyelinating process, but demyelination may be primarily T cell-mediated, with complement activation serving to enhance the T-cell response. Our previous results showed that an inflammatory state existed in the CNS of JHM-infected RAG1−/− mice but that demyelination was not detected in the absence of transferred T cells. As a consequence, this model is excellent for determining whether transfer of anti-virus antibody, in the absence of any transferred T or B cells, is sufficient to mediate demyelination. Pathogen-free C57BL/6 (B6) mice were purchased from the National Cancer Institute (Bethesda, MD). Specific pathogen-free RAG1−/− mice were purchased from Jackson Laboratories (Bar Harbor, ME) and bred at the University of Iowa (Iowa City, IA). FcRγ−/− mice (B6x129; a generous gift from Dr. Timothy Ratliff, University of Iowa) were bred with RAG1−/− mice to generate FcRγ−/−RAG1−/− mice. As a control group for experiments using FcRγ−/−RAG1−/− mice, age-matched littermates that were heterozygous for the FcRγ−/− were used (FcRγ+/−RAG1−/−). Six- to eight-week-old animals were used for all studies. All animal studies were approved by the University of Iowa Animal Care and Use Committee. RAG1−/− mice were inoculated intracerebrally with 1000 plaque forming units (PFU) of the attenuated J2.2-V-1 strain of JHM (a generous gift from Dr. J. Fleming, University of Wisconsin, Madison, WI) in 30 μl of Dulbecco's modified Eagle's medium. Four days later, antibody was delivered intraperitoneally in 500 μl of sterile phosphate-buffered saline (PBS). Rabbit polyclonal anti-JHM antibody was prepared as described previously.27Perlman S Schelper R Bolger E Ries D Late onset, symptomatic, demyelinating encephalomyelitis in mice infected with MHV-JHM in the presence of maternal antibody.Microbiol Pathog. 1987; 2: 185-194Crossref PubMed Scopus (87) Google Scholar The neutralizing titer (NT) of this antibody was 1:1862 plaque reduction units (PRD)/ml. Monoclonal antibodies (mAbs) used in these experiments include: 5B19.2 [anti-surface (S) glycoprotein; IgG1, NT: 1:2410 PRD/mg], 5A13.5 (anti-S; IgG2a, NT: 1:6230 PRD/mg), 5B188.2 [anti-nucleocapsid (N) protein, IgG2a, NT <1:100], 5B93.9 (anti-S; IgA, NT: <1:10028), and 5B11.5 [anti-transmembrane (M) protein, IgG2a, NT: <1:10029] all provided by Dr. M. Buchmeier, Scripps Research Institute, La Jolla, CA, and irrelevant antibody [anti-keyhole limpet hemocyanin (KLH), IgG1; Sigma, St. Louis, MO]. In preliminary experiments, we determined that 5 μg of mAb 5B19.2 administered intraperitoneally 4 days after infection resulted in maximum demyelination. Consequently, 5 μg of mAb per mouse were administered except where noted below. In some experiments, adoptive transfer of splenocytes from B6 mice previously immunized intraperitoneally with JHM to infected RAG1−/− mice was performed as previously described.7Wu 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 Virus was titered as described previously.27Perlman S Schelper R Bolger E Ries D Late onset, symptomatic, demyelinating encephalomyelitis in mice infected with MHV-JHM in the presence of maternal antibody.Microbiol Pathog. 1987; 2: 185-194Crossref PubMed Scopus (87) Google Scholar Briefly, animals were sacrificed and perfused with sterile PBS. The tissues were homogenized in 6 ml of PBS. After two cycles of freeze-thawing, supernatants were obtained by centrifugation at 800 × g for 5 minutes at 4°C. Supernatants were assayed for virus by plaque assay on HeLa cells expressing the cellular receptor for mouse hepatitis virus (HeLa-MHVR). Viral titers are represented as PFU/g of brain ± SEM. To deplete complement in vivo, CVF isolated from Naja naja kaaouthia (0.5 μg/kg; Calbiochem, La Jolla, CA) was administered intraperitoneally in 200 μl of PBS beginning 1 day before antibody injection (3 days after infection with JHM) and every 3 days thereafter until mice were harvested. Control mice received PBS alone. Mice were perfused with PBS. Spinal cords were fixed in 10% zinc formalin (Labesco, Solon, OH) overnight and embedded in paraffin. Eight-μm longitudinal sections of entire spinal cords were prepared and stained with Luxol Fast Blue. For quantification of demyelination, spinal cord sections were photographed and digitalized using a Leitz DMB 100 microscope (Leica Microscope, Wetzler, Germany) and an Optronics digital camera (Optronics, Goleta, CA). Demyelination was quantified using ImageJ software (National Institutes of Health, Bethesda, MD) as described previously.4Wu 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 Data are represented as the fraction of the total white matter of the spinal cord showing demyelination. Average demyelination per group is represented as arithmetic mean value ± SEM. Eight-μm sections were prepared from zinc formalin-fixed spinal cords and permeabilized with 0.1% of Triton X-100. Sections were treated with CAS Block (Zymed Laboratories, South San Francisco, CA), before incubation with primary antibody to detect macrophages/microglia [rat anti-F4/80 (CI:A3-1; Serotec, Oxford, England), 1:200] or viral antigen [mouse anti-N (5B188.2), 1:10,000] overnight at 4°C. After washing, sections were incubated with biotinylated goat anti-rat (F4/80) or anti-mouse (N) antibody (Jackson ImmunoResearch, West Grove, PA) diluted 1:100 for 1 hour at room temperature, followed by avidin-horseradish peroxidase. Sections were developed with 3,3′-diaminobenzidine (Sigma Chemical). For immunofluorescence assays, sections were incubated overnight with a cocktail of mouse anti-phosphoneurofilament mAbs (SMI-312; Sternberger Monoclonals, Lutherville, MD) or biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch). After washing, samples were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson ImmunoResearch) or streptavidin-Cy3 (Jackson ImmunoResearch) and examined by fluorescent microscopy. A two-tailed unpaired Student's t-test was used to analyze differences in mean values between groups. Initially, we investigated whether polyclonal rabbit anti-JHM antibody could mediate demyelination in JHM-infected RAG1−/− mice. JHM-infected RAG1−/− mice survive for 13 to 18 days after inoculation but do not develop significant levels of demyelination.7Wu 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 In preliminary experiments, we determined that 50 μl of a rabbit polyclonal antibody delivered intraperitoneally at 4 days after infection resulted in a significant amount of demyelination whereas administration of normal rabbit sera did not (Table 1 and Figure 1, A and D). Anti-JHM but not normal rabbit sera prolonged survival. Because mice receiving normal sera developed symptoms of severe encephalitis (hunching, lethargy, ruffled fur) by 14 to 16 days after infection, we euthanized all mice at this time. We detected 12.4 ± 2.0% demyelination in mice receiving anti-JHM sera, with 1.9 ± 1.0% detected in recipients of normal rabbit sera (P < 0.002). Demyelination was accompanied by infiltration of macrophages/microglia into the white matter of the spinal cord (Figure 1, B and E). We detected viral antigen throughout the spinal cord in all mice, except in areas of demyelination (Figure 1, C and F). A similar lack of virus antigen in areas of myelin destruction was also observed in other models of JHM-induced demyelination,30Stohlman SA Hinton DR Viral induced demyelination.Brain Pathol. 2001; 11: 92-106Crossref PubMed Scopus (143) Google Scholar, 31Dandekar AA Perlman S Virus-induced demyelination in nude mice is mediated by gamma delta T cells.Am J Pathol. 2002; 161: 1255-1263Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar suggesting that demyelination was a consequence of virus clearance. To begin to understand the mechanism of antibody-induced demyelination, we examined spinal cord sections from antibody-treated, infected RAG1−/− mice for antibody deposition. We were unable to detect antibody in the CNS of mice that received 50 μl of antibody. However, demyelination was also detected in mice that received 500 μl of rabbit anti-JHM antibody and we were able to detect deposited antibody adjacent to sites of demyelination but not elsewhere in these mice (Figure 1; G to I).Table 1Demyelination and Viral Titers in JHM-Infected RAG1−/− Mice after Antibody TransferAntibody (isotype)TargetAmountNumber of mice% Demyelination (mean ± SEM)Viral titer (log10PFU/g brain)*Values are shown as mean ± SEM.Rabbit anti-JHMJHM50 μl512.4 ± 2.0†Demyelination was significantly higher and virus titers lower in mice that received anti-JHM rabbit sera (P < 0.002 and P < 0.05, respectively).5.3 ± 0.1‡Demyelination was significantly higher and virus titers lower in mice that received anti-JHM rabbit sera (P < 0.002 and P < 0.05, respectively).Normal rabbitNone50 μl51.9 ± 1.05.9 ± 0.25B19.2 (IgG1)S5 μg67.7 ± 2.0§Demyelination was significantly different in recipients of mAb 5B19.2 (P < 0.002), mAb 5A13.5 (P < 0.03), or mAb 5B11.5 (P < 0.02) when compared to recipients of anti-KLH mAb.5.3 ± 0.25A13.5 (IgG2a)S5 μg66.0 ± 2.4¶Demyelination was significantly different in recipients of mAb 5B19.2 (P < 0.002), mAb 5A13.5 (P < 0.03), or mAb 5B11.5 (P < 0.02) when compared to recipients of anti-KLH mAb.5.9 ± 0.15B93.9 (IgA)S5 μg30.7 ± 0.36.3 ± 0.15B11.5 (IgG2a)M5 μg57.7 ± 3.4∥Demyelination was significantly different in recipients of mAb 5B19.2 (P < 0.002), mAb 5A13.5 (P < 0.03), or mAb 5B11.5 (P < 0.02) when compared to recipients of anti-KLH mAb.5.7 ± 0.25B188.2 (IgG2a)N5 μg50.9 ± 0.75.5 ± 0.230 μg92.1 ± 1.25.7 ± 0.2Mouse Ig (IgG1)KLH5 μg90.8 ± 0.55.5 ± 0.130 μg50.9 ± 0.65.5 ± 0.2PBSNone060.5 ± 0.25.4 ± 0.1Mice received either rabbit sera or mouse mAbs at 4 days after infection and were harvested at 14 to 16 days after infection.* Values are shown as mean ± SEM.†‡ Demyelination was significantly higher and virus titers lower in mice that received anti-JHM rabbit sera (P < 0.002 and P < 0.05, respectively).§¶∥ Demyelination was significantly different in recipients of mAb 5B19.2 (P < 0.002), mAb 5A13.5 (P < 0.03), or mAb 5B11.5 (P < 0.02) when compared to recipients of anti-KLH mAb. Open table in a new tab Mice received either rabbit sera or mouse mAbs at 4 days after infection and were harvested at 14 to 16 days after infection. If antibody were to function directly in the CNS, it would likely recognize a viral protein on the surface of infected cells. Next, we tested a panel of five anti-JHM mAbs, recognizing the S, N, or M proteins, for their ability to mediate demyelination in infected RAG1−/− mice (Table 1). The S protein is the most abundant JHM protein present on the infected cell surface and would be predicted to be the target for anti-JHM demyelinating antibody. The N and M proteins are predicted to lack surface expression. However, previous reports suggested that N- and M-specific antibody epitopes are expressed on the cell surface.32Kyuwa S Cohen M Nelson G Tahara SM Stohlman SA Modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis.J Virol. 1994; 68: 6815-6819PubMed Google Scholar, 33Fleming JO Shubin RA Sussman MA Casteel N Stohlman SA Monoclonal antibodies to the matrix (E1) glycoprotein of mouse hepatitis virus protect mice from encephalitis.Virology. 1989; 168: 162-167Crossref PubMed Scopus (100) Google Scholar Administration of mAb 5B19.2 (anti-S) protected infected RAG1−/− mice partially from acute encephalitis and prolonged survival from 14 to 16 days after infection in the absence of anti-JHM antibody to 18 to 20 days after infection. mAb 5B19.2-treated mice exhibited clinical signs of demyelination including wobbly gait and limb paresis starting at 12 to 14 days after infection and showed histological evidence of demyelination [7.7 ± 2.0% at 15 days after infection (Figure 1, Figure 2) and 20.5 ± 1.5% at 18 to 20 days after infection]. We determined whether the amount of mAb 19.2 present in the serum of RAG1−/− mice was similar to levels of anti-JHM antibody detected in JHM-infected B6 mice undergoing demyelination. Serum NTs, which primarily measure the anti-S antibody response, were similar between the two groups: 1:32 (range, 1:19 to 1:45; n = 2) at 2 days after mAb 19.2 transfer versus 1:86.2 ± 42.7 (n = 4) at 21 days after infection in JHM-infected B6 mice. Mice that received irrelevant antibody (anti-KLH) or PBS developed only insignificant amounts of demyelination (Table 1). By comparison, JHM-infected RAG1−/− recipients of JHM-immune splenocytes exhibited similar amounts of demyelination, although the kinetics of myelin destruction was more rapid, with demyelination detected by 7 to 8 days after infection (Figure 2).4Wu 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, 7Wu 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 Demyelination in mAb 5B19.2-treated mice did not reflect differences in virus clearance because similar amounts of virus were detected in recipients of anti-JHM mAb, PBS, or irrelevant antibody (Table 1). This inability to detect a difference in virus titer, in the presence of prolonged survival, may reflect a transient reduction in virus titer at early times after infection. As expected, histological examination of sections from mAb 5B19.2-treated mice revealed the presence of activated macrophages/microglia adjacent to demyelinated areas (data not shown). To quantify the number of macrophages/microglia after antibody transfer, we counted the number of macrophages/microglia in the gray and white matter of mid-sagittal sections of spinal cords from three mice that received either mAb 5B19.2 or irrelevant antibody. In these experiments, all of the F4/80+ cells in 1.25-mm-wide cross-sections at 10 levels within spinal cords were counted. The number of macrophages/microglia present per cross-sectional area increased threefold after infusion of anti-JHM antibody compared to those that received control antibody (66.7 ± 3.7 versus 23.3 ± 2.9, P < 0.001). These additional infiltrating F4/80+ cells were localized entirely to the white matter. In other models, JHM-induced demyelination was primary and not secondary to axonal damage.30Stohlman SA Hinton DR Viral induced demyelination.Brain Pathol. 2001; 11: 92-106Crossref PubMed Scopus (143) Google Scholar Similarly, demyelination induced by anti-JHM antibody was primary, because intact axons were detected traversing areas of demyelination (Figure 1, K and L). To examine whether the induction of demyelination after antibody transfer was unique to mAb 5B19.2, four additional mAbs were analyzed: mAb 5A13.5 (anti-S), mAb 5B93.9 (anti-S), mAb 5B11.5 (anti-M), and mAb 5B188.2 (anti-N) (Table 1). None of these antibodies, regardless of their specificity, exhibited a protective effect because all mice were moribund by 14 to 16 days after infection, when 5 μg were administered. This result was not expected, because mAb 5A13.5 has a high neutralizing antibody titer (1:6230 PRD/mg) and has been reported to protect B6 mice from encephalitis.29Buchmeier MJ Lewicki HA Talbot PJ Knobler RL Murine hepatitis virus-4 (strain JHM)-induced neurologic disease is modulated in vivo by monoclonal antibody.Virology. 1984; 132: 261-270Crossref PubMed Scopus (145) Google Scholar We found, however, that mAb 5A13.5 was inefficient compared to mAb 5B19.2 at protecting B6 mice from acute encephalitis after inoculation with neurovirulen
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