Elevated Neuronal Expression of CD200 Protects Wlds Mice from Inflammation-Mediated Neurodegeneration
2007; Elsevier BV; Volume: 170; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.060677
ISSN1525-2191
AutoresTanuja Chitnis, Jaime Imitola, Yue Wang, Wassim Elyaman, Prianka Chawla, Maia Sharuk, Khadir Raddassi, Roderick T. Bronson, Samia J. Khoury,
Tópico(s)Immune Cell Function and Interaction
ResumoAxonal damage secondary to inflammation is likely the substrate of chronic disability in multiple sclerosis and is found in the animal model of experimental autoimmune encephalomyelitis (EAE). Wlds mice have a triplication of the fusion gene Ube4b/Nmnat and a phenotype of axon protection. Wlds mice develop an attenuated disease course of EAE, with decreased demyelination, reduced axonal pathology, and decreased central nervous system (CNS) macrophage and microglial accumulation. We show that attenuated disease in Wlds mice was associated with robust constitutive expression of the nonsignaling CD200 molecule on neurons in the CNS compared with control mice. CD200 interacts with its signaling receptor CD200R, which we found to be expressed on microglia, astrocytes, and oligodendrocytes at similar levels in control and Wlds mice. Administration of blocking anti-CD200 antibody to Wlds mice abrogated disease attenuation and was associated with increased CNS inflammation and neurodegeneration. In vitro, Wlds neuronal cultures were protected from microglial-induced neurotoxicity compared with control cultures, but protection was abrogated by anti-CD200 antibody. The CD200-CD200R pathway plays a critical role in attenuating EAE and reducing inflammation-mediated damage in the CNS. Strategies that up-regulate the expression of CD200 in the CNS or molecules that ligate the CD200R may be relevant as neuroprotective strategies in multiple sclerosis. Axonal damage secondary to inflammation is likely the substrate of chronic disability in multiple sclerosis and is found in the animal model of experimental autoimmune encephalomyelitis (EAE). Wlds mice have a triplication of the fusion gene Ube4b/Nmnat and a phenotype of axon protection. Wlds mice develop an attenuated disease course of EAE, with decreased demyelination, reduced axonal pathology, and decreased central nervous system (CNS) macrophage and microglial accumulation. We show that attenuated disease in Wlds mice was associated with robust constitutive expression of the nonsignaling CD200 molecule on neurons in the CNS compared with control mice. CD200 interacts with its signaling receptor CD200R, which we found to be expressed on microglia, astrocytes, and oligodendrocytes at similar levels in control and Wlds mice. Administration of blocking anti-CD200 antibody to Wlds mice abrogated disease attenuation and was associated with increased CNS inflammation and neurodegeneration. In vitro, Wlds neuronal cultures were protected from microglial-induced neurotoxicity compared with control cultures, but protection was abrogated by anti-CD200 antibody. The CD200-CD200R pathway plays a critical role in attenuating EAE and reducing inflammation-mediated damage in the CNS. Strategies that up-regulate the expression of CD200 in the CNS or molecules that ligate the CD200R may be relevant as neuroprotective strategies in multiple sclerosis. Multiple sclerosis (MS) is an immune-mediated demyelinating and degenerative disease of the central nervous system (CNS). Axonal damage and demyelination are present in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE), and are implicated as the primary determinants of irreversible neurological deficits.1Trapp BD Peterson J Ransohoff RM Rudick R Mork S Bo L Axonal transection in the lesions of multiple sclerosis.N Engl J Med. 1998; 338: 278-285Crossref PubMed Scopus (3531) Google Scholar, 2Brown A McFarlin DE Raine CS Chronologic neuropathology of relapsing experimental allergic encephalomyelitis in the mouse.Lab Invest. 1982; 46: 171-185PubMed Google Scholar Axonal damage is a consequence of both immune-mediated damage as well as activation of degenerative pathways; however, the underlying mechanisms are not well understood. The Wlds mouse is a spontaneously occurring mutant with the unique phenotype of protection against several forms of axonal injury. Degeneration of the distal portion of the axon or Wallerian degeneration has been shown to be delayed in the Wlds mouse after both peripheral3Lunn ER Perry VH Brown MC Rosen H Gordon S Absence of Wallerian degeneration does not hinder regeneration in peripheral nerve.Eur J Neurosci. 1989; 1: 27-33Crossref PubMed Scopus (546) Google Scholar, 4Mack TG Reiner M Beirowski B Mi W Emanuelli M Wagner D Thomson D Gillingwater T Court F Conforti L Fernando FS Tarlton A Andressen C Addicks K Magni G Ribchester RR Perry VH Coleman MP Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.Nat Neurosci. 2001; 4: 1199-1206Crossref PubMed Scopus (513) Google Scholar and CNS nerve transections.5Perry VH Brown MC Lunn ER Very slow retrograde and Wallerian degeneration in the CNS of C57BL/Ola mice.Eur J Neurosci. 1991; 3: 102-105Crossref PubMed Scopus (120) Google Scholar In addition, axons have been shown to remain viable after apoptosis of the neuronal cell body.6Deckwerth TL Johnson Jr, EM Neurites can remain viable after destruction of the neuronal soma by programmed cell death (apoptosis).Dev Biol. 1994; 165: 63-72Crossref PubMed Scopus (137) Google Scholar The Wlds gene has also been shown to be protective in models of vincristine- and paclitaxel-induced neuropathy, suggesting that it has multifaceted neuroprotective effects.7Wang MS Davis AA Culver DG Glass JD WldS mice are resistant to paclitaxel (taxol) neuropathy.Ann Neurol. 2002; 52: 442-447Crossref PubMed Scopus (134) Google Scholar, 8Wang MS Fang G Culver DG Davis AA Rich MM Glass JD The WldS protein protects against axonal degeneration: a model of gene therapy for peripheral neuropathy.Ann Neurol. 2001; 50: 773-779Crossref PubMed Scopus (87) Google Scholar Several studies have demonstrated reduced microglial responses after axonal transection in the Wlds model.9Steward O Trimmer PA Genetic influences on cellular reactions to CNS injury: the reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration.J Comp Neurol. 1997; 380: 70-81Crossref PubMed Scopus (25) Google Scholar, 10Schauwecker PE Steward O Genetic influences on cellular reactions to brain injury: activation of microglia in denervated neuropil in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration.J Comp Neurol. 1997; 380: 82-94Crossref PubMed Scopus (19) Google Scholar, 11Fujiki M Zhang Z Guth L Steward O Genetic influences on cellular reactions to spinal cord injury: activation of macrophages/microglia and astrocytes is delayed in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration.J Comp Neurol. 1996; 371: 469-484Crossref PubMed Scopus (66) Google Scholar, 12Lawson LJ Frost L Risbridger J Fearn S Perry VH Quantification of the mononuclear phagocyte response to Wallerian degeneration of the optic nerve.J Neurocytol. 1994; 23: 729-744Crossref PubMed Scopus (80) Google Scholar, 13Shamash S Reichert F Rotshenker S The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-1beta.J Neurosci. 2002; 22: 3052-3060Crossref PubMed Google Scholar Experiments using bone marrow chimaeras have proven that this is a property that affects “cell populations intrinsic to the Wlds nerve and is not attributable to an anomaly in circulating monocytes.”14Perry VH Brown MC Lunn ER Tree P Gordon S Evidence that very slow Wallerian degeneration in C57BL/Ola mice is an intrinsic property of the peripheral nerve.Eur J Neurosci. 1990; 2: 802-808Crossref PubMed Scopus (158) Google Scholar We have recently shown that compared with wild-type (WT) mice, Wlds mice, when immunized to induce chronic EAE, developed a delayed onset and an attenuated disease course,15Kaneko S Wang J Kaneko M Yiu G Hurrell JM Chitnis T Khoury SJ He Z Protecting axonal degeneration by increasing nicotinamide adenine dinucleotide levels in experimental autoimmune encephalomyelitis models.J Neurosci. 2006; 26: 9794-9804Crossref PubMed Scopus (133) Google Scholar which was associated with a reduction in both axonal loss and demyelination in spinal cord sections. Axonal protection in Wlds mice was associated with increased nicotinamide adenine dinucleotide (NAD) levels; however, the molecular mechanisms mediating axon protection in Wlds mice have not been elucidated. In this study, we explored molecular mediators of neuroprotection in the Wlds EAE model with the goal of identifying potential therapeutic targets for MS. Although there was no difference in T-cell infiltrates in the CNS,15Kaneko S Wang J Kaneko M Yiu G Hurrell JM Chitnis T Khoury SJ He Z Protecting axonal degeneration by increasing nicotinamide adenine dinucleotide levels in experimental autoimmune encephalomyelitis models.J Neurosci. 2006; 26: 9794-9804Crossref PubMed Scopus (133) Google Scholar we found that microglia and macrophage accumulation and activation in the CNS were diminished in Wlds mice compared with WT mice. Microglia and macrophages have been associated with axonal damage within MS lesions,16Bitsch A Schuchardt J Bunkowski S Kuhlmann T Bruck W Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation.Brain. 2000; 123: 1174-1183Crossref PubMed Scopus (766) Google Scholar as well as in diffuse axonal damage in the normal appearing white matter.17Kutzelnigg A Lucchinetti CF Stadelmann C Bruck W Rauschka H Bergmann M Schmidbauer M Parisi JE Lassmann H Cortical demyelination and diffuse white matter injury in multiple sclerosis.Brain. 2005; 128: 2705-2712Crossref PubMed Scopus (1396) Google Scholar Because of these observations, we explored the differential expression of molecules associated with microglial regulation. We found that disease protection in Wlds mice as well as in neuronal cultures was associated with enhanced neuronal and glial expression of CD200, a nonsignaling molecule that has previously been described on neurons18Barclay AN Brown MH Heterogeneity of interactions mediated by membrane glycoproteins of lymphocytes.Biochem Soc Trans. 1997; 25: 224-228PubMed Google Scholar, 19Clark MJ Gagnon J Williams AF Barclay AN MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.EMBO J. 1985; 4: 113-118Crossref PubMed Scopus (126) Google Scholar, 20McCaughan GW Clark MJ Barclay AN Characterization of the human homolog of the rat MRC OX-2 membrane glycoprotein.Immunogenetics. 1987; 25: 329-335Crossref PubMed Scopus (48) Google Scholar and belongs to the immunoglobulin superfamily of glycoproteins. Interaction of CD200 with its ligand, CD200R, has been shown to initiate tyrosine phosphorylation.21Wright GJ Puklavec MJ Willis AC Hoek RM Sedgwick JD Brown MH Barclay AN Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.Immunity. 2000; 13: 233-242Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar Thus, the effects of CD200 are mediated through cells expressing the CD200 receptor (CD200R), including microglia/macrophages.21Wright GJ Puklavec MJ Willis AC Hoek RM Sedgwick JD Brown MH Barclay AN Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.Immunity. 2000; 13: 233-242Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 22Preston S Wright GJ Starr K Barclay AN Brown MH The leukocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages.Eur J Immunol. 1997; 27: 1911-1918Crossref PubMed Scopus (72) Google Scholar, 23Wright GJ Cherwinski H Foster-Cuevas M Brooke G Puklavec MJ Bigler M Song Y Jenmalm M Gorman D McClanahan T Liu MR Brown MH Sedgwick JD Phillips JH Barclay AN Characterization of the CD200 receptor family in mice and humans and their interactions with CD200.J Immunol. 2003; 171: 3034-3046PubMed Google Scholar Macrophage/microglial responses to nerve trauma and EAE were accelerated in mice deficient for CD200.24Hoek RM Ruuls SR Murphy CA Wright GJ Goddard R Zurawski SM Blom B Homola ME Streit WJ Brown MH Barclay AN Sedgwick JD Down-regulation of the macrophage lineage through interaction with OX2 (CD200).Science. 2000; 290: 1768-1771Crossref PubMed Scopus (846) Google Scholar These and other studies25Gorczynski RM Transplant tolerance modifying antibody to CD200 receptor, but not CD200, alters cytokine production profile from stimulated macrophages.Eur J Immunol. 2001; 31: 2331-2337Crossref PubMed Scopus (41) Google Scholar suggest that ligation of CD200R delivers a negative signal for microglia/macrophage activation. CD200R has been found to also be expressed on dendritic cells, mast cells, granulocytes, and to a limited extent on CD8+ T cells, natural killer (NK) cells, NKT cells, and CD4+ cells of the Th2 phenotype.23Wright GJ Cherwinski H Foster-Cuevas M Brooke G Puklavec MJ Bigler M Song Y Jenmalm M Gorman D McClanahan T Liu MR Brown MH Sedgwick JD Phillips JH Barclay AN Characterization of the CD200 receptor family in mice and humans and their interactions with CD200.J Immunol. 2003; 171: 3034-3046PubMed Google Scholar Four isoforms of CD200R have been described, and at least in one study, all four have been shown to bind CD200.26Gorczynski R Chen Z Kai Y Lee L Wong S Marsden PA CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules.J Immunol. 2004; 172: 7744-7749PubMed Google Scholar More recently CD200R agonists have been shown to inhibit proinflammatory cytokine secretion by macrophage cell lines, including interleukin (IL)-17-induced IL-6 production.27Jenmalm MC Cherwinski H Bowman EP Phillips JH Sedgwick JD Regulation of myeloid cell function through the CD200 receptor.J Immunol. 2006; 176: 191-199PubMed Google Scholar CD200R ligation on mast cells inhibits degranulation and cytokine production.28Zhang S Cherwinski H Sedgwick JD Phillips JH Molecular mechanisms of CD200 inhibition of mast cell activation.J Immunol. 2004; 173: 6786-6793PubMed Google Scholar CD200R ligation induces regulatory dendritic cell populations capable of secreting indolamine dioxygenase.29Fallarino F Asselin-Paturel C Vacca C Bianchi R Gizzi S Fioretti MC Trinchieri G Grohmann U Puccetti P Murine plasmacytoid dendritic cells initiate the immunosuppressive pathway of tryptophan catabolism in response to CD200 receptor engagement.J Immunol. 2004; 173: 3748-3754PubMed Google Scholar Moreover, in animal models, CD200R agonists have been shown to ameliorate collagen-induced arthritis30Gorczynski RM Chen Z Lee L Yu K Hu J Anti-CD200R ameliorates collagen-induced arthritis in mice.Clin Immunol. 2002; 104: 256-264Crossref PubMed Scopus (59) Google Scholar and prolong graft survival.31Gorczynski RM Hu J Chen Z Kai Y Lei J A CD200FC immunoadhesin prolongs rat islet xenograft survival in mice.Transplantation. 2002; 73: 1948-1953Crossref PubMed Scopus (22) Google Scholar Using the Wlds model, we studied the effects of neuronal CD200 overexpression in models of inflammation-induced neurotoxicity. We show that increased expression of CD200 is capable of protecting neurons and axons from microglia-induced damage in vitro and in vivo. Moreover, we demonstrated that the CD200 receptor is expressed on CNS glial cells as well as peripheral splenocytes, suggesting that the CD200-CD200R pathway can play a regulatory role in both the CNS and the periphery. Thus, strategies to enhance the CNS expression of CD200 or to ligate its receptor may suppress inflammation-mediated neurodegeneration present in diseases including multiple sclerosis. Female C57BL/6O1aHSD-Wlds and wild-type C57BL/6O1aHSD (WT) from Harlan UK Limited (Bicester, Oxon, UK) were obtained for EAE studies. C57BL/6O1aHSD-Wlds mice are homozygous mutants. Mice were 6 to 10 weeks old at the time of immunization. Myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55) (MEVGWYRSPFSRVVHLYRNGK) corresponding to mouse sequence was synthesized by QCB Inc. (Division of BioSource International, Hopkinton, MA) and purified to >99% by high-performance liquid chromatography. Mice were immunized with 150 μg/75 μl of MOG peptide emulsified with an equal volume of complete Freund's adjuvant containing Mycobacterium tuberculosis (H37RA; Difco, Detroit, MI) at a final concentration of 2 mg/ml. Two hundred ng of pertussis toxin was injected intraperitoneally (List Laboratories, Campbell, CA) on day 0 and day 2 after immunization, EAE was scored by a blinded observer on a scale from 0 to 5, as previously described32Chitnis T Najafian N Benou C Salama AD Grusby MJ Sayegh MH Khoury SJ Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis.J Clin Invest. 2001; 108: 739-747Crossref PubMed Scopus (189) Google Scholar: grade 1, limp tail or isolated weakness of gait without limp tail; grade 2, partial hind leg paralysis; grade 3, total hind leg or partial hind and front leg paralysis; grade 4, total hind leg and partial front leg paralysis; and grade 5, moribund or dead animal. A subgroup of Wlds and WT mice were treated with 200 μg/100 μl of blocking anti-CD200 antibody (clone 10A5, anti-mouse-CD200 rat IgG1κ; Trillium Therapeutics Inc., Toronto, ON, Canada)25Gorczynski RM Transplant tolerance modifying antibody to CD200 receptor, but not CD200, alters cytokine production profile from stimulated macrophages.Eur J Immunol. 2001; 31: 2331-2337Crossref PubMed Scopus (41) Google Scholar, 30Gorczynski RM Chen Z Lee L Yu K Hu J Anti-CD200R ameliorates collagen-induced arthritis in mice.Clin Immunol. 2002; 104: 256-264Crossref PubMed Scopus (59) Google Scholar injected intravenously every other day from days 10 to 20. Control WT and Wlds mice were treated with phosphate-buffered saline (PBS) alone or rat IgG control (Sigma, St. Louis, MO). Delayed-type hypersensitivity responses were assessed by the measurement of ear thickness using calipers (IDC series 543; Mitutoyo, Tokyo, Japan), 48 hours after intradermal injection with MOG peptide (50 μg in 50 μl of PBS) in one ear and an equal volume of PBS in the contralateral ear. Results were reported as fold change in ear thickness of MOG-injected ear/PBS-injected ear. Results from four to six mice per strain were averaged. Mice were euthanized using CO2 and perfused with PBS followed by 4% paraformaldehyde or Bouin's solution (Electron Microscopy Sciences, Fort Washington, PA). Spinal cords and brains were collected at specified time points, using three to four mice in each experimental group. For paraffin embedding, tissues were stored in Bouin's solution for minimum of 48 hours, and paraffin sections were prepared. For immunofluorescence staining, tissues were kept in 4% paraformaldehyde for 48 hours, placed in a 30% sucrose gradient, and then embedded in O.C.T. (Electron Microscopy Sciences), quick-frozen in liquid nitrogen and stored at −80°C until sectioning. Sections cut from paraffin-embedded tissue, were placed in a 20% silver nitrate solution at 37°C. Sections were washed in ammonia, and then a developer solution was added for 3 to 5 minutes until sections were black. Slides were rewashed in ammonia water, dH2O, fixed in 5% thiosulfate for 1 minute, washed, dehydrated, and then mounted in Permount. Sections were cut from paraffin-embedded tissue. Slides were placed in Luxol fast blue solution overnight at 55°C, differentiated in alcohol, dipped in 0.05% lithium carbonate solution, and then counterstained with cresyl violet. Axon loss and demyelination were quantified as follows. Transverse spinal cord sections at the cervical, thoracic, and lumbar levels from WT and Wlds mice at day 60 after immunization were stained with Bielschowsky or Luxol fast blue, as described. Photomicrographs (×100) were taken of sections from the anterior, lateral, and posterior sections of each spinal cord level, using specific landmarks for orientation. The area of regions with >50% axon density or demyelinated areas were quantified, and percent axon loss or demyelination was calculated in comparison to total white matter per section using the NIH Image Analyzer program (Bethesda, MD). Using perfused frozen sections mounted in O.C.T., 30-μm free-floating sections were cut using a cryotome. Sections were blocked in PBS containing 4% goat serum, 0.3% bovine serum albumin, and 0.3% Triton X-100 and incubated with primary antibodies at 4°C overnight, followed by fluorescein- or rhodamine-labeled secondary antibodies 1:250 to 1:500 (Molecular Probes, Eugene, OR) for 2 hours in blocking solutions. The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide,26Gorczynski R Chen Z Kai Y Lee L Wong S Marsden PA CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules.J Immunol. 2004; 172: 7744-7749PubMed Google Scholar clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes). The following isotype controls were used: rabbit polyclonal IgG isotype control (Abcam Inc., Cambridge, MA), rat IgG2a isotype control (eBioscience, San Diego, CA), mouse IgG1 isotype control (eBioscience), mouse IgG2a isotype control (eBioscience), and mouse IgG2b isotype control (eBioscience). Spinal cord sections from WT and Wlds mice were incubated with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia isolectin B4 (LB4) 1:100 (Vector Laboratories, Burlingame, CA) using the standard immunofluorescence protocol described above. Secondary reagent Alexa 488-conjugated anti-FITC antibody (1:500) (Molecular Probes) was used to visualize LB4 staining. Two spinal cord sections from each of five mice per strain per time point were stained for LB4 (for a total of 10 sections per strain per time point). The number of CD4+ or CD8+ LB4+ foci in five adjacent fields per section was quantified. Perimeningeal foci were defined as those limited to the meninges or subpial region, whereas parenchymal foci were defined as those beyond the subpial region. Confocal microscopy was performed using a Zeiss LSM equipped with argon-Kr/HeNe lasers (Zeiss, Heidelburg, Germany), and Zeiss 3D analysis software. Three-dimensional images were obtained using Z-series stacking. Animals with EAE and naïve animals were perfused and fixed with 2.5% paraformaldehyde/2.5% glutaraldehyde solution in 0.1 mol/L sodium cacodylate. The spinal cord was postfixed, dehydrated through serial ethanol concentrations, and embedded in EPON. Thick sections were stained with toluidine blue and examined for regions of interest. Sections were thin cut (1 μm), stained with 2% uranyl acetate in 0.1 mol/L sodium acetate, and followed by lead citrate. Sections were then placed on a carbon-coated formvar grid and viewed with a Hitachi 600 transmission electron microscope (Harvard EM Facility). For proliferation and cytokine measurement, splenocytes were cultured in 96-well plates (Costar, Cambridge, MA). Media used for proliferation and cytokine assays consisted of serum-free Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) containing 75 mmol/L/ml l-glutamine, 100 U/ml penicillin and streptomycin, 1 ml/100 ml of media of a 100× concentrated nonessential amino acid solution, 0.1 mmol/L HEPES/ml, 1 mmol/L/ml sodium pyruvate (all BioWhittaker), and 0.05 mmol/L/ml 2-mercaptoethanol (Sigma). Cells were incubated at 37°C in humidified air containing 7% CO2. For proliferation assay, cells were cultured at 2 × 106 cells/ml and 200 μl/well with various antigen concentrations. After 48 hours of culture, 1 μCi of [3H]thymidine (NEN, Boston, MA) was added in 10 μl of media to each well for another 16 hours. Cells were harvested on filter mats, dried, and counted. For ELISA, cytokine assay cells were cultured at 4 × 106 cells/ml in 200 μl of media at various antigen concentrations. Supernatants for ELISA were collected after 48 hours of culture. Quantitative ELISAs for IL-5, IL-6, IL-10, and interferon (IFN)-γ were performed on 96-well Nunc-Immuno plates (Nalge Nunc International, Rochester, NY) using paired antibodies and recombinant cytokines from Pharmingen, according to the manufacturer's recommendations. Splenocytes from either wild-type or Wlds mice were washed and resuspended in PBS to a concentration of 107 cells/ml. Cells were incubated on ice with 5 μg/106 cells of the appropriate cellular marker (phycoerythrin-conjugated; Pharmingen) and 5 μg/106 of rat anti-mouse CD200 FITC-conjugated antibody (Cedarlane Laboratories) or anti-CD200R1 FITC-conjugated antibody (Serotec) when indicated for 20 minutes on ice. Cells were then washed and analyzed by flow cytometry on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). The percentage of double-positive cells per sample group was calculated. Mice were sacrificed and perfused intracardially with 20 ml of ice-cold PBS. Spinal cords were isolated and passed through a 70-μm nylon filter, spun down, and resuspended in Hanks' balanced salt solution with 10 mmol/L HEPES and 2 mmol/L ethylenediaminetetraacetic acid and incubated on a rotating shaker for 1 hour at 4°C. The pellet was resuspended in 5 ml of isotonic 37% Percoll and spun down. The supernatant was removed, and the pellet was resuspended in PBS containing 1% bovine serum albumin for flow cytometric studies. Antibodies used for flow cytometric studies included FITC- or phycoerythrin-conjugated antibodies to CD11b, CD4, CD8, CD11c, NK1.1, TCRαβ, CD19, Gr1, and allophycocyanine-conjugated CD45 as well as isotype controls (Pharmingen). Allophycocyanine-conjugated FoxP3 antibody was obtained from eBioscience. Tissues were dissected and homogenized in lysis buffer [25 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 0.5% Triton X-100, 10% glycerol, and one tablet of protease inhibitors (Boehringer, Indianapolis, IN)]. Lysates were centrifuged, the resulting supernatants were collected, and protein concentrations were determined by bicinchoninic acid assay (Pierce, Rockford, IL). Samples were mixed with 3× Laemmli's buffer and heated at 99°C for 5 minutes, and equal amounts of total protein was loaded onto 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed for 2 hours at room temperature or overnight at 4°C with primary monoclonal antibodies CD200 (1:10,000) and β-actin (1:5000), rinsed in phosphate-buffered saline/Tween 20, incubated for 1 hour at room temperature with horseradish peroxidase-conjugated goat anti-mouse (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:10,000) or anti-rat antibodies (Caltag, Burlingame, CA) (1:10,000). Membranes were washed in Tris-buffered saline/Tween 20, and immunoreactive proteins were detected using the enhanced chemiluminescence method (Amersham, Piscataway, NJ). Immunoreactivity was quantified using the NIH Image analyzer program. Spinal cord lysates from WT and Wlds mice were incubated with anti-CD200 antibody for 18 hours at 4°C, immunoprecipitated with protein G agarose suspension, and separated on 10% polyacrylamide gel. Samples were immunoblotted with either ubiquitin or CD200 using the same protocol described above. CD22 expression in spinal cord lysates was assessed by immunoblot using anti-CD22 antibody (clone MYG13; Santa Cruz Biotechnology). We followed the protocol outlined by Huang and colleagues.33Huang D Shi FD Jung S Pien GC Wang J Salazar-Mather TP He TT Weaver JT Ljunggren HG Biron CA Littman DR Ransohoff RM The neuronal chemokine CX3CL1/fractalkine selectively recruits NK cells that modify experimental autoimmune encephalomyelitis within the central nervous system.FASEB J. 2006; 20: 896-905Crossref PubMed Scopus (236) Google Scholar Spinal cords were manually homogenized in 1 ml of lysis buffer (150 mmol/L NaCl, 0.01 mol/L Tris, 1.0 mmol/L ethylenediaminetetraacetic acid, 1.0 μg/ml aprotinin, and 100 μg/ml phenylmethyl sulfonyl fluoride) and centrifuged at 500 × g for 10 minutes. Protein concentration in the supernatants was measured, and four samples per group, each containing 2.0 μg/ml total protein in 50 μl of PBS, were assayed for fractalkine concentration using a fractalkine ELISA assay (DY472; R&D Systems Inc., Minneapolis, MN), which contains anti-CX3CL1 and conjugated anti-CX3CL1, as well as a recombinant fractalkine standard. Cortices were dissected from P1 C57BL/6 mice, and trypsin was added for 15 minutes, followed by dissociation by trituration through a fire-polished pipette. Cells were counte
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