Absence of the Cellular Prion Protein Exacerbates and Prolongs Neuroinflammation in Experimental Autoimmune Encephalomyelitis
2008; Elsevier BV; Volume: 173; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2008.071062
ISSN1525-2191
AutoresShigeki Tsutsui, Jennifer Hahn, Trina Johnson, Zenobia Ali, Frank R. Jirik,
Tópico(s)Multiple Sclerosis Research Studies
ResumoAlthough the physiological roles of the cellular prion protein (PrPC) remain to be fully elucidated, PrPC has been proposed to represent a potential regulator of cellular immunity. To test this hypothesis, we evaluated the consequences of PrPC deficiency on the course of experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein peptide. Consistent with augmented proliferative responses and increased cytokine gene expression by myelin oligodendrocyte glycoprotein-primed Prnp−/− T cells, PrPC-deficient mice demonstrated more aggressive disease onset and a lack of clinical improvement during the chronic phase of experimental autoimmune encephalomyelitis. Acutely, Prnp−/− spinal cord, cerebellum, and forebrain exhibited higher levels of leukocytic infiltrates and pro-inflammatory cytokine gene expression, as well as increased spinal cord myelin basic protein and axonal loss. During the chronic phase, a remarkable persistence of leukocytic infiltrates was present in the forebrain and cerebellum, accompanied by an increase in interferon-γ and interleukin-17 transcripts. Attenuation of T cell-dependent neuroinflammation thus represents a potential novel function of PrPC. Although the physiological roles of the cellular prion protein (PrPC) remain to be fully elucidated, PrPC has been proposed to represent a potential regulator of cellular immunity. To test this hypothesis, we evaluated the consequences of PrPC deficiency on the course of experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein peptide. Consistent with augmented proliferative responses and increased cytokine gene expression by myelin oligodendrocyte glycoprotein-primed Prnp−/− T cells, PrPC-deficient mice demonstrated more aggressive disease onset and a lack of clinical improvement during the chronic phase of experimental autoimmune encephalomyelitis. Acutely, Prnp−/− spinal cord, cerebellum, and forebrain exhibited higher levels of leukocytic infiltrates and pro-inflammatory cytokine gene expression, as well as increased spinal cord myelin basic protein and axonal loss. During the chronic phase, a remarkable persistence of leukocytic infiltrates was present in the forebrain and cerebellum, accompanied by an increase in interferon-γ and interleukin-17 transcripts. Attenuation of T cell-dependent neuroinflammation thus represents a potential novel function of PrPC. The cellular prion protein (PrPC), a highly conserved glycosylphosphatidylinositol-anchored cell surface glycoprotein concentrated in lipid rafts,1Taylor DR Hooper NM The prion protein and lipid rafts.Mol Membr Biol. 2006; 23: 89-99Crossref PubMed Scopus (217) Google Scholar is abundantly expressed in the central nervous system (CNS).2Kretzschmar HA Prusiner SB Stowring LE DeArmond SJ Scrapie prion proteins are synthesized in neurons.Am J Pathol. 1986; 122: 1-5PubMed Google Scholar, 3Brown DR Besinger A Herms JW Kretzschmar HA Microglial expression of the prion protein.Neuroreport. 1998; 9: 1425-1429Crossref PubMed Scopus (74) Google Scholar PrPC may serve as a receptor for a variety of putative ligands, including: heparan sulfate,4Pan T Wong BS Liu T Li R Petersen RB Sy MS Cell-surface prion protein interacts with glycosaminoglycans.Biochem J. 2002; 368: 81-90Crossref PubMed Scopus (124) Google Scholar laminin,5Graner E Mercadante AF Zanata SM Forlenza OV Cabral AL Veiga SS Juliano MA Roesler R Walz R Minetti A Izquierdo I Martins VR Brentani RR Cellular prion protein binds laminin and mediates neuritogenesis.Brain Res Mol Brain Res. 2000; 76: 85-92Crossref PubMed Scopus (258) Google Scholar neural cell adhesion molecule,6Schmitt-Ulms G Legname G Baldwin MA Ball HL Bradon N Bosque PJ Crossin KL Edelman GM DeArmond SJ Cohen FE Prusiner SB Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein.J Mol Biol. 2001; 314: 1209-1225Crossref PubMed Scopus (301) Google Scholar various synaptic proteins,7Spielhaupter C Schatzl HM PrPC directly interacts with proteins involved in signaling pathways.J Biol Chem. 2001; 276: 44604-44612Crossref PubMed Scopus (188) Google Scholar and stress-inducible protein-1.8Zanata SM Lopes MH Mercadante AF Hajj GN Chiarini LB Nomizo R Freitas AR Cabral AL Lee KS Juliano MA de Oliveira E Jachieri SG Burlingame A Huang L Linden R Brentani RR Martins VR Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.EMBO J. 2002; 21: 3307-3316Crossref PubMed Scopus (371) Google Scholar These ligand-receptor interactions suggest that PrPC could have a role in diverse processes, including neurodevelopment, synaptic function, neurite outgrowth, and neuronal survival. Evidence for the latter has supported the notion that neuroprotection is one physiological function of PrPC. For example, deletion of PrPC increased neuronal predisposition to damage by modulating susceptibility to apoptosis9Kurschner C Morgan JI Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion protein.Brain Res Mol Brain Res. 1996; 37: 249-258Crossref PubMed Google Scholar, 10Kuwahara C Takeuchi AM Nishimura T Haraguchi K Kubosaki A Matsumoto Y Saeki K Matsumoto Y Yokoyama T Itohara S Onodera T Prions prevent neuronal cell-line death.Nature. 1999; 400: 225-226Crossref PubMed Scopus (374) Google Scholar and the negative consequences of oxidative stress.11Brown DR Nicholas RS Canevari L Lack of prion protein expression results in a neuronal phenotype sensitive to stress.J Neurosci Res. 2002; 67: 211-224Crossref PubMed Scopus (199) Google Scholar, 12Wong BS Liu T Li R Pan T Petersen RB Smith MA Gambetti P Perry G Manson JC Brown DR Sy MS Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein.J Neurochem. 2001; 76: 565-572Crossref PubMed Scopus (152) Google Scholar, 13Rachidi W Vilette D Guiraud P Arlotto M Riondel J Laude H Lehmann S Favier A Expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper delivery.J Biol Chem. 2003; 278: 9064-9072Crossref PubMed Scopus (181) Google Scholar Furthermore, in vivo studies demonstrated that PrPC-deficient mice were more prone to seizure induction,14Walz R Amaral OB Rockenbach IC Roesler R Izquierdo I Cavalheiro EA Martins VR Brentani RR Increased sensitivity to seizures in mice lacking cellular prion protein.Epilepsia. 1999; 40: 1679-1682Crossref PubMed Scopus (168) Google Scholar and exhibited an increased extent of cerebral damage following an ischemic challenge.15Weise J Sandau R Schwarting S Crome O Wrede A Schulz-Schaeffer W Zerr I Bahr M Deletion of cellular prion protein results in reduced Akt activation, enhanced postischemic caspase-3 activation, and exacerbation of ischemic brain injury.Stroke. 2006; 37: 1296-1300Crossref PubMed Scopus (139) Google Scholar In contrast, adenovirus-mediated PrPC overexpression reduced CNS damage in a rat model of cerebral ischemia.16Shyu WC Lin SZ Chiang MF Ding DC Li KW Chen SF Yang HI Li H Overexpression of PrPC by adenovirus-mediated gene targeting reduces ischemic injury in a stroke rat model.J Neurosci. 2005; 25: 8967-8977Crossref PubMed Scopus (105) Google Scholar While the mechanism(s) underlying these phenomena remain unclear, such in vivo findings have lent strong support to the idea that PrPC may have a neuroprotective function. PrPC is expressed on the surface of cells of the human and murine lympho-hematopoietic system, including dendritic cells (DCs), follicular dendritic cells, macrophages/microglia, and in humans, T-lymphocytes.17Isaacs JD Jackson GS Altmann DM The role of the cellular prion protein in the immune system.Clin Exp Immunol. 2006; 146: 1-8Crossref PubMed Scopus (68) Google Scholar, 18Dodelet VC Cashman NR Prion protein expression in human leukocyte differentiation.Blood. 1998; 91: 1556-1561Crossref PubMed Google Scholar, 19Burthem J Urban B Pain A Roberts DJ The normal cellular prion protein is strongly expressed by myeloid dendritic cells.Blood. 2001; 98: 3733-3738Crossref PubMed Scopus (73) Google Scholar, 20Li R Liu D Zanusso G Liu T Fayen JD Huang JH Petersen RB Gambetti P Sy MS The expression and potential function of cellular prion protein in human lymphocytes.Cell Immunol. 2001; 207: 49-58Crossref PubMed Scopus (86) Google Scholar With regard to the latter, in mice PrPC was only detected in a relatively small subset of mature B and T lymphocytes.21Liu T Li R Wong BS Liu D Pan T Petersen RB Gambetti P Sy MS Normal cellular prion protein is preferentially expressed on subpopulations of murine hemopoietic cells.J Immunol. 2001; 166: 3733-3742Crossref PubMed Scopus (58) Google Scholar, 22Ford MJ Burton LJ Morris RJ Hall SM Selective expression of prion protein in peripheral tissues of the adult mouse.Neuroscience. 2002; 113: 177-192Crossref PubMed Scopus (170) Google Scholar Recent studies have concluded that PrPC may play a role in T cell activation,23Mabbott NA Brown KL Manson J Bruce ME T-lymphocyte activation and the cellular form of the prion protein.Immunology. 1997; 92: 161-165Crossref PubMed Scopus (100) Google Scholar the phagocytic ability of macrophages,24de Almeida CJ Chiarini LB da Silva JP PM ES Martins MA Linden R The cellular prion protein modulates phagocytosis and inflammatory response.J Leukoc Biol. 2005; 77: 238-246Crossref PubMed Scopus (96) Google Scholar and T cell-DC interactions.25Ballerini C Gourdain P Bachy V Blanchard N Levavasseur E Gregoire S Fontes P Aucouturier P Hivroz C Carnaud C Functional implication of cellular prion protein in antigen-driven interactions between T cells and dendritic cells.J Immunol. 2006; 176: 7254-7262PubMed Google Scholar The interaction between T cells and DCs represents a critical event for the initiation of primary immune responses, and hence the finding that both T cells and DCs express PrPC, raised the possibility that PrPC plays a role in immune system homeostasis. Precisely how PrPC might regulate the in vivo activities of cells of the immune system during normal or autoimmune T cell-mediated responses, however, remains nebulous. Since PrPC is expressed in cells of the murine immune system, we hypothesized that mice lacking this molecule might show an alteration in their response to an induced T cell-mediated autoimmune disease. We report that mice lacking PrPC develop earlier onset, more severe EAE, and also that they fail to recover during the chronic phase of EAE. This novel phenotype was accompanied by histopathological evidence of greater involvement of cerebellum and forebrain, more extensive spinal cord damage, as well as a striking persistence of monocytic and T cell infiltrates in the CNS. PrPC thus appears to be an important regulator of T cell-mediated neuroinflammation. Mice with a targeted disruption of the prion gene (Prnp) of the Zurich I strain26Bueler H Fischer M Lang Y Bluethmann H Lipp HP DeArmond SJ Prusiner SB Aguet M Weissmann C Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein.Nature. 1992; 356: 577-582Crossref PubMed Scopus (1441) Google Scholar and their controls (of a mixed 129 and Friend Leukemia virus B background) were obtained from the European Mouse Mutant Archive (EM:0158, EMMA-Rome Italy) and interbred to generate Prnp−/− and Prnp+/+ littermates used in the experiments. The Zurich I mice, backcrossed for multiple generations (N = 7 to 8) into a C57BL/6 genetic background, were also used in some of the experiments. To induce EAE, 11 to 14 week-old females were injected subcutaneously at the base of the tail with 50 μg of myelin oligodendrocyte glycoprotein (MOG35–55)27Brundula V Rewcastle NB Metz LM Bernard CC Yong VW Targeting leukocyte MMPs and transmigration: minocycline as a potential therapy for multiple sclerosis.Brain. 2002; 125: 1297-1308Crossref PubMed Scopus (389) Google Scholar emulsified in complete Freund's adjuvant CFA (Difco Laboratories, Sparks, MD), together with 300 ng of reconstituted lyophilized pertussis toxin (List Biological Laboratories, Campbell, CA) administered intraperitonealy. Pertussis toxin injection was repeated after 48 hours.28Liu J Marino MW Wong G Grail D Dunn A Bettadapura J Slavin AJ Old L Bernard CC TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination.Nat Med. 1998; 4: 78-83Crossref PubMed Scopus (498) Google Scholar Animals were assessed for EAE clinical severity for 60 days (Prnp+/+ and Prnp−/− animals) using a 0 to 5 rating scale28Liu J Marino MW Wong G Grail D Dunn A Bettadapura J Slavin AJ Old L Bernard CC TNF is a potent anti-inflammatory cytokine in autoimmune-mediated demyelination.Nat Med. 1998; 4: 78-83Crossref PubMed Scopus (498) Google Scholar as follows: 0 = no disease; 1 = limp tail; 2 = partial paralysis of one or two hind limbs; 3 = complete paralysis of hind limbs; 4 = hind limb paralysis and fore limb paraparesis; and 5 = moribund. Mice were euthanized by cardiac puncture while under methoxyfluorane anesthesia at 60 days post-EAE induction. Animals were maintained in accordance with Canadian Council on Animal Care and University of Calgary Animal Care Committee regulations. Spleens were obtained from non-immunized C57BL/6 Prnp+/+ and Prnp−/− animals. DC-enriched populations and CD4+ T cells were then isolated from dissociated splenocytes by negative selection using two magnetic separation systems: StemSep mouse dendritic cell enrichment kit and EasySep mouse CD4+ T cell enrichment kit, in accordance with the manufacturer's instructions (StemCell Technologies Inc., Vancouver, BC, Canada). CD4+ T cells were cultured in serum-free AIM V media containing 3% IL-2 conditioned media and 2 μmol/L β-mercaptoethanol; cells were stimulated with 1 μg/ml anti-CD3 antibody for 48 hours. For flow cytometric analysis, 1 × 106 cells/ml of either DCs or CD4+ T cells were resuspended in 1% fetal bovine serum in PBS and incubated with anti-mouse CD16/32 (24G2, FcR block, BD Biosciences PharMingen, San Diego, CA) to prevent nonspecific staining. Cells were incubated with 5 μg/ml of anti-mouse PrPC antibody (SAF-83, Cayman Chemical Company, Ann Arbor, MI) or mouse IgG1 isotype control (BD Biosciences, San Jose, CA), and then incubated with 5 μg/ml of fluorescein isothiocyanate-conjugated goat anti-mouse Ig (BD Biosciences). Live cells were collected and gated using a FACSCalibur with CellQuest software (BD Biosciences) and quantified using FlowJo software (version 3.6; TreeStar, Ashland, OR). DC-enriched cells isolated from non-immunized C57BL/6 Prnp+/+ and Prnp−/− mice were irradiated, suspended at a density of 1 × 106 cells/ml, and pulsed with 40 μg/ml MOG35–55 peptide for 30 minutes. DC-enriched cells incubated with vehicle served as the ‘No MOG’ controls. Draining lymph nodes were removed from MOG peptide-immunized (using the same protocol as for EAE induction described above) C57BL/6 Prnp+/+ and Prnp−/− animals at 10 days post-immunization (dpi). Lymph nodes were homogenized in Roswell Park Memorial Institute (RPMI) 1640 media and total T cells were isolated from dissociated lymph nodes, using the EasySep mouse T cell enrichment kit, and suspended at a density of 2.5 × 106 cells/ml. DCs and T cells were plated 1:1 in 96-well U-bottom microtiter plates containing enriched RPMI 1640 media [RPMI 1640, 10% fetal calf serum, 1% l-glutamine, 1% minimum essential medium-nonessential amino acids, 2 μmol/L β- mercaptoethanol, 1% penicillin-streptomycin, and 1% sodium pyruvate]. Cells were then incubated at 37°C for 48 hours before adding 1 μCi [3H] thymidine (MP Biomedicals Inc., Irvine, CA) to each well. Cells were harvested 24 hours later and counted on a liquid scintillation counter (LS3801, Beckman Instruments, Fullerton, CA). Brains and spinal cords were removed from euthanized animals, immersed in 10% neutral buffered formalin and embedded in paraffin wax as described previously.27Brundula V Rewcastle NB Metz LM Bernard CC Yong VW Targeting leukocyte MMPs and transmigration: minocycline as a potential therapy for multiple sclerosis.Brain. 2002; 125: 1297-1308Crossref PubMed Scopus (389) Google Scholar Sections (4 μm) taken from cervical and lumbosacral spinal cords were stained by Bielschowsky's silver impregnation method. Axonal number was quantified by counting silver-positive axonal fibers in four fields in white matter from each spinal cord section and scanned using a Leica DMLB upright microscope and QI Cam digital imaging system (Q Imaging, Pleasanton, CA) to provide digital images. Quantitative analysis of axonal damage was performed using the Adobe Photoshop and the public domain program, Image J as described previously.29Tsutsui S Schnermann J Noorbakhsh F Henry S Yong VW Winston BW Warren K Power C A1 adenosine receptor up-regulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis.J Neurosci. 2004; 24: 1521-1529Crossref PubMed Scopus (271) Google Scholar Immunohistochemistry was performed on sections (4 μm) taken from hippocampi, cerebella, and lumbar spinal cords. Deparaffinized sections were pre-incubated with 10% normal goat serum, 2% bovine serum albumin, and 0.2% Triton X-100 overnight at 4°C to prevent nonspecific binding.29Tsutsui S Schnermann J Noorbakhsh F Henry S Yong VW Winston BW Warren K Power C A1 adenosine receptor up-regulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis.J Neurosci. 2004; 24: 1521-1529Crossref PubMed Scopus (271) Google Scholar Antigen retrieval was achieved as previously reported.30Johnston JB Silva C Gonzalez G Holden J Warren KG Metz LM Power C Diminished adenosine A1 receptor expression on macrophages in brain and blood of patients with multiple sclerosis.Ann Neurol. 2001; 49: 650-658Crossref PubMed Scopus (89) Google Scholar Double staining was performed using Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500 dilution; Molecular Probes, Eugene, OR) to detect the ionized calcium-binding adapter molecule-1 (Iba-1) antibody (1:500; Wako Chemicals, Richmond, VA, Wako, Japan), and Cy-3-conjugated goat anti-mouse secondary antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) to detect the mouse anti-myelin basic protein (MBP) (1:1000 dilution; Sternberger Monoclonals, Lutherville, MD) and anti-CD3 (CD3-ξ, 6B10.2, 1:100 dilution; Santa Cruz Biotech. Inc., Santa Cruz, CA) monoclonal antibodies. Control stains omitted the primary antibody. Images from each spinal cord section were scanned using a Laser Scanning System (LSM 510, Carl Zeiss Canada, Burlington, ON). The quantitative analysis of Iba-1 cell counts per square millimeter and the percentage of MBP-positive area in the white matter of spinal cords were performed as previously described.29Tsutsui S Schnermann J Noorbakhsh F Henry S Yong VW Winston BW Warren K Power C A1 adenosine receptor up-regulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis.J Neurosci. 2004; 24: 1521-1529Crossref PubMed Scopus (271) Google Scholar Animals were euthanized at the onset (12 dpi), peak of clinical disease (17 to 22 dpi), and the chronic phase (60 dpi) of EAE. CNS tissues were dissected-out, homogenized and then lysed in TRIzol (Invitrogen Canada, Burlington, ON) according to the manufacturer's guidelines. Total cellular RNA was isolated, dissolved in diethylpyrocarbonate-treated water; 1 μg of RNA was used for the synthesis of cDNA, and then the real-time PCR reactions were performed as described previously.29Tsutsui S Schnermann J Noorbakhsh F Henry S Yong VW Winston BW Warren K Power C A1 adenosine receptor up-regulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis.J Neurosci. 2004; 24: 1521-1529Crossref PubMed Scopus (271) Google Scholar All mouse primer sequences were previously reported.31Overbergh L Valckx D Waer M Mathieu C Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.Cytokine. 1999; 11: 305-312Crossref PubMed Scopus (523) Google Scholar, 32Martin-Saavedra FM Flores N Dorado B Eguiluz C Bravo B Garcia-Merino A Ballester S Beta-interferon unbalances the peripheral T cell proinflammatory response in experimental autoimmune encephalomyelitis.Mol Immunol. 2007; 44: 3597-3607Crossref PubMed Scopus (40) Google Scholar Semiquantitative analysis was performed by monitoring in real-time the increase of fluorescence of the SYBR-green dye on a Light Cycler (Roche, Canada, Mississauga, ON). Real-time fluorescence measurements were performed and a threshold cycle value for each gene of interest was determined. All data were normalized to GAPDH mRNA expression and expressed as the relative fold-change in mRNA level. Statistical analyses were performed using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA) for both parametric and nonparametric comparisons; P values of less than 0.05 were considered significant. To examine the effects of PrPC deficiency on MOG-induced EAE, we compared disease onset and severity between Prnp−/− mice on a mixed genetic background and their Prnp+/+ littermates. Relative to Prnp+/+ mice, onset of detectable neurological dysfunction occurred earlier in Prnp−/− animals (2.1 ± 0.7 days earlier, P < 0.05 (Figure 1A). No significant difference in clinical disease severity was observed between Prnp+/+ and Prnp−/− mice starting from 12 dpi, and through the first peak of disease and up to approximately 26 dpi (Figure 1B). Prnp+/+ mice reached the peak of disease at 17 dpi, exhibited a partial remission until ∼25 dpi, and this was followed by a second cycle of relapse and remission (Figure 1B). Prnp−/− mice, in contrast, not only failed to recover after the initial peak of disease, but showed increasing EAE severity, leading to sustained neurological impairment that started at approximately 28 dpi and was maintained out to 60 dpi (Figure 1B). The Prnp+/+ and Prnp−/− control mice that were injected with complete Freund's adjuvant plus pertussis toxin alone, and the ‘no EAE’ controls that received no treatment, did not show any signs of neurological disease. To determine whether the phenotypic difference in EAE observed between Prnp+/+ and Prnp−/− mixed background mice would also be observed when mice were more genetically homogenous, we induced EAE in mice that had been backcrossed onto a C57BL/6 genetic background (N = 7 to 8). Mice lacking the prion gene again exhibited earlier onset of EAE, and a more severe clinical disease course than littermate controls, out to 50 dpi (Figure 1C). These two sets of observations, made in mice that differed in their genetic backgrounds, demonstrated that the lack of PrPC was associated not only with worsening of clinical EAE, but particularly with chronic neurological signs suggestive of irreversible CNS damage and/or dysfunction stemming from persistent neuroinflammation. Generation of myelin component-reactive T cells and the subsequent infiltration of autoreactive T cells into the CNS represents a key pathogenic event in EAE.33Hemmer B Archelos JJ Hartung HP New concepts in the immunopathogenesis of multiple sclerosis.Nat Rev Neurosci. 2002; 3: 291-301Crossref PubMed Scopus (497) Google Scholar The finding that PrPC is expressed on cells of the human immune system19Burthem J Urban B Pain A Roberts DJ The normal cellular prion protein is strongly expressed by myeloid dendritic cells.Blood. 2001; 98: 3733-3738Crossref PubMed Scopus (73) Google Scholar, 20Li R Liu D Zanusso G Liu T Fayen JD Huang JH Petersen RB Gambetti P Sy MS The expression and potential function of cellular prion protein in human lymphocytes.Cell Immunol. 2001; 207: 49-58Crossref PubMed Scopus (86) Google Scholar has suggested the possibility of a role for prion protein in the regulation of T cell responses.34Kubosaki A Nishimura-Nasu Y Nishimura T Yusa S Sakudo A Saeki K Matsumoto Y Itohara S Onodera T Expression of normal cellular prion protein (PrP(c)) on T lymphocytes and the effect of copper ion: analysis by wild-type and prion protein gene-deficient mice.Biochem Biophys Res Commun. 2003; 307: 810-813Crossref PubMed Scopus (18) Google Scholar, 35Bainbridge J Walker KB The normal cellular form of prion protein modulates T cell responses.Immunol Lett. 2005; 96: 147-150Crossref PubMed Scopus (37) Google Scholar However, a number of studies of PrPC expression in the murine immune system have shown that while follicular dendritic cells, DCs, and activated lymphocytes in skin, gut- and bronchus-associated and secondary lymphoid tissues express the prion protein, most T and B cells obtained from peripheral lymphoid organs do not express detectable cell surface PrPC.21Liu T Li R Wong BS Liu D Pan T Petersen RB Gambetti P Sy MS Normal cellular prion protein is preferentially expressed on subpopulations of murine hemopoietic cells.J Immunol. 2001; 166: 3733-3742Crossref PubMed Scopus (58) Google Scholar, 22Ford MJ Burton LJ Morris RJ Hall SM Selective expression of prion protein in peripheral tissues of the adult mouse.Neuroscience. 2002; 113: 177-192Crossref PubMed Scopus (170) Google Scholar To assess PrPC expression on cells relevant to EAE pathogenesis, we performed flow cytometric analysis on purified populations of DCs and CD4+ T lymphocytes. After gating (using the isotype control plus secondary antibody), we detected PrPC surface expression on freshly isolated Prnp+/+ DCs (Figure 2, A and B), but not on the negative control population, Prnp−/− DCs. With the anti-PrPC antibody that we used, cell surface expression of PrPC was undetectable on freshly-isolated resting Prnp+/+ CD4+ T cells. However, 48 hours after anti-CD3 antibody stimulation the majority (>80%) of CD4+ cells expressed PrPC (Figure 2, A and B). Thus, although resting naïve CD4+ T cells from wild type mice failed to express detectable PrPC, following antigen receptor-mediated activation these cells clearly express this molecule. These results are consistent with PrPC being an activation marker in murine CD4+ cells, and they demonstrate the potential for this protein to regulate some aspect of CD4+ T cell activation, proliferation, or differentiation. To functionally evaluate PrPC-deficient lymph node T cells, CD4+ T cells from MOG-primed Prnp−/− and Prnp+/+ animals (C57BL/6 background) were co-cultivated with Prnp−/− or Prnp+/+ MOG peptide-pulsed irradiated DCs (isolated from non-immunized mice having a C57BL/6 background) as a source of antigen-presenting cells. T lymphocyte proliferation, as assessed by [3H]-thymidine incorporation, was significantly higher in the MOG-primed Prnp−/− CD4+ lymph node T cells, regardless of whether these were cultivated in the presence of Prnp+/+ or Prnp−/− MOG-pulsed DCs (Figure 2C). The source of the MOG-loaded DCs did not appear to have an effect on the proliferation of the MOG-primed T cells. These results suggested that immunization of Prnp−/− mice with MOG peptide led to the generation of higher numbers of MOG-reactive T cells. Although this in vitro assay failed to reveal a difference in Prnp−/− versus Prnp+/+ DC function in terms of cell proliferation, it did not exclude the possibility of a Prnp−/−-specific DC defect being exerted during the in vivo priming of naïve Prnp−/− T cells. Increased proliferation of Prnp−/− T cells may have accounted in part for the increased numbers of anti-CD3ε-positive cells observed in 12 dpi spinal cords of Prnp−/− mice with EAE (see Supplemental Figure S1 at http://ajp.amjpathol.org). We next determined whether the increased proliferation of Prnp−/− T cells in vitro would be accompanied by the altered expression levels of specific cytokines. We selected two cytokines that correspond to key T cell subsets involved in EAE pathogenesis: (i) interferon (IFN)-γ, a product of Th1 CD4+ T cells, and (ii) interleukin (IL)-17A, a cytokine elaborated by Th17 CD4+ T cells.36Komiyama Y Nakae S Matsuki T Nambu A Ishigame H Kakuta S Sudo K Iwakura Y IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis.J Immunol. 2006; 177: 566-573PubMed Google Scholar, 37Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3280) Google Scholar, 38Park H Li Z Yang XO Chang SH Nurieva R Wang YH Wang Y Hood L Zhu Z Tian Q Dong C A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.Nat Immunol. 2005; 6: 1133-1141Crossref PubMed Scopus (3494) Google Scholar Using real-time RT-PCR, we found that expression of both IFN-γ and IL-17A mRNAs were significantly up-regulated in MOG-primed Prnp−/− CD4+ T cells co-cultivated with either Prnp+/+ or Prnp−/− MOG-pulsed DCs (Figure 2, D and E). Interestingly, IFN-γ and IL-17A transcripts both showed a trend toward greater up-regulation when Prnp−/− CD4+ T cells were cultivated with MOG-pulsed Prnp−/− DCs, however, this did not reach statistical significance. To monitor the effectiveness of MOG-induced T cell activation in the co-cultures we used expression levels of IL-2Rα (CD25) mRNA as an indicator (Figure 2F). Thus, besides showing an increase in both MOG-pulsed DC-induced proliferation and effector cytokine mRNA generation by MOG-primed Prnp−/− T cells, the results suggest that Prnp−/− DCs might have a role in regulating cytokine gene expression by T cells (Figure 2, D and E), although further studies are needed to support this notion. The increased proliferation and cytokine expression of Prnp−/− T cells in response to MOG provides a plausible explanation for disease exacerbation in the prion-deficient mice. Inflammatory cell infiltrates and the cytokines they produce are re
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