Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Modulates Experimental Autoimmune Encephalomyelitis via an iNKT Cell-Dependent Mechanism
2009; Elsevier BV; Volume: 175; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.090265
ISSN1525-2191
AutoresMayumi Fujita, Takao Otsuka, Miho Mizuno, Chiharu Tomi, Takashi Yamamura, Sachiko Miyake,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoCarcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a CEA family member that has been reported to have an important role in the regulation of Th1-mediated colitis. In this study, we examined the role of CEACAM1 in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Treatment of C57BL/6J mice with CEACAM1-Fc fusion protein, a homophilic ligand of CEACAM1, inhibited the severity of EAE and reduced myelin oligodendrocyte glycoprotein-derived peptide (MOG35–55)-reactive interferon-γ and interleukin-17 production. In contrast, treatment of these animals with AgB10, an anti-mouse CEACAM1 blocking monoclonal antibody, generated increased severity of EAE in association with increased MOG35–55-specific induction of both interferon-γ and interleukin-17. These results indicated that the signal elicited through CEACAM1 ameliorated EAE disease severity. Furthermore, we found that there was both a rapid and enhanced expression of CEACAM1 on invariant natural killer T cells after activation. The effect of CEACAM1-Fc fusion protein and anti-CEACAM1 mAb on both EAE and MOG35–55-reactive cytokine responses were abolished in invariant natural killer T cell–deficient Jα18−/− mice. Taken together, the ligation of CEACAM1 negatively regulates the severity of EAE by reducing MOG35–55-specific induction of both interferon-γ and interleukin-17 via invariant natural killer T cell-dependent mechanisms. Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a CEA family member that has been reported to have an important role in the regulation of Th1-mediated colitis. In this study, we examined the role of CEACAM1 in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Treatment of C57BL/6J mice with CEACAM1-Fc fusion protein, a homophilic ligand of CEACAM1, inhibited the severity of EAE and reduced myelin oligodendrocyte glycoprotein-derived peptide (MOG35–55)-reactive interferon-γ and interleukin-17 production. In contrast, treatment of these animals with AgB10, an anti-mouse CEACAM1 blocking monoclonal antibody, generated increased severity of EAE in association with increased MOG35–55-specific induction of both interferon-γ and interleukin-17. These results indicated that the signal elicited through CEACAM1 ameliorated EAE disease severity. Furthermore, we found that there was both a rapid and enhanced expression of CEACAM1 on invariant natural killer T cells after activation. The effect of CEACAM1-Fc fusion protein and anti-CEACAM1 mAb on both EAE and MOG35–55-reactive cytokine responses were abolished in invariant natural killer T cell–deficient Jα18−/− mice. Taken together, the ligation of CEACAM1 negatively regulates the severity of EAE by reducing MOG35–55-specific induction of both interferon-γ and interleukin-17 via invariant natural killer T cell-dependent mechanisms. Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), also known as CD66a, is one of the carcinoembryonic antigen family members and is expressed in epithelial cells, endothelial cells, and hematopoietic cells such as monocytes, dendritic cells, natural killer (NK) cells, B cells, and activated T cells.1Prall F Nollau P Neumaier M Haubeck HD Drzeniek U Helmchen U Loning T Wagener C CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.J Histochem Cytochem. 1996; 44: 35-41Crossref PubMed Scopus (173) Google Scholar, 2Kammerer R Hahn S Singer BB Luo JS von Kleist S Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation.Eur J Immunol. 1998; 28: 3664-3674Crossref PubMed Scopus (119) Google Scholar, 3Gray-Owen SD Blumberg RS CEACAM1: contact-dependent control of immunity.Nat Rev Immunol. 2006; 6: 433-446Crossref PubMed Scopus (390) Google Scholar, 4Hammarstrom S The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues.Semin Cancer Biol. 1999; 9: 67-81Crossref PubMed Scopus (963) Google Scholar It is involved in intercellular adhesion through homophilic or heterophilic interactions and mediates regulatory functions in cellular growth and differentiation. Several splice variants of CEACAM1 have been detected, that differ with respect to the number of extracellular immunoglobulin-like domains, membrane anchorage, and the length of their cytoplasmic tail.3Gray-Owen SD Blumberg RS CEACAM1: contact-dependent control of immunity.Nat Rev Immunol. 2006; 6: 433-446Crossref PubMed Scopus (390) Google Scholar Isoforms of CEACAM1 with a long cytoplasmic tail (CEACAM1-L) contain two immunoreceptor tyrosine-based inhibitory motifs and have been shown to negatively regulate epithelial cell activation and tumor cell growth.3Gray-Owen SD Blumberg RS CEACAM1: contact-dependent control of immunity.Nat Rev Immunol. 2006; 6: 433-446Crossref PubMed Scopus (390) Google Scholar, 4Hammarstrom S The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues.Semin Cancer Biol. 1999; 9: 67-81Crossref PubMed Scopus (963) Google Scholar, 5Izzi L Turbide C Houde C Kunath T Beauchemin N Cis-determinants in the cytoplasmic domain of CEACAM1 responsible for its tumour inhibitory function.Oncogene. 1999; 18: 5563-5572Crossref PubMed Scopus (73) Google Scholar Recently, the specific function of CEACAM1 as a regulator of T cells has been reported in vitro and in vivo.6Morales VM Christ A Watt SM Kim HS Johnson KW Utku N Texieira AM Mizoguchi A Mizoguchi E Russell GJ Russell SE Bhan AK Freeman GJ Blumberg RS Regulation of human intestinal intraepithelial lymphocyte cytolytic function by biliary glycoprotein (CD66a).J Immunol. 1999; 163: 1363-1370Crossref PubMed Google Scholar, 7Nakajima A Iijima H Neurath MF Nagaishi T Nieuwenhuis EES Raychowdhury R Glickman J Blau DM Russell S Holmes KV Blumberg RS Activation-induced expression of carcinoembryonic antigen-cell adhesion molecule 1 regulates mouse T lymphocyte function.J Immunol. 2002; 168: 1028-1035Crossref PubMed Scopus (84) Google Scholar, 8Boulton IC Gray-Owen SD Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes.Nat Immunol. 2002; 3: 229-236Crossref PubMed Scopus (243) Google Scholar, 9Markel G Wolf D Hanna J Gazit R Goldman-Wohl D Lavy Y Yagel S Mandelboim O Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions.J Clin Inv. 2002; 110: 943-953Crossref PubMed Scopus (112) Google Scholar, 10Chen D Iijima H Nagaishi T Nakajima A Russell S Raychowdhury R Morales V Rudd CE Utku N Blumberg RS Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulated human T cell function.J Immunol. 2004; 172: 3535-3543Crossref PubMed Scopus (74) Google Scholar, 11Chen CJ Shively JE The cell-cell adhesion molecule carcinoembryonic antigen-related cellular adhesion molecule 1 inhibits IL-2 production and proliferation in human T cells by association with Src homology protein-1 and down-regulates IL-2 receptor.J Immunol. 2004; 172: 3544-3552Crossref PubMed Scopus (58) Google Scholar, 12Iijima H Neurath MF Nagaishi T Glickman JN Nieuwenhuis EE Nakajima A Chen D Fuss IJ Utku N Lewicki DN Becker C Gallagher TM Holmes KV Blumberg RS Specific regulation of T helper cell 1- mediated murine colitis by CEACAM1.J Exp Med. 2004; 199: 471-482Crossref PubMed Scopus (97) Google Scholar Mice treated with CEACAM1-Fc fusion protein, a homophilic ligand for CEACAM1 that stimulates the signal from CEACAM1, exhibited an immunosuppressive effect on Th1-mediated colitis in vivo, with reduced interferon (IFN)-γ production and T-bet activation.12Iijima H Neurath MF Nagaishi T Glickman JN Nieuwenhuis EE Nakajima A Chen D Fuss IJ Utku N Lewicki DN Becker C Gallagher TM Holmes KV Blumberg RS Specific regulation of T helper cell 1- mediated murine colitis by CEACAM1.J Exp Med. 2004; 199: 471-482Crossref PubMed Scopus (97) Google Scholar However, the significance of CEACAM1 on other inflammatory autoimmune disease models remains unclear. Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease induced by sensitization against central nervous system (CNS) components such as myelin oligodendrocyte glycoprotein (MOG).13Wekerle H Experimental autoimmune encephalomyelitis as a model of immune-mediated CNS disease.Curr Opin Neurobiol. 1993; 3: 779-784Crossref PubMed Scopus (74) Google Scholar Because the neurological signs of paralysis can be monitored continuously, and demyelinating lesions resemble those found in multiple sclerosis, EAE is considered an animal model of the human demyelinating disease multiple sclerosis.13Wekerle H Experimental autoimmune encephalomyelitis as a model of immune-mediated CNS disease.Curr Opin Neurobiol. 1993; 3: 779-784Crossref PubMed Scopus (74) Google Scholar, 14Steinman 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 (217) Google Scholar, 15Hemmer B Archelos JJ Hartung HP New concepts in the immunopathogenesis of multiple sclerosis.Nat Rev Neurosci. 2002; 3: 291-301Crossref PubMed Scopus (501) Google Scholar, 16Sospedra M Martin R Immunology of multiple sclerosis.Annu Rev Immunol. 2005; 23: 683-747Crossref PubMed Scopus (1826) Google Scholar Numerous studies have reported that EAE is mediated by CD4+ Th1 cells that produce IFN-γ.13Wekerle H Experimental autoimmune encephalomyelitis as a model of immune-mediated CNS disease.Curr Opin Neurobiol. 1993; 3: 779-784Crossref PubMed Scopus (74) Google Scholar, 14Steinman 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 (217) Google Scholar, 15Hemmer B Archelos JJ Hartung HP New concepts in the immunopathogenesis of multiple sclerosis.Nat Rev Neurosci. 2002; 3: 291-301Crossref PubMed Scopus (501) Google Scholar, 16Sospedra M Martin R Immunology of multiple sclerosis.Annu Rev Immunol. 2005; 23: 683-747Crossref PubMed Scopus (1826) Google Scholar Recently, this idea was questioned because animals deficient in IFN-γ, IFN-γ receptor, or signal transducer and activator of transcription 1 were still found to develop EAE.17Willenborg DO Fordham S Bernard CC Cowden WB Ramshaw IA IFN-γ plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.J Immunol. 1996; 157: 3223-3227Crossref PubMed Google Scholar, 18Krakowski M Owens T Interferon-γ confers resistance to experimental allergic encephalomyelitis.Eur J Immunol. 1996; 26: 1641-1646Crossref PubMed Scopus (423) Google Scholar, 19Willenborg DO Fordham SA Staykova MA Ramshaw IA Cowden WB IFN-γ is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide.J Immunol. 1999; 163: 5278-5286Crossref PubMed Google Scholar, 20Tran EH Prince EN Owens T IFN-γ shapes immune invasion of the central nervous system via regulation of chemokines.J Immunol. 2000; 164: 2759-2768Crossref PubMed Scopus (272) Google Scholar, 21Bettelli E Sullivan B Szabo SJ Sobel RA Glimcher LH Kuchroo VK Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis.J Exp Med. 2004; 200: 79-87Crossref PubMed Scopus (400) Google Scholar These data led the identification of an interleukin (IL)-23 derived population of Th cells, IL-17-producing Th17 cells, as alternative potent inducers of severe autoimmunity, including EAE.22Cua DJ Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.Nature. 2003; 421: 744-748Crossref PubMed Scopus (2403) Google Scholar, 23Steinman L A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell mediated tissue damage.Nat Med. 2007; 13: 139-145Crossref PubMed Scopus (1135) Google Scholar, 24Komiyama 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: 566-573Crossref PubMed Scopus (1325) Google Scholar However, mice deficient in T-bet and signal transducer and activator of transcription 4, which thus lack Th1 cells, but have large numbers of Th17 cells, are still resistant to EAE.21Bettelli E Sullivan B Szabo SJ Sobel RA Glimcher LH Kuchroo VK Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis.J Exp Med. 2004; 200: 79-87Crossref PubMed Scopus (400) Google Scholar, 25Chitinis T 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 Additionally, Th1 and Th17 cells are observed in the CNS at the peak of EAE and diminish after the recovery.26Korn T Myelin-specific regulatory T-cells accumulate in the central nervous system, but fail to suppress pathogenic effector T-cells at the peak of autoimmune inflammation.Nat Med. 2007; 13: 423-431Crossref PubMed Scopus (685) Google Scholar It has now been described that Th1 and Th17 cells might cooperate to induce the development of EAE.27Bettelli E Oukka M Kuchroo VK TH-17 cells in the circle of immunity and autoimmunity.Nat Immunol. 2007; 8: 345-350Crossref PubMed Scopus (1285) Google Scholar, 28Stromnes IM Cerretti LM Liggitt D Harris RA Goverman JM Differential regulation of central nervous system autoimmunity by Th1 and Th17 cells.Nat Med. 2007; 14: 337-342Crossref Scopus (491) Google Scholar, 29Kroenke MA Carlson TJ Andjelkovic AV Segal BM IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology. CNS chemokine profile, and response to cytokine inhibition.J Exp Med. 2008; 205: 1535-1541Crossref PubMed Scopus (491) Google Scholar Thus, elucidation of the mechanisms that regulate the production of both Th1 and Th17 cytokines is important in relation to the regulation of EAE. In this study, we investigated the role of CEACAM1 in EAE either by CEACAM1 ligation with a homophilic ligand for CEACAM1 (CEACAM1-Fc fusion protein), or by blocking with a CEACAM1-specific antibody, AgB10. Here, we demonstrate that signaling through CEACAM1 suppressed MOG-derived peptide (MOG35–55)-induced EAE associated with a reduction in MOG35–55-specific T cell production of IFN-γ and IL-17. Moreover, we have identified invariant natural killer T (iNKT) cells as a critical component in CEACAM1-mediated suppression of EAE. iNKT cells are an unique subset of CD1-restricted T cells that express an invariant T cell receptor (TCR) α chain, composed of Vα14-Jβ18 segments in mice and Vα14-Jβ18 segments in humans, and use a restricted set of Vβ genes.30Bendela A Savage PB Teyton L The biology of NKT cells. 2007; 25: 297-336Google Scholar, 31Kronenberg M Toward an understanding of NKT cell biology: progress and paradoxes.Annu Rev Immunol. 2005; 23: 877-900Crossref PubMed Scopus (890) Google Scholar Due to the ability to produce a wide variety of cytokines, iNKT cells are thought to play regulatory roles in autoimmune diseases.32Miyake S Yamamura T Therapeutic potential of CD1d-restricted invariant natural killer T cell-based treatment for autoimmune diseases.Int Rev Immunol. 2007; 26: 73-94Crossref PubMed Scopus (9) Google Scholar CEACAM1-mediated suppression of EAE was not observed in iNKT cell-deficient Jα18−/− mice, and MOG35–55-specific T cell production of IFN-γ and IL-17 was not modified in Jα18−/− mice when treated with either CECAM1-Fc fusion protein or AgB10. C57BL/6J (B6) mice were obtained from CLEA Japan Inc. (Tokyo, Japan). Jα18−/− mice were kindly provided by Dr. M. Taniguchi (RIKEN, Tokyo, Japan). All animals were maintained in specific pathogen-free conditions in accordance with institutional guidelines of National Institute of Neuroscience, Tokyo, Japan. MOG35–55 (amino acid sequence, MEVGWYRSPFSRVVHLYRNGK) was synthesized at Toray Research Center (Tokyo, Japan). Incomplete Freund’s adjuvant and heat-killed mycobacterium tuberculosis (H37Ra) were obtained from Difco Laboratories (Detroit, Michigan), and pertussis toxin was obtained from List Biological Laboratories (California). The hybridoma producing CEACAM1-specific antibody, AgB10,33Kuprina NI Baranov VN Yazova AK Rudinskaya TD Escribano M Cordier J Gleiverman AS Goussev AI The antigen of bile canaliculi of the mouse hepatocyte: identification and ultrastructural localization.Histchemistry. 1990; 94: 179-186Crossref PubMed Scopus (15) Google Scholar was kindly provided by Nicole Beauchemin (McGill Cancer Center), and 293 EBNA cells transfected pCEP4-N-CEACAM-Fc, which produce a homophilic ligand of CEACAM1, CEACAM1-Fc fusion protein were kindly provided by Thomas M. Gallagher (Loyola University Medical Center).34Gallagher TM A role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor.J Virol. 1997; 71: 3129-3137Crossref PubMed Google Scholar EAE was induced in mice as described previously.35Miyamoto K Miyake S Mizuno M Oka N Kusunoki S Yamamura T Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyelitis through COX-2-independent pathway.Brain. 2006; 129: 1984-1992Crossref PubMed Scopus (63) Google Scholar Briefly, mice were immunized subcutaneously with 100 μg of MOG35–55 emulsified in incomplete Freund’s adjuvant containing 500 μg of M. tuberculosis. Directly after the immunization and 48 hours later, mice were injected intraperitoneally with 200 ng of pertussis toxin. Clinical signs of EAE were assessed daily with a 0 to 6 scoring system (0, no signs; 1, partial loss of tail tonicity; 2, completely limp tail and abnormal gait; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, fore- and hindlimb paralysis or moribund state; 6, dead). The hybridomas producing AgB10 were cultured in a humidified atmosphere with 5% CO2 at 37°C in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L l-glutamine, and 100 U/ml penicillin/streptomycin. The supernatants were collected and AgB10 was affinity-purified using a protein A column according to the manufacturer’s instructions (Millipore, MA). 293 EBNA cells transfected pCEP4-N-CEACAM1-Fc were cultured in DMEM containing 10% heat-inactivated fetal calf serum, 2 mmol/L l-glutamine, and 100 U/ml penicillin/streptomycin. CEACAM1-Fc fusion protein was affinity-purified using protein G column from the collected supernatants (Amersham Bioscience, NJ). After immunization with MOG35–55, mice were treated intraperitoneally with the indicated compounds, either 250 μg of AgB10 or 250 μg of control rat IgG antibody (Jackson Immuno Research, PA), or either 250 μg of CEACAM1-Fc fusion protein or 250 μg of a chimeric (mouse/human) anti-human CD20 mAb (rituximab) every second day from the day of immunization, day 0, to day 11. The animals were sacrificed at day 11 and inguinal and popliteal lymph nodes (LN) were sampled. Total LN cells were suspended in RPMI 1640 medium containing 2% syngeneic mouse serum, 2 mmol/L l-glutamine, 5 × 10−5 M/L 2-mercaptoethanol, and 100 U/ml penicillin/streptomycin, and were incubated in 96-well plates with 1 × 106 cells/well in the presence of 0, 1, 10, 30, or 100 mg/ml of MOG35–55. Culture supernatant was collected 48 hours after stimulation, and IFN-γ and IL-17 in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) using OptEIA kit (BD Bioscience, CA) and IL-17 ELISA kit (R&D systems), respectively. Sixteen days after the immunization with MOG35–55, the spinal cords were sampled and stored in 10% formaldehyde. Paraffin-embedded spinal cords were stained with either H&E or luxol fast blue. Liver mononuclear cells from B6 mice were isolated by Percoll density-gradient centrifugation. 1 × 106 cells/well were stimulated with 1 mg/ml plate-bound anti-CD3 mAb and 2.5 mg/ml Concanavalin A (ConA) in 96-well plates and collected for the use of flow cytometry. Cells were stained with α-galactosylceramide (α-GC) loaded dimeric mouse CD1 days followed by fluorescein isothiocyanate-conjugated AgB10, phycoerythrin-conjugated mAb A85-1, and allophycocyanin-conjugated anti-TCR β-chain. iNKT cells were gated as α-GC loaded CD1 days dimmer and TCRβ double-positive cells, and T cells were gated as TCRβ single-positive cells. Stained cells were analyzed using a FACSCalibur with CellQuest Software (Becton Dickinson, CA). B6 mice were treated intraperitoneally with either 500 μg of AgB10 or 500 μg of control rat IgG antibody. Four days after the treatment, 250 μl of blood was collected at 2 or 6 hours after intravenous injection with 0.6 μg α-GC/dimethyl sulfoxide or control dimethyl sulfoxide. Blood samples were centrifuged at 3000 rpm for 30 minutes at 4°C, and serum was collected and IFN-γ and IL-4 were determined using ELISA kit (BD Bioscience, CA). EAE clinical scores for groups of mice are presented as the mean group clinical score ± SEM, and statistical differences were analyzed by the Mann-Whitney U nonparametric ranking test. Data for cytokines were analyzed with the two-way analysis of variance. In appropriate cases, post hoc comparisons were made. To assess the role of CEACAM1 on EAE, we first examined the effect of CEACAM1-Fc fusion protein encoding the extracellular portion of the mCEACAM1-4L. CEACAM1-Fc fusion protein has been demonstrated to homophilically ligate the CEACAM1 molecule, which has been shown to inhibit IFN-γ production.12Iijima H Neurath MF Nagaishi T Glickman JN Nieuwenhuis EE Nakajima A Chen D Fuss IJ Utku N Lewicki DN Becker C Gallagher TM Holmes KV Blumberg RS Specific regulation of T helper cell 1- mediated murine colitis by CEACAM1.J Exp Med. 2004; 199: 471-482Crossref PubMed Scopus (97) Google Scholar As shown in Figure 1A, administration of CEACAM1-Fc fusion protein significantly inhibited the development and the progression of EAE compared with control mice. To characterize the immunosuppressive effect of CEACAM1, we performed the pathological analysis of CNS inflammation and demyelination in EAE-induced mice treated with CEACAM1-Fc fusion protein (Figure 1B). Histological examination of the spinal cord 16 days after EAE induction revealed less cellular infiltration and demyelination in CEACAM1-Fc fusion protein-treated mice, as compared with control mice. We next examined the effects of CEACAM1 specific antibody, AgB10, on the development and progression of MOG35–55-induced EAE in B6 mice (Figure 1C). Ligation of CEACAM1, either homophilically by CEACAM1-Fc fusion protein or heterophilically by microbial components such as the spike glycoprotein of murine hepatitis virus, has been demonstrated to inhibit the proliferation and cytokine production of T cells.6Morales VM Christ A Watt SM Kim HS Johnson KW Utku N Texieira AM Mizoguchi A Mizoguchi E Russell GJ Russell SE Bhan AK Freeman GJ Blumberg RS Regulation of human intestinal intraepithelial lymphocyte cytolytic function by biliary glycoprotein (CD66a).J Immunol. 1999; 163: 1363-1370Crossref PubMed Google Scholar, 7Nakajima A Iijima H Neurath MF Nagaishi T Nieuwenhuis EES Raychowdhury R Glickman J Blau DM Russell S Holmes KV Blumberg RS Activation-induced expression of carcinoembryonic antigen-cell adhesion molecule 1 regulates mouse T lymphocyte function.J Immunol. 2002; 168: 1028-1035Crossref PubMed Scopus (84) Google Scholar, 8Boulton IC Gray-Owen SD Neisserial binding to CEACAM1 arrests the activation and proliferation of CD4+ T lymphocytes.Nat Immunol. 2002; 3: 229-236Crossref PubMed Scopus (243) Google Scholar, 9Markel G Wolf D Hanna J Gazit R Goldman-Wohl D Lavy Y Yagel S Mandelboim O Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions.J Clin Inv. 2002; 110: 943-953Crossref PubMed Scopus (112) Google Scholar, 10Chen D Iijima H Nagaishi T Nakajima A Russell S Raychowdhury R Morales V Rudd CE Utku N Blumberg RS Carcinoembryonic antigen-related cellular adhesion molecule 1 isoforms alternatively inhibit and costimulated human T cell function.J Immunol. 2004; 172: 3535-3543Crossref PubMed Scopus (74) Google Scholar, 11Chen CJ Shively JE The cell-cell adhesion molecule carcinoembryonic antigen-related cellular adhesion molecule 1 inhibits IL-2 production and proliferation in human T cells by association with Src homology protein-1 and down-regulates IL-2 receptor.J Immunol. 2004; 172: 3544-3552Crossref PubMed Scopus (58) Google Scholar, 12Iijima H Neurath MF Nagaishi T Glickman JN Nieuwenhuis EE Nakajima A Chen D Fuss IJ Utku N Lewicki DN Becker C Gallagher TM Holmes KV Blumberg RS Specific regulation of T helper cell 1- mediated murine colitis by CEACAM1.J Exp Med. 2004; 199: 471-482Crossref PubMed Scopus (97) Google Scholar In contrast, AgB10 has been reported to enhance the T cell proliferation, indicating that AgB10 acts as a blocking antibody. As expected, the clinical scores of EAE were augmented in the mice treated with AgB10 compared with those of control mice. These results indicate that signals through CEACAM1 suppressed both the clinical and the pathological severity of EAE. Since MOG35–55 induced EAE is thought to be mediated by MOG35–55-specific Th1 and Th17 cells, we next examined MOG35–55-specific T cell responses in CEACAM1-Fc fusion protein-treated (Figure 2A), or AgB10-treated mice (Figure 2B). We immunized mice with MOG35–55 and treated them with either AgB10 or CEACAM1-Fc fusion protein. Twelve days later, we harvested LN cells and re-stimulated them with MOG35–55 peptide in vitro to examine cytokine production and proliferation. Compared with cells from the control mice, LN cells obtained from CEACAM1-Fc fusion protein treated mice were significantly inhibited in IFN-γ and IL-17 production in responses to MOG35–55 re-stimulation (Figure 2A). IL-4 was not detected in the supernatant. On the other hand, in vivo treatment with AgB10 showed an enhancement of IFN-γ and IL-17 production in response to MOG35–55 stimulation (Figure 2B). Proliferative responses were not significantly different between control mice, CEACAM1-Fc protein-treated, or AgB10-treated mice (data not shown). These results indicate that the suppressive effect of CEACAM1 on EAE was associated with reduction of MOG35–55-specific IFN-γ and IL-17 production. It has been reported that CEACAM1 is expressed on T cells early after activation, and its ligation directly inhibits IFN-γ production by such T cells. We therefore examined the time course of CEACAM1 expression by T cells in vitro. As reported previously, CEACAM1 expression was observed on T cells several hours after activation with ConA and anti-CD3 mAb in vitro. Moreover, we observed that there was a rapid and higher expression of CEACAM1 by CD1-restricted iNKT cells after activation (Figure 3A). The log fluorescence intensity of CEACAM1 on surface of iNKT and T cells and the percentage of CEACAM1 expressed cells within total iNKT or T cells showed a rapid and also enhanced expression of CEACAM1 on iNKT cells compared with T cells after activation (Figure 3B). iNKT cells possess the ability to produce a wide variety of cytokines. Activation of iNKT cells is known to lead to either suppressive or stimulatory immune responses depending on the type of cytokine they produce.30Bendela A Savage PB Teyton L The biology of NKT cells. 2007; 25: 297-336Google Scholar We have demonstrated the rapid and enhanced expression of CEACAM1 specifically on iNKT cells (Figure 3A). Thus we next examined whether or not the administration of AgB10 has an effect on cytokine production by iNKT cells. Mice were injected intravenously with iNKT cell-specific ligand, α-GC, or vehicle, and serum levels of IFN-γ and IL-4 were measured. Mice pretreated with AgB10 and injected with α-GC showed significantly increased level of IFN-γ, as compared with mice treated with control antibody and injected with α-GC (Figure 4A). No significant difference was observed in IL-4 production (Figure 4B). The level of IL-12 in serum was not altered in AgB10-treated mice, and IL-17, IL-21, or IL-23 were not detected in the serum (data not shown). The results suggest that the signal from CEACAM1 have a role in IFN-γ production by iNKT cells. Since iNKT cells highly express CEACAM1 after activation, it was of interest to investigate whether the iNKT cells are involved in CEACAM1-mediated amelioration of EAE. To address this question, we examined the effect of CEACAM1-Fc fusion protein on the development of MOG35–55-induced EAE in Jα18−/− mice, which genetically lack iNKT cells. In contrast to B6 mice, no alteration in the severity of EAE was observed in CEACAM1-Fc fusion protein treated Jα18−/− mice, as compared with control mice (Figure 5A). To further determine the effect of the ligation of CEACAM1 on EAE in Jα18−/− mice, we analyzed the CNS inflammation and demyelination in EAE-induced Jα18−/− mice treated with CEACAM1-Fc fusion protein. In contrast to wild-type B6 mice, histological examination of the spinal cord of Jα18−/− mice showed cellular infiltration and demyelination to a similar extent as sham-treated mice (Figure 5B). We next induced EAE in Jα18−/− mice treated with either AgB10 or control antibody. Again, no suppression of clinical EAE was observed in AgB10- treated Jα18−/− mice, as compared with the control mice (Figure 5C). These data show that CEACAM1 signal modulation does not affect on the severity of clinical and pathological EAE in mice lacking iNKT cells. The suppression of EAE by the ligation of CECAM1 in B6 mice was associated with a reduction in MOG35–55-specific IFN-γ and IL-17 production. We next examined MOG35–55-specific T cell responses in CEACAM1-Fc fusion protein-treated (Figure 6A), or AgB10-treated Jα18−/− mice (Figure 6B) by ex vivo re-challenge with MOG35–55 on day 11 after the immunization of MOG35–55. In contrast to B6 mice, LN cells from CEACAM1-Fc fusion protein-treated Jα18−/− mice exhibited no significant reduction of MOG35–55 specific IFN-γ and IL-17 production compared with the control mice (Figure 6A). Additionally, in vivo treatment of Jα18−/− mice with AgB10 also did not significantly enhance of MOG35–55-specific T cell IFN-γ and IL-17 production (Figure 6B). These results indicate that iNKT cells play an important role in CEACAM1-mediated reduction of MOG-specific IFN-γ and IL-17 production. The present study demonstrated that the signal through CEACAM1 suppressed EAE in association with a reduction in MOG35–55-specific production of IFN-γ and IL-17. Moreover, we showed that CEACAM1 was expressed at an early time point by iNKT cells after activation and CEACAM1 also affected the cytokine production by iNKT cells, including IFN-γ, but not IL-4. Finally, we demonstrated that CEACAM1-mediated modulation of EAE and MOG35–55-specific cytokine production required iNKT cells. Since both IFN-γ and IL-17 are known as potent inducers of EAE,21Bettelli E Sullivan B Szabo SJ Sobel RA Glimcher LH Kuchroo VK Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis.J Exp Med. 2004; 200: 79-87Crossref PubMed Scopus (400) Google Scholar, 27Bettelli E Oukka M Kuchroo VK TH-17 cells in the circle of immunity and autoimmunity.Nat Immunol. 2007; 8: 345-350Crossref PubMed Scopus (1285) Google Scholar, 28Stromnes IM Cerretti LM Liggitt D Harris RA Goverman JM Differential regulation of central nervous system autoimmunity by Th1 and Th17 cells.Nat Med. 2007; 14: 337-342Crossref Scopus (491) Google Scholar, 29Kroenke MA Carlson TJ Andjelkovic AV Segal BM IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology. CNS chemokine profile, and response to cytokine inhibition.J Exp Med. 2008; 205: 1535-1541Crossref PubMed Scopus (491) Google Scholar CEACAM1-mediated reduction of these cytokines is thought to have a significant role in ameliorating EAE. Although the mechanisms of IFN-γ and IL-17 reduction in CEACAM1-mediated suppression of EAE are not clearly defined so far, we found that the effects of AgB10 and CEACAM1-Fc fusion proteins on EAE and MOG35–55-reactive cytokine responses were abolished in iNKT cell-deficient Jα18−/− mice. Thus we concluded that CEACAM1-mediated suppression of EAE was mediated via iNKT cells. Activation of iNKT cells are known to modulate dendritic cell functions, and Kammerer et al reported that AgB10 triggered release of IL-12 from dendritic cells and facilitated priming of naive CD4+ T cells with a Th1-like phenotype.36Kammerer R Stober D Singer BB Obrink B Reimann J Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells in a potent regulator or T cell stimulation.J Immunol. 2001; 166: 6537-6544Crossref PubMed Scopus (76) Google Scholar In contrast, Iijima et al showed that CEACAM1-mediated inhibition of Th1-mediated colitis was not dependent on the modulation of IL-12, consistent with this finding, IL-12 was not affected in EAE-induced mice by the in vivo treatment of AgB10. Since iNKT cells have been shown to produce IL-21, which promotes the development of Th17 cells,37Korn T Bettelli E Gao W Awasthi A Jager A Strom TB Oukka M Kuchroo VK IL-21 initiates an alternative pathway to induce proinflammatory TH17 cells.Nature. 2007; 448: 484-487Crossref PubMed Scopus (1535) Google Scholar CEACAM1 expression by iNKT cells may have a regulatory role in IL-17 production by Th17 cells via IL-21. However, the production of IL-21 upon iNKT cell activation was not altered by treatment with AgB10. In addition, production of IL-23, which promotes Th17 cell maintenance by activated iNKT cells was not altered in mice treated with AgB10, as compared with control mice. Therefore, the mechanisms how CEACAM1-treated iNKT cells modulate MOG35–55 reactive Th1 and Th17 cells remain to be elucidated. Recently, Mars et al reported that activation of iNKT cells with α-GarCer during priming of the CD4+ T cell response prevents the differentiation of naïve CD4+ T cells toward the Th17 lineage, and the cytokine neutralization experiments indicated that IL-4, IL-10, and IFN-γ are involved in the iNKT cell-mediated regulation of T cell lineage development.38Mars LT Araujo L Kerschen P Diem S Bourgeois E Van LP Carrie N Dy M Liblau RS Herbelin A Invariant NKT cells inhibit development of the Th17 lineage.Proc Natl Acad Sci USA. 2009; 106: 6238-6243Crossref PubMed Scopus (61) Google Scholar Although the direct mechanisms of iNKT cells in regulating the Th17 compartment are still in question, iNKT cells were shown to have a regulatory role in development of the Th17 lineage. Our laboratory reported that antibiotic treatment alters the composition of gut flora, resulting in amelioration of EAE in a iNKT cell-dependent manner.39Yokote H Miyake S Croxford JL Oki S Mizusawa H Yamamura T NKT cell-dependent amelioration of a mouse model of multiple sclerosis by altering gut flora.Am J Pathol. 2008; 173: 1714-1723Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar iNKT cell-dependent amelioration of EAE was associated with the suppression of MOG35–55- reactive Th17 cells, although the mechanism by which iNKT cells modulate MOG35–55-reactive Th17 cells remained unclear. It was speculated that altering the compositions of gut flora by antibiotic treatment critically influences the function of iNKT cells, which resulted in a reduction of MOG35–55-reactive Th17 cells. Since various bacterial and viral pathogens trans-ligate CEACAM1 and suppresses the activation and proliferation of T cells, it is possible that the alteration of cytokine production in physiological or pathological conditions is partly dependent on the way of trans-ligation of pathogens and CEACAM1 on iNKT cells.3Gray-Owen SD Blumberg RS CEACAM1: contact-dependent control of immunity.Nat Rev Immunol. 2006; 6: 433-446Crossref PubMed Scopus (390) Google Scholar, 12Iijima H Neurath MF Nagaishi T Glickman JN Nieuwenhuis EE Nakajima A Chen D Fuss IJ Utku N Lewicki DN Becker C Gallagher TM Holmes KV Blumberg RS Specific regulation of T helper cell 1- mediated murine colitis by CEACAM1.J Exp Med. 2004; 199: 471-482Crossref PubMed Scopus (97) Google Scholar, 40Gray-Owen SD Lorenzen DR Hude A Meyer TF Dehio C Differential Opa specificities for CD66 receptors influence tissue interactions and cellular response to Neisseria Gonorrhoeae.Mol Microbiol. 1997; 26: 971-980Crossref PubMed Scopus (129) Google Scholar, 41Virji M Makepeace K Ferguson DJP Watt SM Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae.Mol Microbiol. 1996; 22: 941-950Crossref PubMed Scopus (251) Google Scholar, 42Hill DJ Toleman MA Evans DJ Villullas S Van AL Virji M The variable P5 proteins of typeable and non-typeable Haemophilus influenzae target human CEACAM1.Mol Microbiol. 2001; 39: 850-862Crossref PubMed Scopus (98) Google Scholar, 43Toleman M Aho E Virji M Expression of pathogen-like Opa adhesions in commensal Neisseria: genetic and functional analysis.Cell Microbiol. 2001; 3: 33-44Crossref PubMed Scopus (51) Google Scholar, 44Virji M Evams D Griffith J Hill D Serino L Hadfield A Watt SM Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzae.Mol Microbiol. 2000; 36: 784-795Crossref PubMed Scopus (95) Google Scholar, 45Berger CN Billker O Meyer TF Servin AL Kansau I Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia clo (Afa/Dr DAEC).Mol Microbiol. 2004; 52: 963-983Crossref PubMed Scopus (106) Google Scholar In conclusion, this study demonstrates for the first time that CEACAM1 negatively regulates the severity of EAE via an iNKT cell-dependent mechanism. Considering that the selective induction of cytokines by iNKT cells by synthetic ligands has been reported to suppress EAE,32Miyake S Yamamura T Therapeutic potential of CD1d-restricted invariant natural killer T cell-based treatment for autoimmune diseases.Int Rev Immunol. 2007; 26: 73-94Crossref PubMed Scopus (9) Google Scholar, 46Miyamoto K Miyake S Yamamura T A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells.Nature. 2001; 413: 531-534Crossref PubMed Scopus (781) Google Scholar CEACAM1 may prove to be a novel target for immunotherapy of multiple sclerosis. We thank Masaru Taniguchi at Riken Research Center for Allergy and Immunology (Yokohama, Japan) for providing Jα18−/− mice. We thank Thomas M. Gallagher at Loyola University Medical Center (Maywood, IL) for providing 293 EBNA cells transfected with pCEP4-N-CEACAM1-Fc. We thank Nicole Beauchemin (McGill Cancer Center). We are grateful to Ben J.E. Raveney for critical reading of the manuscript.
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