Disproportionate Recruitment of CD8+ T Cells into the Central Nervous System by Professional Antigen-Presenting Cells
1999; Elsevier BV; Volume: 154; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65294-7
ISSN1525-2191
AutoresMonica J. Carson, Christina R. Reilly, J. Gregor Sutcliffe, David Lo,
Tópico(s)T-cell and B-cell Immunology
ResumoInappropriate immune responses, thought to exacerbate or even to initiate several types of central nervous system (CNS) neuropathology, could arise from failures by either the CNS or the immune system. The extent that the inappropriate appearance of antigen-presenting cell (APC) function contributes to CNS inflammation and pathology is still under debate. Therefore, we characterized the response initiated when professional APCs (dendritic cells) presenting non-CNS antigens were injected into the CNS. These dendritic cells expressed numerous T-cell chemokines, but only in the presence of antigen did leukocytes accumulate in the ventricles, meninges, subarachnoid spaces, and injection site. Within the CNS parenchyma, the injected dendritic cells migrated preferentially into the white matter tracts, yet only a small percentage of the recruited leukocytes entered the CNS parenchyma, and then only in the white matter tracts. Although T-cell recruitment was antigen specific and thus mediated by CD4+ T cells in the models used here, CD8+ T cells accumulated in numbers equal to or greater than that of CD4+ T cells. Few of the recruited T cells expressed activation markers (CD25 and VLA-4), and those that did were primarily in the meninges, injection site, ventricles, and perivascular spaces but not in the parenchyma. These results indicate that 1) the CNS modulates the cellular composition and activation states of responding T-cell populations and that 2) myelin-restricted inflammation need not be initiated by a myelin-specific antigen. Inappropriate immune responses, thought to exacerbate or even to initiate several types of central nervous system (CNS) neuropathology, could arise from failures by either the CNS or the immune system. The extent that the inappropriate appearance of antigen-presenting cell (APC) function contributes to CNS inflammation and pathology is still under debate. Therefore, we characterized the response initiated when professional APCs (dendritic cells) presenting non-CNS antigens were injected into the CNS. These dendritic cells expressed numerous T-cell chemokines, but only in the presence of antigen did leukocytes accumulate in the ventricles, meninges, subarachnoid spaces, and injection site. Within the CNS parenchyma, the injected dendritic cells migrated preferentially into the white matter tracts, yet only a small percentage of the recruited leukocytes entered the CNS parenchyma, and then only in the white matter tracts. Although T-cell recruitment was antigen specific and thus mediated by CD4+ T cells in the models used here, CD8+ T cells accumulated in numbers equal to or greater than that of CD4+ T cells. Few of the recruited T cells expressed activation markers (CD25 and VLA-4), and those that did were primarily in the meninges, injection site, ventricles, and perivascular spaces but not in the parenchyma. These results indicate that 1) the CNS modulates the cellular composition and activation states of responding T-cell populations and that 2) myelin-restricted inflammation need not be initiated by a myelin-specific antigen. Because it is essential for an organism's survival, the central nervous system (CNS) must be defended from pathogens and other insults. However, inappropriate immune responses within the CNS are thought to initiate or exacerbate neurotoxic and neurodegenerative symptoms observed in a wide variety of neuropathologies. Until recently, the active participation of the CNS in these immune processes was largely ignored. The CNS was assumed to be isolated passively behind the blood-brain barrier (BBB) and to be uninvolved in regulation of immune responses. It has now been realized that leukocyte migration into the CNS is not blocked by the BBB, and the CNS itself suppresses or fails to support the initiation of immune responses. For example, allografts transplanted into the CNS, unlike those transplanted into peripheral sites, are not rejected.1Medawar PB Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to anterior chamber of the eye.Br J Exp Pathol. 1948; 29: 58-69PubMed Google Scholar, 2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar Similarly, rapid T cell responses are mounted against bacillus Calmette-Guerin (BCG) injected into peripheral sites but not sites in the CNS.3Matyszak MK Townsend MJ Perry VH Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen.Neuroscience. 1997; 78: 549-560Crossref PubMed Scopus (28) Google Scholar The CNS is not completely impervious to T-cell responses; peripheral initiation of allograft- or BCG-directed immune responses does lead to T-cell recruitment in the CNS.1Medawar PB Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to anterior chamber of the eye.Br J Exp Pathol. 1948; 29: 58-69PubMed Google Scholar, 2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar, 3Matyszak MK Townsend MJ Perry VH Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen.Neuroscience. 1997; 78: 549-560Crossref PubMed Scopus (28) Google Scholar Yet, as in experimental autoimmune encephalomyelitis (EAE), demyelinating perivenous encephalomyelitis, and multiple sclerosis, T-cell recruitment is anatomically restricted.4Owens T Sriram S The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis.Neurol Clin. 1995; 13: 51-73PubMed Google Scholar, 5Sriram S Rodriguez M Indictment of the microglia as the villain in multiple sclerosis.Neurology. 1997; 48: 464-470Crossref PubMed Scopus (135) Google Scholar, 6Bell MD Taub DD Perry VH Overriding the brain's intrinsic resistance to leukocyte recruitment with intraparenchymal injections of recombinant chemokines.Neuroscience. 1996; 74: 283-292Crossref PubMed Scopus (156) Google Scholar, 7Wekerle H CD4 effector cells in autoimmune diseases of the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 460-492Google Scholar, 8Steinman L A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry.Proc Natl Acad Sci USA. 1996; 93: 2253-2256Crossref PubMed Scopus (194) Google Scholar, 9Gay FW Drye TJ Dick GWA Esri MM The application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis: identification and characterization of the primary demyelinating lesion.Brain. 1997; 120: 1461-1483Crossref PubMed Scopus (278) Google Scholar, 10Berger T Weerth S Kojima K Linington C Wekerle H Lassmann H Experimental autoimmune encephalomyelitis. The antigen specificity of T lymphocytes determines the topography of lesions in the central and peripheral nervous system.Lab Invest. 1997; 76: 355-364PubMed Google Scholar In the early stages of disease, lymphocyte recruitment is enriched in the perivascular, meningeal, and ventricular spaces as compared with the parenchyma. Thus, although some T cells infiltrate the CNS parenchyma, most do not.4Owens T Sriram S The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis.Neurol Clin. 1995; 13: 51-73PubMed Google Scholar, 5Sriram S Rodriguez M Indictment of the microglia as the villain in multiple sclerosis.Neurology. 1997; 48: 464-470Crossref PubMed Scopus (135) Google Scholar, 6Bell MD Taub DD Perry VH Overriding the brain's intrinsic resistance to leukocyte recruitment with intraparenchymal injections of recombinant chemokines.Neuroscience. 1996; 74: 283-292Crossref PubMed Scopus (156) Google Scholar, 7Wekerle H CD4 effector cells in autoimmune diseases of the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 460-492Google Scholar, 8Steinman L A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry.Proc Natl Acad Sci USA. 1996; 93: 2253-2256Crossref PubMed Scopus (194) Google Scholar, 9Gay FW Drye TJ Dick GWA Esri MM The application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis: identification and characterization of the primary demyelinating lesion.Brain. 1997; 120: 1461-1483Crossref PubMed Scopus (278) Google Scholar, 10Berger T Weerth S Kojima K Linington C Wekerle H Lassmann H Experimental autoimmune encephalomyelitis. The antigen specificity of T lymphocytes determines the topography of lesions in the central and peripheral nervous system.Lab Invest. 1997; 76: 355-364PubMed Google ScholarSeveral possible mechanisms are thought to contribute collectively to the high threshold for the activation and infiltration of T cells into the CNS parenchyma. First, the presence of a BBB coupled with the absence of draining lymphatics reduces the number of immune cells and the rate at which immune cells interact with the CNS as compared with other tissues.1Medawar PB Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to anterior chamber of the eye.Br J Exp Pathol. 1948; 29: 58-69PubMed Google Scholar, 2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar, 4Owens T Sriram S The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis.Neurol Clin. 1995; 13: 51-73PubMed Google Scholar Second, major histocompatibility (MHC) class I and MHC class II proteins are expressed at very low levels in the CNS parenchyma as compared with other tissues.2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar, 4Owens T Sriram S The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis.Neurol Clin. 1995; 13: 51-73PubMed Google Scholar, 5Sriram S Rodriguez M Indictment of the microglia as the villain in multiple sclerosis.Neurology. 1997; 48: 464-470Crossref PubMed Scopus (135) Google Scholar, 11Hickey W Kimura H Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.Science. 1988; 239: 290-292Crossref PubMed Scopus (1235) Google Scholar, 12Talmor M Mirza A Turley S Mellman I Hoffman LA Steinman RM Generation of large numbers of immature and mature dendritic cells from rat bone marrow cultures.Eur J Immunol. 1998; 28: 811-817Crossref PubMed Scopus (116) Google Scholar, 13Sedgwick JD Hickey WF Antigen presentation in the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 364-418Google Scholar As a consequence, under normal, nonpathological circumstances, the CNS lacks a resident cell population that is equipped to present antigen to T cells.11Hickey W Kimura H Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.Science. 1988; 239: 290-292Crossref PubMed Scopus (1235) Google Scholar, 13Sedgwick JD Hickey WF Antigen presentation in the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 364-418Google Scholar, 14Carson MJ Reilly CR Sutcliffe JG Lo D Mature microglia resemble immature antigen-presenting cells.Glia. 1998; 22: 72-85Crossref PubMed Scopus (290) Google Scholar By contrast, macrophages in the perivascular spaces, meninges, and choroid plexus do express MHC class I and II proteins and thus may be able to act as antigen-presenting cells (APCs).2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar, 11Hickey W Kimura H Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.Science. 1988; 239: 290-292Crossref PubMed Scopus (1235) Google Scholar, 12Talmor M Mirza A Turley S Mellman I Hoffman LA Steinman RM Generation of large numbers of immature and mature dendritic cells from rat bone marrow cultures.Eur J Immunol. 1998; 28: 811-817Crossref PubMed Scopus (116) Google Scholar, 13Sedgwick JD Hickey WF Antigen presentation in the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 364-418Google Scholar Hence, allografts can be rejected and T-cell responses can be initiated in these latter regions.2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar, 3Matyszak MK Townsend MJ Perry VH Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen.Neuroscience. 1997; 78: 549-560Crossref PubMed Scopus (28) Google Scholar, 11Hickey W Kimura H Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.Science. 1988; 239: 290-292Crossref PubMed Scopus (1235) Google Scholar, 13Sedgwick JD Hickey WF Antigen presentation in the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 364-418Google Scholar Third, the CNS environment may inhibit T-cell activation directly. Ceramides, transforming growth factor (TGF)-β1 production, and antigen-specific interactions between T cells and either astrocytes or microglia have all been suggested to suppress T-cell activation or even to induce T-cell apoptosis in the CNS.15Irani DN Lin KI Griffin DE Regulation of brain-derived T cells during acute central nervous system inflammation.J Immunol. 1997; 158: 2318-2326PubMed Google Scholar, 16Gold R Hartung H-P Lassmann H T cell apoptosis in autoimmune diseases: termination of inflammation in the nervous system and other sites with specialized immune-defense mechanisms.Trends Neurosci. 1997; 20: 399-404Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Gold R Schmied M Tontsch U Hartung HP Wekerle H Toyka KV Lassmann H Antigen presentation by astrocytes primes rat T lymphocytes for apoptotic cell death.Brain. 1996; 119: 651-659Crossref PubMed Scopus (86) Google Scholar, 18Ford AL Foulcher E Lemckert FA Sedgwick JD Microglia induce CD4 T lymphocyte final effector function and death.J Exp Med. 1996; 184: 1737-1745Crossref PubMed Scopus (224) Google Scholar, 19Bonetti B Pohl J Gao YL Raine CS Cell death during autoimmune demyelination. Effector but not target cells are eliminated by apoptosis.J Immunol. 1997; 159: 5733-5741PubMed Google Scholar Thus, although antigen-specific responses may be generated in the perivascular spaces, meninges, ventricles, or even within allografts that contain their own resident APCs, T-cell responses may have difficulty spreading into the CNS parenchyma.In vivo, the relative contributions of these factors have been difficult to ascertain because it is rarely possible to manipulate the parameters individually without global activation of the peripheral immune system or gross changes in CNS physiology. It is unclear whether the development of spontaneous CNS pathologies such as multiple sclerosis are due more to the failures of the CNS to limit immune responses or to the overactivation of leukocytes outside the CNS. Studies of early cases of multiple sclerosis favor the former possibility because the initial demyelinating lesions are composed primarily of activated microglia/macrophages and not lymphocytes.4Owens T Sriram S The immunology of multiple sclerosis and its animal model, experimental allergic encephalomyelitis.Neurol Clin. 1995; 13: 51-73PubMed Google Scholar, 5Sriram S Rodriguez M Indictment of the microglia as the villain in multiple sclerosis.Neurology. 1997; 48: 464-470Crossref PubMed Scopus (135) Google Scholar, 9Gay FW Drye TJ Dick GWA Esri MM The application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis: identification and characterization of the primary demyelinating lesion.Brain. 1997; 120: 1461-1483Crossref PubMed Scopus (278) Google Scholar Lymphocyte accumulation appears fairly restricted to perivascular cuffs in the early stages of disease.Similarly, it is unclear whether the higher failure rate of xenografts as compared with allografts is primarily T cell mediated. Suggestively, allografts in the CNS fail with an increasing rate correlating with increasing antigenic differences between donor and host.1Medawar PB Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to anterior chamber of the eye.Br J Exp Pathol. 1948; 29: 58-69PubMed Google Scholar, 2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar However, in most types of CNS graft failure, very few lymphocytes are found; rather, activated microglia/macrophages appear to mediate most of the graft damage. It is unclear whether the few lymphocytes recruited are sufficient to direct the microglia/macrophage-inflicted damage or whether their contribution to graft failure is of low significance.2Poltorak MP Freed WJ Transplantation into the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 611-641Google Scholar, 8Steinman L A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry.Proc Natl Acad Sci USA. 1996; 93: 2253-2256Crossref PubMed Scopus (194) Google Scholar, 20Tran EH Hoekstra K van Rooijen N Dijkstra CD Owens T Immune invasion of the central nervous system parenchyma and experimental allergic encephalomyelitis, but not leukocyte extravasation from blood, are prevented in macrophage-depleted mice.J Immunol. 1998; 161: 3767-3775PubMed Google ScholarHere, we have tested whether CNS immune privilege results primarily from the absence of an effective resident APC population by injecting bone-marrow-derived dendritic cells directly into the CNS. These dendritic cells are 100- to 1000-fold more potent APCs than macrophages.12Talmor M Mirza A Turley S Mellman I Hoffman LA Steinman RM Generation of large numbers of immature and mature dendritic cells from rat bone marrow cultures.Eur J Immunol. 1998; 28: 811-817Crossref PubMed Scopus (116) Google Scholar In contrast to the CNS, most tissues have similar resident populations of tissue dendritic cells that can efficiently process and present antigen and that frequently recirculate to the lymph nodes.12Talmor M Mirza A Turley S Mellman I Hoffman LA Steinman RM Generation of large numbers of immature and mature dendritic cells from rat bone marrow cultures.Eur J Immunol. 1998; 28: 811-817Crossref PubMed Scopus (116) Google Scholar, 13Sedgwick JD Hickey WF Antigen presentation in the central nervous system.in: Keane RW Hickey WF Immunology of the Nervous System. Oxford University Press, Oxford1997: 364-418Google Scholar Many of the myelin and neural antigens usually used as T-cell targets in EAE are also expressed at very high levels in the thymus and spleen where their presence has been suggested to regulate ongoing T-cell responses.21Bernier L Alvare F Norgard EM Raible DW Mentaberry A Schembri JG Sabatini DD Colman DR Molecular cloning of a 2′,3′-cyclic nucleotide 3′-phosphodiesterase. mRNAs with different 5′ ends encode the same set of proteins in nervous and lymphoid tissues.J Neurosci. 1987; 7: 2703-2710Crossref PubMed Google Scholar, 22Landry CF Ellison JA Pribyl TM Campagnoni C Kampf K Campagnoni AT Myelin basic protein gene expression in neurons.J Neurosci. 1996; 16: 2452-2462PubMed Google Scholar, 23Birnbaum G Kotilinek L Schievert P Clark HB Trotter J Horvath E Gao E Cox M Braun PE Heat shock proteins and experimental autoimmune encephalomyelitis (EAE). I. Immunization with a peptide of the myelin protein 2′,3′ cyclic nucleotide 3′ phosphodiesterase that is cross reactive with a heat shock protein alters the course of EAE.J Neurosci Res. 1996; 44: 381-396Crossref PubMed Scopus (27) Google Scholar, 24Fritz RB Zhao ML Thymic expression of myelin basic protein (MBP): activation of MBP-specific T cells by thymic cells in the absence of exogenous MBP.J Immunol. 1996; 157: 5249-5253PubMed Google Scholar, 25Mackenzie-Graham AJ Pribyl TM Kim S Porter VR Campagnoni AT Voskuhl RR Myelin protein expression is increased in lymph nodes of mice with relapsing experimental autoimmune encephalomyelitis.J Immunol. 1997; 159: 4602-4610PubMed Google Scholar, 26Pribyl TM Campagnoni CW Kampf K Kashima T Handley VW McMahon J Campagnoni AT The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems.Proc Natl Acad Sci USA. 1997; 90: 10695-10699Crossref Scopus (182) Google Scholar Consequently, to study CNS-specific modulation of immune responses independent of CNS epitopes, we used two types of stimuli that have been well characterized in both in vitro assays and peripheral tissue immune responses: 1) allogeneic stimuli in which fewer than 5% to 10% of the T-cell population are potential responders and 2) the moth cytochrome C peptide, in a transgenic mouse model in which greater than 90% of the T cells are potential responders. We found that, whereas potent APCs expressed numerous T-cell chemokines, T cells accumulated in the CNS only in an antigen-specific manner. The antigenic stimuli used were not myelin related and should have triggered primarily CD4+ T cell responses. Surprisingly, although leukocyte recruitment occurred predominantly in the ventricles, meninges, subarachnoid spaces, and the injection site, parenchymal leukocyte infiltration occurred almost exclusively in white matter tracts and consisted of equal numbers of CD4+ and CD8+ cells.Materials and MethodsMiceDendritic-cell cultures were prepared from C57Bl/6, B10.(A)5R, B10.D2, and 107KO mouse strains and injected into C57Bl/6, 107KO, or ANDB6 mice. The AND transgenic mice express a transgenic TCR specific for moth cytochrome c peptide 88–102 presented on class II I-Eb.27Kaye J Hsu L Sauron ME Jameson SC Gascoigne NRJ Hedrick SM Selective development of CD4+ T cells in transgenic mice expressing class II MHC-restricted antigen receptor.Nature. 1989; 341: 746-749Crossref PubMed Scopus (588) Google Scholar The mice are maintained on a C57Bl/6 background; therefore, the TCR-transgenic T cells are positively selected on I-Ab even in the absence of the restricting element I-Eb. Thus, these mice contain clonotype-positive CD4+ T cells (>90% of all T cells) but do not contain APCs that can stimulate antigen-specific responses. Dendritic cells prepared from either the B10.(A)5R and 107KO mouse strains express I-Eb and can act as APCs to transgenic AND T cells.28Lo D Reilly C Burkly L Dekoning J Laufer T Glimcher L Thymic stromal cell specialization and the T cell receptor repertoire.Immunol Res. 1997; 16: 3-14Crossref PubMed Scopus (45) Google Scholar The 107KO mouse line is maintained on a C57Bl/6 background and does not express any MHC class II other than I-Eb.14Carson MJ Reilly CR Sutcliffe JG Lo D Mature microglia resemble immature antigen-presenting cells.Glia. 1998; 22: 72-85Crossref PubMed Scopus (290) Google Scholar, 28Lo D Reilly C Burkly L Dekoning J Laufer T Glimcher L Thymic stromal cell specialization and the T cell receptor repertoire.Immunol Res. 1997; 16: 3-14Crossref PubMed Scopus (45) Google ScholarDendritic Cell IsolationDendritic cells were isolated from bone marrow cultures essentially as described.12Talmor M Mirza A Turley S Mellman I Hoffman LA Steinman RM Generation of large numbers of immature and mature dendritic cells from rat bone marrow cultures.Eur J Immunol. 1998; 28: 811-817Crossref PubMed Scopus (116) Google Scholar Briefly, marrow from femur bones was eluted in RPMI 1640. Cells were recovered by centrifugation and cultured at 1 mouse equivalent per 150-mm plate in RPMI 1640 plus 10% fetal bovine serum (FBS), 25 mmol/L Hepes, 1 mmol/L glutamine, 50 μmol/L 2-mercaptoethanol, 50 U/ml granulocyte/macrophage colony-stimulating factor (GM-CSF), and 100 U/ml interleukin (IL)-4. After 2 days, non-adherent cells were transferred into a new 150-mm plate. Five days after the initiation of the bone marrow cultures, the cells were placed in AIM V media (Gibco/BRL, Gaithersburg, MD), and 48 hours later, dendritic cells were isolated from the non-adherent population in both 150-mm plates by flow cytometric sorting. Dendritic cells were identified by size, side scatter, and high B7.2 expression using fluorescein isothiocyanate (FITC)-conjugated antibodies against B7.2 (Pharmingen, San Diego, CA) and using a FACS Vantage or FACStar Plus with CellQuest acquisition software (Becton Dickinson, Mountain View, CA). FACs analysis demonstrated that these sorted dendritic cell preparations consisted of greater than 95% N418/CD11c+ and NLDC145/Dec 205+ cells.Intrathecal InjectionsAfter isolation, dendritic cells were allowed to recover for 20 minutes at 37°C in RPMI plus 2% FBS, 25 mmol/L Hepes, 50 μmol/L 2-mercaptoethanol in the presence or absence of 2 μg/ml moth cytochrome c peptide 88–102. Cells were washed and resuspended at approximately 2000 to 5000 cells per μl in the same media plus or minus peptide. Approximately 10 μl were injected intrathecally using a 26-gauge needle into metophane-anesthetized mice.Fluorescent Cell LabelingDendritic cells were incubated in RPMI plus 10% FBS and 25 μmol/L Cell Tracker Green (Molecular Probes, Eugene, OR) at 37°C for 20 minutes. As the dye must be cleaved intracellularly to become fluorescent, only viable cells are labeled. By this measure, greater than 90% of the sorted dendritic cells were viable.Microglia Isolation from Mixed Glial CulturesMixed glial cultures were prepared as previously described.29Raible DW McMorris FA Induction of oligodendrocyte differentiation by activators of adenylate cyclase.J Neurosci Res. 1990; 27: 43-46Crossref PubMed Scopus (67) Google Scholar Briefly, CNS from newborn mice were stripped of meninges, mechanically dissociated, seeded into T-75 flasks, and maintained in OM5 media with 10% FBS. After 2 to 4 weeks, cultures were trypsinized and incubated in RPMI (10% FBS, without phenol red) in suspension for 60 minutes at 37°C to allow for the re-expression of trypsinized surface markers. Microglia were then purified by flow cytometry using phycoerythrin (PE)-conjugated antibodies against FcR/CD16/CD32 (Pharmingen) as previously described.14Carson MJ Reilly CR Sutcliffe JG Lo D Mature microglia resemble immature antigen-presenting cells.Glia. 1998; 22: 72-85Crossref PubMed Scopus (290) Google Scholar An extensive characterization of the antigenic phenotype and APC potential of microglia prepared by this method has been previously described.14Carson MJ Reilly CR Sutcliffe JG Lo D Mature microglia resemble immature antigen-presenting cells.Glia. 1998; 22: 72-85Crossref PubMed Scopus (290) Google Scholar The cells were suspended in RPMI plus 2% FBS, 25 mmol/L Hepes, 50 μmol/L 2-mercaptoethanol at approximately 2000 to 5000 cells per μl before intrathecal injection.Preparation of Brain Cell SuspensionsThe brains were removed from halothane-euthanized mice, the meninges were rapidly removed, and the brain tissue was mechanically dissociated into a single-cell suspension in PBS plus 10% serum. The cell suspension was washed twice to remove small debris and was suspended in RPMI plus 2% FBS, 25 mmol/L Hepes, 50 μmol/L 2-mercaptoethanol at approximately 2000 to 5000 cells per μl before intrathecal injection.ImmunohistochemistryMice were sacrificed by halothane inhalation. The brains were rapidly removed and snap-frozen in OCT (Miles Laboratories, Elkhart, IN). Cryostat sections (25 μm) were post-fixed in ice-cold 4.5% paraformaldehyde for 1 minute and then blocked in serial incubations with avidin, biotin, 5% goat serum, and 0.01% Triton X-100. Sections were incubated with primary antibodies overnight at 4°C, followed by incubations with secondary biotin-conjugated antibodies and tertiary streptavidin-horseradish peroxidase-conjugated antibodies. Immunoreactivity was visualized by using 3-amino-9-ethyl carbazole as the chromogen. For GFAP immunoreactivity, CNS tissue was prepared from mice that were perfused through the heart with saline followed by 4.5% paraformaldehyde. Brains were removed and post-fixed overnight in 4.5% paraformaldehyde at 4°C before cryosectioning. Sections were processed as described above.Reverse Transcription Polymerase Chain ReactionRNA was isolated from cells as previously described,30Schibler K Tosi M Pittet AC Fabiani L Wellauer PK Tissue-specific expression of mouse α-amylase genes.J Mol Biol. 1980; 142: 93-116Crossref PubMed Scopus (129) Google Scholar and cDNA was prepared using the Pharmacia first-strand kit according to the manufacturer's directions. Polymerase chain reactions (PCRs) were performed in a Perkin Elmer 9600, with a denaturing temperature of 94°C (15 seconds
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