RESUMO
Microglia, a type of tissue macrophage, are the only cells in the central nervous system (CNS) parenchyma to express some major histocompatibility complex (MHC) class II constitutively or to upregulate expression readily. They are thought to play a role in CD4 T cell activation in autoimmune diseases such as multiple sclerosis, as well as in neurodegenerative conditions, Alzheimer's disease in particular. We show here that highly purified MHC class II+ microglia when tested directly ex vivo do indeed support an effector response by an encephalitogenic myelin basic protein-reactive CD4 T cell line from which production of the proinflammatory cytokines, interferon gamma and tumor necrosis factor, is elicited, but not interleukin (IL)-2 secretion or proliferation. After this interaction, the T cells die by apoptosis. Other nonmicroglial but CNS-associated macrophages isolated in parallel stimulate full T cell activation, including IL-2 production, proliferation, and support T cell survival. Neither CNS-derived population expresses B7.1/B7.2. Resident macrophages that terminate effector T cells in tissues constitute a novel and broadly applicable regulatory measure of particular relevance to processes of self-tolerance against sequestered antigens.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária , Macrófagos/imunologia , Microglia/imunologia , Animais , Células Apresentadoras de Antígenos , Antígenos CD/análise , Doenças Autoimunes/etiologia , Antígeno B7-1/análise , Antígeno B7-2 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Sistema Nervoso Central/citologia , Anergia Clonal , Doença Enxerto-Hospedeiro , Antígenos de Histocompatibilidade Classe II , Interferon gama/biossíntese , Interleucina-2/farmacologia , Antígenos Comuns de Leucócito , Glicoproteínas de Membrana/análise , Proteína Básica da Mielina/imunologia , Ratos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
OBJECTIVE: To define a clear ex vivo flow cytometric phenotype for adult human microglia that would distinguish it from all other macrophage lineage cells in the central nervous system (CNS) or blood, and to utilize this phenotype to examine the activation state and CD4 expression of microglia freshly derived from CNS tissue of HIV-positive patients and those with other neurological diseases. DESIGN: Fresh human CNS tissue from both HIV-uninfected and HIV-infected individuals was obtained by biopsy or resection, and cells isolated immediately, labelled for flow cytometry and analysed. METHODS: A Percoll density gradient isolation technique and phenotypic characteristics used for rodent microglia were applied and modified. RESULTS: Resident microglia could clearly be defined by the flow cytometric phenotype CD45low CD4- CD11b+ CD11chigh major histocompatibility complex (MHC) class II+ CD26- CD14-. Assuming normally low-level MHC class II expression in the healthy CNS, it was likely that MHC class II positivity reflected underlying pathology necessitating biopsy or resection and appeared to be a 'leaky' activation marker. Microglia activation was observed in specimens from only six (35%) out of 17 HIV-uninfected but all four (100%) HIV-infected patients, defined strictly as any level of upregulation of CD4 expression, to produce the phenotype CD45low/medium CD4low CD11b+ CD1.1Chigh MHC class II+/+2 CD26- CD14-. Where examined by immunohistology, CD68 was also upregulated in these cases. CONCLUSIONS: When activated in situ, microglia express low levels of CD4 and this is always seen in tissue from HIV-infected patients. Using the flow cytometric phenotype established here, microglia from HIV-infected tissue can now be isolated in pure form and studied directly ex vivo.
Assuntos
Encéfalo/metabolismo , Antígenos CD4/biossíntese , Citometria de Fluxo/métodos , Soropositividade para HIV/metabolismo , Microglia/metabolismo , Adulto , Animais , Encéfalo/citologia , Soropositividade para HIV/patologia , Humanos , Antígenos Comuns de Leucócito/biossíntese , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Camundongos , Microglia/citologia , FenótipoRESUMO
Dispase was used to digest central nervous system (CNS) tissue for isolation of the fixed tissue macrophage population (CNS microglia) and other leucocytes present. An apparent reduction in expression of some leucocyte cell surface markers was investigated further by addition of CD45b allotype-marked post-activated CD4+ T cells of known phenotype to CNS tissue preparations derived from a CD45a-expressing rat strain, before enzymatic treatment. Assessment of these T cells for expression of a range of surface molecules after completion of the isolation procedure revealed almost complete loss of expression of CD4 and CD25 and reduced expression of a number of other surface antigens.
Assuntos
Antígenos CD/análise , Endopeptidases/farmacologia , Macrófagos/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD4/análise , Feminino , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Masculino , Ratos , Ratos Endogâmicos Lew , Receptores de Interleucina-2/análiseRESUMO
Central nervous system (CNS)-resident macrophages (microglia) normally express negligible or low level MHC class II, but this is up-regulated in graft-vs-host disease (GvHD), in which a sparse CNS T cell infiltrate is observed. Relative to microglia from the normal CNS, those from the GvHD-affected CNS exhibited a 5-fold up-regulation of characteristically low CD45, MHC class II expression was increased 10- to 20-fold, and microglial cell recoveries were enhanced substantially. Immunohistologic analysis revealed CD4+ alphabetaTCR+CD2+ T cells scattered infrequently throughout the CNS parenchyme, 90% of which were blast cells of donor origin. An unusual clustering of activated microglia expressing strongly enhanced levels of CD11b/c and MHC class II was a feature of the GvHD-affected CNS, and despite the paucity of T lymphocytes present, activated microglial cell clusters were invariably intimately associated with these T cells. Moreover, 70% of T cells in the CNS were associated with single or clustered MHC class II+ microglia, and interacting cells were predominantly deep within the tissue parenchyme. Approximately 3.7% of the microglia that were freshly isolated from the GvHD-affected CNS were cycling, and proliferating cell nuclear Ag-positive microglia were detected in situ. Microglia from GvHD-affected animals sorted to purity by flow cytometry and cultured, extended long complex processes, exhibited spineous processes, and were phagocytic and highly motile. These outcomes are consistent with direct tissue macrophage-T cell interactions in situ that lead to activation, proliferation, and expansion of the responding tissue-resident cell.
Assuntos
Movimento Celular/imunologia , Sistema Nervoso Central/imunologia , Ativação Linfocitária , Microglia/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apoptose , Adesão Celular/imunologia , Comunicação Celular/imunologia , Divisão Celular/imunologia , Sistema Nervoso Central/citologia , Cruzamentos Genéticos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Imunofenotipagem , Ativação de Macrófagos , Microglia/citologia , Microglia/fisiologia , Fagócitos/imunologia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Baço/citologia , Subpopulações de Linfócitos T/citologiaRESUMO
Fibrosis in the lung directly underlying the field of irradiation is an almost universal long term sequelae of thoracic irradiation. It is assumed to represent the consequence of direct damage to local tissues and/or vascular endothelium by ionizing radiation. This view, however, is not in keeping with our current understanding of fibrotic processes, which suggest that growth factors for fibroblasts (including platelet-derived growth factor (PDGF), insulin-like growth factor I (IGF-I)) and cytokines stimulating collagen synthesis (notably transforming growth factor-beta) are largely responsible for this process. Since a major source of these factors is the macrophage, present in large numbers within the lung, it appeared possible that radiation-induced fibrosis might be mediated by similar mechanisms. Therefore, a study was designed to determine, first, whether in vitro irradiation of mononuclear phagocytes could induce the release of growth factors for fibroblasts. Second, we wished to ascertain whether these same growth factors might also be secreted by bronchoalveolar cells from humans who had undergone in vivo thoracic irradiation. The results of this study indicate that irradiation of a number of different types of mononuclear phagocytes resulted in the dose-dependent synthesis and release of several growth factors for fibroblasts, including PDGF, tumour factor-alpha (TNF-alpha) and IGF-I. Further, cells obtained by bronchoalveolar lavage from patients undergoing thoracic radiation spontaneously released PDGF following irradiation. These findings strongly support the contention that synthesis and release of macrophage-derived growth factors for fibroblasts (particularly PDGF and IGF-I) occur after thoracic irradiation and play a significant role in the pathogenesis of irradiation-induced pulmonary fibrosis in humans.
Assuntos
Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/efeitos da radiação , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Neoplasias da Mama/radioterapia , Líquido da Lavagem Broncoalveolar/imunologia , Carcinoma/radioterapia , Ensaio de Imunoadsorção Enzimática , Feminino , Fatores de Crescimento de Fibroblastos/química , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Linfoma Difuso de Grandes Células B , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/efeitos da radiação , Células Tumorais CultivadasRESUMO
OBJECTIVE: To determine if unilateral thoracic irradiation results in a lymphoid alveolitis in both irradiated and unirradiated lung fields. DESIGN: A prospective, nonrandomized study. PATIENTS: Women receiving postoperative radiotherapy for carcinoma of the breast were evaluated both before and 4 to 6 weeks after radiotherapy. Findings after radiotherapy in 15 asymptomatic patients were compared with findings in a group of patients with clinical radiation pneumonitis. MEASUREMENTS: History, physical examination, chest radiograph, quantitative gallium lung scanning, respiratory function tests, bronchoalveolar lavage, and lavage lymphocyte subset analysis. RESULTS: After irradiation, lavage lymphocytes increased significantly (34.5% versus 46.8%; P = 0.01) in the 17 patients studied prospectively. There was an associated reduction in vital capacity (102.5% versus 95.5%; P = 0.04). Comparison of results in patients before treatment, after treatment without clinical pneumonitis, and after treatment with clinical pneumonitis showed a dramatic increase in total lymphocytes after irradiation (6.3 versus 9.4 versus 35.2 million, respectively; P = 0.005), particularly in those with clinical pneumonitis. Only in those with clinical pneumonitis was this accompanied by an increase in the gallium index (3.7 versus 3.4 versus 9.0, respectively; P < 0.001). Vital capacity was also progressively reduced (102.5% versus 96.9% versus 76.7%, respectively; P = 0.04), as was diffusing capacity (98.6% versus 91.4% versus 72.6%, respectively; P = 0.003). No statistical differences existed between irradiated and unirradiated sides of the chest in either lavage or gallium lung scan studies. CONCLUSION: In most patients, a lymphocytic alveolitis develops in both lung fields after strictly unilateral thoracic irradiation; this is more pronounced in patients developing clinical pneumonitis. These findings suggest that radiotherapy may cause a generalized lymphocyte-mediated hypersensitivity reaction.