RESUMO
Monoblasts, promonocytes, and macrophages in in vitro cultures of murine bone marrow were studied ultrastructurally, with special attention to peroxidatic activity. Monoblasts show peroxidatic activity in the rough endoplasmic reticulum and nuclear envelope as well as in the granules. The presence of peroxidatic activity in the Golgi apparatus could not be determined. Promonocytes have peroxidase-positive rough endoplasmic reticulum, Golgi apparatus, nuclear envelope, and granules, as previously reported. During culture, cells are formed with peroxidatic activity similar to that of monocytes or exudate macrophages (positive granules; negative Golgi apparatus, RER, and nuclear envelope); we call these cells early macrophages. In addition, transitional macrophages with both positive granules and positive RER, nuclear envelope, negative Golgi apparatus (as in exudate- resident macrophages in vivo), and mature macrophages with peroxidatic activity only in the RER and nuclear envelope (as in resident macrophages in vivo) were found. A considerable number of cells without detectable peroxidatic activity were also encountered. Our finding that macrophages with the peroxidatic pattern of monocytes (early macrophages), exudate-resident macrophages (transitional macrophages), and resident macrophages (mature macrophages), develop in vitro from proliferating precursor cells deriving from the bone marrow, demonstrates once again that resident macrophages in tissues originate from precursor cells in the bone marrow. Therefore, this conclusion can no longer be challenged on the basis of a cytochemical difference between monocytes and exudate macrophages on the one hand and resident macrophages on the other.
Assuntos
Células da Medula Óssea , Peroxidases/metabolismo , Fagócitos/ultraestrutura , Animais , Células Cultivadas , Meios de Cultura , Exsudatos e Transudatos/citologia , Macrófagos/enzimologia , Camundongos , Mitose , Monócitos/enzimologia , Organoides/enzimologia , Fagócitos/enzimologiaRESUMO
The process of demyelination occurring in diseases as multiple sclerosis is usually investigated in animal models. A major drawback of animal models is that only one condition can be tested per animal, necessitating many animals and systemic effects are factors to be considered. The aim of the study was to develop a reproducible in vitro model for de- and remyelination using whole brain spheroid cultures and lysophosphatidyl choline (LPC). In spheroid cultures, single cell suspensions of embryonic day 15 rodent brain cells reaggregate under constant rotation. Three-dimensional contacts form between the central nervous system cell types present. Multilayered myelin is maximal in four-week old cultures. A week of repeated exposure to LPC led to 30% loss of MBP protein concentration and 2',3'-cyclic nucleotide 3'-phosphodiesterase activity measurements in both rat and mouse spheroids and 56% loss in the number of myelin sheets, with partial remyelination after a week of recovery. The number of dividing cells was increased after LPC exposure and oligodendrocytes were shown to be among the dividing cells. Microglia and astrocytes were not affected and neurons were relatively spared. This suggests that LPC toxicity is specific for myelin and oligodendrocytes. LPC toxicity could be decreased using cholesterol and simvastatin, suggesting that LPC works through altering membrane composition. Thus, in different rodent species and using different read-outs, we could reproducibly show de- and remyelination in spheroid cultures after LPC exposure. This model for demyelination with potential for remyelination offers possibilities for testing novel therapies and studying mechanisms of remyelination.
Assuntos
Encéfalo/fisiologia , Doenças Desmielinizantes , Lisofosfatidilcolinas , Modelos Neurológicos , Bainha de Mielina/fisiologia , Esferoides Celulares/fisiologia , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase , Animais , Encéfalo/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/farmacologia , Colesterol/farmacologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/fisiopatologia , Lisofosfatidilcolinas/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Sinvastatina/farmacologia , Esferoides Celulares/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
CD163 is a member of the group B scavenger receptor cysteine-rich (SRCR) superfamily. This study describes aspects of the tissue distribution, the regulation of expression, and signal transduction after cross-linking of this receptor at the cell surface of macrophages. CD163 showed an exclusive expression on resident macrophages (e.g., red pulp macrophages, alveolar macrophages). The expression was inducible on monocyte-derived macrophages by glucocorticoids but not by interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma. The combination of IL-4 or GM-CSF with glucocorticoids resulted in a further increase. Subcellular analysis of alveolar macrophages by immunoelectron microscopy showed a plasma membrane localization of the antigen. Cross-linking of CD163 with monoclonal antibody induced a protein tyrosine kinase-dependent signal that resulted in (1) slow-type calcium mobilization, (2) inositol triphosphate production, and (3) secretion of IL-6 and GM-CSF. The data suggest a function for the SRCR-superfamily receptor CD163 in the regulation of inflammatory processes by macrophages.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Receptores de Superfície Celular , Transdução de Sinais , Animais , Antígenos de Diferenciação Mielomonocítica/genética , Reagentes de Ligações Cruzadas , Citocinas/biossíntese , Humanos , Immunoblotting , Líquido Intracelular , Macrófagos/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Depuradores , Frações Subcelulares/metabolismo , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Células U937RESUMO
In vivo in the animal model peritoneal macrophages show four different peroxidatic activity (PA) patterns characterizing exudate macrophages, exudate-resident macrophages, resident macrophages, and PA-negative macrophages. Cultured in vitro, rat and human blood monocytes and rat, mouse and human exudate macrophages acquire the characteristic PA pattern of resident macrophages via the transitional stage of cells with the characteristics of exudate-resident macrophages. The cytochemistry, the occurrence of this cell type in vivo, and the kinetics in vitro, indicate that exudate-resident macrophages represent a transitional form between the exudate and the resident macrophages. The results obtained in vivo and in vitro strongly suggest that divergent PA patterns of mononuclear phagocytes represent differences in the stage of development of these cells in the sequence, monoblast, promonocyte, monocyte, exudate macrophage, exudate-resident macrophage, (PA-negative macrophage), resident macrophage. This means that in general resident macrophages are provided by recruitment from the blood via the transitional stage of exudate-resident macrophages.
Assuntos
Exsudatos e Transudatos/citologia , Macrófagos/classificação , Doença Aguda , Animais , Animais Recém-Nascidos , Líquido Ascítico/citologia , Doença Crônica , Cobaias , Humanos , Inflamação/induzido quimicamente , Inflamação/etiologia , Inflamação/patologia , Macrófagos/enzimologia , Macrófagos/metabolismo , Masculino , Camundongos , Monócitos/metabolismo , Parafina , Peroxidases/metabolismo , Ratos , Ratos Endogâmicos , TioglicolatosRESUMO
Langerhans cells (LC) are known to be present in squamous epithelia of the human body. They are dendritic cells (DC) and characterized by the presence of Birbeck granules (BG). In previous studies, DC positive for CD1a and HLA-DR were found in the cylindrical epithelium and the lamina propria of the nasal mucosa. In our study, more CD1a cells occurred in the allergic patients than in the non-allergic controls. In a combined light microscopy (LM) and electron microscopy (EM) study, biopsies of nasal mucosa in allergic patients were studied. We used monoclonal antibodies against CD1a and HLA-DR, to identify DC in LM cryostat sections. The presence of BG identified most of the intra-epithelial DC as LC on the EM level, whereas a minority of DC in the lamina propria also contained BG. The ultrastructure of LC and DC in the ciliated cylindrical epithelium and the lamina propria is compared.
Assuntos
Células de Langerhans/ultraestrutura , Mucosa Nasal/patologia , Rinite Alérgica Sazonal/patologia , Adulto , Antígenos CD/biossíntese , Antígenos CD1 , Biópsia , Células Dendríticas/ultraestrutura , Feminino , Antígenos HLA-DR/biossíntese , Humanos , Células de Langerhans/imunologia , Masculino , Microscopia EletrônicaRESUMO
Class II molecules are a prerequisite for antigen presentation. We studied whether class II molecules can be found in the endocytic and/or lysosomal route of dendritic cells (DC), which are very potent antigen-presenting cells. Therefore first immunolabelling for HLA-DR alpha chain was applied on ultrathin cryosections of cells of which plasma membrane HLA-DR/DQ molecules were labelled in suspension, followed by incubation with the endocytic marker BSA-gold. Second, immunolabelling for HLA-DR alpha chains was applied on ultrathin cryosections of cells on which enzyme cytochemistry for acid phosphatase (APh) was performed to see whether the class II positive vesicles belong to the lysosomal compartment. Third, this immunolabelling was applied on cryosections of cells pretreated with the protein synthesis inhibitor cycloheximide (CHX) to see whether the class II positive vesicles are derived from biosynthesis. We found limited uptake of BSA-gold into endosomes and lysosomes, some of which also contained endocytozed HLA-DR/DQ. APh and HLA-DR were observed in the same vesicles but also vesicles containing either HLA-DR or APh were found. However, many class II positive vesicles were found, which were apparently not accessible to exogenous molecules. Moreover, the amount of class II positive vesicles decreased after treatment of the cells with CHX, suggesting that these vesicles form part of the biosynthetic route. These results imply that there is a cluster of class II positive vesicles, probably a storage compartment, that has connections with the lysosomal system. The concentration of lysosomes and class II positive vesicles in the juxtanuclear area of DC is probably of crucial importance in the processing of antigens.
Assuntos
Células Dendríticas/imunologia , Antígenos HLA-D/metabolismo , Fosfatase Alcalina/metabolismo , Células Dendríticas/enzimologia , Células Dendríticas/ultraestrutura , Endocitose , Humanos , Imuno-Histoquímica , Lisossomos/imunologia , Microscopia ImunoeletrônicaRESUMO
The localization of histocompatibility antigens of the HLA-D locus in dendritic cells (DC) and monocytes (Mo) isolated from human peripheral blood was investigated. Functionally DC were characterized by their capacity to act as strong stimulators in an allogeneic mixed leucocyte reaction. In cytospins, DC were differentiated from Mo by dendritic morphology, strong HLA class II and moderate RFD1 expression on the plasma membrane and acid phosphatase activity in a juxtanuclear spot. Ultrathin cryosections showed that DC had a heavily labelled plasma membrane for HLA-D. In addition, these antigens were found in intracellular vesicles predominantly located in the juxtanuclear zone. This pattern of labelling was not seen in Mo. Obviously, DC concentrate intracellular class II antigens in the same area as lysosomal activity. These results may indicate that this juxtanuclear area is a center of antigen processing in DC.
Assuntos
Células Dendríticas/imunologia , Antígenos HLA-D/metabolismo , Fosfatase Ácida/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/imunologia , Células Dendríticas/enzimologia , Células Dendríticas/ultraestrutura , Histocitoquímica , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Teste de Cultura Mista de Linfócitos , Monócitos/enzimologia , Monócitos/imunologia , Frações Subcelulares/enzimologia , Frações Subcelulares/imunologiaRESUMO
Immunolabeling of cryo-sections of human anterior pituitaries obtained at autopsy, and of cryo-sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC-class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX62 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC-class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo-stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immuno-electron microscopy of rat anterior pituitaries fixed with periodate-lysine-paraformaldehyde, we were able to distinguish non-granulated cells expressing MHC-class II determinants, whereas no MHC-class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo-sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC-class II-expressing and S100 protein-expressing cells. Furthermore, MHC-class II-expressing and S100-positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo-stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100-positive cells was found immunopositive for the MHC-class II marker OX6. In the human AP, S100-positive folliculo-stellate cells and cells expressing the leukocyte common antigen CD45 were found to occupy predominantly different tissue compartments in the human anterior pituitary, namely the epithelial parenchyme cords and perivascular compartments respectively. A proportion of CD45+ cells was found in the parenchyme compartment and, vice versa, indicating an overlap of the tissue compartments in which both cell types occur. However, at the light microscopical level we could not find cells expressing both the S100 and CD45 marker. The present finding of a proportion of S100-positive pituitary cells with ultrastructural and immunohistochemical characteristics of both dendritic cells and folliculo-stellate cells, confirms the suggested heterogeneity of the latter cell group with respect to their ultrastructural phenotype and putative function. The possibility of a myeloid origin of part of the folliculo-stellate cell group in the AP, is discussed and might elucidate some of the discrepancies in the literature concerning the embryological origin of this cell group.
Assuntos
Células Dendríticas/química , Células Dendríticas/citologia , Adeno-Hipófise/química , Adeno-Hipófise/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais , Células Dendríticas/ultraestrutura , Feminino , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Imuno-Histoquímica , Lactente , Macrófagos/química , Macrófagos/citologia , Macrófagos/ultraestrutura , Masculino , Pessoa de Meia-Idade , Adeno-Hipófise/ultraestrutura , Ratos , Ratos Wistar , Valores de Referência , Proteínas S100/análiseRESUMO
In an immature state, dendritic cells (DC) can capture antigen via at least two mechanisms. First, DC use macropinocytosis for continuous uptake of large amounts of soluble antigens. Second, they express high levels of mannose receptor that can mediate internalization of glycosylated ligands. We found that dendritic cells can present mannosylated antigen 100-1000 fold more efficiently than non-mannosylated antigen. Immunocytochemistry as well as subcellular fractionation demonstrated that the mannose receptor and MHC class II molecules were located in distinct subcellular compartments. These results demonstrate that the mannose receptor endows DC with a high capacity to present glycosylated antigens at very low concentrations.
Assuntos
Apresentação de Antígeno/fisiologia , Antígenos/metabolismo , Células Dendríticas/imunologia , Lectinas Tipo C , Lectinas de Ligação a Manose , Receptores de Superfície Celular/imunologia , Transporte Biológico Ativo , Comunicação Celular , Compartimento Celular , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Glicosilação , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Técnicas In Vitro , Ativação Linfocitária , Receptor de Manose , Microscopia Imunoeletrônica , Receptores de Superfície Celular/metabolismo , Frações Subcelulares/imunologia , Linfócitos T/imunologiaAssuntos
Células Dendríticas/imunologia , Antígenos HLA-DR/análise , Antígenos/metabolismo , Compartimento Celular , Separação Celular , Grânulos Citoplasmáticos/fisiologia , Grânulos Citoplasmáticos/ultraestrutura , Células Dendríticas/ultraestrutura , Endocitose , Humanos , Células de Langerhans/imunologia , Células de Langerhans/ultraestrutura , Lisossomos/fisiologia , Lisossomos/ultraestrutura , Microscopia Imunoeletrônica , Pele/citologiaRESUMO
Only a small proportion of thymocytes survive T cell selection in the thymus and leave the thymus as mature T cells. The vast majority of thymocytes undergo cell death during selection, either due to failure to undergo positive selection on self peptide-MHC presented by thymic antigen presenting cells (APC) or due to negative selection. In the murine thymus it has been shown that most thymocytes that fail selection undergo apoptosis in the thymic cortex and are removed by cortical macrophages. However, it is unknown how apoptotic thymocytes are cleared from the cortex of the human thymus. Here we report the identification of antigen-presenting cells of haematopoietic origin (hAPCs) by expression of dendritic cell (DC) specific C-type lectin DC-SIGN (CD209) in the cortex of the human thymus, and show that these cells exhibit features of both immature DCs and macrophages. The analysis of cellular markers, in particular the expression of the molecular chaperone HLA-DM, on cortical hAPCs further suggests that these hAPCs may participate in selection of thymocytes in the cortex. Using in situ terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), we demonstrated that these cortical hAPCs are surrounded by apoptotic, TUNEL(+) thymocytes in situ. Futhermore, in situ immuno-cryo-electron microscopy suggests that cortical hAPCs take up and remove apoptotic thymocytes. Thus, DC-SIGN(+) hAPCs in the human thymic cortex appear to function in thymocyte selection and removal of apoptotic thymocytes from the thymic cortex.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T/imunologia , Timo/imunologia , Células Apresentadoras de Antígenos/ultraestrutura , Apoptose/imunologia , Moléculas de Adesão Celular/metabolismo , Pré-Escolar , Células Dendríticas/imunologia , Humanos , Imunofenotipagem , Marcação In Situ das Extremidades Cortadas/métodos , Lactente , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Microscopia Imunoeletrônica , Receptores de Superfície Celular/metabolismo , Linfócitos T/ultraestrutura , Timo/ultraestruturaRESUMO
Dendritic cells (DC) are highly potent activators of the immune response. The precise mechanisms that give rise to the DC phenotype are not known. To investigate the mechanisms that contribute to the generation of the DC phenotype, precursor DC were freshly isolated from human blood and allowed to mature in vitro. These matured DC showed the phenotypical and functional characteristics of DC. Analysis of the MHC class II and invariant chain (li) biosynthesis revealed that upon maturation, class II synthesis was induced whereas li synthesis was significantly up-regulated. In mature DC, despite the presence of large amounts of li, export of MHC class II molecules from the endoplasmic reticulum was incomplete, up to 4 h after biosynthesis. Thus, MHC class II-li synthesis and transport in DC is highly regulated during maturation of DC. Analysis of the regulatory mechanisms may contribute to a better understanding of antigen-presenting capacities during the differentiation of DC.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/biossíntese , Transporte Biológico , Western Blotting , Diferenciação Celular , Separação Celular , Eletroforese , Endocitose , Antígenos HLA-DR , Antígenos de Histocompatibilidade Classe II/biossíntese , Humanos , Microscopia Eletrônica , Testes de PrecipitinaRESUMO
In this study, double labelling for major histocompatibility complex (MHC) class I and class II molecules and for MHC molecules and the lysosomal membrane protein lamp-1 on ultrathin cryosections of dendritic cells isolated from human peripheral blood was performed. The plasma membrane proved to be positive for both MHC class I and MHC class II molecules and was labelled for only a very few lamp-1 molecules. MHC class I and MHC class II molecules did not co-localize intracellularly except in some peripherally located vesicles. However, many MHC class II-labelled vesicles were present in a juxtanuclear position but only some of them were co-labelled for lamp-1. These results indicate the presence of a separate, non-lysosomal compartment for class II molecules in dendritic cells.
Assuntos
Antígenos CD , Células Dendríticas/química , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe I/análise , Glicoproteínas de Membrana/análise , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Secções Congeladas , Humanos , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/imunologia , Microscopia ImunoeletrônicaRESUMO
The role that milky spots in the greater omentum play in tumour cell spread in the peritoneal cavity is presently not fully understood. To study whether intraperitoneally injected tumour cells appear preferentially in milky spots of the greater omentum and to study the changes in the greater omentum, and especially in the cell population of milky spots after tumour cell infiltration, the following study was performed. A detailed temporal sequences of changes in morphology and cellular composition in milky spots of the greater omentum of Wag/Rij rats 5, 15, 30, 60 min, 2, 4, 8, 16, 24 h, 2, 4, 8 days and 2 and 4 weeks after intraperitoneal administration of 2.0 x 10(6) CC 531 tumour cells was investigated by light microscopy and electron microscopy (pre-embedding labelling). Our data showed that the milky spots in the greater omentum were the sites to which tumour cells migrated preferentially from the peritoneal cavity. The tumour cells infiltrated the milky spots and formed clusters within. The cellular population in milky spots reacted by a very rapid influx of young macrophages during the first hour and an increase of the total number of cells (P < 0.01). After 4 h tumour cells were also located on the greater omentum outside the area of the milky spots. Around these tumour cell deposits, new milky spots are formed, which increased the total number of milky spots. The cells present in milky spots are not capable of reversing the growth of tumours and finally a solid omental cake of tumour cells is formed.