RESUMO
Stimulation of T cells by antigen activates many signalling pathways. The capacity for this range of biochemical responses may reside in the complex structure of the seven-chain T-cell antigen receptor (TCR). In addition to the complexity shared by all TCRs, coexpression of zeta (zeta) and the distinct but related eta (eta) chains creates structural diversity among the TCR complexes expressed on a given cell. In most murine T cells that we have studied, about 90% of the heptameric receptor complexes contain a zeta zeta disulphide homodimer, whereas 10% contain a zeta eta disulphide heterodimer. Recent studies suggest that zeta has a critical role in allowing antigen to activate the cell, whereas eta expression has been correlated with the capacity for antigen-induced phosphoinositide turnover. A third zeta-related protein, the gamma (gamma) chain of the Fc epsilon and some Fc gamma receptors, exists as a disulphide homodimer in those complexes. The structural relatedness of zeta and gamma is emphasized by the recent demonstration of zeta zeta in association with CD16 in TCR-negative natural killer cells. Here we identify T cells lacking Fc receptors but coexpressing zeta, gamma, and eta, document the formation of novel heterodimers between zeta and gamma and between eta and gamma and show their association with the TCR. A greater range of homologous coupling structures than previously thought may be one way of achieving the variety of TCR-mediated (and possibly Fc receptor-mediated) biochemical responses and effector functions.
Assuntos
Dissulfetos , Receptores de Antígenos de Linfócitos T/análise , Receptores Fc/análise , Linfócitos T/imunologia , Animais , Linhagem Celular , Sondas de DNA , Eletroforese em Gel Bidimensional , Humanos , Hibridomas/análise , Immunoblotting , Técnicas de Imunoadsorção , Substâncias Macromoleculares , Mastócitos/análise , Camundongos , Peso Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Receptores de Antígenos de Linfócitos T/genética , Receptores Fc/genética , Linfócitos T Citotóxicos/análiseRESUMO
Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above-mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers. These results demonstrate microenvironmental effects exerted by the MESF and HSF monolayers on IL-3-dependent hBMMCs similar to those reported in the conversion of murine mast cell phenotype.
Assuntos
Células da Medula Óssea , Sulfatos de Condroitina/análise , Condroitina/análogos & derivados , Fibroblastos/fisiologia , Heparina/biossíntese , Mastócitos/metabolismo , Azul Alciano , Animais , Antineoplásicos , Células Cultivadas , Histocitoquímica/métodos , Humanos , Interleucina-3/farmacologia , Mastócitos/análise , Mastócitos/efeitos dos fármacos , Camundongos , Fenazinas , Fenótipo , Proteoglicanas/biossíntese , Proteínas Recombinantes/farmacologia , Pele/citologia , Timidina/metabolismo , TrítioRESUMO
Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.
Assuntos
Colina/análogos & derivados , Colina/análise , Mastócitos/análise , Neutrófilos/análise , Fosforilcolina/análise , Animais , Etanolamina , Etanolaminas/análise , Humanos , Ratos , Serina/análise , Cloreto de Sódio , SolubilidadeRESUMO
Respiratory epithelial mast cells are an expression of airway inflammatory processes. Nasal epithelial mast cells are known to be increased in allergic rhinitis and have now been examined in patients with nasal polyps. Metachromatic cell counts (mean +/- standard error) expressed as the sum of large mast cells, atypical mast cells and basophils in epithelial scrapings of the inferior turbinates, assessed after Carnoy's fixation and toluidine blue staining (pH 0.5), were 37.5 +/- 29 in non-allergic normal control subjects (n = 11), 435 +/- 130 in polyp patients who were allergic (n = 18), and 699 +/- 267 in polyp patients who were not allergic (n = 8). Metachromatic cell counts in epithelial scrapings obtained in vivo from nasal polyps of allergic patients (n = 8) were 1769 +/- 962, and 2308 +/- 1544 from polyps of non-allergic patients (n = 5); metachromatic counts were 2089 +/- 633 in epithelial scrapings from excised polyps of allergic patients (n = 14) and 2214 +/- 640 from polyps of non-allergic patients (n = 13). It is concluded that the number of metachromatic cells in the epithelium of nasal polyps and the adjacent nasal mucosa is elevated compared with normal nasal epithelium and the increased number does not depend upon allergy.
Assuntos
Hipersensibilidade/patologia , Mastócitos/análise , Pólipos Nasais/patologia , Conchas Nasais/patologia , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/análise , Inflamação , Mastócitos/imunologia , Mucosa Nasal/imunologia , Mucosa Nasal/patologia , Pólipos Nasais/imunologia , Testes Cutâneos , Conchas Nasais/imunologiaRESUMO
By an indirect immunohistochemical method with fluorescein-isothiocyanate (FITC) and horse radish peroxidase as markers (HRP) the presence of vasopressin was shown in cells of dura mater in white rats. Mast cells were identified after staining with methylene blue by the metachromatic granularity of the cytoplasm. It was shown that the number of cells found by means of FITC luminescence corresponds with their number found by means of methylene blue. The use of conjugate with HRP unveils a lesser number of vasopressin-containing cells.
Assuntos
Dura-Máter/análise , Vasopressinas/análise , Animais , Dura-Máter/citologia , Fluoresceína-5-Isotiocianato , Fluoresceínas , Peroxidase do Rábano Silvestre , Imuno-Histoquímica , Medições Luminescentes , Mastócitos/análise , Azul de Metileno , Ratos , TiocianatosRESUMO
Los mastocitos son células que participan en actividades de hipersensibilidad inmediata y tardía, inmunorregulación e inflamación. Recientemente se han detectado dos grupos de ellos: a) los que se localizan en tejido conjuntivo y b) los de la mucosa intestinal. Entre ellos hay diferencias morfológicas y funcionales. Los de mucosa intestinal son T dependientes y proliferan durante las parasitosis así como en los procesos de hipersensibilidad intestinal a diversos antígenos. Las placas de Peyer (PP) son los sitios principales donde se captan antígenos e inician las respuestas inmunitarias en el intestino, por lo que en este trabajo investigamos la relación morfológica entre las células de las PP y los mastocitos. Las PP de segmentos proximal, medial y distal del intestino delgado de ratones Balb/c se procesaron histológicamente, tiñeron con azul de toluidina y cuantificaron los mastocitos en las diferentes capas del intestino. Los datos se analizaron mediante una prueba de doble T pareada para diferencia de medios. Se observó mayor cantidad de mastocitos en la zona marginal de la PP en comparación con las capas: muscular resistente, submucosa y corion. La abundancia de mastocitos en relación con la PP sugiere que probablemente tenga influencia moduladora sobre la unción de las células linfoides de la PP
Assuntos
Camundongos , Animais , Masculino , Técnicas In Vitro , Mastócitos/análise , Nódulos Linfáticos Agregados/análiseRESUMO
The nuclear factor GF-1 (also known as NF-E1, Eryf-1; refs 1-3 respectively) is important in regulation of the transcription of globin and other genes that are specifically expressed in erythroid cells. We have previously shown that GF-1 of both mouse and human origin is a 413-amino-acid polypeptide with two novel zinc-finger domains whose expression is restricted to erythroid cells. Using in situ hybridization of mouse bone marrow cells and northern blot analysis of purified cell populations and permanent cell lines, we show here that GF-1 is expressed in two other hematopoietic lineages, megakaryocytes and bone marrow-derived mast cells. Our findings are consistent with results from hematopoietic progenitor culture which suggest a relationship between erythroid, megakaryocytic and mast cell lineages, and imply that GF-1 is expressed in committed multipotential cells and their progeny. Hence, the mere presence of this transcription factor is unlikely to be sufficient to programme differentiation of a single haematopoietic lineage. GF-1 may regulate the transcription of not only erythroid genes, but also many genes characteristic of megakaryocytes and mast cells, or genes shared among these lineages.
Assuntos
Proteínas de Ligação a DNA/genética , Eritrócitos/metabolismo , Expressão Gênica , Mastócitos/metabolismo , Megacariócitos/metabolismo , Fatores de Transcrição , Animais , Células da Medula Óssea , Diferenciação Celular , Clonagem Molecular , DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Globinas/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Eritroblástica Aguda , Mastócitos/análise , Megacariócitos/análise , Camundongos , Hibridização de Ácido Nucleico , Sondas RNA , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Three different polypeptides capable of stimulating histamine release from mast cells were isolated from human whole saliva, using heparin-gel chromatography followed by reversed-phase HPLC. The amino acid sequences of these peptides were shown to be identical to those of histatins 1, 3 and 5.
Assuntos
Glicoproteínas/isolamento & purificação , Proteínas/isolamento & purificação , Saliva/análise , Proteínas e Peptídeos Salivares/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Glicoproteínas/análise , Histamina/análise , Humanos , Masculino , Mastócitos/análise , Dados de Sequência Molecular , Proteínas/análise , Ratos , Ratos Endogâmicos , Proteínas e Peptídeos Salivares/análiseRESUMO
Surgical human thymus, upper respiratory tract, lung and small and large bowel specimens were analyzed for the presence of interleukin 2 receptor (IL2-R)-positive cells. Histochemical (toluidine) and immunologic (anti-IL2-R monoclonal antibody) staining procedures revealed a distinct anti-IL2-R positivity in most of metachromatically staining cells. These positive cells were observed not only in tissues showing strong inflammatory reaction and mast cell hyperplasia, as in Crohn's disease, but also in those not histologically affected by pathologic conditions. This finding suggested that human mast cells, like T blast cells, express the p55 chain of IL2-R on their surface. To see whether IL2-R was being actively synthesized, a cell preparation rich in peripheral blood basophils (PBB), which are cells closely related to mast cells, was obtained. Ultrastructural analysis of PBB after indirect immunogold procedure revealed that the vast majority expressed the IL2-R. Moreover, the presence of intracellular reaction products in the cytoplasm of most membrane-positive PBB was indicative of active antigen synthesis. Furthermore, Northern blot analysis evidenced specific IL2-R mRNA in PBB, while its expression was augmented several times when PBB were cultured in the presence of stimulated T cell supernatant.
Assuntos
Basófilos/análise , Mastócitos/análise , Receptores de Interleucina-2/análise , Animais , Anticorpos Monoclonais , Antígenos de Superfície/análise , Basófilos/ultraestrutura , Citoplasma/análise , Humanos , Imuno-Histoquímica , Lactente , Camundongos , RNA Mensageiro/isolamento & purificação , Coelhos , Receptores de Interleucina-2/imunologia , Linfócitos T/análiseRESUMO
We describe a patient with fever and multiple osteolytic bone lesions accompanied by hypercalcemia, a duodenal ulcer, anemia, and thrombocytopenia. Bone marrow showed a dense infiltration by abnormal cells characterized by small basophil granula, erythrophagocytosis and nuclear atypia. These cells were positive for toluidine blue and partly for myeloperoxidase and chloroacetate esterase, expressed myeloid differentiation markers, and exhibited multiple numerical and structural chromosome aberrations. Molecular genetic analysis showed no breakpoint cluster region rearrangement. Electron microscopy demonstrated granula both of basophil and mast cell type. Concluding, in this patient an acute hematopoietic malignancy with many features of malignant mastocytosis but also with signs of a basophil differentiation. This is further support for a hematopoietic stem cell origin of human mast cells.
Assuntos
Basófilos/patologia , Leucemia de Mastócitos/genética , Mastócitos/patologia , Cromossomo Filadélfia , Adulto , Basófilos/análise , Basófilos/ultraestrutura , Southern Blotting , Medula Óssea/análise , Medula Óssea/patologia , Medula Óssea/ultraestrutura , Diferenciação Celular , Feminino , Humanos , Imunoquímica , Imuno-Histoquímica , Cariotipagem , Leucemia de Mastócitos/metabolismo , Leucemia de Mastócitos/patologia , Mastócitos/análise , Mastócitos/ultraestruturaRESUMO
This study was undertaken to characteize the different phases of the allergic pleurisy induced by ovalbumin in actively sinsitized rats. The reaction was triggered by the intrathoracic injection of ovalbumin (12 microng/cavity) into animals sensitized 14 days before. The challenge caused, at 30 mjin, a drastic mast cell degranulation and exudation which peaked within 4h. At this time, an intense pleural leucocyte recruitment also occurred, accounted for by an increase in the mononuclear cell counts and by a predominant influyx of neutrophils. After 24h, the mast cell counts stated to reover, accompanied by a long-lasting (96 h) accumaltion of pleural eosinophils. Forty-eight hour later, the exudation and neutrophils were at basal levels, whereas mast cell counts increased progressively to reach control values at 120 h. This study describes the time course of the exudatory and cellular alterations observed during pleural inflammation induced by low antigen concentrations
Assuntos
Ratos , Animais , Masculino , Feminino , Hidróxido de Alumínio/farmacologia , Leucócitos/análise , Mastócitos/análise , Ovalbumina/farmacologia , Derrame Pleural/patologia , Pleurisia/fisiopatologia , Doença Aguda , Cinética , Ratos WistarRESUMO
Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.
Assuntos
Hipófise/análise , Inibidor da Tripsina Pancreática de Kazal/análise , Inibidores da Tripsina/análise , Animais , Bovinos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Imunoglobulinas/análise , Imuno-Histoquímica , Isomerismo , Pulmão/análise , Masculino , Mastócitos/análise , Mastócitos/ultraestrutura , Neuro-Hipófise/análiseRESUMO
The present study has examined the catabolism of 1-O-[3H]hexadecyl-2-acetyl-GPC (C16-PAF) and of 1-O-octadecyl-2-acetyl-GPC (C18-PAF) in spleen-derived PT-18 murine mast cells (mast cells). Mast cells catabolized exogenous PAF into two inactive metabolites, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC). The rate of conversion of C16-PAF to metabolites was more rapid than that of C18-PAF. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC formed during the metabolism of PAF revealed that arachidonic acid (20:4) was the major fatty acyl chain incorporated at the sn-2 position. However, 25% of newly formed 1-O-alkyl-2-acyl-GPC was reacylated with docosahexaenoic acid (22:6). The influence of cellular fatty acid content on PAF catabolism was further explored in mast cells in which the ratio of fatty acids within cellular phosphoglycerides had been altered by supplementing the cells with various fatty acids in culture. Mast cells supplemented with 20:4 or 22:6 converted PAF to 1-O-alkyl-2-acyl-GPC at a significantly higher rate than non-supplemented cells. In contrast, cells supplemented with linoleic acid (18:2) metabolized PAF at rates similar to non-supplemented cells. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC derived from the metabolism of PAF in 20:4-supplemented cells indicated that 20:4 was incorporated exclusively into the sn-2 position. Conversely, 22:6-supplemented cells incorporated predominantly 22:6 at the sn-2 position of 1-alkyl-2-lyso-GPC. Supplementation with 18:2 had no effect on the acylation pattern seen in newly formed 1-O-alkyl-2-acyl-GPC. Activation of passively sensitized mast cells with antigen or with ionophore A23187 significantly enhanced the rate of catabolism of exogenously-provided PAF but had no effect on the acylation pattern of 1-O-alkyl-2-acyl-GPC. Experiments performed with the soluble fraction of the cells showed that acetyl hydrolase activity was increased in mast cells stimulated with antigen. In addition, supernatant fluids from antigen or ionophore-treated mast cells converted PAF to lysoPAF, suggesting that acetyl hydrolase activity was released during cell activation. These data indicate that the ability of mast cells to catabolize PAF to inactive metabolites is influenced by cell activation and by the cellular levels of certain fatty acids.
Assuntos
Ácidos Graxos/análise , Fator de Ativação de Plaquetas/metabolismo , Animais , Antígenos/imunologia , Calcimicina/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Glicerofosfatos/metabolismo , Imunização , Mastócitos/análise , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Palmitoil-CoA Hidrolase/metabolismoRESUMO
Beta-adrenergic agonists can prevent mediator release from guinea pig pulmonary mast cells. By pharmacologic characterization, this response is mediated through a beta-2 receptor. Structural characterization of this receptor on the lung mast cell, however, has been limited by methods for isolation of this pulmonary cell. In this study, the guinea pig lung mast cell was isolated to greater than 90% purity, and its beta-adrenergic receptor identified by photoaffinity labeling with [125I]iodoazidobenzylpindolol (125IABP) and separation of membrane proteins by SDS-PAGE. We found the guinea pig pulmonary mast cell beta-adrenergic receptor to electrophorese as a heterogeneous protein between 68 and 116 kD. Photoaffinity labeling with 125IABP was protectable by alprenolol and isoproterenol but not by phentolamine and norepinephrine. Using subtype-selective compounds, the pulmonary mast cell receptor was established to be of a beta-2 subtype. This is the first report of the structural identification of a lung mast cell beta-adrenergic receptor and the first report of a beta-adrenergic receptor of approximately 100 kD in mass. This mast cell receptor is considerably larger than the 65 kD beta-adrenergic receptors that have been identified in whole lung and other tissues. Data we have obtained using Northern blot analysis of mast cell RNA suggest a protein message of 45 kD for this beta-adrenergic receptor and a high degree of glycosylation most likely accounts for the large molecular size observed.
Assuntos
Pulmão/análise , Mastócitos/análise , Receptores Adrenérgicos beta/análise , Marcadores de Afinidade , Animais , Autorradiografia , Northern Blotting , Membrana Celular/análise , Cobaias , Fotólise , RNA Mensageiro/análise , Receptores Adrenérgicos beta/genéticaRESUMO
High-(Fc epsilon RI) and low-(Fc epsilon RII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet-P40-solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. Fc epsilon RII were isolated by affinity chromatography using IgE-Sepharose or by anti-Fc epsilon RII antisera and protein A-Sepharose. The surface-exposed, IgE-binding alpha subunits of Fc epsilon RI [Fc epsilon RI alpha] were isolated by affinity chromatography using IgE and anti-IgE-Sepharose. Fc epsilon RI alpha on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL-2H3 cells (51 kDa) or RBL-CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine-HCl, pH 3, prior to iodination removed much of the surface-bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid-treated PMC, upon anti-IgE-Sepharose isolation acid-treated IMMC yielded an intensely radioactive Fc epsilon RI alpha band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of Fc epsilon RII isolated; 50-kDa and 58-kDa forms of Fc epsilon RII were obtained from IMMC, whereas PMC yielded most often a single 56-kDa Fc epsilon RII band. These results were mimicked by the two RBL cell sublines: RBL-2H3 cells yielded two Fc epsilon RII (46 kDa and 55 kDa), but only one form of Fc epsilon RII (54-kDa) was obtained from RBL-CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Mucosa Intestinal/análise , Mastócitos/análise , Receptores Fc/isolamento & purificação , Animais , Cromatografia de Afinidade , Mucosa Intestinal/citologia , Peso Molecular , Cavidade Peritoneal/citologia , Ratos , Receptores de IgGRESUMO
In a patient with eosinophilic fasciitis, a biopsy specimen obtained within 4 weeks of the onset of symptoms showed infiltration of the subcutis and fascia with mast cells, and there was up to a 19-fold increase in plasma histamine levels. The patient improved and experienced softening of the skin when treated with systemic corticosteroids and a histamine2-receptor antagonist, and her plasma histamine level returned to normal. Tissue mast cell infiltration and excessive plasma histamine levels were not present in two otherwise similar patients with eosinophilic fasciitis who were studied 7 months after disease onset. It is possible that mast cells play a pathogenic role in some patients with eosinophilic fasciitis.
Assuntos
Eosinofilia/sangue , Fasciite/sangue , Histamina/sangue , Adulto , Idoso , Biópsia , Fasciite/diagnóstico , Fasciite/tratamento farmacológico , Feminino , Humanos , Mastócitos/análise , Pessoa de Meia-Idade , Prednisona/uso terapêuticoRESUMO
Trypstatin, a new cellular Kunitz-type protease inhibitor purified from rat mast cells, inhibited syncytium formation in human immunodeficiency virus type 1 (HIV-1)-infected CCRF-CEM and uninfected Molt-4 clone 8 at a concentration of 1 microM. Anti-rat tongue mast cell tryptase antibodies reacted with Molt-4 clone 8 cells, as determined by Western blot and by immunofluorescence. In addition, the antibody inhibited syncytium formation. These findings along with homologous sequences with trypstatin and a neutralizing epitope of gp120 of HIV-1 suggest that a tryptase-like cellular enzyme(s) is involved in HIV-1 infection.
Assuntos
Síndrome da Imunodeficiência Adquirida/enzimologia , Anticorpos/farmacologia , HIV-1/enzimologia , Peptídeo Hidrolases/imunologia , Inibidores de Proteases/farmacologia , Proteínas/farmacologia , Linfócitos T/efeitos dos fármacos , alfa-Globulinas , Animais , Anticorpos/análise , Antígenos de Superfície/análise , Western Blotting , Células Cultivadas , Epitopos/análise , Humanos , Mastócitos/análise , Testes de Neutralização , Proteínas/análise , Ratos , Linfócitos T/imunologia , Linfócitos T/microbiologia , Língua/enzimologiaRESUMO
A series of permanent IL-3-dependent cell lines have been established from normal BALB/c or C3H bone marrow using alpha-thioglycerol-supplemented culture medium and PWM-stimulated spleen cell-conditioned medium as a source of IL-3. The cell lines and derivatives cloned in agar resembled "mucosal type" mast cells with respect to phenotypic and functional properties. In this report we demonstrate that in vitro growth of these mast cell lines was not only dependent on IL-3 and synergistically enhanced by IL-4, but in addition regulated by alpha-thioglycerol which could be replaced by 2-ME or cysteamine. We show that these thiol-sensitive mast cell lines respond to a mast cell growth enhancing activity (MEA) present in spleen cell-conditioned medium and acting in concert with IL-3. Partially purified MEA was not able to stimulate the growth of IL-3-dependent 32Dcl.23 cells, IL-2-dependent CTLL-2 cells or the mouse T cell line F4/4K.6 (L3T4+) adapted to grow in purified IL-4. Moreover, 11B11 hybridoma-derived anti-IL-4 mAb specifically neutralizing mouse Il-4 were unable to abolish the bioactivity of MEA. PWM, CSF-1, GM-CSF, IL-1, IL-2, IL-5, IL-6, IL-7, IFN-gamma, TGF-alpha, TNF-alpha, NGF, or EPO did not substitute for MEA in our standard proliferation assay.
Assuntos
Células da Medula Óssea , Interleucina-3/fisiologia , Interleucinas/farmacologia , Mastócitos/fisiologia , Compostos de Sulfidrila , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura/análise , Feminino , Interleucina-3/análise , Interleucina-3/farmacologia , Interleucina-4 , Masculino , Mastócitos/análise , Mastócitos/classificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , FenótipoRESUMO
We have identified a late, committed stage in the differentiation of the mast cell progenitor just before granulation. Mast cell committed progenitors (MCCP) are nongranulated cells with a density of 1.060 to 1.070 g/ml which can be harvested from the mesenteric lymph node of mice infected with Nippostrongylus brasiliensis. Mast cell-committed progenitors are able to proliferate and differentiate in the absence of IL-3 or IL-4 when cultured on a monolayer of embryonic skin or 3T3 fibroblasts and can form colonies in methylcellulose supplemented with fibroblast conditioned medium. Fibroblast conditioned medium appears to contain a soluble MCCP proliferation factor that maintains biologic activity when heated to 56 degrees C for 45 min but is destroyed by incubation with either trypsin or chymotrypsin. It can be selectively precipitated with 60 to 70% saturated ammonium sulfate. The factor is not absorbed by immobilized antibodies to nerve growth factor. The MCCP proliferation activity of the factor could not be mimicked by IL-1, IL-2, IL-4, granulocyte-macrophage-CSF, granulocyte-CSF, macrophage-CSF, IFN-alpha/beta, IFN-gamma, nerve growth factor, epidermal growth factor, serum fibronectin, heparin, or a number of glycosaminoglycans. At high salt concentrations, the factor passes through a 50-kDa membrane and can be concentrated above a 5-kDa membrane. MCCP acquire a connective tissue phenotype when cultured on a fibroblast monolayer and a mucosal phenotype when cloned in the presence of conditioned medium from PWM-stimulated spleen cells. When cultured in the absence of IL-3 on a monolayer of embryonic skin or 3T3 fibroblasts, mast cell-committed progenitors produce mast cells which stain with berberine sulfate suggesting a connective tissue phenotype; however, the mast cells that develop when mast cell-committed progenitors are cultured in the presence of IL-3 or conditioned media from PWM-stimulated spleen cells do not stain with berberine sulfate. MCCP intercalate into monolayers of embryonic skin or 3T3 fibroblasts, but T cells are not able to associate with the monolayer and can be completely washed away. Attempts to enrich mast cell-committed progenitors by intercalation and elution from embryonic skin monolayers proved unsuccessful, but some enrichment of mast cell-committed progenitors could be achieved by discontinuous Percoll gradients. Thus, we have identified a way to obtain late-stage, mast cell-committed progenitors in an environment that is virtually uncontaminated with other hematopoietic progenitors.