RESUMO
Natural autoantibodies play an important regulatory role in the maintenance of immune homeostasis. They act as a first line of defense against environmental pathogens like toxins, bacteria and erythrocytes. In humans they are mainly produced by CD5+ B cells that are under the control of a regulatory T cell population. Fc-gamma receptors are involved in antigen recognition and signal transduction and tuning, and some of the members of the FcR family have structural similarity to MHC molecules; they may interact with multiple Ig ligands and with non-Ig ligands. We discuss the interactions between immune-complexes formed with natural autoantibodies and Fc-gamma receptors and suggest that such interactions may affect self-recognition in the thymus and regulate immune homeostasis.
Assuntos
Complexo Antígeno-Anticorpo , Autoanticorpos , Homeostase/imunologia , Modelos Imunológicos , Receptores Fc , Antígenos CD5 , Regulação para Baixo , Antígenos Comuns de LeucócitoRESUMO
This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10(+) when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4(+) and CD8(+) T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV(+) subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10(+) as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.
Assuntos
Apoptose/imunologia , Neprilisina/biossíntese , Linfócitos T/imunologia , Linfócitos T/patologia , Células Cultivadas , Citometria de Fluxo , Soropositividade para HIV/sangue , Soropositividade para HIV/imunologia , Humanos , Neprilisina/imunologiaRESUMO
We have previously characterized mouse H4 (mH4), a surface glycoprotein recognized by the C398.4A monoclonal antibody. We now show that C398.4A also binds its human putative homolog (hpH4). Both hpH4 and mH4 (1) are selectively expressed by activated T cells and mature thymocytes, (2) are disulfide-linked dimers of two chains (29/37 kDa in humans, 25/29 kDa in mice), whose N-deglycosylation produces a single band at 20 - 21 kDa, and (3) display a low association with CD4 and the TCR. The expression pattern of hpH4 and its biochemical features showed that it is different from other known activation molecules, and this was confirmed when analysis of the tryptic digest of the hpH4 29-kDa band by peptide mass searching using matrix-assisted laser desorption ionization mass spectrometry did not reveal any significant homology with other molecules. In normal lymphoid tissue, hpH4 is expressed by T cells located at the periphery of lymph node germinal centers and paracortical areas. In T cell neoplasia, expression of hpH4 clusters with a subset of peripheral T cell lymphomas with a large-cell component, and with cases of angioimmunoblastic T cell lymphomas. Overall, these data provide evidence for a novel T cell activation molecule that could help in the phenotypic categorization of T cell malignancies.
Assuntos
Antígenos de Diferenciação de Linfócitos T/química , Linfoma de Células T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Timo/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Humanos , Ativação Linfocitária/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Linfoma de Células T/imunologia , Camundongos , Especificidade de Órgãos/imunologia , Homologia de Sequência de Aminoácidos , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Células Tumorais CultivadasRESUMO
Most normal and neoplastic cell types are resistant to tumor necrosis factor (TNF) cytotoxicity unless cotreated with protein or RNA synthesis inhibitors, such as cycloheximide and actinomycin D. Cellular resistance to TNF requires TNF receptor-associated factor 2 (TRAF2), which has been hypothesized to act mainly by mediating activation of the transcription factors nuclear factor kB (NFkB) and activator protein 1 (AP1). NFkB was proposed to switch on transcription of yet unidentified anti-apoptotic genes. To test the possible existence of NFkB-independent cytoprotective pathways, we systematically compared selective trans-dominant inhibitors of the NFkB pathway with inhibitors of TRAF2 signaling for their effect on TNF cytotoxicity. Blockade of TRAF2 function(s) by signaling-deficient oligomerization partners or by molecules affecting TRAF2 recruitment to the TNF receptor 1 complex completely abrogated the cytoprotective response. Conversely, sensitization to TNF cytotoxicity induced by a selective NFkB blockade affected only a fraction of TNF-treated cells in an apparently stochastic manner. No cytoprotective role for c-Jun amino-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), which are activated by TRAF2 and contribute to stimulation of activator protein 1 activity, could be demonstrated in the cellular systems tested. Although required for cytoprotection, TRAF2 is not sufficient to protect cells from TNF + cycloheximide cytotoxicity when overexpressed in transfected cells, thus indicating an essential role of additional TNF receptor 1 complex components in the cytoprotective response. Our results indicate that TNF-induced cytoprotection is a complex function requiring the integration of multiple signal transduction pathways.
Assuntos
Apoptose , Proteínas I-kappa B , NF-kappa B/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/metabolismo , Dimerização , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Oligodendroglia/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/antagonistas & inibidores , Proteínas/genética , Ratos , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF , Dedos de ZincoRESUMO
In this paper we show that in viral hepatitis most Kupffer cells (KCs) are activated and express high levels of CD80, CD40, and class-II MHC molecules, thus acquiring the phenotype of professional antigen presenting cells (APCs). Activated KCs display a close contact with CD4+ T lymphocytes and form KCs-T lymphocyte clusters. Clusters are found within the sinusoids, across the sinusoid wall, and within the liver parenchyma as well, as a consequence of transendothelial migration (TEM). The positivity of activated KCs for hepatitis C virus (HCV) antigens, which likely reflects phagocytosis of infected hepatocytes, suggests that KCs-T cell clusters represent the morphological expression of the functional interaction between KCs acting as professional APCs and antigen-experienced CD4+ T lymphocytes within the liver. These phenotypic and morphological changes are distinct features of livers in chronic hepatitis patients compared with controls.
Assuntos
Antígeno B7-1/biossíntese , Antígenos CD40/biossíntese , Hepacivirus/isolamento & purificação , Hepatite C Crônica/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Células de Kupffer/imunologia , Adulto , Idoso , Apresentação de Antígeno , Antígenos Virais/imunologia , Antígeno B7-1/imunologia , Antígenos CD40/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/patologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Células de Kupffer/patologia , Pessoa de Meia-IdadeRESUMO
Peripheral blood monocyte-derived multinucleated giant cells are a well-known feature of chronic inflammatory conditions. Similarly, virus-induced syncytia derived from CD4+ cells are considered to be typical of human immunodeficiency virus infection under culture conditions. Here, Stefano Fais and colleagues summarize recent experimental results comparing the mechanisms underlying the formation and fate of these two different polykaryons, discussing their putative role in the immune response.
Assuntos
Células Gigantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Fusão Celular , HIV/imunologia , HIV/fisiologia , Humanos , Imunidade , Monócitos/imunologia , Monócitos/patologiaRESUMO
The human hepatitis B virus (HBV) protein pX is a multifunctional regulatory protein that is known to affect both transcription and cell growth. Here we describe induction of apoptosis in NIH 3T3 polyclonal cell lines upon stimulation of pX expression from a dexamethasone inducible mouse mammary tumor virus (MMTV)-X expression vector. The effect of long-term pX expression on the cell survival of mouse fibroblasts was confirmed in colony generation assays. This effect is not shared either by the other HBV products and it is c-myc mediated, as shown by the use of a dominant negative deletion mutant of c-myc. pX also sensitize cells to programmed cell death after exposure to DNA damaging agents. Taking advantage of stable transfectants carrying the p53val135 temperature-sensitive allele, we directly demonstrate that induction of apoptosis by pX requires p53. In p53 null mouse embryo fibroblasts pX activates transcription and confers an evident growth advantage without loss of cell viability. Although pX protein was not detectable in the experimental conditions we used, our results indicate that its expression affects both cell growth and cell death control.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica/genética , Antígenos da Hepatite B/genética , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro , Replicação do DNA/genética , Camundongos , Camundongos Endogâmicos BALB C , Ativação Transcricional , Proteínas Virais Reguladoras e AcessóriasRESUMO
Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Músculo Esquelético/metabolismo , Proteína MyoD/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Quinase 2 Dependente de Ciclina , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F4 , Fase G1/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Músculo Esquelético/citologia , Proteína MyoD/genética , Fase de Repouso do Ciclo Celular/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Regulação para CimaRESUMO
In this study, we investigated the c-myc expression by tonsillar germinal center (GC) B cells using reverse transcriptase-polymerase chain reaction, flow cytometry, Western blot and in situ immunohistochemical methods. The results obtained demonstrate elevated levels of c-myc mRNA and of Myc protein in GC B cells compared to those of the other resting or activated tonsillar B cells. Separation of GC B cells into centroblasts and centrocytes revealed that, while differing in their cell cycle status, surface marker expression and morphology, the two cell types had the same propensity to apoptosis and elevated Myc protein expression, thus reinforcing the notion of a close correlation between these two events. Based upon these observations and other considerations it is proposed that elevation of Myc proteins confers to GC B cells a particular propensity to apoptosis, while the subsequent decision between progression into the cell cycle or programmed cell death is dictated by other signals that are delivered in the GC and perhaps operate at the level of other proto-oncogenes.
Assuntos
Apoptose , Linfócitos B/imunologia , Centro Germinativo/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Ciclo Celular , Expressão Gênica , Humanos , Tonsila Palatina/citologia , RNA Mensageiro/genética , Triexosilceramidas/análiseRESUMO
BACKGROUND AND OBJECTIVE: The lymph nodes involved in classic Hodgkin's disease (HD), i.e. mixed cellularity (MC) and nodular sclerosis (NS) subtypes, usually contain few (1-2%) Reed-Sternberg (RS) cells scattered in a background of lymphocytes, eosinophils, plasma cells and neutrophils. CD4+ T-lymphocytes are increased in number, express activation markers and cluster around RS cells. The presence of eosinophilia in most HD patients and the presence of hyper-IgE in a subset of them may suggest that activated lymph node T cells release large amounts of IL5 and IL4, respectively. METHODS: The expression of four T-cell-associated cytokine genes, i.e. interleukin (IL)2, IL4, IL5 and interferon (IFN)-gamma, in frozen sections of 14 HD (7 MC, 7 NS) and 10 reactive lymph nodes was investigated by qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). T-cell clones were also raised from purified CD4+ lymphocytes of one HD lymph node and one reactive lymph node and tested for IL2, IL4, IL5 and IFN-gamma secretion in culture supernatants by immunoassays. RESULTS: The transcripts of all the cytokine genes were detected in every lymph node irrespective of the HD or reactive nature. HD or reactive lymph node-derived CD4+ T-cell clones released the four cytokines according to a predominant T-helper (Th)0-type pattern. In more than half of the lymph nodes of either HD or reactive nature, there was a predominance of IL4 over IFN-gamma mRNA production (Th2-type pattern). In the remaining HD or reactive lymphadenopathies, either a balanced IL4/IFN-gamma mRNA ratio (Th0-type pattern) or a predominance of IFN-gamma over IL4 mRNA expression (Th1-type pattern) was observed. INTERPRETATION AND CONCLUSIONS: The overall pattern of cytokine gene expression in classic HD is similar to that detected in reactive lymph nodes. Further studies are needed to determine whether differences in the absolute concentrations of cytokines released in HD versus reactive lymph nodes and the long-standing course of HD versus the self-limiting nature of reactive adenopathies may explain certain peculiar features of HD, such as eosinophilia, for example.
Assuntos
Expressão Gênica , Doença de Hodgkin/metabolismo , Interferon gama/genética , Interleucinas/genética , Doenças Linfáticas/metabolismo , Adolescente , Adulto , Idoso , Feminino , Doença de Hodgkin/genética , Humanos , Interleucina-2/genética , Interleucina-4/genética , Interleucina-5/genética , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5-10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5- B cells had a surface phenotype (IgM+, IgD+, CD23-, CD38+/-, CD10-, CD44+) that was different from that of FM (IgM+, IgD+, CD23+, CD39+, CD38-, CD10-, CD44+2) and of germinal center (GC) (CD23-, CD39-, CD38+, CD10+, CD44+/-, IgG+) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5- B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5- B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5- B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5- B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP+, while GC cells were consistently ALP-. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5- B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5- B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.
Assuntos
Linfócitos B/imunologia , Tonsila Palatina/imunologia , Fosfatase Alcalina/metabolismo , Linfócitos B/ultraestrutura , Antígenos CD5/análise , Criança , Humanos , Imuno-Histoquímica , Imunofenotipagem , Palato/imunologiaRESUMO
This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike FM B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-mu antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-mu-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-mu antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.
Assuntos
Linfócitos B/fisiologia , Tonsila Palatina/imunologia , Animais , Formação de Anticorpos , Antígenos CD/análise , Apoptose , Linfócitos B/imunologia , Humanos , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Palato/imunologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2 , Trinitrobenzenos/imunologiaRESUMO
BACKGROUND & AIMS: Transmural perivascular mononuclear cell infiltrates are a feature of Crohn's disease. The aim of this study was a molecular characterization of the mechanisms leading to the formation of these infiltrates. METHODS: Endothelial cell and leukocyte expression of the adhesion molecules directing leukocyte transendothelial migration were studied in situ by immunohistochemical analysis of 10 samples from patients with Crohn's disease and 10 samples from normal controls. Double-staining methods were used to characterize the cells forming the infiltrates. RESULTS: CD11a+ and L-selectin-positive mononuclear cells seemed to be the major component of perivascular infiltrates. The vast majority of these cells were CD68+, CD31+ monocytes/macrophages surrounded by CD3+, L-selectin-positive, CD31+, CD45RA+, and/or CD45RO+ T lymphocytes. T lymphocytes within the vessels expressed both CD45RA and CD45RO markers. Endothelial cells were intercellular adhesion molecule 1 positive and mostly CD34+. Strong adhesion between L-selectin-positive and CD11a+ intravascular mononuclear cells and CD34+ and intercellular adhesion molecule 1-positive endothelial cells were observed. CONCLUSIONS: Data indicate that peripheral mononuclear cells are actively recruited in the submucosa of Crohn's disease tissue; endothelial cells express adhesion molecules highly permissive for transendothelial migration of monocytes and both naive and memory T cells contributing to infiltrates generation; and close membrane contact between migrated macrophages and naive T cells leads to the T-cell transition from naive to memory phenotype within Crohn's disease.
Assuntos
Doença de Crohn/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Complexo CD3/análise , Movimento Celular , Doença de Crohn/patologia , Humanos , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/análise , Mucosa Intestinal/patologia , Selectina L/análise , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/análiseRESUMO
Angiotensin II (Ang-II) receptor engagement activates many immediate early response genes in both vascular smooth muscle cells and cardiomyocytes whether a hyperplastic or hypertrophic response is taking place. Although the signaling pathways stimulated by Ang-II in different cell lines have been widely characterized, the correlation between the generation of different second messengers and specific physiological responses remains relatively unexplored. In this study, we report how in both C2C12 quiescent myoblasts and differentiated myotubes Ang-II significantly stimulates AP1-driven transcription and c-Jun.c-Fos heterodimer DNA binding activity. Using a set of different protein kinase inhibitors, we could demonstrate that Ang-II-induced increase in AP1 binding is not mediated by the cAMP-dependent pathway and that both protein kinase C and tyrosine kinases are involved. The observation that in quiescent myoblasts Ang-II increase of AP1 binding and induction of DNA synthesis and, in differentiated myotubes, Ang-II stimulation of protein synthesis are abolished by the cysteine-derivative and glutathione precursor N-acetyl-L-cysteine strongly suggests a role for reactive oxygen intermediates in the intracellular transduction of Ang-II signals for immediate early gene induction, cell proliferation, and hypertrophic responses.
Assuntos
Angiotensina II/fisiologia , Proteínas de Ligação a DNA/metabolismo , Músculos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Acetilcisteína/farmacologia , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Expressão Gênica/efeitos dos fármacos , Isoquinolinas/farmacologia , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Piperazinas/farmacologia , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , Transdução de SinaisRESUMO
Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.
Assuntos
Angiotensina II/farmacologia , Expressão Gênica/efeitos dos fármacos , Músculo Esquelético/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transdução de Sinais/fisiologia , Animais , Antioxidantes/farmacologia , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Genes Precoces/efeitos dos fármacos , Coração/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Oligodesoxirribonucleotídeos , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
BACKGROUND: Multinucleated giant cells (MGC), interferon-gamma (IFN-gamma) production, and increased expression of adhesion molecules are features of granulomatous reactions. IFN-gamma induces the fusion of macrophages and the subsequent MGC generation in vitro. Moreover, IFN-gamma increases ICAM-1 expression on lymphoid cells and an important role for adhesion molecules in MGC generation has been proposed. EXPERIMENTAL DESIGN: The time course of the IFN-gamma-driven MGC generation was investigated in slide-chamber cultures of adherence-purified human monocytes. The fusion index, the monocytes clustering the total number of MGC were determined. The expression of intercellular cell adhesion molecule-1 (ICAM-1), LFA-1 and HLA-DR was investigated by immunohistochemistry. The effect of anti-ICAM-1, anti-LFA-1 and anti-HLA-DR monoclonal antibodies on IFN-gamma-induced MGC generation was also examined. RESULTS: IFN-gamma enhanced the generation of MGC in a dose- and time-dependent fashion. In all experiments, MGC formation was preceded by a sequence of changes in the morphology of cultured monocytes. Cell clustering occurred as early as 3 days after IFN-gamma stimulation and was followed by the adhesion of cells that eventually fused. Immunohistochemistry showed that ICAM-1 was increased by IFN-gamma and constantly polarized on a cell uropode. When monocytes clustered, ICAM-1 was localized on the membrane where the cell-to-cell contact occurred. In newly formed MGC, ICAM-1 stained in the center of the giant cell. The cellular distribution of LFA-1 on cultured monocytes was not modified by IFN-gamma. HLA-DR expectedly enhanced by IFN-gamma was mostly cytoplasmic and tended to disappear when MGC formed. Finally, anti-LFA-1 and anti-ICAM-1 monoclonal antibodies variably inhibited IFN-gamma-induced MGC generation. CONCLUSIONS: Taken together, these data add support to the concept that IFN-gamma is essential for MGC generation by promoting cell clustering and cell-to-cell adhesion. The present data also indicate that among the mechanisms by which IFN-gamma exert such a promoting effect, changes in the ICAM-1 expression and cellular distribution are included. The observation that ICAM-1 appears to be trapped in the cytoplasm of IFN-gamma-driven MGC and that HLA-DR tend to disappear from macrophages undergoing MGC formation, also suggest changes in the functional properties of these cells.
Assuntos
Células Gigantes de Corpo Estranho/citologia , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/farmacologia , Macrófagos/citologia , Fusão Celular , Células Gigantes de Corpo Estranho/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Técnicas Imunoenzimáticas , Técnicas In Vitro , Antígeno-1 Associado à Função Linfocitária/metabolismo , Macrófagos/metabolismoRESUMO
BACKGROUND AND METHODS: A significant correlation between bone marrow (BM) histology, survival and disease progression (DP) probability has been observed by several authors in chronic lymphocytic leukemia (CLL). The prognostic value of BM histologic patterns on survival and disease progression probability was investigated in 335 B-CLL patients. RESULTS: Actuarial survival probability estimated by univariate analysis proved to be significantly influenced by stage (p < 0.0001), BM histology (p = 0.01), and by the following parameters: anemia (p = 0.0005), splenomegaly (p < 0.001), CLL-related symptoms (p < 0.01), thrombocytopenia (p < 0.01), number of involved nodal areas (p = 0.01) and peripheral lymphocyte count over 30 x 10(9)/L (p < 0.05). In this series we did not detect a discriminating prognostic effect for BM histology within the individual stages. Cox multivariate regression analysis failed to demonstrate a significant value for BM histology, while stage and anemia emerged as the best prognostic variables. Actuarial DP free probability in 294 untreated patients with A and B stages was significantly related to stage (p < 0.00001) and to BM pattern (p = 0.01). CONCLUSIONS: Despite the clear correlation between the D pattern of BM involvement and advanced and early progressive disease, we were unable to demonstrate an independent prognostic value for BM histology. These findings suggest that stage emerged as the best predictive factor of survival probability in our B-CLL patients.
Assuntos
Exame de Medula Óssea , Leucemia Linfocítica Crônica de Células B/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
EN4 MoAb was originally described as a MoAb that reacts specifically with human endothelial cells, and the reagent was not assigned to any of the presently known CD. Here, we provide evidence indicating that EN4 reacts with the CD31 antigen. Thus, EN4 stains strongly murine fibroblasts transfected with the human CD31 gene. Furthermore, SDS-PAGE analysis of immunoprecipitates of cell lysates from surface-iodinated Jurkart T cells demonstrated that EN4 and reference CD31 MoAb recognized the same antigen, of 130 kD mol. wt. Finally, both EN4 and CD31 gave the same pattern of reactivity when tested on tonsillar or peripheral blood lymphoid cells by FACS analysis or by immunohistochemistry on sections of a variety of human tissues. EN4, however, proved consistently more efficient than the reference anti-CD31 MoAb as judged by both the intensity of fluorescence or of tissue staining. This property has thus allowed a better characterization of the tissue and cellular distribution of CD31.