RESUMO
Plasmacytoid dendritic cells (pDCs) link innate to acquired immune responses by producing high levels of type I IFN upon infection. In order to identify the specific genes that control pDC, we compared serial analysis of gene expression libraries from human pDCs, herpes simplex virus-stimulated pDCs and monocytes. We found that Ig-like transcript ILT7 is specifically expressed on pDC cell surfaces and is down-regulated when pDC mature in response to viral or bacterial stimulation. ILT7 expression on the cell surface required association with the Fc epsilon RI gamma adaptor molecule. Although treatment with one anti-ILT7-specific mAb suppressed type I IFN production in response to cytosine-phosphate-guanosice (CpG) stimulation, another anti-ILT7 mAb up-regulated type I IFN production. We conclude that ILT7 is a key regulator of human pDC function.
Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica , Biblioteca Gênica , Interferon Tipo I/biossíntese , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Interferon Tipo I/genética , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , TransfecçãoRESUMO
Plasmacytoid dendritic cells (pDCs) play an important primary role for antiviral innate immunity by rapidly producing large amounts of type 1 interferon (IFN) upon viral infection. To study pDC biology, we generated a monoclonal antibody, termed 2E6, that recognizes pDCs. Molecular cloning of a cDNA encoding the 2E6 antigen revealed that it is a type II C-type lectin, Ly49Q, that consists of 247 amino acids with high homology to the natural killer (NK) receptor family Ly49, with an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic domain. Ly49Q is expressed on pDCs but not on NK cells or myeloid dendritic cells. B220+, CD11c+, CD11b- pDCs in bone marrow were divided into Ly49Q+ and Ly49Q- subsets. While both subsets produced IFN-alpha upon cytosine-phosphate-guanosine (CpG) and herpes simplex virus stimulation, Ly49Q- pDCs responded poorly to influenza virus. In addition, Ly49Q- pDCs produced inflammatory cytokines such as interleukin 6 (IL-6), IL-12, and tumor necrosis factor alpha (TNF-alpha) upon stimulation at lower levels than those produced by Ly49Q+ pDCs. In contrast to bone marrow, Ly49Q+ pDCs were only found in peripheral blood, lymph nodes, and spleen. These results indicate that Ly49Q is a specific marker for peripheral pDCs and that expression of Ly49Q defines 2 subsets of pDCs in bone marrow.
Assuntos
Antígenos Ly/genética , Antígenos Ly/imunologia , Biomarcadores , Células Dendríticas/classificação , Células Dendríticas/citologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos Ly/metabolismo , Células da Medula Óssea/classificação , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Células Cultivadas , Clonagem Molecular , DNA Complementar , Células Dendríticas/metabolismo , Feminino , Interferon-alfa/metabolismo , Lectinas Tipo C , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Ratos , Ratos Wistar , Receptores Semelhantes a Lectina de Células NK , Baço/citologiaRESUMO
Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA , Genoma Viral , Hepacivirus/genética , Fígado/virologia , Proteínas Quinases/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Western Blotting , Caseína Quinases , Ciclo Celular , Divisão Celular , Linhagem Celular , Separação Celular , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , DNA Complementar/metabolismo , Fatores de Transcrição E2F , Citometria de Fluxo , Regulação Viral da Expressão Gênica , Genes Reporter , Hepatócitos/metabolismo , Humanos , Immunoblotting , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Telomerase, the ribonucleoprotein enzyme that synthesizes telomeric DNA, is thought to be necessary for cellular immortality and carcinogenesis. Telomerase activity is associated with the majority of malignant human cancers. The mRNA that encodes the telomerase catalytic subunit (human telomerase repeat transcriptase; hTERT) has recently been identified, and the expression of the hTERT gene is thought to regulate the activation of telomerase. However, the expression of hTERT mRNA in esophageal tissues has not been reported. We investigated hTERT gene expression in cancerous and noncancerous esophageal tissues, and determined the relationship between hTERT mRNA expression and telomerase activity. METHODS: Tissues from esophageal carcinomas in 14 patients, reflux esophagitis in 12 patients, esophageal acanthosis in 2 patients, esophageal papilloma in 1 patient, radiation esophagitis in 1 patient, and normal esophageal epithelium in 11 patients (including 3 specimens of normal epithelium from patients with esophageal carcinoma) were examined. All specimens were taken endoscopically. hTERT gene expression was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Quantitative analysis of telomerase activity was analyzed by fluorescence telomeric repeat amplification protocol (F-TRAP) assay. RESULTS: Thirteen of the 14 (93%) esophageal carcinoma specimens expressed hTERT mRNA and revealed detectable telomerase activity. Noncancerous esophageal lesions had not only hTERT mRNA expression with a high frequency (14 of 16 cases; 88%) but also detectable telomerase activity (12 of 13 cases; 92%). Normal esophageal epithelium also highly expressed hTERT mRNA (10 of 11 cases; 91%) and revealed detectable telomerase activity (all 9 cases; 100%). In 32 of the 35 specimens analyzed for both hTERT mRNA and telomerase activity (91%), the expression of hTERT mRNA was consistent with detectable telomerase activity. CONCLUSIONS: The expression of hTERT mRNA was detected not only in cancerous but also in noncancerous esophageal tissues at a high frequency. This result was different from that reported for other gastrointestinal epithelium. Moreover, telomerase activity in esophageal carcinoma was significantly stronger than that in reflux esophagitis and normal epithelium. In addition, there was a strong relation ship between the detection of telomerase activity and the expression of hTERT mRNA in cancerous and noncancerous esophageal tissues. Thus, the qualitative analysis of hTERT mRNA expression may not be useful as a biomarker of carcinoma in esophageal tissues. Nevertheless, the quantitative analysis of telomerase activity may be somewhat useful.