RESUMEN
Interleukin 4 (IL-4) can suppress delayed-type hypersensitivity reactions (DTHRs), including organ-specific autoimmune diseases in mice and humans. Despite the broadly documented antiinflammatory effect of IL-4, the underlying mode of action remains incompletely understood, as IL-4 also promotes IL-12 production by dendritic cells (DCs) and IFN-γ-producing T(H)1 cells in vivo. Studying the impact of IL-4 on the polarization of human and mouse DCs, we found that IL-4 exerts opposing effects on the production of either IL-12 or IL-23. While promoting IL-12-producing capacity of DCs, IL-4 completely abrogates IL-23. Bone marrow chimeras proved that IL-4-mediated suppression of DTHRs relies on the signal transducer and activator of transcription 6 (STAT6)-dependent abrogation of IL-23 in antigen-presenting cells. Moreover, IL-4 therapy attenuated DTHRs by STAT6- and activating transcription factor 3 (ATF3)-dependent suppression of the IL-23/T(H)17 responses despite simultaneous enhancement of IL-12/TH1 responses. As IL-4 therapy also improves psoriasis in humans and suppresses IL-23/T(H)17 responses without blocking IL-12/T(H)1, selective IL-4-mediated IL-23/T(H)17 silencing is promising as treatment against harmful inflammation, while sparing the IL-12-dependent T(H)1 responses.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Silenciador del Gen , Inflamación/fisiopatología , Interleucina-23/genética , Interleucina-4/fisiología , Células Th17/inmunología , HumanosRESUMEN
Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell-mediated autoimmune diseases. By dissecting Th1 cell-mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into Kit(W)/Kit(W-v), TNF(-/-), and TNFR1(-/-) mice showed that the signaling defect exclusively affects mast cell-EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1(-/-) mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1(-/-) mice. As substitution of TNF(-/-) mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.
Asunto(s)
Endotelio Vascular/patología , Inflamación/etiología , Mastocitos/fisiología , Receptor Cross-Talk/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Haptenos/efectos adversos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/genética , Hipersensibilidad Tardía/inmunología , Inflamación/genética , Inflamación/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Cloruro de Picrilo/efectos adversos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Selective skewing of autoreactive interferon-gamma (IFN-gamma)-producing T helper cells (Th1) toward an interleukin-4 (IL-4)-producing (Th2) phenotype can in experimental animals alleviate autoimmune disease without inducing general immunosuppression. In a prospective dose escalation study, we assessed treatment with human IL-4 (rhuIL-4) in 20 patients with severe psoriasis. The therapy was well tolerated, and within six weeks all patients showed decreased clinical scores and 15 improved more than 68%. Stable reduction of clinical scores was significantly better at 0.2-0.5 microg rhuIL-4 than at < or =0.1 microg rhuIL-4 (P = 0.009). In psoriatic lesions, treatment with 0.2-0.5 microg/kg rhuIL-4 reduced the concentrations of IL-8 and IL-19, two cytokines directly involved in psoriasis; the number of chemokine receptor CCR5+ Th1 cells; and the IFN-gamma/IL-4 ratio. In the circulation, 0.2-0.5 microg/kg rhuIL-4 increased the number of IL-4+CD4+ T cells two- to three-fold. Thus, IL-4 therapy can induce Th2 differentiation in human CD4+ T cells and has promise as a potential treatment for psoriasis, a prototypic Th1-associated autoimmune disease.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Interleucina-4/inmunología , Interleucina-4/uso terapéutico , Psoriasis/tratamiento farmacológico , Psoriasis/inmunología , Células Th2/inmunología , Adolescente , Adulto , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Preescolar , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-18/metabolismo , Interleucina-4/administración & dosificación , Interleucina-4/farmacología , Interleucinas , Masculino , Persona de Mediana Edad , Fenotipo , Psoriasis/patología , Psoriasis/fisiopatología , Receptores CCR5/metabolismo , Índice de Severidad de la Enfermedad , Piel/citología , Piel/efectos de los fármacos , Piel/patología , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/metabolismoRESUMEN
Tissue neovascularization involves recruitment of circulating endothelial progenitor cells that originate in the bone marrow. Here, we show that a class of embryonic endothelial progenitor cells (Tie-2+, c-Kit+, Sca-1+, and Flk-1-/low), which were isolated at E7.5 of mouse development at the onset of vasculogenesis, retain their ability to contribute to tumor angiogenesis in the adult. Using intravital fluorescence videomicroscopy, we further defined the multistep process of embryonic endothelial progenitor cell (eEPC) homing and incorporation. Circulating eEPCs are specifically arrested in "hot spots" within the tumor microvasculature, extravasate into the interstitium, form multicellular clusters, and incorporate into functional vascular networks. Expression analysis and in vivo blocking experiments provide evidence that the initial cell arrest of eEPC homing is mediated by E- and P-selectin and P-selectin glycoprotein ligand 1. This paper provides the first in vivo insights into the mechanisms of endothelial progenitor cell recruitment and, thus, indicates novel ways to interfere with pathological neovascularization.
Asunto(s)
Endotelio Vascular/citología , Glioma/irrigación sanguínea , Neovascularización Patológica , Células Madre/fisiología , Animales , Biomarcadores , Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Adhesión Celular/fisiología , Embrión de Mamíferos , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Glioma/metabolismo , Glioma/patología , Hemodinámica , Glicoproteínas de Membrana/metabolismo , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Selectinas/metabolismoRESUMEN
The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-kappaB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-kappaB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.
Asunto(s)
Linfocitos B/virología , Herpesvirus Humano 4/fisiología , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Latencia del Virus , Linfocitos B/metabolismo , Línea Celular Transformada , Proliferación Celular , Transformación Celular Viral , Análisis por Conglomerados , ADN/biosíntesis , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Humanos , FN-kappa B/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Activation induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching in the immunoglobulin genes of B-lymphocytes. AID expression by itself is sufficient to induce a GC-basepair biased mutator phenotype in lymphoid and non-lymphoid cell lines. Nevertheless a network of cis-regulatory elements and additional trans-factor proteins seems to govern the molecular mechanism of somatic hypermutation. To address the nature of mutation rate changes observed in the hypermutating pre-B cell line 18-81, we extended our previously described green fluorescent protein (GFP) reversion-system. Introducing an additional mutation reporter transgene enables us to discriminate between cis- and trans-factor caused alterations in the mutator phenotype. We show here that in cell line 18-81 the mutation rate declines upon prolonged periods of cell culture. The gradual loss of the mutator phenotype in cell line 18-81 is due to the downregulation of endogenous AID expression and can be reconstituted by overexpression of human AID protein. A correlation between AID mRNA levels and mutation rates is evident and even small changes in AID expression levels cause a significant effect on the mutability of the reporter transgenes.
Asunto(s)
Linfocitos B/citología , Hipermutación Somática de Inmunoglobulina , Factores de Transcripción , Animales , Línea Celular , Citidina Desaminasa/genética , Regulación hacia Abajo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Cinética , Ratones , Fenotipo , ARN Mensajero/análisis , TransfecciónRESUMEN
A defined and balanced immunomodulatory response is crucial for the protection of mucosal surfaces being in contact with pathogenic microorganisms. This study examined the local host response mechanisms of epithelial cells in experimental Candida albicans, C. tropicalis, and C. glabrata infections by measuring the expression of cytokines at the mRNA and protein level. During the course of infection with active but not with heat-killed C. albicans stimulation of the gene expression levels for interleukin-1alpha, interleukin-1beta, tumor necrosis factor, Exodus-2, P-selectin ligand, granulocyte-monocyte colony-stimulating factor, and interleukin-8 was observed by standard and quantitative reverse transcription-polymerase chain reaction. This cytokine pattern may favor a chemotactic and a T helper 1 response. Initial moderate or weak upregulation of these cytokine genes by reverse transcription-polymerase chain reaction was also observed in epithelial infection with the less virulent species C. tropicalis and C. glabrata. Heat-killed C. albicans failed to induce an epithelial immune response. At the protein level, expression of interleukin-8 protein was strongly enhanced during the course of C. albicans infection, whereas lower levels were seen with C. tropicalis and C. glabrata. The different expression patterns of cytokines were associated with differences in virulence of the Candida strains. This study's data, therefore, show a correlation between the virulence potential of pathogenic fungi, possibly mediated by specific virulence factors (such as proteinases), and the secretion of epithelial cytokines and chemokines, which may initiate in vivo a protective T helper 1 immunologic response and contribute to the recruitment of activated leukocytes and lymphocytes to the site of mucosal infection.
Asunto(s)
Candida albicans/patogenicidad , Candidiasis Bucal/inmunología , Citocinas/genética , Candida albicans/inmunología , Candidiasis Bucal/fisiopatología , Quimiocina CCL21 , Quimiocinas CC/genética , Epitelio/microbiología , Expresión Génica/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Calor , Humanos , Técnicas In Vitro , Interleucina-1/genética , Interleucina-8/genética , Interleucina-8/metabolismo , Glicoproteínas de Membrana/genética , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/genética , VirulenciaRESUMEN
Skin equivalents based on reconstituted human epidermis have been used recently to establish models for allergic/irritant contact dermatitis and cutaneous candidosis. In the present study the cytokine expression pattern and the morphological alterations in experimental cutaneous candidosis were investigated by RT-PCR and histological analysis. In experimental cutaneous C albicans infection the mRNA expression levels of interleukin (IL)-1a, IL-1beta, IL-8, GM-CSF, Exodus-2, tumour necrosis factor-alpha and PSL (P-selectin ligand) were upregulated. Cytokine profile and histological features of infected skin (separation of keratinocytes, oedema, vacuolisation) were comparable to that seen in experimental contact dermatitis. These immunomodulatory and morphological similarities might reflect a common pathogenesis factor in both diseases.
Asunto(s)
Candida albicans/inmunología , Candidiasis Cutánea/inmunología , Citocinas/biosíntesis , Candida albicans/genética , Candidiasis Cutánea/microbiología , Candidiasis Cutánea/patología , Citocinas/genética , Regulación de la Expresión Génica/inmunología , Humanos , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immune responses may arrest tumor growth by inducing tumor dormancy. The mechanisms leading to either tumor dormancy or promotion of multistage carcinogenesis by adaptive immunity are poorly characterized. Analyzing T antigen (Tag)-induced multistage carcinogenesis in pancreatic islets, we show that Tag-specific CD4+ T cells home selectively into the tumor microenvironment around the islets, where they either arrest or promote transition of dysplastic islets into islet carcinomas. Through combined TNFR1 signaling and IFN-gamma signaling, Tag-specific CD4+ T cells induce antiangiogenic chemokines and prevent alpha(v)beta(3) integrin expression, tumor angiogenesis, tumor cell proliferation, and multistage carcinogenesis, without destroying Tag-expressing islet cells. In the absence of either TNFR1 signaling or IFN-gamma signaling, the same T cells paradoxically promote angiogenesis and multistage carcinogenesis. Thus, tumor-specific T cells can directly survey multistage carcinogenesis through cytokine signaling.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Transformación Celular Neoplásica/inmunología , Insulinoma/inmunología , Interferón gamma/metabolismo , Neoplasias Pancreáticas/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Animales , Antígenos Virales de Tumores/genética , Antígenos Virales de Tumores/metabolismo , Glucemia/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Movimiento Celular , Supervivencia Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Inmunoterapia/métodos , Insulinoma/irrigación sanguínea , Insulinoma/genética , Insulinoma/metabolismo , Insulinoma/patología , Insulinoma/terapia , Integrina alfaVbeta3/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Ratones Transgénicos , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Células TH1/inmunología , Células TH1/patología , Factores de Tiempo , Irradiación Corporal TotalRESUMEN
RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.
Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa II/metabolismo , Serina/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Epítopos , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Humanos , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Fosforilación , Subunidades de Proteína/química , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Polimerasa II/inmunologíaRESUMEN
OBJECTIVE: Induction of arthritis with autoantibodies against glucose-6-phosphate isomerase (GPI) is entirely independent of T cells and B cells but is strictly dependent on the presence of mast cells. Here, we used this disease model to analyze whether exclusive intraarticular mast cell reconstitution is sufficient for disease induction and whether targeted mast cell silencing can prevent neoangiogenesis and joint destruction, 2 hallmarks of rheumatoid arthritis. METHODS: Ankle swelling and clinical index scores were determined after injection of either K/BxN mouse-derived serum or control serum in wild-type Kit(+)/Kit(+) mice, congenic mast cell-deficient Kit(W)/Kit(W-v) mice, or mast cell-deficient Kit(W)/Kit(W-v) mice reconstituted with mast cells, either by intraperitoneal or selective intraarticular injection. Angiogenesis was quantified in vivo by measuring activated alphavbeta3 integrin using (18)F-galacto-RGD and positron emission tomography. In addition, staining of joint tissue with hematoxylin and eosin, Giemsa, beta3, and alpha-actin was performed. The effect of mast cell stabilization by treatment with cromolyn or salbutamol was investigated in C57BL/6 or BALB/c mice. RESULTS: Comparing wild-type mice, mast cell-deficient Kit(W)/Kit(W-v) mice, and mast cell-reconstituted Kit(W)/Kit(W-v) mice, we first showed that intraarticular and intraperitoneal mast cell engraftment fully restores susceptibility to antibody-induced arthritis, angiogenesis, and alphavbeta3 integrin activation. Importantly, selective mast cell silencing with either salbutamol or cromolyn prevented alphavbeta3 integrin activation, angiogenesis, and joint destruction. CONCLUSION: Mast cell engraftment fully restores susceptibility to alphavbeta3 integrin activation, angiogenesis, and joint destruction in GPI antibody-induced arthritis. Importantly, selective mast cell stabilization prevents alphavbeta3 integrin activation, angiogenesis, and joint destruction.
Asunto(s)
Artritis Reumatoide/fisiopatología , Artropatías/prevención & control , Mastocitos/fisiología , Neovascularización Patológica/prevención & control , Albuterol/farmacología , Animales , Anticuerpos Antiidiotipos/efectos adversos , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/patología , Cromolin Sódico/farmacología , AMP Cíclico/fisiología , Modelos Animales de Enfermedad , Femenino , Glucosa-6-Fosfato Isomerasa/inmunología , Integrina alfaVbeta3/fisiología , Artropatías/patología , Artropatías/fisiopatología , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatologíaRESUMEN
Deregulation of the proto-oncogene c-myc is a key event in the pathogenesis of many tumors. A paradigm is the activation of the c-myc gene by chromosomal translocations in Burkitt lymphoma (BL). Despite expression of a restricted set of Epstein-Barr viral (EBV) antigens, BL cells are not recognized by antigen-specific cytotoxic T cells (CTLs) because of their inability to process and present HLA class I-restricted antigens. In contrast, cells of EBV-driven posttransplant lymphoproliferative disease (PTLD) are recognized and rejected by EBV-specific CTLs. It is not known whether the poor immunogenicity of BL cells is due to nonexpression of viral antigens, overexpression of c-myc, or both. To understand the basis for immune recognition and escape, we have compared the mRNA expression profiles of BL and EBV-immortalized cells (as PTLD model). Among the genes expressed at low level in BL cells, we have identified many genes involved in the NF-kappaB and interferon response that play a pivotal role in antigen presentation and immune recognition. Using a cell line in which EBNA2 and c-myc can be regulated at will, we show that c-MYC negatively regulates STAT1, the central player linking the Type-I and Type-II interferon response. Switching off c-myc expression leads to STAT1 induction through a direct and indirect mechanism involving induction of Type-I interferons. c-MYC thus masks an interferon-inducing activity in these cells. Our findings imply that immune escape of tumor cells is not only a matter of in vivo selection but may be additionally promoted by activation of a cellular oncogene.
Asunto(s)
Linfoma de Burkitt/etiología , Regulación Neoplásica de la Expresión Génica/fisiología , Genes myc/fisiología , Interferón beta/farmacología , FN-kappa B/genética , Proteínas de Neoplasias/genética , Western Blotting , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Humanos , Análisis por Micromatrices , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Células Tumorales CultivadasRESUMEN
Inflammatory diseases at epithelial borders develop from aberrant interactions between resident cells of the tissue and invading immunocytes. Here, we unraveled basic functions of epithelial cells and immune cells and the sequence of their interactions in an inflammatory skin disease. Ubiquitous deficiency of the IkappaBalpha protein (Ikba(Delta)(/Delta)) as well as concomitant deletion of Ikba specifically in keratinocytes and T cells (Ikba(K5Delta/K5Delta lckDelta/lckDelta)) resulted in an inflammatory skin phenotype that involved the epithelial compartment and depended on the presence of lymphocytes as well as tumor necrosis factor and lymphotoxin signaling. In contrast, mice with selective ablation of Ikba in keratinocytes or lymphocytes showed inflammation limited to the dermal compartment or a normal skin phenotype, respectively. Targeted deletion of RelA from epidermal keratinocytes completely rescued the inflammatory skin phenotype of Ikba(Delta)(/Delta) mice. This finding emphasizes the important role of aberrant NF-kappaB activation in both keratinocytes and lymphocytes in the development of the observed inflammatory skin changes.
Asunto(s)
Dermatitis/inmunología , Proteínas I-kappa B/fisiología , Queratinocitos/inmunología , Piel/inmunología , Linfocitos T/inmunología , Absceso/genética , Absceso/inmunología , Absceso/patología , Animales , Comunicación Celular , Dermatitis/genética , Dermatitis/patología , Epidermis/inmunología , Epidermis/patología , Eliminación de Gen , Proteínas I-kappa B/genética , Queratinocitos/patología , Queratinas/metabolismo , Linfotoxina-alfa/metabolismo , Ratones , Ratones Mutantes , Inhibidor NF-kappaB alfa , Piel/patología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.
Asunto(s)
Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Linfocitos B/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitt's lymphoma and Hodgkin's disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization.
Asunto(s)
Linfocitos B/virología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Linfocitos B/citología , Secuencia de Bases , Transformación Celular Viral , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Expresión Génica , Genes myc , Herpesvirus Humano 4/patogenicidad , Herpesvirus Humano 4/fisiología , Humanos , Técnicas In Vitro , Biosíntesis de Proteínas , Proteoma , Proto-Oncogenes Mas , Proteínas ViralesRESUMEN
Conserved molecular patterns derived from pathogenic microorganisms prime antigen-presenting dendritic cells (DC) to induce adaptive T cell responses. In contrast, virus-infected or tumor cells that express low levels of major histocompatibility complex (MHC) class I activate natural killer (NK) cells for direct killing. It is unknown whether NK cell recognition of MHC class I(low) targets can also induce adaptive T cell responses. Here, we show that MHC class I(low) targets initiate a cascade of immune responses, starting with the immediate activation of NK cells. The activated NK cells then prime DC to produce IL-12 and to induce highly protective CD8 T cell memory responses. Therefore, sensing of MHC class I(low) targets by NK cells can link innate and adaptive immunity to induce protective T cell responses and may alarm the immune system during early infection with noncytopathic viruses.