RESUMEN
Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.
Asunto(s)
Asma/inmunología , Plaquetas/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Células Th2/inmunología , Proteínas Wnt/antagonistas & inhibidores , Animales , Antígenos Dermatofagoides/inmunología , Antígenos de Protozoos/inmunología , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Pyroglyphidae , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Cancer cells can develop an immunosuppressive tumor microenvironment to control tumor-infiltrating lymphocytes. The underlying mechanisms still remain unclear. Here, we report that mouse and human colon cancer cells acquire lymphocyte membrane proteins including cellular markers such as CD4 and CD45. We observed cell populations harboring both a tumor-specific marker and CD4 in the tumor microenvironment. Sorted cells from these populations were capable of forming organoids, identifying them as cancer cells. Live imaging analysis revealed that lymphocyte membrane proteins were transferred to cancer cells via trogocytosis. As a result of the transfer in vivo, cancer cells also acquired immune regulatory surface proteins such as CTLA4 and Tim3, which suppress activation of immune cells [T. L. Walunas et al, Immunity 1, 405-413 (1994) and L. Monney et al., Nature 415, 536-541 (2002)]. RNA sequencing analysis of ex vivo-cocultured splenocytes with trogocytic cancer cells showed reductions in Th1 activation and natural killer cell signaling pathways compared with the nontrogocytic control. Cancer cell trogocytosis was confirmed in the patient-derived xenograft models of colorectal cancer and head and neck cancer. These findings suggest that cancer cells utilize membrane proteins expressed in lymphocytes, which in turn contribute to the development of the immunosuppressive tumor microenvironment.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Antígeno CTLA-4/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Linfocitos Infiltrantes de Tumor/citología , Animales , Células CACO-2 , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Madre Hematopoyéticas/citología , Humanos , Sistema Inmunológico , Inmunosupresores , Células Jurkat , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Organoides/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Trogocitosis , Microambiente TumoralRESUMEN
Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.
RESUMEN
Foxp3 is a key transcription factor for differentiation and function of regulatory T (Treg) cells that is critical for maintaining immunological self-tolerance. Therefore, increasing Treg function by Foxp3 transduction to regulate an inflammatory immune response is an important goal for the treatment of autoimmune and allergic diseases. Here we have generated a cell-permeable Foxp3 protein by fusion with the unique human HHph-1-PTD (protein transduction domain), examined its regulatory function in T cells, and characterized its therapeutic effect in autoimmune and allergic disease models. HHph-1-Foxp3 was rapidly and effectively transduced into cells within 30 min and conferred suppressor function to CD4(+)CD25(-) T cells as well as directly inhibiting T-cell activation and proliferation. Systemic delivery of HHph-1 Foxp3 remarkably inhibited the autoimmune symptoms of scurfy mice and the development of colitis induced by scurfy or wild-type CD4 T cells. Moreover, intranasal delivery of HHph-1-Foxp3 strongly suppressed ovalbumin-induced allergic airway inflammation. These results demonstrate the clinical potential of the cell-permeable recombinant HHph-1-Foxp3 protein in autoimmune and hypersensitive allergic diseases.
Asunto(s)
Asma/terapia , Enfermedades Autoinmunes/terapia , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/uso terapéutico , Enfermedades Inflamatorias del Intestino/terapia , Animales , Asma/inmunología , Enfermedades Autoinmunes/inmunología , Permeabilidad de la Membrana Celular , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/administración & dosificación , Factores de Transcripción Forkhead/genética , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T Reguladores/inmunologíaRESUMEN
MicroRNAs are negative gene regulators and play important roles in cardiac development and disease. As evident by cardiomyopathy following cardiac-specific Dicer knockdown they also are required for maintaining normal cardiac contractile function but the specific role of miR-1 in the process is poorly understood. To characterize the role of miR-1 in particular and to identify its specific targets we created a tamoxifen-inducible, cardiac-specific Dicer knockdown mouse and demonstrated that Dicer downregulation results in a dramatic and rapid decline in cardiac function concurrent with significantly reduced levels of miR-1. The importance of miR-1 was established by miR-1 antagomir treatment of wild-type mice, which replicated the cardiac-specific Dicer knockdown phenotype. Down-regulation of miR-1 was associated with up-regulation of its predicted target Sorcin, an established modulator of calcium signaling and excitation-contraction coupling, subsequently verified as a miR-1 target with luciferase constructs. siRNA-mediated knockdown of Sorcin effectively rescued the cardiac phenotypes after Dicer or miR-1 knockdown affirming Sorcin as a critical mediator of the acute cardiomyopathy observed. The regulatory relationship between miR-1 and Sorcin was further confirmed in cultured mouse cardiomyocytes where modulation of miR-1 was associated with discordant Sorcin levels and dysregulation of calcium signaling. Pathological relevance of our findings included decreased miR-1 and increased Sorcin expression in end-stage cardiomyopathy. These findings demonstrate the importance of miR-1 in cardiac function and in the pathogenesis of heart failure via Sorcin-dependent calcium homeostasis.
Asunto(s)
Señalización del Calcio/genética , Proteínas de Unión al Calcio/genética , MicroARNs/fisiología , Contracción Miocárdica/genética , Interferencia de ARN , Animales , Secuencia de Bases , Proteínas de Unión al Calcio/metabolismo , Volumen Cardíaco/genética , Cardiomiopatías/metabolismo , Línea Celular , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Corazón/fisiopatología , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Miocardio/metabolismo , Miocardio/patología , ARN Interferente Pequeño/genética , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Regulación hacia ArribaRESUMEN
There is a need for prognostic markers to select patients most likely to benefit from antibody-drug conjugate (ADC) therapy. We quantified the relationship between pretreatment PET imaging of glycoprotein nonmetastatic melanoma B (gpNMB) with 89Zr-labeled anti-gpNMB antibody ([89Zr]ZrDFO-CR011) and response to ADC therapy (CDX-011) in triple-negative breast cancer. First, we compared different PET imaging metrics and found that standardized uptake values (SUV) and tumor-to-heart SUV ratios were sufficient to delineate differences in radiotracer uptake in the tumor of four different cell- and patient-derived tumor models and achieved high standardized effect sizes. These tumor models with varying levels of gpNMB expression were imaged with [89Zr]ZrDFO-CR011 followed by treatment with a single bolus injection of CDX-011. The percent change in tumor volume relative to baseline (% CTV) was then correlated with SUVmean of [89Zr]ZrDFO-CR011 uptake in the tumor. All gpNMB-positive tumor models responded to CDX-011 over 6 weeks of treatment, except one patient-derived tumor regrew after 4 weeks of treatment. As expected, the gpNMB-negative tumor increased in volume by 130 ± 59% at endpoint. The magnitude of pretreatment SUV had the strongest inverse correlation with the % CTV at 2-4 weeks after treatment with CDX-011 (Spearman ρ = -0.8). However, pretreatment PET imaging with [89Zr]ZrDFO-CR011 did not inform on which tumor types will regrow over time. Other methods will be needed to predict resistance to treatment.
Asunto(s)
Melanoma , Neoplasias de la Mama Triple Negativas , Glicoproteínas , Humanos , Melanoma/tratamiento farmacológico , Glicoproteínas de Membrana , Tomografía de Emisión de Positrones , Radioisótopos/uso terapéutico , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Circonio/uso terapéuticoRESUMEN
Zika virus (ZIKV) can generate a number of neurological dysfunctions in infected humans. Here, we tested the potential of human immune cells to protect against ZIKV infection in genetically humanized MISTRG mice. FACS analysis showed robust reconstitution of the mouse spleen with human T cells. Peripheral ZIKV inoculation resulted in infection within the brains of MISTRG mice. Mice that were reconstituted with human peripheral blood mononuclear cells (PBMC) showed a more rapid lethal response to ZIKV than the control mice lacking these immune cells. Immunocytochemical analysis of T cell markers CD3, CD45, or CD8 showed strong T cell presence in the brain, together with robust infection by ZIKV particularly in the excitatory pyramidal and granule neurons of the hippocampus. Infection was also found in cortex, striatum, the dopamine neurons of the substantia nigra, and other brain loci. Infection was considerably less in other regions such as the septum and hypothalamus. These data support the perspective that, rather than exerting a protective function, T cells may underlie some ZIKV-mediated neuropathology in the brain.
Asunto(s)
Enfermedades del Sistema Nervioso , Infección por el Virus Zika , Virus Zika , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Leucocitos Mononucleares/patología , Ratones , Infección por el Virus Zika/patologíaRESUMEN
Enhanced stemness in colorectal cancer has been reported and it contributes to aggressive progression, but the underlying mechanisms remain unclear. Here we report a Wnt ligand, Dickkopf-2 (DKK2) is essential for developing colorectal cancer stemness. Genetic depletion of DKK2 in intestinal epithelial or stem cells reduced tumorigenesis and expression of the stem cell marker genes including LGR5 in a model of colitis-associated cancer. Sequential mutations in APC, KRAS, TP53, and SMAD4 genes in colonic organoids revealed a significant increase of DKK2 expression by APC knockout and further increased by additional KRAS and TP53 mutations. Moreover, DKK2 activates proto-oncogene tyrosine-protein kinse Src followed by increased LGR5 expressing cells in colorectal cancer through degradation of HNF4α1 protein. These findings suggest that DKK2 is required for colonic epithelial cells to enhance LGR5 expression during the progression of colorectal cancer.
RESUMEN
Many mechanisms of and treatments for type 1 diabetes studied in the NOD mouse model have not been replicated in human disease models. Thus, the field of diabetes research remains hindered by the lack of an in vivo system in which to study the development and onset of autoimmune diabetes. To this end, we characterized a system using human CD4(+) T cells pulsed with autoantigen-derived peptides. Six weeks after injection of as few as 0.5 × 10(6) antigen-pulsed cells into the NOD-Scid Il2rg(-/-) mouse expressing the human HLA-DR4 transgene, infiltration of mouse islets by human T cells was seen. Although islet infiltration occurred with both healthy and diabetic donor antigen-pulsed CD4(+) T cells, diabetic donor injections yielded significantly greater levels of insulitis. Additionally, significantly reduced insulin staining was observed in mice injected with CD4(+) T-cell lines from diabetic donors. Increased levels of demethylated ß-cell-derived DNA in the bloodstream accompanied this loss of insulin staining. Together, these data show that injection of small numbers of autoantigen-reactive CD4(+) T cells can cause a targeted, destructive infiltration of pancreatic ß-cells. This model may be valuable for understanding mechanisms of induction of human diabetes.
Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Islotes Pancreáticos/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Diabetes Mellitus Tipo 1/etiología , Humanos , Células Secretoras de Insulina/inmunología , Ratones , Ratones Endogámicos NODRESUMEN
A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLB gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol ß, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol ß activity results in SLE, we constructed a mouse model of POLB that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLB allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol ß activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol ß in humans is an underlying cause of SLE.
Asunto(s)
ADN Polimerasa beta/genética , Lupus Eritematoso Sistémico/genética , Mutación Missense , Animales , Ratones , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido Simple , Recombinación V(D)JRESUMEN
Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-gamma-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-gamma-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-gamma sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-gamma. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-gamma were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-gamma via STAT3 that promotes the death of human VSMCs.
Asunto(s)
Proteínas F-Box/metabolismo , Regulación de la Expresión Génica , Interferón gamma/fisiología , Músculo Liso Vascular/patología , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Biológicos , Transducción de Señal , Trasplante de TejidosRESUMEN
Mature B cells, upon activation, progressively differentiate through centroblasts into centrocytes and finally to plasmacytes that express large amounts of selected immunoglobulins. A significant part of this maturation is thought to involve induction of the unfolded protein response (UPR). We have compared gene expression in normal germinal center centroblasts, centrocytes, lymphoblastoid cells undergoing induced UPR, and the CCL155 plasmacytoma cell line. In the centroblast to centrocyte transition there is a change in the expression of a relatively small number of genes. These include a limited subset of the genes upregulated by a fully activated UPR as well as a small number of other transcription factors, some disulphide isomerases, and other genes. This is consistent with a model in which this transition is mediated by changes in the levels of expression of transcription factor B-lymphocyte-induced maturation protein 1 (Blimp1) (PRDM1), BACH2, X-box binding protein 1 (XBP1), interferon regulatory factor 4 (IRF4), and possibly vitamin D receptor (VDR) expression, together with post-transcriptional changes and a limited induction of aspects of the UPR.
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Linfocitos B/fisiología , Diferenciación Celular/genética , Centro Germinal/citología , Animales , Linfocitos B/citología , Línea Celular , Niño , Preescolar , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ditiotreitol/metabolismo , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Pliegue de Proteína , Factores de Transcripción del Factor Regulador X , Factores de Transcripción , Tunicamicina/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
Hematopoietic cell-specific disruption of the STAT3 gene induces hyperproliferation of cells of the myeloid lineage. Osteoclasts (OC), the bone-resorbing cells, are generated from the same precursors as monocyte/macrophages. STAT3 mutant mice exhibit decreased bone density, bone volume, and increased numbers of TRAP-positive OC. In vitro generation of OC showed significantly greater numbers of OC in mutant mice. The increased numbers of Mac1+ cells and c-kit+ cells were detected by FACS analysis, suggesting an increased number of OC precursors. Treatment of splenocytes with CSF-1 and RANKL significantly increased the Mac-1+RANK+ cells, with much higher levels observed in cells from mutant mice compared with littermate controls. Besides enhanced number of Mac1+ OC precursors, we also identified an OC-generating Mac1- c-kit+ population in mutant mice which was absent in littermate controls. The Mac1- c-kit- cells did not generate OC. Finally, we determined that protein expression and mRNA level of c-fos, a transcription factor which is essential for OC differentiation, were enhanced in OC precursors of mutant mice, which may contribute to the osteopenic phenotype.
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Remodelación Ósea , Resorción Ósea/metabolismo , Resorción Ósea/patología , Proteínas de Unión al ADN/deficiencia , Osteoclastos/patología , Osteoporosis/metabolismo , Osteoporosis/patología , Transactivadores/deficiencia , Animales , Resorción Ósea/etiología , Proteínas Portadoras/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Madre Hematopoyéticas , Glicoproteínas de Membrana/metabolismo , Ratones , Osteoporosis/complicaciones , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Factor de Transcripción STAT3RESUMEN
Most hemopoietic cells express one or more members of the Ly-6 supergene family of small glycosylphosphatidylinositol-linked proteins. Although levels of Ly-6 proteins vary with stages of differentiation and activation, their function largely remains unknown. To ascertain whether ligands for Ly-6 proteins exist, chimeric proteins were constructed in which Ly-6E, Ly-6C, and Ly-6I were fused to the murine IgM heavy chain. These chimeras specifically stained both developing and mature B lymphocytes, as assessed by flow cytometry. Analysis of variants of the CH27 B cell lymphoma revealed that Ly-6A/E and Ly-6I recognized different molecules. CH27 cells with low levels of Ly-6A/E ligand activity also lost expression of CD22, and cells transfected with CD22 gained the ability to bind the Ly-6A/E chimera and, to a lesser extent, the Ly-6C and Ly-6I chimeric proteins. As many mature B cells coexpress Ly-6A/E and CD22, the function of Ly-6 molecules may be to associate with other membrane proteins, possibly concentrating these ligands in lipid rafts, rather than acting directly as cell:cell adhesion molecules.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos Ly/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Antígenos CD59/metabolismo , Moléculas de Adhesión Celular , Lectinas/metabolismo , Proteínas de la Membrana/metabolismo , Familia de Multigenes/inmunología , Animales , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos Ly/genética , Antígenos CD59/biosíntesis , Antígenos CD59/genética , Antígenos CD59/aislamiento & purificación , Células CHO , Células COS , Cricetinae , Variación Genética/inmunología , Regiones Constantes de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Lectinas/genética , Ligandos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Células Mieloides/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Transfección , Células Tumorales CultivadasRESUMEN
STAT4 signaling, activated by either interleukin 12 (IL12) or interferon alpha (IFNalpha), promotes T(H)1 responses in CD4(+) T cells. Vascular endothelial cells (EC) may also become polarized in response to various cytokines, favoring recruitment and activation of T(H)1 or T(H)2 effector cells. Here we have investigated the role of the STAT4 pathway in EC. Cultured human umbilical vein EC (HUVEC) express low levels of STAT4, which may be tyrosine-phosphorylated by treatment with IFNalpha but not IL12. This is because HUVEC lack both subunits of the IL12 receptor (IL12Rbeta1 and IL12Rbeta2), even following treatment with various cytokines. IL12 phosphorylation of STAT4 can be observed in HUVEC that have been transduced to express the IL12R. To identify STAT4-induced genes we pursued three approaches: analysis by DNA microarray and quantitative RT-PCR (Q-PCR) of the IL12 responses in IL12R-transduced EC; analysis by Q-PCR of IFNalpha responses in STAT4-overexpressing EC; and analysis of IFNalpha responses in U3A neuroblastoma cell lines that express either STAT1 or STAT4, but not both. In all three instances we observe STAT4-mediated induction of the chemokine monocyte chemoattractant protein 1 (MCP1) and suppressor of cytokine signaling 3 (SOCS3) mRNA, and we confirm the production of each protein in both IL12R-transduced EC and STAT4-transduced U3A cells. These observations reveal that there is a STAT4 response of EC, activated by IFNalpha but not IL12, and that it may modulate the pro-inflammatory behavior of EC.