RESUMEN
Directional migration of T-lymphocytes is a key process during immune activation and is tightly regulated both temporally and spatially. The initial cell membrane protrusion at a particular site is critical for determining the direction of cell migration. In this study, we found that ZAP-70 protein appeared not only at the margin of the spreading areas of polarized Jurkat T cells but also formed clusters near the center of the cell body on a fibronectin plate. Specifically, some pZAP-70 was located at the lamellipodia/filopodia and was closely associated with the most extended membrane contact. To visualize the dynamic distribution of ZAP-70 on migrating Jurkat T cells, we generated a fluorescent ZAP-70-EGFP fusion protein (hZAP70G). Expression of the hZAP70G in P116 cells, a ZAP-70 defective Jurkat derivative, restored its chemotactic migration toward SDF-1, adhesion to fibronectin matrix, and integrin activation. In addition, the distribution of hZAP70G protein is associated with changes in cell shape, specifically the membrane protrusion step, forming filopodia/lamellipodia and a retracting uropod. Furthermore, SDF-1 stimulated the formation of ZAP-70 and CXCR4 complex. CXCR4 was observed mainly at the leading edge of migrating cell. The localization of ZAP-70 at the very front edge of protruding lamellipodia was close to CXCR4 and a part of them were overlapped. Collectively, our data describe the critical early step of directional cell movement toward SDF-1 that ZAP-70 is recruited to the CXCR4 at the leading edge of membrane and consequently modulates lamellipodia/filopodia formation and integrin activation.
Asunto(s)
Quimiocina CXCL12/metabolismo , Quimiotaxis de Leucocito/fisiología , Seudópodos/metabolismo , Receptores CXCR4/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Adhesión Celular/genética , Línea Celular Tumoral , Quimiotaxis de Leucocito/genética , Fibronectinas/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos , Integrinas/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Células MCF-7 , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/fisiología , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
Cancer-associated fibroblasts play a crucial role in accelerating tumor progression, but there is a knowledge gap regarding the chemotactic signal activated in a tumor microenvironment. In this study, the expression of type IV collagen was knocked down using a lentiviral-mediated short hairpin RNA strategy. Although there was no obvious effect on cell growth in vitro, silencing the Col4-α1 gene decreased the tumorigenicity of B16F10 in C57BL/6 mice, which was accompanied by a reduction in the infiltration of alpha-smooth muscle actin-positive (α-SMA+) fibroblasts. Silencing the Col4-α1 gene or disrupting integrin engagement by blocking the antibody reduced the expression of platelet-derived growth factor A (PDGF-A), a potent chemotactic factor for fibroblasts. Furthermore, ectopic expression of the autoclustering integrin mutant significantly stimulated PDGF-A expression in murine B16F10 and human U118MG and Huh7 cells. PDGF-A-specific sh-RNA and neutralizing anti-PDGF-A antibody effectively inhibited the transwell migration of fibroblasts. Adding recombinant PDGF-A back to shCol cell-conditioned media restored the fibroblast-attraction ability indicating that PDGF-A is a major chemotactic factor for fibroblasts in the current study model. The integrin-associated PDGF-A production correlated with the activation of Src and ERK. High type IV collagen staining intensity colocalized with elevated PDGF-A expression was observed in tumor tissues obtained from hepatoma and glioma patients. The integrin signal pathway was activated by collagen engagement through Src and ERK, leading to enhanced PDGF-A production, which serves as a key regulator of fibroblast recruitment.
Asunto(s)
Colágeno Tipo IV/metabolismo , Fibroblastos/metabolismo , Integrina beta1/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Animales , Carcinogénesis/patología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Ratones Endogámicos C57BL , Modelos Biológicos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Inhibidores de Proteínas Quinasas/farmacología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human ß1 integrin or α2ß1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of ß1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.
Asunto(s)
Colágeno Tipo IV/metabolismo , Integrinas/metabolismo , Melanoma Experimental/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Colágeno Tipo IV/genética , Módulo de Elasticidad , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Melanoma Experimental/secundario , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
T helper 17 (Th17) cells, which produce interleukin 17 (IL-17), are involved in the pathogenesis of autoimmune diseases and inflammatory conditions. Th17 cells have been detected in many Fas ligand-positive tumors. This study investigates the expression of Th17-related genes in PHA/IL-2-activated human T cells upon Fas ligation. Activated T cells transiently express RORγt, IL-17A, and IL-17F. A subsequent Fas receptor stimulation or contact with FasL-expressing glioma cells significantly prolongs the induction of RORγt and Th17-related cytokines. Treatments with inhibitors of caspase-1 and Stat3 reduce the Fas-signal-associated induction of RORγt, IL-17A, and IL-17F, as well as the phosphorylation of Stat3. Although the ligation of Fas results in caspase-8 cleavage and ERK1/2 phosphorylation, inhibitors for caspase-8 and MEK have no effect on the expressions of RORγt, IL-17A, and IL-17F. The results suggest that the Fas signal favors the Th17-phenotypic features of human T cells through the caspase-1/Stat3 signaling pathway.
Asunto(s)
Caspasa 1/inmunología , Factor de Transcripción STAT3/inmunología , Linfocitos T/inmunología , Células Th17/inmunología , Receptor fas/inmunología , Western Blotting , Caspasa 1/metabolismo , Caspasa 8/inmunología , Caspasa 8/metabolismo , Línea Celular Tumoral , Células Cultivadas , Proteína Ligando Fas/inmunología , Proteína Ligando Fas/metabolismo , Expresión Génica/inmunología , Humanos , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucina-2/inmunología , Interleucina-2/farmacología , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Inmunológicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Fitohemaglutininas/inmunología , Fitohemaglutininas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Células Th17/metabolismo , Receptor fas/metabolismoRESUMEN
Many late-stage cancer cells express Fas ligand (FasL) and show high malignancy with metastatic potential. We report here a novel signaling mechanism for FasL that hijacks the Met signal pathway to promote tumor metastasis. FasL-expressing human tumor cells express a significant amount of phosphorylated Met. The down-regulation of FasL in these cells led to decreased Met activity and reduced cell motility. Ectopic expression of human FasL in NIH3T3 cells significantly stimulated their migration and invasion. The inhibition of Met and Stat3 activities reverted the FasL-associated phenotype. Notably, FasL variants activated the Met pathway, even though most of their intracellular domain or Fas binding sites were deleted. FasL interacted with Met through the FasL(105-130) extracellular region in lipid rafts, which consequently led to Met activation. Knocking down Met gene expression by RNAi technology reverted the FasL-associated motility to basal levels. Furthermore, treatment with synthetic peptides corresponding to FasL(117-126) significantly reduced the FasL/Met interaction, Met phosphorylation, and cell motility of FasL(+) transfectants and tumor cells. Finally, the transfectants of truncated FasL showed strong anchorage-independent growth and lung metastasis potential in null mice. Collectively, our results establish the FasL-Met-Stat3 signaling pathway and explains the metastatic phenotype of FasL-expressing tumors.
Asunto(s)
Proteína Ligando Fas/metabolismo , Neoplasias Pulmonares/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proteína Ligando Fas/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Microdominios de Membrana/genética , Microdominios de Membrana/patología , Ratones , Células 3T3 NIH , Metástasis de la Neoplasia/genética , Fosforilación/genética , Proteínas Proto-Oncogénicas c-met/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Eliminación de SecuenciaRESUMEN
Inflammation contributes to leukocyte migration, termed insulitis, and ß-cell loss in type 1 diabetes (T1D). Naturally occurring anthraquinones are claimed as anti-inflammatory compounds; however, their actions are not clear. This study aimed to investigate the effect and mechanism of catenarin on the inflammatory disease, T1D. Catenarin and/or its anthraquinone analogs dose-dependently suppressed C-X-C chemokine receptor type 4 (CXCR4)- and C-C chemokine receptor type 5 (CCR5)-implicated chemotaxis in leukocytes. Catenarin, the most potent anthraquinone tested in the study, prevented T1D in nonobese diabetic mice. Mechanistic study showed that catenarin did not act on the expression of CCR5 and CXCR4. On the contrary, catenarin inhibited CCR5- and CXCR4-mediated chemotaxis via the reduction of the phosphorylation of mitogen-activated protein kinases (p38 and JNK) and their upstream kinases (MKK6 and MKK7), and calcium mobilization. Overall, the data demonstrate the preventive effect and molecular mechanism of action of catenarin on T1D, suggesting its novel use as a prophylactic agent in T1D.
RESUMEN
Dense accumulations of T cells are often found in peritumoral areas, which reduce the efficiency of contact-dependent lysis of tumor cells. We demonstrate in this study that the extracellular matrix (ECM) produced by tumors can directly regulate T cell migration. The transmigration rate of several T cells including peripheral blood primary T cell, Jurkat, and Molt-4 measured for glioma cells or glioma ECM was consistently low. Jurkat cells showed reduced amoeba-like shape formation and delayed ERK activation when they were in contact with monolayers or ECM of glioma cells as compared with those in contact with HepG2 and MCF-7 cells. Phospho-ERK was located at the leading edge of migrating Jurkat cells. Glioma cells, but not MCF-7 and HepG2 cells, expressed tenascin-C. Knocking down the tenascin-C gene using the short hairpin RNA strategy converted glioma cells to a transmigration-permissive phenotype for Jurkat cells regarding ERK activation, transmigration, and amoeba-like shape formation. In addition, exogenous tenascin-C protein reduced the amoeba-like shape formation and transmigration of Jurkat cells through MCF-7 and HepG2 cell monolayers. A high level of tenascin-C was visualized immunohistochemically in glioma tumor tissues. CD3(+) T cells were detected in the boundary tumor area and stained strongly positive for tenascin-C. In summary, glioma cells can actively paralyze T cell migration by the expression of tenascin-C, representing a novel immune suppressive mechanism achieved through tumor ECM.
Asunto(s)
Inhibición de Migración Celular/inmunología , Polaridad Celular/inmunología , Matriz Extracelular/inmunología , Glioblastoma/inmunología , Tolerancia Inmunológica , Subgrupos de Linfocitos T/inmunología , Tenascina/fisiología , Línea Celular Tumoral , Movimiento Celular/inmunología , Células Cultivadas , Activación Enzimática/genética , Activación Enzimática/inmunología , Matriz Extracelular/enzimología , Matriz Extracelular/patología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/inmunología , Glioblastoma/enzimología , Glioblastoma/patología , Células Hep G2 , Humanos , Tolerancia Inmunológica/genética , Células Jurkat , Microscopía Confocal , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/patología , Tenascina/deficiencia , Tenascina/genéticaRESUMEN
Aberrant lymphocyte infiltration is crucial for many disorders such as tumor immune escape and autoimmunity. In this study, we have investigated T-cell migration in a three-dimensional collagen matrix containing tumor spheroids and by using micro-Slide chemotaxis and found that Zap70 regulates directionality during cell chemotaxis. Jurkat cells actively migrated toward SDF-1, nutrition, and spheroids of MCF-7 breast carcinoma cells embedded in collagen matrix. Inhibition of Zap70 activity impaired transmigration and mu-Slide chemotaxis but not the random movement of T cells in the collagen/fibronectin matrix. P116 cells, a Zap70 deficient variant of Jurkat, showed active random movement but failed to migrate against chemoattractants. P116 cells exhibited a reduced polarization of cell morphology, showing less lamellipodia formation accompanied with a fast pseudopod turnover rate. Instead of direct interacting with F-actin, Zap70 formed a complex with talin which is an integrin scaffold for F-actin. SDF-1 enhanced Zap70 phosphorylation and also stimulated binding of talin and beta1 integrin activation. P116 cells showed reduced complex of talin and beta1 integrin in parallel with impaired integrin activation. Collectively, Zap70 modulates integrin activation by interacting with talin, which contributes to directionality of T-cell migration, severing as a potential target for anti-inflammation therapy.
Asunto(s)
Quimiotaxis de Leucocito , Integrinas/fisiología , Linfocitos T/inmunología , Talina/fisiología , Proteína Tirosina Quinasa ZAP-70/fisiología , Células Cultivadas , Adhesiones Focales , Humanos , Inflamación/etiología , Activación de LinfocitosRESUMEN
Delayed Fas-mediated apoptosis in T cells is associated with inflammatory diseases including rheumatoid arthritis (RA). CD3(+) T cells in RA synovia expressed high amounts of phospho-p38 MAPK. Exposure to RA synovial fluid or soluble collagen, a degradation product of extracellular matrix abundant in RA synovium, induced the phosphorylation of p38 MAPK in Jurkat T cells accompanied by resistance against Fas-mediated apoptosis. Blocking beta1 integrin by antibody diminished this effect. In addition, ectopic expression of auto-activated beta1 integrin variant in T cells profoundly induced the phosphorylation of p38 MAPK. Suppression of p38 MAPK sensitized T cells to Fas-mediated apoptosis and increased caspase-8 and caspase-3 cleavage. A physical interaction of p38 MAPK and caspase-8 was demonstrated by using confocal microscopic imaging and co-immunoprecipitation assay. RA synovial fluid markedly increased the formation of phospho-p38 MAPK/caspase-8 complex in Jurkat T cells. In conclusion, abnormal activation of p38 MAPK to prevent Fas-mediated apoptosis may represent a common survival mechanism of RA synovial T cells contributing to the persistent inflammation of affected synovium.
Asunto(s)
Apoptosis/inmunología , Artritis Reumatoide/inmunología , Integrina beta1/inmunología , Linfocitos T/inmunología , Receptor fas/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Caspasa 3/inmunología , Caspasa 8/inmunología , Colágeno/inmunología , Humanos , Células Jurkat , Fosforilación/inmunología , Líquido Sinovial/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND/PURPOSE: Chlamydophila pneumoniae infection has been associated with several pulmonary and cardiac diseases. However, it has not been explored for its ability to activate the same immunopathologic mechanisms of asthma, namely, a predominant Th2 immune response and structural changes that are termed airway remodeling. This study evaluated immune responses in the lung and airway pathology of BALB/c mice with chronic and repeated C. pneumoniae infections. METHODS: Mice were inoculated intranasally with 5 x 10(6) inclusion-forming units of C. pneumoniae TWAR strain, and re-inoculated at 14 and 42 days after the primary inoculation. Cytokine gene expression in bronchoalveolar lavage (BAL) cells was analyzed by RT-PCR on day 70. Airway pathology was also evaluated by morphometric measurements. RESULTS: A significant increase of interleukin (IL)-4 mRNA was detected in BAL cells in infected mice, and a significant increase in subepithelial basement membrane thickness of the airways was also noted in infected mice as compared with control mice (8.95 +/- 0.28 microm vs. 5.54 +/- 0.22 microm, p < 0.0001). We further analyzed the correlation between IL-4 cytokine expression and the increased subepithelial basement membrane thickness of airways in infected mice. We found that mice with increased IL-4 mRNA expression had significant increases in the thickness of subepithelial basement membrane as compared with mice without increased IL-4 mRNA expression (9.87 +/- 0.51 microm vs. 6.49 +/- 0.52 microm, p < 0.0001). CONCLUSION: It is believed that our results demonstrated for the first time that chronic and repeated infections with C. pneumoniae increased IL-4 gene expression and thickness of airway subepithelial basement membrane in mice.
Asunto(s)
Membrana Basal/patología , Infecciones por Chlamydophila/inmunología , Chlamydophila pneumoniae/inmunología , Interleucina-4/inmunología , Pulmón/inmunología , Sistema Respiratorio/inmunología , Adyuvantes Inmunológicos/genética , Análisis de Varianza , Animales , Membrana Basal/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/aislamiento & purificación , Interleucina-4/genética , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , ARN Mensajero/inmunología , Sistema Respiratorio/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Caspase-3 is known as a cysteine protease that primarily executes the cell death program. However, some tumors express higher levels of caspase-3 in positive correlation with malignancy. Here, we showed that caspase-3 can promote tumor metastasis in a protease-independent mechanism. Ectopic expression of caspase-3 enhanced lung metastasis and cell motility of caspase-3 deficient MCF-7 cells. By contrast, caspase-3 siRNA reduced the invasiveness and metastasis ability of A549 cells that express high level of caspase-3. Moreover, caspase-3 induced ERK activation. Alteration of caspase-3 by introducing non-processable mutation at its cleavage site or treatment of caspase-3 inhibitor did not diminish the caspase-3-associated increases in ERK phosphorylation and cell migration. Confocal microscopy study showed that caspase-3 was not physically associated with ERK. Inhibiting ceramide formation by blockage of the ceramide synthase or acid sphingomyelinase activity resulted in significant reduction of ERK phosphorylation and cell migration. In summary, caspase-3 induces ERK activation through a ceramide-dependant, protease activity-independent mechanism, which represents a novel role of caspase-3 in tumor metastasis.
Asunto(s)
Caspasa 3/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Pulmonares/enzimología , Invasividad Neoplásica , Péptido Hidrolasas/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Caspasa 3/genética , Línea Celular , Movimiento Celular/fisiología , Ceramidas/metabolismo , Citometría de Flujo , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Microscopía Confocal , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Fosforilación , ARN Interferente Pequeño , Esfingomielina Fosfodiesterasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
It has recently become apparent that the microenvironment made up of the extracellular matrix may affect cell signaling. In this study, we evaluated Fas-triggered apoptosis in T cells in contact with tumor cells, which resembles the cell-to-cell interactions found in tumor regions. Jurkat cells were less susceptible to the Fas-mediated apoptosis when cocultured with U118, HeLa, A549, and Huh-7 tumor cells. This was indicated by less plasma membrane alteration, an amelioration of the loss of mitochondria membrane potential, a decrease in caspase-8 and caspase-3 activation, a decrease in DNA fragmentation factor-45/35 cleavage, and a reduction in the breakage of DNA when compared with Jurkat cells cultured alone. In contrast, the tumor cell lines MCF-7 and HepG2 produced no such protective effect. This protective event was independent of the expression of Fas ligand on the tumor cells. Interrupting the beta integrins-matrix interaction diminished the coculture effect. In Jurkat cells, cell matrix contact reduced the assembly of the Fas death-inducing signaling complex and Bcl-x(L) cleavage, but enhanced the phosphorylation of ERK1/2, p38 MAPK, and Akt. Only PI3K inhibitor, but not kinase inhibitors for MEK, ERK1/2, p38 MAPK, JNK, protein kinase C, and protein kinase A, completely abolished this tumor cell contact-associated protection and in parallel restored Fas-induced Bcl-x(L) cleavage as well as decreasing the phosphorylation of Bad at serine 136. Together, our results indicate that stimulation of the beta integrin signal of T cells by contact with tumor cells may trigger a novel protective signaling through the PI3K/Akt pathway of T cells against Fas-mediated apoptosis.
Asunto(s)
Integrinas/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Receptor fas/metabolismo , Animales , Anticuerpos/inmunología , Apoptosis , Línea Celular , Técnicas de Cocultivo , Activación Enzimática , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias/patología , Fosforilación , Linfocitos T/inmunología , Proteína Letal Asociada a bcl/metabolismo , Proteína bcl-X/metabolismo , Receptor fas/inmunologíaRESUMEN
Although lead and lipopolysaccharide (LPS), both important environmental pollutants, activate cells through different receptors and participate in distinct upstream signaling pathways, Pb increases the amount of LPS-induced tumor necrosis factor-alpha (TNF-alpha). We examined the cells responsible for the excess production of Pb-increased LPS-induced TNF-alpha and liver injury, and the roles of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (MAPK) in the induction of TNF-alpha. Peritoneal injection of Pb alone (100 micromol/kg) or a low dose of LPS (5 mg/kg) did not affect serum TNF-alpha or liver functions in A/J mice. In contrast, coexposure to these noneffective doses of Pb plus LPS (Pb+LPS) strongly induced TNF-alpha expression and resulted in profound liver injury. Direct inhibition of TNF-alpha or functional inactivation of monocytes/macrophages significantly decreased the level of Pb+LPS-induced serum TNF-alpha and concurrently ameliorated liver injury. Pb+LPS coexposure stimulated the phosphorylation of p42/44 MAPK and the expression of TNF-alpha in CD14+ cells of cultured mouse whole blood, peritoneal macrophages, and RAW264.7 cells. Moreover, blocking PKC or MAPK effectively reduced Pb+LPS-induced TNF-alpha expression and liver injury. In summary, monocytes/macrophages were the cells primarily responsible for producing, through the PKC/MAPK pathway, the excess Pb-increased/LPS-induced TNF-alpha that caused liver injury. alpha.
Asunto(s)
Plomo/toxicidad , Lipopolisacáridos/toxicidad , Hígado/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Proteína Quinasa C/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Activación Enzimática , Hígado/enzimología , Macrófagos/enzimología , Ratones , Monocitos/enzimologíaRESUMEN
In this study, we investigated the interaction between lipopolysaccharide (LPS) and lead (Pb) and the involvement of tumor necrosis factor-alpha (TNF-alpha) and oxidative stress in Pb-plus-LPS (Pb/LPS)-induced liver damage in rats. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), TNF-alpha, nitric oxide (NO), and lipid peroxidation (LPO) were determined in rats treated with Pb and/or LPS. Pb ranging from 0 to 15 mg/kg dose dependently increased AST, ALT, NO, or LPO in LPS-treated rats. Pretreatment with iNOS inhibitor 1400W reduced NO, LPO, TNF-alpha, AST, and ALT in Pb/LPS-treated rats. Thus, Pb increased LPS-induced liver damage, which might be associated with increased NO-initiated oxidative stress and TNF-alpha in rats.
Asunto(s)
Plomo/toxicidad , Lipopolisacáridos/toxicidad , Hígado/efectos de los fármacos , Hígado/lesiones , Óxido Nítrico/metabolismo , Estrés Oxidativo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Alanina Transaminasa/sangre , Amidinas/farmacología , Animales , Aspartato Aminotransferasas/sangre , Bencilaminas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Plomo/metabolismo , Plomo/farmacología , Peroxidación de Lípido , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Masculino , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/sangre , Compuestos Organometálicos/farmacología , Ratas , Ratas WistarRESUMEN
The therapeutic efficacy of HER2/c-erbB-2/neu DNA immunization on mouse tumor cells expressing exogenous human or rat p185neu but not on mouse tumor cells naturally expressing mouse p185neu has been demonstrated. We investigated the feasibility of using N-terminal rat neu DNA immunization on mouse tumor overexpressing endogenous p185neu and enhancing the therapeutic efficacy of this vaccine by fusion to various cytokine genes, including interleukin-2 (IL-2), interleukin-4 (IL-4), or granulocyte-macrophage colony-stimulating factor. In a therapeutic model, N'-neu-IL-2 DNA vaccine was significantly better than N'-neu DNA vaccine, and N'-neu DNA vaccine was significantly better than control DNA or N'-neu-IL-4 DNA vaccine. The therapeutic efficacy of DNA vaccines was correlated with tumor infiltration of CD8+ T cells. Depletion of CD8+ T cells completely abolished the therapeutic effects of N'-neu-IL-2 DNA vaccine and N'-neu DNA vaccine. Depletion of CD4+ T cells after tumor implantation had no influence on N'-neu-IL-2 DNA vaccine, but enhanced the therapeutic efficacy of N'-neu DNA vaccine. Our results demonstrate that rat N'-neu DNA vaccine has a therapeutic effect on established tumor through the CD8+ T-cell-dependent pathway. Depletion of CD4+ T cells or fusion to the IL-2 gene can thus further enhance the therapeutic effects of N'-neu DNA immunization on mouse tumor expressing endogenous p185neu.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Citocinas/genética , Genes erbB-2/genética , Neoplasias Experimentales/tratamiento farmacológico , Animales , Suero Antilinfocítico/inmunología , Fusión Artificial Génica , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/genética , Supresión Clonal/inmunología , Citocinas/metabolismo , Femenino , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones , Trasplante de Neoplasias , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Ratas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Vacunas de ADN/genética , Vacunas de ADN/uso terapéuticoRESUMEN
Both lead (Pb) and lipopolysaccharide (LPS) damage nervous system, partly, by the induction of tumor necrosis factor-alpha (TNF-alpha) in glia origin. In this study, we examined the Pb- and LPS-triggered signal leading to TNF-alpha expression in a glioma cell line, U-373MG. Both Pb and LPS increased the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), which depended on the activation of MAPK kinase (MEK) and protein kinase C (PKC). Selective p42/44 MAPK inhibitor could reduce the Pb- and LPS-triggered TNF-alpha expression in U-373MG cells. Suppressing PKC by chelerythrine chloride completely diminished the Pb- and LPS-induced TNF-alpha expression in glial cells in the mouse brain. Thus, our results indicated that PKC-MEK-p42/44 MAPK is a common signaling pathway for Pb- and LPS-induced TNF-alpha expression in glial cells.
Asunto(s)
Encéfalo/efectos de los fármacos , Glioma/metabolismo , Plomo/toxicidad , Lipopolisacáridos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Alcaloides , Animales , Benzofenantridinas , Encéfalo/enzimología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Inhibidores Enzimáticos/farmacología , Glioma/tratamiento farmacológico , Humanos , Masculino , Ratones , Ratones Endogámicos , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Fenantridinas/farmacología , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/biosíntesis , Transducción de SeñalRESUMEN
PROBLEM: To establish an in vitro culture model of the endometrium and endometriotic lesions, and demonstrate the different expressions of intercellular adhesion molecule-1 (ICAM-1) in these lesions. METHODS: Eight women with moderate to severe stages of endometriosis were enrolled. The specimens were collected from their eutopic endometrium, visually normal peritoneum, ovarian endometrioma and peritoneal endometriotic spots during the follicular phase of the menstrual cycle. ICAM-1 mRNA and protein were expressed by using reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblot, and immunocytochemistry. RESULTS: The results demonstrate that visually normal peritoneal cells and ovarian endometriomas of endometriotic patients can express high ICAM-1 mRNA. Normal peritoneal cells further expressed significant soluble ICAM-1 protein levels without cytokine stimulation. The eutopic endometrium expressed less soluble ICAM-1 protein, and ICAM-1 expressions increased in cultured stromal cells of eutopic endometrium, ovarian endometrioma, and peritoneal endometriotic spots under interferon-gamma (INF-gamma) stimulation. CONCLUSION: The ICAM-1 expression in visually normal peritoneal cells from women with endometriosis may play a role in the early implantation of peritoneal endometriosis. Peritoneal INF-gamma stimulation is significantly associated with ICAM-1 expression in endometriosis. Therefore, the differential expression and changes of ICAM-1 may be involved in the mechanism that can escape immunosurveillance and allow refluxed endometrial cells to spread and invade other location.
Asunto(s)
Endometriosis/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/farmacología , Células Cultivadas , Endometriosis/etiología , Endometriosis/genética , Endometrio/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fase Folicular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-10/farmacología , Interleucina-4/farmacología , Ovario/citología , Peritoneo/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Elevated expression of IL-10 has been frequently observed in tumor tissues and tumor-infiltrating cells. We show herein that transcription of the IL-10 gene in primary peripheral T cells and T cell lines is up-regulated upon contact with glioma cells without an induction of apoptosis in those T cells. Glioma-associated IL-10 induction was suppressed by interrupting the engagement of Fas and its ligand (Fas-L) with the antagonistic Ab, ZB4, by reducing Fas-L expression of glioma cells using the Fas-L-specific ribozyme, or by preventing cell-to-cell contact in a Transwell culture setting. Cross-linking of Fas with the agonistic Ab, CH-11, triggered apoptosis and enhanced the expression of IL-10 in Jurkat cells at the transcriptional and translational levels. Inhibiting caspase activities by caspase inhibitors, Z-VAD (Z-Val-Ala-Asp(Ome)-fluoromethylketone) and Z-IETD (Z-Ile-Glu(Ome)-Thr(Ome)-Asp(Ome)-fluoromethylketone), abolished this IL-10 induction in Jurkat cells. Intracellular staining detected IL-10 proteins in Fas-cross-linked Jurkat cells and in PHA-activated T cells. However, few IL-10 proteins were detectable in Jurkat cells cocultured with glioma cells, indicating a requirement of other factors for IL-10 production. Direct activation of protein kinase A (PKA) by forskolin elevated the transcription of IL-10 in Jurkat cells. However, KT5720, a selective PKA inhibitor, reduced neither anti-Fas-triggered nor glioma-associated IL-10 expression. Phosphorylation of cAMP response element binding protein and activating transcription factor-1 in Jurkat cells was not affected by coculturing with glioma cells or by anti-Fas treatment, further suggesting a PKA-independent pathway. In summary, our results demonstrate nonlethal cross-talk between tumor and immune cells leading to IL-10 dysregulation in T cells, which might contribute to Fas-L(+) tumor-associated immunosuppression.
Asunto(s)
Comunicación Celular/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Glioma/inmunología , Interleucina-10/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Transcripción Genética/inmunología , Regulación hacia Arriba/inmunología , Receptor fas/fisiología , Anticuerpos Bloqueadores/farmacología , Apoptosis/genética , Apoptosis/inmunología , Caspasas/metabolismo , Caspasas/fisiología , Comunicación Celular/genética , Línea Celular Tumoral , Técnicas de Cocultivo , Activación Enzimática/inmunología , Proteína Ligando Fas , Regulación Neoplásica de la Expresión Génica/inmunología , Glioma/genética , Glioma/patología , Humanos , Interleucina-10/biosíntesis , Interleucina-10/sangre , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Células Jurkat , Ligandos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Transducción de Señal/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/metabolismo , Regulación hacia Arriba/genética , Receptor fas/inmunología , Receptor fas/metabolismoRESUMEN
Angiostatin consisting of the first four-kringle domains of the plasminogen potently inhibits angiogenesis in vitro and in vivo. However, the molecular mechanism of action whereby angiostatin mediates its inhibitory effect on proliferating endothelial cells remains elusive. We therefore used the proliferating cultured human umbilical vein endothelial cells (HUVECs) promoted by vascular endothelial growth factor A to identify the endogenous signaling elements that mediate the antiangiogenic effect of angiostatin. Treatment of HUVEC with angiostatin at a concentration known to inhibit cell proliferation and induce apoptosis resulted in induction of p53-, Bax-, and tBid-mediated release of cytochrome c into the cytosol. In addition, angiostatin also activated the Fas-mediated apoptotic pathway in part via up-regulation of FasL mRNA, down-regulation of c-Flip, and activation of caspase 3. These results suggest that the anti-angiogenic action of angiostatin is likely mediated by two distinct signaling pathways, one intrinsic mediated by p53 while the other extrinsic involved in FasL engagement and mitochondria dysfunction.
Asunto(s)
Angiostatinas/fisiología , Endotelio Vascular/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Angiostatinas/metabolismo , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Southern Blotting , Western Blotting , Proteínas Portadoras/metabolismo , División Celular , Células Cultivadas , Citocromos c/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Endotelio Vascular/citología , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo , Venas Umbilicales/citología , Regulación hacia Arriba , Proteína X Asociada a bcl-2RESUMEN
The expression of Fas ligand (FasL) on tumor cells (tumor FasL) has been implicated in their evasion of immune surveillance. In this study, we investigated the cellular mechanism for FasL-associated immune escape using melanoma B16F10-derived cells as a model. Transfectants carrying FasL-specific ribozymes expressed low levels of FasL (FasL(low) tumor cells) as compared with those carrying enhanced green fluorescent protein-N1 plasmids (FasL(high) tumor cells). When injected s.c. into C57BL/6 mice, FasL(low) tumor cells grew more slowly than did FasL(high) melanoma cells. FasL(high) tumor cells showed more intensive neutrophilic infiltration accompanied by multiple necrotizing areas than did FasL(low) tumor cells. The average size of FasL(low) tumors, but not of FasL(high) tumors, was significantly enhanced in mice depleted of neutrophils. Consistently, a local injection of LPS to recruit/activate neutrophils significantly delayed tumor formation by FasL(low) tumor cells, and slightly retarded that of FasL(high) tumor cells in both C57BL/6 and nonobese diabetic/SCID mice. Neutrophils killed FasL(low) melanoma cells more effectively than FasL(high) melanoma cells in vitro. The resistance of FasL(high) melanoma cells to being killed by neutrophils was correlated with impaired neutrophil activation, as demonstrated by reductions in gelatinase B secretion, reactive oxygen species production, and the surface expression of CD11b and the transcription of FasL. Local transfer of casein-enriched or PMA-treated neutrophils delayed tumor formation by melanoma cells. Taken together, inactivation of neutrophils by tumor FasL is an important mechanism by which tumor cells escape immune attack.