RESUMEN
OBJECTIVES: Decreased phagocytosis of apoptotic cells plays an important role in the pathogenesis of SLE. This can lead to secondary necrosis and release of nuclear proteins, such as high mobility group box 1 (HMGB1). We hypothesized that increased HMGB1 levels, as present in SLE, skew macrophage differentiation towards M1-like phenotypes and thereby diminish uptake of apoptotic cells. The aim of this study was to investigate the effect of HMGB1 on macrophage polarization and on phagocytic capacity of differentiated macrophages. METHODS: SLE patients with quiescent disease (SLEDAI ⩽4) and healthy controls (HCs) were included. Monocytes and differentiated M1 and M2 macrophages were assessed for expression of M1 and M2 markers and for phagocytic capacity. HMGB1 was added during differentiation and during phagocytosis. RESULTS: Expression of CD86 (M1) was not different, whereas CD163 (M2) was significantly lower on SLE monocytes. After differentiation, no differences regarding surface receptor expression and phagocytic capacity were observed between M1 and M2 macrophages from SLE patients and HCs. Addition of HMGB1 during M2 differentiation resulted in high IL-6 and TNF-α mRNA expression and reduced phagocytic capacity of apoptotic cells. Furthermore, adding HMGB1 to apoptotic Jurkat cells diminished phagocytosis of these cells. CONCLUSION: Circulating monocytes from SLE patients display an M1-like phenotype compared with HCs, but in vitro differentiation abolishes this difference. HMGB1 skews differentiation of M2-like macrophages towards an M1-like phenotype and, subsequently, reduces phagocytosis of apoptotic cells. These data imply that the phenotype of monocytes or macrophages is determined by their environment, such as the presence of cytokines and HMGB1.
Asunto(s)
Proteína HMGB1/fisiología , Macrófagos/fisiología , Fagocitosis/fisiología , Adulto , Apoptosis/fisiología , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Femenino , Proteína HMGB1/farmacología , Humanos , Técnicas In Vitro , Células Jurkat/fisiología , Leucocitos Mononucleares/fisiología , Activación de Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Necrosis , Fagocitosis/efectos de los fármacos , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
Persistent Helicobacter pylori infection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylori facilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pylori MV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pylori strains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pylori MV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pylori MV in the stimulation of innate pro- and anti-inflammatory responses and in the suppression of T cell immunity.
Asunto(s)
Apoptosis/fisiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Apoptosis/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Células Cultivadas , Interacciones Huésped-Patógeno/fisiología , Humanos , Células Jurkat/fisiologíaRESUMEN
The identification of a neutralizing mAb against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication but also as an extracellular toxin able to disrupt the immune system. We showed previously that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi and selected a mAb named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat as an unfolded protein. mAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Epítopos/inmunología , VIH-1/inmunología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Apoptosis , Proliferación Celular/efectos de los fármacos , Mapeo Epitopo , VIH-1/genética , Células HeLa , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4(+)CD25(+) regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.
Asunto(s)
Células Jurkat/metabolismo , Análisis por Matrices de Proteínas/métodos , Transducción de Señal/fisiología , Complejo CD3/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Células Jurkat/fisiología , Cinética , Proteínas de la Leche/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-raf/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Linfocitos T/metabolismo , Transactivadores/metabolismoRESUMEN
Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4ß1 and α5ß1 and show that the low-affinity states bind substantially faster than the high-affinity state. On- and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low-affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high-affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by 'inside-out signaling.'
Asunto(s)
Sitios de Unión , Adhesión Celular/fisiología , Citoesqueleto/fisiología , Integrinas/química , Células Jurkat/fisiología , Células K562/fisiología , Ligandos , Humanos , Cinética , Modelos MolecularesRESUMEN
Microparticles (MPs) are small membrane-bound vesicles released from cells undergoing activation or cell death. These particles display potent biological activities that can impact on physiologic and pathologic processes. Previous studies with the Jurkat T leukemia cell line demonstrated that staurosporine (STS) induces the release of MPs as cells undergo apoptosis. To investigate further this process, we tested the effects of STS, its analogue, 7-hydroxystaurosporine (UCN-01), and other protein kinase C (PKC) and cyclin-dependent kinase (CDK) inhibitors. FACS analysis was used to assess MP release. Results of these studies indicate that STS and UCN-01 induce MP release by Jurkat cells; in contrast, other PKC and CDK inhibitors failed to induce comparable release, suggesting that release does not result from simple inhibition of either kinase alone. Time course experiments indicated that STS-induced particle release occurred as early as 2 h after treatment, with the early release MPs displaying low levels of binding of annexin V and propidium iodide (PI). Early-release MPs, however, matured in culture to an annexin V- and PI-positive phenotype. Together, these results indicate that STS and UCN-01 induce MPs that are phenotypically distinct and reflect specific patterns of kinase inhibition during apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Inhibidores Enzimáticos/farmacología , Células Jurkat , Estaurosporina/farmacología , Animales , Apoptosis/fisiología , Micropartículas Derivadas de Células/química , Citometría de Flujo , Humanos , Células Jurkat/citología , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Ácidos Nucleicos/análisis , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismoRESUMEN
Genetically susceptible rodents exposed to low nontoxic levels of inorganic mercury (Hg(2+)) develop idiosyncratic autoimmune disease associated with defective T-cell function. However, the molecular mechanisms underlying this phenomenon remain mostly unexplained. Brief exposure of T cells to micromolar concentrations of Hg(2+) leads to physiologically relevant nontoxic cellular mercury burdens, and as we have previously reported, attenuates T-cell receptor (TCR) signal strength by approximately 50%. We have found this to be the result of an inadequate activation of the tyrosine kinase ZAP-70, which is hypophosphorylated following TCR stimulation in Hg(2+) burdened cells when compared to untreated controls. In T cells, ZAP-70 phosphorylation is dependent on lymphocyte-specific protein tyrosine kinase (Lck) activity, which in turn is either positively or negatively regulated by the phosphorylation of specific Lck tyrosine residues. In particular, the general belief is that Lck is negatively regulated by phosphorylation of tyrosine 192 (Y192). We now demonstrate by Western blotting that, in Jurkat T cells, TCR signal transduction (and ZAP-70 phosphorylation) was positively associated with a rapid transient phosphorylation of Y192, which was inhibited in cells that were briefly (5 min) exposed to 5 microM Hg(2+). Thus, Hg(2+) inhibits a critical activating role played by Lck Y192 during the most proximal events of the TCR-induced cell signaling.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Mercurio/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/fisiología , Animales , Complejo CD3/genética , Complejo CD3/metabolismo , Activación Enzimática , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Mercurio/farmacología , Fosforilación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
BACKGROUND: Whether microgravity might influence tumour growth and carcinogenesis is still an open issue. It is not clear also if and how normal and transformed cells are differently solicited by microgravity. The present study was designed to verify this issue. METHODS: Two normal, LB and HSC93, and two transformed, Jurkat and 1310, lymphoblast cell lines were used as representative for the two conditions. Two lymphoblast lines from Fanconi's anemia patients group A and C (FA-A and FA-C, respectively), along with their isogenic corrected counterparts (FA-A-cor and FA-C-cor) were also used. Cell lines were evaluated for their proliferative ability, vitality and apoptotic susceptibility upon microgravity exposure in comparison with unexposed cells. Different parameters correlated to energy metabolism, glucose consumption, mitochondrial membrane potential (MMP), intracellular ATP content, red-ox balance and ability of the cells to repair the DNA damage product 8-OHdG induced by the treatment of the cells with 20 mM KBrO3 were also evaluated. RESULTS: Transformed Jurkat and 1310 cells appear resistant to the microgravitational challenge. On the contrary normal LB and HSC93 cells display increased apoptotic susceptibility, shortage of energy storages and reduced ability to cope with oxidative stress. FA-A and FA-C cells appear resistant to microgravity exposure, analogously to transformed cells. FA corrected cells did shown intermediate sensitivity to microgravity exposure suggesting that genetic correction does not completely reverts cellular phenotype. CONCLUSIONS: In the light of the reported results microgravity should be regarded as an harmful condition either when considering normal as well as transformed cells. Modeled microgravity and space-based technology are interesting tools in the biomedicine laboratory and offer an original, useful and unique approach in the study of cellular biochemistry and in the regulation of metabolic pathways.
Asunto(s)
Anemia de Fanconi/fisiopatología , Linfocitos/fisiología , Ingravidez/efectos adversos , 8-Hidroxi-2'-Desoxicoguanosina , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Apoptosis/fisiología , Línea Celular Transformada , Proliferación Celular , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Metabolismo Energético/fisiología , Glucosa/análisis , Humanos , Células Jurkat/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
The use of indigo naturalis to treat psoriasis has proved effective in our previous clinical studies. The present study was designed to examine the anti-inflammatory effect of indigo naturalis in primary cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of cells with indigo naturalis extract attenuated TNF-α-induced increase in Jurkat T cell adhesion to HUVECs as well as decreased the protein and messenger (m)RNA expression levels of vascular cell adhesion molecule-1 (VCAM-1) on HUVECs. Indigo naturalis extract also inhibited the protein expression of activator protein-1 (AP-1)/c-Jun, a critical transcription factor for the activation of VCAM-1 gene expression. Since the reduction of lymphocyte adhesion to vascular cells by indigo naturalis extract could subsequently reduce the inflammatory reactions caused by lymphocyte infiltration in the epidermal layer and help to improve psoriasis, this study provides a potential mechanism for the anti-inflammatory therapeutic effect of indigo naturalis extract in psoriasis.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales/química , Factor de Necrosis Tumoral alfa/fisiología , Venas Umbilicales/citología , Molécula 1 de Adhesión Celular Vascular/análisis , Adhesión Celular/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Psoriasis/tratamiento farmacológico , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Factor de Transcripción AP-1/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Ligation of major histocompatability complex class I (MHC-I) molecules expressed on T cells leads to both growth arrest and apoptosis. The aim of the current study was to investigate the intracellular signal pathways that mediate these effects. MHC-I ligation of human Jurkat T cells induced a morphologically distinct form of apoptosis within 6 h. A specific caspase inhibitor, which inhibited Fas-induced apoptosis, did not affect apoptosis induced by MHC-I ligation. Furthermore, MHC-I-induced apoptosis did not involve cleavage and activation of the poly(ADP- ribose) polymerase (PARP) endonuclease or degradation of genomic DNA into the typical fragmentation ladder, both prominent events of Fas-induced apoptosis. These results suggest that MHC-I ligation of Jurkat T cells induce apoptosis through a signal pathway distinct from the Fas molecule. In our search for other signal pathways leading to apoptosis, we found that the regulatory 85-kD subunit of the phosphoinositide-3 kinase (PI-3) kinase was tyrosine phosphorylated after ligation of MHC-I and the PI-3 kinase inhibitor wortmannin selectively blocked MHC-I-, but not Fas-induced, apoptosis. As the c-Jun NH2-terminal kinase (JNK) can be activated by PI-3 kinase activity, and has been shown to be involved in apoptosis of lymphocytes, we examined JNK activation after MHC-I ligation. Strong JNK activity was observed after MHC-I ligation and the activity was completely blocked by wortmannin. Inhibition of JNK activity, by transfecting cells with a dominant-negative JNKK- MKK4 construct, led to a strong reduction of apoptosis after MHC-I ligation. These results suggest a critical engagement of PI-3 kinase-induced JNK activity in apoptosis induced by MHC-I ligation.
Asunto(s)
Apoptosis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Antígenos de Histocompatibilidad Clase I/fisiología , Células Jurkat/fisiología , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T/fisiología , Androstadienos/farmacología , Anticuerpos/farmacología , Núcleo Celular/ultraestructura , Citoplasma/ultraestructura , Fragmentación del ADN , Heterocromatina/ultraestructura , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Células Jurkat/citología , Células Jurkat/inmunología , Microscopía Electrónica , Fosfatidilinositol 3-Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Transfección , WortmaninaRESUMEN
Chemokines play pivotal roles in regulating a wide variety of biological processes by modulating cell migration and recruitment. Deregulation of chemokine signaling can alter cell recruitment, contributing to the pathogenic states associated with autoimmune disease, inflammatory disorders, and sepsis. During chemotaxis, lipid rafts and their resident signaling molecules have been demonstrated to partition to different parts of the cell. Herein, we investigated the role of lipid raft resident Src-family kinases (SFK) in stromal cell-derived factor 1/CXCL12-mediated chemotaxis. We have shown that Lck-deficient J.CaM 1.6 cells are defective in CXCL12-mediated chemotaxis in contrast to their parental counterpart, Jurkat cells. Ectopic expression of the SFK hematopoietic cell kinase (Hck) in J.CaM 1.6 cells reconstituted CXCL12 responsiveness. The requirement of lipid raft association of SFK was assessed using both isoforms of Hck: the dually acylated p59(Hck) isoform that is targeted to lipid rafts and the monoacylated p61(Hck) isoform that is nonraft-associated. We have shown using several gain and loss of acylation alleles that dual acylation of Hck was required for CXCL12-mediated chemotaxis in J.CaM 1.6 cells. These results highlight the importance of the unique microenvironment provided by lipid rafts and their specific contribution in providing specificity to CXCL12 signaling.
Asunto(s)
Quimiocina CXCL12/fisiología , Quimiotaxis/fisiología , Células Jurkat/fisiología , Familia-src Quinasas/metabolismo , Acilación , Citometría de Flujo , Humanos , Indoles/farmacología , Microdominios de Membrana/fisiología , Plásmidos , Proteínas Proto-Oncogénicas c-hck/genética , Proteínas Proto-Oncogénicas c-hck/fisiología , Sulfonamidas/farmacología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.
Asunto(s)
Apoptosis/fisiología , Membrana Celular/fisiología , Epítopos/fisiología , Membranas Intracelulares/fisiología , Lípidos de la Membrana/fisiología , Fosfatidilserinas/fisiología , Animales , Apoptosis/efectos de la radiación , Tamaño de la Célula , Células Cultivadas , Retículo Endoplásmico/fisiología , Citometría de Flujo , Humanos , Células Jurkat/patología , Células Jurkat/fisiología , Ratones , Microscopía Fluorescente , Neutrófilos/patología , Neutrófilos/fisiología , Coloración y EtiquetadoRESUMEN
BACKGROUND: Currently, there is clear evidence that apoptosis plays an important role in the development and progression of tumors. One of the best characterized apoptosis triggering systems is the CD95/Fas/APO-1 pathway; previous reports have demonstrated high levels of soluble CD95 (sCD95) in serum of patients with some types of cancer. Cervical cancer is the second most common cancer among women worldwide. As a first step in an attempt to design a minimally invasive test to predict the risk of developing cervical cancer in patients with precancerous lesions, we used a simple assay based on the capacity of human serum to induce apoptosis in Jurkat cells. We evaluated the relationship between sCD95 levels and the ability to induce apoptosis in Jurkat cells in cervical cancer patients and controls. METHODS: Jurkat cells were exposed to serum from 63 women (20 healthy volunteers, 21 with cervical intraepithelial neoplasia grade I [CIN 1] and 22 with cervical-uterine carcinoma). The apoptotic rate was measured by flow cytometry using Annexin-V-Fluos and Propidium Iodide as markers. Serum levels of sCD95 and soluble CD95 ligand (sCD95L) were measured by ELISA kits. RESULTS: We found that serum from almost all healthy women induced apoptosis in Jurkat cells, while only fifty percent of the sera from women with CIN 1 induced cell death in Jurkat cells. Interestingly, only one serum sample from a patient with cervical-uterine cancer was able to induce apoptosis, the rest of the sera protected Jurkat cells from this killing. We were able to demonstrate that elimination of Jurkat cells was mediated by the CD95/Fas/Apo-1 apoptotic pathway. Furthermore, the serum levels of sCD95 measured by ELISA were significantly higher in women with cervical cancer. CONCLUSION: Our results demonstrate that there is a strong correlation between low levels of sCD95 in serum of normal women and higher apoptosis induction in Jurkat cells. We suggest that an analysis of the apoptotic rate induced by serum in Jurkat cells and the levels of sCD95 in serum could be helpful during the prognosis and treatment of women detected with precancerous lesions or cervical cancer.
Asunto(s)
Apoptosis , Células Jurkat/fisiología , Displasia del Cuello del Útero/fisiopatología , Neoplasias del Cuello Uterino/fisiopatología , Receptor fas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Femenino , Humanos , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Factores de Riesgo , Neoplasias del Cuello Uterino/sangre , Displasia del Cuello del Útero/sangreRESUMEN
BACKGROUND: A delicate balance between positive and negative regulatory mechanisms during T-cell activation determines the specificity and magnitude of an immune response. Phosphoinositide 3-kinase (PI 3-kinase) is activated by a diverse set of receptors that determine T-cell function, including the T-cell antigen receptor (TCR), the costimulatory receptor CD28, and negative regulators of T-cell activation such as CTLA-4. PI 3-kinase is also regulated by the haematopoietic cytokines that determine T-cell differentiation and lymphocyte proliferation. PI 3-kinase can thus dynamically influence the outcome of the immune reactions at various stages. In this study, we investigated the importance of PI 3-kinase in TCR-directed T-cell activation using activated or inhibitory versions of PI 3-kinase. RESULTS: Certain aspects of TCR responses such as the induction of transcriptional activity of AP1 and serum response factor were not affected by expression of the mutant forms of PI 3-kinase. We found, however, that PI 3-kinase profoundly influenced the transactivation capacity of 'nuclear factor of activated T cells' (NF-AT) elicited by the TCR: expression of an activated form of PI 3-kinase inhibited TCR-mediated NF-AT responses, whereas expression of a dominant negative mutant of PI 3-kinase potently enhanced TCR-controlled NF-AT induction. These effects of PI 3-kinase were not mediated by previously identified PI 3-kinase effectors, such as protein kinase B, a positive regulator of PI 3-kinase, or the GTPase Rac, and are therefore likely to involve a novel, as yet unknown, effector molecule. CONCLUSIONS: Our results establish that PI 3-kinase can both positively and negatively regulate T-cell function, and uncover a previously unrecognized function for PI 3-kinase in T cells as a selective negative regulator of TCR-signalling events and therefore as a determinant of T-cell homeostasis.
Asunto(s)
Inmunoconjugados , Células Jurkat/fisiología , Activación de Linfocitos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/fisiología , Transcripción Genética , Abatacept , Antígenos CD , Antígenos de Diferenciación/metabolismo , Apoptosis , Antígenos CD28/fisiología , Antígeno CTLA-4 , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas de Unión al ADN/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Factor de Respuesta Sérica , Linfocitos T/inmunología , Factor de Transcripción AP-1/metabolismo , Transfección , Proteínas de Unión al GTP racRESUMEN
Syncytia formation in HIV infections is driven by the virus fusion-active molecules (Env) interacting with membrane components of hosts cells. HIV-syncytia are usually interpreted as pathogenic entities and although they may potentially vary in size, numbers and types of constituent cells, little is known about the extent and significance of their diversity. Here, we describe numerically the cell population dynamics and the diversity of syncytia produced in the in vitro cell-fusion between two Jurkat T cell lines, one CD4(+) and the other Env(+). Cell-fusion partners were differentially stained with the lipophilic DiI and DiO, or with the cytoplasmic CMFDA and CMTMR tracers and syncytia showing double fluorescence were counted in a flow cytometer. The total number of syncytia formed, their size, cellular complexity and ratio of CD4(+)/Env(+) cells recruited, varied significantly in relation with time of reaction and initial proportions of fusion partners. The considerable structural diversity of syncytia formed, in so limited an in vitro cell fusion reaction, suggests that a greater heterogeneity may be formed in the natural course of disease. Identification of the main determinants of syncytia diversity allows for a detailed study of the relation between the syncytia structure and function.
Asunto(s)
Antígenos CD4/metabolismo , Células Gigantes/citología , VIH-1/química , Proteínas del Envoltorio Viral/metabolismo , Recuento de Células , Fusión Celular , Tamaño de la Célula , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Células Jurkat/fisiologíaRESUMEN
T lymphocytes undergo apoptosis in response to a variety of stimuli, including exposure to UV radiation and gamma-irradiation. While the mechanism by which stress stimuli induce apoptosis is not well understood, we have previously shown that the induction of Fas ligand (FasL) gene expression by environmental stress stimuli is dependent on c-Jun N-terminal kinase (JNK) activation. Using inducible dominant-active (DA) JNK kinase kinase (MEKK1) expression in Jurkat cells, we map a specific MEKK1-regulated response element to positions -338 to -316 of the Fas ligand (FasL) promoter. Mutation of that response element abrogated MEKK1-mediated FasL promoter activation and interfered in stress-induced activation of that promoter. Using electrophoretic mobility shift assays, we demonstrate that activator protein 1 (AP-1) binding proteins, namely, activating transcription factor 2 (ATF2) and c-Jun, bind to the MEKK1 response element. Transient transfection of interfering c-Jun and ATF2 mutants, which lack the consensus JNK phosphorylation sites, abrogated the transcriptional activation of the FasL promoter, demonstrating the involvement of these transcription factors in the regulation of the FasL promoter. Taken together, our data indicate that MEKK1 and transcription factors regulated by the JNK pathway play a role in committing lymphocytes to undergo apoptosis by inducing FasL expression via a novel response element in the promoter of that gene.
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Citocinas/farmacología , Quinasa 1 de Quinasa de Quinasa MAP , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/fisiología , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Activación Enzimática , Inducción Enzimática , Proteína Ligando Fas , Rayos gamma , Genes Reporteros , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Células Jurkat/efectos de la radiación , Luciferasas/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Estrés Fisiológico , Linfocitos T/efectos de los fármacos , Linfocitos T/efectos de la radiación , Transfección , Rayos UltravioletaRESUMEN
OBJECTIVE: Cardiolipin (CL) is found in membranes of bacteria, in the inner membrane of mitochondria and in plasma low-density lipoprotein (LDL). Anticardiolipin antibodies (aCL) are associated with disease states, and we have suggested that many aCL bind to oxidized CL (oxCL) but not native CL. To determine the immunogenicity and origins of oxCL in vivo, we cloned a natural antibody to oxCL. METHODS AND RESULTS: A monoclonal IgM antibody to oxCL (LRO1) was cloned from a nonimmunized LDLR-/- mouse. The V(H) sequence originated from the V(H)Gam3.8 germline with one nucleotide difference, and the Vkappa was 100% identical to Vkappa19-20 germline gene, making LRO1 a natural antibody. LRO1 bound specifically to oxCL and oxidized-LDL, but not to native CL or native LDL. LRO1 epitopes were demonstrated in apoptotic, but not in viable, Jurkat cells by flow cytometry, immunofluorescence and deconvolution microscopy. Human and rabbit atherosclerotic lesions contained LRO1 epitopes. Human LDL (n=113) showed LRO1 immunoreactivity, which correlated with aCL IgG titers (r=0.32, P=0.0004). CONCLUSIONS: These data demonstrate that some aCL antibodies are highly conserved natural antibodies binding to oxCL in oxLDL, apoptotic cells, and atherosclerotic lesions. This suggests that oxCL is one of the pathogen-associated molecular patterns of innate immunity and gives insight into the pathogenic events of diseases with increased titers of aCL antibodies.
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Anticuerpos Monoclonales/inmunología , Apoptosis , Aterosclerosis/inmunología , Cardiolipinas/inmunología , Células Jurkat/inmunología , Células Jurkat/fisiología , Lipoproteínas LDL/inmunología , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Aterosclerosis/patología , Epítopos , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Familia de Multigenes , ConejosRESUMEN
A new class of antigen-specific killer cells that combines the features of antibody-mediated and cell-mediated immunity was designed. The principle and feasibility of this strategy are illustrated by creating an antigen-specific killer cell to produce and secrete targeted antibody-toxin molecules that recognize and kill human immunodeficiency virus (HIV)-1-infected cells. The transduced lymphocytes, which were able to produce and secrete the targeted toxin proteins, remained viable due to the lack of the target antigen on the cell surface. These transduced cells were found to have selective and potent cytotoxicity to the HIV-infected cells. This approach combines the specificity of antibodies, extreme potency of toxins, and effector-cell properties of T-cells to generate a new class of potent antigen-specific killer cells, which may have broad applications for the treatment of viral infection and other diseases. This study demonstrates the principle that mammalian cells can be genetically modified to produce targeted toxins, indicating that in vivo production of targeted toxins can be achieved to locally or systematically destroy targeted cells.
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Anticuerpos Monoclonales/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Inmunotoxinas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Células COS/metabolismo , Muerte Celular/efectos de los fármacos , Técnicas de Cocultivo , Quimioterapia/métodos , Ensayo de Inmunoadsorción Enzimática , Exotoxinas/genética , Exotoxinas/inmunología , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Células Jurkat/fisiología , Células Jurkat/virología , Pseudomonas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/metabolismo , TransfecciónRESUMEN
PURPOSE: The heme oxygenase-1 (HO-1) system is associated with the rate-limiting step of conversion of heme, one of the most critical roles in cytoprotective mechanisms. Our study investigated its potential role in protection of endothelial cells from T cells. METHODS: The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells. Indirect fluorescent staining was used to examine the expression of HO-1 protein. Then endothelial cells primed by INF-gamma were mixed in culture with Jurkat T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The number of adhesive Jurkat T cells was determined using FACS to evaluate the adhesion effect. After being cultured with endothelial cells, the cell cycle of Jurkat T cells was detected using FACS. RESULTS: Expression of HO-1 on endothelial cells conferred significant protection against Jurkat T-cell-mediated adhesion. The rate of Jurkat T-cell adhesions was reduced to 19.06%, in contrast with 31.42% in the control group (P<.05). After using ZnPP, an inhibitor of HO-1, the rate of Jurkat T-cell adhesion recovered to 29.08%. The binding activities between endothelial cells and Jurkat T cells was blocked by HO-1 expression. The proliferation of Jurkat T cells was inhibited after culture with endothelial cells, which had been transfected with HO-1, which blocked cell cycle entry of T cells. More than 60% of Jurkat T cells remained in G0/G1 compared with 40% among the control group. CONCLUSION: HO-1 directly protected endothelial cells primed by INF-gamma from Jurkat T cells and down-regulated the expression of HLA-DR on the surface of endothelial cells. These results indicated that transgenic expression of HO-1 may be useful to prevent lymphocytes from responding to endothelial cells.
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Adhesión Celular/efectos de los fármacos , Endotelio Vascular/fisiología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/uso terapéutico , Interferón gamma/farmacología , Células Jurkat/fisiología , Animales , Cartilla de ADN , Fluoresceínas , Regulación de la Expresión Génica/inmunología , Antígenos HLA-DR/genética , Humanos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Bazo/enzimología , SuccinimidasRESUMEN
The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10kDa, extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement using DLE during treatment of allergies, cancer,immunodeficiencies, and in mycotic and viral infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.