RESUMEN
The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37 degrees C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37 degrees C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.
Asunto(s)
Interleucina-17/análisis , Interleucina-17/metabolismo , Linfocitos T/metabolismo , Animales , Citometría de Flujo/métodos , Ionomicina/farmacología , Ratones , Tasa de Secreción/efectos de los fármacos , Bazo/citología , Bazo/efectos de los fármacos , Coloración y Etiquetado/métodos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10(+/+), IL-10(+/-), and IL-10(-/-) mice were chronically irradiated with UV. IL-10(+/+) and IL-10(+/-) mice developed skin cancer to similar extents, whereas IL-10(-/-) mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10(-/-) mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10(-/-) mice showed impaired suppressor function when cocultured with conventional CD4+CD25- T cells. CD4+CD25- T cells from IL-10(-/-) mice produced increased amounts of IFN-gamma and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10(-/-) mice, suggesting strong Th1-driven immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10(-/-) mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10(-/-) animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.
Asunto(s)
Interleucina-10/fisiología , Neoplasias Inducidas por Radiación/inmunología , Neoplasias Cutáneas/inmunología , Rayos Ultravioleta , Animales , Citocinas/biosíntesis , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/efectos de la radiación , Interleucina-10/deficiencia , Interleucina-10/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Neoplasias Inducidas por Radiación/genética , Neoplasias Inducidas por Radiación/patología , Neoplasias Inducidas por Radiación/prevención & control , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/prevención & control , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Linfocitos T Reguladores/efectos de la radiaciónRESUMEN
Tumor necrosis factor (TNF) binds to two different receptors. Although most of its functions are attributed to TNF receptor 1 (TNFR1), the independent role of TNFR2 is still largely unknown. Using TNFR single or double knock-out mice, we show here that the expression of TNFR2 alone on host cells was sufficient to suppress the growth of TNF-secreting tumors in both immune competent and T/B lymphocyte-deficient severe combined immunodeficiency (SCID) mice. Histologic studies showed that TNF recruited, via TNFR2, large numbers of macrophages and efficiently inhibited angiogenesis in the tumor. In vitro, TNF activated TNFR1-deficient macrophages to produce nitric oxide (NO). Treatment of TNFR1 knock-out mice with L-NAME, a specific NO synthase inhibitor, almost completely eliminated TNF-induced angiostasis and tumor suppression. Moreover, L-NAME acted only during the first few days of tumor growth. Our results show for the first time that TNFR2 expressed on host innate immune cells is sufficient to mediate the antitumor effect of TNF, and NO is necessary for this process, possibly by inhibition of angiogenesis in the tumor.
Asunto(s)
Óxido Nítrico/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Animales , Linfocitos B/inmunología , Femenino , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Masculino , Sarcoma de Mastocitos/irrigación sanguínea , Sarcoma de Mastocitos/genética , Sarcoma de Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Neovascularización Patológica/inmunología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Plasmacitoma/irrigación sanguínea , Plasmacitoma/genética , Plasmacitoma/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/biosíntesis , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The CC chemokine receptor CCR7 has been identified as a key regulator of homeostatic B and T cell trafficking to secondary lymphoid organs. Data presented here demonstrate that CCR7 is also an essential mediator for entry of both dermal and epidermal dendritic cells (DC) into the lymphatic vessels within the dermis while this receptor is dispensable for the mobilization of Langerhans cells from the epidermis to the dermis. Moreover, a distinct population of CD11c(+)MHCII(high) DC showing low expression of the costimulatory molecules CD40, CD80, and CD86 in wild-type animals was virtually absent in skin-draining lymph nodes of CCR7-deficient mice under steady-state conditions. We provide evidence that these cells represent a semimature population of DC that is capable of initiating T cell proliferation under conditions known to induce tolerance. Thus, our data identify CCR7 as a key regulator that governs trafficking of skin DC under both inflammatory and steady-state conditions.
Asunto(s)
Movimiento Celular/fisiología , Células Dendríticas/metabolismo , Inflamación/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Antígeno CD11c/inmunología , Antígeno CD11c/metabolismo , División Celular/inmunología , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inflamación/inmunología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Ratones , Receptores CCR7 , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation in the course of adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Nicotine has been shown to increase the growth of atherosclerotic lesions. Therefore, we investigated whether nicotine can stimulate APCs and their T cell-stimulatory capacity using human monocyte-derived DCs and murine bone marrow-derived DCs as APCs. METHODS AND RESULTS: Nicotine dose-dependently (10(-8) to 10(-4) mol/L) induced DC expression of costimulatory molecules (ie, CD86, CD40), MHC class II, and adhesion molecules (ie, LFA-1, CD54). Moreover, nicotine induced a 7.0-fold increase in secretion of the proinflammatory T(H)1 cytokine interleukin-12 by human DCs. These effects were abrogated by the nicotinic receptor antagonist alpha-bungarotoxin and mecamylamine, respectively. The effects of nicotine were mediated in part by the phosphorylation of the PI3 kinase downstream target Akt and the mitogen-activated kinases ERK and p38 MAPK. Nicotine-stimulated APCs had a greater capacity to stimulate T-cell proliferation and cytokine secretion, as documented by mixed lymphocyte reactions and ovalbumin-specific assays with ovalbumin-transgenic DO10.11 mice. In a murine model of atherosclerosis, nicotine significantly enhanced the recruitment of DCs to atherosclerotic lesions in vivo. CONCLUSIONS: Nicotine activates DCs and augments their capacity to stimulate T-cell proliferation and cytokine secretion. These effects of nicotine may contribute to its influence on the progression of atherosclerotic lesions.