RESUMEN
Dendritic cells (DCs) are a unique leukocyte type consisting of different subsets of professional antigen-presenting cells. Since DCs initiate and govern the immune response, they represent an ideal target for intervention aimed at modulating and potentiating immune responses against cancer and infectious diseases. We recently described and characterized, at a functional level, a novel DC subset, interferon (IFN)-DCs, derived from blood monocytes after a short exposure to type I IFN and GM-CSF. Here, we review our recent studies on IFN-DCs and discuss their possible use in clinical immunotherapeutic strategies.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Inmunoterapia/métodos , Interferón Tipo I/farmacología , Monocitos/efectos de los fármacos , Diferenciación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Relación Dosis-Respuesta a Droga , Humanos , Inmunoterapia/tendencias , Monocitos/citología , Monocitos/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
The migration capability of dendritic cells (DCs) is regulated by their response to factors, namely chemokines, that characterize maturation stage and shape their functional activities. This study examines the morphology, expression of chemokines/chemokine receptors, and migration properties of DCs generated after treatment of monocytes with type I interferon (IFN) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (IFN-DCs). IFN-DCs showed phenotypical and morphologic features undetectable in DCs generated in the presence of interleukin 4 (IL-4) and GM-CSF, such as expression of CD83 and CD25 and the presence of CD44+, highly polarized, thin, and long dendrites. IFN-DCs markedly migrated in response to beta-chemokines (especially MIP-1beta) and expressed the Th-1 chemokine IP-10. Notably, IFN-DCs showed an up-regulation of CCR7 as well as of its natural ligand MIP-3beta, characteristics typical of mature DCs. Of interest, IFN-DCs exhibited a marked chemotactic response to MIP-3beta in vitro and strong migratory behavior in severe combined immunodeficient (SCID) mice. In SCID mice reconstituted with human peripheral blood leukocytes, IFN-DCs induced a potent primary human antibody response and IFN-gamma production, indicative of a Th-1 immune response. These results define the highly specialized maturation state of IFN-DCs and point out the existence of a "natural alliance" between type I IFN and monocyte/DC development, instrumental for ensuring an efficient connection between innate and adaptive immunity.
Asunto(s)
Quimiocinas CC/biosíntesis , Quimiotaxis , Células Dendríticas/efectos de los fármacos , Linfocinas/biosíntesis , Receptores de Quimiocina/biosíntesis , Animales , Anticuerpos Heterófilos/biosíntesis , Presentación de Antígeno , Movimiento Celular , Extensiones de la Superficie Celular/ultraestructura , Quimiocina CCL19 , Quimiocinas CC/genética , Quimiocinas CC/farmacología , Quimiotaxis/efectos de los fármacos , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Células Dendríticas/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Anticuerpos Anti-VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Humanos , Inmunización , Inmunofenotipificación , Interferón-alfa/farmacología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-4/farmacología , Linfocinas/genética , Ratones , Ratones SCID , Microscopía Electrónica de Rastreo , Monocitos/citología , Receptores CCR7 , Receptores de Quimiocina/genética , Factores de Tiempo , Trasplante HeterólogoRESUMEN
Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)-treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-expressing DCs endowed with potent functional activities, superior with respect to the interleukin (IL)-4/GM-CSF treatment, as shown by FACS((R)) analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1-pulsed autologous DCs. Type I IFN induced IL-15 production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1-pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Interferón Tipo I/farmacología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Citocinas/genética , Cartilla de ADN/genética , Células Dendríticas/citología , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Anticuerpos Anti-VIH/biosíntesis , Antígenos VIH/genética , VIH-1/genética , VIH-1/inmunología , Humanos , Técnicas In Vitro , Transfusión de Leucocitos , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Monocitos/citología , Proteínas Recombinantes , Trasplante HeterólogoRESUMEN
In a previous study, we reported that a single injection of cyclophosphamide (CTX) in tumor-bearing mice resulted in tumor eradication when the animals were subsequently injected with tumor-sensitized lymphocytes. Notably, CTX acted by inducing bystander effects on T cells, and the response to the combined CTX/adoptive immunotherapy regimen was inhibited in mice treated with antibodies to mouse interferon (IFN)-alpha/beta. In the present study, we have investigated whether CTX induced the expression of type I IFN, and we have characterized the CTX effects on the phenotype of T cells in normal mice. CTX injection resulted in an accumulation of type I IFN messenger RNA in the spleen of inoculated mice, at 24 to 48 hours, that was associated with IFN detection in the majority of the animals. CTX also enhanced the expression of the Ly-6C on spleen lymphocytes. This enhancement was inhibited in mice treated with anti-type I IFN antibodies. Moreover, CTX induced a long-lasting increase in in vivo lymphocyte proliferation and in the percentage of CD44(hi)CD4(+) and CD44(hi)CD8(+ )T lymphocytes. These results demonstrate that CTX is an inducer of type I IFN in vivo and enhances the number of T cells exhibiting the CD44(hi) memory phenotype. Since type I IFN has been recently recognized as the important cytokine for the in vivo expansion and long-term survival of memory T cells, we suggest that induction of this cytokine may explain at least part of the immunomodulatory effects observed after CTX treatment. Finally, these findings provide a new rationale for combined treatments with CTX and adoptive immunotherapy in cancer patients. (Blood. 2000;95:2024-2030)
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Ciclofosfamida/farmacología , Receptores de Hialuranos/metabolismo , Interferón Tipo I/metabolismo , Linfocitos T/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Bromodesoxiuridina/metabolismo , Citometría de Flujo , Inmunoterapia , Cinética , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inmunología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.
Asunto(s)
Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Proteínas Proto-Oncogénicas/farmacología , Proteínas Tirosina Quinasas Receptoras/farmacología , Antígenos CD34 , Células Cultivadas , Citometría de Flujo , Humanos , Inmunofenotipificación , Recién Nacido , Tirosina Quinasa 3 Similar a fmsRESUMEN
Umbilical cord blood (UCB) transplant represents a promising therapeutic approach, nevertheless this procedure has been so far almost exclusively used in pediatric patients because of the reduced volume of UCB units. The availability of larger numbers of early and late hematopoietic progenitors by ex vivo amplification procedure may allow the use of UCB in adults and improve the rate and time to engraftment. We describe a stroma-free liquid culture system that induces a 10-fold increase of CD34+ cells and hematopoietic progenitors after 8 days in vitro amplification. The presence of flt3L is essential to preserve and amplify the early stem cell compartment identified by the phenotype CD34+Thy-1+CD45RO+.
Asunto(s)
Antígenos CD34/análisis , Compartimento Celular/efectos de los fármacos , Sangre Fetal/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas de la Membrana/farmacología , Adulto , Recuento de Células Sanguíneas , Células Cultivadas , Sangre Fetal/química , Células Madre Hematopoyéticas/citología , Humanos , Antígenos Comunes de Leucocito/análisis , Fenotipo , Antígenos Thy-1/análisisRESUMEN
The effects of basic fibroblast growth factor (bFGF) on differentiation of human fetal microglial cells were investigated. Human ramified microglial cells treated with human recombinant bFGF underwent a morphological change which resulted in a round-shape phenotype. bFGF was also able to induce a dose- and time-dependent increase in cell proliferation and enhanced phagocytic and non-specific esterase activity. These results indicate that ramified microglia, when properly stimulated, are regulated in their physiological and proliferative activities and are transformed into amoeboid forms. Growth factors, such as bFGF, are likely to play a key role in microglial transformation in both normal developing brain and in central nervous system injury.
Asunto(s)
Encéfalo/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Feto/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Microglía/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In VitroRESUMEN
This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.
Asunto(s)
Escherichia coli , Lipopolisacáridos/farmacología , Microglía/enzimología , Óxido Nítrico/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Bases , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Escherichia coli/química , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Microglía/efectos de los fármacos , Datos de Secuencia Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisisRESUMEN
The effect of basic fibroblast growth factor (bFGF) on inducible nitric oxide synthase (iNOS) mRNA expression in human cultured ramified microglial cells was investigated. Using RT-PCR and Southern blot analysis, we found that bFGF prevented the iNOS gene expression as induced by LPS/TNF alpha. Also, bFGF dose-dependently inhibited nitrite levels in treated cell supernatants. That the early presence of bFGF during LPS/TNF alpha induction was essential indicates that iNOS gene expression can be transcriptionally regulated.