RESUMEN
In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.
Asunto(s)
Comunicación Celular , Células Dendríticas/fisiología , Activación de Linfocitos , Linfocitos T/fisiología , Anticuerpos Monoclonales/inmunología , Antígenos CD/fisiología , Antígenos CD13/fisiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/fisiología , Células Cultivadas , Proteína-1 Reguladora de Fusión , Humanos , Inmunofenotipificación , Antígenos Comunes de Leucocito/análisis , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Receptores de Superficie Celular/fisiología , Receptores del Activador de Plasminógeno Tipo UroquinasaRESUMEN
1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)VD(3)) and retinoic acid (RA) modulate the activation of monocytes (MO) and their differentiation into macrophages (MAC). As these effects are mostly mediated by heterodimers or homodimers of the specific nuclear receptors for 1,25(OH)(2)VD(3) and RA, we investigated the expression of the retinoic acid receptors (RAR) alpha, beta, and gamma and the retinoid X-receptor (RXR) alpha in MO during differentiation into MAC or dendritic cells (DC). The mRNA of all investigated receptors except RARbeta was detected in short-term cultured MO. During differentiation of MO to MAC the mRNA expression of the RA receptors decreased. In contrast, along the differentiation pathway of MO to DC, only the mRNA expression of RARgamma declined, whereas RARalpha and RXRalpha were constantly expressed at a high level. Despite the strong expression of RARalpha and RXRalpha at mRNA level in MO-derived DC, the protein expression of the receptors was low in these cells. However, MO and MO-derived MAC showed a strong expression of these receptors at protein level. This suggests that a posttranscriptional or posttranslational mechanism of receptor regulation is occurring in these cells, and in particular in the DC. The inverse regulation of RA receptor expression and protein levels between MAC and DC may control the responsiveness of these cells to 1,25(OH)(2)VD(3) and RA.
Asunto(s)
Células Dendríticas/citología , Macrófagos/citología , Monocitos/citología , Receptores de Ácido Retinoico/genética , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , ARN Mensajero/análisis , Receptores de Calcitriol/biosíntesis , Receptores de Calcitriol/genética , Receptores de Ácido Retinoico/biosíntesis , Receptores X Retinoide , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
U937 cells provide a co-stimulatory signal for CD3-mediated T-cell activation which is independent of the CD28/CD80/CD86 interaction. This study set out to identify which molecules contribute to this co-stimulatory activity. Monoclonal antibodies (mAb) to the known accessory molecules CD11a, CD18, CD54 and CD45, all inhibited T-cell proliferation. Although CD11a/18 mAb inhibited U937/T-cell cluster formation as well as proliferation, CD45 enhanced the size of the clusters formed, suggesting that this was not the only mechanism of inhibition. The alternative co-stimulatory pathway provided by U937 cells preferentially stimulated a response in the CD18+ T-cell population, and this reflected the reduced sensitivity of CD8+ T cells to CD28-mediated activation. Monoclonal antibodies to three molecules, CD53, CD98 and CD147, also inhibited U937-dependent T-cell proliferation. The mAb to CD98 and CD147 were inhibitory when prepulsed on to the U937 cells, suggesting an effect mediated by these molecules on the antigen-presenting cell.
Asunto(s)
Activación de Linfocitos , Linfocitos T/citología , Linfocitos T/inmunología , Células U937/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD18/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/inmunología , Adhesión Celular/inmunología , División Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Proteína-1 Reguladora de Fusión , Humanos , Molécula 1 de Adhesión Intercelular/inmunología , Antígenos Comunes de Leucocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Tonsila Palatina/inmunologíaRESUMEN
Although retinoic acid (RA) has been known for many years to be a modulating agent that plays a role in generating both granulocytes and monocytes, the molecular mechanism underlying this role has not been defined in the monoblast lineage. In particular, the part played by the retinoid X receptors (RXRs), which are members of the steroid/thyroid hormone nuclear receptor family, has not been explored. In this study, therefore, the human monoblastic leukemia cell line U937 has been used as a model system to investigate the role of one of the RXRs, RXR-alpha, in monoblast differentiation. RXR-alpha mRNA was present in untreated U937 cells, and levels increased after induction of differentiation with phorbol ester. The same was found for RXR-beta mRNA. Using plasmids containing sense or antisense RXR-alpha sequences under the control of an inducible promoter, we generated stably transfected cell lines which expressed either increased or decreased levels of RXR-alpha, respectively. The sense cell lines (U alpha S and its clonal derivative alpha G2S) showed increased sensitivity to RA, while the antisense cell lines (U alpha A and its clonal derivative alpha B5A) showed decreased sensitivity to RA, as demonstrated by growth inhibition and by regulation of an RA-responsive reporter gene. Both U alpha A and alpha B5A also failed to respond to another modulating agent, 1 alpha,25-dihydroxycholecalciferol (DHCC), but only U alpha S and not alpha G2S showed an enhanced response to DHCC. The combination of RA and DHCC together inhibited growth of both sense and antisense cell lines. In addition, alpha G2S exhibited increased expression of CD11b and CD54, while alpha B5A cells showed increased expression of CD102, suggesting that RXR-alpha has a role in regulating expression of cell adhesion molecules in U937 cells. These results demonstrate that RXR-alpha has a role in mediating growth inhibition and cell adhesion during myelomonocytic differentiation, and suggest that different species of heterodimers involving RXR-alpha may control the acquisition of different features of mature monocyte/macrophage function.
Asunto(s)
Antineoplásicos/farmacología , ADN sin Sentido/genética , Receptores de Ácido Retinoico/genética , Factores de Transcripción/genética , Tretinoina/farmacología , Vitamina D/farmacología , Antígenos CD/genética , Calcitriol/farmacología , Moléculas de Adhesión Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/genética , Linfoma de Células B Grandes Difuso , Antígeno de Macrófago-1/genética , Monocitos/citología , ARN Mensajero/metabolismo , Receptores X Retinoide , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Activating germline mutations of the RET receptor tyrosine kinase are found in the majority of cases of inherited cancer syndrome MEN 2, and inactivating mutations in some cases of dominantly inherited Hirschsprung disease. Using RET activated by a MEN 2 mutation, we show that both the SH2 and PTB domains of the adaptor protein Shc interact with RET, and we identify the PTB domain interaction site. Interaction with both the SH2 and PTB domains of Shc contributes to the transcriptional activation of a serum response element. RET alternate splicing affects the strength of interaction with both the Shc SH2 and PTB domains. In addition, a splice isoform-specific HSCR missense mutation, which does not inactivate the RET kinase activity, decreases the strength of the PTB domain interaction and the level of RET-dependent Shc phosphorylation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Empalme Alternativo , Proteínas de Drosophila , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Activación Enzimática , Proteína Adaptadora GRB2 , Enfermedad de Hirschsprung/genética , Humanos , Datos de Secuencia Molecular , Neoplasia Endocrina Múltiple Tipo 2a/genética , Mutación , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , TransfecciónRESUMEN
The RET proto-oncogene encodes a receptor tyrosine kinase. We and others have recently shown that distinct germline mutations of the RET proto-oncogene account for the majority of cases of the dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes, and can cause a dominantly inherited form of Hirschsprung disease, a disorder of development of the autonomic innervation of the gut. RET is also oncogenically activated in some sporadic thyroid and adrenal tumours. Here we report the characterisation of multiple mRNA isoforms of RET generated by alternate splicing. Two isoforms are predicted to encode membrane-spanning receptors with a truncated extracellular ligand-binding domain. A third isoform is predicted to encode a soluble, secreted form of the receptor. These mRNA isoforms are expressed in both normal and tumour tissues.
Asunto(s)
Proteínas de Drosophila , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Empalme Alternativo , Secuencia de Bases , Cartilla de ADN/química , Expresión Génica , Humanos , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/genética , Distribución TisularRESUMEN
Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.
Asunto(s)
Carcinoma Medular/genética , Proteínas de Drosophila , Neoplasia Endocrina Múltiple/genética , Mutación Puntual , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/genética , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Tiroides/genética , Secuencia de Bases , Codón/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-retRESUMEN
We have investigated the binding of short (10 base) acridine-linked triplex-forming oligonucleotides to the target sequence A6G6.C6T6 by DNase I footprinting. Specific binding is detected at low pH (< 6.0) for 5'-Acr-T5C5 and 5'-Acr-5BrU5(5Me)C5. The sequence T5C5, lacking the acridine modification, binds less strongly, though specific binding is still evident. 5'-Acr-T5C5 produces footprints at slightly lower concentrations than 5'-Acr-5BrU5(5Me)C5. All three oligonucleotides produce enhanced DNase I digestion at the 3'-end of the target purine strand, suggesting that there is a DNA structural change at the triplex-duplex boundary. Target sequences AnG4A and TAC3Tn, containing one and two triplex mismatches, show no interaction with the acridine-free oligonucleotide, but bind the acridine-linked oligonucleotides. In these secondary binding modes the third strand is positioned so that the mismatches are located at the 3'-end of the oligonucleotide. Mithramycin and distamycin, binding in the minor groove to GC- and AT-rich sequences respectively, abolish triple helix formation.