RESUMEN
We have identified a novel lysosome-associated membrane glycoprotein localized on chromosome 3q26.3-q27, DC-LAMP, which is homologous to CD68. DC-LAMP mRNA is present only in lymphoid organs and DC. A specific MAb detects the protein exclusively in interdigitating dendritic cells. Expression of DC-LAMP increases progressively during in vitro DC differentiation, but sharply upon activation with LPS, TNFalpha, or CD40L. Confocal microscopy confirmed the lysosomal distribution of the protein. Furthermore, DC-LAMP was found in the MHC class II compartment immediately before the translocation of MHC class II molecules to the cell surface, after which it concentrates into perinuclear lysosomes. This suggests that DC-LAMP might change the lysosome function after the transfer of peptide-MHC class II molecules to the surface of DC.
Asunto(s)
Antígenos CD/biosíntesis , Células Dendríticas/citología , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Glicoproteínas de Membrana/biosíntesis , Secuencia de Aminoácidos , Anticuerpos Monoclonales/análisis , Antígenos CD/genética , Antígenos CD/inmunología , Secuencia de Bases , Diferenciación Celular/fisiología , División Celular/fisiología , ADN Complementario/análisis , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/química , Humanos , Inmunohistoquímica , Linfa/citología , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , ARN Mensajero/biosíntesisRESUMEN
In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-alpha. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1alpha, IL-1beta, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-alpha, TGF-beta, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-gamma transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+ CD11c+ CD3- DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.
Asunto(s)
Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Antígenos CD1/análisis , Antígenos CD34/análisis , Células Cultivadas , Niño , Citocinas/genética , Células Dendríticas/clasificación , Sangre Fetal/citología , Sangre Fetal/inmunología , Células Madre Hematopoyéticas/inmunología , Humanos , Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-13/biosíntesis , Interleucina-18 , Interleucina-7/biosíntesis , Interleucina-7/genética , Ionomicina/farmacología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Receptores de Lipopolisacáridos/análisis , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.
Asunto(s)
Células Dendríticas/inmunología , Proteínas Inflamatorias de Macrófagos/metabolismo , Receptores de Quimiocina , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Mapeo Cromosómico , Clonación Molecular , Cricetinae , ADN Complementario/genética , Células Dendríticas/metabolismo , Expresión Génica , Humanos , Células Híbridas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CCR6 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
This paper describes an antibody (mAb 7D6) that specifically recognizes human follicular dendritic cells (FDCs). By expression cloning, a cDNA clone encoding for the long human CR2/ CD21 isoform (CD21L) that contains an additional exon (10a) was isolated. We demonstrated that FDCs selectively express CD21L, while B cells selectively express the short CR2/CD21 lacking exon 10a (CD21S). By screening mouse Ltk- cells transfected with the CD21L cDNA, we further showed that the other two anti-human FDC mAbs DRC-1 and KiM4 also recognize CD21L. Thus, CD21L represents the first characterized human FDC-specific molecule, which may confer unique functions of FDCs in germinal center development.
Asunto(s)
Antígenos CD/biosíntesis , Linfocitos B/inmunología , Células Dendríticas/inmunología , Receptores de Complemento 3d/biosíntesis , Animales , Anticuerpos Monoclonales , Antígenos CD/genética , Secuencia de Bases , Niño , Clonación Molecular , Cartilla de ADN , ADN Complementario , Exones , Humanos , Células L , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Tonsila Palatina/inmunología , Reacción en Cadena de la Polimerasa , Receptores de Complemento 3d/genética , Proteínas Recombinantes/biosíntesis , TransfecciónAsunto(s)
Antígenos CD34/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Sangre Fetal/citología , Sangre Fetal/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Sangre Fetal/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Técnicas In Vitro , Recién Nacido , Receptores de Lipopolisacáridos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.
Asunto(s)
Antígenos CD34/análisis , Células Dendríticas/citología , Sangre Fetal/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Células Madre Hematopoyéticas/citología , Factor de Necrosis Tumoral alfa/fisiología , Antígenos CD1/análisis , Diferenciación Celular , División Celular , Células Cultivadas , Citometría de Flujo , Humanos , Inmunofenotipificación , Receptores de Lipopolisacáridos/análisis , Activación de Linfocitos , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/citología , Linfocitos T/inmunologíaRESUMEN
Phenotypic alterations occur when resting human B lymphocytes become germinal center (GC) cells. These include the induction of surface CD38, CD95 (FAS/APO-1), and carboxy-peptidase-M (CPM), a recently described GC marker. However, the factors that govern the in vivo induction of these surface molecules on B cells remain unknown. Here, we purified resting (CD38-) human B lymphocytes from tonsils in an attempt to establish culture conditions resulting in the induction of these three GC markers. We show that interferon (IFN) alpha or IFN-gamma, as well as antibodies against the B cell antigen receptor (BCR), could induce CD38 on resting B lymphocytes, a phenomenon further enhanced by CD40 stimulation. Concomitantly, CD95 was upregulated by CD40 ligation and, to a lesser extent, by IFN-gamma. By contrast, CPM expression could be upregulated only through BCR triggering. This CPM induction was specifically enhanced by CD19 or CD40 ligation. CD40 + BCR stimulation of resting B cells with CD40 ligand-transfected fibroblastic cells in the presence of cross-linked anti-BCR monoclonal antibodies resulted in the coexpression of CD38, CD95, and CPM. As GC cells, these cells also expressed CD71, CD80 (B7.1), and CD86 (B7.2), but not CD24. However, CD10+ or CD44- B cells could not be detected in these culture conditions, suggesting that yet other signals are required for the induction of these GC markers. Consistent with a GC phenotype, CD40 + BCR-stimulated cells exhibited reduced viability when cultured for 20 h in the absence of stimulus. These results first demonstrate that cotriggering of resting B cells through BCR and CD40 induces both phenotypic and functional GC features. They also show that IFN and CD19 triggering of resting B cells specifically modulate the expression of GC markers.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Linfocitos B/inmunología , Antígenos CD40/metabolismo , Centro Germinal/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Antígenos de Diferenciación/biosíntesis , Linfocitos B/efectos de los fármacos , Separación Celular , Células Cultivadas , Citometría de Flujo , Proteínas Ligadas a GPI , Centro Germinal/citología , Humanos , Memoria Inmunológica , Interferón gamma/farmacología , Glicoproteínas de Membrana , Metaloendopeptidasas/biosíntesis , N-Glicosil Hidrolasas/biosíntesis , Tonsila Palatina/citología , Fenotipo , Receptor fas/biosíntesisRESUMEN
Monoclonal antibody (MoAb) M27 was generated after immunization of mice with the human B-lineage acute lymphoblastic leukemia cell line Pre-ALP. Under reducing conditions, MoAb M27 precipitated a 60-kD surface-membrane molecule from Pre-ALP cells. Expression cloning of Pre-ALP cDNA showed that M27 recognizes carboxypeptidase M (CPM), a cell-surface, zinc-dependent protease known to cleave off basic C-terminal amino acids from peptide hormones. Using M27 antibody, CPM was detected only at discrete B lymphocyte developmental stages, namely on committed precursors and on germinal center cells. CPM was also expressed on mature T cells, mainly after activation. These results provide the first description of a carboxy-peptidase on lymphoid cells. In addition, CPM was found on granulocytes and monocytes, but not on their progenitors. Strikingly, CPM was present only on CD38+ cells, irrespective of lineage affiliation. Of interest, CPM displayed a largely overlapping distribution with the CD10 and CD13 peptidases, with which it shares common substrates (enkephalins, bradykinin). Collectively, the present data show a previously unrecognized distribution pattern of CPM on lymphoid and myeloid cells and suggest that CPM may cooperate with CD10 and CD13 to regulate biologic activity of peptide hormones on leukocytes.
Asunto(s)
Antígenos CD , Médula Ósea/enzimología , Antígenos CD13/metabolismo , Linfocitos/enzimología , Metaloendopeptidasas/metabolismo , Neprilisina/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/análisis , Linfocitos B/citología , Linfocitos B/enzimología , Médula Ósea/embriología , Células de la Médula Ósea , Diferenciación Celular , Niño , Proteínas Ligadas a GPI , Humanos , Inmunofenotipificación , Activación de Linfocitos , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos BALB C , N-Glicosil Hidrolasas/análisis , Linfocitos T/enzimología , Zinc/fisiologíaRESUMEN
The present study was aimed at identifying surface-membrane molecules involved in the regulation of human B-cell ontogeny. For this purpose, murine monoclonal antibodies (MoAbs) were generated against Pre-Alp, a pre-B acute lymphoblastic leukemia (ALL) cell line, and MoAb R34.34 was selected for further characterization. R34.34 recognized a molecule expressed on normal B-cell precursors (BCP) but not on mature B cells. The antibody also reacted with T lymphocytes, a subpopulation of monocytes from peripheral blood, and a subset of CD34+ cells. Immunoprecipitation analysis indicated that R34.34 recognizes an 80-kD molecular weight antigen. Antibody R34.34 was further found to be directed against an epitope interfering with binding of interleukin-7 (IL-7) to Pre-Alp cells. Expression cloning from a Pre-Alp cDNA library showed that R34.34 antigen is CDw127, the 75- to 80-kD IL-7 receptor. Proliferation of the B-lineage ALL cell lines Reh and Mieliki was inhibited by IL-7, and this effect was specifically reverted by MoAb R34.34. In addition, antibody R34.34 specifically inhibited IL-7-dependent proliferation of normal BCP, Pre-Alp cells, and peripheral T cells. These results imply that both inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. R34.34 antibody should be important for the analysis of signal transduction through CDw127.
Asunto(s)
Inhibidores de Crecimiento/farmacología , Interleucina-7/farmacología , Receptores de Interleucina/fisiología , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Humanos , Interleucina-7/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores de Interleucina-7 , Transducción de SeñalRESUMEN
To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgG1. The CD40-Fc fusion protein was able to specifically bind to CD4+ and various CD8+ T cell clones activated with immobilized anti-CD3. The 125I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4+ T cell clone (MT9) with an equilibrium dissociation constant (Kd) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of approximately 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8+ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.