RESUMO
In a search for genes expressed by dendritic cells (DC), we have cloned cDNAs encoding different forms of an asialoglycoprotein receptor (ASGPR). The DC-ASGPR represents long and short isoforms of human macrophage lectin, a Ca(2+)-dependent type II transmembrane lectin displaying considerable homology with the H1 and H2 subunits of the hepatic ASGPR. Immunoprecipitation from DC using an anti-DC-ASGPR mAb yielded a major 40-kDa protein with an isoelectric point of 8.2. DC-ASGPR mRNA was observed predominantly in immune tissues. Both isoforms were detected in DC and granulocytes, but not in T, B, or NK cells, or monocytes. DC-ASGPR species were restricted to the CD14-derived DC obtained from CD34(+) progenitors, while absent from the CD1a-derived subset. Accordingly, both monocyte-derived DC and tonsillar interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type cells did not. Furthermore, DC-ASGPR is a feature of immaturity, as expression was lost upon CD40 activation. In agreement with the presence of tyrosine-based and dileucine motifs in the intracytoplasmic domain, mAb against DC-ASGPR was rapidly internalized by DC at 37 degrees C. Finally, intracellular DC-ASGPR was localized to early endosomes, suggesting that the receptor recycles to the cell surface following internalization of ligand. Our findings identify DC-ASGPR/human macrophage lectin as a feature of immature DC, and as another lectin important for the specialized Ag-capture function of DC.
Assuntos
Células Dendríticas/imunologia , Endocitose , Lectinas Tipo C , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/fisiologia , Sequência de Aminoácidos , Animais , Receptor de Asialoglicoproteína , Antígenos CD40/metabolismo , Células Cultivadas , Clonagem Molecular , Endossomos/química , Granulócitos/imunologia , Humanos , Lectinas/genética , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Monócitos/imunologia , Filogenia , RNA Mensageiro/biossíntese , Ratos , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Células-Tronco/imunologiaRESUMO
We have measured the cross section for quasielastic 1p-shell proton knockout in the 16O(e,e(')p) reaction at omega = 0.439 GeV and Q2 = 0.8 (GeV/c)(2) for missing momentum P(miss)=355 MeV/c. We have extracted the response functions R(L+TT), R(T), R(LT), and the left-right asymmetry, A(LT), for the 1p(1/2) and the 1p(3/2) states. The data are well described by relativistic distorted wave impulse approximation calculations. At large P(miss), the structure observed in A(LT) indicates the existence of dynamical relativistic effects.
RESUMO
We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection of Langerin cDNA into fibroblasts created a compact network of membrane structures with typical features of BG. Langerin is thus a potent inducer of membrane superimposition and zippering leading to BG formation. Our data suggest that induction of BG is a consequence of the antigen-capture function of Langerin, allowing routing into these organelles and providing access to a nonclassical antigen-processing pathway.
Assuntos
Antígenos de Superfície/fisiologia , Endocitose/fisiologia , Células de Langerhans/fisiologia , Lectinas Tipo C , Lectinas de Ligação a Manose , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Citoplasma/metabolismo , DNA Complementar , Epitopos de Linfócito B/imunologia , Expressão Gênica , Humanos , Líquido Intracelular/imunologia , Células de Langerhans/citologia , Células de Langerhans/metabolismo , Camundongos , Dados de Sequência Molecular , Prolina , RNA Mensageiro , Ratos , TransfecçãoRESUMO
Using a cDNA subtraction technique, a novel member of the immunoglobulin superfamily was isolated from human Dendritic cells (DC). This cDNA which we named DORA, for DOwn-Regulated by Activation encodes a protein belonging to the CD8 family of receptors containing a single V type loop domain with an associated J chain region, a transmembrane region containing an atypical tyrosine residue and a cytoplasmic domain containing three putative tyrosine phosphorylation sites. The hDORA gene has been localised to chromosome 16. From database searches a rat cDNA was identified that encoded a polypeptide with 63% identity to hDORA. The expression of the human cDNA was studied in detail. Northern blot analysis revealed 1.0 kb and 2.5 kb mRNAs in peripheral blood lymphocytes, spleen and lymph node, while low levels were observed in thymus, appendix, bone marrow and fetal liver. No signal was noted in non-immune system tissues. By RT-PCR analysis of hDORA revealed expression in cells committed to the myeloid lineage but not in CD34+ precursors or B cells and low expression in T cells. Expression was also observed in DC, purified ex vivo or generated in vitro from either monocytes or CD34+ progenitors. This was down-regulated following activation both by PMA and Ionomycin treatment and also by CD40L engagement. In situ hybridisation performed on tonsil sections showed the presence of hDORA in cells within Germinal Centers. This structure and expression suggests a function as a co-receptor, perhaps in an antigen uptake complex, or in homing or recirculation of DC.
Assuntos
Células Dendríticas/imunologia , Imunoglobulinas/biossíntese , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Apresentação de Antígeno , Sequência de Bases , Ligante de CD40 , Antígenos CD8 , Clonagem Molecular , DNA Complementar/genética , Regulação para Baixo , Centro Germinativo/química , Células-Tronco Hematopoéticas/química , Humanos , Sistema Imunitário/química , Imunoglobulinas/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição TecidualRESUMO
Using a cDNA subtraction technique, a novel member of the ubiquitin family was isolated from human dendritic cells. This gene encodes a diubiquitin protein containing tandem head to tail ubiquitin-like domains, with the conservation of key functional residues. Expression of this 777-bp mRNA was restricted to dendritic cells and B cells, with strong expression in mature B cells. Southern blot analysis indicated that a single copy of this gene is present. In situ hybridization on tonsillar tissue showed expression in epithelial cells and isolated cells within the germinal center. With respect to an expressed-sequence tag (EST) this cDNA could be localized to the major histocompatibility complex class I region of chromosome 6. Comparative analysis and the expression pattern of this gene suggests a function in antigen processing and presentation.
Assuntos
Linfócitos B/metabolismo , Células Dendríticas/metabolismo , Ubiquitinas/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular , Sequência Consenso , DNA Complementar/genética , Sangue Fetal/citologia , Expressão Gênica , Genes , Humanos , Dados de Sequência Molecular , Tonsila Palatina/citologia , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Técnica de Subtração , Ubiquitinas/biossíntese , Ubiquitinas/genéticaRESUMO
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.