RESUMO
The discovery of dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN) as a DC-specific ICAM-3 binding receptor that enhances HIV-1 infection of T cells in trans has indicated a potentially important role for adhesion molecules in AIDS pathogenesis. A related molecule called DC-SIGNR exhibits 77% amino acid sequence identity with DC-SIGN. The DC-SIGN and DC-SIGNR genes map within a 30-kb region on chromosome 19p13.2-3. Their strong homology and close physical location indicate a recent duplication of the original gene. Messenger RNA and protein expression patterns demonstrate that the DC-SIGN-related molecule is highly expressed on liver sinusoidal cells and in the lymph node but not on DCs, in contrast to DC-SIGN. Therefore, we suggest that a more appropriate name for the DC-SIGN-related molecule is L-SIGN, liver/lymph node-specific ICAM-3-grabbing nonintegrin. We show that in the liver, L-SIGN is expressed by sinusoidal endothelial cells. Functional studies indicate that L-SIGN behaves similarly to DC-SIGN in that it has a high affinity for ICAM-3, captures HIV-1 through gp120 binding, and enhances HIV-1 infection of T cells in trans. We propose that L-SIGN may play an important role in the interaction between liver sinusoidal endothelium and trafficking lymphocytes, as well as function in the pathogenesis of HIV-1.
Assuntos
Antígenos CD , Antígenos de Diferenciação , Moléculas de Adesão Celular/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Lectinas Tipo C , Lectinas/fisiologia , Fígado/metabolismo , Receptores de Antígenos/fisiologia , Receptores de HIV/fisiologia , Receptores Virais/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Mapeamento Cromossômico , DNA Complementar , Células Dendríticas , Endotélio/citologia , Éxons , HIV-1/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polimorfismo Genético , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de HIV/genética , Receptores de HIV/metabolismoRESUMO
Dendritic cells (DC) have been implicated in the pathogenesis of both human and simian immunodeficiency viruses (HIV and SIV, respectively). The DC-specific HIV-1 trans-receptor DC-SIGN is thought to be essential for viral dissemination by DC. Abundant expression in lymphoid tissues also implies a function for DC-SIGN in chronic HIV-1 infections, in facilitating persistent infection of T cells. We have therefore isolated the rhesus macaque and chimpanzee homologues of DC-SIGN to investigate their function in a primate model. Both rhesus macaque and chimpanzee DC-SIGN are highly similar to the human homologue. Three monoclonal antibodies against human DC-SIGN, AZN-D1, -D2 and -D3, cross-react with rhesus macaque DC-SIGN, whereas AZN-D2 does not cross-react with chimpanzee DC-SIGN. The primate homologues are abundantly expressed in lymphoid tissues such as lymph nodes, as well as in mucosal tissues involved in sexual transmission of HIV-1, and are functionally similar to human DC-SIGN. They have a high affinity for the immunological ligands of DC-SIGN: ICAM-2 and -3. Moreover, both homologues bind the HIV-1 envelope glycoprotein gp120 and therefore can act as a HIV-1 trans-receptor in the same way as human DC-SIGN. These data demonstrate that primate models are suitable to further dissect the role of DC-SIGN in the transmission and pathogenesis of infection with immunodeficiency viruses.
Assuntos
Moléculas de Adesão Celular , Lectinas Tipo C , Lectinas/imunologia , Macaca mulatta/imunologia , Glicoproteínas de Membrana , Pan troglodytes/imunologia , Receptores de Superfície Celular/imunologia , Receptores de HIV/imunologia , Vírus da Imunodeficiência Símia/imunologia , Proteínas do Envelope Viral , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Reações Cruzadas , DNA Complementar/genética , Células Dendríticas/imunologia , Expressão Gênica , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Lectinas/genética , Ligantes , Macaca mulatta/genética , Dados de Sequência Molecular , Pan troglodytes/genética , Receptores de Superfície Celular/genética , Receptores de HIV/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Dendritic cells (DC) capture micro-organisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present in antigenic form to resting T cells and thus initiate adaptive immune responses. Here we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC, but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. The interaction of DC-SIGN with HIV gp120 may be an important target for therapeutic intervention and vaccine development.
Assuntos
Moléculas de Adesão Celular , HIV-1/metabolismo , Lectinas Tipo C , Lectinas/metabolismo , Placenta/metabolismo , Complicações Infecciosas na Gravidez , Receptores de Superfície Celular/metabolismo , Receptores de HIV/metabolismo , Linfócitos T/metabolismo , Proteínas Virais/metabolismo , Feminino , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Linfonodos/metabolismo , Linfonodos/virologia , Mucosa/metabolismo , Mucosa/virologia , Placenta/virologia , Gravidez , Linfócitos T/virologiaRESUMO
Continuous production of polyhedra or baculovirus-expressed proteins in insect cell cultures is limited to about 4 weeks. The decrease in production has been ascribed to the interference of defective deletion mutants with wild-type baculoviruses. The deleted genome sequences include the polyhedrin gene (or the heterologous gene of interest); in the remaining part, the major late p10 gene is always maintained. In the present study, the productivity of a recombinant baculovirus with the lacZ gene from Escherichia coli cloned downstream of the p10 promoter at the p10 locus was investigated. It was hypothesized that this p10 promoter driven gene is preserved over a longer period of time in a continuously operated two-stage bioreactor system than foreign genes behind the polyhedrin promoter at the polyhedrin locus. In two separate runs, beta-galactosidase production with the p10-lacZ recombinant reached quasi-steady-state levels of 30 and 60 units/cm3. Polyhedron production was about 3 x 10(6) and 6 x 10(6) polyhedra/cm3, respectively. However, both polyhedron and beta-galactosidase production decreased after about 30 days of relatively constant production. In the infection reactor, deletion mutants of the virus, which contained both the polyhedrin and lacZ gene, were predominant. Therefore, the presence of the polyhedrin or p10 gene alone in deletion mutants is not sufficient for prolonged expression; other genes involved in major late gene expression and not present in the deleted virus are probably necessary.
Assuntos
Baculoviridae/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , beta-Galactosidase/biossíntese , Animais , Vetores Genéticos , Óperon Lac , MariposasRESUMO
The leukocyte-specific beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)/beta(2)) mediates activation-dependent adhesion to intercellular adhesion molecule (ICAM)-1. In leukocytes, LFA-1 requires activation by intracellular messengers to bind ICAM-1. We observed malfunctioning of LFA-1 activation in leukemic T cells and K562-transfected cells. This defective inside-out integrin activation is only restricted to beta(2) integrins, since beta(1) integrins expressed in K562 readily respond to activation signals, such as phorbol 12-myristate 13-acetate. To unravel these differences in inside-out signaling between beta(1) and beta(2) integrins, we searched for amino acids in the beta(2) cytoplasmic domain that are critical in the activation of LFA-1. We provide evidence that substitution of a single amino acid (L732R) in the beta(2) cytoplasmic DLRE motif, creating the DRRE motif, is sufficient to completely restore PMA responsiveness of LFA-1 expressed in K562. In addition, an intact TTT motif in the C-terminal domain is necessary for the acquired PMA responsiveness. We observed that restoration of the PMA response altered neither LFA-1 affinity nor the phosphorylation status of LFA-1. In contrast, strong differences were observed in the capacity of LFA-1 to form clusters, which indicates that inside-out activation of LFA-1 strongly depends on cytoskeletal induced receptor reorganization that was induced by activation of the Ca(2+)-dependent protease calpain.
Assuntos
Aminoácidos/química , Citoplasma/química , Antígeno-1 Associado à Função Linfocitária/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Calpaína/química , Adesão Celular , Linhagem Celular Transformada , Células Cultivadas , Citoplasma/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismo , Ativação Enzimática , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células K562 , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Mutagênicos , Mutação , Fosforilação , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol , TransfecçãoRESUMO
Contact between dendritic cells (DC) and resting T cells is essential to initiate a primary immune response. Here, we demonstrate that ICAM-3 expressed by resting T cells is important in this first contact with DC. We discovered that instead of the common ICAM-3 receptors LFA-1 and alphaDbeta2, a novel DC-specific C-type lectin, DC-SIGN, binds ICAM-3 with high affinity. DC-SIGN, which is abundantly expressed by DC both in vitro and in vivo, mediates transient adhesion with T cells. Since antibodies against DC-SIGN inhibit DC-induced proliferation of resting T cells, our findings predict that DC-SIGN enables T cell receptor engagement by stabilization of the DC-T cell contact zone.
Assuntos
Apresentação de Antígeno , Antígenos CD , Antígenos de Diferenciação , Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C , Lectinas/fisiologia , Ativação Linfocitária/fisiologia , Receptores de Superfície Celular/fisiologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/metabolismo , Cálcio/fisiologia , Adesão Celular , Moléculas de Adesão Celular/isolamento & purificação , Moléculas de Adesão Celular/metabolismo , Comunicação Celular , Células Cultivadas , Citometria de Fluxo , Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/fisiologia , Células K562 , Lectinas/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Mananas/farmacologia , Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Peso Molecular , Receptores de Superfície Celular/imunologia , Receptores de HIV/isolamento & purificação , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T/citologia , TransfecçãoRESUMO
Dendritic cells (DCs) are recruited from blood into tissues to patrol for foreign antigens. After antigen uptake and processing, DCs migrate to the secondary lymphoid organs to initiate immune responses. We now show that DC-SIGN, a DC-specific C-type lectin, supports tethering and rolling of DC-SIGN-positive cells on the vascular ligand ICAM-2 under shear flow, a prerequisite for emigration from blood. The DC-SIGN-ICAM-2 interaction regulates chemokine-induced transmigration of DCs across both resting and activated endothelium. Thus, DC-SIGN is central to the unusual trafficking capacity of DCs, further supported by the expression of DC-SIGN on precursors in blood and on immature and mature DCs in both peripheral and lymphoid tissues.
Assuntos
Antígenos CD/imunologia , Moléculas de Adesão Celular/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C , Lectinas/imunologia , Receptores de Superfície Celular/imunologia , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Humanos , Imunidade CelularRESUMO
Dendritic cells (DC) capture microorganisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present these in antigenic form to resting T cells and thus initiate adaptive immune responses. Here, we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We propose that DC-SIGN efficiently captures HIV-1 in the periphery and facilitates its transport to secondary lymphoid organs rich in T cells, to enhance infection in trans of these target cells.