RESUMO
Respiratory allergic disorders are a global public health problem that are responsible for substantial morbidity and healthcare expenditure. Despite the availability of allergen immunotherapy (AIT), its efficacy is suboptimal and regimens are lengthy, with a significant risk of potentially severe side effects. Studies on the recognition of allergens by immune cells through carbohydrate-lectin interactions, which play a crucial role in immune modulation and pathogenesis of allergy, have paved the way for improvements in AIT. We highlight innovative approaches for more effective and safer AIT, including the use of allergens conjugated to specific carbohydrates that bind to C-type lectins (CLRs) and sialic acid-binding immunoglobulin-type lectins (Siglecs) on immune cells to induce suppressive responses.
Assuntos
Hipersensibilidade , Imunoglobulina E , Alérgenos , Carboidratos , Dessensibilização Imunológica , HumanosRESUMO
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
Intracellular signals are required to activate the leukocyte-specific adhesion receptor lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) to bind its ligand, intracellular adhesion molecule-1 (ICAM-1). In this study, we investigated the role of the cytoskeleton in LFA-1 activation and demonstrate that filamentous actin (F-actin) can both enhance and inhibit LFA-1-mediated adhesion, depending on the distribution of LFA-1 on the cell surface. We observed that LFA-1 is already clustered on the cell surface of interleukin-2/phytohemagglutinin-activated lymphocytes. These cells bind strongly ICAM-1 and disruption of the actin cytoskeleton inhibits adhesion. In contrast to interleukin-2/phytohemagglutinin-activated peripheral blood lymphocytes, resting lymphocytes, which display a homogenous cell surface distribution of LFA-1, respond poorly to intracellular signals to bind ICAM-1, unless the actin cytoskeleton is disrupted. On resting peripheral blood lymphocytes, uncoupling of LFA-1 from the actin cytoskeleton induces clustering of LFA-1 and this, along with induction of a high-affinity form of LFA-1, via "inside-out" signaling, results in enhanced binding to ICAM-1, which is dependent on intact intermediate filaments, microtubules, and metabolic energy. We hypothesize that linkage of LFA-1 to cytoskeletal elements prevents movement of LFA-1 over the cell surface, thus inhibiting clustering and strong ligand binding. Release from these cytoskeletal elements allows lateral movement and activation of LFA-1, resulting in ligand binding and "outside-in" signaling, that subsequently stimulates actin polymerization and stabilizes cell adhesion.
Assuntos
Actinas/fisiologia , Adesão Celular/fisiologia , Citoesqueleto/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Citocalasina D/farmacologia , Fibroblastos , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Filamentos Intermediários/metabolismo , Células L , Leucócitos/metabolismo , Melanoma , Camundongos , Microtúbulos/metabolismo , Ligação Proteica , Células Tumorais CultivadasRESUMO
The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562.
Assuntos
Citoplasma/metabolismo , Cadeias beta de Integrinas , Integrinas/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Antígenos CD18/efeitos dos fármacos , Antígenos CD18/genética , Antígenos CD18/metabolismo , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Fibronectinas/metabolismo , Humanos , Integrina beta1/efeitos dos fármacos , Integrina beta1/genética , Integrina beta1/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucemia Eritroblástica Aguda/tratamento farmacológico , Leucemia Experimental , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/genética , Linfócitos/fisiologia , Dados de Sequência Molecular , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
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
Earlier we observed that calcium phosphate (Ca-P)-coated implant substrates stimulated the differentiation of osteoblast-like cells compared to uncoated substrates. This suggests that this difference in osteogenic induction is due to the chemical composition of the substratum. We hypothesized that Ca-P coatings modulate integrin expression patterns, because those receptors are the sensors of the cell. Therefore, in the present study we quantitatively analyzed integrin expression of osteosarcoma cells and their proliferation behavior on various well-defined Ca-P substrates. For this study we used the osteosarcoma cell line U2OS. Five groups of substrates were used: thermanox (Th), uncoated titanium (Ti), dense sintered hydroxyapatite (HA), and two Ca-P-coated titanium discs (TiHA-O% and TiHA-5%). At day 5, cell numbers were significantly lower (p < 0.05) for both types of Ca-P-coated titanium substrates compared to the other substrates. There were no significant differences between HA and uncoated titanium. From day 5 to 8, accumulated cell number was ranking highest to lowest HA > Th = Ti > TiHA-0% > TiHA-5%. Integrin expression at day 5 and day 8 of incubation was analyzed by flow cytometry for integrin subunits beta 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v. Fluorescence-activated cell sorting (FACS) analysis showed that the cells express high levels of beta 1, low levels of alpha 4, alpha 5, and alpha 6, and moderate levels of alpha 3 and alpha v integrin subunits on the various biomaterial substrates. Minor differences in integrin expression between the various substrates were seen. Therefore, the observed differences in proliferation between the coatings may reside in modulating the functional properties of integrins.
Assuntos
Engenharia Biomédica/métodos , Fosfatos de Cálcio/química , Adesão Celular/fisiologia , Cerâmica , Materiais Revestidos Biocompatíveis/química , Matriz Extracelular/metabolismo , Integrinas/análise , Osteoblastos/metabolismo , Fosfatase Alcalina/análise , Fosfatase Alcalina/biossíntese , Fosfatos de Cálcio/farmacologia , Divisão Celular/fisiologia , Citometria de Fluxo , Humanos , Hidroxiapatitas/análise , Hidroxiapatitas/química , Integrinas/metabolismo , Microscopia Eletrônica de Varredura , Propriedades de Superfície , Fatores de Tempo , Titânio/análise , Titânio/química , Células Tumorais CultivadasRESUMO
To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)beta(2)), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete beta(2) cytoplasmic tail and/or the conserved KVGFFKR sequence in the alpha(L) cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Animais , Adesão Celular , Células Cultivadas , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Antígeno-1 Associado à Função Linfocitária/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Although ICAM-3 is implicated in both adhesion and signal transduction events of leukocytes, its low affinity for LFA-1 compared to other ligands of LFA-1 has puzzled many investigators. Here we investigated the role of ICAM-3 in supporting LFA-1-mediated ICAM-1 binding and subsequently cell signaling. We observed that although ICAM-3 binds poorly to LFA-1 expressed on resting T cells, it specifically facilitates and increases LFA-1-mediated adhesion to the high affinity ligand of LFA-1, ICAM-1. We demonstrate that low-affinity binding of LFA-1 to ICAM-3 together with ICAM-1 alters the cell surface distribution of LFA-1 dramatically, inducing large clusters of LFA-1 that facilitate ICAM-1 binding after LFA-1 activation. We found that LFA-1-mediated ICAM-1 cell-cell interactions such as T cell proliferation greatly depend on low affinity LFA-1/ICAM-3 interactions that enhance stable LFA-1/ICAM-1 cell-cell contact. Taken together, these data demonstrate that low affinity LFA-1 binding to ICAM-3 regulates strong LFA-1/ICAM-1-mediated adhesion by driving LFA-1 into clusters to facilitate cell-cell interactions that take place in the immune system.
Assuntos
Antígenos CD , Antígenos de Diferenciação , Moléculas de Adesão Celular/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Linfócitos T/citologia , Adesão Celular , Moléculas de Adesão Celular/farmacologia , Divisão Celular , Células Cultivadas , Humanos , Células K562 , Ativação Linfocitária , Proteínas de Neoplasias/fisiologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
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
The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg2+, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A beta2 cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.
Assuntos
Movimento Celular/imunologia , Molécula 1 de Adesão Intercelular/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Cátions Bivalentes/farmacologia , Adesão Celular/genética , Adesão Celular/imunologia , Comunicação Celular/genética , Comunicação Celular/imunologia , Movimento Celular/genética , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Células K562/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos , Microscopia Confocal , Microscopia de Contraste de Fase , Microscopia de Vídeo , Ligação Proteica/imunologia , Reologia , Deleção de Sequência/imunologia , Estresse Mecânico , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
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
A three-dimensional single-particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA-1 forms a connection to the cytoskeleton after binding to its ligand ICAM-1. LFA-1 is an integrin consisting of an alpha and a beta chain. Thus far, only the cytoplasmic tail of the beta chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete beta cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single-particle tracking measurements using beads coated with the ligand ICAM-1 indicate that this mutant form of LFA-1 does not move freely within the cell membrane, suggesting that LFA-1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA-1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the beta chain.
Assuntos
Citoesqueleto/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Citocalasina D/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Células K562 , Antígeno-1 Associado à Função Linfocitária/genética , MutagêneseRESUMO
Aberrant proliferation, differentiation, and/or migration of progenitors observed in various hematological malignancies may be caused by defects in expression and/or function of integrins. In this study, we have developed a new fluorescent beads adhesion assay that facilitates flow cytometric investigation of lymphocyte function-associated antigen 1 (LFA-1)- and very late activation antigen-4 (VLA-4)-mediated functional adhesion in B-lineage acute lymphoblastic leukemia (ALL) of both the CD10(-) and CD10(+) (leukemic) cell population within one blood or bone marrow sample. Surprisingly, of the 20 B-lineage ALL patients investigated, 17 contained a leukemic cell population with LFA-1- and/or VLA-4-mediated adhesion defects. Five patients contained CD10(+) cells that did not exhibit any LFA-1-mediated adhesion due to the lack of LFA-1 surface expression. The CD10(+) cells from 10 ALL patients expressed LFA-1 that could not be activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA), whereas the CD10(-) cells expressed a functional LFA-1. Seven patients contained CD10(+) cells that expressed a PMA-unresponsive form of VLA-4. The PMA unresponsiveness of the integrins LFA-1 and VLA-4 expressed by the CD10(+) cells may be due to mutations in the integrins itself, in protein kinases, or in other intracellular molecules involved in integrin adhesion. These data clearly demonstrate the importance of investigating integrin function in addition to integrin surface expression. The strikingly high frequency (85%) of adhesion defects in ALL could suggest a causal relationship between integrin-mediated adhesion and B-lineage ALL.
Assuntos
Linfoma de Burkitt/patologia , Adesão Celular , Integrinas/deficiência , Antígeno-1 Associado à Função Linfocitária/análise , Proteínas de Neoplasias/deficiência , Receptores de Retorno de Linfócitos/deficiência , Adolescente , Adulto , Linfócitos B/química , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Medula Óssea/patologia , Linfoma de Burkitt/sangue , Citometria de Fluxo , Humanos , Integrina alfa4beta1 , Integrinas/análise , Molécula 1 de Adesão Intercelular/metabolismo , Microesferas , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Neprilisina/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras B/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Receptores de Retorno de Linfócitos/análise , Acetato de Tetradecanoilforbol/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
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.