RESUMEN
Evidence for immunologic contribution to glaucoma pathophysiology is steadily increasing in ophthalmic research. Particularly, an altered abundance of circulating autoantibodies to ocular antigens is frequently observed. Here, we report an analysis of autoantibody abundancies to selected antigens in sera of open-angle glaucoma patients, subdivided into normal-tension glaucoma (N = 31), primary open-angle glaucoma (N = 43) and pseudoexfoliation glaucoma (N = 45), vs. a non-glaucomatous control group (N = 46). Serum samples were analyzed by protein microarray, including 38 antigens. Differences in antibody levels were assessed by ANOVA. Five serological antibodies showed significantly altered levels among the four groups (P < 0.05), which can be used to cluster the subjects in groups consisting mainly of PEXG or POAG/NTG samples. Among the altered autoantibodies, anti-Clathrin antibodies were identified as most important subgroup predictors, enhancing prospective glaucoma subtype prediction. As a second aim, we wanted to gain further insights into the characteristics of previously identified glaucoma-related antigens and their role in glaucoma pathogenesis. To this end, we used the bioinformatics toolset of Metascape to construct protein-protein interaction networks and GO enrichment analysis. Glaucoma-related antigens were significantly enriched in 13 biological processes, including mRNA metabolism, protein folding, blood coagulation and apoptosis, proposing a link of glaucoma-associated pathways to changes in the autoantibody repertoire. In conclusion, our study provides new aspects of the involvement of natural autoimmunity in glaucoma pathomechanisms and promotes advanced opportunities toward new diagnostic approaches.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoinmunidad , Clatrina/inmunología , Susceptibilidad a Enfermedades/inmunología , Síndrome de Exfoliación/diagnóstico , Síndrome de Exfoliación/etiología , Autoanticuerpos/sangre , Biomarcadores , Estudios de Casos y Controles , Biología Computacional/métodos , Síndrome de Exfoliación/sangre , Glaucoma de Ángulo Abierto/sangre , Glaucoma de Ángulo Abierto/diagnóstico , Glaucoma de Ángulo Abierto/etiología , Humanos , Análisis por Matrices de Proteínas/métodosRESUMEN
BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a cell-surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec-8 surface expression. OBJECTIVES: We sought to determine whether Siglec-8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec-8-bearing cells to kill these cells. METHODS: Siglec-8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec-8-transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec-8 intracellular trafficking was followed by confocal microscopy. The ribosome-inhibiting protein saporin was conjugated to a Siglec-8-specific antibody to examine the targeting of an agent to these cells through Siglec-8 endocytosis. RESULTS: Siglec-8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C activities, and both clathrin and lipid rafts. Internalized Siglec-8 localized to the lysosomal compartment. Maximal endocytosis in Siglec-8-transduced HEK293T cells required an intact immunoreceptor tyrosine-based inhibitory motif. Siglec-8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec-8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec-8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC-1.2. CONCLUSIONS: Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec-8 endocytic pathway.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Eosinófilos/inmunología , Lectinas/inmunología , Mastocitos/inmunología , Actinas/inmunología , Basófilos/inmunología , Muerte Celular/inmunología , Línea Celular , Línea Celular Tumoral , Clatrina/inmunología , Endocitosis/inmunología , Citometría de Flujo/métodos , Células HEK293 , Humanos , Ligandos , Microdominios de Membrana/inmunología , Proteína Quinasa C/inmunología , Proteínas Tirosina Quinasas/inmunologíaRESUMEN
Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Clatrina/inmunología , Dinaminas/inmunología , Células Endoteliales/inmunología , Proteínas de Neoplasias/inmunología , Fracciones Subcelulares/inmunología , Células Cultivadas , Células Endoteliales/ultraestructura , HumanosRESUMEN
In α1-antitrypsin-deficient HIV patients, an accelerated decline of CD4(+) T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4(+) T cells. Thus, primary CD4(+) T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4(+) T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4(+) T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4(+) T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1-mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome-lysosome-cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , VIH-1/fisiología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/inmunología , Activación Viral/inmunología , alfa 1-Antitripsina/inmunología , Clatrina/inmunología , Citosol/inmunología , Endocitosis/inmunología , Endosomas/inmunología , Femenino , Humanos , Lisosomas/inmunología , Masculino , FN-kappa B/inmunologíaRESUMEN
Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence-dependent endocytotic process. Both clathrin- and caveolae/lipid raft-mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Transporte de Proteínas/genética , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos P2X7/inmunología , Bacterias/inmunología , Línea Celular , Clatrina/inmunología , Endocitosis/inmunología , Humanos , Lisosomas/inmunología , Macrófagos/metabolismo , Microdominios de Membrana/metabolismo , Neutrófilos/inmunología , Antagonistas del Receptor Purinérgico P2X/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Receptores Purinérgicos P2X7/genética , CatelicidinasRESUMEN
We have employed a proteomic approach to study the immune response to human sperm in an infertile female patient suffering from systemic lupus erythematosus (SLE). Human sperm antigenic extracts were resolved by means of two-dimensional electrophoresis and electroblotted onto nitrocellulose membranes. The membranes were incubated with serum from the SLE patient. Sperm antigens that were reactive to polyclonal antibodies were next visualized on X-ray film, using the enhanced chemiluminescence (ECL). Three spots corresponding to the positions of sperm immunoreactive antigens on a nitrocellulose membrane were localized in a silver stained gel and subjected to mass spectrometry. A database search of the sequences recognized by the analyzed SLE serum revealed its homology to the clathrin heavy chain (CHC). Further analysis revealed that anti-CHC antibody reacted with multiple sperm antigenic determinants, resolved by either one- or two-dimensional electrophoresis. When studied by immunofluorescence, we demonstrated anti-CHC antibody reactivity with the sperm tail tip (corresponding to the sperm agglutination pattern), also with the principal piece and with cytoplasmic droplets around the sperm midpiece. Live sperm clearly exhibited reactivity with the midpiece. This study demonstrates clathrin heavy chain on human sperm using serum of an infertile individual with a concomitant autoimmune disease.
Asunto(s)
Clatrina/metabolismo , Infertilidad Femenina/inmunología , Lupus Eritematoso Sistémico/inmunología , Adulto , Clatrina/inmunología , Reacciones Cruzadas , Epítopos/metabolismo , Femenino , Humanos , Infertilidad Femenina/sangre , Infertilidad Femenina/complicaciones , Isoanticuerpos/sangre , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/complicaciones , Masculino , Espectrometría de Masas , Unión Proteica , Proteómica , Aglutinación Espermática/inmunología , Cola del Espermatozoide/metabolismoRESUMEN
Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped to elucidate the physical and molecular mechanisms involved in membrane transport. To gain a better understanding of endocytosis, we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked by GTPgammaS. Triggering fission by replacing GTPgammaS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17-dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.
Asunto(s)
Proteínas Portadoras/metabolismo , Estructuras de la Membrana Celular/fisiología , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Endocitosis/fisiología , Actinas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas Portadoras/inmunología , Bovinos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Estructuras de la Membrana Celular/efectos de los fármacos , Estructuras de la Membrana Celular/ultraestructura , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/fisiología , Clatrina/inmunología , Invaginaciones Cubiertas de la Membrana Celular/efectos de los fármacos , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Citosol/metabolismo , Dinaminas/metabolismo , Endocitosis/efectos de los fármacos , Proteínas de Unión a Ácidos Grasos , Fibroblastos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/farmacología , Humanos , Imagenología Tridimensional/métodos , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Modelos Biológicos , Potoroidae , Ratas , Receptores de Transferrina/metabolismo , Tiazolidinas/farmacologíaRESUMEN
Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-beta-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.
Asunto(s)
Antígenos CD36/inmunología , Clatrina/inmunología , Diglicéridos/farmacología , Endocitosis/inmunología , Receptores de Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Oligopéptidos/farmacología , Receptor Toll-Like 2/inmunología , Animales , Antígenos CD36/genética , Línea Celular , Clorpromazina/farmacología , Clatrina/genética , Clatrina/metabolismo , Diglicéridos/inmunología , Diglicéridos/metabolismo , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Endocitosis/genética , Humanos , Ionóforos/farmacología , Receptores de Lipopolisacáridos/genética , Macrófagos Peritoneales/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Nistatina/farmacología , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.
Asunto(s)
Apoptosis/inmunología , Membrana Celular/inmunología , Clatrina/inmunología , Dinaminas/inmunología , Endocitosis/inmunología , Granzimas/inmunología , Perforina/inmunología , Animales , Apoptosis/efectos de los fármacos , Membrana Celular/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/inmunología , Endosomas/metabolismo , Granzimas/farmacología , Células HeLa , Humanos , Perforina/metabolismo , RatasRESUMEN
Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin beta-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)(2)GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.
Asunto(s)
Arrestina/química , Clatrina/química , Complejos Multiproteicos/química , Secuencias de Aminoácidos , Animales , Arrestina/genética , Arrestina/metabolismo , Bovinos , Clatrina/genética , Clatrina/inmunología , Endocitosis/fisiología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Relación Estructura-ActividadRESUMEN
Recognition of MHC class I-related chain (MIC) molecules on the surface of target cells by the activating receptor NKG2D leads to their lysis by immune effector cells. Up-regulation of NKG2D ligands is broadly related to stress, although the detailed molecular mechanisms that control the presence of these molecules at the plasma membrane are unclear. To investigate the posttranslational mechanisms that control surface expression of the human NKG2D ligand MICB, we studied the subcellular localization and trafficking of this molecule. We found that in several cellular systems, the expression of MICB molecules on the cell surface is accompanied by an intracellular accumulation of the molecule in the trans-Golgi network and late endosome-related compartments. Surprisingly, MICB has a much shorter half-life at the plasma membrane than MHC molecules and this depends on both recycling to internal compartments and shedding to the extracellular medium. Internalization of MICB depends partially on clathrin, but importantly, the lipid environment of the membrane also plays a crucial role in this process. We suggest that the brief residence of MICB at the plasma membrane modulates, at least in part, the function of this molecule in the immune system.
Asunto(s)
Membrana Celular/inmunología , Colesterol/inmunología , Clatrina/inmunología , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Línea Celular Tumoral , Endosomas/inmunología , Humanos , Factores de TiempoRESUMEN
Muramyl dipeptide (MDP), the NOD2 agonist, induces NF-kappaB and MAPK activation leading to the production of antimicrobial and proinflammatory molecules. MDP is internalized into acidified vesicles in macrophages. However, the endocytic mechanism of MDP uptake that induces NOD2 signaling is unknown. We now report the identification of an endocytosis pathway dependent on clathrin and dynamin that mediates MDP internalization and NOD2 activation. Intracellular MDP uptake was inhibited by chlorpromazine, a drug that disrupts clathrin-dependent endocytosis, but not by compounds that block pinocytosis or cellular entry via scavenger or mannose receptors. In contrast, MDP uptake and NOD2-dependent signaling were unimpaired in macrophages deficient in PepT1, a peptide transporter previously implicated in MDP internalization. Both chlorpromazine and knockdown of clathrin expression by RNA interference attenuated MDP-induced NF-kappaB and MAPK activation. Furthermore, MDP uptake and NOD2-dependent signaling were impaired by inhibition of dynamin, a GTPase required for budding of clathrin-coated vesicles from the plasma membrane. Finally, bafilomycin A, a specific inhibitor of the vacuolar proton pump, blocked MDP accumulation in acidified vesicles and cytokine responses, suggesting that vacuolar maturation is important for MDP-induced NOD2 signaling. These studies provide evidence for a clathrin- and dynamin-dependent endocytosis pathway that mediates MDP uptake and NOD2 activation.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Adyuvantes Inmunológicos/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitosis/fisiología , Proteína Adaptadora de Señalización NOD2/metabolismo , Acetilmuramil-Alanil-Isoglutamina/inmunología , Animales , Línea Celular , Clatrina/inmunología , Vesículas Cubiertas por Clatrina/inmunología , Vesículas Cubiertas por Clatrina/metabolismo , Dinaminas/inmunología , Citometría de Flujo , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Proteína Adaptadora de Señalización NOD2/inmunología , Transportador de Péptidos 1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Simportadores/deficiencia , Simportadores/genética , Vacuolas/metabolismoRESUMEN
TLR3 and cytoplasmic RIG-I-like receptor (RLR) recognize virus-derived dsRNA and induce type I IFN production in a distinct manner. Human TLR3 localizes to the endosomal compartments in myeloid dendritic cells (mDCs), while it localizes to both the cell surface and interior in fibroblasts and epithelial cells. TLR3 signaling arises in the intracellular compartment in both cell types and requires endosomal maturation. The mechanisms by which extracellular dsRNA is delivered to the TLR3-containing organelle remain largely unknown. Among various synthetic dsRNAs, poly(I:C) is preferentially internalized and activates TLR3 in mDCs. In vitro transcribed dsRNAs hardly induce IFN-beta production in mDCs. In this study, we demonstrate that the clathrin-dependent endocytic pathway mediates cell entry of poly(I:C) to induce IFN-beta gene transcription. Furthermore, poly(I:C)-induced IFN-beta production is inhibited by pretreatment of cells with B- and C-type oligodeoxynucleotides (ODNs) but not with TLR7/8 ligands. The binding and internalization of B-type ODNs by mDCs was reduced in the presence of poly(I:C), suggesting that poly(I:C) shares the uptake receptor with B- and C-type ODNs. Hence, foreign dsRNA is recognized by differently categorized receptors, cytoplasmic RIG-I-like receptor, membrane-bound TLR3 and cell-surface RNA capture. The endocytic pathway is critical for dsRNA-induced TLR3-mediated cell activation.
Asunto(s)
Clatrina/inmunología , Células Dendríticas/inmunología , Endosomas/inmunología , Interferón beta/biosíntesis , Células Mieloides/inmunología , ARN Bicatenario/inmunología , ARN Viral/inmunología , Receptor Toll-Like 3/inmunología , Línea Celular , Membrana Celular/inmunología , Células Epiteliales/inmunología , Fibroblastos/inmunología , Humanos , Inductores de Interferón/inmunología , Inductores de Interferón/farmacología , Ligandos , Oligonucleótidos/inmunología , Oligonucleótidos/farmacología , Poli I-C/inmunología , Poli I-C/farmacología , ARN Bicatenario/farmacología , ARN Viral/farmacología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/inmunología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
C-type lectin receptors (CLRs) fulfill multiple functions within the immune system by recognition of carbohydrate moieties on foreign or (altered) self-structures. CLRs on myeloid dendritic cells (DCs) have been well characterized as pattern-recognition receptors (PRRs) combining ligand internalization with complex signaling events. Much less is known about CLR expression and function in human plasmacytoid DCs (pDCs), the major type I interferon (IFN) producers. In this study, we demonstrate that, next to the CLR BDCA-2, human pDCs express DC immunoreceptor (DCIR), a CLR with putative immune-inhibitory function, but not dectin-1, mannose receptor, or DC-specific ICAM-3-grabbing nonintegrin. DCIR surface levels are reduced on pDC maturation after TLR9 triggering. Interestingly, DCIR triggering inhibits TLR9-induced IFN-alpha production while leaving up-regulation of costimulatory molecule expression unaffected. Furthermore, DCIR is readily internalized into pDCs after receptor triggering. We show that DCIR internalization is clathrin-dependent because it can be inhibited by hypertonic shock and dominant-negative dynamin. Importantly, antigens targeted to pDCs via DCIR are presented to T cells. These findings indicate that targeting DCIR on pDCs not only results in efficient antigen presentation but also affects TLR9-induced IFN-alpha production. Collectively, the data show that targeting of DCIR can modulate human pDC function and may be applied in disease prevention and treatment.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/biosíntesis , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Animales , Células CHO , Diferenciación Celular , Línea Celular , Clatrina/inmunología , Cricetinae , Cricetulus , Células Dendríticas/citología , Endocitosis , Hemocianinas/inmunología , Humanos , Linfocitos/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
HIV-1 strains use C-C-chemokine receptor 5, CCR5, as a coreceptor for host transmission. Human CCR5 chemokine ligands inhibit binding and infection, whereas CCR5 mutations also inhibit infection by preventing surface expression, resulting in delayed progression to AIDS. Here, we describe a human herpesvirus 6 (HHV-6A) chemokine, U83A, which binds CCR5 with higher affinity than human chemokines, displacing their binding and leading to inhibition of chemotaxis of human leukocytes. Similarly, U83A inhibits infection by HIV-1 strains which use CCR5, but not the CXCR4, coreceptor. Unlike human CCR5 chemokine ligands which induce rapid CCR5 internalization mediated via clathrin, treatment with U83A prevents internalization. A spliced truncated U83A isoform, U83A-N, also binds CCR5 albeit with lower affinity, and this correlates with lower HIV-1 infection inhibition, whereas further truncation abolishes binding and any inhibition. Confocal microscopy confirms CCR5 internalization inhibition by U83A treatment, whereas labeled transferrin uptake shows that endocytosis via clathrin is unaltered. Previous results show that, although U83A-N is an antagonist, U83A is an agonist for CCR1, CCR4, CCR6, and CCR8 present on immune effector and antigen-presenting cells and here also shown for CCR5. Thus, U83A could act as a novel inhibitor of HIV-1 infection while also stimulating local immunity to the virus.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Antagonistas de los Receptores CCR5 , Quimiocinas/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , VIH-1/inmunología , Receptores CCR5/agonistas , Proteínas Virales/farmacología , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Empalme Alternativo/genética , Animales , Células COS , Quimiocinas/genética , Quimiocinas/inmunología , Chlorocebus aethiops , Clatrina/inmunología , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Humanos , Inmunidad Celular/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/farmacología , Receptores CCR5/inmunología , Receptores de Quimiocina/agonistas , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Chemokines are key controllers of cell trafficking and are involved in numerous pathologic and inflammatory conditions. However, the fate of a chemokine ligand, once it is endocytosed with its receptor, remains obscure. Here, using chemokine-tumor antigen fusion constructs, we demonstrate for the first time that chemokines are internalized to early/late endosomal and lysosomal compartments through a clathrin-dependent process and subsequently delivered to the cytosol for proteasomal processing, facilitating efficient cross-presentation to the TAP-1-dependent MHC class I processing pathway. These data not only elucidate the intracellular fate of chemokine ligands upon receptor uptake, but also demonstrate the superior carrier potency of chemokines for delivering self-antigens to both class I and II processing pathways to induce CD8(+) and CD4(+) T-cell responses.
Asunto(s)
Presentación de Antígeno/inmunología , Autoantígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Receptores de Quimiocina/inmunología , Transducción de Señal/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Animales , Presentación de Antígeno/genética , Autoantígenos/genética , Linfocitos T CD4-Positivos , Quimiocinas/genética , Quimiocinas/inmunología , Clatrina/genética , Clatrina/inmunología , Endocitosis/genética , Endocitosis/inmunología , Endosomas/genética , Endosomas/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II , Humanos , Ligandos , Lisosomas/genética , Lisosomas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Receptores de Quimiocina/genética , Transducción de Señal/genéticaRESUMEN
Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.
Asunto(s)
Presentación de Antígeno/inmunología , Endocitosis/inmunología , Inmunidad Innata/inmunología , Lipopolisacáridos/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Presentación de Antígeno/efectos de los fármacos , Transporte Biológico Activo/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Clatrina/inmunología , Dinaminas/inmunología , Endocitosis/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/farmacología , Fosfoproteínas/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
The identification of host factors that control susceptibility to infection has been hampered by a lack of amenable genetic systems. We established an in vivo model to determine the host factors that control pathogenesis and identified viral entry as a rate-limiting step for infection. We infected Drosophila melanogaster cells and adults with drosophila C virus and found that the clathrin-mediated endocytotic pathway is essential for both infection and pathogenesis. Heterozygosity for mutations in genes involved in endocytosis is sufficient to protect flies from pathogenicity, indicating the exquisite sensitivity and dependency of the virus on this pathway. Thus, this virus model provides a sensitive and efficient approach for identifying components required for pathogenesis.
Asunto(s)
Drosophila melanogaster/inmunología , Drosophila melanogaster/virología , Virus de Insectos/inmunología , Virus de Insectos/patogenicidad , Infecciones por Picornaviridae/inmunología , Animales , Western Blotting , Línea Celular , Clatrina/inmunología , Efecto Citopatogénico Viral , Modelos Animales de Enfermedad , Dinaminas/inmunología , Endocitosis/inmunología , Virus de Insectos/genética , Microscopía Fluorescente , Picornaviridae/genética , Picornaviridae/inmunología , Picornaviridae/patogenicidad , Infecciones por Picornaviridae/virologíaRESUMEN
The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.