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• W2140697776 abstract "Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-α. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-α-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response. Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-α. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-α-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response. Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. They are present in trace amounts in most tissues, but they are particularly abundant and act as sentinels in organs with an environmental interface, such as the skin, the respiratory system, and the gastrointestinal tract. Due to their location, immature dendritic cells are profoundly influenced by the environment and transmit danger signals to cells of the adaptive immune system. The presence of pathogens activates immature dendritic cells and triggers their maturation, resulting in enhanced expression of costimulatory molecules such as CD86 and CD80, and of maturation markers such as CD83. Once activated, DCs migrate to lymph nodes where antigen presentation leads to the maturation and proliferation of specific T-cell clones, which in turn migrate to the injured tissue.1Banchereau J Briere F Caux C Davoust J Lebecque S Liu YJ Pulendran B Palucka K Immunobiology of dendritic cells.Annu Rev Immunol. 2000; 18: 767-811Crossref PubMed Scopus (5566) Google ScholarDepending on their location, DCs are able to release a specific array of cytokines to amplify the innate response. In addition, we would like to suggest that the innate and adaptive immune response may also be amplified through the release of tiny DC-derived microparticles. At least two types of vesicles released from DCs into the extracellular medium have been described. The first type are membrane vesicles, or exovesicles, which are between 0.1 and 1 μm in diameter; they are produced by membrane surface shedding, and released through a process similar to viral budding.2Obregon C Rothen-Rutishauser B Gitahi SK Gehr P Nicod LP Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.Am J Pathol. 2006; 169: 2127-2136Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Heijnen HF Schiel AE Fijnheer R Geuze HJ Sixma JJ Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar, 4Segura E Amigorena S Thery C Mature dendritic cells secrete exosomes with strong ability to induce antigen-specific effector immune responses.Blood Cells Mol Dis. 2005; 35: 89-93Crossref PubMed Scopus (218) Google Scholar The second type of vesicle is defined as an exosome, ie, microvesicle of endocytic origin, cup-shaped, and ∼0.05 μm in diameter; exosomes are released through exocytosis of multivesicular bodies.4Segura E Amigorena S Thery C Mature dendritic cells secrete exosomes with strong ability to induce antigen-specific effector immune responses.Blood Cells Mol Dis. 2005; 35: 89-93Crossref PubMed Scopus (218) Google Scholar, 5Thery C Zitvogel L Amigorena S Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3631) Google ScholarInitially, the secretion of these tiny microparticles was described as a process designed to regulate membrane functions and eliminate unnecessary membrane proteins.5Thery C Zitvogel L Amigorena S Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3631) Google Scholar However, exosomes have raised immunological interest because they originate from compartments of the endocytic pathway, which are sites of peptide loading on major histocompatibility complex (MHC) class II molecules. Indeed, both exovesicles and exosomes have been shown to be highly immunogenic, expressing on their surface not only MHC II molecules, but also costimulatory molecules such as CD86,5Thery C Zitvogel L Amigorena S Exosomes: composition, biogenesis and function.Nat Rev Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3631) Google Scholar, 6Andre F Chaput N Schartz NE Flament C Aubert N Bernard J Lemonnier F Raposo G Escudier B Hsu DH Tursz T Amigorena S Angevin E Zitvogel L Exosomes as potent cell-free peptide-based vaccine. I Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells.J Immunol. 2004; 172: 2126-2136Crossref PubMed Scopus (374) Google Scholar, 7Thery C Boussac M Veron P Ricciardi-Castagnoli P Raposo G Garin J Amigorena S Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.J Immunol. 2001; 166: 7309-7318Crossref PubMed Scopus (1172) Google Scholar and specific proteins lacking secretory signals sequence, such as interleukin (IL)-1β.8Qu Y Franchi L Nunez G Dubyak GR Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.J Immunol. 2007; 179: 1913-1925Crossref PubMed Scopus (428) Google Scholar, 9Pizzirani C Ferrari D Chiozzi P Adinolfi E Sandona D Savaglio E Di Virgilio F Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells.Blood. 2007; 109: 3856-3864Crossref PubMed Scopus (197) Google Scholar, 10MacKenzie A Wilson HL Kiss-Toth E Dower SK North RA Surprenant A Rapid secretion of interleukin-1beta by microvesicle shedding.Immunity. 2001; 15: 825-835Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar Recently, we were able to quantify, on a per cell basis, the release of exovesicles from activated DCs; these exovesicles represent the most relevant microparticles released by DCs. Using double vital staining, we demonstrated that exovesicles released from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to T-lymphocytes.2Obregon C Rothen-Rutishauser B Gitahi SK Gehr P Nicod LP Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.Am J Pathol. 2006; 169: 2127-2136Abstract Full Text Full Text PDF PubMed Scopus (86) Google ScholarIn the present study, we analyzed the composition and the fate of exovesicles regarding the epithelium. We were able to show that exovesicles from lipopolysaccharide (LPS)-activated DCs are important carriers of tumor necrosis factor (TNF)-α. Using double vital staining, we demonstrated that they are internalized by epithelial cells (ECs), and that this process induces the release of inflammatory mediators such as IL-8, Monocyte chemotactic protein-1 (MCP-1), Macrophage inflammatory protein 1β (MIP-1β), Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), and TNF-α. Furthermore, we demonstrate that the TNF-α cascade is one of the pathways involved in the activation of these cytokines. In contrast to the well-characterized transfer of alloantigens of exovesicles to heterologous resting DCs, exovesicles in the co-culture with ECs do not transfer an antigen presenting capacity to ECs. Our results demonstrate the potential role of exovesicles not only in adaptive immunity, as a relevant source of antigens fusing with the cytoplasmic membrane of resting DCs, allowing them to present antigens, but also in innate immunity by triggering ECs to release cytokines-chemokines, thereby amplifying the magnitude of the innate immune response.Materials and MethodsMonocyte Isolation and Differentiation to Dendritic CellsMonocytes generated from peripheral blood mononuclear cells of healthy human donors were isolated by Ficoll-Hypaque density gradient centrifugation of buffy coats as described previously,11Obregon C Dreher D Kok M Cochand L Kiama GS Nicod LP Human alveolar macrophages infected by virulent bacteria expressing SipB are a major source of active interleukin-18.Infect Immun. 2003; 71: 4382-4388Crossref PubMed Scopus (32) Google Scholar following spontaneous aggregation12Mentzer SJ Guyre PM Burakoff SJ Faller DV Spontaneous aggregation as a mechanism for human monocyte purification.Cell Immunol. 1986; 101: 312-319Crossref PubMed Scopus (94) Google Scholar and rosetting.13Armant M Rubio M Delespesse G Sarfati M Soluble CD23 directly activates monocytes to contribute to the antigen-independent stimulation of resting T cells.J Immunol. 1995; 155: 4868-4875PubMed Google Scholar Briefly, Ficoll-Paque-purified peripheral blood mononuclear cells were suspended in RPMI 1640 medium (Invitrogen Life Technologies, Basel, Switzerland) supplemented with 10% fetal calf serum (Biochrome AG, Berlin, Germany), 2 mmol/L glutamine, 100 U of penicillin per ml, and 100 U streptomycin per ml, referred to as complete culture medium, containing 2 μg polymyxin B sulfate/ml (Sigma-Aldrich, Buchs, Switzerland). Cells were incubated for 40 minutes at 4°C for aggregation. Rosetting was applied to deplete contaminant lymphocytes. Monocyte enriched fractions were incubated overnight with sheep red blood cells (BioMérieux, Geneva, Switzerland). Monocyte fractions characterized by high expression of CD14 (more than 85%) and low expression of CD83 and CD86 (less than 5%) were then isolated by Ficoll-Hypaque density gradient centrifugation. Differentiation of DCs from monocytes was performed as originally described by Sallusto and Lanzavecchia,14Sallusto F Lanzavecchia A Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4475) Google Scholar by culture cells in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interleukin-4 (10 ng/ml) for 6 days. The cells were kept at 37°C in a 5% CO2 humidified atmosphere. On day 3, the culture medium was replaced with fresh medium.Labeling and Stimulation of DCsAfter 6 days in culture, DCs were washed and suspended at a density of 1 × 106 cells/ml in serum-free medium (RPMI 1640 medium). Cells were labeled with VIBRANT cell labeling DiI (1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate) (Molecular Probes, Leiden, The Netherlands) for 10 minutes at 37°C and 5% CO2. After labeling, cells were washed three times with RPMI 1640 in 37°C pre-warmed media, and cultured in RPMI 1640 supplemented with 1% glutamine and 1% microvesicle-free human serum obtained by ultracentrifugation (110,000 × g) of the serum for 2 hours. Cells were stimulated with 100 ng LPS or left unstimulated and incubated for 12 hours at 37°C in 5% CO2.Labeling and Stimulation of ECsHuman A549 (ATCC#CCL185) alveolar epithelial cells were grown in complete culture medium. Eighty-percent confluent cells were harvested by trypsination, washed twice and resuspended in pre-warmed RPMI 1640 serum-free medium at a density of 1 × 106 cells/ml. Cells were labeled with VIBRANT cell labeling DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) (Molecular Probes, Leiden, The Netherlands) for 10 minutes at 37°C and 5% CO2. After labeling, cells were washed three times with RPMI 1640 in 37°C pre-warmed medium and resuspended in complete culture medium. For the analysis of endosomal compartments, ECs were labeled with transferrin Alexa 633, 20 μg/ml (Molecular Probes, Leiden, The Netherlands), 30 minutes at 37°C and 5% CO2 after DiO labeling.For blocking experiments, exovesicles were pre-incubated 1 hour at 37°C with a blocking anti TNF-α, which is a human/mouse chimeric antibody of IgG1κ isotope (Inflimab; Remicade). Afterward, pre-treated exovesicles were incubated with ECs for 24 hours. Supernatants were harvested and stored at −80°C until used. A panel of 14 cytokines were measured using the Luminex system from BioRad according to manufacture’s recommendation. The respective human IgG1κ was used as a control (Sigma-Aldrich, Buchs, Switzerland).Purification of ExovesiclesExovesicles were isolated using the standard process of a series of differential ultracentrifugation and filtration described previously.7Thery C Boussac M Veron P Ricciardi-Castagnoli P Raposo G Garin J Amigorena S Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.J Immunol. 2001; 166: 7309-7318Crossref PubMed Scopus (1172) Google Scholar, 15Raposo G Nijman HW Stoorvogel W Liejendekker R Harding CV Melief CJ Geuze HJ B lymphocytes secrete antigen-presenting vesicles.J Exp Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2383) Google Scholar The supernatant of 10 × 106 DCs was collected and exovesicles were purified by centrifugation at 250 × g for 8 minutes as described previously,2Obregon C Rothen-Rutishauser B Gitahi SK Gehr P Nicod LP Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.Am J Pathol. 2006; 169: 2127-2136Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar then run trough 0.45-μm filters to eliminate large debris. The filtered supernatant was ultracentrifuged at 110,000 × g for 1 hour. Exovesicles were washed once with RPMI 1640 and pelleted by ultracentrifugation at 110,000 × g for 1 hour. Then the pellet was resuspended in 150 μl RPMI 1640 medium. The exovesicles were either used for immunofluorescence labeling or co-cultured with ECs.Laser Scanning MicroscopyLaser scanning microscopy (LSM) analysis was performed with a Zeiss LSM 510 Meta with an inverted Zeiss microscope (Axiovert 200M, Lasers: HeNe 543 nm and Ar 488 nm). Optical sections were taken with a 63x/1.4 Plan-Apochromat objective. In combination with digital zoom, this resulted in a voxel dimension of 0.1 × 0.1 × 0.25 μm. Images were processed and visualized with IMARIS, a three-dimensional multichannel image processing software for laser scanning microscope images (Bitplane AG, Zurich, Switzerland). To quantify the intracellular exovesicles, the IsoSurface mode of the Surpass module in IMARIS was used, and an intensity threshold was applied to create a model of the data visualized as a solid surface. The intracellular objects were then counted by the software. For this quantification, all microscope settings were kept constant during one experiment, ie, for control as well as treated cultures. All settings used for the image single restoration were also equal and the cells were chosen at random. Co-localization analysis was performed with the IMARIS co-localization module. For all control stainings the same imaging parameters were used as for the specific antibodies.ImmunolabelingDCs or exovesicles isolated from DCs unstimulated or stimulated with LPS were resuspended in complete culture medium containing 2% of alginate.16Page AM Lagnado JR Ford TW Place G Calcium alginate encapsulation of small specimens for transmission electron microscopy.J Microsc. 1994; 175: 166-170Crossref Scopus (13) Google Scholar Drops of the media were suspended carefully in a CaCL2 solution (50 mmol/L) for 1 hour to allow the matrix formation and exovesicle immobilization. DCs or exovesicles in alginate drops were fixed in 3% paraformaldehyde containing 0.1 M/L sodium cacodylate and 7 mmol/L CaCL2 buffer and subsequently washed in 0.1 M/L glycine, 0.1 M/L sodium cacodylate, and 7 mmol/L CaCL2 buffer. Drops were stored in 0.1 M/L glycine, 0.1 M/L sodium cacodylate, and 7 mmol/L CaCL2 buffer at 4°C until immunolabeling.Cell surface markers were analyzed using pure antibodies CD83 (clone HB15a), CD40 (clone mAb89), HLA-DR (CR3/43), TNFRI (clone IP05), TNFRII (clone 22210)anti-TNF-α (MAb11), and the specific IgG1 isotype control (MOPC-21). Rhodamine-conjugated or Cy3-conjugated goat anti-mouse was used as secondary antibody (Chemicon International, Zug, Switzerland).Protein Preparation and ElectrophoresisProteins recovered from exovesicles were quantified with the ATTO-TAG CBQCA kit according to the manufacturer’s recommendations (Molecular Probes, Leiden, The Netherlands). The fluorescence emission was measured at 550 nm (filter 530 ± 30 nm) with excitation at 465 nm (filter 485 ± 20 nm) in a CytoFluor 4000 fluorescence microplate reader (gain 40) (PerSeptive Biosystems, Foster City, CA). Sample preparation was done with 8 mol/L urea (Sigma-Aldrich Chemie BV), 2% CHAPS (Amersham Pharmacia Biotech), 20 mmol/L dithiothreitol (Sigma-Aldrich Chemie BV), 0.01% bromophenol blue (Sigma-Aldrich Chemie BV). Twelve percent SDS-polyacrylamide gel (1.0 mm 16 cm) electrophoresis was run according to manufacturer’s recommendations (Protean II xi Cell; Bio Rad Laboratories, Hemel Hempstead, UK), under reducing conditions.Western Blot AnalysisProteins were electroblotted after one-dimensional electrophoresis onto an Immobilon P membrane (Millipore Corp.), and then incubated with a rabbit anti-human TNF-α antibody (Leinco Technologies, Inc.), followed by horseradish peroxidase-conjugated secondary antibodies. Using SuperSignal West Pico chemiluminescent substrate (Pierce Perbio Science) detection was performed on a chemiluminiscense film (Amersham, Hyperfilm). As a control, the Immobilon membrane was stripped using Restore reagent (Pierce Perbio Science) and then incubated with a mouse anti-human Actin (clone MAB1501) as previously described.StatisticsData are expressed as mean values with the SEM. The statistical significance was determined using Student’s t-test. P < 0.05 was considered to be significant.ResultsDC-Derived Exovesicles Express MHC-II and Costimulatory Molecules Such as HLA-II, CD40, and CD83As we have shown recently, exovesicles released from LPS-stimulated allo-DCs are able to elicit T-cell proliferation after 6 days of incubation.2Obregon C Rothen-Rutishauser B Gitahi SK Gehr P Nicod LP Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.Am J Pathol. 2006; 169: 2127-2136Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar Therefore, in the initial steps of these experiments, we wanted to identify the molecules that could be expressed on the surface of these exovesicles. Exovesicles in culture supernatants of LPS-activated DCs were purified using the standard ultracentrifugation and filtration process described in the Materials and Methods. DCs and isolated exovesicles were then immobilized in the alginate matrix, fixed, and immunolabeled for HLA-DR-DQ, CD40, and CD83. The fluorescence was analyzed by LSM (Figure 1). The results showed that exovesicles carried large amounts of MHC-II, CD40 and, a smaller but nonetheless clearly positive amount of CD83. These results suggest that the molecules that were already expressed on the surface of DCs (Figure 1, A, C, E) were also present on the exovesicles (Figure 1, B, D, F), thus corroborating the hypothesis that exovesicles can play a role as immunological messengers of DCs, and that costimulatory molecules are present on their surface. Control cells were labeled with irrelevant IgG1 antibody resulting in no fluorescence (Figure 1, G and H).DC-Derived Exovesicles Are Important Carriers of TNF-αTNF-α is a pro-inflammatory cytokine and is up-regulated by LPS in DCs.17Sullivan KE Regulation of inflammation.Immunol Res. 2003; 27: 529-538Crossref PubMed Scopus (23) Google Scholar, 18Locksley RM Killeen N Lenardo MJ The TNF and TNF receptor superfamilies: integrating mammalian biology.Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (2965) Google Scholar We have therefore investigated to what extent TNF-α can be carried on these exovesicles isolated from DCs. DC-derived exovesicles, unstimulated or stimulated with 100 ng LPS, were purified using the standard ultracentrifugation and filtration process described in the Materials and Methods. Western blot analysis revealed for the first time that exovesicles from activated DCs are important carriers of TNF-α molecules, whereas under control conditions, exovesicles carry none (Figure 2A). Additionally, when we resuspended DCs in alginate previously labeled with DiO and immunolabeled with anti-TNF-α (Figure 2B), we were able to visualize a large amount of TNF-α co-localizing with the cytoplasmic membrane of immature DCs, whereas with LPS matured DCs, the internal TNF content was decreased and some TNF-α was found co-localized in structures budding from plasma membrane. Interestingly, when we resuspended exovesicles in alginate from DCs untreated or treated with LPS immunolabeled with anti-TNFRI and TNFRII (Figure 2C), we were able to visualize both receptors on the membrane of the isolated exovesicles, suggesting that exovesicles are able to specifically carry TNF-α molecules on their surface when they are activated with LPS. Control exovesicles were labeled with irrelevant IgG1 antibody resulting in no fluorescence. Thus, exovesicles might play a role not only in adaptive immunity, transferring molecules involved in the activation of T-cells,2Obregon C Rothen-Rutishauser B Gitahi SK Gehr P Nicod LP Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.Am J Pathol. 2006; 169: 2127-2136Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar but also in innate immunity, transferring TNF-α molecules during inflammation.Figure 2Analysis of TNF-α in exovesicles. DCs were stimulated with LPS or not treated. Exovesicles were then isolated by ultracentrifugation. A: Western blot analysis of TNF-α in exovesicles isolated from line 1: immature DCs, line 2: LPS-activated DC-s and labeled against TNF-α (upper panel) or against Actin (lower panel). B: DiO-labeled DCs (green) were immunolabeled with anti-TNF-α (red). TNF-α is very abundant inside the cells (left panel), in contrast with LPS-DCs (right panel) where the cells are nearly empty of TNF-α molecules. The images represent single optical sections, the insets are volume renderings. C: LSM of isolated exovesicles from resting and LPS DCs resuspended in alginate matrix and immunolabeled for the expression of TNFRI (upper panel), TNFRII (middle panel), and isotype control (lower panel). The images represent volume renderings.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DC-Derived Exovesicles Are Mostly Internalized by ECsRecently, we demonstrated that DC-derived exovesicles are able to fuse with the plasma membrane of resting DCs in their vicinity. Therefore, we have looked to what extent DC-derived exovesicles fuse with the plasma membrane of ECs or are internalized by them. DCs were stained with the fluorescent probe DiI (red), stimulated for 10 hours with 100 ng LPS, and then exovesicles were isolated. In parallel, ECs were labeled with the DiO (green) fluorescent probes and ECs were co-cultured with exovesicles from control DCs or LPS-stimulated DCs. Cells were investigated after 1, 6, and 24 hours and the incorporation of the red fluorescence (DC-exovesicles) in green labeled cells (ECs) was analyzed (Figure 3). These exovesicles were mostly internalized by ECs, whereas in resting DCs, exovesicles mostly fused with the plasma membrane.2Obregon C Rothen-Rutishauser B Gitahi SK Gehr P Nicod LP Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.Am J Pathol. 2006; 169: 2127-2136Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar The amount of internalized exovesicles observed was higher in the early hours (1 hour) and decreased over 24 hours, probably due to a process of degradation and recycling of membrane proteins and lipids. At first, to find out to what extent this internalization is due to an active process, ECs were treated with 10 μg/ml cytochalasin D, a fungal metabolite known to block phagocytosis through the depolymerization of the actin filament network. Cytochalasin D inhibited most of the internalization of exovesicles (data not shown). However, some exovesicles were still internalized and co-localization voxels were detected. These results suggest that the exovesicles might be internalized through different modes, including phagocytosis. Thus, we analyzed to what extent exovesicles are internalized through receptor-mediated endocytosis.Figure 3Incorporation of red-labeled exovesicles into green-labeled ECs. Fluorescent signals of DiO-labeled ECs (green) co-cultured with isolated exovesicles from DiI-labeled LPS-treated DCs (red) were investigated with LSM. Exovesicles from LPS-stimulated DCs are internalized by ECs, as shown in the intracellular content of ECs. An important internalization of ECs membrane can be observed co-localizing with exovesicles. Images represent xy- and xz-projections; yellow arrowhead marks the position of projections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exovesicles Are Partially Internalized by ECs through Receptor-Mediated EndocytosisInternalization of exovesicles can be performed through different mechanisms, such as active phagocytosis or using receptor-mediated endocytosis, such as clathrin-coated pits, caveole, or transferrin receptors. After internalization, ligands can be recycled back to the plasma membrane or go to lysosomes or other compartments such as the Golgi apparatus, where lipids can be recycled. To determine whether exovesicles are internalized through a receptor-mediated endocytosis, isolated exovesicles from LPS-stimulated DCs or from resting DCs (labeled in red) were co-cultured with ECs labeled with DiO (green) and transferrin conjugate (blue). Cells were analyzed after 1, 3, and 6 hours and the incorporation of red fluorescence (DC-exovesicles) in green labeled ECs, or the incorporation of the red in blue endosomal compartments were analyzed using the co-localization module in IMARIS (co-localization color purple), as shown in Figure 4A. As shown previously, internalization of exovesicles was observed after 1 hour. Interestingly, some exovesicles fused with the endosomal compartments (purple), but other internalized exovesicles remained in red. When the number of co-localized voxels was analyzed (Figure 4B), an increased fusion with the endosomal compartment in a time-dependent manner was found with exovesicles originating from resting DCs (approximately 50% of internalized exovesicles), whereas endosomal fusion was reduced with exovesicles originating from LPS-DCs. These results could imply either that the turnover of exovesicles from LPS-DC is faster and exovesicles are processed and recycled or destroyed more rapidly, or that exovesicles from LPS-DCs decrease the active uptake of particles by ECs. This latter hypothesis highlights the important role of sorting during the internalization process, showing that exovesicles involved in inflammation processes are able to modulate a particular response once they are internalized.Figure 4Exovesicles from immature dendritic cells are internalized through receptor-mediated endocytosis. A: Volume rendering of ECs membrane DiO-labeled (green), DiI-labeled exovesicles from LPS-stimulated DCs (red) internalized by ECs, and co-localization of exovesicles in endosome compartment labeled with transferrin (purple). B: Diagram showing total voxel amount of the intracellular exovesicles (black bars) and co-localized voxels of exovesicles and transferrin (white bars) at 1, 3, and 6 hours. Results are expressed as means ± SEM. The asterisk represents a statistically significant difference (*P < 0.05) between LPS treated and control groups.View Large" @default.
• W2140697776 created "2016-06-24" @default.
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• W2140697776 date "2009-08-01" @default.
• W2140697776 modified "2023-09-30" @default.
• W2140697776 title "Active Uptake of Dendritic Cell-Derived Exovesicles by Epithelial Cells Induces the Release of Inflammatory Mediators through a TNF-α-Mediated Pathway" @default.
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