Intestinal epithelial cells secrete exosome–like vesicles Guillaume Van Niel, Graça Raposo, Céline Candalh, Muriel Boussac, Robert Hershberg, Nadine Cerf–Bensussan, Martine Heyman  Gastroenterology  Volume 121, Issue 2, Pages 337-349 (August 2001) DOI: 10.1053/gast.2001.26263 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.1 Electron microscopy characterization of the pellets obtained after ultracentrifugation of 48-hour (A) apical and (B) basolateral T84-DRB1*0401/CIITA cell-conditioned media.Single vesicles of 30–90-nm diameter were observed in apical pellets.In contrast, the vesicles derived from the basolateral compartment appeared more aggregated and heterogeneous in shape and size.The same morphologic pattern was observed with the HT29-19A cell line (see Figure 4). Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.2 Quantification of exosome protein release in apical and basolateral compartments of filter-grown T84-DRB1*0401/CIITA and HT29-19A cell monolayers.Results (means ± SD) are expressed as nanograms of exosome proteins released by 106 intestinal cells in 48 hours (n = 3–8 experiments).The release was observed from both the apical and basolateral side of the cells.Total release was increased in the presence of IFN-γ (P < 0.001 and P < 0.07 in T84-DRB1*0401/CIITA and HT29-19A, respectively). Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.3 Flow cytometry analysis of surface molecules expressed on HT29-19A and T84-DRB1*0401/CIITA intestinal cell lines.Cells were cultured for 3 days on tissue culture flasks in the absence (light line) or presence (bold line) of 100 U/mL IFN-γ for 72 hours.Cells were incubated with FITC-labeled anti–MHC class I or PE-labeled anti–MHC class II antibodies and analyzed by flow cytometry.The gate was set up to eliminate nonspecific autofluorescence.Negative isotype-matched control antibodies are indicated by the shaded areas.Note that the constitutive expression of MHC class II molecules on T84-DRB1*0401/ CIITA cells was approximately 10 times higher than that observed in IFN-γ–stimulated HT29-19A cells. Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.4 Expression of antigen-presenting molecules in HT29-19A cell–derived exosomes stimulated or not stimulated with IFNγ.(A) MHC class I molecules were analyzed by Western blot in whole-cell lysates (CL) and apical or basolateral exosomes (ApE and BasE, respectively).Each lane was loaded with 2 μg proteins derived from CL or exosome preparations.MHC class I molecules were expressed in lysates and exosomes in basal conditions, and their expression was highly increased in the presence of IFN-γ.MHC class II molecules were not detectable in basal conditions; their expression was induced in the presence of IFN-γ, and an ~2.5-fold enrichment was observed in BasE compared with CL.MHC class II molecules were detectable with an antibody recognizing the α chain (1B5) under-reducing (30–34-kilodalton form) or nonreducing (α/β complex, 56-kilodalton form) conditions.(B) Invariant chain (Ii) and Na+,K+-ATPase were expressed in CL, but were almost undetectable in ApE and BasE.(C) CD26 (dipeptidyl peptidase IV) and (D) CD63 were also found in both types of exosomes, as also confirmed by immunogold electron microscopy. Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.5 Detection of antigen-presenting molecules in T84-DRB1*0401/CIITA cell–derived exosomes and whole-cell lysates (CL) by Western blot and immunogold electron microscopy.(A) Each lane was loaded with 5 μg of proteins derived either from CL or from exosome preparations.MHC class II molecules were constitutively expressed in CL and in apical (ApE) and basolateral (BasE) exosomes.MHC class II molecules were loaded with peptides because they were detected by the 1B5 antibody recognizing the 56-kilodalton α/β compact form under nonreducing conditions.Densitometric analysis indicated a 7- and 9-fold enrichment in ApE and BasE, respectively.(B) Transferrin receptor and Na+,K+-ATPase were expressed in CL but only weakly in exosomes, suggesting that the latter are not plasma membrane fragments.(C) MHC class I and class II molecules were colocalized in the same vesicles (MHC class I, 10-nm gold particles, arrowhead; MHC class II [anti–HLA-DR α chain], 5-nm gold particles, small arrow).(D) Analysis of exosomes on sucrose gradient confirmed their vesicular nature and their labeling with MHC class II molecules. Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.6 Expression of CD63 and CD26 in T84-DRB1*0401/CIITA cell–derived exosomes.CD63 and CD26 were expressed on apical (ApE) and basolateral (BasE) exosomes.(A) CD63, analyzed under nonreducing conditions by Western blot, was highly enriched in ApE and BasE exosomes compared with cell lysates (CL).CD26, detected by Western blot, was highly enriched in ApE and was also detected by immunogold electron microscopy, but in a lower proportion than in HT29-19A–derived exosomes.CD63 and CD26 sometimes colocalized with MHC class II molecules (double labeling, CD63, 15-nm gold particles, arrowhead; MHC class II, 10-nm gold particles, small arrow).(B) Evaluation of the percentage of exosomes labeled with CD63, CD26, or MHC class II molecules or doubly labeled.Results are expressed as the percentage of total exosomes.#P < 0.0001, *P < 0.002, significantly different from apical exosomes. Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.7 Protein profile of apical and basolateral exosomes as seen by Coomasie blue staining.Twenty micrograms of exosomes were separated on a 10% SDS gel and stained.The major bands were analyzed by trypsin digestion and MALDI-TOF-MS (see Table 1). Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig.8 A model for the molecular structure of epithelial-derived exosomes.Ubiquitously expressed molecules such as enzymes of the intracellular metabolism (pyruvate kinase M2, creatine kinase, α-enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase) and cytoskeleton proteins (actin, tubulin), as well as molecules possibly involved in antigen presentation (MHC class I, MHC class II, CD63), were found in both apical and basolateral exosomes.Apical exosomes also carried molecules involved in apical addressing of endosomes (syntaxin 3, syntaxin-binding protein 2), whereas basolateral exosomes had molecules that might act as adhesion or costimulatory molecules (A33 antigen and epithelial cell surface antigen). Gastroenterology 2001 121, 337-349DOI: (10.1053/gast.2001.26263) Copyright © 2001 American Gastroenterological Association Terms and Conditions