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*
Institut National de la Santé et de la Recherche Médical, Unité 520, Institut Curie, Paris, France;
Commissariat à l’Energie Atomique, Laboratoire de Chimie des Protéines, Grenoble, France;
Department of Biotechnology and Biological Sciences, Second University of Milan, Milan, Italy; and
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 144, Institut Curie, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and intracellular membrane
transport and signaling factors (such as several annexins, rab 7 and
11, rap1B, and syntenin). Importantly, we also identified a novel
category of exosomal proteins related to apoptosis: thioredoxin
peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to
analyze possible structural relationships between exosomes and
microvesicles released by apoptotic cells. We show that although they
both represent secreted populations of membrane vesicles relevant to
immune responses, exosomes and apoptotic vesicles are biochemically and
morphologically distinct. Therefore, in addition to cytokines,
dendritic cells produce a specific population of membrane vesicles,
exosomes, with unique molecular composition and strong
immunostimulating properties. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent functional studies suggested that secreted membranes may indeed
play specific roles in immune responses. Apoptotic blebs are
efficiently phagocytosed by DCs and macrophages through specific
receptors, including
v
3/v5
and CD36 (9). Phagocytosis by macrophages results in
degradation and clearance of apoptotic material, whereas phagocytosis
by DCs results in efficient processing and presentation of Ags
expressed in the apoptotic cell to CD4+ and
CD8+ T lymphocytes (10).
Exosome’s biological functions, on the other hand, are starting to be unraveled. In reticulocytes, secretion of exosomes eliminates proteins that are not necessary for the function of differentiated RBC (11). B lymphocyte-derived exosomes bear abundant MHC class II molecules and stimulate CD4+ T lymphocytes in vitro (3). B lymphocyte-derived exosomes also concentrate high amounts of tetraspanins (i.e., CD63, CD81, CD37, and CD82), a protein family that also accumulates in late endocytic compartments and whose biological functions are unclear (12). Interestingly, recent results show that B lymphocyte-derived exosomes bind selectively to follicular DCs in vivo, suggesting a possible function for exosomes in humoral immune responses (13).
Exosomes produced by DCs bear not only MHC class II molecules, but also MHC class I and CD86, an important T cell costimulatory molecule (4). Tumor peptide-loaded DC-derived exosomes stimulate strong cytotoxic T lymphocyte-mediated anti tumor immune responses in vivo and induce the rejection of established tumors (4). The mechanism of action of exosomes in vivo is poorly understood. Exosomes could stimulate T cells directly, through the MHC-peptide complexes they harbor, or they could be captured by other professional APC, which could then use peptide-loaded MHC molecules, Ags, or peptides present in exosomes to stimulate T cells.
To define the identity and the modes of action of DC-derived exosomes, we recently undertook an analysis of their protein composition (14). Using trypsin digestion and peptide mapping by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, we identified nine major protein components of exosomes. Potentially interesting exosomal components thus identified are hsc70, a heat shock protein with potent immune stimulatory activity, and several membrane-associated proteins with affinity for ligands on other cell membranes that may target exosomes to their effector cells.
Here we have identified a new set of 21 proteins specifically enriched in exosomes, thus establishing an extensive molecular map of DC-derived exosomes. Exosomal proteins include molecules initially described in the endocytic pathway, at the plasma membrane, or in the cytosol, but not in mitochondria, Golgi apparatus, or the ER. Interestingly, four identified proteins play a role in apoptosis. These findings led us to re-examine the possible relationship between exosomes and the plasma membrane, and between exosomes and membranes produced by DCs undergoing apoptosis (apoptotic microvesicles). By direct comparison of the biochemical composition of exosomes, plasma membrane, endocytic compartments, and microvesicles released by apoptotic cells, we provide new evidence of the biochemical similarities between exosomes and endocytic compartments and of the distinct nature of exosomes and membrane microvesicles released by cells undergoing apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The spleen-derived murine DC line D1 (15) was cultured in complete medium: IMDM (Sigma, St. Quentin, France) supplemented with 10% endotoxin-free FCS (Valbiotech, ABCYS, Paris, France) and 30% conditioned medium from J558 (a GM-CSF-secreting plasmacytoma, provided by Dr. D. Gray, Hammersmith Hospital, London, U.K.) (16). Cells were split twice a week in 145-mm non-tissue culture-treated petri dishes (5 x 106 cells/dish).
Apoptosis was induced in D1 cells by UV treatment. On day 3 after passage, culture medium was replaced with 2 ml PBS, and cells in petri dishes were irradiated for 50 s with 2 mJ/cm2/s, using a 6 x 15W TFX-UV table (Vilber-Lourmat, Marne la Vallée, France). Control cells were treated identically, except for UV irradiation. Fresh medium was added, and cells further cultured for up to 24 h.
For exosome production, cells were cultured in complete medium depleted of contaminating vesicles and protein aggregates by overnight centrifugation at 110,000 x g (14). Supernatants were collected either 3 days after passage or 24 h after changing the medium of 3-day-old D1 cells culture. After UV treatment, exosome purification was performed as previously described (3) by three successive centrifugations at 300 x g (5 min), 1,200 x g (20 min), and 10,000 x g (30 min) to pellet cells and debris, followed by centrifugation for 1 h at 110,000 x g. For large scale preparations of exosomes (biochemical analysis), the 1,200 and 10,000 x g centrifugations were replaced by filtration on 0.22 µm to eliminate large debris. As assessed by electron microscopy (EM), Western blotting with known exosomal markers (14), and protein pattern on Coomassie blue-stained acrylamide gel, exosomes obtained this way are quantitatively and qualitatively similar to those obtained after successive centrifugations (P. Véron, unpublished observations).
Protein identification by peptide mass mapping and tandem mass spectrometry
After separation of 50 µg of exosomal proteins on 10 or 15% SDS-PAGE, the Coomassie-stained protein bands were excised from the gel, trypsin digested, and analyzed essentially as previously described (14, 17). Mass spectra of the peptide mixtures were acquired on a Biflex (Bruker-Franzen Analytik, Bremen, Germany) MALDI-TOF mass spectrometer equipped with a gridless delayed extraction. The instrument was operated in the reflector mode. A mass list of peptides was obtained for each protein digest, and the appropriate software was used to identify the proteins (usually MS-FIT: http://prospector.ucsf.edu/ucsfhtml3.2/msfit.htm).
When a protein could not be confidently identified from its peptide mass map, the trypsin digest was extracted with acetonitrile and a 5% formic acid solution. The digest solution and the extracts were then pooled, dried in a vacuum centrifuge, and desalted with ZipTip C18 (Millipore, Bedford, MA) before the nanospray tandem mass spectrometry (MS/MS) analysis (18). A Q-TOF instrument (Micromass, Manchester, U.K.) was used with a Z-Spray ion source working in the nanospray mode. About 3–5 µl of the desalted sample was introduced into a needle (medium sample needle, PROTANA, Odense, Denmark) to run MS and MS/MS experiments. The capillary voltage was set at an average voltage of 1000 V, and the sample cone was set at 50 V. Glufibrinopeptide was used to calibrate the instrument in the MS/MS mode. Amino acid sequences, sequence tags, or peptide ion fragments that could be determined were used to screen the protein databases with dedicated software: Pepfrag (http://prowl1.rockefeller.edu/prowl/pepfragch.html), peptide search (http://www.mann.embl-heidelberg.de/Services/PeptideSearch/PeptideSearchIntro.html), or BLAST for homology searches (http://www.ncbi.nlm.nih.gov/blast/blast.cgi).
Antibodies
The Abs used were: for FACS analysis: FITC-conjugated anti mouse
CD11b (M1/70 clone), CD86 (GL1), H-2Kb (AF6-88.5
clone), I-Ab (AF6-120.1 clone), CD11c (HL3), and
the corresponding FITC-conjugated isotype-matched controls rat IgG2b,
mouse IgG2a, and hamster IgG (all from PharMingen, San Diego, CA), and
rat anti-mouse Fc
R type II/III (2.4G2 clone), followed by
FITC-conjugated donkey anti-rat IgG (Jackson ImmunoResearch, West
Grove, PA); for FACS analysis and Western blotting: rat anti-mouse
CD9 (KMC8 clone) and Lamp2 (ABL-93 clone), both from PharMingen,
followed by FITC-conjugated donkey anti-rat IgG (for FACS, Jackson
ImmunoResearch) or HRP-conjugated donkey anti-rat IgG (for Western
blotting, Pierce, Rockford, IL); and for Western blotting:
rabbit antisera anti-mouse MHC class II -chain C terminus
(14), anti-FcR type II/III (provided by Dr. C.
Sautes, Institut National de la Santé et de la Recherche
Médical Centre, Unit 255, Paris, France), and anti-AIP1/Alix
(provided by R. Sadoul, Centre Hospitalier Universitaire, Grenoble,
France), followed by HRP-conjugated donkey anti-rabbit IgG
(Pierce).
FACS analysis of cells and exosomes
For FACS analysis, 30 µg of exosomes (or 30 of µg FCS proteins for negative control) were incubated with 10 µl of 4-µm diameter aldehyde/sulfate latex beads (Interfacial Dynamics, Portland, OR) for 15 min at room temperature in a 30–100 µl final volume, followed by 2 h with gentle shaking in 1 ml PBS. The reaction was stopped by incubation for 30 min in 100 mM glycine. Exosome- or FCS-coated beads were washed three times in FACS wash (3% FCS and 0.1% NaN3 in PBS) and resuspended in 500 µl FACS wash. In parallel, D1 cells were washed twice in FACS wash. Cells (105) or 10 µl coated beads were incubated for 1 h with each primary Ab, followed when necessary by incubation in FITC-conjugated secondary Ab, washed, and analyzed on a FACSCalibur (Becton Dickinson, San Diego, CA).
Detection of apoptosis, as evidenced by annexin V binding to phosphatidylserine exposed at the cell surface, was performed by FACS using the Early Apoptosis Detection Kit (Kamiya Biomedical, Seattle, WA). At various times after UV irradiation, cells were flushed from the tissue culture dish, washed once, and resuspended in 500 µl binding buffer. Cells were incubated for 5 min in the dark with 0.25 µg/ml FITC-labeled annexin V and analyzed on a FACSCalibur (Becton Dickinson) immediately after addition of 0.25 µg/ml propidium iodide (PI).
Subcellular fractionation
Subcellular fractionation of D1 cells was performed as
previously described (14), on a free flow electrophoresis
(FFE) chamber (Dr. Werber, Ismaning, Germany). Fractions were
collected, pooled pairwise, and analyzed for protein content (Bradford
assay; Bio-Rad, Hercules, CA) and -hexosaminidase activity
(19). Fifteen pools of fractions, within 10 fractions of
the protein and -hexosaminidase activity peaks, were kept for
further analysis. They were centrifuged at 10,000 x g
for 1 h, and the pellets were resuspended in SDS-sample buffer
with or without (for CD9 detection) 100 mM DTT and run on SDS-PAGE for
Western blot analysis. Alternatively, eight fractions corresponding to
the -hexosaminidase activity peak were pooled and centrifuged for
1 h at 10,000 x g, and the pellet was loaded on
12% SDS-PAGE for Coomassie blue staining and protein analysis by
trypsin digestion and MALDI-TOF mass spectrometry.
Western blotting
The same amount of proteins, as measured by Bradford assay, from control and UV-treated cells and pellets of the successive centrifugations were separated on 12% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore), and incubated with specific Abs, followed by HRP-conjugated secondary Abs, detected using an enhanced chemiluminescence kit (Roche Diagnostics, Meylan, France).
Sucrose gradient
Floatation of vesicles released by apoptotic cells on a continuous sucrose gradient was performed as described for exosomes (3, 14). Fractions of the gradient (1 ml each) were diluted in 2 ml PBS, centrifuged for 1 h at 100,000 x g, separated on a 12% SDS gel, and stained with Coomassie brilliant blue.
Electron microscopy
For EM observation of whole mounts of exosomes or apoptotic vesicles, pellets obtained after 110,000 x g centrifugation were fixed in 2% paraformaldehyde, loaded on Formwar/carbon-coated EM grids, postfixed in 1% glutaraldehyde, and contrasted successively in 2% uranyl acetate, pH 7, and 2% methylcellulose/0.4% uranyl acetate, pH 4. Observations were made with a CM20 Twin Phillips electron microscope (Phillips Electronic Instruments, Mahway, NJ).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proteins identified to date in DC-derived exosomes are the
major components of these vesicles and consist mostly of
membrane-associated proteins (4, 14). The proposed model
for exosome biogenesis, however, predicts that a small amount of
cytosol is trapped inside exosomes. To further define their molecular
identity, 50 µg of exosomes from a growth factor-dependent DC line
(14, 15) were loaded on either 10 or 15% SDS gels (Fig. 1
). All bands obtained were subjected to
trypsin digestion and peptide mass mapping by MALDI-TOF mass
spectrometry as previously described (14, 17), followed by
tandem mass spectrometry (MS/MS) when necessary (18).
Bands A–H in Fig. 1 correspond to major exosomal proteins identified
previously (14), whereas numbers correspond to newly
identified proteins. Table I gives a
summary of the proteins identified.
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-chain (also called CR3 or
CD18) had been coimmunoprecipitated with its counterpart (also
called CD11b) from radiolabeled exosomes (14). Western
blotting for various heat shock proteins had shown the presence of
hsp84 in exosomes (14).
Whereas many of the major exosomal proteins identified before are
transmembrane or peripherally associated with membranes
(14), most of the newly identified proteins are cytosolic:
cytoskeleton and cytoskeleton-binding proteins (tubulin, actin,
cofilin, profilin I, elongation factor-1), membrane-associated
proteins involved in intracellular transport (annexins I, II, IV, V,
and VII; small GTPase family members or related proteins: rab7, rab11,
rap1B, and rab GDP dissociation inhibitor), or cytosolic proteins
involved in signal transduction (Gi2,
syntenin, and 14-3-3) or in protein translation (elongation factor-1
and elongation initiation factor-4A). Importantly, several of the newly
identified proteins are related to apoptosis, either as markers
specifically released by cells undergoing apoptosis (histones H2A–H4),
or as putative pro- or anti-apoptosis factors (respectively,
AIP1/Alix, thioredoxin peroxidase II (TPxII), 14-3-3, and
galectin-3).
This analysis therefore provides an extended set of proteins
specifically targeted to exosomes in DCs. A schematic representation of
DC-derived exosomes, as observed in this and our previous study, is
given in Fig. 2.
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Consistent with the proposed late endosomal origin of exosomes
(3, 4, 5), several proteins identified in these vesicles are
associated with endosomes and lysosomes: annexin II (20),
Gi2 (18), hsc73
(21), MHC class II (22), MHC class I
(4), and CD86 (23). This is not the case,
however, for two of the major transmembrane proteins, CD9 and Mac-1,
which have to date only been described at the cell surface.
To determine whether these two molecules are also present in endocytic
compartments, endosomes and lysosomes of D1 cells were purified by FFE
as previously described (14). As shown in Fig. 3A, membrane fractions
deviated toward the anode of the electrophoresis chamber contain a very
small fraction of the total proteins (fractions 30–35, proteins curve,
lower panel). Most of the -hexosaminidase (a lysosomal
enzyme) activity (betaHex curve, lower panel), but no
ER-resident proteins (14), are present in these fractions,
which therefore represent an enriched population of endosomes and
lysosomes.
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A,
upper panels) showed that MHC class II and CD9 are present
in the endocytic fractions. The presence in these fractions of Mac-1,
for which no Ab working in Western blotting is available, was revealed
by SDS-PAGE of pooled lysosomal fractions (Fig. 3B), and
peptide mass mapping of two bands running at 175 and 90 kDa, which
corresponded, respectively, to the - and -chains of Mac-1.
Another major exosome protein, milk fat globule-EGF factor 8
(MFG-E8)/lactadherin, was also found in purified endocytic compartments
by peptide mass mapping (not shown).
Conversely, if exosomes are derived from endocytic compartments,
certain membrane proteins expressed at the cell surface should be
absent from exosomes. To examine surface expression of membrane
proteins in cells and exosomes, we set up a FACS assay for exosomes.
Exosomes were covalently linked to 3- to 4-µm aldehyde-activated
latex beads, and the presence of membrane proteins was revealed by
immunofluorescence. As expected from our proteomic analysis, MHC class
I and II, CD86, CD9, and Mac-1 can be readily detected using this
assay, as efficiently as on live cells (Fig. 4A). Importantly, Lamp-2, a
lysosomal resident not expressed at the plasma membrane, was also
readily detected on exosomes (Fig. 4, B and C).
Furthermore, abundant plasma membrane proteins, such as type I (data
not shown) and types II/III Fc receptor (FcR II/III), were not
detected in exosomes (Fig. 4, B and C).
Enrichment of Lamp2 and absence of FcR II/III in exosomes were also
confirmed by Western blot (Fig. 4C). Consistent with their
absence in exosomes, FcR II/III were not detected in endocytic
compartments (Fig. 3A, upper panel).
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Comparison of exosomes and vesicles released by cells undergoing apoptosis
Another striking observation from our proteomic analysis is that exosomes contain proteins associated with apoptosis: histones are released by apoptotic cells as chromatin fragments (24), AIP-1/Alix, a protein of unknown function, interacts with the proapoptosis protein ALG-2 (25, 26), and TPxII (27, 28), 14-3-3 (29), and galectin-3 (30) can protect cells from apoptosis. Since apoptotic cells are known to release membrane microvesicles in vitro, it was important to determine whether exosome production is somehow related to apoptosis.
To induce apoptosis, D1 cells were submitted to UV irradiation and then
cultured for up to 24 h in fresh culture medium. At different
times after irradiation, cells were collected, stained with
FITC-labeled annexin V, an early marker of apoptosis, and PI, a DNA
intercalating compound that only stains cells with permeabilized
membrane (i.e., necrotic cells). FACS analysis (Fig. 5) shows that between 3 and 6 h
after UV treatment, cells transiently enter an early apoptosis stage
characterized by annexin V staining and low PI staining
(lower right quadrant in Fig. 5 contains 4% of cells
at 3 h, 10% at 6 h, and 18% at 9 h). At later time
points, cells undergo secondary necrosis, characterized by both annexin
V and PI staining (upper right quadrant contains 32% of
cells at 3 h, 58% at 6 h, 71% at 9 h, and 97% at
24 h).
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A).
Thirty or 10 µg of proteins from whole cells or from the three
successive pellets (1,200, 10,000, and 110,000 x g)
were loaded on 10% SDS-PAGE and subjected to Western blotting using
Abs specific for MHC class II, a major exosomal protein, or for the
apoptosis-related protein AIP-1/Alix. As expected, MHC class II
molecules are abundant in exosomes (i.e., 110,000 x g
pellet from control cells; Fig. 6B), but are hardly detected
in the 1,200 and 10,000 x g pellets of control cells.
Apoptotic cells, on the other hand (Fig. 6B, +UV), release
abundant MHC class II molecules associated to both low speed (1,200 and
10,000 x g) and high speed (110,000 x
g) pellets. Confirming our proteomic analysis, we detected
AIP-1/Alix by Western blot in the 110,000 x g pellet
from both control and UV-treated cells, where it is roughly 3 times
more concentrated than in the cells (Fig. 6B). Alix is also
present in the 1,200 x g pellet from UV-treated cells,
but not from control cells (Fig. 6B, 1,200 x
g). This pellet probably represents large membrane blebs
released by apoptotic cells, whereas the 110,000 x g
pellet contains smaller vesicles.
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C
shows that the protein patterns of the control and apoptotic
microvesicles are distinct; whereas the 110,000 x g
pellet produced by non-UV-treated cells contains the typical exosomal
major proteins (bands B–G, Fig. 6C), the same
pellet from apoptotic cells also contains major proteins that are
absent from exosomes (bands 1–4, Fig. 6C).
The four major bands running around 20 kDa (no. 1–4) in apoptotic
110,000 x g pellet were trypsin-digested and
identified by peptide mass mapping (Table II); they correspond to four types of
mammalian histones, known to be released as complexes with DNA
(chromatin) by cells undergoing apoptosis (24, 31).
Interestingly, these histones have been identified in the proteomic
analysis of control exosomes (Table I). Fig. 6C, however,
shows that they are present at hardly detectable levels when 30 µg of
total proteins from control exosomes are run on SDS-PAGE (50 µg had
been used for the analysis in Table I). This suggests that exosome
preparations may contain some material coming from the few cells
undergoing spontaneous apoptosis in the culture, but that this material
is scarce compared with exosomes produced by live cells. In contrast,
vesicles produced by apoptotic cells contain some regular exosomes,
most likely produced by the cells before they enter apoptosis, plus a
large proportion of histone-containing material. Interestingly, as
shown in Fig. 6D, membranes secreted by apoptotic cells
contain two different populations of vesicles characterized by
different densities on a sucrose gradient. Histones are associated to
membranes that float at a density of 1.24–1.28 g/ml (bands 1, 3, and 4
in Fig. 6D), whereas most other proteins are associated with
membranes floating at a density of 1.18 g/ml. This latter density is
slightly higher than the usual exosomal density (1.15 g/ml) (3, 12, 14). Finally, observation by EM of the material obtained
showed that apoptotic vesicles (Fig. 7B) are much larger and denser
and do not present the characteristic cup shape of exosomes (Fig. 7A), making them easily distinguishable.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unexpectedly, our proteomic analysis revealed a novel category of exosomal proteins, composed of several molecules implicated in apoptosis. This observation led us to explore possible structural relationships between exosomes and apoptotic blebs. Indeed, both exosomes from DCs (4) and apoptotic blebs and bodies from monocytes (10) have been shown to induce immune responses mediated by DCs. Consistent with previous observations showing that apoptotic cells release membrane particles (7), we obtained larger amounts of membrane-bound material from dying than from healthy cells. UV-treated cells release particularly abundant large membrane particles, probably corresponding to apoptotic corpses. In addition, we also purified, from apoptotic cell supernatants, smaller membrane particles that pellet only at high speed, like exosomes from healthy cells. These small vesicles from apoptotic cells are distinct from exosomes, since they contain very abundant histones associated with membranes floating at a high sucrose density (1.24–1.28 g/ml), and they are very heterogeneous in size and morphology when observed by EM. In exosomal preparations from healthy, non-UV-treated cells, some large dense vesicles can occasionally be observed by EM, and some histones can be detected, but they represent a very minor subset of vesicles, most likely resulting from the small number of apoptotic cells present in the culture. Exosomes, as defined by their protein composition (no ER- or nuclear-resident proteins), their density on sucrose gradient (1.15–1.18 g/ml), and their cup-shaped morphology in EM, are therefore a defined subcellular compartment released by living DCs as a physiological process.
Therefore, exosomes and apoptotic blebs are different in nature. It is
most likely that the small amount of histones found in exosome
preparations come from some apoptotic material present in the
preparations. This is probably also true for the nuclear and
Golgi-associated protein, the transcription factor tumor susceptibility
protein (32), and the translation initiation factor
elongation initiation factor-4A, which is normally associated with
ribosomes. The other apoptosis-related proteins identified in exosomes,
however, are most likely real exosome components. AIP-1/Alix is a
cytosolic and membrane-associated protein binding to the proapoptosis
factor ALG-2 in the presence of Ca2+ (25, 26). We confirmed here by Western blot (Fig. 6B) that
AIP-1/Alix is abundant in exosomes, and, most importantly, that, even
if it is also present in vesicles produced by apoptotic cells, it is
not as enriched therein. The presence of Alix in exosomes could be
related to its reported association with internal membranes
(26) and phagosomes (18). It will be
interesting to analyze precisely the intracellular compartments with
which AIP-1/Alix associates and their relation to late endosomes and
lysosomes from which exosomes originate. AIP-1/Alix is homologous to
yeast and fungus genes participating in signal transduction pathways
(25, 26); it may therefore have other functions, unrelated
to its binding to the pro-apoptotic molecule ALG-2, important for
exosome’s biology. The other proteins found in exosomes and related to
apoptosis have antiapoptotic activities. TPxII (27, 28)
and galectin-3 (30) protect cells against oxidative
damages, and 14-3-3 inhibits the proapoptosis effect of the protein Bad
(29). The presence in exosomes of these apoptosis-related
proteins suggests unexpected structural and/or functional relationships
between the endocytic pathway and the apoptotic process, which remain
to be defined.
Our results also strongly support the previous model of exosome
biogenesis in the endocytic pathway. Indeed, most exosomal compounds
have been previously shown, or are shown herein, to be present in or
associated with endosomes and lysosomes. This is true for membrane
proteins, such as tetraspanins (Ref. 2 and this study),
Lamp2, MHC molecules (22), or Mac-1 (this study), and for
cytosolic proteins, such as hsc73 (21), syntenin
(33), rab7 (34), rab11 (35),
rap1B (36), and several annexins (37). Both
actin and tubulin interact with endosomes and/or lysosomes
(38), and it is therefore not surprising that we also
found several actin-binding proteins associated with exosomes: cofilin
(39), profilin I (40), and elongation
factor-1 (41). Interestingly, cofilin promotes actin
depolymerization (42), which may, in turn, induce membrane
invagination at the plasma membrane (43). A similar actin
depolymerization/invagination-coupled process may also be involved in
the formation of exosomes from the limiting membrane of late endosomes.
Importantly, several proteins identified in exosomes were previously
reported by some of us (J. Garin) to be present in macrophage
phagosomes: Gi2, galectin 3, 14-3-3, Alix,
syntenin, rab7, rab11, rap1-B, annexin V, hsc70, hsp84, and
MFG-E8/lactadherin (18).
Several proteins identified in exosomes play roles in different signal transduction pathways. The many isoforms of 14-3-3, four of which are present in exosomes, are ligands for various intracellular proteins, especially serine-phosphorylated transmembrane receptors or actors of signaling cascades (44). Syntenin also functions as an adaptor molecule between transmembrane receptors and signaling pathways (45). The signal transduction factors that accumulate in exosomes are most likely involved either in endocytic transport through late endosomes or in the biogenesis of internal vesicles within multivesicular endosomes.
Another category of exosome-associated proteins are those involved in membrane traffic. Annexins bind to intracellular membranes and are generally involved in intracellular membrane fusion (46). Association of annexins with exosomes could be a consequence of the presence of phosphatidylserine in these vesicles (P. Véron, unpublished observation). The small GTP-binding protein rab7 associates with endosomes upon GTP binding, and the cycle between GDP-bound cytosolic and GTP-bound membrane-associated forms of rab proteins is regulated by the GDP-dissociating inhibitor rabGDI (47). Association of rab7 to late endosomes is necessary for fusion with lysosomes (48). Rap1B is also a late endosome-associated GTP-binding protein (36), but its role and the compartments it regulates are not yet known (49). All these proteins could be involved in budding of vesicles from the external membrane of the multivesicular endosome to form the exosomes and/or in fusion of these compartments with the plasma membrane that result in exosome secretion. Interestingly, two cytosolic proteins found in exosomes have been described in the extracellular environment: galectin-3, which modulates cell interaction with laminin (50, 51), and annexin II (52). These proteins do not bear a signal sequence responsible for secretion through the constitutive pathway; it would therefore be interesting to determine whether exosomes represent an unconventional secretion pathway for some proteins (53).
Besides proteins potentially implicated in the process of exosome
formation, we have also evidenced many proteins that may be involved in
the biological functions of exosomes. Several proteins exposed at the
surface of exosomes bind ligands on other membranes: adhesion molecules
ICAM-1 and -2 for Mac-1 (54), integrins
v3 and
v5 for
MFG-E8/lactadherin (55, 56), and an EGF-like growth factor
receptor for CD9 (57, 58). CD9 also has an essential role
in sperm-oocyte docking and/or fusion during fecundation (59, 60). These proteins could be involved in exosome targeting,
docking, and/or fusion with other cells. Exosomes could thus represent
a new way of communication, i.e., exchange of antigenic information,
between cells in the immune system. This idea is consistent with recent
reports showing that exchange of membranes bearing MHC-peptide
complexes occurs between APCs (61) or between APCs and T
cells (62). It could also account for older observations
that described shedding of membrane vesicles from spleen
(63) or tumor cells (64), giving rise to
antigenic material, or soluble MHC molecules in the serum of transplant
patients (65).
Thus, besides direct cell-cell contact and the secretion of soluble proteins, exosomes could represent an additional means of communication between cells of the immune system. Exosomes could deliver integrated signals through different surface receptors on target cells and, if exosomes fuse with acceptor cells, they could also transfer membrane and cytosolic proteins between different cells. In vivo, exosomes have been evidenced in tonsil B follicles (13) or in serum (our unpublished observations). The cellular source of these exosomes, however, is probably heterogeneous, and formal demonstration that DCs secrete exosomes in vivo awaits further analyses.
Finally, although a physiological role for exosomes has yet to be demonstrated, their use in tumor immunotherapy is currently being implemented. This study should also allow to improve exosome-based immunotherapy strategies and help in defining new vaccination strategies.
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Acknowledgments |
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Footnotes |
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2 C.T. and M.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Clotilde Théry, Institut National de la Santé et de la Recherche Médical, Unité 520, Institut Curie, 12 rue Lhomond, 75005 Paris, France. E-mail address: clotilde.thery{at}curie.fr
4 Abbreviations used in this paper: DC, dendritic cell; FFE, free flow electrophoresis; ER, endoplasmic reticulum; MALDI-TOF, matrix-assisted laser desorption-ionization-time of flight; TPxII, thioredoxin peroxidase II; EM, electron microscopy; PI, propidium iodide; MS/MS, tandem mass spectrometry; MFG-E8, milk fat globule-EGF factor 8; EF, elongation factor.
Received for publication February 1, 2001. Accepted for publication April 9, 2001.
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References |
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v5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.: implication in Rho1p-regulated reorganization of the actin cytoskeleton in Saccharomyces cerevisiae. Oncogene 16:2011.[Medline]
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31 at cell-cell contact sites. J. Cell Biol. 129:1691.This article has been cited by other articles:
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R. Gastpar, M. Gehrmann, M. A. Bausero, A. Asea, C. Gross, J. A. Schroeder, and G. Multhoff Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells Cancer Res., June 15, 2005; 65(12): 5238 - 5247. [Abstract] [Full Text] [PDF]
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F. Bianco, E. Pravettoni, A. Colombo, U. Schenk, T. Moller, M. Matteoli, and C. Verderio Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1{beta} Release from Microglia J. Immunol., June 1, 2005; 174(11): 7268 - 7277. [Abstract] [Full Text] [PDF]
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S.-H. Kim, E. R. Lechman, N. Bianco, R. Menon, A. Keravala, J. Nash, Z. Mi, S. C. Watkins, A. Gambotto, and P. D. Robbins Exosomes Derived from IL-10-Treated Dendritic Cells Can Suppress Inflammation and Collagen-Induced Arthritis J. Immunol., May 15, 2005; 174(10): 6440 - 6448. [Abstract] [Full Text] [PDF]
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J. Roy, G. Martin, J.-F. Giguere, D. Belanger, M. Petrin, and M. J. Tremblay HIV Type 1 Can Act as an APC upon Acquisition from the Host Cell of Peptide-Loaded HLA-DR and CD86 Molecules J. Immunol., April 15, 2005; 174(8): 4779 - 4788. [Abstract] [Full Text] [PDF]
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J. Sun, M. Walsh, A. V. Villarino, L. Cervi, C. A. Hunter, Y. Choi, and E. J. Pearce TLR Ligands Can Activate Dendritic Cells to Provide a MyD88-Dependent Negative Signal for Th2 Cell Development J. Immunol., January 15, 2005; 174(2): 742 - 751. [Abstract] [Full Text] [PDF]
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A. E. Morelli, A. T. Larregina, W. J. Shufesky, M. L. G. Sullivan, D. B. Stolz, G. D. Papworth, A. F. Zahorchak, A. J. Logar, Z. Wang, S. C. Watkins, et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells Blood, November 15, 2004; 104(10): 3257 - 3266. [Abstract] [Full Text] [PDF]
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N. Amzallag, B. J. Passer, D. Allanic, E. Segura, C. Thery, B. Goud, R. Amson, and A. Telerman TSAP6 Facilitates the Secretion of Translationally Controlled Tumor Protein/Histamine-releasing Factor via a Nonclassical Pathway J. Biol. Chem., October 29, 2004; 279(44): 46104 - 46112. [Abstract] [Full Text] [PDF]
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T. Pisitkun, R.-F. Shen, and M. A. Knepper Identification and proteomic profiling of exosomes in human urine PNAS, September 7, 2004; 101(36): 13368 - 13373. [Abstract] [Full Text] [PDF]
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M. D. Stuchell, J. E. Garrus, B. Muller, K. M. Stray, S. Ghaffarian, R. McKinnon, H.-G. Krausslich, S. G. Morham, and W. I. Sundquist The Human Endosomal Sorting Complex Required for Transport (ESCRT-I) and Its Role in HIV-1 Budding J. Biol. Chem., August 20, 2004; 279(34): 36059 - 36071. [Abstract] [Full Text] [PDF]
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A. R. Mantegazza, M. M. Barrio, S. Moutel, L. Bover, M. Weck, P. Brossart, J.-L. Teillaud, and J. Mordoh CD63 tetraspanin slows down cell migration and translocates to the endosomal-lysosomal-MIICs route after extracellular stimuli in human immature dendritic cells Blood, August 15, 2004; 104(4): 1183 - 1190. [Abstract] [Full Text] [PDF]
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F. Aline, D. Bout, S. Amigorena, P. Roingeard, and I. Dimier-Poisson Toxoplasma gondii Antigen-Pulsed-Dendritic Cell-Derived Exosomes Induce a Protective Immune Response against T. gondii Infection Infect. Immun., July 1, 2004; 72(7): 4127 - 4137. [Abstract] [Full Text] [PDF]
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B. Fevrier, D. Vilette, F. Archer, D. Loew, W. Faigle, M. Vidal, H. Laude, and G. Raposo Cells release prions in association with exosomes PNAS, June 29, 2004; 101(26): 9683 - 9688. [Abstract] [Full Text] [PDF]
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M. Sachse, G. J. Strous, and J. Klumperman ATPase-deficient hVPS4 impairs formation of internal endosomal vesicles and stabilizes bilayered clathrin coats on endosomal vacuoles J. Cell Sci., May 1, 2004; 117(9): 1699 - 1708. [Abstract] [Full Text] [PDF]
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J. P.J.J. Hegmans, M. P.L. Bard, A. Hemmes, T. M. Luider, M. J. Kleijmeer, J.-B. Prins, L. Zitvogel, S. A. Burgers, H. C. Hoogsteden, and B. N. Lambrecht Proteomic Analysis of Exosomes Secreted by Human Mesothelioma Cells Am. J. Pathol., May 1, 2004; 164(5): 1807 - 1815. [Abstract] [Full Text] [PDF]
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J. A. Coppinger, G. Cagney, S. Toomey, T. Kislinger, O. Belton, J. P. McRedmond, D. J. Cahill, A. Emili, D. J. Fitzgerald, and P. B. Maguire Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions Blood, March 15, 2004; 103(6): 2096 - 2104. [Abstract] [Full Text] [PDF]
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A. W. Purcell and J. J. Gorman Immunoproteomics: Mass Spectrometry-based Methods to Study the Targets of the Immune Response Mol. Cell. Proteomics, March 1, 2004; 3(3): 193 - 208. [Abstract] [Full Text] [PDF]
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F. Andre, N. Chaput, N. E. C. Schartz, C. Flament, N. Aubert, J. Bernard, F. Lemonnier, G. Raposo, B. Escudier, D.-H. Hsu, et al. 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., February 15, 2004; 172(4): 2126 - 2136. [Abstract] [Full Text] [PDF]
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G. Greicius, L. Westerberg, E. J. Davey, E. Buentke, A. Scheynius, J. Thyberg, and E. Severinson Microvilli structures on B lymphocytes: inducible functional domains? Int. Immunol., February 1, 2004; 16(2): 353 - 364. [Abstract] [Full Text] [PDF]
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Y. Trioulier, S. Torch, B. Blot, N. Cristina, C. Chatellard-Causse, J.-M. Verna, and R. Sadoul Alix, a Protein Regulating Endosomal Trafficking, Is Involved in Neuronal Death J. Biol. Chem., January 16, 2004; 279(3): 2046 - 2052. [Abstract] [Full Text] [PDF]
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Y. Yan, L. Phan, F. Yang, M. Talpaz, Y. Yang, Z. Xiong, B. Ng, N. A. Timchenko, C. J. Wu, J. Ritz, et al. A Novel Mechanism of Alternative Promoter and Splicing Regulates the Epitope Generation of Tumor Antigen CML66-L J. Immunol., January 1, 2004; 172(1): 651 - 660. [Abstract] [Full Text] [PDF]
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S. Taverna, G. Ghersi, A. Ginestra, S. Rigogliuso, S. Pecorella, G. Alaimo, F. Saladino, V. Dolo, P. Dell'Era, A. Pavan, et al. Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells J. Biol. Chem., December 19, 2003; 278(51): 51911 - 51919. [Abstract] [Full Text] [PDF]
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A. de Gassart, C. Geminard, B. Fevrier, G. Raposo, and M. Vidal Lipid raft-associated protein sorting in exosomes Blood, December 15, 2003; 102(13): 4336 - 4344. [Abstract] [Full Text] [PDF]
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E. Nikko, A.-M. Marini, and B. Andre Permease Recycling and Ubiquitination Status Reveal a Particular Role for Bro1 in the Multivesicular Body Pathway J. Biol. Chem., December 12, 2003; 278(50): 50732 - 50743. [Abstract] [Full Text] [PDF]
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K. Katoh, H. Shibata, H. Suzuki, A. Nara, K. Ishidoh, E. Kominami, T. Yoshimori, and M. Maki The ALG-2-interacting Protein Alix Associates with CHMP4b, a Human Homologue of Yeast Snf7 That Is Involved in Multivesicular Body Sorting J. Biol. Chem., October 3, 2003; 278(40): 39104 - 39113. [Abstract] [Full Text] [PDF]
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S. J. Gould, A. M. Booth, and J. E. K. Hildreth The Trojan exosome hypothesis PNAS, September 16, 2003; 100(19): 10592 - 10597. [Abstract] [Full Text] [PDF]
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R. Wubbolts, R. S. Leckie, P. T. M. Veenhuizen, G. Schwarzmann, W. Mobius, J. Hoernschemeyer, J.-W. Slot, H. J. Geuze, and W. Stoorvogel Proteomic and Biochemical Analyses of Human B Cell-derived Exosomes. POTENTIAL IMPLICATIONS FOR THEIR FUNCTION AND MULTIVESICULAR BODY FORMATION J. Biol. Chem., March 21, 2003; 278(13): 10963 - 10972. [Abstract] [Full Text] [PDF]
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I. D. Davis, M. Jefford, P. Parente, and J. Cebon Rational approaches to human cancer immunotherapy J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29. [Abstract] [Full Text] [PDF]
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A. R. Bateman, K. J. Harrington, T. Kottke, A. Ahmed, A. A. Melcher, M. J. Gough, E. Linardakis, D. Riddle, A. Dietz, C. M. Lohse, et al. Viral Fusogenic Membrane Glycoproteins Kill Solid Tumor Cells by Nonapoptotic Mechanisms That Promote Cross Presentation of Tumor Antigens by Dendritic Cells Cancer Res., November 15, 2002; 62(22): 6566 - 6578. [Abstract] [Full Text] [PDF]
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B. Fritzsching, B. Schwer, J. Kartenbeck, A. Pedal, V. Horejsi, and M. Ott Release and Intercellular Transfer of Cell Surface CD81 Via Microparticles J. Immunol., November 15, 2002; 169(10): 5531 - 5537. [Abstract] [Full Text] [PDF]
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C. Chatellard-Causse, B. Blot, N. Cristina, S. Torch, M. Missotten, and R. Sadoul Alix (ALG-2-interacting Protein X), a Protein Involved in Apoptosis, Binds to Endophilins and Induces Cytoplasmic Vacuolization J. Biol. Chem., August 2, 2002; 277(32): 29108 - 29115. [Abstract] [Full Text] [PDF]
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H. Vincent-Schneider, P. Stumptner-Cuvelette, D. Lankar, S. Pain, G. Raposo, P. Benaroch, and C. Bonnerot Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells Int. Immunol., July 1, 2002; 14(7): 713 - 722. [Abstract] [Full Text] [PDF]
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K. E. Pace, T. Lebestky, T. Hummel, P. Arnoux, K. Kwan, and L. G. Baum Characterization of a Novel Drosophila melanogaster Galectin. EXPRESSION IN DEVELOPING IMMUNE, NEURAL, AND MUSCLE TISSUES J. Biol. Chem., April 5, 2002; 277(15): 13091 - 13098. [Abstract] [Full Text] [PDF]
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) or apoptotic cells (+UV, 
) were
purified by successive centrifugations at 1,200, 10,000, and
110,000 x g. The amount of proteins obtained in
each pellet in three independent experiments was quantified by Bradford
assay. Dying cells released 2–4 times more material in their culture
medium than healthy cells, and this material consisted of both big
(1,200 x g pellet) and small vesicles (10,000 and
110,000 x g pellets). B, Thirty and
10 µg of proteins from each pellet (cells 1,200, 10,000, and
110,000 x g), from control (-UV) and UV-treated
(+UV) cells were separated on a 10% SDS gel and analyzed by Western
blotting using Abs specific for MHC class II (MHC II) and AIP-1/Alix
(Alix). Exosomes (110,000 x g pellet, -UV) as
well as apoptotic vesicles (110,000 x g pellet,
+UV) are rich in MHC class II molecules. Both contain AIP-1/Alix, which
is roughly 3 times more concentrated than in the cells. For the
intermediate pellets (1,200 and 10,000 x g), only
those produced by apoptotic cells contain abundant MHC class II and
some AIP-1/Alix (1,200 x g, +UV).
C, Thirty micrograms of proteins from the cells or
110,000 x g pellets were separated on 12%
SDS-PAGE, and stained by Coomassie Brilliant Blue. Bands B–G are the
major exosomal proteins shown in Fig. 1