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*
Unité d’Immuno-Allergie, Institut Pasteur, Paris, France;
Institut Jacques Monod, Unité Mixte de Recherche 7592, Paris, France; and
Laboratoire de Chimie Structurale des Macromolécules (Unité de Recherche Associeé 2185, Centre National de la Recherche Scientifique), Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, but
no detectable IL-4. Similar data were obtained by injecting exosomes
into naive mice. In contrast to mast cell lines, a pretreatment with
IL-4 is required for bone marrow-derived mast cells to secrete active
exosomes. Structurally, exosomes were found to harbor immunologically
relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These
findings indicate that mast cells can represent a critical component of
the immunoregulatory network through secreted exosomes that display
mitogenic activity on B and T lymphocytes both in vitro and in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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completely abrogated this
function. With respect to interactions between mast cells and B cells,
human mast and basophilic cell lines as well as purified human lung
mast cells and blood basophils can provide the cell contact signals to
purified B cells to produce IgE (6). This B cell-mast cell
interaction occurred in the absence of T cells and the human cell lines
HMC-1 (mast) and KU812 (basophilic), used as B cell counterparts, both
have been shown to express the ligand for CD40, which is known in
conjunction with IL-4 to be responsible for IgE production.
In a previous study, we provided evidence indicating that mast
cells have a direct effect on B cell stimulation through a T
cell-independent mechanism (7). Mast cell-dependent B cell
activation resulted, within 48 h of incubation, in blast
formation, proliferation, and IgM production. More recently, we have
shown that T cells that cannot be directly activated by mast cells get
stimulated when B cells were present in the coculture. This
Ag-independent stimulation that occurs when mast cells are cocultured
with spleen cells resulted in large clusters of T cell and B cell
blasts as well as IL-2, IL-12, and IFN- production (8).
This mast cell-mediated heterotypic aggregation as well as cytokine
production was completely inhibited by anti-LFA-1 and
anti-ICAM-1 mAb (8). Other investigators have shown
that activated mast cells were able to form heterotypic aggregates with
activated but not with resting T lymphocytes. This coculture, which
resulted in histamine release, was adhesion-dependent because the
addition of anti-LFA-1 and anti-ICAM-1 inhibited the
adhesion-induced mast cell degranulation (9). Based on our
previous demonstration that mast cell supernatant could replace the
cell-to-cell contacts to generate B and T lymphocyte activation, we
sought in the present report to characterize this mast cell mediator.
Combining biochemistry and immunoelectron microscopy methods, we found
that the mast cell-derived B and T lymphocyte stimulatory activity was
constitutively secreted and consisted of membrane vesicles termed
exosomes, originally stored in the mast cell cytoplasmic granules. In
addition to MHC class II molecules, several proteins including adhesion
and costimulatory molecules were found to be associated with exosomes.
A characteristic feature of mast cell-derived exosomes is their
potential to induce in vitro and in vivo lymphocyte activation with
IL-2 and IFN- production, and no detectable IL-4.
These results reveal a previously unrecognized mechanism by which mast cells express their inflammatory and immunoregulatory functions. Mast cells may, in addition to cell-to-cell contacts and cytokine release, use a third intercellular communication vector that consists of secreted exosomes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DBA/2 (6–8 wk old) were purchased from Janvier (Laval, France). STAT6-knockout (KO) and p47phox-KO mice were kindly provided by J. Ihle (St Jude Children’s Hospital, Memphis, TN) and S. Holland (National Institutes of Health, Bethesda, MD), respectively.
Reagents and Abs
BSA was purchased from Sigma (St. Louis, MO). Mouse recombinant
IL-3, IL-4, IFN- and GM-CSF were purchased from Immugenex (Los
Angeles, CA). Unlabeled anti IFN- (clone AN18) and biotinylated
anti-IFN- (clone R46A2) were purchased from PharMingen (San
Francisco, CA). Rat anti-mouse CD80 (clone 1G10) and anti-CD86
(clone 2D10) mAb were obtained from Dr. Gordon Powers (Hoffmann-La
Roche, Nutley, NJ). Rat anti-mouse LFA-1 and anti-ICAM-1 mAbs
were kindly provided by Dr Genevieve Milon (Institut Pasteur, Paris,
France). Mouse anti-mouse I-Ab/d mAb was
prepared from clone 25-9-17S (American Type Culture Collection,
Manassas, VA). Rabbit anti-mouse CD40 and CD40 ligand (CD40L) were
kindly provided by Dr. Elaine K. Thomas (Immunex, Seattle, WA).
Peroxydase-labeled goat anti-rabbit IgG and anti-rat IgG were
purchased from Biosys (Compiegne, France). The peroxydase-labeled
anti-mouse IgG was purchased from Diagnostic Pasteur (Marnes la
Coquette, France).
Mast cell lines
P815 is a mastocytoma cell line from DBA/2 mice. The IL-3-dependent mast cell line MC/9, derived from the A/J x C57Bl/6 F1 mouse, was kindly provided by Dr. T. Hara (DNAX, Palo Alto, CA).
Preparation of BMMC
BMMC from DBA/2, STAT6-KO, and p47phox-KO mice were prepared as described by Razin (10) and modified by us. After 3 wk of culture using RPMI 1640 supplemented with 10% FCS (American Type Culture Collection) and in the presence of 3 U/ml of rIL-3 (Diaclone, Besançon, France), the cells were harvested after 21 days of culture and consisted of 98% pure mast cells as assessed by toluidine blue staining. Consistent with our previous reports, nonspecific esterase staining and immunofluorescence staining for Mac-1, NLDC-145, and B220 cell surface Ag indicated that mast cell preparations were not contaminated with macrophages, dentritic cells, or B cells, respectively. BMMC were cultured for the last 48 h before harvest in the presence of 3 U/ml rIL-3 and 100 U/ml IL-4 in insulin-transferrin-sodium selenite supplement (ITS; Boehringer Mannheim, Indianapolis, IN)-complemented RPMI 1640 in the absence of FCS. Because FCS has been reported to contain exosomes, this culture procedure provides us with supernatants that contain exosomes exclusively from mast cell origin.
BMMC and cell lines were tested for contamination by mycoplasma using a highly sensitive mycoplasma PCR ELISA performed according to the manufacturer procedure (Roche Diagnostics, Meylan, France).
Exosome purification
Exosomes were prepared from the supernatant of 3-wk-old BMMC cultures. During the last 48 h, BMMC were cultured at 3 x 106 cells/ml in IL-3- and IL-4-containing RPMI 1640 supplemented with 1/1000 dilution of ITS in the absence of FCS. Supernatants were then subjected to two successive centrifugations at 300 x g for 5 min and at 1,200 x g for 20 min to eliminate cells and debris, followed by a centrifugation for 2 h at 70,000 x g. Two fractions were obtained: a high-density (pellet) and a low-density (hypodense) fraction. The exosomes concentrated in the pellet were washed twice in a large volume of PBS centrifuged at 70,000 x g for 1.5 h. The amount of exosomal proteins recovered was measured by Bradford assay (Bio-Rad, Richmond, CA). Immunization was conducted by injecting rabbits with 200 µg exosomes in 1 ml PBS emulsified in an equal volume of CFA (Difco, Detroit, MI). Three booster immunizations with the same dose were given at 3-wk intervals in incomplete Freund’s adjuvant, and the rabbits were bled 1 wk after the last injection. All immunizations were administered in multiple 100-µl intradermal and s.c. injections. The purification of the rabbit anti-exosome Abs was conducted by using protein A-SepharoseCL4B immunosorbent column (Pharmacia, Piscataway, NJ). Exosomes were specifically recognized by the rabbit anti-exosome Abs using solid phase ELISA.
Splenocyte activation and IL-2 assay
Spleen cells harvested from DBA/2 mice were incubated at 107 cells/ml for 4 min at 4°C in Gey’s solution, which allowed the lysis of RBC. After two washes, cells were resuspended in RPMI 1640 medium supplemented with 10% FCS. The spleen cell stimulation assay was conducted in flat-bottom 96-, or 24-well plates (Life Technologies, Cergy-Sontoise, France) using RPMI 1640 supplemented with 10% (v/v) FCS. To a final volume of 200 µl/well (5 x 105 splenocytes) or 1 ml/well (3 x 106 splenocytes), mast cells were added at a mast cell/spleen cell ratio of 1/100 previously established as the optimal mast cell concentration for lymphocyte activation. For a strict comparison, unfractionated and fractionated supernatants harvested from a 3 x 106 cells/ml were routinely tested for their activity at 1/100 dilution. Pellets containing purified exosomes were also used at 1/100 dilution after reconstitution to the initial volume before separation. After 48 h of incubation, 50-µl aliquots of supernatants were collected and assayed for IL-2. IL-2 was titrated using the IL-2-dependent CTLL-2 cell line. Supernatants were incubated for 18 h with 104 CTLL-2 cells and 0.25 mCi [methyl-3H]dThd/well was added 8 h before cell harvest. Spleen cell proliferation was measured by thymidine uptake following the same procedure. The results are given as the mean cpm of duplicate cultures.
Detection of IL-4 and IFN-
Flat-bottom 96-well plates (Nunc, Copenhagen, Denmark) were
coated for 2 h at 37°C with anti-IL-4 mAb (Interchim, Ann
Arbor, MI) at 1 µg/ml or with anti-IFN- mAb AN18 at 3 µg/ml.
After 3 washes in 0.1% PBS-Tween 20, plates were saturated for 1
h at 37°C with 0.1% PBS-BSA. After 3 washes, standards and culture
supernatants were then incubated overnight at 4°C. Plates were then
incubated for 2 h at 37°C with 1 µg/ml of biotinylated
anti-IL-4 (Interchim) or with biotinylated anti-IFN- R46A2
mAbs, followed by a further 2-h incubation with streptavidin-peroxydase
conjugate. After washing, O-phenylendiamine (Sigma) was
added and absorbance was determined by OD at 490 nm.
FACS analysis
After 48 h of spleen cell culture in the presence of mast cells or mast cell-derived exosomes, cells were collected and centrifuged at 300 x g for 5 min. Cells were resuspended in 0.5 ml of PBS, kept on ice, and analyzed in a FACScan cytofluorograph (Becton Dickinson, San Jose, CA). Blast cells refer to activated cells exhibiting large size, and the percentage was determined by an arbitrary gate that distinguished resting cells from activated cells. The percentage of blast cells was calculated as follows: the percent of blast cells = (number of large cells/number of total cells) x 100.
Electron microscopy and immunogold labeling
Formvar-coated copper grids were incubated with bacitracin for 2 min. Vesicular preparation (15 µl at 100 µg/ml) were dropped on grids for 10 min and fixed with 0.5% glutaraldehyde (Euromedex, Souffelweyersheim, France) and then washed with ammonium acetate and negatively stained in 1% uranyl acetate (Merck, West Point, PA) for 5 min. Excess stain was removed and the grids were air dried before visualization. For negative stain immunogold labeling, 15 µl of the preparation was placed on formvar-coated nickel grids wet with bacitracin for 2 min and then fixed with 3% paraformaldehyde for 5 min. Grids were washed in PBS, blocked with 0.5% PBS-BSA for 30 min and then incubated with anti-exosomes rabbit Abs (1/200) in 0.1% PBS BSA for 45 min. Grids were washed again and incubated with protein A conjugated to 10-nm gold particles (purchased from Dr. Slot, Medical School, Utrecht University, Utrecht, The Netherlands) diluted in 0.1% PBS-BSA for 45 min at room temperature. Grids were washed with PBS, fixed with 0.5% glutaraldehyde, rinsed with ammonium acetate 0.2 M and then negatively stained with uranyl acetate and examined under a Philips EM410 electron microscope. In addition, normal rabbit Igs were used for immunocytochemistry in control experiments.
Electron microscopy and immunocytochemistry
Mast cells were fixed with 4% formaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Cells were prepared as previously described (11, 12, 13), embedded in 10% gelatin for 30 min on ice and cut into small gelatin blocks that were infiltrated with 2.3 M sucrose and frozen in liquid nitrogen. Ultrathin cryosections were performed in a Leica (Deerfield, IL) Ultracut E ultramicrotome with a model RMC cryochamber attachment. For immunolabeling, the sections were incubated with anti-exosome rabbit Abs diluted in 1/200 in 0.1% PBS BSA, after preincubation in phosphate buffer containing 0.05 M ammonium chloride and 1% BSA. The sections were then incubated with protein A and 10 nm colloidal gold for 45 min. The sections were washed with PBS, fixed with 1% glutaraldehyde (in PBS) for 5 min, rinsed with PBS and then water, and stained with 2% neutral uranyl acetate and 4% aqueous uranyl acetate. The sections were picked up in a mixture of 50% sucrose and 50% methylcellulose as previously described (14) and studied in a Philips (Eindhoven, The Netherlands) EM410 electron microscope.
Detection of Ags associated with exosomes
Flat-bottom 96-well plates (Nunc) were coated overnight at 4°C with 10 µg/ml of exosome solution. After three washes in 0.1% PBS-Tween, plates were saturated for 1 h at 37°C with 0.1% PBS-BSA followed by the addition of different mAbs including anti-LFA-1, anti-ICAM-1, anti-IAb/d, anti-CD80, anti-CD86, anti-CD-40, anti-CD-40L and control Abs from rabbit, mouse, and rat. After 2 h of incubation at 37°C, plates were washed and incubated for 2 h at 37°C with peroxydase-labeled anti-mouse, anti-rabbit, or anti-rat IgG. After washing, O-phenylendiamine (Sigma) was added. Absorbance was determined by OD at 490 nm.
Labeling of cells and exosomes
For starvation, BMMC were incubated at 4 x 106/ml for 1 h at 37°C in cysteine/methionine-free medium supplemented with 0.1% ITS. The cells were then incubated overnight in the same medium containing 10 µCi/ml [35S]methionine/cysteine (Promix35S; ICN Pharmaceuticals, Costa Mesa, CA). The cells were washed and reincubated further for 24 h at 37°C in fresh complete medium. Exosomes were purified from the supernatant and their protein content analyzed by a 5–15% SDS-PAGE. The gel was silver stained, dried, and autoradiographed.
Analysis of exosomal proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS)
Exosome preparation (50 µg) was diluted in reducing sample
buffer and boiled for 5 min. Separation of exosomal proteins was
performed by electrophoresis using a 5–15% SDS-PAGE. For in-gel
digestion, sample preparation is performed as described by Shevchenko
(15). Briefly the band was excised from the gel, washed,
in-gel reduced, S-alkylated with iodoacetamide, and in-gel
digested with bovine trypsin (sequencing grade, Roche Molecular
Biochemicals, Meylan, France) at 37°C overnight. Peptides were
extracted, dried with SpeedVac and resolubilized in 8 µl of 0.1%
trifluoroacetic acid. ZipTips (Millipore, Bedford, MA) were used to
desalt samples. Mass peptide mapping was performed using 1 µl of the
tryptic digest mixture using 
-cyano-4-hydroxy cinnamic acid (Sigma).
The samples were analyzed by MALDI-TOF-MS on a Voyager DE STR
(PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser
(337 nm). The instrument was operated in the delayed extraction mode
and the delay time was 150 ns. Each mass spectrum was an average of 250
laser shots. Calibration was performed with four proteins.
For the database search on NCBInr, monoisotopic masses were assigned, using MS-FIT from Protein Prospector. The parameters were set as follows: no restriction on the isoelectric point of proteins, 100 ppm were allowed as the maximum mass error, mass protein was adapted to each apparent molecular mass ± 25% for each band according to the SDS-PAGE analysis, and one incomplete cleavage per peptide was considered.
Statistical analysis
Results are expressed as the means ± SEM from two to four experiments. The Mann-Whitney U test was used to compare the mean values between different groups, and the significance was set at a p value of <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the optimal conditions for mast cells to induce
lymphocyte activation, mouse BMMC were cultured 19 days in the presence
of IL-3 followed by treatment with different cytokine combinations for
the last 48 h, including IL-4 and IFN-. Mast cells were washed
before their coculture with the spleen cells to eliminate any residual
IL-4 or IFN-, and during this incubation period no cytokine was
added. Lymphocyte activation that occurred after 48 h of culture
at the optimal mast cell/spleen cell ratio of 1/100 gave rise to a
characteristic heterotypic adhesion of B and T lymphocytes (as shown by
specific labeling with anti-B220 and anti-CD4 mAbs,
respectively; data not shown) that underwent blast formation and
proliferation. As shown in Fig. 1
, IL-4
was found to be the only cytokine that was able to prime BMMC before
their coculture with spleen cells to provide them with
lymphocyte-stimulating activity. IL-3 alone, or in combination with
IFN-, or GM-CSF (not shown) did not induce any activity.
Subsequently, in the next experiments and throughout the manuscript the
same experimental set up was used, which consisted of the pretreatment
of BMMC with IL-4 48 h before their coculture with spleen
cells.
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Earlier evidence (7) indicated that physical contact
between mast cells and spleen cells was not required and that mast cell
supernatants were fully active. To isolate the bioactive material,
supernatant from 48-h cultures of IL-4-treated BMMC was subjected to
differential centrifugation. After two centrifugation steps at 300
x g and 1200 x g to eliminate cells and
debris followed by a final 70,000 x g
ultracentrifugation, a dense fraction was pelleted and the resulting
hypodense fractions were separated and tested for their capacity to
induce lymphocyte stimulation. As shown in Fig. 2, most of the activity of the initial
supernatant was recovered into the pellet while the hypodense fraction
lacked activity. This dense fraction was able to induce both blast
formation and proliferation of splenocytes and was as effective as the
initial unfractionated supernatant. Unfractionated supernatants as well
as 70,000 x g pellets obtained from two mast cell
lines, P815 and MC/9, were similarly effective in inducing splenocyte
activation. However, in contrast to BMMC, the lymphocyte-stimulating
activity induced by the two mast cell lines is constitutively expressed
and did not require pretreatment with IL-4 (Fig. 2).
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To characterize the structural features of the biologically active
high-density fraction of mast cell supernatant, the 70,000 x
g pellet was analyzed by negative staining. As shown in Fig. 3, the pellet consisted of a population
of 60- to 100-nm vesicles called exosomes some of them with an
electron-dense core. The term exosome was originally used to describe
small membrane vesicles (50–100 nm of diameter) released by
reticulocytes during their final stage of maturation into RBC
(16). To determine the intracellular localization of
exosomes, we used Abs raised in rabbits immunized with purified
exosomes. The specificity of these Abs was assessed by the lack of
reactivity with exosomes purified from human mast cells. BMMC were
fixed and ultrathin cryosections were immunogold labeled with
anti-exosome Abs coupled to 10-nm gold particles. Fig. 4A shows that almost all gold
particles accumulate into the intracytoplasmic granules, suggesting
that these compartments are the storage site of exosomes. As shown in
Fig. 4B, exosomes appeared at the external leaflet of the
plasma membrane indicating that exosomes may be externalized from mast
cells most likely through an exocytic profile based on the fusion of
the limiting membrane of granules with the plasma membrane. Control
preparations incubated with preimmune rabbit Ig did not show any
immunogold labeling (not shown). Positive labeling of secreted exosomes
demonstrates that these vesicles are similar to those found in the
granules. Based on the protein content of exosomes, we consistently
obtained 0.5–0.8 µg/106 cells of exosomal
proteins from the 48-h culture supernatants of 3 x
106 BMMC/ml. Altogether, these data demonstrate
that the mast cell-dependent lymphocyte-stimulating activity as shown
in Fig. 2
is very likely mediated by intracytoplasmic
granule-associated exosomes, which are constitutively released into the
extracellular medium.
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We have previously reported that mast cells cultured with spleen
cells not only induced B and T lymphocyte proliferation but also
resulted in IL-2 and IFN- production (8). To
investigate whether exosomes purified from BMMC-derived supernatant
were endowed with similar biological activities as BMMC, spleen cells
were incubated in the presence of unfractionated BMMC supernatant
(1/100), exosome-depleted supernatant (hypodense fraction) (1/100),
exosomes (15 ng/ml), and BMMC (at a BMMC/spleen cell ratio of 1/100).
After 48 h of culture, cell proliferation and cytokine production
were used as a read-out for lymphocyte stimulation. As shown in Fig. 5, unfractionated mast cell supernatant
was able to induce lymphocyte proliferation and IL-2 and IFN-
production. More interestingly, the whole biological activity of the
unfractionated supernatant was found to be associated with exosomes,
whereas the exosome-depleted supernatant completely lost its activity.
None of the cultures contained detectable IL-4 as assessed by ELISA
(not shown). These data demonstrate that mouse BMMC constitutively
release exosomes that are endowed with a full biological activity
similar to that obtained by intact mast cells and preferentially induce
Th1-type responses.
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Based on previous demonstration (8) that
IL-4-treated BMMC induce in vivo B and T lymphocyte activation and
cytokine production and that this activity is mediated by exosomes,
mice were injected i.p. with 5 µg, or 0.5 µg of BMMC-derived
exosomes. Control mice were injected with 107
BMMC, 5 x 106 BMMC, or with PBS alone.
Spleen and lymph node cells were harvested 6 days later and cultured
for 48 h after which lymphocyte activation was assessed. Fig. 6 shows that, in addition to a
characteristic heterotypic cell aggregation (data not shown), mice
injected with exosomes displayed cell proliferation and IL-2 and
IFN- production. Data also indicate that exosomes were as potent as
mast cells in inducing lymphocyte activation. These results suggest
that exosome-induced lymphocyte activation in vivo may have physiologic
significance.
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We have previously reported that membrane vesicles or exosomes
present in type I and type II mast cell granules contain
lysosomal-associated protein (lamp)1 and 2 as well as invariant chain
and MHC class II molecules (17). Here, we extended the
investigation to other immunologically relevant molecules. As shown in
Fig. 7, using a sandwich ELISA method, we
demonstrate that, in addition to MHC class II molecules, CD86, CD40,
CD40L, LFA-1, and ICAM-1 molecules were found to be associated with
exosomes. These results may ascribe to exosomes a possible Ag
presenting function or a capacity to transfer this property to
other APCs.
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A), demonstrating that all
the proteins in the exosomes are of mast cell origin. As shown in Fig. 8B, preparative electrophoresis of exosomes displayed about
14 major bands ranging from about 150 to 6 kDa. To identify these
proteins, 10 major bands indicated by arrows in Fig. 8 were excised and
trypsin-digested, and the resulting peptide fragments were analyzed by
MALDI-TOF-MS. The profiles of the generated tryptic peptides were
compared with the theoretical profiles of tryptic digests from known
proteins available in the databases (18). As shown in
Table I, of 10 bands analyzed, 7 proteins
associated with exosomes were identified as follows: CD13, ribosomal
protein S6 kinase, annexin VI, CDC25, -actin-like protein,
-actin, and cytoplasmic -actin. According to the reported
functions of these proteins, one possible candidate for the
exosome-related lymphocyte-stimulating activity could be the CDC25
molecule, a phosphatase directly involved in the progression of the
cell cycle. It should be pointed out that other components among
several minor proteins contained in the exosome extract that could not
be picked up by the MALDI-TOF-MS analysis may well be responsible for
such biological activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retrospectively, while we were investigating possible expression
by mouse mast cells of mouse mammary tumor virus-derived superantigens,
cocultures of mast cells from different H-2 haplotypes with syngeneic
or autologous spleen cells resulted in vigorous heterotypic cell
adhesion and cytokine production. It turned out to be an Ag- or
superantigen-independent mast cell-mediated B and T lymphocyte
activation (7). More recently, we found that this mast
cell-induced lymphocyte activation occurs in vitro as well as in vivo
and could be mediated by cultured mast cells and peritoneal resident
mature mast cells as well (8). Furthermore, the lymphocyte
activation is mediated by mast cell-derived factor(s), and cell surface
contact between mast cells and lymphocytes is not required. Since then,
we set out to characterize the mast cell mediator released in the
culture medium. As a first step toward the characterization of the
bioactive material secreted by mast cells, culture supernatant
subjected to differential centrifugation completely lost its activity,
which was part of a supramolecular material recovered in a 70,000
x g pellet. Ultrastructural analysis of the pelleted
material revealed the presence of membrane vesicles called exosomes
known to be secreted by many cells of hematopoietic origin. Exosome
release represents a common feature to reticulocytes, several B cell
lines, B lymphocytes (19), and dendritic cells
(20). Cells such as CTL and NK cells, or mast cells and
basophils contain cytoxic granules and secretory granules,
respectively. Much evidence indicates that these intracellular
compartments display intralumenal membrane vesicles named
multivesicular bodies, which fuse with the plasma membrane during the
exocytosis process. The mast cell granules’ function as intracellular
storage sites for exosomes was confirmed in this study by specific
labeling of granules in ultrathin mast cell cryosections using
anti-exosome-specific Abs. In a previous report, we found that the
intracellular localization of MHC class II molecules in mast cells was
restricted to the membranes of 60- to 80-nm exosomes localized in type
I multivesicular and type II intermediate granules (17).
More interestingly, MHC class II-containing exosomes could be released
following mast cell activation. Combining immunoenzymatic and
electrophoretic analysis followed by MALDI-TOF-MS, exosome-associated
proteins consisted of a fairly great number of molecules among which we
identified MHC class II, CD86, CD40, CD40L, LFA-1, ICAM-1, CD13,
annexin VI, actins, and CDC25. Similarly, it has been recently reported
that dendritic cells secrete exosomes that contain several proteins
including MHC class II, Mac-1, CD9, MHC class I, and CD86
(21). Moreover, when dendritic cell-derived exosomes were
pulsed with tumor-derived peptides, they were able to eradicate or
suppress growth of established murine tumors in a T cell-dependent
manner (20). In contrast to exosomes derived from various
cell types including B lymphocytes and macrophages (data not shown),
only BMMC- and peritoneal mast cell-derived exosomes have the potential
to induce B and T lymphocyte activation. In addition, no
immunostimulatory activity could be expressed by exosomes unless mast
cells were pretreated with IL-4, indicating that this function is a
cytokine-regulated process. Induction of bioactivity by IL-4 is not
unique for cultured bone marrow mast cells because peritoneal mouse
mast cells have also been shown to acquire their lymphocyte-stimulating
activity only after IL-4 treatment (8). In contrast to
BMMC and peritoneal mast cells, mast cell lines P815 and MC/9 release
active exosomes independently of IL-4. This finding raises the question
as to the relationship between IL-4 receptor signaling pathways and the
constitutively expressed bioactivity in mast cell lines. One can
postulate that in these cell lines, IL-4R downstream signals are
constitutively turned on; in particular the IL-4 transcription factor
STAT6 may be continuously up-regulated. Other IL-4R-mediated signaling
pathways could be involved as well. The IL-4R -chain was found to be
associated with p47phox, an activator of the
NADPH oxidase in B cells (22). To investigate the possible
implication of these two IL-4R-dependent signaling pathways, STAT6-
(23) and p47phox-KO mice
(24) have been used to assess the potential of their BMMC
to induce lymphocyte activation. We found no alteration of BMMCs nor
their respective exosomes in their capacity to activate B and T
lymphocytes (data not shown), suggesting alternative signaling pathways
associated with IL-4R. To identify the molecular mechanism by which
exosomes from IL-4-treated BMMC induce B and T lymphocyte activation,
among several molecules we have examined including MHC class II, CD86,
CD40, CD40L, LFA-1, and ICAM-1, only Abs against LFA-1 and ICAM-1 were
able to completely block cell activation and cytokine production (data
not shown). These data are in agreement with those obtained when
stimulation was conducted by using BMMC (8). However,
LFA-1- and ICAM-1-dependent cell activation may participate only at a
distal level and is a consequence of proximal events initiated by
distinct molecules. One of the possible candidates among proteins
associated with exosomes that could be involved in the
lymphoproliferative response is a member of the mouse CDC25 gene family
expressed in mammalian cells. CDC25 gene encodes a Thr/Tyr phosphatase,
which activates cyclin-dependent kinase directly involved in the
mitosis process (25, 26). However, CDC25 should not be the
exclusive molecule that mediates this biological effect. Considering
the relatively wide spectrum of exosomal proteins, other mast cell
exosome-associated molecules, unidentified as yet, may well be
potentially immunostimulatory. Alternatively, heat shock proteins that
have not been picked up by our MALDI-TOF-MS analysis, a member of which
(hsc73) was recently found to be selectively associated with exosomes
derived from dendritic cells (21) may account for the
immunostimulatory activity observed in our system. Indeed, heat shock
proteins are known to be potent inducers of Ag-dependent
(27) and Ag-independent (28) T cell
responses. Based on previous work (17), accumulation of
some proteins in mast cell exosomes did not occur fortuitously because
MHC class II molecules, mannose phosphate receptor, lysosomal membrane
proteins (lamp1 and lamp2) are colocalized and enriched in these
vesicles. Whether this preferential localization applies to all
exosomal proteins, this remains to be investigated. Besides
cell-to-cell contacts, and soluble mediators such as cytokines,
exchange of exosomes may be considered as an alternative for
intercellular communication. Such a mechanism of protein transfer to a
naive cell via exosomes has been proposed to explain transfer of MHC
molecules between different cells of the immune system. Follicular
dendritic cells in tonsil germinal centers do not synthesize MHC II
molecules but express them at the cell surface and act as APCs once
exposed to supernatants of B cells that contain MHC II-bearing exosomes
(29). Recently, it has been demonstrated that MHC
II-bearing exosomes derived from MHC II-positive cells were found
attached to the follicular dendritic cell surface
(30).
What could be the pathophysiological relevance of exosome release
by mast cells? The ubiquitous tissue distribution of mast cells and
their potential to release several inflammatory and immunoregulatory
cytokines provide these cells with a unique role in host defense
mechanisms (31). Several pathophysiological conditions
indicate that mast cells and T lymphocytes are found in close
apposition in inflammatory sites (32, 33). Based on their
dual potential to release cytokines and immunologically active
exosomes, mast cells may play a role in the recruitment and activation
of B and T lymphocytes in inflamed tissues. Evidence that exosomes were
active in vivo was provided by the ability of BMMC-derived exosomes to
induce lymphocyte activation and cytokine release from lymph nodes and
spleen cells obtained from exosome-injected mice. What remains to be
investigated is whether exosome secretion by normal tissue mast cells
occurs constitutively or is under the control of a regulatory process.
It is consistently observed that mast cells and mast cell-derived
exosomes preferentially induce Th1-type responses as evidenced by the
production of IL-2, IFN-, and IL-12 (8). In support of
these findings, we recently demonstrated that in vivo transfer of
Ag-pulsed mast cells to syngeneic mice induced Ag-specific IgG1 and
IgG2a Abs but no IgE response could be elicited (Villa et al.,
unpublished results). It can be postulated that mast cells, directly or
through exosomes, may down-regulate allergic responses. The unique
property of exosomes from mast cells and mast cell lines to activate B
and T lymphocytes suggest that mast cells may contribute to the
development and the amplification of specific and nonspecific
inflammatory responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Salaheddine Mécheri, Unité d’Immuno-Allergie, Institut Pasteur, 28 rue Dr Roux, 75724 Paris Cedex, France.
3 Abbreviations used in this paper: BMMC, bone marrow-derived mast cells; KO, knockout; ITS, insulin-transferrin-sodium selenite supplement; MALDI-TOF-MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; lamp, lysosomal-associated protein; CD40L, CD40 ligand.
Received for publication July 25, 2000. Accepted for publication October 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI-dependent mast cell degranulation following coculture with activated T cells: dependency on ICAM-1-and leukocyte function-associated antigen (LFA)-1-mediated heterotypic aggregation. J. Immunol. 160:4026.-chain with p47phox, an activator of the phagocyte NADPH oxidase in B cells. Mol. Immunol. 36:45.[Medline]
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) and 5 x 106 (
) IL-4-treated BMMC, or with 5
µg (
) were
injected with PBS. Six days later, spleen and lymph node cells were
harvested and cultured at indicated concentrations in the absence of
any stimulus. After 72 h of incubation, cell proliferation
(A) was monitored by thymidine uptake, and culture
supernatants were tested for their content in IL-2 (
) and IFN-
) (B) using CTLL-2 proliferation assay and ELISA,
respectively. Results are presented as mean values ± SEM from two
different experiments.
