T cell exosomes induce cholesterol ... - Wiley Online Library

ORIGINAL ARTICLE Journal of

T Cell Exosomes Induce Cholesterol Accumulation in Human Monocytes Via Phosphatidylserine Receptor LIUDMILA ZAKHAROVA, MARIA SVETLOVA,

AND

Cellular Physiology

ALLA F. FOMINA*

Department of Physiology and Membrane Biology, University of California, Davis, Davis, California Activated T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4þ activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma-associated cells are not known. We report here that exosomes isolated from the supernatants of activated human CD4þ T cells enhance cholesterol accumulation in cultured human monocytes and THP-1 cells. Lipid droplets found in the cytosol of exosome-treated monocytes contained both cholesterol ester and free cholesterol. Anti-phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome-induced cholesterol accumulation, indicating that exosome internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF-a enhanced in parallel with monocyte cholesterol accumulation. Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor. J. Cell. Physiol. 212: 174–181, 2007. ß 2007 Wiley-Liss, Inc.

The release of small vesicles, termed exosomes, originating from the endocytic compartments is a general feature of cells of the hematopoietic lineage. Exosomes are formed by invagination of the limiting membrane of late endosomes, which results in the formation of multivesicular bodies (MVB). Exosomes are released into the extracellular space upon fusion of MVB with the plasma membrane. Because of the abundance of signaling proteins and adhesion molecules at the exosome surface, it was suggested that they may serve as vehicles for long-range cell–cell interactions that shape the immune responses (Denzer et al., 2000; Stoorvogel et al., 2002; Thery et al., 2002). In T lymphocytes, exosome biogenesis within the endocytic pathway requires the stimulation of T cell receptors and is a specific function of activated T cells (Blanchard et al., 2002; Fomina et al., 2003). T cells secrete exosomes in substantial quantities and externalized exosomes form large clusters at the surface of the activated T cells (Fomina et al., 2003). The biological functions of T cell exosomes are generally unclear, although FasL and APO2/TRAIL death ligands found in T cell exosomes have been shown to suppress the immune response in vitro (Martinez-Lorenzo et al., 1999; Monleon et al., 2001). During exosome biogenesis within the endocytic compartments, certain proteins and lipids are specifically recruited to the exosome membranes. T cell exosomes display strong positive staining with the cholera toxin B (CTB), a marker for the lipid raft-associated GM1 glycosphingolipid (Fomina et al., 2003), which is likely a result of the lipid rafts segregation into exosomes due to raft-mediated endocytosis (Deckert et al., 1996; Nabi and Le, 2003). Another prominent feature of the exosome membranes is that they express phosphatidylserine (PS) at their outer membrane leaflet (Heijnen et al., 1999; Fomina et al., 2003), which appears to be an essential requirement for exosome budding within the late endosomes. Exposure of PS triggers the phagocytosis of apoptotic lymphocytes by macrophages (Krahling et al., 1999). Multiple PS-binding proteins were found at the surface of phagocytes, which include several classes of scavenger receptors, integrins, complement receptors, and CD14 (Platt et al., 1998; Fadok et al., 2001; Lauber et al., 2004). In addition, a putative specific ß 2 0 0 7 W I L E Y - L I S S , I N C .

PS receptor (PSR) has been cloned in human macrophages using monoclonal antibodies that block the engulfment of apoptotic cells by professional and amateur phagocytes (Fadok et al., 2000). It was shown that low affinity binding of the putative PSR to the PS-expressing targets stimulated engulfment (Hoffmann et al., 2001). Preincubation with anti-PSR antibodies prevented uptake of subsequently added apoptotic cells (Fadok et al., 2000). We hypothesized that the PS on the exosome surface may trigger phagocytosis in macrophages via PS-recognition pathway. Given the high cholesterol content of exosome membranes, their internalization may affect cholesterol metabolism in macrophages. Activated CD4þ T lymphocytes constitute approximately 10% to 20% of the cell population in advanced human atherosclerotic plaques (Jonasson et al., 1986; Hansson, 2005). The pathogenic potential of CD4þ T cells has been demonstrated in several experimental models of atherosclerosis (Zhou et al., 2000; Laurat et al., 2001; Song et al., 2001). However, the specific mechanisms by which atheroma-associated T cells may exert their atherogenic effects remain unclear. Within the early atherosclerotic lesions and at the shoulders of more mature lesions, T cells colocalize with monocytes and macrophages (Fan and Watanabe, 2003). Macrophage transformation into foam cells, a hallmark of atherosclerosis, can be profoundly altered by activated T cells (Hansson, 2005; Langheinrich and Bohle, 2005). It is believed

Contract grant sponsor: Philip Morris USA Inc. and Philip Morris International. Contract grant sponsor: NIH NCRR Research Facilities Improvement Program Grant; Contract grant number: C06 RR-12088-01. *Correspondence to: Alla F. Fomina, Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA 95616. E-mail: [email protected] Received 19 October 2006; Accepted 8 December 2006 DOI: 10.1002/jcp.21013

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that T cells promote atherogenesis via the release of proinflammatory cytokines, particularly INF-g, which activates macrophages and vascular cells (Zhou, 2003). In addition to cytokines, activated T cells may secrete a substantial amount of exosomes (Blanchard et al., 2002; Fomina et al., 2003). However, the effects of T cell exosomes on monocytes and macrophages have not been investigated. Blood-born monocytes are recruited to the sites of atheroma formation where they subsequently differentiate into macrophages (Hansson, 2005). Similarly to other mammalian cells, monocytes and macrophages store and transport lipids in spherical droplets primarily composed of neutral lipids: triacylglycerol and esterified cholesterol (EC) (Murphy and Vance, 1999). Internalization of the extracellular cholesterol cargo alters the macrophage lipid homeostasis, which in turn affects macrophage viability and functions (Tabas, 2002; Maxfield and Tabas, 2005). Macrophages handle the external cholesterol load by esterification of free cholesterol (FC) and transport the EC into intracellular depots. Excessive loading with exogenous cholesterol results in formation of numerous cytosolic droplets composed of neutral lipids and accumulation of FC within the internal membranes (Feng et al., 2003). Accumulation of both FC and EC in atheroma-associated monocytes/macrophages is a crucial feature in the development and progression of atherosclerosis (Tabas, 2002; Maxfield and Tabas, 2005). In addition, excessive cholesterol-loading in macrophages stimulates release of a proinflammatory cytokine TNF-a that further promotes atherogenesis (Li et al., 2005). In this study, we have investigated the effects of T cell exosomes on lipid accumulation and cytokine production in cultured human monocytes and phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1-cells, a human monocytic cell line. We have employed fluorescent markers Nile Red and filipin, which have been shown to predominantly stain neutral lipids and FC, respectively, in variety of cells and tissues (Kruth et al., 1979; Kruth and Fry, 1984; Fowler and Greenspan, 1985; Greenspan et al., 1985; Brown et al., 1988; Klinkner et al., 1997; Prattes et al., 2000; Pham et al., 2005). We found that T cell exosomes significantly enhance the cholesterol accumulation and production of TNF-a in human monocyte cell cultures, implying that within the atherosclerotic plaque, T cell exosomes may promote atherogenesis. Materials and Methods Cell cultures and chemicals

Unless indicated, all chemicals were from Sigma-Aldrich (St. Louis, MO). For this study, 13 healthy volunteers were recruited from adult population (21–45 years old) of both sexes and different ethnic backgrounds. Monocytes or resting CD4þ T lymphocytes were isolated from peripheral blood using RosetteSepTM human monocyte or CD4þ T cell enrichment cocktails following the manufacturer’s instructions (Stem Cell Tech, Vancouver, Canada). Monocytes were resuspended in RPMI 1640 (Cambrex, Walkersville, MD) supplemented with 1% MEM, 1% Naþ-pyruvate, 1% glutamine, 0.035 ml/L b-mercaptoethanol, and 10% lipoprotein deficient serum (LPDS) (Biomedical, Stoughton, MA). THP-1 human monocytic leukemia and Jurkat E6-1 T cell lines were obtained from the American Type Culture Collection and maintained in culture according to ATCC recommendations. Before the experiment, THP-1 cells were washed twice with PBS, resuspended in RPMI 1640 (Cambrex) supplemented with 1% LPDS, and activated with PMA (50 nM, Calbiochem, San Diego, CA) to induce their differentiation into macrophages (Auwerx, 1991). For cholesterol and cytokine assays, human monocytes or PMA-activated THP-1 cells were plated into 96-well plates (Primaria, Fisher, Pittsburgh, PA) at density of 0.3 106 cells/ml (0.1 106 cells/well). For fluorescence studies, monocytes were plated onto glass-bottomed chambers 0.5 106 cells/ml (0.25 106 cells/chamber). The T cell growth media was prepared as described previously (Blanchard et al., 2002). Briefly, RPMI 1640 medium (Cambrex) JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

supplemented with 10% FBS (Omega, Tarzana, CA) was ultracentrifuged overnight at 100,000 g at 48C. Supernatant was then supplemented with 1% MEM, 1% Naþ-pyruvate, 2% Glutamax (Life Technology, Rockville, MD), 1 RPMI 1640 Vitamins Solution, 1 RPMI 1640 Amino Acids Solution, and 0.035 ml/L b-mercaptoethanol. Isolated T cells were resuspended at 0.5–0.7 106 cells/ml in the T cell growth media and activated with 20 mg/ml phytohaemagglutinin P (PHA) for 3–4 days. Exosome purification and staining

Exosomes were purified as described previously (Blanchard et al., 2002). Briefly, activated T cells were centrifuged at 200 g for 10 min and supernatant was filtered using 0.22-mm filter. The 100 ml of the filtrate were saved to use in control experiments, whereas the rest was ultracentrifuged at 100,000 g for 1.5 h at 48C; pellet was collected and resuspended in 50 ml PBS supplemented with protease inhibitors cocktail (Roshe, Mannheim, Germany), and high speed centrifugation was repeated two more times. Final pellet was reconstituted in 50–100 ml PBS with protease inhibitors and considered as a purified exosome preparation. For some experiments, exosome isolation procedure was performed using a cell-free T cell culture medium; pellets were collected and their effect on monocyte lipid accumulation investigated. EM Imaging

Exosome preparations were fixed with 2.5% glutaraldehyde (Electron Microscopy, Fort Washington, PA) and 2% PFA (Electron Microscopy) in 0.08 M sodium phosphate buffer. A drop of exosome preparation was placed on carbon-stabilized coated copper grid and contrasted with OsO4 vapors (2% OsO4 in H2O). For negative staining, 10 ml phosphotungstic acid (Electron Microscopy; 2% in H2O, pH 5.8) were added to the grids and immediately wicked off. Grids were viewed using Philips EM400 transmission electron microscope (Goniometer, FEI Company, Hillsboro, OR). Fluorescence staining and imaging

For exosome staining, annexin V-Alexa Fluor 568 conjugate solution (5 ml/100 ml, Molecular Probes, Eugene, OR) and FITC-conjugated CTB (10 mg/ml) were added to the pellet obtained after first high-speed centrifugation of T cell supernatant. After 20 min incubation, the pellet was resuspended in 50 ml centrifugation solution and dyes were washed out during two consequent high-speed centrifugations. Stained exosome preparations were placed onto poly-l-lysine-coated glass-bottom chambers and images were taken using LSM 510 confocal imaging system (Carl Zeiss, Thornwood, NY). For staining of neutral lipids and FC, stock solutions of Nile Red (1 mM) or filipin (0.25%) were prepared in ethanol and DMSO correspondingly. The adherent monocytes were washed with PBS and then fixed for 30 min with 3% PFA and 0.2% glutaraldehyde in PBS. After washing, cells were stained for 5 min with Nile Red (1 mM in PBS). For double staining, fixed cells were stained for 2 h with filipin (0.005% in PBS supplemented with 10% FBS), washed with PBS, and then stained with Nile Red. For anti-PSR Ab antigen staining, adherent cells were fixed with 4% PFA, washed three times with 2 PBS and were either immediately incubated with primary antibodies for surface staining or permeabilized for 1 h in saponin solution (0.075% w/v saponin, 1% bovine serum albumin, 1% goat serum in 2 PBS) for intracellular staining. Primary rabbit anti-human polyclonal anti-PSR Ab (1:200, Sigma) were applied for 45 min in 2 PBS containing 2% goat serum. After washing, cells were incubated with chicken-anti rabbit Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes), then washed and mounted in Anti Fade mounting solution (Molecular Probes). Stained exosome or cell preparations were visualized and recorded either with LSM 510 laser scanning confocal imaging system or with Apotom imaging system (Carl Zeiss) using a 63/1.4 n.a. oil immersion objective. Image acquisition settings were identical for different experiments. Western blot

Cells or exosomes were lysed with PBS supplemented with 1% SDS, 5 mM EDTA, and protease inhibitor cocktail (Roshe, Mannheim, Germany). Lysates were separated by 4–12% SDS–PAGE, transferred to nitrocellulose membrane (Amersham, Buckinghamshire, UK), and processed for immunoblotting. Rabbit anti-human polyclonal

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anti-CD81 (Santa-Cruz Biotech., Santa-Cruz, CA), or anti-CD45 (Santa-Cruz Biotech.), or anti-PSR antibodies (Sigma) were used as the primary; and HRP-conjugated goat-anti rabbit antibodies (Santa-Cruz Biotech.) were used as the secondary antibodies. Membranes were developed by ECL Western Detection reagent (Amersham). Total cholesterol and protein measurements

Monocytes from the same donor or THP-1 cells simultaneously activated with PMA were used for different conditions in each experiment. Prior exosome application, the monocyte culture medium was substituted with the medium containing 1% LPDS and then 20 ml of exosome preparation (0.3–1 mg of protein/ml), or equal volume T cell supernatant (control) were added to each well with adherent monocytes. Anti-PSR Ab (100 mg/ml, Sigma) were added to some wells 20 min prior addition of exosome preparation. To account for the nonspecific effects of sodium azide present in the stock solution of anti-PSR Ab, 1 mM of sodium azide was added to control and exosomestimulated cells. All conditions were run in triplicates in each experiment and all incubations were run in parallel. After overnight incubation, cells were washed twice with PBS, then lysed with PBS containing 5 mM EDTA, 1.5% CHAPS, and a protease inhibitor cocktail (Roshe). Total cholesterol content was determined in whole cell lysates from each sample using the cholesterol oxidase-based Amplex Red Cholesterol Assay Kit (Molecular Probes) according to manufacturer’s protocol. The Amplex Red fluorescence was excited at 530 nm and detected at 590 nm wavelengths using a Cytofluor 2300 plate reader (Millipore, Betford, MA). The amount of total cholesterol was normalized to the total protein content for each sample. The total protein contents in exosome preparations and in whole cell lysates were determined using BCA Protein Assay Kit (Pierce Biotech, Rockford, IL) according to manufacturer’s protocol. The color reactions were read on VERSAmax plate reader (Molecular Devices, Sunnyvale, CA). To ensure that non-internalized exosomes did not contaminate the measurements from cell lysates, exosomes were also added to cell-free wells. No measurable amounts of the protein or cholesterol were detected in lysates from cell-free wells incubated with exosomes alone (n ¼ 4, not shown), indicating that non-internalized exosomes were washed out from the wells and did not affect the measurements of the protein and cholesterol levels in monocyte cultures.

Fig. 1. Characteristics of T Cell Exosome Preparation. A, B: Transmitted electron micrographs of exosome clusters contrasted with OsO4 (A) and negatively stained with phosphotungstic acid (B). Note that negatively stained exosomes are not uniformly electronlucent. C, D: Fluorescent images of exosome clusters stained with CTB (green) and annexin V (red). E: Superimposed images from C and D overlaid on transmitted light image. A 488-nm excitation laser line and 505–550-nm emission filter, or 543-nm excitation laser line and 585 LP emission filter were used for FITC-CTB or Alexa Fluor 568-annexin V respectively. Fluorescence was collected from 1 mm optical slice. Bars are 100 nm in A and B, and 10 mm in C–E. F: Western blot performed on exosome preparation (3 mg) and T cell lysate (3 mg) using anti-CD81 and anti-CD 45 antibodies.

Cytokine assay

For cytokine assays supernatants from cultured monocytes were collected from monocyte cultures after overnight incubation in the absence or presence of T cell exosomes, and contents of TNF-a and TGF-b1 were determined by ELISA using corresponding Quantikine kits (R&D systems, Minneapolis, MN). Data analysis

Statistical analysis was performed using Microcal Origin software and web-based calculator. Exosome size analysis was performed in Photoshop 7.0 software. All results are expressed as mean standard error (SE); n—number of experiments.

Results Characterization of T cell exosome preparation

We have shown previously that activation of peripheral bloodderived CD4þ T cells with PHA stimulates secretion of the exosomes (Fomina et al., 2003). For this study, exosomes from supernatants of activated CD4þ human T lymphocytes were isolated using the high speed centrifugation method, which has been shown to produce a pure preparation of exosomes (Blanchard et al., 2002). Electron microscopic study revealed that exosome preparations were composed of clusters of clear vesicles with an average diameter of 50.0 1.0 nm (n ¼ 97, Fig. 1A,B). Consistent with previous observations (Fomina et al., 2003), exosome clusters were positively stained with CTB and annexin V (Fig. 1C–E). Western blot analysis demonstrated the presence of characteristic exosome marker CD81 (Escola et al., 1998; Fritzsching et al., 2002) in lysates of exosome preparations, but not in whole T cell lysates (Fig. 1F). In contrast, CD45, a plasma membrane marker (Blanchard JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

et al., 2002), was not detectable in exosomes (Fig. 1F). These data indicate that our procedure yielded the exosome preparation with characteristics consistent with previously described properties of T cell exosomes. T cell exosomes promote neutral lipids accumulation in human monocytes

Next, we explored the effect of T cell exosomes on monocyte lipid accumulation. Human monocytes were isolate from the peripheral blood mononuclear cells (PBMC) and placed into culture to induce their differentiation into macrophages (Davies and Gordon, 2005). Following an overnight incubation in lipoprotein-deficient medium, the aliquots of T cell supernatant (20 ml, control) or exosome preparation (0.3–1 mg of protein/ml) were added to monocyte cell cultures. After an overnight incubation, monocytes were fixed and stained with Nile Red. Confocal imaging revealed that control monocytes were roundish in shape and displayed small bright spots of the cytosolic Nile Red fluorescence (Fig. 2A,B). In contrast, the monocytes incubated in the presence of T cell exosomes spread on the coverslip and displayed large cytosolic droplets brightly stained with Nile Red (Fig. 2C,D). On average, the size of the area outlined by the external cell perimeter on transmitted light images (Fig. 2A,C) increased twofold after incubation with the exosomes (Fig. 2G, and supplementary Fig. S1 A), which likely reflects the increase in cell size. Cell enlargement correlated


Fig. 2. T cell exosomes stimulate the accumulation of neutral lipids and FC in human monocytes. A, B: Confocal images of human monocytes after overnight incubation in the absence (A, B) or presence (C, D) of T cell exosomes, or in the presence of T cell exosomes and anti- PSR Ab (E, F). Red staining corresponds to Nile Red fluorescence. B, D, and F are Z projections of the fluorescent stacks obtained from 17 optical planes. Each optical plane was 1 mm in depth. In A, C, and E, the Z projections of the fluorescent stacks are overlaid on transmitted light images. Note that brightest Nile Red fluorescence originates from the spherical droplets. For quantitative assessment of the amount of neutral lipids within the cytosolic droplets, the external cell perimeter of each cell was outlined on transmitted light images (as shown in A, C, and E), and then both area and mean fluorescence intensity were measured for each cell (as shown in B, D, and F). Diffused cytosolic Nile Red fluorescence was considered as nonspecific background fluorescence, and was subtracted before determining the mean Nile Red fluorescence. G, H: Average area (G) and mean Nile Red fluorescence (H) measured within the single cell perimeter of monocytes cultured overnight in the absence (C, n ¼ 145) or presence of T cell exosomes (E, n ¼ 103), or in the presence of T cell exosomes and anti-PSR Ab (ERanti-PSR Ab, n ¼ 58). n is the number of cells analyzed for each condition from images obtained in 4 (Control or Exosomes) and 2 (Exosomes R anti-PSR Ab) experiments. Stars and brackets indicate that differences between means are significant ( P < 0.01 independent Student’s t-test). I–K: A typical monocyte co-cultured with T cell exosomes and stained with Nile Red (I, red) and filipin (J, green). K: Superimposed images from I and J overlaid on transmitted light image. Images were recorded using Apotom imaging system (Carl Zeiss). Arrows in J and K indicate filipin staining surrounding Nile Red-stained droplets. Note that some droplets display weak Nile Red staining but strong filipin fluorescence (arrowhead). Note that in part I the diffused fluorescence is more prominent than in parts B, D, F due to residual out-of-focus fluorescence remained after Apotom Image processing. These images were not taken for the statistical analysis presented in G and H. A 543-nm excitation wavelength and 575–640 nm emission filter, or 360 nm excitation wavelength and 420 nm LP emission filter were used for Nile Red or filipin fluorescences respectively. Bars are 10 mm.

with threefold increase in the mean fluorescence intensity of Nile Red within the area outlined by the external cell perimeter in exosome-treated cells (Fig. 2H, and supplementary Fig. S1 B). Similar effects were observed in PMA-activated THP-1 cells (not shown). The Z stack projections shown in Figure 2 (parts B, D, and F) demonstrate that bright Nile Red fluorescence mainly originates from the spherical droplets, which is consistent with Nile Red staining of droplets composed of neutral lipids. Nile Red can also bind to phospholipid-rich regions (Brown et al., 1988, 1992) and this may account for diffused cytosolic fluorescence (Fig. 2B,D,F). The intensity of the diffused fluorescence, however, was more than five times lower than fluorescence intensity originated from the spherical droplets in confocal projections. Therefore, we have considered this JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

fluorescence as non-specific to neutral lipids and this signal was not taken into consideration. It has been established that an excessive loading with exogenous cholesterol causes accumulation of FC within macrophage internal membranes (Maxfield and Tabas, 2005). Therefore, we stained the exosome-treated monocytes with both Nile Red and filipin. We found that in monocytes co-cultured with T cell exosomes, large Nile Red-stained droplets were surrounded by strong filipin staining (Fig. 2I–K), indicating the formation of structurally complex droplets composed of both neutral lipids and FC. Interestingly, some droplets were predominantly composed of FC. Taken together, these data strongly indicate that exosomes were internalized by monocytes/immature macrophages, resulting in enhanced accumulation of both EC and FC. Since exosomes expose PS at

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their outer membrane leaflet, we next explored whether exosome internalization was mediated via a PS-recognition pathway. Anti-PSR antibodies abolish exosome-induced cholesterol accumulation

We have examined the effect of anti-phosphatidylserine receptor antibodies (anti-PSR Ab) on exosome-induced cholesterol accumulation in monocytes. Western blot analysis of lysates of PMA-activated THP-1 cells and human monocytes cultured overnight revealed that anti-PSR Ab reacted with a protein of apparent molecular weight of 70 kDa (Fig. 3A). The anti-PSR immunoreactivity was absent in lysates of Jurkat T lymphocytes (Fig. 3A). Immunostaining of non-permeabilized PMA-activated THP-1 cells demonstrated that anti-PSR Ab antigen was exposed at the plasma membrane (Fig. 3B–D). Staining of permeabilized THP-1 cells with anti-PSR Ab revealed that the antigen was also abundant in the cytosol but excluded from the nuclei (Fig. 3E–G). Similar results were obtained in human monocytes (supplementary Fig. S2). Consistent with the Western blot analysis, no specific anti-PSR staining was detected in Jurkat T cells (supplementary Fig. S2). We conclude that, at the time prior to the stimulation with T cell exosomes, both human monocytes and PMA-activated THP-1 cells expressed PSR at the cell surface, and therefore appeared to be primed for PSR-mediated phagocytosis. Application of anti-PSR Ab to monocytes cell cultures prior to addition of T cell exosomes significantly reduced monocyte enlargement (Fig. 2E,G) and cytosolic Nile Red staining (Fig. 2F,H) compared to monocytes incubated with T cell exosomes alone. In addition, the enzymatic cholesterol detection assay performed on whole cell lysates demonstrated a significant increase in total cholesterol content in human monocytes and PMA-activated THP-1 cells incubated in the presence of T cell exosomes (Fig. 4A,B). The anti-PSR Ab abolished the exosome-induced cholesterol accumulation, indicating that exosome internalization was mediated via activation of the endogenous PSR-mediated signaling pathway.

Exosomes stimulate TNF-a production

Since FC accumulation induces the production of inflammatory cytokines in macrophages (Li et al., 2005), we measured the cytokine content in supernatants from macrophage cell cultures incubated in the presence or absence of T cell exosomes. We found that the concentration of the proinflammatory cytokine TNF-a increased dramatically in the supernatants of exosome-stimulated human monocytes and PMA-activated THP-1 cells (Fig. 4C). Unexpectedly, the anti-PSR Ab also potently stimulated TNF-a secretion (Fig. 4C). However, monocytes pretreated with anti-PSR-Ab prior to the application of T cell exosomes produced a larger TNF-a response than monocytes incubated with either exosomes or anti-PSR Ab alone (Fig. 4C), although in human monocytes, this difference was not statistically significant. The anti-PSR Ab or T cell exosomes failed to induce detectable changes in the levels of anti-inflammatory cytokine TGF-b1 (n ¼ 7, data not shown). Discussion

In the present study, we investigated the effects of exosomes isolated from supernatants of activated human CD4þ T cells on cultured human monocytes and PMA-activated THP-1 cells. Our principal finding was that T cell exosomes induced accumulation of neutral lipids and FC as well as enhanced TNF-a production in monocyte cell cultures. These effects, at least partially, were mediated via the PSR signaling pathway. We conclude that within an atherosclerotic plaque, T cell exosomes may promote cholesterol accumulation and production of proinflammatory cytokines in monocytes and macrophages, thereby promoting atherogenesis. The vesicles isolated from T cell supernatants used in our study displayed all the characteristics of T cell exosomes. The exosome clusters were composed of membrane delimited vesicles (Fig. 1A) with a diameter of 50 nm typical for T cell exosomes (Blanchard et al., 2002). Exosomes were morphologically different from plasma lipoproteins (Fig. 1B),

Fig. 3. Antigen recognition by anti-PSR Ab in monocyte cell cultures. A: Western blot performed on whole cell lysates from Jurkat T cells and PMA-activated (16 h) THP-1 cells (left part), and human monocytes 16 h after isolation (right part). Equal amounts of protein (5 mg) were used for each blot. Note bands of anti-PSR immunoreactivity (arrow), which corresponds to the size of the glycosylated PSR (70 kD) in human monocytes and PMA-activated THP-1 cells. B–G: Confocal images of non-permeabilized (B–D) and permeabilized (E–G) THP-1 cells activated with PMA for 16 h. B, E: Anti-PSR immunofluorescence images overlaid on transmitted light images of the same cells. C and F are anti-PSR immunofluorescence recorded from 0.5 mm optical slice. D and G are orthogonal Z sections reconstructed from Z stacks of 35 optical slices of 0.5 mm in depth. Thin lines in C and F show the positions of the orthogonal Z sections shown in D and G correspondingly. Note the luck of nuclear staining in F and G. A 488 nm excitation laser line and 505–530 nm BP emission filter were used for anti-PSR-Alexa Fluor 488 fluorescence. Bars are 10 mm.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP


Fig. 4. Anti-PSR Ab abolish exosome-induced cholesterol accumulation but promote TNF-a production. Total cholesterol content in lysates of human monocytes (A) and PMA-activated THP-1 cells (B) incubated overnight in the absence (Control), or presence of T cell exosomes (Exosomes), or in the presence of exosomes and anti-PSR Ab (Exosomes R anti-PSR Ab). Each data point represents the average of three measurements. In each individual experiment, cells were plated and incubated at different conditions simultaneously. Data points from an individual experiment are connected by lines. Horizontal bars indicate average values from five experiments for each treatment condition. Stars indicate that differences between groups are significant ( P < 0.05, Wilcoxon directional test; n ¼ 5). C: Average content of TNF-a in supernatants from human monocyte (shaded bars) and PMA-activated THP-1 cell cultures (open bars). Numbers above bars represent number of experiments. Cells were incubated overnight in the absence (Control) or presence of the T cell exosomes (Exosomes); or in the presence of Anti-PSR Ab (Anti-PSR Ab); or in the presence of exosomes and anti-PSR Ab (Exosomes R anti-PSR Ab). Stars and brackets indicate that differences between means are significant ( P < 0.05 independent Student’s t-test).

which usually appear as uniformly electron-lucent particles at negative staining conditions (Nordestgaard et al., 1995). In addition, the exosome preparations were positively stained with the CTB and annexin V (Fig. 1D–F) consistently with staining of exosome clusters in cultures of activated T cells (Fomina et al., 2003). Moreover, our exosome preparations bore the specific exosome marker CD81 (Escola et al., 1998) but lacked the CD45, a marker present in the plasma membranes of viable and apoptotic cells but excluded from the exosomes (Blanchard et al., 2002). Taken together, these data indicate that our procedure yielded the purified preparation of T cell exosomes. The PBMC-derived monocytes spontaneously differentiate into macrophages when placed in culture (Davies and Gordon, 2005). Application of T cell exosomes to cultured human monocytes at the early stage of their differentiation into macrophages (16 h in culture) resulted in cytosolic accumulation of neutral lipids as revealed by enhanced Nile Red fluorescence. Interestingly, the large neutral lipid droplets were surrounded by FC (Fig. 2C–E), indicating the formation of structurally complex droplets composed of neutral lipids (EC and triacylglycerol) and FC. The formation of similar droplets was previously observed in foam cells derived from murine J774.1 cells or peritoneal macrophages incubated in hyperlipidemic serum (Mori et al., 2001). Formation of EC-FC droplets was attributed to the metabolic modification of EC into FC presumably due to the imbalance between neutral EC hydrolase activity and compensatory re-esterification and/or cell capacity for FC export (Tabas, 2002). This process may eventually result in the complete conversion of neutral lipid droplets into FC-containing droplets and massive intracellular FC accumulation within internal membranes leading to macrophage apoptosis (Feng et al., 2003). Since the development of macrophage-derived foam cells that contain massive amounts of EC is a hallmark of atherosclerotic lesions (Li and Glass, 2002; Tabas, 2002), and because the lesional instability is linked to macrophage apoptosis, we concluded that T cell exosomes may promote atherogenesis by inducing intracellular EC and FC accumulation in monocytes/immature macrophages. JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

Previous studies demonstrated that monoclonal antibodies mAb 217 prevented the engulfment of the apoptotic cells by human macrophages in vitro (Fadok et al., 2000). The gene encoding the antigenic target of mAb 217 was denoted as PSR and proposed to be a putative receptor for PS binding in both professional and amateur phagocytes. Although several studies have reported crucial role of the PSR in phagocytosis, other studies demonstrated essential role of PSR in embryogenesis but not in apoptotic cell clearance (Williamson and Schlegel, 2004; Gardai et al., 2006). In addition, in expression systems the recombinant PSR was targeted to the nuclei (Cikala et al., 2004; Cui et al., 2004; Mitchell et al., 2006). Thus, until now the data on PSR localization and function obtained in native and genetically altered systems remain controversial. In our study, Western blot analysis performed on lysates of human monocytes and PMA-activated THP-1 cells revealed that polyclonal anti-PSR Ab specific for 361–381 aa region of a synthetic PSR protein reacted with an endogenous protein with an apparent molecular weight of 70 kDa (Fig. 3A), which corresponds to the size of the endogenous glycosylated PSR recognized by mAb 217 in PMA-activated THP-1 cells (Fadok et al., 2000). In accordance with the previous report (Fadok et al., 2000), the anti-PSR Ab immunoreactivity was absent in Jurkat T cells. In addition, the antigen recognized by anti-PSR Ab used in our study was targeted to the plasma membrane (Fig. 3B–D) and expressed in the cytosol but not in the nuclei (Fig. 3E–G). We conclude that, anti-PSR Ab used in our study recognize an endogenous PSR, that is specifically expressed at the surface of human macrophages and PMA-activated THP-1 cells. Our further study demonstrated that the anti-PSR Ab inhibited exosome-induced monocyte cholesterol accumulation (Figs 2H, and 4A,B). Taken together with the previous report that anti-PSR mAb 217 antibodies inhibit phagocytosis of the PSexpressing targets (Fadok et al., 2000), these results indicate that anti-PSR Ab prevent phagocytosis of PS-expressing exosomes. We conclude that monocytes and/or macrophages engulf T cell exosomes by a mechanism that involves the endogenous surface PSR. Previous studies performed on dendritic cells demonstrated that internalized exosomes were

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processed within the endocytic pathway (Morelli et al., 2004). After engulfment by macrophages, the cholesterol-rich exosomes are most likely to undergo the conventional endosomal processing (Tabas, 2002; Maxfield and Tabas, 2005), which results in the formation of the cytosolic EC droplets. Thus, T cell exosomes may contribute to the macrophage EC loading and may promote their differentiation into the foam cells. It is established that FC accumulation is a potent inducer of inflammatory cytokine production in macrophages (Li et al., 2005). In addition, FC-loaded apoptotic cells may, in turn, stimulate production of proinflammatory cytokines in competent phagocytes, further amplifying the inflammation. Consistent with this, we found that production of TNF-a, a proinflammatory cytokine abundantly expressed in atherosclerotic lesions (Tipping and Hancock, 1993), increased in parallel with cholesterol accumulation in exosomestimulated human monocytes and PMA-activated THP-1 cells (Fig. 4C). The unexpected finding was that the anti-PSR Ab also potently stimulated TNF-a secretion. The anti-PSR mAb 217 have been reported to block lipopolysaccharide-induced TNF-a release in mature macrophages, although the effects of these antibodies alone on proinflammatory cytokine production in either mature or immature macrophages have not been shown (Fadok et al., 2000). Our data suggest the PSR stimulation may directly activate second messenger pathway(s) leading to TNF-a release. Regardless of the nature of proinflammatory response to anti-PSR Ab, the combined application of T cell exosomes and anti-PSR Ab produced a larger TNF-a response than exosomes or anti-PSR Ab alone (Fig. 4C). These data indicate that T cell exosomes may utilize different, PSR-independent signaling pathway to stimulate TNF-a production in monocytes. For example, FasL expressed on T cell exosomes (Martinez-Lorenzo et al., 1999; Monleon et al., 2001) may stimulate a Fas pathway, which may induce a proinflammatory response in monocytes/macrophages (Park et al., 2003). Further studies are necessary to unravel PSRdependent and PSR-independent pathways that stimulate proinflammatory responses in monocytes and macrophages. In atherosclerotic lesion, TNF-a may further promote atherogenesis by local actions on the endothelial cells and the recruitment of circulating blood monocytes (Hansson, 2005). The actions of T cell exosomes within the atherosclerotic plaque may not be limited to the stimulation of monocyte cholesterol accumulation and TNF-a production. For example, T cell exosomes have a potential to directly induce apoptosis in atheroma-associated cells via FasL and APO2 pathways (Martinez-Lorenzo et al., 1999). A broader array of cellular and cytokine responses from different atheroma-associated cells is required to fully evaluate the role of T cell exosomes in the development of proatherogenic responses within the atherosclerotic plaque. Acknowledgments

We thank Sepehr Dadsetan and Grete Adamson for valuable technical assistance, Hiroaki Misono for assistance with Apotom imaging system, Martha O’Donnell and Anna Shyrokova for critical reading and comments on the manuscript. Research described in this article was supported by Philip Morris USA, Inc. and Philip Morris International. This investigation was conducted in part in a facility constructed with support from the NIH NCRR Research Facilities Improvement Program Grant C06 RR-12088-01. Literature Cited Auwerx J. 1991. The human leukemia cell line, THP-1: A multifacetted model for the study of monocyte-macrophage differentiation. Experientia 47:22–31.


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