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The Journal of Immunology, 1999, 163: 1274-1281.
Copyright © 1999 by The American Association of Immunologists

Activated Human T Cells Release Bioactive Fas Ligand and APO2 Ligand in Microvesicles1

María José Martínez-Lorenzo*, Alberto Anel2,*, Susana Gamen*, Inmaculada Monleón*, Pilar Lasierra{dagger}, Luis Larrad{dagger}, Andrés Piñeiro*, María A. Alava* and Javier Naval*

* Departamento de Bioquímica y Biología Molecular y Cellular, Facultad de Ciencias, and {dagger} Servicio de Inmunología, Hospital Clínico Universitario, Universidad de Zaragoza, Zaragoza, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100–200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (<=1 h) after PHA stimulation, well before the cell enters apoptosis. FasL- and APO2L-containing vesicles are also present in supernatants from PHA-activated fresh human PBMC. These observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Once a cellular immune response has taken place, most activated T cells are eliminated by a process called activation-induced cell death (AICD)3to prevent potential autoimmune damage (1). Interaction of Fas with its natural ligand, FasL, mediates AICD of T cell hybridomas (2, 3, 4) and mature normal T lymphocytes (5, 6), playing a key role in regulation of peripheral tolerance and lymphocyte homeostasis (7, 8). Other molecules can also be involved in this process; so AICD of mature CD8+ T cells is mediated by the TNF/TNFR system (9).

Fas is a type I transmembrane glycoprotein that belongs to the TNF receptor superfamily and induces apoptosis when cross-linked with agonist Abs or by FasL (10, 11). FasL is a type II membrane glycoprotein, a member of the TNF family, and is primarily expressed in activated T cells (11, 12).

APO2L (also known as TNF-related apoptosis-inducing ligand (TRAIL)) is another recently discovered member of the TNF family, which induces cell death in a Fas-independent fashion (13, 14). Several receptors for APO2L have been cloned that also belong to the TNF receptor family, some of which transduce death signals, while others act as decoy receptors (see Ref. 15 for a recent review). We have recently shown the implication of APO2L in AICD of Jurkat and normal human T cell blasts (16).

It was demonstrated that culture supernatants from anti-TCR/CD3-activated Jurkat cells (2) or FasL-transfected COS cells (17) induced apoptosis in Fas-expressing cells. A soluble 26-kDa protein, present in supernatants from FasL-transfected COS cells or PMA/ionomycin-activated human T cells (18, 19) was identified as the soluble form of human FasL (sFasL). Similarly to TNF, it has been reported that the cleavage of human FasL and generation of sFasL by T cells is mediated by Zn2+-dependent metalloproteinases (19, 20, 21). In a previous work we demonstrated that AICD of the T cell leukemia Jurkat was mainly due to the rapid release of preformed FasL to the culture medium upon TCR stimulation (22), which was attributed to the generation of its soluble form. We later characterized the additional contribution of secreted APO2L to AICD in Jurkat and normal human T cell blasts (16). In the present work we present evidence of FasL and APO2L being released to the supernatant in the form of whole, nonproteolyzed proteins, associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100–200 nm in diameter. These microvesicles are released from Jurkat and normal human T cell blasts shortly after PHA stimulation (<=1 h), well before the cell enters apoptosis. Our observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture

The human T cell leukemia Jurkat (American Type Culture Collection, Manassas, VA; clone E6.1) was cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (hereafter, complete medium). Human PBMC were obtained from the blood of healthy donors by Ficoll-Paque density centrifugation, as indicated previously (23). In some experiments PBMC were activated by treatment with PHA (50 µg/ml) for 15 min, washing, and incubation in complete medium supplemented with 30 U/ml of human rIL-2 for 24 h. T cell blasts were generated as follows. PBMC (2 x 106 cells/ml) were pulse-stimulated with PHA for 15 min and washed, as indicated. Then cells were resuspended in complete medium supplemented with rIL-2 and cultured for 6 days (day 6 T cell blasts), with medium changes every 48 h.

Cytotoxicity assays

Sensitivity to AICD was tested by pulse stimulation with 50 µg/ml PHA for 5 min and incubation for 16 h as previously described (22). Cell viability was determined by a modification of the MTT reduction method (24, 25) and is expressed as percentage of the corresponding values for control cells. The toxicity of supernatants from PHA-stimulated cells was tested as previously described (22). Briefly, cells (4 x 105 cells/ml) were prestimulated with 50 µg/ml PHA for 5 min; after PHA removal by centrifugation and washing, they were resuspended in complete medium and cultured at 37°C for different times. Cells were recovered after brief centrifugation, and supernatant cytotoxicity was assayed on nonactivated Jurkat cells (5 x 104) resuspended in 100-µl aliquots of supernatants. After incubation for 16 h, cytotoxicity was determined by the MTT assay or trypan blue staining. Supernatants from untreated cells were used as controls.

Determination of mitochondrial membrane potential ({Delta}{Psi}m)

To evaluate {Delta}{Psi}m, 2.5 x 105 cells were incubated with the specific probe DiOC6(3) (40 nM; Molecular Probes, Eugene, OR) at 37°C for 15 min essentially as described previously (26). As a negative control, cells were treated in parallel cultures with the protonophore carbonyl cyanide m-chloro-phenylhydrazone (50 µM). After incubations, cells were diluted to 1 ml with PBS, and fluorescence intensity was analyzed by flow cytometry.

Immunoblotting

Detection of FasL in cell lysates or culture supernatants was performed with a rabbit polyclonal Ab (N-20, Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes the intracellular N-terminal region of FasL, whose specificity was validated in previous studies (16, 27). Rabbit anti-human APO2L polyclonal Ab pAb3 was produced by immunizing rabbits with the peptide CEKALGRKINSWESSRS, corresponding to aa 144–160 of the extracellular region of APO2L. This Ab recognized a 41-kDa band in extracts from Jurkat and human T cell blasts (16) and was provided by Dr. Avi Ashkenazi, Genentech (South San Francisco, CA). Analysis of caspase-3 activation was performed using an mAb (clone 19, Transduction Laboratories, Lexington, KY) raised against the 32-kDa proenzyme as indicated previously (28). Nonactivated cells (5 x 106 in 100 µl) or cells prestimulated with 50 µg/ml PHA for 5 min were resuspended in fresh culture medium and incubated at 37°C for different times (ranging from 30 min to 24 h). Then cells were lysed at 4°C in a buffer containing 1% Triton X-100 and protease and phosphatase inhibitors (22). The presence of FasL and APO2L in supernatants was analyzed as follows. Cells (2 x 107) were resuspended in 1 ml of RPMI 1640 and treated with 50 µg/ml of PHA for 5 min. Then, cells were washed, resuspended in 60 µl of RPMI 1640 medium, and cultured for 1 h. Aliquots of culture supernatants were diluted 10-fold, and cytotoxicity was assayed on nonactivated Jurkat cells as described. Diluted supernatants exhibited a toxicity comparable to those obtained from 4 x 105 cells/ml, as indicated above. For protein analysis, supernatants were mixed with 30 µl of 3x SDS sample buffer and subjected to 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and serially incubated with 1 µg/ml of anti-FasL, anti-APO2L, or anti-CPP32 Abs and 0.2 µg/ml of the corresponding secondary Abs coupled to alkaline phosphatase and revealed with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium.

Ultracentrifugation

Aliquots (600 µl) of supernatants from culture medium were obtained by low speed centrifugation (800 x g, 10 min) from 2 x 108 cells after treatment with PHA for different periods and further clarified by centrifugation at 10,000 x g for 20 min to eliminate cell debris. Supernatants from PHA-stimulated or control cells were diluted to a final volume of 6 ml (~10-fold), and an aliquot was used for cytotoxicity testing. Supernatants were ultracentrifuged at 100,000 x g for 18 h as previously described (29). Supernatant from ultracentrifugation was recovered, and the pellet was resuspended in 1.5 ml of RPMI 1640. The toxicity of both fractions on nonactivated Jurkat cells was analyzed by the MTT assay, and the presence of FasL and APO2L was determined by immunoblotting.

Flow cytometric analysis

Labeling of microvesicles was performed by directly adding 4 µl of a 1 mg/ml solution of the FITC-labeled anti-FasL mAb (H11, Bender MedSystems) to 100 µl of microvesicle suspension and incubation at 4°C for 1 h. For APO2L labeling, 1 µl of a 5 mg/ml solution of the anti-APO2L mAb 5C2 (16), provided by Dr. Avi Ashkenazi (Genentech), was added to 100 µl of microvesicle suspension and incubated under similar conditions. Then, 1 µl of a 1 mg/ml solution of an FITC-labeled goat anti-mouse IgG (Caltag, South San Francisco, CA) was added to the microvesicle suspension and incubated for 30 min at 4°C. Labeled microvesicle suspension was diluted to 1 ml with PBS, and fluorescence was analyzed by flow cytometry (EPICS XL-MCL, Becton Dickinson, Mountain View, CA). Microvesicles were gated using calibrated polystyrene latex beads (0.05 and 0.1 µm) bound to a yellow-green fluorescent dye (Sigma, St. Louis, MO). For intracellular labeling, cells (1 x 106 cells in 50 µl) were permeabilized by treatment with 1% paraformaldehyde in PBS for 10 min, washed twice with 0.03% saponin in PBS, labeled with 1 µl of a 1 mg/ml anti-FasL-FITC in 0.3% saponin in PBS containing 5% FCS, washed, and analyzed by flow cytometry.

Scanning electronic microscopy

Samples of microvesicles for analysis by scanning electron microscopy were prepared essentially as previously described (30). Glutaraldehyde was added to supernatants from control or PHA-activated T cells up to a final concentration of 1%. A drop of these solutions was placed onto microscope glass slides previously treated with 3-aminopropyl-triethoxysilane (Sigma) for 30 min. Then, samples were fixed with 1% OsO4 for 30 min, washed with PBS, dehydrated in a graded series of ethanol (30–100%), critical point dried in a CO2 system, mounted on specimen, and gold coated in a sputtering device. Samples were examined using a Zeiss scanning electron microscope (DSM 940A, Zeiss, New York, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FasL and APO2L are present in supernatants from PHA-stimulated human T cells as nonproteolyzed proteins.

In a previous study we demonstrated the presence of significant amounts of FasL in the cytoplasm of Jurkat cells. Within 15 min after anti-CD3 or PHA stimulation, these cells begin to release molecules toxic for nonactivated Jurkat cells. A first peak of toxic activity was observed after 1 h of stimulation, diminishing thereafter and giving a second peak at around 7 h. The first peak was not blocked by inhibitors of transcription or translation, while the second peak was due to de novo mRNA and protein synthesis. The toxicity associated with supernatants was completely prevented by a combination of neutralizing anti-Fas and anti-APO2L Abs (16, 22). The presence of preformed FasL in several types of activated human T cells has been confirmed in a recent study (31).

Previous reports indicated that FasL, like TNF, could be processed to a soluble 26-kDa form through the action of metalloproteases (18, 19, 20, 21), and it was assumed that supernatant’s toxicity was due to the generation of soluble FasL. In fact, in Jurkat cells, the divalent cation chelator 1,10-phenanthroline, which was shown to inhibit FasL proteolytic processing (20), prevented the release of toxicity to the culture medium (22). However, phenanthroline was also highly toxic for Jurkat cells (22).

To verify the molecular form under which FasL and APO2L were liberated, Jurkat cells were pulsed-stimulated with 50 µg/ml PHA for 5 min, washed, and cultured in fresh medium for 1 h (22). Supernatants from 1-h stimulated Jurkat cells were collected, and the presence of FasL was analyzed by immunoblot using a polyclonal Ab raised against the intracellular N-terminal region of FasL. This Ab does not recognize the C-terminal region of the protein, corresponding to soluble FasL. In whole cell extracts, this Ab recognizes a major band of 40 kDa (16, 22), and other bands at lower molecular mass (Fig. 1Goa, lane 1). Between them, a 35- to 36-kDa broad band could correspond to partially glycosylated forms of FasL as indicated previously (22). Surprisingly, a readily detectable band of 40 kDa, corresponding to the m.w. of the nonproteolyzed protein, was observed in supernatants from stimulated Jurkat cells (Fig. 1Goa, lane 3). No immunoreactivity was detected in supernatants from nonactivated Jurkat cells (Fig. 1Goa, lane 2). This result was consistently obtained in at least 10 individual experiments, suggesting that preformed FasL could have been released in intact form.



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  		FIGURE 1. Western blot analysis of FasL and APO2L in Jurkat cells and their culture supernatants. a, Anti-FasL immunoblot of: lane 1, Jurkat cells (2 x 106 cells); lane 2, supernatant from 2 x 107 nonstimulated Jurkat cells; lane 3, supernatant from 2 x 107 Jurkat cells stimulated with 50 µg/ml PHA for 5 min, washed, and cultured in RPMI 1640 for 1 h at 37°C. b, Lanes 1–3, Anti-APO2L immunoblot of the same samples as in a. Lane 4, Soluble rAPO2L (100 ng) was included in the gel to verify the specificity of the anti-APO2L Ab. The positions of the molecular mass markers are indicated on the left. The positions of FasL, rAPO2L, and native APO2L are marked by arrows on the right. The gels shown are representative of 10 and 5 experiments for FasL and APO2L, respectively.

 
Recently, we have characterized the intracellular presence of APO2L in Jurkat cells as well as in normal human T cell blasts. Upon TCR stimulation, APO2L is released to the culture medium, contributing to the AICD process, although to a lesser extent than FasL (16). The anti-APO2L polyclonal Ab pAb3, raised against the extracellular region of APO2L, preferentially recognizes a 41-kDa band corresponding to the fully glycosylated protein and, to a lesser extent, a 35-kDa band corresponding to the predicted molecular mass of the peptide moiety of APO2L (16) (Fig. 1Gob, lane 1). The 41-kDa band, but not the 35-kDa band, was readily detected also in supernatants from PHA-stimulated Jurkat cells (Fig. 1Gob, lane 3). The 24-kDa form of soluble APO2L (13) was not detected in supernatants, although this Ab recognized soluble recombinant APO2L by immunoblot (Fig. 1Gob, lane 4). These data indicate that preformed APO2L is released by Jurkat cells upon PHA stimulation mainly in its whole, nonproteolyzed form.

It is worth noting that at this time no morphological signs of apoptosis were observed in Jurkat cells, which showed no trypan blue staining, the absence of membrane blebbing, and normal nuclear morphology. One of the first biochemical events noticed in the apoptotic process is the loss of mitochondrial membrane potential ({Delta}{Psi}m), which is followed by activation of effector caspases (26). As shown in Fig. 2Goa, no significant changes in {Delta}{Psi}m were observed in Jurkat cells stimulated with PHA for 1 h, as measured by the specific probe DiOC6 (3) (26). Activation of caspase-3 was analyzed by Western blot with an Ab that preferentially recognizes the 32-kDa proenzyme. Caspase-3 activation is associated with a loss in the amount of the proenzyme and the apparition of active proteolytic fragments (28). Using this approach, we did not detect any significant caspase-3 activation after treatment of Jurkat cells with PHA for 1 h; this activation was evident only at longer (8-h) activation times (Fig. 2Gob). By 8 h other apoptotic events, such as chromatin condensation, were also observed (data not shown).



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  		FIGURE 2. Effect of pulse stimulation with PHA on {Delta}{Psi}m and caspase-3 activation. Jurkat cells were stimulated with PHA (50 µg/ml) for 5 min, washed, and incubated in complete medium for 1 h. a, {Delta}{Psi}m was measured by flow cytometric analysis of cells stained with DiOC6(3). R1 gating corresponds to cells with high {Delta}{Psi}m values, characteristic of viable cells, and R2 corresponds to cells with low {Delta}{Psi}m values. Cells displaying low {Delta}{Psi}m (negative control) were obtained by treatment with 50 µM of the protonophore carbonyl cyanide m-chloro-phenylhydrazone (CCCP). Numbers on R2 correspond to the percentage of cells with low {Delta}{Psi}m values. The experiment shown is representative of three different assays, in which the mean percentage of cells in R2 was 2.4 ± 1.3% for control cells and 4.8 ± 1.8% for PHA-treated cells, giving a difference that is not statistically significant. b, Western blot analysis of caspase-3 activation. Jurkat cells were left untreated or were pulse-stimulated with PHA, as indicated, for 1 or 8 h. The positions of the molecular mass markers and of the 32-kDa protein band are indicated on the left. The experiments shown are representative of four different assays.

 
Because Jurkat is a T cell leukemia, the observations made could be a specific characteristic of its particular AICD process. To evaluate the physiological significance of our findings, we generated T cell blasts from PBMC obtained from the blood of healthy donors and followed a similar experimental approach. Day 6 blasts, obtained by PHA/IL-2 stimulation, were restimulated with 50 µg/ml PHA for 5 min, washed, resuspended in fresh complete medium, and cultured for 1 h. T cell blasts expressed intracellular FasL and APO2L, as previously described (16) and shown in Fig. 3Go, a and b. As shown in Fig. 3Go, most FasL and APO2L present in supernatants from PHA-activated, normal T cell blasts were also in their whole, nonproteolyzed forms of 40 and 41 kDa, respectively. In the case of FasL, a 12-kDa band was consistently observed only in supernatants from activated T cell blasts, but not in whole cell extracts. This molecular mass corresponds to the intracellular N-terminal portion of FasL after proteolytic cleavage of the extracellular region, and it was not detected in supernatants from PHA-pulsed Jurkat cells (see Fig. 1Goa, lane 3). Soluble APO2L (Mr, 24 kDa) was not detected by immunoblot in supernatants from PHA-activated day 6 T cell blasts (Fig. 3Gob, lane 3).



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  		FIGURE 3. Western blot analysis of day 6 T cell blasts and their culture supernatants. a, Lane 1, Anti-FasL immunoblot of day 6 T cell blasts (2 x 106 cells); lane 2, supernatants from 2 x 107 control T cell blasts; lane 3, supernatants from 2 x 107 T cell blasts restimulated with 50 µg/ml PHA for 5 min, washed, and incubated for 1 h at 37°C. b, Anti-APO2L immunoblot of samples similar to those in a. The positions of the molecular mass markers are indicated on the left. The positions of FasL, its 12-kDa N-terminal proteolytic fragment, and APO2L are marked by arrows on the right. The gels shown are representative of four different experiments for both FasL and APO2L.

 
Cytochalasin B inhibits the release of cytotoxic molecules from PHA-stimulated human T cells

The fact that FasL and APO2L were released from activated T cells in their whole, nonproteolyzed form was hardly compatible with their known character of type II integral membrane proteins. One possibility would be that FasL and APO2L were released associated with small membrane structures. The functional release of small membrane structures (microvesicles) has been described during platelet, dendritic cells, and B cell activation (32, 33, 34). In the case of platelets, microvesicle generation is dependent on actin cytoskeleton reorganization and is inhibited by cytochalasins (35). On the other hand, endoplasmic reticulum protein synthesis, vesicular traffic to the plasma membrane of integral proteins, and metalloprotease activity are not dependent on microfilament integrity (35). In consequence, we tested in our model the effect of cytochalasin B on the toxicity of supernatants from PHA-activated Jurkat T cells. As shown in Fig. 4Go, treatment with cytochalasin B blocked the release of toxic molecules to the 1-h supernatant from Jurkat cells pulse-stimulated with PHA.



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  		FIGURE 4. Effect of cytochalasin B on the release of toxic molecules to supernatants. Jurkat cells were treated with 50 µg/ml PHA for 5 min, washed, and incubated at 37°C for 1 h in the presence (1 h ss + Cyt B) or the absence (1 h ss) of 5 µg/ml cytochalasin B. Culture supernatants (ss) from those cells were collected, and their toxicity was tested using nonactivated Jurkat cells as targets. Cell viability was determined after incubation for 16 h by the MTT reduction assay and are the mean ± SD of quadruplicate determinations in three different experiments. As a control, culture supernatants from Jurkat cells treated with 5 µg/ml cytochalasin B for 1 h, without PHA stimulation, were collected, and their toxicity was tested in parallel (Cyt B).

 
Released FasL and APO2L are associated with microvesicles

To verify the hypothesis that FasL and APO2L are released from PHA-activated human T cells associated with microvesicles, 1-h supernatants from Jurkat cells pulse-stimulated with PHA were subjected to ultracentrifugation. It has been reported that microvesicles with an approximate size of 100 nm, generated from endotoxin-stimulated monocytes (29), can be separated by ultracentrifugation. We used a similar protocol and analyzed next the presence of FasL and APO2L by immunoblot and the toxicity of the different fractions obtained. As shown in Fig. 5Goa, the diluted supernatant induced the death of 25% of the nonactivated Jurkat cells before ultracentrifugation. The ultracentrifugation supernatant showed no toxicity, while the pellet fraction caused ~60% cell death (Fig. 5Goa). The toxicity in the pellet fraction was higher than that observed in the initial supernatant before ultracentrifugation because of the concentration of vesicles in the pellet (see Materials and Methods). Similar results were obtained with supernatants from PHA-activated day 6 T cell blasts (Fig. 5Goa). Again, the toxicity patterns were associated with the presence of the whole FasL and APO2L in the pellet fractions, but not in the ultracentrifugation supernatants, as detected by immunoblot (Fig. 5Go, b and c, for FasL and APO2L, respectively). These results suggest that bioactive FasL and APO2L are released from PHA-activated human T cells associated with small membrane vesicles. The 12-kDa band was consistently detected in anti-FasL immunoblots of supernatants from PHA-pulsed T cell blasts, as shown in Fig. 3Goa. This band remained associated with the particulate fraction after ultracentrifugation (Fig. 5Gob, right panel, lane 3). The presence of this proteolytic fragment of FasL in the particulate fraction of the supernatants implicates that a considerable portion of FasL is proteolyzed to its soluble form upon normal T cell activation, in agreement with previous studies (18, 19). However, only FasL associated with microvesicles is bioactive, because the supernatants of the ultracentrifugation completely lost its toxic potential (Fig. 5aGo).



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  		FIGURE 5. Ultracentrifugation of supernatants from Jurkat and T cell blasts. a, Six milliliters of supernatants (SS) from Jurkat cells or normal human T cell blasts pulse-stimulated with PHA, as indicated, were subjected to ultracentrifugation at 100,000 x g for 18 h. Supernatants of the ultracentrifuges were recovered (Ultra-ss), and the pellet was resuspended in 1.5 ml of culture medium (Ultra-pellet). The toxicities of the initial supernatant (white bars), the ultra-ss (filled bars), and the ultrapellet (hatched bars) were assayed on nonactivated Jurkat cells as indicated. Cell viability was determined by the MTT reduction method, and the results shown correspond to the same supernatants analyzed by immunoblot (below). b, Anti-FasL immunoblot of the same supernatants as those in a before ultracentrifugation (lane 1) and of the supernatant (lane 2) or the resuspended pellet (lane 3) after their ultracentrifugation. c, Anti-APO2L immunoblot of the same supernatants as that in a before ultracentrifugation (lane 1) and of the supernatant (lane 2) or of the resuspended pellet (lane 3) after their ultracentrifugation. Left panels correspond to Jurkat cell supernatants, and right panels correspond to T cell blasts supernatants. The positions of the molecular mass markers are indicated on the left. The positions of FasL and APO2L are marked by arrows. The experiment shown is representative of four different experiments for Jurkat and three for T cell blasts.

 
As an initial approach to calibrate the sizes of these microvesicles, toxic supernatants were gravity-filtered through 0.22- and 0.1-µm pore size filters, and the toxicity of the filtered solution was tested on nonactivated Jurkat cells. Filtration through the 0.1-µm pore size filter eliminated most of the toxicity from the supernatants, but this was not the case after filtration through the 0.22-µm pore size filter, indicating that the size of the particles should range between 100–220 nm.

Scanning electronic microscopy of microvesicles

The presence of microvesicles in supernatants from PHA-activated Jurkat and T cell blasts was further analyzed by scanning electronic microscopy. As a control, a preparation from supernatants of nonactivated cells was also analyzed. As shown in Fig. 6GoA, very few particulate objects could be observed in these control preparations. In contrast, a considerable amount of round vesicles was clearly detected in preparations obtained from PHA-activated Jurkat (Fig. 6GoB) or day 6 T cell blasts (Fig. 6GoC). Diameter size of these vesicles ranged between 100–200 nm. In some instances, aggregates of several microvesicles could be observed (Fig. 6Go, B and C).



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  		FIGURE 6. Scanning electron microscopy of microvesicles secreted from PHA-stimulated Jurkat and human T cell blasts. Culture supernatants from control Jurkat cells (A), Jurkat cells pulse-stimulated with PHA for 1 h (B), and human T cell blasts pulse-stimulated with PHA for 1 h (C), as indicated in the text, were analyzed by scanning electron microscopy in a Zeiss DSM 940A microscope and photographed (bar = 500 nm). Original magnification, x20,000. The pictures shown are representative of two different experiments.

 
Detection of FasL and APO2L in microvesicles by flow cytometry

By using calibrated fluorescent latex beads, we optimized the gating conditions, allowing the simultaneous flow cytometric analysis of both cells and microvesicles (Fig. 7Goa) and the analysis of microvesicles in supernatants. Significant percentages of microvesicles found in supernatants from PHA-stimulated Jurkat (Fig. 7Go, d and g) or day 6 T cell blasts (Fig. 7Go, e and h) contained both FasL and APO2L, according to their toxic potential. The cytotoxicity of these supernatants on nonactivated Jurkat cells was substantially attenuated by anti-Fas and anti-APO2L neutralizing Abs (not shown), as previously described (16, 22). FasL- and APO2L-containing microvesicles were also detected in supernatants from PBMC committed to proliferate by treatment with PHA/IL-2, although the proportion of positive vesicles was lower (Fig. 7Go, f and i), according to the lower toxicity of these supernatants (data not shown) (16). Of note, we could not detect the surface expression of FasL and APO2L on Jurkat, normal T cells, PHA-activated PBMC, or T cell blasts. However, these cells were stained by the anti-FasL and anti-APO2L mAbs after saponin permeabilization (Fig. 7Gob and data not shown). This result agrees with the reported intracellular FasL localization in Jurkat cells by immunofluorescence (22). No FasL- or APO2L-bearing vesicles were detected in supernatants from unstimulated Jurkat cells and resting PBMC. The presence of FasL and APO2L in the cell cytoplasm and in released microvesicles, but not in plasma membrane, suggests that probably the origin of microvesicles is intracellular, rather than plasma membrane.



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  		FIGURE 7. Flow cytometric analysis of FasL and APO2L expression in microvesicles secreted by PHA-stimulated Jurkat or T cell blasts. a, Light-scatter patterns of microvesicles (lower box) secreted from Jurkat cells pulse-stimulated with PHA and cells (upper box). b, FasL labeling of permeabilized Jurkat cells (black peak), performed with the anti-FasL mAb H11, compared with labeling using an irrelevant FITC-labeled IgG (white peak). c, Labeling of Jurkat-derived microvesicles using an irrelevant IgG plus FITC-labeled secondary Ab (negative control). d–i, FasL; d–f, or APO2L; g–i, labeling of microvesicles obtained from 1-h supernatants of Jurkat cells (d and g) and T cell blasts pulse-stimulated with PHA (e and h) as indicated or from fresh PBMC pulse-stimulated with PHA for 15 min and cultured in IL-2-supplemented medium for 24 h (f and i). The diagrams shown are representative of at least five different experiments for each experimental condition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The generation of human sFasL has been characterized in cell transfectants and in human PBMC activated with PMA/ionomycin (18, 19). It has been also demonstrated that, similarly to TNF, sFasL can be produced through the action of matrix metalloproteinases (19, 20, 21). Human sFasL was shown to be active against murine W4 cells (18) and human Jurkat cells (36), which are highly susceptible to Fas-induced apoptosis. Also, recombinant soluble APO2L, generated from the cDNA sequence corresponding to the extracellular region of the protein (13), showed efficient toxicity against Jurkat cells (16).

In the present work we show that FasL and APO2L, two main mediators of AICD in overactivated T cells, can be released from activated T cells in the form of whole, membrane-bound proteins. FasL and APO2L were released from PHA-activated T cells associated with microvesicles with a size ranging between 100–200 nm. That these microvesicles are distinct from apoptotic blebs is supported by several observations. First, we have shown that cells that actively produce microvesicles after 1 h of PHA stimulation, do not show any significant loss of {Delta}{Psi}m or activation of caspase-3, the main effector caspase, or any morphological sign of apoptosis. The loss of {Delta}{Psi}m is an early biochemical event that occurs during apoptosis, preceding effector caspase activation (26). On the contrary, the blebbing process typical of apoptosis is a late event and occurs after caspase-3 activation (37). In addition, FasL- and APO2L-positive microvesicles have been detected in culture supernatants from fresh PBMC stimulated with PHA and IL-2 for 24 h (Fig. 7Go). These cells are not committed to apoptosis, and the result of their stimulation is proliferation and generation of T cell blasts (16, 38). Moreover, the typical apoptotic blebs are much greater in size (1–3.4 µm) (39).

The results presented here agree with recent observations indicating that the membrane-bound form of FasL is the functional form of the molecule; the toxic activity of the cleaved soluble form (sFasL) is much lower (40, 41, 42). From these studies, it was also inferred that FasL proteolytic processing was a mechanism of functional down-regulation (41). In fact, the toxic activity of the culture supernatant from FasL-transfected Neuro-2a cells was associated in one of these studies with the constitutive secretion to the culture medium of FasL-bearing membrane fragments or vesicles (42). A similar constitutive secretion of Fas- and FasL-positive vesicles has been recently observed in some tumor cell lines (43). By contrast, the microvesicle secretion process described in the present work is clearly regulated by the activation state of T cells.

Because the level of AICD induced by immobilized anti-CD3 Abs is much lower than that induced by PHA (2, 16, 22), this lectin was used in this study rather than anti-CD3 Abs as an optimal AICD inducer. In this connection, it has been recently observed that efficient T cell activation needs the formation of supramolecular activation clusters between regions of the plasma membrane of the APC enriched in MHC molecules and regions of the T cell plasma membrane enriched in Ag receptors and other costimulatory molecules such as LFA-1 (44). Anti-TCR/CD3 Abs, although triggering the same biochemical events as Ag, are in many instances not enough to induce full functional responses in T cells, probably because they are unable to induce the formation of these supramolecular structures (44). In this sense, PHA would probably better mimic the role of APC than anti-CD3 Abs, by inducing the cross-linking and colocalization of glycoproteins important for signal transduction in addition to TCR/CD3.

Generation of functional microvesicles has been described in platelets, erythrocytes, monocytes, dendritic cells, and B cells, although in processes not related to AICD. Platelets generate microvesicles when activated with thrombin and collagen or calcium ionophores, and their probable function is to increase the contact surface and the density of binding sites for prothrombinase enzyme complexes, favoring blood coagulation (32, 35). Erythrocytes secrete similar microvesicles when activated with calcium ionophores, which are also associated with a procoagulant activity (45). Monocytes generate microvesicles with a size between 0.05–0.3 µm upon endotoxin stimulation, also favoring blood coagulation (29).

More related with our present observations may be the degranulation process that takes place during CTL effector function. It has been described that the preformed cytoplasmic CTL granules are stored inside a post-Golgi, prelysosomal multivesicular structure called secretory lysosomes (46). After CTL activation by Ag, the external membrane of this compartment can fuse with the plasma membrane, leaving at least a portion of the small internal vesicles to be secreted intact (46). More recently, this type of multivesicular bodies has been detected in professional APC, and the secretion of microvesicles expressing high amounts of Ag-loaded MHC class II, named exosomes, has been described in B cells (33) and in dendritic cells (34). Exosomes contain membrane proteins correctly oriented, and this has led to the proposal that multivesicular bodies are formed by inward vesiculation of the limiting membrane of an organelle with the characteristics of a lysosomal compartment (33). The existence of these secretory lysosomes seems to be a special characteristic of hemopoietic cells (47). The microvesicles characterized in the present study are secreted intact from T cells shortly after activation, suggesting that they could be preformed inside the cells. Because microvesicles contain FasL and APO2L correctly inserted in their membranes, they could be generated as MHC class II-containing exosomes in APC. Obviously, further studies are needed to better characterize their generation pathway. In this connection, the presence of preformed FasL in lysosomal compartments of activated human T cells that migrate to the plasma membrane upon short term stimulation with ionomycin has been reported in a very recent work (31).

Our present data unveil a mechanism of potential relevance in the regulation of the immune system through which apoptosis inducers such as FasL and APO2L are released from activated T cells in their membrane-bound forms, preserving their whole cytotoxic potential. Microvesicle-bound FasL and APO2L have a very high cross-linking efficiency, even greater than that of cell surface molecules. Microvesicles offer a great surface increase for the expression of these ligands if compared with the surface of a single cell. On the other hand, this form of release offers evident advantages over the secretion of soluble proteins. FasL- and APO2L-containing microvesicles have a lower mobility than their corresponding soluble forms, thus keeping a high local concentration while limiting their action to close neighbor cells.


   Acknowledgments
 
We thank Dr. Avi Ashkenazi (Genentech, South San Francisco, CA) for providing anti-APO2L Abs pAb3 and 5C2 and for critical reading of the manuscript. We thank Dr. Jacek Wierzchos and Xavier Calomarde (Electron Microscopy Service, University of Lleida) for their technical expertise in scanning electron microscopy; Dr. José Antonio Brieva (Hospital Puerta del Mar, Cádiz) for his advice regarding ultracentrifugation of microvesicles; and Dr. Ramón Merino (University of Cantabria) for the protocol for flow cytometric analysis of permeabilized cells.


   Footnotes
 
1 This work was supported by Grant PB 95-0079 from Dirección General de Enseñanza Superior (Spain). M.J.M.L. and S.G. were supported by fellowships from Asociación Española Contra el Cancer. Back

2 Address correspondence and reprint requests to Dr. Alberto Anel, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, E-50009 Zaragoza, Spain. E-mail address: Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; sFasL, soluble FasL; APO2L, APO2 ligand; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; {Delta}{Psi}m, mitochondrial membrane potential. Back

Received for publication January 29, 1999. Accepted for publication May 20, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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