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*
Departamento de Bioquímica y Biología Molecular y Cellular, Facultad de Ciencias, and
Servicio de Inmunología, Hospital Clínico Universitario, Universidad de Zaragoza, Zaragoza, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
(
m)
To evaluate 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
a, 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. 1a,
lane 3). No immunoreactivity was detected in supernatants
from nonactivated Jurkat cells (Fig. 1a, 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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b, lane 1). The 41-kDa band, but not the 35-kDa
band, was readily detected also in supernatants from PHA-stimulated
Jurkat cells (Fig. 1b, 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. 1b, 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
(m), which is followed by activation of effector caspases
(26). As shown in Fig. 2a, no significant changes in
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. 2b). By 8 h other
apoptotic events, such as chromatin condensation, were also observed
(data not shown).
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,
a and b. As shown in Fig. 3, 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. 1a, lane 3).
Soluble APO2L (Mr, 24 kDa) was not
detected by immunoblot in supernatants from PHA-activated day 6 T cell
blasts (Fig. 3b, lane 3).
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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. 4, 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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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. 5a,
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. 5a). 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. 5a). 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. 5, 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. 3a. This band remained associated
with the particulate fraction after ultracentrifugation (Fig. 5b, 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. 5a).
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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. 6A, 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. 6B) or day 6 T cell blasts (Fig. 6C). Diameter
size of these vesicles ranged between 100–200 nm. In some instances,
aggregates of several microvesicles could be observed (Fig. 6, B and C).
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By using calibrated fluorescent latex beads, we optimized the
gating conditions, allowing the simultaneous flow cytometric analysis
of both cells and microvesicles (Fig. 7a) and the analysis of
microvesicles in supernatants. Significant percentages of microvesicles
found in supernatants from PHA-stimulated Jurkat (Fig. 7, d
and g) or day 6 T cell blasts (Fig. 7, 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. 7, 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. 7b
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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
m or activation of caspase-3, the main
effector caspase, or any morphological sign of apoptosis. The loss of
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. 7). 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.
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Acknowledgments |
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Footnotes |
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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:
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; m, mitochondrial membrane potential.
Received for publication January 29, 1999. Accepted for publication May 20, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 W. Baum, V. Kirkin, S. B. M. Fernandez, R. Pick, M. Lettau, O. Janssen, and M. Zornig Binding of the Intracellular Fas Ligand (FasL) Domain to the Adaptor Protein PSTPIP Results in a Cytoplasmic Localization of FasL J. Biol. Chem., December 2, 2005; 280(48): 40012 - 40024. [Abstract] [Full Text] [PDF]
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 S. L. Straszewski-Chavez, V. M. Abrahams, and G. Mor The Role of Apoptosis in the Regulation of Trophoblast Survival and Differentiation during Pregnancy Endocr. Rev., December 1, 2005; 26(7): 877 - 897. [Abstract] [Full Text] [PDF]
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R. Alonso, M. C. Rodriguez, J. Pindado, E. Merino, I. Merida, and M. Izquierdo Diacylglycerol Kinase {alpha} Regulates the Secretion of Lethal Exosomes Bearing Fas Ligand during Activation-induced Cell Death of T Lymphocytes J. Biol. Chem., August 5, 2005; 280(31): 28439 - 28450. [Abstract] [Full Text] [PDF]
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S.-H. Kim, E. R. Lechman, N. Bianco, R. Menon, A. Keravala, J. Nash, Z. Mi, S. C. Watkins, A. Gambotto, and P. D. Robbins Exosomes Derived from IL-10-Treated Dendritic Cells Can Suppress Inflammation and Collagen-Induced Arthritis J. Immunol., May 15, 2005; 174(10): 6440 - 6448. [Abstract] [Full Text] [PDF]
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J. M. Martin-Fernandez, J. A. Cabanillas, M. Rivero-Carmena, E. Lacasa, J. Pardo, A. Anel, P. R. Ramirez-Duque, F. Merino, C. Rodriguez-Gallego, and J. R. Regueiro Herpesvirus saimiri-transformed CD8+ T cells as a tool to study Chediak-Higashi syndrome cytolytic lymphocytes J. Leukoc. Biol., May 1, 2005; 77(5): 661 - 668. [Abstract] [Full Text] [PDF]
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A. Bosque, J. Pardo, M{a} J. Martinez-Lorenzo, M. Iturralde, I. Marzo, A. Pineiro, M{a} A. Alava, J. Naval, and A. Anel Down-regulation of normal human T cell blast activation: roles of APO2L/TRAIL, FasL, and c- FLIP, Bim, or Bcl-x isoform expression J. Leukoc. Biol., April 1, 2005; 77(4): 568 - 578. [Abstract] [Full Text] [PDF]
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C. Monari, E. Pericolini, G. Bistoni, A. Casadevall, T. R. Kozel, and A. Vecchiarelli Cryptococcus neoformans Capsular Glucuronoxylomannan Induces Expression of Fas Ligand in Macrophages J. Immunol., March 15, 2005; 174(6): 3461 - 3468. [Abstract] [Full Text] [PDF]
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N. Askenasy, E. S. Yolcu, I. Yaniv, and H. Shirwan Induction of tolerance using Fas ligand: a double-edged immunomodulator Blood, February 15, 2005; 105(4): 1396 - 1404. [Abstract] [Full Text] [PDF]
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 L. Frangsmyr, V. Baranov, O. Nagaeva, U. Stendahl, L. Kjellberg, and L. Mincheva-Nilsson Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level Mol. Hum. Reprod., January 1, 2005; 11(1): 35 - 41. [Abstract] [Full Text] [PDF]
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 N Dainiak, S K Schreyer, and J Albanese The search for mRNA biomarkers: global quantification of transcriptional and translational responses to ionising radiation Br. J. Radiol., January 1, 2005; Supplement_27(1): 114 - 122. [Abstract] [Full Text] [PDF]
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A. E. Morelli, A. T. Larregina, W. J. Shufesky, M. L. G. Sullivan, D. B. Stolz, G. D. Papworth, A. F. Zahorchak, A. J. Logar, Z. Wang, S. C. Watkins, et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells Blood, November 15, 2004; 104(10): 3257 - 3266. [Abstract] [Full Text] [PDF]
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S. Xiao, U. S. Deshmukh, S. Jodo, T. Koike, R. Sharma, A. Furusaki, S.-s. J. Sung, and S.-T. Ju Novel Negative Regulator of Expression in Fas Ligand (CD178) Cytoplasmic Tail: Evidence for Translational Regulation and against Fas Ligand Retention in Secretory Lysosomes J. Immunol., October 15, 2004; 173(8): 5095 - 5102. [Abstract] [Full Text] [PDF]
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V. M. Abrahams, S. L. Straszewski-Chavez, S. Guller, and G. Mor First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis Mol. Hum. Reprod., January 1, 2004; 10(1): 55 - 63. [Abstract] [Full Text] [PDF]
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 V. M. Abrahams, S. L. Straszewski, M. Kamsteeg, B. Hanczaruk, P. E. Schwartz, T. J. Rutherford, and G. Mor Epithelial Ovarian Cancer Cells Secrete Functional Fas Ligand Cancer Res., September 1, 2003; 63(17): 5573 - 5581. [Abstract] [Full Text] [PDF]
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R. Greil, G. Anether, K. Johrer, and I. Tinhofer Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools J. Leukoc. Biol., September 1, 2003; 74(3): 311 - 330. [Abstract] [Full Text] [PDF]
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 Y. Yang, I. Tikhonov, T. J. Ruckwardt, M. Djavani, J. C. Zapata, C. D. Pauza, and M. S. Salvato Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4+ Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism J. Virol., June 15, 2003; 77(12): 6700 - 6708. [Abstract] [Full Text] [PDF]
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S. A. Renshaw, J. S. Parmar, V. Singleton, S. J. Rowe, D. H. Dockrell, S. K. Dower, C. D. Bingle, E. R. Chilvers, and M. K. B. Whyte Acceleration of Human Neutrophil Apoptosis by TRAIL J. Immunol., January 15, 2003; 170(2): 1027 - 1033. [Abstract] [Full Text] [PDF]
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 G. Andreola, L. Rivoltini, C. Castelli, V. Huber, P. Perego, P. Deho, P. Squarcina, P. Accornero, F. Lozupone, L. Lugini, et al. Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles J. Exp. Med., May 20, 2002; 195(10): 1303 - 1316. [Abstract] [Full Text] [PDF]
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N. Blanchard, D. Lankar, F. Faure, A. Regnault, C. Dumont, G. Raposo, and C. Hivroz TCR Activation of Human T Cells Induces the Production of Exosomes Bearing the TCR/CD3/{zeta} Complex J. Immunol., April 1, 2002; 168(7): 3235 - 3241. [Abstract] [Full Text] [PDF]
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I. Monleon, M. J. Martinez-Lorenzo, L. Monteagudo, P. Lasierra, M. Taules, M. Iturralde, A. Pineiro, L. Larrad, M. A. Alava, J. Naval, et al. Differential Secretion of Fas Ligand- or APO2 Ligand/TNF-Related Apoptosis-Inducing Ligand-Carrying Microvesicles During Activation-Induced Death of Human T Cells J. Immunol., December 15, 2001; 167(12): 6736 - 6744. [Abstract] [Full Text] [PDF]
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D. Siegmund, A. Hausser, N. Peters, P. Scheurich, and H. Wajant Tumor Necrosis Factor (TNF) and Phorbol Ester Induce TNF-related Apoptosis-inducing Ligand (TRAIL) under Critical Involvement of NF-kappa B Essential Modulator (NEMO)/IKKgamma J. Biol. Chem., November 16, 2001; 276(47): 43708 - 43712. [Abstract] [Full Text] [PDF]
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S. Jodo, S. Xiao, A. Hohlbaum, D. Strehlow, A. Marshak-Rothstein, and S.-T. Ju Apoptosis-inducing Membrane Vesicles. A NOVEL AGENT WITH UNIQUE PROPERTIES J. Biol. Chem., October 19, 2001; 276(43): 39938 - 39944. [Abstract] [Full Text] [PDF]
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P. Secchiero, P. Mirandola, D. Zella, C. Celeghini, A. Gonelli, M. Vitale, S. Capitani, and G. Zauli Human herpesvirus 7 induces the functional up-regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) coupled to TRAIL-R1 down-modulation in CD4+ T cells Blood, October 15, 2001; 98(8): 2474 - 2481. [Abstract] [Full Text] [PDF]
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T. M. Baetu, H. Kwon, S. Sharma, N. Grandvaux, and J. Hiscott Disruption of NF-{kappa}B Signaling Reveals a Novel Role for NF-{kappa}B in the Regulation of TNF-Related Apoptosis-Inducing Ligand Expression J. Immunol., September 15, 2001; 167(6): 3164 - 3173. [Abstract] [Full Text] [PDF]
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A. Renz, W. E. Berdel, M. Kreuter, C. Belka, K. Schulze-Osthoff, and M. Los Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo Blood, September 1, 2001; 98(5): 1542 - 1548. [Abstract] [Full Text] [PDF]
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R. Di Pietro, P. Secchiero, R. Rana, D. Gibellini, G. Visani, K. Bemis, L. Zamai, S. Miscia, and G. Zauli Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by selective up-regulation of TRAIL-R1 Blood, May 1, 2001; 97(9): 2596 - 2603. [Abstract] [Full Text] [PDF]
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Y. Miura, N. Misawa, N. Maeda, Y. Inagaki, Y. Tanaka, M. Ito, N. Kayagaki, N. Yamamoto, H. Yagita, H. Mizusawa, et al. Critical Contribution of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (Trail) to Apoptosis of Human Cd4+T Cells in HIV-1-Infected Hu-Pbl-Nod-Scid Mice J. Exp. Med., March 5, 2001; 193(5): 651 - 660. [Abstract] [Full Text] [PDF]
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 A. D'Alessio, A. Riccioli, P. Lauretti, F. Padula, B. Muciaccia, P. De Cesaris, A. Filippini, S. Nagata, and E. Ziparo Testicular FasL is expressed by sperm cells PNAS, February 22, 2001; (2001) 51566098. [Abstract] [Full Text]
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S. Jodo, A. M. Hohlbaum, S. Xiao, D. Chan, D. Strehlow, D. H. Sherr, A. Marshak-Rothstein, and S.-T. Ju CD95 (Fas) Ligand-Expressing Vesicles Display Antibody-Mediated, FcR-Dependent Enhancement of Cytotoxicity J. Immunol., November 15, 2000; 165(10): 5487 - 5494. [Abstract] [Full Text] [PDF]
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M. Austin Taylor, M. Bennett, V. Kumar, and J. D. Schatzle Functional Defects of NK Cells Treated with Chloroquine Mimic the Lytic Defects Observed in Perforin-Deficient Mice J. Immunol., November 1, 2000; 165(9): 5048 - 5053. [Abstract] [Full Text] [PDF]
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C. Traidl, S. Sebastiani, C. Albanesi, H. F. Merk, P. Puddu, G. Girolomoni, and A. Cavani Disparate Cytotoxic Activity of Nickel-Specific CD8+ and CD4+ T Cell Subsets Against Keratinocytes J. Immunol., September 15, 2000; 165(6): 3058 - 3064. [Abstract] [Full Text] [PDF]
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L. Zamai, P. Secchiero, S. Pierpaoli, A. Bassini, S. Papa, E. S. Alnemri, L. Guidotti, M. Vitale, and G. Zauli TNF-related apoptosis-inducing ligand (TRAIL) as a negative regulator of normal human erythropoiesis Blood, June 15, 2000; 95(12): 3716 - 3724. [Abstract] [Full Text] [PDF]
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S. Jodo, D. Strehlow, and S.-T. Ju Bioactivities of Fas Ligand-Expressing Retroviral Particles J. Immunol., May 15, 2000; 164(10): 5062 - 5069. [Abstract] [Full Text] [PDF]
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E. S. Bergmann-Leitner and S. I. Abrams Differential Role of Fas/Fas Ligand Interactions in Cytolysis of Primary and Metastatic Colon Carcinoma Cell Lines by Human Antigen-Specific CD8+ CTL J. Immunol., May 1, 2000; 164(9): 4941 - 4954. [Abstract] [Full Text] [PDF]
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A. D'Alessio, A. Riccioli, P. Lauretti, F. Padula, B. Muciaccia, P. De Cesaris, A. Filippini, S. Nagata, and E. Ziparo Testicular FasL is expressed by sperm cells PNAS, March 13, 2001; 98(6): 3316 - 3321. [Abstract] [Full Text] [PDF]
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