Expression of the functional soluble form of human fas ligand in activated lymphocytes.

Abstract

Fas is a type I membrane protein which mediates apoptosis. Fas ligand (FasL) is a 40 kDa type II membrane protein expressed in cytotoxic T cells upon activation that belongs to the tumor necrosis factor (TNF) family. Here, we found abundant cytotoxic activity against Fas-expressing cells in the supernatant of COS cells transfected with human FasL cDNA but not with murine FasL cDNA. Using a specific polyclonal antibody against a peptide in the extracellular region of human FasL, a protein of 26 kDa was detected in the supernatant of the COS cells. The signal sequence of granulocyte colony-stimulating factor was attached to the extracellular region of human FasL. COS cells transfected with the cDNA coding for the chimeric protein efficiently secreted the active soluble form of human FasL (sFasL). Chemical crosslinking and gel filtration analysis suggested that human sFasL exists as a trimer. Human peripheral T cells activated with phorbol myristic acetate and ionomycin also produced functional sFasL, suggesting that human sFasL works as a pathological agent in systemic tissue injury.

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The EMBO Journal vol.14 no.6 pp.1129-1135, 1995
Expression of the functional soluble form of human
Fas ligand in activated lymphocytes
Masato Tanaka, Takashi Suda,
Tomohiro Takahashi and Shigekazu Nagata
Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565, Japan
Communicated by C.Weissmann
Fas is a type I membrane protein which mediates
apoptosis. Fas ligand (FasL) is a 40 kDa type II
membrane protein expressed in cytotoxic T cells upon
activation that belongs to the tumor necrosis factor
(TNF) family. Here, we found abundant cytotoxic
activity against Fas-expressing cells in the supernatant
of COS cells transfected with human FasL cDNA but
not with murine FasL cDNA. Using a specific polyclonal
antibody against a peptide in the extracellular region
of human FasL, a protein of 26 kDa was detected in
the supernatant of the COS cells. The signal sequence
of granulocyte colony-stimulating factor was attached
to the extracellular region of human FasL. COS cells
transfected with the cDNA coding for the chimeric
protein efficiently secreted the active soluble form of
human FasL (sFasL). Chemical crosslinking and gel
filtration analysis suggested that human sFasL exists
as a trimer. Human peripheral T cells activated with
phorbol myristic acetate and ionomycin also produced
functional sFasL, suggesting that human sFasL works
as a pathological agent in systemic tissue injury.
Key words: apoptosis/cytokine/cytotoxicity/Fas/Fas ligand
Introduction
Fas, also called APO-1, is a type I membrane protein of
45 kDa which is expressed in various tissues and cells
including the thymus, liver, ovary and lung (Itoh et al.,
1991; Oehm et al., 1992; Watanabe-Fukunaga et al.,
1992b). Fas is a member of the tumor necrosis factor
(TNF)/nerve growth factor receptor family (Beutler and
van Huffel, 1994; Nagata, 1994; Smith et al., 1994), and
mediates apoptosis when crosslinked with agonistic anti-
Fas or anti-APO-1 antibody (Trauth et al., 1989; Yonehara
et al., 1989), or Fas ligand (FasL; Suda et al., 1993). FasL
is a type II membrane protein of Mr 40 kDa and is a
member of the TNF family which includes TNFax, ax- and
,B-chains of lymphotoxin (LT), CD40 ligand and CD30
ligand (Suda et al., 1993). The amino acid sequences of
human and murine FasL are 76.9% identical, and they
are not species-specific (Takahashi et al., 1994a,b). The
expression of FasL mRNA is most prominent in cells of
the T cell lineage, except for those of the testis (Suda
et al., 1993). The activation of mature T cells with phorbol
myristic acetate (PMA) and ionomycin, concanavalin A
(Con A) or anti-CD3, induces FasL gene expression
(Vignaux et al., 1995; Suda et al., 1995).
Molecular and genetic analyses of the Fas and FasL
genes indicated that murine spontaneous, autosomal and
recessive lpr and gld mutations are mutations of Fas and
FasL, respectively (Watanabe-Fukunaga et al.,
Nagata and Suda, 1995; Takahashi et al., 1994a). Since lpr
and gld mice develop lymphadenopathy and splenomegaly,
and suffer from autoimmune disease by producing auto-
antibodies including anti-DNA and rheumatoid factors
(Cohen and Eisenberg, 1991),
system is involved in the apoptotic process of T cell
development. Recent studies of lpr and gld mice suggested
that the Fas system plays an important role in the clonal
deletion of autoreactive T cells in the periphery or activa-
tion-induced suicide of T cells (Russell and Wang, 1993;
Russell et al., 1993; Singer and Abbas, 1994; Watanabe
et al., 1995). In addition to the development of T cells,
FasL works as an effector molecule of cytotoxic T cells
(Kagi et al., 1994; Kojima et al., 1994; Lowin et al.,
1994). Accordingly, various T cell clones express FasL
upon activation, and then kill the target cells in a Fas-
dependent manner (Rouvier et al., 1993; Hanabuchi et al.,
1994; Stalder et al., 1994; Vignaux et al., 1995; Suda
et al., 1995).
TNFx, a prototype of the TNF family, is produced as a
membrane-bound form (Kriegler et al., 1988; Kinkhabwala
et al., 1990; Perez et al., 1990) and is processed to an
active soluble form by proteolytic shedding. Both forms
of TNFa mediate a range of inflammatory and cellular
immune responses, including tumor regression, septic
shock and cachexia (Beutler and Cerami, 1989; Fiers,
1991). Biochemical and biophysical analyses of TNFa
revealed that the soluble form of TNFa is a homotrimer
(Smith and Baglioni, 1987; Eck and Sprang, 1989; Jones
et al., 1989), and that it binds to the receptor at a ratio of
3:3 (Banner et al., 1993).
Here we have identified the active soluble form of
human FasL (sFasL) in the supernatant of COS cells
transfected with the full-length FasL cDNA, as well as in
the supernatant of activated human peripheral T cells.
Functional sFasL was also produced in COS cells using
a cDNA, in which the signal sequence was attached to
the N-terminus of the extracellular domain of FasL.
Chemical crosslinking, followed by Western blotting using
anti-FasL antibody, as well as gel filtration indicated that
sFasL has a trimeric structure.
1992a;
it is clear that the Fas
Results
The soluble form of human FasL in COS cells
transfected with FasL cDNA
Monkey COS cells were transfected withpEF-BOSvector
alone or vector carrying the full-lengthcDNA for murine
FasL or human FasL. After 72 h, the supernatants were
assayed for cytotoxic activity againstmurine WR19L or
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M.Tanaka et aL
.01
1
Concentration of Supernatant (fold)
B
kD
123
4
200 -
5 6 7 8
97.4
69 -
46 -
30-
21.5-
c
1
23
4
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97.4-
69 -
46-
30 -
21.5
as
ft
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Fig. 1. The production of active human sFasL in COS cells transfected with human FasL cDNA. (A) Cytotoxic activity of the COS cell
supernatants. COS cells were transfected with pEF-BOS vector ([O), pEF-BOS carrying the full-length human FasL cDNA (0) or murine FasL
cDNA (0). At 72 h after transfection, the cytotoxic activity of the supernatants was determined by means of the MTT assay using W4 cells, as
described in Materials and methods. The cell viability is expressed as the percentage of that observed without the COS cell supernatant.
(B) Production of human FasL protein in COS cells. COS cells were transfected with pEF-BOS (lanes 1, 2, 5 and 6) or pEF-BOS carrying the full-
length human FasL cDNA (lanes 3, 4, 7 and 8). At 72 h after transfection, the cell lysates (lanes 1, 3, 5 and 7) or supernatants (lanes 2, 4, 6 and 8)
were immunoprecipitated with mFas-Fc. The immunoprecipitates were heated at 95°C for 2 min in Laemmli's sample buffer containing 0.1 M
P-mercaptoethanol,and resolved by electrophoresis on a 10-20% gradient polyacrylamide gel. Human FasL proteins were then detected by Western
blotting using anti-human FasL antibody in the absence (lanes 1-4) or presence (lanes 5-8) of 30gg/mlof the peptide CPSPPPEKKELRKVAH. As
size markers, molecular weight standards (RainbowTI, Coloured Protein Marker, Pharmacia) were electrophoresed in parallel and the sizes of the
standard proteins are shown in kDa. (C) Effects of endoglycosidase on human FasL. COS cells were transfected with pEF-BOS carrying the full-
length human FasL cDNA. At 72 h after transfection, the cell lysates (lanes
mFas-Fc. The immunoprecipitates were incubated for 5 min at 90°C in PBS containing 0.5% SDS and 0.1 MP-mercaptoethanol. After
centrifugation, the supernatants were diluted 3.5-fold with 50 mM phosphate buffer (pH 6.0) and incubated for 16 h at 37°C with (lanes 2 and 4) or
without (lanes
containing 0.1 MP-mercaptoethanoland analyzed by Western blotting using the anti-human FasL antibody as described above.
1 and 2) or supernatants (lanes 3 and 4) were immunoprecipitated with
1 and 3) 4 mU endoglycosidase H (Genzyme). The samples were then incubated for 5 min at 90°C in Laemmli's sample buffer
its transformant W4, expressing murine Fas. As reported
for rat FasL (Suda et al., 1993), the supernatant of COS
cells transfected with murine FasL expression plasmid
contained
a
expressing cells (<20 U/ml; Figure IA). On the other
hand, transfection with the human FasL expression plasmid
gave rise to a large amount of FasL activity in the
supernatant (-1200 U/ml). The cytotoxic activity was
specific
observed against WR19L cells which do not express Fas.
To confirm that the cytotoxic substance in the super-
natant of the COS cells transfected with human FasL
cDNA is sFasL, and to analyze its molecular properties,
a polyclonal antibody against human FasL was prepared
by immunizing rabbits with a peptide of human FasL.
The peptide carries the sequence from 134 to 148 of
human FasL, which corresponds to the N-terminal region
of the soluble TNFa (Suda et al., 1993; Takahashi et al.,
1994b). The peptide of human TNFa corresponding to
this region has high antigenicity (Socher et al., 1987).
The antibody was affinity-purified from the serum of the
immunized rabbits using peptide-conjugated resin. The
antibody specifically recognized human but not murine
FasL (data not shown).
The supernatant and the cell lysates of the transfected
COS cells were immunoprecipitated with
protein consisting of the extracellular region of murine
Fas and the Fc region of human immunoglobulin (mFas-
Fc; Suda and Nagata, 1994). The immunoprecipitates were
resolved by electrophoresis on a polyacrylamide gel, and
little cytotoxic activity against the Fas-
to the Fas-expressing
cells, since none was
a chimeric
analyzed by Western blotting using the anti-human FasL
antibody. As shown in Figure iB, both the cell lysates
and the supernatant of the COS cells transfected with the
human FasL cDNA gave intensive bands recognized by
anti-human FasL antibody. All bands recognized by the
antibody are specific because these bands disappeared on
the inclusion of 30 ,ug/ml of the human FasL peptide in
the solution for Western blotting. The protein ofMr40 kDa
in the lysates seems to be human FasL expressed on the
membrane. The lower bands have Mrs identical to those
found in the supematant, indicating that they are probably
sFasL. Such a soluble form in the cells was reported
previously in the TNF system (Kriegler et al., 1988). The
supernatant of the COS cells contained two bands of Mr
26 and 23 kDa which are significantly larger than the
calculatedMrfor the extracellular region of human FasL.
To examine whether human FasL is glycosylated, the
immunoprecipitates were treated with endoglycosidase H
and analyzed by Western blotting. As shown in Figure
IC, the sizes of human FasL in the lysates were reduced
to 33, 22 and 17 kDa. Whereas, a single protein of
17 kDa was detected in the immunoprecipitates of the
supernatant, the molecular weights of 33 and 17 kDa
agree reasonably with the calculated Mr for the membrane-
bound (Mr 31 486) and soluble forms (Mr 17 695) of
human FasL. The 22 kDa protein may be sFasL containing
the residual sugar moiety. These results indicate that the
N-glycosylated active sFasL can be produced in COS cells
transfected with human FasL cDNA.
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Human Fas ligand soluble form
~~~~~~~RA
1% _
Fig. 2. Schematic diagrams of the human FasL construct carrying the
G-CSF signal sequence. The structures of human FasL and its
chimeric proteins containing the murine G-CSF signal sequence are
shown schematically. CYT, TM and EXT represent the cytoplasmic,
transmembrane and extracellular regions of human FasL, respectively.
In the S1 construct, the G-CSF signal sequence replaces the
cytoplasmic and transmembrane regions of human FasL, whereas the
signal sequence was attached to Prol37 of human FasL in the S2
construct. The amino acid sequence at the junction between the murine
G-CSF signal sequence and human FasL is shown. The arrow
indicates the cleavage site of the murine G-CSF precursor protein.
Production of human sFasL using a signal
sequence
The attachment of the signal sequence to the N-terminus
of the extracellular region of human TNFa exclusively
produces the soluble form of TNFa (Perez et al., 1990).
To confirm that human sFasL is functional, two expression
plasmids for sFasL were constructed using the signal
sequence of murine granulocyte colony-stimulating factor
(G-CSF; Tsuchiya et al., 1986). In the first construct (SI
construct), the signal sequence of G-CSF was attached
immediately downstream of the transmembrane region of
human FasL (Figure 2). In the second construct (S2
construct), the signal sequence was joined to the 34
amino acids downstream of the transmembrane region,
the position corresponding to where soluble TNF begins
(Pennica et al., 1984). Both constructs, as well as the
expression plasmid for the full-length human FasL cDNA,
were transfected into COS cells, and the supernatants
were harvested 72 h later. As shown in Figure 3A and B,
the supernatants of COS cells transfected with SI, S2 and
the full-length human FasL cDNA contained -800, 3800
and 1200 U/ml of cytotoxic activity against W4 cells but
not against WR19L cells. The supernatants were then
analyzed directly by Western blotting using anti-human
FasL antibody (Figure 3C). The COS cells transfected
with the SI construct produced a protein of Mr 33 kDa
recognized by the antibody, whereas the supernatants of
COS cells transfected with either the S2 construct or full-
length cDNA showed bands of similar Mr The difference
in the Mr between the SI and S2 constructs can be
explained by the 34 extra amino acids at the N-terminus
of the S1 construct and glycosylation of the putative N-
glycosylation site in this extra sequence. The proteins
produced by the SI and S2 constructs are supposed to
carry nine amino acids from mature G-CSF and one from
the linker sequence, in addition to the sequence of human
FasL. Considering these points, the similar Mr of the
soluble proteins produced by the full-length cDNA and
the S2 construct suggestedthat the membrane-bound FasL
coded by the full-length cDNA was cleaved at around
amino acid 127. This position is slightly upstream of
where
the corresponding membrane-bound TNFac
cleaved (Perez et al., 1990).
The soluble form of TNF functions as a trimer (Smith
and Baglioni, 1987). To examine whether sFasL also
exists as a trimer, the supernatant ofCOS cells transfected
with the full-length FasL cDNA was concentrated and
treated with the chemical
suberate (DSS). After mixing with 0.5% SDS, the samples
were boiled for 5 min under reducing conditions, resolved
by electrophoresis at room temperature and analyzed by
Western blotting using the anti-FasL antibody. As shown
in Figure 4, the treatment with the crosslinker produced
an intensive band at Mr 50 kDa and faint bands at Mr
-80 kDa, which may correspond to a dimer and trimer of
human FasL. It is known that the oligomeric structures of
some proteins are conserved if electrophoresis is carried
out under milder conditions (Fukunaga et al., 1990).
Therefore, the concentrated COS cell supernatants were
mixed at 4°C with 0.5% SDS under non-reducing condi-
tions and resolved by electrophoresis at 4'C. Western
blotting using the anti-FasL antibody indicated that sFasL,
even without crosslinking, has MrS of 26, 50 and 82 kDa
under these conditions (Figure 4). The intensity of the
two upper bands was slightly increased by the chemical
crosslinker, and no forms larger than the trimer were
detected, suggesting that human sFasL is a trimer.
To confirm that human sFasL is a trimer, the supernatant
of COS cells transfected with the full-length FasL cDNA
was concentrated and analyzed by gel filtration. As shown
in Figure 5A, the FasL activity was eluted at the position
corresponding to a Mr of -70 kDa. When each fraction
was analyzed by Western blotting using the anti-FasL
antibody, the FasL protein was found in fractions which
showed the FasL activity, and no FasL protein was detected
in the fractions where a monomer of sFasL (Mr 26 kDa)
should be eluted (Figure SB). These results indicated that
most human sFasL exists as a trimer, and this trimer has
cytotoxic activity.
is
crosslinker,
disuccinimidyl
Production of the soluble form of Fas ligand by
activated lymphocytes
The activation of mature T cells induces the expression
of the FasL gene, and the cells express the functional
FasL on their surface (Suda and Nagata, 1994; Vignaux
et al., 1995; Suda et al., 1995). To examine whether
human lymphocytes produce sFasL, Con A blasts were
prepared from human peripheral blood lymphocytes (PBL)
and expanded in the presence of interleukin (IL)-2. The
cells were then activated with PMA and ionomycin for
24 h. The supernatant was concentrated and its cytotoxic
activity was examined, with WR19L and W4 cells as
target cells. As shown in Figure 6A, the supernatant of
the activated T cells showed cytotoxic activity (30 U/ml)
against the Fas-expressing W4 cells, but not WR19L cells.
This activity was inhibited by soluble murine Fas (mFas-
Fc) in a dose-dependent manner. On the other hand, the
soluble form of the TNF receptor (TNFR,-Fc) did not
affect the cytotoxic activity of the supernatant (Figure
6B). To confirm that the activated human lymphocytes
produce sFasL, the supernatant of the activated PBL was
immunoprecipitated with mFas-Fc. Western blotting of
the immunoprecipitates revealed a small amount of sFasL
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M.Tanaka et al.
c
A
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1301
120-f
-Oi
110
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70f
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50-
40-
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.10
.001
.01
B
140-
130-
1201
110
-
701
60-
501
401
30-
20-
10a
-10
.01
Concentration of Supernatant (fold)
20
1 2 3 4
"
kD
0o
97.4 -
69 -
46 -
MO
l-i
30-
-S
21.5 -
14.3 -
Fig. 3. Production of human sFasL using the G-CSF signal sequence. (A and B) Cytotoxic activity of the supernatants of COS cells transfected with
the human FasL expression plasmids. COS cells were transfected with pEF-BOS-SIG (0), SI construct (0), S2 construct(Cl) orpEF-BOS carrying
the full-length human FasL cDNA (-). At 72 h after transfection, the cytotoxic activity of supernatants was assayed using W4 (A) or WR19L (B) as
the target cells. (C) Western blotting. 16 p1 of the supernatant of COS cells transfected with pEF-BOS-SIG (lane 1), S1 construct (lane 2), S2
construct (lane 3) or pEF-BOS carrying the full-length human FasL cDNA (lane 4) were heated in Laemmli'ssample buffer without f-
mercaptoethanol at 90°C for 2 min, and resolved by electrophoresis on a 10-20% gradient polyacrylamide gel.Afterblottingto a PVDF membrane,
human sFasL was detected using anti-human FasL antibody as described in Materials and methods. The sizes of molecularweight standards are
shown in kDa on the left.
in the medium conditioned for 24 h with Con A blasts of
human PBL (Figure 6C). Activation of PBL with PMA
and ionomycin greatly increased the amount of sFasL in
the medium. The Mr of the sFasL produced by the
activated PBL was similar to that found in the supernatant
of COS cells transfected with the full-length FasL cDNA.
These results indicated that human peripheral T cells can
produce functional sFasL upon activation.
Discussion
TNFa is expressed initially in activated T cells and
macrophages as a membrane-bound form which is then
proteolytically processed into
retains
activity (Kriegler
Kinkhabwala et al., 1990). In this report we described
that human FasL, a member of the TNF family, also
becomes a soluble form. Human sFasL produced in COS
cells by transfection with the full-length human FasL
cDNA,
PBL, was active;
Fas-expressing cells. Various groups have reported that
metalloproteases are responsible for the processing of the
membrane-bound TNFa (Gearing et al., 1994; McGeehan
et al., 1994; Mohler et al., 1994). Although the region of
human FasL around amino acid 127, where the proteolytic
cleavage should occur, does not show significant similarity
to the cleavage site of TNFa (Pennica et al., 1984; Perez
et al., 1990), a similar protease(s) may be involved in the
processing of membrane-type FasL. In contrast to the
supernatant of COS cells transfected with human FasL
cDNA, very little Fas-dependent cytotoxic activity was
detected in the supematant of COS cells transfected with
murine FasL cDNA. This finding agrees with the findings
that activation of ratXmouse T cell hybridoma PC60-
dIOS with PMA and ionomycin produced little sFasL
(Rouvier et al., 1993; Suda and Nagata,
raised a specific polyclonal antibody against murine FasL
a soluble cytokine that
et al.,
its biological
1988;
as well
as that produced in activated human
it specifically induced cytolysis of
1994). We
1
2
kD
200 -
97.4-
69
46 -
3
4
kD
a .-
200-1
.
wue
97.4 -
_
69
46-
30-
30
21.5-
14.3
14.3-
Fig. 4. Chemical crosslinking of human sFasL. COS cells were
transfected with pEF-BOS vector carrying the full-length human FasL
cDNA. At 72 h after transfection, the supernatant was concentrated
-40-fold and an aliquot was left on ice for 10 min without (lanes
and 3) or with 0.15 mM DSS (lanes 2 and 4). In lanes
samples were heated at 90°C for 5 min in Laemmli's sample buffer
containing 0.1 M P-mercaptoethanol. Samples were resolved by
electrophoresis at room temperature on a 4-20% gradient
polyacrylamide gel. In lanes 3 and 4 samples were treated at 4°C for
10 min in Laemmli's sample buffer without P-mercaptoethanol and
resolved by electrophoresis at 4°C on a 4-20% polyacrylamide gel.
After electrophoresis, proteins were analyzed by Western blotting
using the anti-FasL antibody as described in Materials and methods.
1
1 and 2,
(M.Tanaka,
results). Western blotting of the supernatant of COS cells
transfected with murine FasL cDNA revealed a similar
amount of murine sFasL which has a Mr similar to that
of human sFasL found in the COS cell supematant. These
results indicated that murine FasL is also proteolytically
processed to the soluble form, but is non-functional or
very unstable. In this regard, it is worth noting that an
inactive form of murine TNFa is produced by inappro-
priate processing in the murine macrophage cell line
RAW264.7 (Cseh and Beutler, 1989). Normally, mature
T.Takahashi
and
S.Nagata,
unpublished
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Human Fas ligand soluble form
2500
-
0.1
BD
IgGTF
OVA
CTA
2000
-0.08
1500 -
-0.06
1000 -
-0.04
500-
-0.02
10
15
20
25
30
35
Fraction Number
B
kD
17 18 19 20 21 22 23 24 25 26 27 28 29 30
200-
97.4 -
69-
46-
0
o
30 -
21.5-
14.3-
Fig. 5. Gel filtration of human sFasL. COS cells were transfected with pEF-BOS carrying the full-length of human FasL cDNA. At 72 h after
transfection, the supernatant was concentrated 20-fold and 80glaliquots were loaded onto a SuperdexTm 200 HR10/30 column (Pharmacia) attached
to the FPLC system (Pharmacia). The column was developed with PBS containing 0.1 mg/ml BSA at a flow rate of 0.5 ml/min; 0.5 ml fractions
were collected. (A) FasL activity. The cytotoxic activity ([) in the fractions was determined by MTT assay as described in Materials and methods.
The profile of the OD at 280 nm is shown by a thin line. The positions of molecular weight marker proteins are indicated by arrows. BD, blue
dextran 2000; IgG, human immunoglobulin (160 kDa); TF, transferrin (81 kDa); OVA, ovalbumin (43 kDa); CTA, chymotrypsinogen A (25 kDa).
(B) The FasL protein. 16glaliquots of each fraction were analyzed by Western blotting with the anti-FasL antibody, as described in Materials and
methods.
Concentration of the supernatant (fold)
kD
1 2 3 4
200-
=
97.4-
69-
46-
30-
21.5-
14.3 -
..-..
.01
....... .. .
I..--
.1
100
1000
Concentratiom of chimeric proteins (ugIml)
11 0
Fig. 6. Production of active human sFasL from activated human PBLs. (A) Cytotoxic activity in the supernatant of activated human PBL. Con A
blasts of human PBL were incubated at 37°C for 24 h with 10 ng/ml PMA and 500 ng/ml ionomycin. After centrifugation, the cytotoxic activity of
the supernatant was determined by the MTT assay using W4 (0) or WR19L cells (0) as the target cells. (B) Effect of mFas-Fc on the cytotoxic
activity of the supernatant of activated human PBL. The cytotoxic activity of the 1.25-fold concentrated supernatant of activated human PBL was
determined by the MTT assay using W4 cells in the presence of indicated concentrations of mFas-Fc (0) or hTNFR3-Fc (0). (C) Detection of
sFasL in the supernatant of activated human PBL. Con A blasts of human PBL were incubated with (lane 4) or without (lane 3) PMA and
ionomycin, as described above. 1 ml of the supernatant was immunoprecipitated with mFas-Fc and the immunoprecipitate was analyzed by Western
blotting with anti-human FasL antibody, as described in Materials and methods. As controls, COS cells were transfected with pEF-BOS (lane 1) or
pEF-BOS carrying human FasL cDNA (lane 2). 40
and Western blotting as described above. The sizes of the molecular weight standard proteins are indicated as kDa on the left.
tl aliquots of the supernatant of the transfected COS cells were analyzed by immunoprecipitation
T cells express FasL upon activation (Suda et al., 1995).
Whether or not murine mature T cells produce non-
functional sFasL remains to be studied.
Active TNFax has a homotrimeric structure (Smith and
Baglioni, 1987). The X-ray diffraction analysis of human
TNFcx and TNFfi (LTcx) indicates that these cytokines are
compact trimers with a 'bell-shaped appearance' (Eckand
Sprang, 1989; Jones et al., 1989; Eck et al., 1992). Since
the primary structure of the extracellular region of FasL
is highly homologous to that of TNFa and TNFf, especi-
ally in the regions where the TNFs are known to form a
,B-strand, we postulated that FasL forms a homotrimer
(Suda et al., 1993). This assumption was confirmed here
by gel filtration and chemical crosslinking analyses of
human sFasL. Since LT exists as a heterotrimer consisting
of one LTa and twoLTPmolecules on the cell surface
(Androlewicz et al., 1992), the membrane-bound form of
human FasL may also be a trimer. Several monoclonal
antibodies against Fas have agonistic
original anti-Fas (CH-1 1; Yonehara et al., 1989)and 2D1
antibodies (Takahashi et al., 1993) are of the immuno-
globulin M type, while the anti-APO-1 antibody is an
activities. The
1133
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