(Received for publication, February 28, 1995; and in revised form, June 5, 1995)
From the
Tumor necrosis factor (TNF) is produced as a membrane-bound,
26-kDa proform from which the mature, 17-kDa TNF subunit is released by
proteolytic cleavage. In order to compare the biological activity of
membrane-bound versus soluble TNF, mutational analysis of
potential cleavage sites in murine TNF was carried out. The biological
activity was assessed after transfection in L929 cells. Deletion of the
first nine codons of the mature part of the murine TNF gene still led
to the production of secretable TNF, indicating alternative cleavage
sites separate from the -1/+1 junction. However, an
additional deletion of 3 amino acids, generating TNF
The cytokine tumor necrosis factor (TNF) ( The mature, secreted 17-kDa TNF subunit is derived
from a 26-kDa TNF precursor. It has been demonstrated that the proform
of TNF is present on the cell surface as a type II transmembrane
protein (Kriegler et al., 1988). The latter is then
proteolytically cleaved by a membrane-associated Ser protease (Scuderi et al., 1989; Kim et al., 1993) or metalloprotease
(Mohler et al., 1994; Gearing et al., 1994; McGeehan et al., 1994) in order to release mature 17-kDa TNF (52 kDa in
the native, trimeric form). At present, there is only indirect
evidence that the membrane-bound precursor of mTNF, presumably as a
trimer, is biologically active. It was found that
paraformaldehyde-fixed murine macrophages still exhibit TNF-mediated
cytotoxicity (Decker et al., 1987). Likewise, immunoreactive
TNF, detected in the course of mouse embryogenesis, was not secreted
but rather membrane-bound, suggesting that membrane-bound TNF may
function during that process (Osawa and Natori, 1989). Moreover,
TNF-expressing tumor cells display reduced tumorigenicity in
vivo. Considering the absence of detectable, soluble TNF in the
serum of such mice, membrane-bound TNF might be responsible for this
effect (Vanhaesebroeck et al., 1991). In order to develop a
system allowing us to study the biological activity of membrane-bound
TNF, we investigated the proteolytic conversion of the 26-kDa mTNF
proform to a 17-kDa, secreted TNF subunit by site-directed mutagenesis.
The mTNF mutants generated were expressed in the murine fibrosarcoma
cell line L929 and analyzed for their biochemical and biological
characteristics. This approach permitted us to generate a
nonsecretable, biologically active mutant of mTNF. L929 cells
expressing this mutant induced proliferation of the murine CT6 cell
line and GM-CSF secretion by a rat thymoma cell line; moreover, they
were cytotoxic to U937 cells. These results directly demonstrate that
the membrane-bound form of TNF is able to trigger biological activities
mediated by TNF-R55 as well as by TNF-R75. However, depending on the
receptor signal transduction system, different bioactivities of
membrane-bound versus secreted mTNF could be observed.
In
order to remove the authentic cleavage site between presequence and
mature mTNF, a mutant was generated in which the first 9 amino acids of
mature TNF were deleted (referred to as TNF
Figure 1:
Overview of TNF mutants generated. An invertedfilledtriangle shows the known
cleavage site between the TNF presequence and mature TNF
(-1/+1). The potential N-glycosylation site Asn at
position 7 is indicated by an asterisk. Openbars indicate deleted amino acid sequences. mTNF
Figure 2:
Expression of TNF by L929 cells
transfected with various TNF secretion mutants as detected in cell
lysates and culture supernatants. Open and closedarrowheads point to 26- and 17-kDa TNF species,
respectively. A, immunoprecipitation followed by immunoblot
analysis with antiserum to 17-kDa mTNF on cell lysates of L929 cells
transfected either with the neo
Figure 3:
Flow-cytometric analysis of expression of
cell-associated TNF by L929 cells transfected with TNF secretion
mutants. Cells were transfected either with the neo
In order to identify the cleavage
site responsible for this alternative processing, the deletion was
extended to the first 12 amino acids of mature mTNF, generating the
mutant TNF Lys is frequently present in protease recognition sites (Harris,
1989) and is present at position 11 of mature mTNF (Fig. 1).
Since this Lys is a possible candidate for the alternative cleavage
site of mTNF, we replaced in TNF
Figure 4:
Cytotoxic activity present in the
supernatant of L929 cells transfected with various TNF mutants. L929
cells were transfected either with the neo
The biological activity of uncleavable
TNF
Figure 5:
Determination of the bioactivity of
membrane-bound TNF mutants by cellular coculture experiments. A, TNF-induced proliferation of CT6 cells incubated for 24 h
with L929 transfectants pretreated with mitomycin C. Control
experiments showed that mTNF clearly induced CT6 to proliferate; CT6
alone or cocultured with L929-neo
Whether cell
surface-expressed TNF could induce the production of GM-CSF in
PC60-R55/R75 cells (Vandenabeele et al., 1995), was also
investigated. Induction of GM-CSF secretion by PC60-R55/R75 cells
involves signaling by both types of TNF-R in a cooperative effect
between TNF-R55 and TNF-R75. ( Finally, TNF-dependent induction of
apoptosis in U937 cells was studied. These cells die from apoptosis
after an incubation of 4 h in the presence of TNF and cycloheximide.
Selective triggering of hTNF-R55 in U937 cells with the agonistic htr-1
monoclonal antibody induced apoptosis (Fig. 5C, inset). But apoptosis could not be induced via selective
triggering of hTNF-R75 using utr-1 monoclonal antibodies (Fig. 5C, inset). Moreover, this utr-1
monoclonal antibody, which is agonistic on transfected PC60 cells
(Vandenabeele et al., 1992, 1995), did not enhance the
cytotoxicity on U937 cells exerted by htr-1. These findings indicate
that only hTNF-R55 is signal-transducing in U937 cells, which is in
agreement with previously reported data (Shalaby et al., 1990;
Barbara et al., 1994). But hTNF-R75 might play an important
role by facilitating binding of TNF to hTNF-R55, presumably through a
ligand-passing mechanism (Tartaglia et al., 1993b). This
phenomenon is supported by our observation that blocking of hTNF-R75 by
pretreatment with a monoclonal antibody (utr-1) reduced the induction
of apoptosis in U937 cells by TNF, but not by htr-1 (which only
interacts directly with hTNF-R55) (Fig. 5C, inset). After coculture with U937 cells,
L929-TNF
The biosynthesis of TNF is a complex phenomenon. The 26-kDa
proform of TNF, which presumably forms a trimer, is present as a type
II transmembrane protein on the cell surface. The 17-kDa TNF subunit is
then proteolytically cleaved off and released still as a trimer in the
extracellular milieu (Kriegler et al., 1988). The proteolytic
cleavage of mTNF normally takes place between the presequence and
mature mTNF (-1/+1; Fig. 1) as shown by amino acid
sequencing of the NH In
this study we eliminated the standard TNF cleavage site by mutating the
N-terminal moiety of mature mTNF. We found that removal of the first 9
amino acids of mature TNF was not sufficient to prevent TNF processing
and still led to the release of biologically active TNF in the
supernatant. However, by deleting 3 additional amino acids, creating
TNF The biological activity of uncleavable TNF mutants was evaluated by
coculturing TNF-expressing L929 transfectants with various
TNF-responsive target cell lines. These included murine CT6, rat/mouse
PC60-R55/R75 and human myeloid U937 cells, which respond to TNF by
proliferation, GM-CSF production, and apoptosis, respectively. The
proliferation of CT6 cells is exclusively mediated by mTNF-R75
signaling (Tartaglia et al., 1993a), presumably through
interaction with TNF-R-associated factors 1 and 2 (Rothe et
al., 1994), whereas the induction of GM-CSF production by
PC60-R55/R75 involves the two TNF-Rs. Our data indicate
that cleavage of the 26-kDa mTNF precursor is not aimed at generating
an active TNF form from an inactive one, but rather at switching
between two active TNF forms, one membrane-bound and the other
diffusible. The proteolytic cleavage of TNF might therefore be an
important regulatory step to switch from TNF effects mediated at the
local level through cell-cell contact to effects mediated at the
paracrine and systemic level. Also the mechanism of TNF signaling by
membrane-bound TNF is different, since it does not involve ligand
passing and therefore raises the threshold concentration for
TNF-R55-signaling responses. Thus, the relative contribution of both
forms of TNF to bioactivity is crucial for understanding the
physiological impact of TNF. Elevated doses of soluble TNF in animals
and humans are severely toxic (Beutler et al., 1985; Tracey et al., 1986). Recently, metalloproteinase inhibitors
impairing TNF processing have been identified and found to protect
animals against a lethal dose of endotoxin-induced TNF (Mohler et
al., 1994). These data suggest that secreted TNF, and not
membrane-bound TNF, is most harmful to the host. Our findings suggest
that membrane-bound TNF in the presence of proteinase inhibitors would
still be biologically active. Injection of TNF-expressing tumor cells
in mice showed a reduced tumorigenicity (Vanhaesebroeck et
al., 1991). In order to investigate the role therein of
membrane-bound versus secreted TNF, the in vivo behavior of L929 cells expressing uncleavable TNF mutants will be
studied. In this respect, biologically active, uncleavable TNF mutants
will allow examination of the physiological relevance of membrane-bound
TNF in vivo.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1-12,
resulted in a membrane-bound form of TNF. Site-directed mutagenesis
revealed Lys
as the critical residue for alternative
cleavage. Mutation of this residue to Glu in a TNF
1-9 mutant
gave rise to uncleavable, membrane-bound TNF with biological activities
similar to wild-type TNF. Induction of apoptosis, proliferation, or
cytokine production by triggering of either 55-kDa or 75-kDa TNF
receptors in appropriate cell lines occurred efficiently both with
soluble and with membrane-bound TNF. The latter was, however, less
active in the cytotoxic assays on U937 cells in which the 75-kDa TNF
receptor is not signaling, but contributes to maximal TNF activity by
ligand passing. This indicates that membrane-bound TNF cannot be passed
from the 75-kDa to the 55-kDa TNF receptor.
)plays a
crucial role in immunological and inflammatory reactions as a result of
an infection or a tumor burden (reviewed in Vassalli(1992),
Fiers(1993), Tracey and Cerami(1994)). TNF interacts with two specific
receptors present on the cell surface of almost every cell type:
TNF-R55 and TNF-R75, corresponding to a molecular mass of 55 and 75
kDa, respectively (Loetscher et al., 1990; Smith et
al., 1990; Lewis et al., 1991; Goodwin et al.,
1991). Since the active form of TNF in solution is a trimer (Wingfield et al., 1987; Schoenfeld et al., 1991) and contains
three TNF-R-binding sites (Van Ostade et al., 1991), signal
transduction after TNF binding presumably occurs by TNF-R clustering
(Espevik et al., 1990; Pennica et al., 1992;
Tartaglia and Goeddel, 1992). Almost the whole spectrum of TNF
activities is mediated by TNF-R55 (Engelmann et al., 1990;
Espevik et al., 1990; Tartaglia et al., 1993c). So
far, the role of TNF-R75 as a signal transducer seems to be restricted
to T lymphocytes (Tartaglia et al., 1991, 1993a; Vandenabeele et al., 1992). Because of the higher affinity and rate of
dissociation, it has also been proposed that TNF-R75 can concentrate
TNF and pass it on to neighboring TNF-R55 molecules (Tartaglia et
al., 1993b).
Cell Lines and Cell Culture
The fibrosarcoma
cell lines L929sA and WEHI 164 cl 13 (Espevik and Nissen-Meyer, 1986)
were cultured as described (Vanhaesebroeck et al., 1992).
PC60-R55/R75 is a rat mouse T cell hybridoma transfected with
both hTNF-R55 and hTNF-R75 cDNA under control of the SV40 early
promoter (Vandenabeele et al., 1995). CT6 is an
interleukin-2-dependent murine cytotoxic T cell line (Ranges et
al., 1989). PC60-R55/R75, CT6, and the human myeloid U937 cell
line were cultured in RPMI 1640 supplemented with 10% fetal calf serum,
antibiotics, 2 mML-glutamine, 1 mM sodium
pyruvate, and 50 µM 2-mercaptoethanol. All cell lines were
repeatedly screened for mycoplasma by a DNA-fluorochrome assay and were
found to be negative.
Cytokines and Antisera
Purified Escherichia
coli-derived mTNF and hTNF were obtained in our laboratory and had
a specific biological activity of 2 10
IU/mg and
8.4
10
IU/mg, respectively. International standards
for TNF quantification (IU/ml) were obtained from the National
Institute for Biological Standards and Control (Potters Bar, United
Kingdom). Recombinant mGM-CSF was kindly provided by Dr. J. DeLamarter
(Glaxo IMB, Geneva, Switzerland). Polyclonal rabbit antiserum against
mTNF was provided by J. Van der Heyden (Roche Research, Gent, Belgium).
Polyclonal rabbit antiserum directed against the tip region of mTNF
(amino acids 99-115; Lucas et al., 1994) was a generous
gift from Dr. R. Lucas (Brussels University, Brussels, Belgium).
Anti-hTNF-R55 and anti-hTNF-R75 monoclonal antibodies were generously
provided by Dr. M. Brockhaus (Hoffmann-La Roche, Basel, Switzerland;
Brockhaus et al., 1990).
Determination of TNF Bioactivity in Culture
Supernatant
Supernatant of transfected cell lines was
concentrated 100-fold by centrifugation in Centriprep-10 and
Centricon-10 microseparation devices (Amicon, Danvers, MA). TNF was
quantified in an 18-h cytotoxicity assay using WEHI 164 cl 13 cells in
the presence of 1 µg of actinomycin D/ml (Espevik and Nissen-Meyer,
1986). The detection limit of this assay was about 1 pg/ml.Site-directed Mutagenesis
Site-directed
mutagenesis was carried out using pMa and pMc phasmids as described
(Stanssens et al., 1989). Mutations were verified by DNA
sequencing.Plasmid Constructions and Transfection
Mutant TNF
or wtTNF genes were inserted in the eukaryotic expression vector pSV23S
under control of the SV40 early promoter (Huylebroeck et al.,
1988). The pSV2neo plasmid encoding the neo gene
under control of the SV40 early promoter was used as a selectable
marker in L929 transfections (Southern and Berg, 1982). Plasmid DNA
used for transfection was purified using pZ523 columns (5 Prime
3 Prime, West Chester, PA). Stable transfection of L929 cells was
carried out by an improved DNA-calcium phosphate coprecipitation method
as described (Vanhaesebroeck et al., 1992).
Immunoprecipitation and Immunoblotting of TNF
TNF
was immunoprecipitated from concentrated cell supernatant or from cell
lysates as described (Vanhaesebroeck et al., 1992).
Immunoprecipitates were separated by electrophoresis in an
SDS-containing 12.5% polyacrylamide gel under reducing conditions.
After electrophoresis, the proteins were blotted on a nitrocellulose
membrane using a Novablot II apparatus (Pharmacia Biotech Inc.)
according to the manufacturer's instructions. Blocking was
performed overnight at 4 °C in 50 mM Tris-HCl, pH 8.0, 2
mM CaCl, 80 mM NaCl, 0.2% (w/v) Nonidet
P-40, 0.02% (w/v) dry milk. The blot was then incubated for 4 h at 4
°C with polyclonal rabbit antiserum to TNF, followed by a 1-h
incubation at room temperature with 0.2 µCi of
I-protein A (Amersham International, Amersham, UK; 30
mCi/mg) in 20 ml of 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20. Radioactivity was detected by
autoradiography.
Flow Fluorocytometry
Membrane-bound TNF was
analyzed by incubation of the cells for 1 h at 4 °C with polyclonal
antiserum to the tip of mTNF (amino acids 99-115; 1 µg of
antibody/4 10
cells/200 µl), 1 h of incubation
at 4 °C with biotinylated donkey anti-rabbit polyclonal antiserum
(Amersham International), and 1 h at 4 °C incubation with
phycoerythrin-conjugated streptavidin. Analysis was performed by flow
fluorocytometry using a Coulter Epics 753 equipped with an argon-ion
laser (Coulter, Hialeah, FL).
Bacterial Expression of TNF
wtTNF and mutant
TNF-K11E genes (both without presequence) were cloned in the vectors
pMaTrp and pMcTrp (Stanssens et al., 1989), respectively,
under control of the inducible Trp promoter. After 3 days of induction
at 18 °C, bacteria were sonicated, and TNF bioactivity was tested
in a WEHI 164 cl 13 cytotoxicity assay. In order to determine the
specific bioactivity of E. coli-derived mTNF-K11E, the
recombinant mutant protein was purified as described previously for
wtTNF (Tavernier et al., 1990).Cellular Coculture
The bioactivity of TNF in
transfected L929 cells was determined by a coculture between
transfected L929 effector cells and TNF-responsive target cells (CT6,
PC60-R55/R75, or U937). Adherent effector cells were seeded in flat
bottomed 96-well microtiter plates at 20,000 cells/well 24 h before the
addition of 25,000 nonadherent target cells/well. The biological
effects of in situ produced TNF on the various target cells
were established as detailed below.CT6 Proliferation Assay
The proliferation of
effector L929 transfectants was inhibited by 4 h of pretreatment with
mitomycin C (50 µg/ml) at 37 °C followed by three washes. After
20 h of coculture with CT6 cells, proliferation was measured by
[H]dThd incorporation for 4 h (Ranges et
al., 1989).
Induction of GM-CSF Production by PC60-R55/R75
Cells
L929 transfectants were cocultured with PC60-R55/R75
cells. These transfectants were used in order to enhance the
TNF-induced response. After 24 h of coculture the supernatants were
harvested and assayed in an FDCp1 cell proliferation assay (DeLamarter et al., 1985; Vandenabeele et al., 1990).Determination of Apoptosis in U937 Cells
U937
cells were pretreated for 1 h at 4 °C or for 3 h at 37 °C in
the presence or absence of 2 µg/ml utr-1 antiserum, which is a
monoclonal antibody against hTNF-R75 (Brockhaus et al., 1990).
U937 cells were incubated with L929 effector cells in the presence of
50 µg/ml cycloheximide. After 4 h, nonadherent U937 cells were
harvested, incubated with propidium iodide, frozen, and analyzed by
flow fluorocytometry. The extent of apoptosis was quantified as the
percentage of cells with a hypoploid DNA profile (Grooten et
al., 1993). Contaminating L929 effector cells were electronically
gated out on the basis of their different forward angle and 90°
angle light-scattering properties.
Biochemical Characterization of TNF Mutants with
Deletions at the N Terminus of Mature TNF
In order to determine
which amino acids are crucial for the proteolytic processing of the
membrane-bound proform of mTNF into a secreted mature form, mutants
were constructed in which coding sequences for the N-terminal part of
mature mTNF were deleted. The mutants were stably expressed in L929
fibroblast cells, which have been shown to become resistant to
autocrine TNF production (Vanhaesebroeck et al., 1992).1-9) (Fig. 1). It is indeed known that deletion of up to 9 amino
acids does not change the biological activity of hTNF (Creasey et
al., 1987). Immunoprecipitation and immunoblotting of
mTNF
1-9 transfectants showed a truncated 25-kDa TNF
precursor in cell lysates (Fig. 2A) and different TNF
species in the supernatant (Fig. 2B). Furthermore, flow
fluorocytometric analysis of L929 transfected with wtTNF or
TNF
1-9 revealed a membrane-bound TNF form (Fig. 3).
Acidic treatment, applied to dissociate soluble TNF from the TNF-R, did
not alter the level of immunofluorescence (data not shown). These
results demonstrate that TNF
1-9 is present not only as a
membrane-bound form but also as a cleaved, soluble form. Thus removal
of the cleavage site between presequence and mature mTNF
(-1/+1) (Fig. 1) (Sherry et al., 1990) did
not abolish mTNF processing and still permitted secretion and membrane
expression of TNF
1-9.
1-9,K(11)E,
mTNF
1-9,K11E.
gene alone (lane1), or combined with plasmid coding for wt-mTNF (lane2), mTNF
1-9 (lane3; a, 25 kDa), mTNF
1-12 (lane4; b, 24.6 kDa), or mTNF
1-9,K11E (lane5; c, 25 kDa). B,
immunoprecipitation followed by immunoblot analysis with anti-mTNF on
the supernatant from clonal L929 cells transfected with either the neo
gene alone (lane1) or
combined with plasmid coding for wt-mTNF (lane2),
mTNF
1-9 (lane3), mTNF
1-12 (lane4), or mTNF
1-9,K11E (lane5).
gene alone (A) or with the neo
gene
combined with plasmid coding for wtTNF (B), mTNF
1-9 (C), mTNF
1-12 (D), or
mTNF
1-9,K11E (E). Cells were treated with secondary
antibody alone (curve1) or with antiserum to the tip
of TNF (amino acids 99-115) and secondary antibody (curve2). No signal other than staining with secondary antibody
alone was found with an irrelevant rabbit IgG (not
shown).
1-12. Although the corresponding cell lysate
contained the truncated TNF proform with a predicted molecular mass of
24.6 kDa (Fig. 2A), protein bands reactive with
antiserum against TNF were not detectable in the supernatant of
TNF
1-12 transfectants, as assayed by immunoprecipitation and
immunoblotting (Fig. 2B). Moreover, flow
fluorocytometric analysis of L929 transfectants with antiserum to the
tip of the TNF molecule (amino acids 99-115) revealed the
presence of membrane-bound TNF at expression levels similar to the ones
observed with wtTNF or TNF
1-9 (Fig. 3). These data
show that TNF
1-12 was expressed at the cell surface but,
unlike TNF
1-9, could not be secreted. Consequently, the
additional cleavage site responsible for processing of
TNF
1-9 is presumably located between amino acids 9 and 13.
1-9 the positively charged
Lys with the negatively charged Glu by site-directed mutagenesis (Fig. 1). Cell lysate from the resulting TNF
1-9,K11E
transfectants clearly showed the truncated 25-kDa TNF proform (Fig. 2A), while the supernatant did not reveal any
TNF-specific band (Fig. 2B). Further analysis by flow
fluorocytometry demonstrated the presence of membrane-bound TNF at a
density similar to that observed for L929-TNF
1-12 or
L929-wtTNF transfectants (Fig. 3). These findings confirm that
Lys
constitutes the alternative cleavage site by which
mTNF is processed in the absence of the proper cleavage site at
-1/+1.
Functional Characterization of Secretable and
Nonsecretable TNF Deletion Mutants
The biological activity of
secreted TNF was assessed on the highly sensitive WEHI 164 cl 13 cells
(Espevik and Nissen-Meyer, 1986). As expected, 100-fold concentrated
culture supernatant of L929 cells transfected with wtTNF or secretable
TNF1-9 had a cytotoxic activity of 16.4 IU/ml and 70 IU/ml
TNF, respectively, in originally unconcentrated supernatant (Fig. 4). This cytotoxicity was mediated by TNF, since it was
abolished by the addition of neutralizing anti-TNF antiserum (data not
shown). But no cytotoxicity could be detected in the supernatant of
TNF
1-12 or TNF
1-9,K11E transfectants (Fig. 4). This is in agreement with the absence of
immunoprecipitable TNF in these culture supernatants and further
supports the absence of production of mature mTNF by such
transfectants.
gene
alone (▴) or with the neo
gene combined with
plasmid coding for wtTNF (
), mTNF
1-9 (
),
mTNF
1-12 (▪), or mTNF
1-9,K11E (
).
The cytotoxic activity was tested on WEHI 164 cl 13
cells.
1-12 and TNF
1-9,K11E was investigated by
coculture of L929 transfectants with various TNF-responsive target cell
lines. A first assay was based on the TNF-R75-mediated proliferation of
CT6 cells, in which TNF-R75 is signal-transducing (Tartaglia et
al., 1993a). As shown in Fig. 5A, L929 cells
producing wtTNF or TNF
1-9,K11E exerted a similar
growth-inducing activity, while TNF
1-12 exerted this
activity to a lower extent. These results show that both uncleavable
TNF mutants trigger mTNF-R75-mediated signaling.
cells hardly
showed CT6 proliferation. Experiments were done in triplicate, and S.D.
is indicated. B, induction of GM-CSF production by
PC60-R55/R75 cells incubated for 24 h with L929 transfectants. GM-CSF
released in the supernatant was assessed in an FDCp1 cell proliferation
assay. 500 pg/ml mTNF induced GM-CSF levels similar to those recorded
after coculture of PC60-R55/R75 cells with L929 wt-mTNF transfectants;
L929 transfectants did not produce more than 10 pg/ml GM-CSF. C, TNF-dependent induction of apoptosis of U937 cells treated
with cycloheximide after cocultivation with TNF-expressing L929
transfectants. Unpretreated U937 cells (filledbars)
or U937 cells pretreated with 2 µg/ml utr-1 (openbars) (utr-1 binds to hTNF-R75 and prevents ligand
passing) were incubated with mTNF-expressing L929 cells for 4 h in the
presence of 50 µg/ml cycloheximide. The inset shows the
induction of apoptosis in U937 cells, either untreated or after
treatment with htr-1 (10 ng/ml) or TNF (0.1 ng/ml); filled and openbars refer to control and utr-1 pretreatment.
Apoptosis in U937 cells was quantified by the percentage of hypoploid
particles. Experiments were done twice in triplicate; bars indicate S.D. TNF
1-9,K(11)E,
TNF
1-9,K11E.
)TNF
1-9,K11E was
found to induce a GM-CSF response similar to that induced by wtTNF,
whereas TNF
1-12 induced a 3-fold lower response (Fig. 5B).
1-9,K11E induced an appreciable level of apoptosis,
though this activity was significantly lower than that induced by
L929-wtTNF transfectants (Fig. 5C), which express
similar amounts of membrane-bound TNF. This is in contrast with CT6 and
PC60-R55/R75 cells, in which the activity patterns induced by
L929-TNF
1-9,K11E and L929-wtTNF were identical (TNF-R75 is
signal-transducing in these two target cells). The different behavior
of these two L929 transfectants may be explained by ligand passing of
soluble TNF produced by L929-wtTNF, since utr-1 pretreatment of U937
cells (which prevented ligand passing) resulted in a wtTNF activity
similar to that of membrane-bound TNF
1-9,K11E. These results
suggest that the membrane-bound mutant, and by extension the
membrane-bound TNF, cannot be passed. The cellular cytotoxicity
observed was TNF-dependent, as evidenced by abrogation of the apoptotic
response by addition of TNF-neutralizing antiserum, while inclusion of
preimmune serum or irrelevant antiserum did not alter the biological
response (data not shown). Furthermore, TNF
1-12 was
completely ineffective in inducing cell death (Fig. 5C). Fig. 5, A-C, shows
that membrane-bound TNF
1-9,K11E is capable of inducing
signal transduction by TNF-R75 and TNF-R55; in the latter case,
however, the efficiency is less than with soluble TNF, which has a
higher efficacy because of ligand passing.
A Postulated Salt Bridge between Lys
As shown above, the bioactivity of
membrane-bound, uncleavable TNFand
C-terminal Leu
Is Not Required for Bioactivity of
Soluble mTNF
1-12 and
TNF
1-9,K11E was lower in certain assays than the bioactivity
of wtTNF. A possible explanation for these differences is that in these
TNF mutants a postulated salt bridge between Lys
and the
C-terminal Leu
of an adjacent subunit in mTNF could no
longer be formed after substitution or deletion of Lys
. It
has indeed been suggested that this salt bridge in the soluble hTNF
trimer may be required for bioactivity (Eck and Sprang, 1989).
Therefore, Lys
was substituted by Glu in full-length,
soluble, mature mTNF. The specific bioactivity of TNF-K11E expressed in
and purified from bacteria was, however, very similar to that of wtTNF, viz. 6.6
10
IU/mg and 2
10
IU/mg, respectively. This finding is in agreement with reported
data that substitution of Lys
(mutant K11Q, K11M, K11T, or
K11N) in mature hTNF, expressed in E. coli, had virtually no
effect on its bioactivity (Zhang et al., 1992). As a
consequence, the almost normal biological activity of TNF-K11E
indicates that removal of the hypothetical salt bridge on itself cannot
explain the differences in bioactivity observed between wtTNF and
uncleavable, membrane-bound TNF mutants in some of the assays. It is
likely that differences in TNF-induced signaling pathways, inherent to
the assays applied, account for the distinct responses observed.
terminus of TNF secreted by
stimulated murine macrophages (Sherry et al., 1990).
1-12, a membrane-bound, uncleavable TNF form was
generated. This result is in agreement with the finding that deletion
of the first 12 amino acids of mature hTNF also leads to an uncleavable
mutant (Perez et al., 1990). The residual cleavage of
TNF
1-9 versus the absence of cleavage with
TNF
1-12 is indicative of the existence of an additional
cleavage site in mature mTNF. This alternative cleavage site seems to
be nonfunctional in wtTNF. When the proper site (-1/+1) is
removed, the second site might become more exposed, facilitating
extracellular cleavage at this noncanonical site. Lys
is
crucial for this cleavage, since cellular expression of
TNF
1-9,K11E resulted in uncleavable, membrane-bound mTNF.
In both types of
assay, cells expressing membrane-bound TNF
1-9,K11E were
found to have a bioactivity roughly similar to that of wtTNF
transfectants and to be more potent than TNF
1-12
transfectants. These results demonstrate that both TNF-R55 and TNF-R75
can be triggered by membrane-bound TNF to the same extent as by soluble
TNF. But different results were obtained for the induction of apoptosis
in U937 cells, where cells expressing uncleavable TNF mutants were less
effective. The lesser activity of the membrane-bound TNF mutant was not
due to removal of the hypothetical salt bridge linking Lys
to Leu
. Rather, it can be explained by the fact
that induction of apoptosis in U937 cells is a hTNF-R55-mediated effect
(Barbara et al., 1994); at low concentrations, hTNF-R75 might
facilitate triggering of TNF-R55 by ligand passing (Tartaglia et
al., 1993b). This mechanism is presumably not possible with the
uncleavable, membrane-bound TNF mutant. The reduced bioactivity of
TNF
1-12 transfectants as compared with
TNF
1-9,K11E transfectants was not due to a lower protein
expression level, since membrane-associated expression of
TNF
1-12 and TNF
1-9,K11E showed similar levels (Fig. 3). The lower bioactivity of TNF
1-12 might be
caused by removal of Pro
, which could lead to a local
structural disturbance, resulting indirectly in lower binding
capacity/affinity for TNF-R. Carlino et al. (1987) also
demonstrated a direct correlation between the bioactivity of hTNF
mutants with N-terminal deletions and the physical binding to TNF-R.
Analysis of the expression levels of uncleavable TNF mutants did not
reveal increased numbers of TNF molecules on the cell membrane, despite
the fact that these molecules are no longer cleaved off. This is
analogous to findings that after inhibition of TNF secretion by
metalloproteinase inhibitors, no accumulation of membrane-bound TNF was
observed (Gearing et al., 1994). This lack of TNF accumulation
might be due to the presence of a homeostatic feedback mechanism, which
ensures that the proform of TNF is rapidly recycled and degraded inside
the cells (Pradines-Figueres and Raetz, 1992).
, neomycin-resistant; TNF-R, TNF receptor;
TNF-R55, 55-kDa TNF-R; TNF-R75, 75-kDa TNF-R; wt, wild type.
We thank A. Raeymaekers for providing TNF
preparations; J. Van der Heyden (Roche Research, Gent, Belgium), Dr. R.
Lucas (Brussels University, Brussels, Belgium), and Dr. M. Brockhaus
(Hoffmann-La Roche, Basel, Switzerland) for antisera; and Dr. J.
DeLamarter (Glaxo IMB, Geneva, Switzerland) for mGM-CSF. A. Meeus, M.
Van den Hemel, W. Burm, D. Ginneberge, and F. Molemans are acknowledged
for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.