From the Institute of Cell Biology and Immunology, University of Stuttgart, 70569 Stuttgart, Germany
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ABSTRACT |
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Tumor necrosis factor (TNF) exists in two
bioactive forms, the membrane-integrated form and the proteolytically
derived soluble cytokine. Cells that produce TNF are often responsive
to TNF, allowing autocrine/juxtacrine feedback loops. However, whether the membrane form of TNF is involved in such regulatory circuits is
unclear. Here we demonstrate that HeLa cells, expressing a permanently
membrane-integrated mutant form of TNF, constitutively express
TNF·TNF receptor complexes at their cell surface. These cells show a
permanent activation of the transcription factor NF- Tumor necrosis factor
(TNF)1 is the prototype
cytokine of a ligand family whose members are typically expressed as
type II transmembrane proteins (1). Action of a metalloproteinase (2, 3) leads to proteolytical release of soluble TNF (sTNF). Both TNF forms
are bioactive and bind to two membrane receptors of 55-60 kDa (TNF-R1)
and 75-80 kDa (TNF-R2). TNF-R1 is constitutively expressed in nearly
all tissues and represents the main mediator of cellular TNF responses
(for review, see Ref. 4). TNF-R2 is more restricted in expression,
e.g. to lymphoid tissue, is tightly regulated in its
expression, and modulates cellular responses to sTNF or transmembrane
TNF in a cooperative manner with TNF-R1. We have recently shown that
TNF-R2, in contrast to TNF-R1, needs stimulation by the membrane form
of the cytokine for full activation (5). The low efficiency of sTNF for
stimulation of TNF-R2 in comparison to TNF-R1 has been explained by the
transient sTNF-TNF-R2 interaction at physiological temperatures
(6).
The cellular response pattern to TNF stimulation is extremely broad and
cell type-dependent. For example, TNF can induce apoptosis and initialize a variety of other signals, including induction of
cytokine production, enhancement of adhesion molecule expression, and
growth stimulation (for review, see Ref. 4). These cellular responses
are caused by different intracellular signaling pathways induced by
TNF-R1. One major signaling pathway of TNF-R1 leads to the activation
of aspartate-directed proteinases (caspases) via the death domain
proteins TNF receptor-associated death domain protein and
Fas-associated death domain protein (7). Another major signaling
pathway leads to the activation of the transcription factor NF- TNF is produced by many different cell types including macrophages, T
lymphocytes, and endothelial cells (for review, see Ref. 4). These
cells often also express TNF receptors and thus display TNF
responsiveness. Auto-/juxtacrine-acting TNF has been implicated in
monocyte-mediated cytotoxicity (14), primary T cell activation
(15),2 and induction of
endothelial tissue factor production by cross-linking of adhesion
molecules (16). A critical involvement of transmembrane TNF signaling
has been revealed in immunological reactions such as
antileishmanial defense in macrophages, T cell/B cell interaction, and tumor cell killing by infiltrating lymphocytes (17, 18, 19).
Moreover, the transgenic expression of a noncleavable and, thus,
permanently membrane-anchored TNF mutein in mice is sufficient to
induce a pathological phenotype similar to rheumatoid arthritis (20),
underlining the high biological potential of local TNF signaling.
Whether and to what extent a juxta-/autotropic signaling loop of
transmembrane TNF plays a role in these models, i.e. whether cell surface-expressed TNF acts predominantly on neighboring cells (juxtatropic) or in a true autotropic signaling mode, is unresolved.
To investigate the selective effects of transmembrane TNF expression on
a TNF-sensitive cell, we have generated HeLa transfectants expressing a
noncleavable, constitutively membrane-bound, biologically active
derivative of human TNF (HeLatmTNF). We show that these cells display a phenotype similar to TNF-stimulated untransfected HeLa
cells. Accordingly, TNF-mediated signaling pathways involving the
transcription factor NF- Antibodies and Reagents--
Recombinant human TNF (specific
activity 2 × 107 units/mg) was kindly provided by
I.-M. von Broen, Knoll AG, Ludwigshafen, Germany. The TNF-R1-specific
mAb H398 has been described elsewhere (21). The TNF-specific mAb T1 was
provided by Dr. Boettinger, University of Stuttgart, Germany. The
anti-human TNF mAb 357-101-4 was kindly provided by A. Meager
(National Institute for Biological Standards and Control; Herts, United
Kingdom). The fluorescein isothiocyanate-labeled goat anti-mouse IgG + IgM Ab was obtained from Dianova, Hamburg, Germany. The construct
coding for a noncleavable membrane form of human TNF (TNF Cells--
HeLa cells and 293 cells were obtained from American
Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 culture medium (Biochrom, Berlin, Germany) supplemented with 10%
heat-inactivated fetal calf serum and 2 mM L-glutamine.
Liposome-mediated transfections were performed using pFx-2 (Invitrogen,
Groningen, The Netherlands) or SuperFect (Qiagen, Hilden, Germany)
according to the protocols of the manufacturers. Stable transfectants
were generated by selection with 400 µg/ml Zeocin (Invitrogen,
Groningen, The Netherlands), and populations of transgene-positive
cells were sorted using immunofluoresent staining with TNF-specific Abs
and a FACStarplus cell sorter (Becton and Dickinson, San
Jose, CA).
Cell Death Assays--
HeLa cells (1.5 × 104/well) were seeded into 96-well microtiter plates
overnight. The next day CHX was titrated in the presence or absence of
additional reagents in a final volume of 150 µl. After 18 h of
culture, supernatants were discarded, and the cells were washed once
with PBS followed by crystal violet staining (20% methanol, 0.5%
crystal violet) for 15 min. The wells were washed with H2O
and air-dried. The dye was resolved with methanol for 15 min, and
optical density at 550 nm was determined with a R5000 ELISA plate
reader (Dynatech, Guernsey, United Kingdom). For cytotoxicity assays
using IFN- Analysis of p38 MAP Kinase Phosphorylation--
Cells (2.5 × 106) were seeded overnight in 100-mm culture dishes. The
next day the cells were stimulated as indicated, washed once with PBS,
lysed in 120 µl of SDS loading buffer, boiled for 10 min, and
electrophoresed on a 12% SDS-polyacrylamid gel (20 µl/lane)
under reducing conditions. After transfer onto nitrocellulose membranes, Western blots were performed according the instructions of
the manufacturer (BioLabs, New England).
Western Blotting--
For preparation of cytosolic extracts,
3 × 106 cells were seeded in 100-mm cell culture
dishes and cultured overnight. After stimulation with the indicated
reagents for 4 h, the cells were washed once with cold PBS,
harvested, and resuspended in 200 µl of buffer A (10 mM
KCl, 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride). 15 µl of 10% Nonidet P-40 were
added, and after 2 min nuclei were removed by centrifugation. Protein
concentrations were determined using the Bio-Rad protein assay kit
(Bio-Rad). Extracts were mixed with 6-fold concentrated Laemmli buffer
and boiled for 5 min, and 100 µg of protein were resolved by
SDS-polyacrylamide gel electrophoresis. Proteins were transferred to
nitrocellulose membranes, and caspase-3 was detected using a monoclonal
anti-caspase-3 antibody (Transduction Laboratories, Dianova, Hamburg, Germany).
Binding Studies--
Human recombinant TNF was labeled with
125I by the chloramine-T method to a specific radioactivity
of 40-60 µCi/µg of protein. The percentage of iodinated ligand
able to specifically bind to receptor-positive cells was 60%. Binding
studies were performed as described (21). For determination of
unspecific binding, a 200-fold excess of unlabeled TNF was added. For
association kinetics, cells (1 × 106) were incubated
for different time periods with 15 ng/ml 125I-TNF at
37 °C. Cell-bound 125I-TNF was determined after
centrifugation of cells through a phthalate oil mixture as described
(6). For equilibrium binding studies at 0 °C, cells (1 × 106) were subjected to a pH 3.0 buffer (125 mM
NaCl, 50 mM glycine/HCl) or a pH 7.4 buffer (PBS) for
90 s before the addition of 15 ng/ml 125I-TNF. After
2 h on ice, cell-bound radioactivity was determined as described above.
IL-6 Assay--
1.5 × 104 cells were seeded in
96-well microtiter plates overnight. After washing the cells once with
PBS, they were cultured for an additional 6 h in the absence or
presence of 10 ng/ml TNF in a total culture volume of 150 µl.
Supernatants were removed and cleared by centrifugation for 10 min at
15,000 rpm. IL-6 concentrations were determined using a commercially
available IL-6 ELISA kit according to the manufacturer's
recommendations (PharMingen, Hamburg, Germany), and color development
was measured at 405 nm using a R5000 ELISA plate reader (Dynatech,
Guernsey, UK). For TNF neutralization experiments, 1 × 103 cells were seeded in a 96-well microtiter plate
overnight. The next day anti-TNF mAbs and control IgG, respectively,
were added. After 5 days of additional culture, cells were washed once
in PBS, and antibody supplemented culture medium was added. After 6 h of culture, IL-6 concentrations were determined as described above.
Electrophoretic Mobility Shift Assays (EMSA)--
For
preparation of nuclear extracts, 3 × 106 cells were
seeded in 100-mm2 cell culture dishes and cultivated
overnight. The next day cells were stimulated with the required
combination of reagents for 30 min. Cells were washed twice with cold
PBS and incubated on ice for 15 min with 5 ml of buffer A (10 mM KCl, 10 mM HEPES, pH 7.9, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). Subsequently, cells were
harvested, pelleted, and resuspended in 100 µl of buffer A. 6 µl of
10% Nonidet P-40 were added for 2 min, and nuclei were pelleted and
resuspended in buffer B (400 mM NaCl, 20 mM
HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol). After 20 min of shaking and subsequent
centrifugation, the lysates containing the nuclear proteins in the
supernatant were used for the electrophoretic mobility shift assay
after protein determination with bovine serum albumin as the standard
(Bio-Rad). High performance liquid chromatography-purified
NF- Luciferase Reporter Assay--
HeLa cells (1 × 104) were seeded in 96-well microtiter plates overnight and
subsequently transfected with a minimal promoter containing a
three-NF- Coexpression of TNF-R1 and tmTNF in
TNF
The lack of detectable amounts of TNF-R1 on HeLatmTNF cells
could represent down-regulation of cell surface-expressed TNF-R1 by
endogenous tmTNF and/or formation of cell surface-expressed tmTNF·TNF-R1 complexes in which the H398 binding epitope is masked. In fact, after treatment of HeLatmTNF cells with an acidic
buffer, pH 3.0, to disrupt putative tmTNF·TNF-R1 complexes, H398
specifically bound to HeLatmTNF (Fig. 1B) in amounts
comparable with untreated (Fig. 1C) or pH 3.0-treated HeLa
cells (Fig. 1D). Equilibrium binding studies performed with
radioiodinated TNF at 0 °C confirmed the absence of free TNF-R1 and
the presence of tmTNF·TNF-R1 complexes at the cell surface of
HeLatmTNF cells (Fig. 1E). No specific binding
of the label was obtained with untreated cells, whereas specific
125I-TNF binding capacity of pH 3.0-treated
HeLatmTNF cells was about 50% that of parental HeLa cells.
The presence of cell surface-expressed tmTNF·TNF-R1 complexes was
further substantiated by ligand association studies at 37 °C. Under
these conditions a rapid and specific association of
125I-TNF to otherwise untreated HeLatmTNF cells
could be observed (Fig. 1F). The association rate was
similar to that observed with HeLa cells (Fig. 1F).
Constitutive IL-6 Production, Activation of NF-
Because IL-6 production has been shown to be critically dependent on
the transcription factor NF-
Next, we looked for activation of the p38 kinase pathway, which has
been demonstrated to be also critically involved in TNF-induced IL-6
production (24). Western blot analyses with antibodies specific for
phosphorylated p38 kinase revealed an constitutive activation of this
signaling pathway in HeLatmTNF cells (Fig. 3D).
HeLatmTNF Cells Are Sensitive to Apoptosis by the
Addition of CHX or IFN- Transmembrane TNF Can Act at the Single Cell Level in an Autotropic
Manner--
In the experimental settings described above, cells had
been cultured as confluent monolayers, which allows in principle both stimulation of TNF-R1 on the neighboring cell (juxtatropic) as well as
on the TNF-expressing cell (autotropic). To investigate whether tmTNF
is able to stimulate TNF-R1 of the very same cell, i.e. in
an autotropic manner, HeLatmTNF cells were seeded at a statistical density of 1 cell/well and then treated with CHX for 18 h. After diluting off the CHX, the percentage of surviving cells was determined by analysis of growing colonies after 14 days of
culture. CHX exerted a strong cytotoxic effect on HeLatmTNF cells but not on HeLa cells (Fig. 5),
similar to the experiments using confluent cultures (Fig.
4A). The addition of zVAD-fmk inhibited CHX-induced
cytotoxicity (Fig. 5), confirming that CHX treatment induces cell death
in HeLatmTNF cells in a caspase-dependent
manner. More important, the results strongly suggest that tmTNF can
stimulate TNF-R1 in an autotropic manner, i.e. by
ligand/receptor interaction at the single cell level.
The aim of the present study was to investigate whether expression
of the membrane-integrated form of TNF in a TNF-R1-positive cell would
induce a constitutive signaling rather than cellular unresponsiveness.
As a model, we have chosen the human cell line HeLa, which expresses
about 3,000 molecules of TNF-R1/cell but only neglectable amounts of
TNF-R2 (28, 12). HeLa cells are largely unresponsive to the cytotoxic
activity of TNF per se but can be rendered sensitive by
treatment with e.g. CHX or IFN- HeLa cells transfected with human Using the TNF-sensitive mouse cell line L929, Decoster et
al. (31) have recently demonstrated that expression of tmTNF
resulted in a total TNF receptor downmodulation paralleled by full TNF unresponsiveness. Obviously, induction of unresponsiveness induced by
the autotropic action of TNF was dependent on the transmembrane localization of the cytokine, as a secretable TNF mutein was
ineffective in this regard (31). These data are clearly at variance to
our results. However, we believe that the TNF-induced cellular
stimulation described here is not cell-specific but rather reflects a
physiological relevant response pattern. First, identical results have
been obtained using two distinct cell lines, HeLa and 293 (Fig. 3, A and C). Second, recent data confirm an
autocrine, TNF-mediated permanent stimulation of NF- With regard to a potential cellular TNF response in vivo, we
suggest that the response pattern observed here could be of
physiological relevance. We show that cells capable of producing TNF
are principally able to autostimulate themselves via a tmTNF-mediated
autotropic signaling pathways and do not enter the apoptotic pathway.
In the case of a prolonged tmTNF-mediated stimulus, as mimicked here by
constitutive expression, these cells enter a status of an enhanced sensitivity for the induction of apoptosis. Such a stressed cellular status would give normal tissue cells the opportunity to recover when
TNF stimulation is abrogated but would alternatively allow the organism
to destroy them more easily when additional stress factors, such as
e.g. virus infections, make a recovery unlikely.
B, exert
constitutive p38 mitogen-activated protein kinase activity, and produce
high amounts of interleukin-6. In parallel, transmembrane
TNF-expressing HeLa cells display high sensitivity to cycloheximide or
interferon-
, similar to untransfected cells treated with these
agents in combination with sTNF. Moreover, cycloheximide-induced
apoptosis in transmembrane TNF transfectants can be blocked by the
caspase inhibitor zVAD-fmk and does not necessarily need cell to cell
contact, indicating a critical role of constitutive autotropic
signaling of TNF·TNF receptor complexes. These data demonstrate that
autotropic signaling loops of membrane TNF can exist, which may be of
importance for cells that express both TNF and TNF receptors, such as T
lymphocytes, macrophages, and endothelial cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B,
N-terminal c-Jun kinase, and p38 kinase. NF-
B is believed to be
activated via TNF receptor-associated protein 2 (TRAF2), a member of
the TRAF family, and/or the death domain-containing protein kinase RIP
(receptor interacting protein) (8, 9). Activation of NF-
B results in
the production of cytokines such as interleukin (IL)-6 (10) but also
induces protective mechanisms by stimulation of the expression of
regulatory proteins with potential antiapoptotic activity, such as
TRAF1, TRAF2, and the inhibitor of apoptosis (IAP) proteins c-IAP1 and
c-IAP2 (11). In addition to this antiapoptotic activity, TRAF2 is also
critically involved in a positive, proapoptotic TNF receptor
cooperation, not dependent on de novo gene
induction (12, 13).
B and the p38 MAP kinase are constitutively stimulated, and high amounts of IL-6 are produced in a
TNF-dependent manner. In addition, HeLatmTNF
cells undergo apoptosis in the presence of cycloheximide (CHX) or
interferon-
(IFN-
), similar to Hela cells treated with a
combination of TNF and one of these reagents.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1-12) (5, 22)) was inserted into the mammalian expression
vector pZEO (Invitrogen, Groningen, The Netherlands). The caspase
inhibitor zVAD-fmk was purchased from Bachem AG (Bubendorf,
Switzerland). All other reagents were obtained from Sigma if not
otherwise stated.
, HeLa cells (4 × 103) were seeded into
96-well microtiter plates overnight. Next day the cells were treated
with IFN-
(20 ng/ml) in the absence or presence of TNF (50 ng/ml) in
a final volume of 150 µl. After 3 additional days of culture, crystal
violet staining was performed as described above.
B-specific oligonucleotides (5'-ATCAGGGACTTTCCGCTG
GGGACTTTCCG-3') obtained from MWG-Biotech (Ebersberg, Germany) were
end-labeled with [
-33P]ATP using T4 polynucleotide
kinase. EMSAs were performed by incubating 10 µg of nuclear extracts
with 5 µg of poly(dI-dC) in a binding buffer (5 mM HEPES,
pH 7.8, 5 mM MgCl2, 50 mM KCl, 0.2 mM EDTA, 5 mM dithiothreitol, 10% glycerol).
The double-stranded, end-labeled, purified oligonucleotide probe
(2 × 104-5 × 104 cpm) was added to
the reaction mixture for 15 min at room temperature. The samples were
then separated by native polyacrylamide gel electrophoresis in low
ionic strength buffer.
B element-driven Firefly luciferase reporter plasmid (23),
kindly provided by Marius Ueffing (Gesellschaft für
Strahlenforschung, Munich, Germany) using the SuperFect reagent (Qiagen). After 1 day of recovery, the cells were treated as indicated, washed once in PBS, and harvested in 50 µl of lysis buffer (Promega, Madison, WI). 45-µl aliquots of cell lysates were mixed with 100 µl
of luciferase assay reagent (Promega), and the luciferase activity was
determined using a Lumat LB9501 (Berthold, Wildbad, Germany). For
normalization of transfection efficacy, a Renilla (control) luciferase-encoding plasmid (pRL-TK, Promega) was co-transfected, and
luciferase activity was determined with a second luciferase substrate
after quenching the light reaction of the first luciferase substrate in
a single step following the the manufacturer's instructions (dual-luciferase reporter assay system, Promega).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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(1-12)-transfected HeLa Cells--
Transfection of
cells with an expression construct for human TNF in which the codons
for the amino acids 1 to 12 of the soluble TNF have been deleted
results in the expression of a bioactive transmembrane
TNF
(1-12) (tmTNF) without production of detectable
amounts of bioactive sTNF (22, 5). To characterize possible
autostimulatory effects of the membrane-expressed form of TNF, we
transfected tmTNF into HeLa cells, a cell line that reacts to TNF with
a variety of cellular responses. Three independent pools of transfected
cells (HeLatmTNF-1,-2,-3) were established by cell sorting.
High levels of cell surface-expressed TNF could be readily detected by
flow cytometry (Fig. 1A).
Virtually no cell surface-expressed TNF-R1 was found on
HeLatmTNF cells in contrast to untransfected or control
vector-transfected HeLa cells using the TNF-R1-specific mAb H398 (Fig.
1C and data not shown).
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Fig. 1.
Coexpression of TNF-R1 and transmembrane TNF
on HeLatmTNF cells. Cell surface expression of TNF-R1
and transmembrane TNF in HeLatmTNF cells (A and
B) and HeLa cells (C and D) was
determined by immunofluorescent staining and cytometric analysis using
the TNF-R1-specific mAb H398 or the TNF-specific mAb T1 as indicated or
a control mAb (mouse IgG1 (shaded histograms)) at 20 µg/ml
each. Cells analyzed in B and D were subjected to
an acidic-washing step, pH 3.0, before immunofluorescent staining.
E, binding of 125I-TNF (15 ng/ml) to
HeLatmTNF cells and HeLa cells at 0 °C was determined by
equilibrium binding studies after washing the cells using an acidic
buffer, pH 3.0 (hatched bars) or a neutral buffer, pH 7.3, (empty bars). Shown are mean values ±S.D. of specific
125I-TNF binding after subtraction of nonspecific binding
determined in the presence of a 200-fold excess of unlabeled TNF.
F, binding of 125I-TNF (15 ng/ml) to
HeLatmTNF cells and HeLa cells at 37 °C was determined
by association kinetics. Cell-bound radioactivity was quantitated by
removal of free ligand by centrifugation at the indicated times.
Nonspecific binding was determined in the presence of a 200-fold excess
of unlabeled ligand and has been subtracted in the figure.
B, and p38 MAP
Kinase in HeLatmTNF Cells--
The permanent presence of
tmTNF·TNF-R1 complexes at the cell surface implies that continuous
TNF signaling in HeLatmTNF cells may occur. A typical
cellular response induced by TNF-R1 triggering is the stimulation of
IL-6 gene expression. In fact, HeLatmTNF cells continuously
produced high amounts of this cytokine, comparable with that of HeLa
cells stimulated with sTNF (Fig.
2A). Addition of exogenous
sTNF did not further enhance IL-6 production. Incubation of
HeLatmTNF cells with TNF-specific neutralizing antibodies
inhibited the constitutive IL-6 production, although the inhibition was only partial at high concentrations of the antibody used (Fig. 2B). This is in agreement with our own findings that
neutralization of the effects of transmembrane TNF versus
sTNF generally requires much higher TNF-specific antibody
concentrations.3 These data
further argue for permanent signaling of cell surface-expressed tmTNF·TNF-R1 complexes in HeLatmTNF cells.
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Fig. 2.
Constitutive IL-6 production in
HeLatmTNF cells. A, the concentration of
IL-6 in the supernatants of the indicated cells cultured in the
presence or absence of TNF (10 ng/ml) for 6 h was determined by an
IL-6-specific ELISA. The results of a representative experiment are
shown (n = 3). B, effects of neutralization
of TNF on the constitutive IL-6 production of HeLatmTNF
cells. Cells were cultured for 5 days with the indicated concentrations
of a neutralizing TNF-specific antibody (mAb 357-101-4) or control
mouse IgG and washed, and IL-6 production was determined after a
further 6-h culture in the presence of the indicated antibodies.
B (10), we used a transient reporter
gene assay in which gene expression is under the control of a NF-
B
minimal promotor to investigate NF-
B activation in HeLatmTNF cells. The data revealed a significant,
constitutive NF-
B activation (Fig.
3A) that could be confirmed by
EMSA, demonstrating a permanent nuclear translocation of NF-
B in all
three HeLatmTNF cell pools (Fig. 3B). NF-
B
activation in HeLatmTNF cells was lower as compared with
TNF-treated HeLa cells, but treatment with exogenous sTNF did not
further enhance NF-
B activation (Fig. 3, A and
B). A permanently activated status of NF-
B, driven by endogenous tmTNF, was confirmed in 2 distinct 293-cell populations stably transfected with TNF
(1-12) (Fig. 3C,
inset) showing a strong constitutive NF-
B activation
comparable with sTNF-treated 293 cells (Fig. 3C).
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Fig. 3.
Constitutive activation of
NF- B and p38 MAP kinase in tmTNF-expressing
cells. A, activation of a NF-
B-dependent
reporter gene was determined 24 h after transient transfection of
the indicated cells with a NF-
B-driven luciferase reporter plasmid
in the absence or presence of soluble TNF (20 ng/ml) for 6 h.
Luciferase activity was determined, and transfection efficiency was
normalized by cotransfection of a control luciferase reporter plasmid.
B, activation of NF-
B in HeLatmTNF cells and
HeLa cells was analyzed by EMSA using nuclear extracts of untreated or
TNF-treated (30 min; 20 ng/ml) cells with a 33P-labeled
NF-
B-specific oligonucleotide probe. C, pools of 293 cells stable-transfected with TNF
(1-12) and
untransfected 293 cells were analyzed for NF-
B activation by
luciferase reporter gene assays as described in A.
Expression of tmTNF was controlled by immunofluorescent staining and
fluorescence-activated cell sorter analysis using the TNF-specific mAb
T1 (inset). D, activation of p38 MAP kinase in
HeLatmTNF cells and HeLa cells was determined by Western
blotting with anti-phospho-p38 MAP kinase antibody (pp38
(top panel)) and with anti-p38 MAP kinase antibody (p38
(bottom panel)) using cell lysates of untreated or
TNF-treated (20 ng/ml, 15 min) cells.
--
HeLa cells are relatively resistant to
the cytotoxic effects of TNF. However, in the presence of the protein
synthesis inhibitor CHX, TNF induces apoptosis, indicative for
protective mechanisms dependent on de novo protein synthesis
(25). We therefore asked whether HeLatmTNF cells are
sensitive to the action of the protein synthesis inhibitor CHX. In
fact, HeLatmTNF cells were highly susceptible, as the
ED50 for CHX-induced cytotoxicity was about 1 µg/ml,
whereas the majority of HeLa cells survived CHX concentrations as high
as 60 µg/ml (Fig. 4A). A
combination of sTNF and CHX did not further enhance cytotoxicity in
HeLatmTNF cells, whereas HeLa cells showed the expected
strong cytotoxic response (not shown). In HeLatmTNF cells,
CHX-induced cytotoxicity could be blocked by the broad spectrum caspase
inhibitor zVAD-fmk (Fig. 4A). TNF is known to induce cell
death synergistically with IFN-
(26). Fig. 4B shows that
the HeLatmTNF cells can be killed by the sole addition of
IFN-
, whereas HeLa cells are resistant to this treatment. To confirm
that the cytotoxic effects in HeLatmTNF cells do reflect apoptosis, we investigated the central apoptosis executioner caspase-3, which is known to be activated by proteolytic cleavage (27). HeLatmTNF cells showed an almost complete caspase-3
activation after treatment with CHX only (Fig. 4C), like
HeLa cells upon combined CHX and sTNF treatment. Together, these data
demonstrate that expression of tmTNF in HeLa cells initiates the
apoptotic signal cascade in a similar way, like sTNF in parental HeLa
cells, which is in both cases counterbalanced by CHX/IFN-
-sensitive factors.
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Fig. 4.
Inhibition of protein synthesis in
HeLatmTNF cells induces cytotoxicity in a
caspase-dependent manner. A, cell viability
of untreated or zVAD-fmk-treated (20 µM)
HeLatmTNF cells and HeLa cells was determined in the
presence of titrated concentrations of CHX after 18 h by crystal
violet staining. B, cell viability of indicated cells was
determined after treatment with IFN- (20 ng/ml) in the presence or
absence of TNF (50 ng/ml) for 3 days by crystal violet staining. The
results given show the mean values ±S.D. from three independent
experiments, each performed in duplicate in percent of viable cells
(100% = untreated controls). C, caspase-3 degradation in
HeLatmTNF cells and HeLa cells was determined by Western
blotting after treatment with CHX and TNF as indicated for 4 h.
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Fig. 5.
Autotropic stimulation of HeLa cells by
transmembrane TNF. HeLatmTNF and HeLa cells were
seeded at a statistical density of 1 cell/well in microtiter plates
with 192 replicates/experimental group and treated with CHX in the
presence or absence of zVAD-fmk (total culture volume, 10 µl; CHX, 2 µg/ml; zVAD-fmk, 20 µM). After 18 h, 290 µl of
culture medium were added to each well to dilute off the reagents, and
after an additional 14 days of culture, the plates were microscopically
investigated for growing colonies. The numbers of growing colonies in
the untreated control groups were in the expected range of 63%,
derived from Poisson statistics (HeLa: 66 and 67%, corresponding to
126 and 128 positive cultures; HeLatmTNF: 49 and 77%,
corresponding to 95 and 148 positive cultures). Shown are the relative
effects of CHX treatment in the absence and presence of zVAD-fmk
(control groups = 100% viable cells). Data from two independent
experiments are shown.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
. This cellular response
pattern resembles that of normal tissue cells, which are typically
resistant to the cytotoxic action of TNF unless metabolically stressed
by e.g. virus infection (29). In addition, HeLa cells are
well known to respond to sTNF with activation of the transcription
factor NF-
B and production of IL-6 (12). To study membrane TNF
action under conditions where effects by soluble TNF can be avoided,
TNF was constitutively expressed as a permanently membrane-anchored
mutein, human
(1-12) TNF (tmTNF) (22). In the mouse system, the
homologue
(1-12) deletion mutant of mouse TNF has a reduced
bioactivity (30) and only partly down-regulates TNF receptor expression
in L929 cells (31). It is therefore important to mention that the
(1-12) deletion mutant of human TNF shows full bioactivity on human
cells. We have recently demonstrated that the naturally occurring
transmembrane form of TNF as well as the
(1-12) deletion mutant
derived thereof have superior bioactivity on TNF-R2 when compared with
sTNF (5). When acting on TNF-R1, both molecules possess a bioactivity
indistinguishable from
sTNF.4
(1-12) TNF
(HeLatmTNF) expressed cell surface TNF in high amounts and
did not release bioactive soluble TNF into the culture supernatants
(data not shown). They further expressed considerable amounts of
tmTNF·TNF-R1 complexes at the cell surface. This is indicated by the
facts that free TNF-R1 was detectable only after a pH 3.0 treatment of
the cells (Fig. 1, B and E) and that constitutive
IL-6 production could be inhibited by TNF-specific antibodies (Fig.
2B). More important, the latter data also indicate that cell
surface-expressed tmTNF·TNF-R1 complexes are functional with respect
to signal transduction, although it cannot be excluded that in
addition, signaling from intracellular complexes might occur.
Accordingly, HeLatmTNF cells show a phenotype similar to
sTNF-stimulated HeLa cells. The transcription factor NF-
B (Fig. 3,
A and B) and the p38 MAP kinase (Fig.
3D) are constitutively activated, and consequently, the
cells produce high amounts of IL-6 (Fig. 2A). In addition,
HeLatmTNF cells undergo apoptosis in the presence of CHX
(Fig. 4A) or IFN-
(Fig. 4B). Together, these
data strongly support the hypothesis that in HeLatmTNF cells, TNF signaling pathways are permanently activated by the action
of endogenous tmTNF. Analysis at the single cell level (Fig. 5)
indicates that tmTNF action can occur in a truly autotropic manner.
Closer analysis of the constitutive activation pattern of
HeLatmTNF cells revealed a differential regulation of the
signaling pathways; NF-
B seems to be only partly activated in
HeLatmTNF cells, whereas IL-6 production and the kinetics
of induction of apoptosis in the presence of CHX were quantitative
similar to sTNF-treated HeLa cells (Fig. 2A and data not
shown). These differences might reflect differential regulation of
responses to the permanent tmTNF stimulation in HeLatmTNF
cells versus short term sTNF treatment of HeLa cells. Long
term sTNF-stimulated HeLa cells show in fact a significant NF-
B
down-regulation (data not shown).
B in HuT-78
cells (32). Third, it is unlikely that full TNF unresponsiveness is the
typical cellular response pattern for normal, TNF-R1-positive tissue
cells that are also capable of producing TNF. For example, human
umbilical cord vein endothelial cells coexpress both TNF receptors and
produce tissue factor upon adhesion molecule cross-linking via
autocrine/juxtacrine TNF action (16).
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ACKNOWLEDGEMENTS |
---|
We thank I.-M. von Broen, Knoll AG, for
recombinant human TNF, G. Kollias, Hellenic Pasteur Institute, Athens,
for the plasmid encoding human TNF(1-12), and O. Weiler
for help with the cloning of expression plasmids. The expert technical
assistance of Gudrun Zimmermann, Gisela Schubert, and Nathalie Peters
was gratefully acknowledged.
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FOOTNOTES |
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* This research was supported by Deutsche Forschungsgemeinschaft Grants Sche 349/5-1, Wa 1025/3-1, and Gr 1307/3-2).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of Cell
Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Tel.: 49 711 685 6987; Fax.: 49 711 685 7484;
E-mail: Peter.Scheurich{at}po.uni-stuttgart.de.
2 M. Grell and P. Scheurich, unpublished observations.
3 M. Grell, unpublished observations.
4 M. Grell, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
TNF-R, TNF receptor;
sTNF, soluble TNF;
tmTNF, transmembrane
TNF;
TRAF, TNF receptor-associated factor;
CHX, cycloheximide;
EMSA, electrophoretic mobility shift assay;
IFN-, interferon-
;
IAP, inhibitor of apoptosis;
IL, interleukin;
ELISA, enzyme-linked
immunosorbent assay;
NF-
B, nuclear factor
B;
PBS, phosphate-buffered saline;
MAP kinase, mitogen-activated protein
kinase;
mAb, monoclonal antibody.
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REFERENCES |
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