Proc. Nati. Acad. Sci. USA
Vol. 88, pp. 9292-92%, October 1991
The two different receptors for tumor necrosis factor mediate
distinct cellular responses
(species specificity/antibodies/manganous superoxide dismutase/cytotoxicity/proliferation)
Louis A. TARTAGLIA*, RICHARD F. WEBER*, IRENE S. FIGARIt, CARMEN REYNOLDSt,
MICHAEL A. PALLADINO, JR.t, AND DAVID V. GOEDDEL*
Departments of *Molecular Biology and tCell Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080
Communicated by Bruce N. Ames, July 25, 1991
type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and
TNF-R2) were investigated utilizing (i) the strong species
specificity of TNF-R2 for murine TNF compared to human
TNF and (it) agonistic rabbit polyclonal antibodies directed
against the individual TNF receptors. Proliferation of mouse
thymocytes and the murine cytotoxic T-cell line CT-6 is stim-
ulated by murine TNF but not by human TNF. Consistent with
this observation, polyclonal antibodies directed against
TNF-R2 induced proliferation in both of these cell types,
whereas polyclonal antibodies directed against TNF-R1 had no
effect. In contrast, cytotoxicity in murine LM cells (which are
sensitive tomurine andhumanTNF) was induced by antibodies
against TNF-R1 but not by antibodies against TNF-R2. Also,
the steady-state level of manganous superoxide dismutase
mRNA in the murine NIH 3T3 cell line was induced by murine
TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2.
These results suggest that TNF-R2 initiates signals for the
proliferation of thymocytes and cytotoxic T cells, whereas
TNF-R1 initiates signals for cytotoxicity and the induction of
the protective activity, manganous superoxide dismutase. The
nonredundant signaling observed for the two TNF receptors
cannot be explained simply by the differential expression ofthe
two TNF receptors in the various cell types, because LM cells
express on their surface higher levels ofTNF-R2 than TNF-R1,
and LM cells, NIH 3T3 cells, and thymus cells all express
mRNA corresponding to both receptor types. It is therefore
likely that the two receptors initiate distinct signalingpathways
that result in the induction of different cellular responses.
The individual roles ofthe murine type 1 and
Tumor necrosis factor (TNF) is a multifunctional cytokine
produced mainly by activated macrophages, T cells, mast
cells, and some epithelial tumor cells (1-3). The wide range
of biological effects elicited by TNF include hemorrhagic
necrosis of transplanted tumors, growth proliferation of
normal cells, cytotoxicity, inflammatory, immunoregulatory
and antiviral responses, and an important role in endotoxic
shock (4-8). The first step in the induction of these various
cellular responses by TNF is the binding to specific cell
surface receptors. TNF receptors have been detected on a
wide variety ofnormal tissues and cell lines that are sensitive
or resistant to TNF (9-12). Two immunologically distinct
TNF receptors of approximately 55 kDa (TNF-R1) and 75
kDa (TNF-R2) have now been identified (13-16), and human
and mouse cDNAs corresponding to both receptor types
have been isolated and characterized (17-20).
A number of recent reports have described initial studies
investigating the individual roles of the two human TNF
receptors. Polyclonal and monoclonal antibodies directed
against human TNF-R1 have been shown to behave as
receptor agonists and elicit several TNF activities, such as
cytotoxicity, fibroblast proliferation, resistance to chlamid-
iae, and synthesis of prostaglandin E2 (15, 21, 22). Monoclo-
nal antibodies against human TNF-R1 that block the binding
ofTNF to TNF-R1 and antagonize several TNF effects have
also been described (21-23). Although no direct signaling role
for TNF-R2 has yet been identified with either receptor
agonists or transfection studies, several reports have de-
scribed monoclonal antibodies directed against TNF-R2 that
can partially antagonize TNF responses (such as cytotoxicity
and activation of NF-KB) and enhance the antagonistic ef-
fects of anti-TNF-R1 monoclonal antibodies (22-24). These
reports suggested thatbothTNF receptors are active in signal
transduction and that there is redundancy in the function of
the two receptors. However, the reported effects of the
TNF-R2 antagonists have been quite small and were ob-
served exclusively at very low TNF concentrations. It is
therefore possible that TNF-R2 is only participating as a
minor accessory component to TNF-R1 in the signaling of
these responses. In this report we describe a direct role of
TNF-R2 in stimulating proliferation of murine thymocytes
and T cells and show that this receptor is distinct from the
receptor (TNF-R1) mediating cytotoxicity.
MATERIALS AND METHODS
Reagents. Recombinant murineTNF (muTNF) and recom-
binant human TNF (hTNF) (specific activity >107 units/mg)
were provided by the Genentech manufacturing group. Rab-
bit anti-murine TNF-R1 and rabbit anti-murine TNF-R2
antibodies were generated against the soluble extracellular
domain of the corresponding receptors (ref. 20; R.F.W. and
D.V.G., unpublished results). The titers of these antisera
were quantitated by a direct antigen-coated ELISA. The
dilutions of anti-TNF-Rl and anti-TNF-R2 giving 50o bind-
ing to the corresponding purified soluble receptor were
1:109,000 and 1:104,000, respectively. The cross-reactive
titers ofTNF-R1 antiserum to soluble TNF-R2 and TNF-R2
antiserum to soluble TNF-R1 were <1:200. C3H/HeJ mice
(The Jackson Laboratory) were used as the source of fresh
Thymocyte Proliferation Assay. C3H/HeJ thymocytes
were cultured in 96-well flat-bottomed culture plates (1.5 X
106 per 0.1 ml) (Costar) in Eagle's minimal essential medium
supplemented with 10%t heat-inactivated fetal bovine serum
(HyClone), 1% L-glutamine, 1% nonessential amino acids,
100 units of penicillin per ml, 100jugof streptomycin per ml,
0.1% gentamicin (GIBCO), and 0.05 mM 2-mercaptoethanol
(Sigma) in the presence of 0.1% phytohemagglutinin P
Abbreviations: TNF, tumor necrosis factor; muTNF, recombinant
murine TNF; hTNF, recombinant human TNF; TNF-R1, TNF
receptortype 1;TNF-R2, TNF receptor type 2; MnSOD, manganous
superoxide dismutase; PHA-P, phytohemagglutinin P; PAM-i, plas-
minogen activator inhibitor 1; IL-6, interleukin 6.
The publication costs ofthis article were defrayed in part by page charge
payment. This article must therefore be herebymarked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. Natl. Acad. Sci. USA 88(1991)
(PHA-P; Difco). PHA-P, muTNF, and antibodies were added
to a final volume of 0.2 ml. After 60 h at 370C, cultures were
pulsed with 1 uCi of [3H]thymidine (5 Ci/mmol; 1 Ci = 37
GBq; New England Nuclear) for 12 h and harvested onto
glass fiber filters (PhD; Cambridge Technology, Watertown,
MA); mean [3H]thymidine incorporation (cpm) of triplicate
cultures was determined using a liquid scintillation counter
CT-6 Proliferation Assay. CT-6 cells (25) were cultured in
96-well flat-bottomed culture plates (5.0 x 104 per 0.1 ml)
(Costar) in RPMI medium supplemented with 10%o fetal calf
serum (HyClone), 1% L-glutamine, 100 units of penicillin per
ml, and 100Agofstreptomycin per ml (GIBCO). muTNF and
antisera were added to a final volume of0.2 ml. After 24 h at
370C, cultures were pulsed with 1ACiof [3H]thymidine (5
Ci/mmol) for 4 h and harvested onto glass fiber filters, and
mean [3H]thymidine incorporation (cpm) of triplicate cul-
tures was determined.
LM Cytotoxicity Assay. LM cells (2 x 104 cells per well)
were seeded into 96-well microtiter plates in 200p1 ofmedium
[RPMI medium supplemented with 10% fetal calf serum, 1%
L-glutamine, 100 units of penicillin per ml, 1001gof strep-
tomycin per ml (GIBCO), and 51ugof insulin per ml] and
incubated 24 h at 370C in a 5% CO2 atmosphere. Medium was
then brought to 10 pug of cycloheximide per ml and the
anti-TNF-R antibodies were added to the wells and serially
diluted. The plates were incubated for an additional 16 h and
the viable cells were stained with 20% methanol containing
0.5% crystal violet. The dye was eluted with 0.1 M sodium
citrate and 50%o ethanol and absorbance was measured at 540
Northern Analysis. Total cytoplasmic RNA was extracted
from cells as described (26), electrophoresed on a 1.2% form-
aldehyde/agarose gel (15AgofRNA per lane), and transferred
to nitrocellulose filters. Filters were hybridized and washed as
described (26). The probes used for filter hybridizations were
either a random-primed 32P-labeled 0.8-kilobase (kb) fiagment
(entire coding region) of the manganous superoxide dismutase
(MnSOD) cDNA (27) or 32P-labeled oligonucleotides corre-
sponding to the murine genes encoding interleukin 6 (IL-6),
plasminogen activator inhibitor 1 (PAI-1), P2-microglobulin,
and c-fos. Autoradiography was done at -70°C using Kodak
Thymocyte and T-Cell Proliferation Is Stimulated by Anti-
bodies to TNF-R2. In a previous report we described the
cloning of cDNAs encoding the two murine TNF receptors
and showed thatTNF-R2 binds muTNF with high affinity but
fails to recognize hTNF (20). This observation suggested that
the various activities ofhTNF reported in mice or in murine
cell lines would be mediated by TNF-R1, whereas those
activities that displayed a strong species specificity for
muTNF might be regulated by TNF-R2. Literature searches
revealed that the majority ofTNF effects reported in mice or
murine cell lines exhibited little species preference. An
exception to this was TNF-induced proliferation of murine
thymocytes and the cytotoxic T-cell line CT6 (25, 28-30).
muTNF concentrations as low as 100 units/ml (<10 ng/ml)
could induce a strong proliferative response in both of these
cell types, whereas hTNF had no effect even at concentra-
tions of >10,000 units/ml (=1 ,ug/ml). To examine more
directly the receptor that mediates proliferation in these cell
types, we tested highly specific polyclonal antibodies di-
rected against the individual receptors for possible agonist
As shown in Fig. lA, muTNF stimulates C3H/HeJ thy-
mocyte proliferation in the presence of a submitogenic dose
of PHA-P. No stimulation is seen with hTNF even at doses
° E 6E
muTNF (A) and antibodies directed against the murine TNF recep-
tors (B. *, muTNF; *, anti-TNF-R1; o, anti-TNF-R2; e, prebleed-
TNF-R1; o, prebleed-TNF-R2. Thymocytes were cultured for 60 h
with a submitogenic dose ofPHA-P and the indicated concentrations
ofmuTNForanti-TNF-R antibody. Cultures were then pulse labeled
with [3H]thymidine for 12 h and mean [3H]thymidine incorporation
(±SEM) of triplicate cultures was determined. The amount of 3H
incorporation in thymocytes treated with PHA-P alone is indicated
by a dashed line. No proliferation was observed in the absence of
Proliferation of murine thymocytes in response to
as high as 105 units/ml (refs. 28 and 29; data not shown).
Thymocyte proliferation was also examined in response to
various dilutions of polyclonal antibodies directed against
murine TNF-R1 and TNF-R2. Antibodies to TNF-R2
strongly stimulated the proliferation ofthe C3H1/HeJ thymo-
cytes even at a dilution factor of 104. Polyclonal antibodies
directed against TNF-R1 had no effect at any concentration
tested despite the similar titer of the two types of antisera
A similar set of experiments was performed with the
interleukin 2 (IL-2)-dependent cytotoxic T-cell line CT6.
When IL-2 is removed from the growth medium, muTNF can
serve as a proliferative signal (Fig. 2A), whereas hTNF
cannot (refs. 25 and 30; data not shown). Polyclonal anti-
bodies against TNF-R2 also stimulated the growth of CT6
cells and the magnitude of the proliferative response was
similar to that seen with muTNF (Fig. 2B). No significant
effect was observed with polyclonal antibodies directed
against TNF-R1. The agonist activity of the anti-TNF-R2
antibodies in the thymocyte and T-cell proliferation assays
indicates that TNF-R2 can signal proliferation in at least
some T-cell populations and also thatTNF is not required for
the signal transmission.
Cytotoxicity in LM Ceils Is Induced by Antibodies to TNF-
Rl.'One trivial explanation for the inability of the TNF-R1
antibodies to induce T-cell and thymocyte proliferation was
that these antibodies do not possess agonist activity even for
those effects that are normally signaled by TNF-R1. It has
been shown previously that polyclonal as well as some
monoclonal antibodies directed against human TNF-R1 can
Immunology: Tartagliaet A
Immunology: Tartaglia et al.
antibodies directed against the murine TNF receptors (B).
muTNF; m, anti-TNF-R1; o, anti-TNF-R2; e, prebleed-TNF-R1; o,
prebleed-TNF-R2. CT6 cells were cultured for24 h with the indicated
concentrations of muTNF or anti-TNF-R antibody. Cultures were
then pulse labeled with [3H]thymidine for 4 h and mean [3H]thymi-
dine incorporation (±SEM) of triplicate cultures was determined.
The amount of 3H incorporation in untreated CT6 cells is indicated
by a dashed line.
Proliferation ofCT6 cells in response to muTNF (A) and
induce cytotoxicity in human cell lines (15, 21, 22), although
no effect ofthe human TNF-R1 antibodies has been reported
in murine cell lines. To determine ifthe anti-murine TNF-R1
antibodies could mimic TNF activity, we assayed them for
on LM cells. o, Anti-TNF-R1; e, anti-TNF-R2. The antisera were
applied for 16 h at the indicated dilutions in the presence of
cycloheximide at 10 ug/ml. Viability of cells was determined (see
text). Preimmune sera and anti-NGF antiserum had no effect in the
range of serum concentrations used in thisstudy.The data shown are
the mean of three experiments (±SEM). Error bars have been
omitted for points with SEM < the size ofsymbol (±2%).
Cytocidal effect of anti-murine TNF receptor antibodies
cytotoxicity against murine LM cells. LM cells were chosen
because they possess TNF-R1 (40%o) and TNF-R2 (60%o) (20)
and show a similar sensitivity to muTNF and hTNF (indic-
ative of a TNF-R1 response) (30). As shown in Fig. 3, LM
cells' were highly sensitive to antibodies against TNF-R1 but
not TNF-R2. These results indicate that the antibodies to
muTNF-R1 are agonistic and that TNF-R1 can mediate
cytotoxicity in murine cells. The resistance ofLM cells to the
TNF-R2 antibodies, despite the cell surface expression of
TNF-R2, suggests that TNF-R2 cannot deliver a cytotoxic
signal in murine LM cells. Similar TNF-R1 specific cytotox-
icity was also seen with the murnine cell lines B6MS5, L929,
and NIH 3T3 (data not shown).
TNF Induction ofMnSOD mRNA Is Mediated by TNF-R1.
The mitochondrial enzyme MnSOD is an important determi-
nant of cellular resistance to the cytotoxic effects of TNF
(27). In addition, MnSOD synthesis is specifically and rapidly
induced by TNF treatment of many cell types (26). We
therefore tested whether induction of the MnSOD gene was
mediated by the same or different TNF receptor as the one
that signals cytotoxicity. To make afirstapproximation ofthe
individual roles of TNF-R1 and TNF-R2 in mediating Mn-
SOD induction, we examined the TNF species specificity of
this response. As shown in Fig. 4, muTNF and hTNF
strongly induced the 1-kb and 4-kb MnSOD transcripts in the
murine NIH 3T3 cell line. Both cytokines also induced the
steady-state mRNA levels ofthe genes encoding IL-6, PAI-1,
.82-microglobulin, and c-fos. MnSOD mRNA levels were
induced by muTNF and hTNF during a short ex'osure (3 h)
in the presence of the protein synthesis inhibitor cyclohexi-
mide and a longer exposure (12 h) in the absence of cyclo-
12h - CHX
three lanes show steady-state levels of mRNA encoding MnSOD,
IL-6, PAI-1, f2-microglobulin, and c-fos after a 3-h treatment period
in the presence of 10 ,g ofcycloheximide (CHX) per ml: control (C),
100 ngofmuTNF per ml, 100 ngofhTNF per ml. The right three lanes
show steady-state mRNA levels after a 12-h treatment period in the
absence ofcycloheximide: control (C), 100 ng ofmuTNF per ml, 100
ng of hTNF per ml.
Induction of mRNA in murine NIH 3T3 cells. The left
Proc. Natl. Acad Sci. USA 88(1991)
Immunology: Tartaglia et al.
cells. Cells were treated with the indicated reagents for 12 h: control,
100 ng ofmuTNF per ml, 1:100 dilution ofanti-TNF-R1 serum, 1:100
dilution of anti-TNF-R2 serum, 1:100 dilution of anti-NGF serum.
Northern analysis ofMnSOD mRNA in murine NIH 3T3
heximide. These results suggested that MnSOD induction is
signaled through TNF-R1. To test this prediction, NIH 3T3
cells were treated with the agonistic anti-TNF-R1 and anti-
TNF-R2 antibodies. As shown in Fig. 5, anti-TNF-R1 anti-
bodies strongly induced MnSOD mRNA, whereas anti-
TNF-R2 antibodies had no effect. These results demonstrate
that the receptor responsible for signaling cytotoxicity (TNF-
R1) also mediates the induction of a key protective activity.
We have previously shown that murine TNF-R1 has a similar
affinity for muTNF and hTNF, whereas murine TNF-R2 is
specific formuTNF (20). The murine system therefore allows
predictions to be made as to which TNF receptor mediates a
given response: TNF-R1 responses should be induced by
muTNF and hTNF, whereas TNF-R2 responses should only
be induced by muTNF. To validate this model and more
directly examine the individual roles of the two TNF recep-
tors, we generated rabbit polyclonal antibodies against sol-
uble forms of both muTNF receptors.
Polyclonal antibodies directed against TNF-R2 were found
to stimulate proliferation of murine thymocytes and the
cytotoxic T-cell line CT6. However, polyclonal antibodies
directed against TNF-R1 had no such proliferative effect.
These results are consistent with the proliferation of these
cell types in response to muTNF, and not hTNF (25, 28-30),
which is also suggestive of a TNF-R2 response.
Cytotoxicity in murine LM cells is standardly used as a
sensitive assay for hTNF (31). Also, LM cells exhibit a
similar sensitivity to muTNF and hTNF (30). This is sugges-
tive of a response mediated by TNF-R1 with little if any
requirement for the binding ofTNF to TNF-R2. In agreement
with this, polyclonal antibodies directed against TNF-R1
induced cytotoxicity in LM cells, even at a serum dilution of
1:104. No cytotoxicity was seen with TNF-R2 antibodies,
despite the ability of these antibodies to behave as agonists
in the T-cell proliferation assays. It is interesting to note that
induction of the mRNA encoding the defense activity Mn-
SOD was also TNF-R1 specific. Overexpression ofMnSOD
mRNA has been previously shown to counteract the cyto-
toxic effects ofTNF, and therefore mitochondrial generation
has been implicated as a key component of TNF-
mediated cell killing (27). Thus, it appears that the cascade of
events that lead to generation of01 and the induction ofa02
scavaging activity are signaled by the same TNF receptor.
The inability ofthe TNF-R2 antibodies to act as agonists in
the LM cytotoxicity assay was not due to the absence of
TNF-R2 on LM cells; we previously showed that the TNF
Proc. Natl. Acad. Sci. USA 88 (1991)
receptors expressed on the surface ofLM cells are about60%
TNF-R2 and 40% TNF-R1 (20). In addition, the NIH 3T3
cells used in the MnSOD induction experiments expressed
transcripts corresponding to TNF-R1 and TNF-R2 (data not
shown). The results ofthe cytotoxicity and MnSOD mRNA
studies therefore suggest that the functions of TNF-R1 and
TNF-R2 are not redundant, but rather that only TNF-R1
signals these two responses.
A similar argument can be made for the different behavior
ofthe two antibody preparations in the T-cell and thymocyte
proliferation assays. Whereas CT6 cells express little or no
TNF-R1mRNAanddo notbind detectable amounts ofhTNF
(20), thymus cells expressmRNAforboth receptortypes (20)
and the thymocytes used in the proliferation assays bind
muTNF and hTNF (29) (indicative of the presence ofTNF-
Ri). Therefore, it would appear that the proliferative signal
for these cells can be mediated by TNF-R2 but not by
TNF-R1. These results indicate that different TNF receptors
signal cytotoxicity and T-cell proliferation and that the two
TNFreceptors are notredundant in signalingthese functions.
The amount of primary sequence similarity between
TNF-R1 and TNF-R2 is also suggestive of distinct functions
for the two TNF receptors. For although the extracellular
ligand-binding domains ofthe twoTNF receptors show some
homology (=20%), their intracellular domains show no sig-
nificant similarities (20). This would be consistent with the
intracellular regions of the two receptors being coupled to
different signal transduction pathways.
Several reports have described antibodies directed against
human TNF-R1 that have agonist properties (15, 21, 22).
These studies demonstrate that a specific conformational
change induced by the TNF molecule itself is probably not
responsible for the activation ofTNF-R1. Rather, a nonspe-
cific perturbation, most likely receptor dimerization or ag-
gregation, can be sufficient to signal through this receptor. In
this report, we show that TNF-R2 can also be activated by
immunoglobulins in the absence ofTNF. Therefore, signaling
via TNF-R2 does not have an absolute requirement forTNF
and may be initiated through a mechanism similar to that
utilized by TNF-R1.
The absence ofreports onTNF-R2 agonists may be a result
of direct TNF-R2 responses being much less numerous than
TNF-R1 responses. In support of this, most activity com-
parisons of hTNF and muTNF in mice or murine cell lines
show relatively small differences, with T-cell and thymocyte
proliferation being the most dramatic exceptions. Why
TNF-R2 effects would be specific for such a small cell
population is not clear, given the near ubiquitous distribution
of this receptor in cell types and tissues. Perhaps a number
ofTNF-R2 responses are yet to be identified. Also, TNF-R2
may play an accessory role in mediating other TNF effects,
even if it is not responsible for the signal transmission.
The experiments described in this study show thatTNF-R1
can initiate signals for cytotoxicity and TNF-R2 can initiate
signals for thymocyte and cytotoxic T-cell proliferation.
However, it should be noted that TNF-induced proliferation
may not be mediated by TNF-R2 in all cell types. Engelmann
et al. (15) have shown that polyclonal antibodies against
human TNF-R1 can stimulate proliferation of human FS11
fibroblasts. Thus, it appears that differentTNF receptors can
signal proliferation in different cell types. It is therefore likely
that many additional studies will be required before a thor-
ough understanding of the individual roles of the two TNF
receptors will be realized.
We thank Bill Kohr and Helga Raab for help and advice on the
purification ofthe soluble TNF receptors and also Greg Bennett and
Roderick Vitangcol for preparation of the polyclonal antisera.
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