Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino Jr MA, Goeddel DVThe two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci USA 88:9292-9296

Abstract

The individual roles of the murine type 1 and 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 (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated 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 to murine and human TNF) 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 of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-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 signaling pathways that result in the induction of different cellular responses.

Full text

Proc.
Nati.
Acad.
Sci.
USA
Vol.
88,
pp.
9292-92%,
October
1991
Immunology
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
ABSTRACT
The
individual
roles
of
the
murine
type
1
and
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
to
murine
and
human
TNF)
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
of
the
two
TNF
receptors
in
the
various
cell
types,
because
LM
cells
express
on
their
surface
higher
levels
of
TNF-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
signaling
pathways
that
result
in
the
induction
of
different
cellular
responses.
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
of
normal
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
of
TNF
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
that
both
TNF
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
murine
TNF
(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
of
TNF-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
thymocytes.
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,
100
jug
of
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
receptor
type
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.
9292
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
88
(1991)
9293
(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
(Beckman).
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
100
Ag
of
streptomycin
per
ml
(GIBCO).
muTNF
and
antisera
were
added
to
a
final
volume
of
0.2
ml.
After
24 h
at
370C,
cultures
were
pulsed
with
1
ACi
of
[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
200
p1
of
medium
[RPMI
medium
supplemented
with
10%
fetal
calf
serum,
1%
L-glutamine,
100
units
of
penicillin
per
ml,
100
1g
of
strep-
tomycin
per
ml
(GIBCO),
and
5
1ug
of
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
nm.
Northern
Analysis.
Total
cytoplasmic
RNA
was
extracted
from
cells
as
described
(26),
electrophoresed
on
a
1.2%
form-
aldehyde/agarose
gel
(15
Ag
of
RNA
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
intensifying
screens.
RESULTS
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
that
TNF-R2
binds
muTNF
with
high
affinity
but
fails
to
recognize
hTNF
(20).
This
observation
suggested
that
the
various
activities
of
hTNF
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
of
TNF
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
activities.
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
.2b
15-
C
E
10
o00
0C
0
-
101
1-2
103
104
muTNF
(U/ml)
12-
CCO
10-
B
°
E
6E
00C.
0C
0
-
10-6
10
10-4
10-3
102
10-1
Serum
Dilution
FIG.
1.
Proliferation
of
murine
thymocytes
in
response
to
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
of
PHA-P
and
the
indicated
concentrations
of
muTNF
or
anti-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
PHA-P.
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
of
the
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
(Fig.
18).
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
that
TNF
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:
Tartaglia
et
A

9294
Immunology:
Tartaglia
et
al.
)
1.
r.
10-6
10-5
104
Serum
Dilution
10-3
10-2
FIG.
2.
Proliferation
of
CT6
cells
in
response
to
muTNF
(A)
and
antibodies
directed
against
the
murine
TNF
receptors
(B).
A,
muTNF;
m,
anti-TNF-R1;
o,
anti-TNF-R2;
e,
prebleed-TNF-R1;
o,
prebleed-TNF-R2.
CT6
cells
were
cultured
for
24
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.
induce
cytotoxicity
in
human
cell
lines
(15,
21,
22),
although
no
effect
of
the
human
TNF-R1
antibodies
has
been
reported
in
murine
cell
lines.
To
determine
if
the
anti-murine
TNF-R1
antibodies
could
mimic
TNF
activity,
we
assayed
them
for
120-
100
0-
80-
60
0
Anti-muTNF-R1
>
40-
\
*
Anti-muTNF-R2
40-
20-
0.
1i-7
10-6
10-4
10-3
10-2
Serum
Dilution
FIG.
3.
Cytocidal
effect
of
anti-murine
TNF
receptor
antibodies
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
this
study.
The
data
shown
are
the
mean
of
three
experiments
(±SEM).
Error
bars
have
been
omitted
for
points
with
SEM
<
the
size
of
symbol
(±2%).
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
of
LM
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
of
MnSOD
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
a
first
approximation
of
the
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
of
the
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-
Ch
-HX
1
2h
-
C
HX
2
.
~~
MnSOD
6X
IL-6
0
w
PAl-1
2-
Microglobulin
IW
c-tos
FIG.
4.
Induction
of
mRNA
in
murine
NIH
3T3
cells.
The
left
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
of
cycloheximide
(CHX)
per
ml:
control
(C),
100
ng
of
muTNF
per
ml,
100
ng
of
hTNF
per
ml.
The
right
three
lanes
show
steady-state
mRNA
levels
after
a
12-h
treatment
period
in
the
absence
of
cycloheximide:
control
(C),
100
ng
of
muTNF
per
ml,
100
ng
of
hTNF
per
ml.
C
CM
.o
2
L-
x
(.)
a)
-n
E
I
°.
ovv
25
-
20
15-
10-
5-
10°
-
idi
102
103
muTNF
(U/ml)
30
CC~j
.2
6
"
x
00
0-
o
C.
'
C
C3
cis=
25
20
15
10
Proc.
Natl.
Acad
Sci.
USA
88
(1991)

Immunology:
Tartaglia
et
al.
0
z
z
0
FIG.
5.
Northern
analysis
of
MnSOD
mRNA
in
murine
NIH
3T3
cells.
Cells
were
treated
with
the
indicated
reagents
for
12
h:
control,
100
ng
of
muTNF
per
ml,
1:100
dilution
of
anti-TNF-R1
serum,
1:100
dilution
of
anti-TNF-R2
serum,
1:100
dilution
of
anti-NGF
serum.
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.
DISCUSSION
We
have
previously
shown
that
murine
TNF-R1
has
a
similar
affinity
for
muTNF
and
hTNF,
whereas
murine
TNF-R2
is
specific
for
muTNF
(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
of
TNF
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
of
MnSOD
mRNA
has
been
previously
shown
to
counteract
the
cyto-
toxic
effects
of
TNF,
and
therefore
mitochondrial
generation
of
0°
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
of
01
and
the
induction
of
a
02
scavaging
activity
are
signaled
by
the
same
TNF
receptor.
The
inability
of
the
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)
9295
receptors
expressed
on
the
surface
of
LM
cells
are
about
60%
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
of
the
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
of
the
two
antibody
preparations
in
the
T-cell
and
thymocyte
proliferation
assays.
Whereas
CT6
cells
express
little
or
no
TNF-R1
mRNA
and
do
not
bind
detectable
amounts
of
hTNF
(20),
thymus
cells
express
mRNA
for
both
receptor
types
(20)
and
the
thymocytes
used
in
the
proliferation
assays
bind
muTNF
and
hTNF
(29)
(indicative
of
the
presence
of
TNF-
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
TNF
receptors
are
not
redundant
in
signaling
these
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
of
the
two
TNF
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
of
TNF-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
of
TNF.
Therefore,
signaling
via
TNF-R2
does
not
have
an
absolute
requirement
for
TNF
and
may
be
initiated
through
a
mechanism
similar
to
that
utilized
by
TNF-R1.
The
absence
of
reports
on
TNF-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
of
TNF-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
that
TNF-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
different
TNF
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
of
the
soluble
TNF
receptors
and
also
Greg
Bennett
and
Roderick
Vitangcol
for
preparation
of
the
polyclonal
antisera.

92%
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