(Received for publication, November 27, 1995)
From the
The role for the two tumor necrosis factor (TNF) receptors in
discriminating TNF and lymphotoxin (LT
) effects has been
studied. TNF and LT
were equally mitogenic in Fs4 fibroblasts,
which express a high amount of the p55 compared to the p75 TNF
receptors (TNFRs). In contrast, TNF was more potent than LT
in
mediating gene regulation and cytotoxicity in SW480-
Gal cells and
KYM-1 cells, which have a high p75/p55 TNFR ratio. Both TNF and LT
showed comparable affinities for the two TNFRs. However, in contrast to
LT
, TNF dissociated rapidly from the p75 TNFR, whereas both
cytokines dissociated slowly from the p55 TNFR. Soluble p55 TNFR was
much more potent than soluble p75 TNFR in inhibiting TNF cytotoxicity,
whereas both soluble receptors moderately decreased LT
-mediated
cytotoxicity with comparable efficacy. Antagonistic monoclonal
antibodies against either TNFR types markedly inhibited TNF effects.
However, only the p55 TNFR antagonistic antibody significantly
decreased LT
-mediated cytotoxicity and cytomegalovirus promoter
activation, whereas blocking of the p75 TNFR enhanced the LT
effects. These data suggest that whereas the p75 TNFR can both directly
propagate TNF signals and ``pass'' TNF to the p55 TNFR, it
attenuates LT
and may serve as a decoy receptor for this cytokine.
Tumor necrosis factor (TNF) (
)and lymphotoxin
(LT
, TNF
) are pleiotropic cytokines which mediate a
large variety of inflammatory, immunostimulatory, and antiviral
responses(1) . They are both members of the TNF ligand and
receptor family, which now contains at least 12 ligand-receptor
pairs(2) . TNF and LT
are unique among this family in
sharing the two TNF receptors, the p55 TNFR (CD120a) and the p75 TNFR
(CD120b), both of which are expressed on most mammalian
cells(3) . Both TNF and LT
exist as homotrimeric
molecules, each with the capability of complexing with three
receptors(4, 5) , and both cytokines are believed to
elicit their effects by the cross-linking of cell surface
receptors(6, 7, 8) . LT
has also been
shown to form a cell surface heterocomplex with LT
(9) .
While the LT
homotrimer binds to the p55 and p75 TNFRs, the
heteromeric LT
LT
complex interacts with a TNFR-related
protein (LT
-R)(10) . Gene knock-out experiments in mice
suggest that the LT
-LT
heteromer is involved in the
development of the peripheral lymphoid tissues(11) , while the
two TNFRs are responsible for the resistance for certain types of
bacterial pathogens(12, 13) .
The ratio of the two receptors on the cell surface varies among cells, i.e. some cell types have a high proportion of the p75 TNFR versus the p55 TNFR, and vice versa. Furthermore, the cell surface expression of the two TNFRs is independently regulated in many cell types. It has been shown, for instance, that activation of B cells results in a marked up-regulation of the p75 but not the p55 TNFR(14) . Also, the generation of soluble receptors differs for the two TNFRs, because in vitro stimulation of monocytes with lipopolysacharide results in the specific secretion of the p75 TNFR, whereas administration of endotoxin to humans leads to increased serum levels of both types of soluble receptors(15, 16) . Both receptors can also be found in soluble forms in body fluids during various pathological and physiological conditions(17) . However, the functional role of soluble TNFRs in vivo is yet to be elucidated since they have been reported to either attenuate or enhance TNF activity in vitro(18, 19) . Recombinant soluble receptors are currently under evaluation in the clinic in attempts to attenuate the harmful effects of TNF.
Despite
strong redundancy, TNF and LT have been reported to differ in
their potency to exert biological effects in many cell types. Thus, TNF
was more potent than LT
in inducing cytotoxicity toward MCF7
carcinoma cells and cytokine secretion by human fibroblasts, monocytes,
and endothelial
cells(20, 21, 22, 23) . However, TNF
and LT
were equally cytotoxic toward mouse WEHI clone 13 and L929
fibroblastic cell lines (24, 25) . LT
, on the
other hand, was more potent than TNF when assayed for the ability to
induce cytotoxicity and apoptosis in oligodendrocyte
cultures(25) . Furthemore, LT
, but not TNF, has been
reported to act as a growth factor for Epstein-Barr virus-infected B
cell lines(26) . It is, however, not entirely clear to what
extent these cytokines have differential physiological
effects(27) .
The specific roles of the two TNFRs in
mediating the TNF and LT signals are currently vividly
debated(28, 29) . In many cell types, studies with
antagonistic antibodies have indicated that both receptors are
important in mediating TNF effects(30) . Furthermore, a unique
``passing model'' has been suggested to explain the role of
the p75 TNFR in mediating TNF responses(31) . The basis for
this model is that although TNF binds to the p75 TNFR with somewhat
higher affinity than to the p55 TNFR, the rate of dissociation of TNF
from the p75 TNFR is higher than from the p55 TNFR. This rapid rate of
dissociation of the TNF
p75 TNFR complex may facilitate
interaction of TNF with the p55 TNFR, suggesting a role of the p75 TNFR
in passing TNF to the p55 TNFR, which has been postulated to be the
main TNF signal transducer. On the other hand, also the p75 TNFR
mediates several TNF effects such as proliferation of T- and
B-cells(14, 32) , activation of the human CMV promoter
and induction of NF-
B(33) , and
cytotoxicity(8, 34, 35) . Studies with
agonistic antibodies specific for either of the receptors have
indicated that at least in some cell types both receptors may induce
similar effects, although possibly by using different intracellular
signaling pathways(33, 34, 35) . At present,
the distinct functional roles of the two TNFRs, as well as their
possible role in defining disparate TNF and LT
effects, are not
clear.
Here we show that cells expressing a high proportion of the
p75 TNFR are relatively resistant to the action of LT and that
LT
, in contrast to TNF, has a slow rate of dissociation from the
p75 TNFR. Blocking of the p75 TNFR with antagonistic antibodies
inhibited TNF effects but enhanced LT
activities in cells
expressing a high p75/p55 TNFR ratio. It suggests a mechanism by which
the high p75 TNFR expression may inhibit LT
effects and indicates
distinct specific roles for the two receptor types in discriminating
between TNF and LT
effects on cells.
Figure 1:
Effects of TNF and LT in Fs4 (A), KYM-1 (B), and SW480-
Gal (C)
cells. Cells were incubated as indicated with TNF (
) and LT
(
). Responses were assayed as described under ``Experimental
Procedures'' and were: incorporation of
[
H]thymidine in Fs4 cells, cytotoxicity measured
as MTT release in KYM-1 cells, and activation of a
-galactosidase
gene under the control of the CMV promoter in SW480-
Gal
cells.
Figure 2:
Mobilization of NF-B in
SW480-
Gal cells by TNF and LT
. Gel-shift assays were
performed on nuclear extracts from SW480-
Gal cells treated with
the indicated concentrations of TNF or LT
for 1 h at 37 °C (left-hand side) or with 10 ng/ml TNF and 100 ng/ml LT
for the indicated time periods in minutes (right-hand side). wt-
B and m-
B represent lanes where the
nuclear extracts and the specific probe were incubated with excess
amounts of unlabeled wild type NF-
B and mutated NF-
B probes,
respectively, as described
earlier(20) .
Taken together, these results indicate that
although TNF and LT are about equally potent in inducing responses
in some cell lines, other cells are more or less resistant to the
action of LT
while still being susceptible to the action of TNF.
Figure 3:
Specific binding of I-TNF (A, C, and E) and
I-LT
(B, D, and F) to Fs4 (A and B), KYM-1 (C and D), and SW480-
Gal (E and F) cells. Cells as described under
``Experimental Procedures'' were incubated with the indicated
concentrations of iodinated TNF or LT
, in the absence or presence
of either 0.5 nM unlabeled TNF or LT
, 10 µg/ml p75
TNFR specific utr-1 antibody, or 10 µg/ml p55 TNFR specific htr-9
antibody.
Scatchard
analysis of the data indicated that all three cell lines used here
express approximately similar numbers of the p55 TNFR per cell
(1000-3000 receptors/cell). On the other hand, these cell lines
express in the range from a few hundreds (Fs4) to more than one hundred
thousands (SW480-Gal) of the p75 TNFR per cell (data not shown).
Taken together, these results suggest that a high specific activity of
LT
correlates with a high p55/p75 TNFR ratio, whereas the specific
activity of TNF is not particularly affected by the p55/p75 ratio. It
should also be noted that in all three cell lines examined, the sum of
the binding of both TNF and LT
in the presence of utr-1 and htr-9
equals the specific binding in the absence of antibodies. Thus, there
is no indication of the existence of a third type of binding sites for
either TNF, or LT
, which could explain the differences in their
biological activity.
Figure 4:
Scatchard plot of the binding of I-LT
to Fs4 (A) and KYM-1 (B)
cells. Binding data from experiments as shown in Fig. 3were
plotted according to the method of
Scatchard.
Taken
together, these data indicate that the difference in specific activity
between TNF and LT in Fs4 and KYM-1 cells cannot be explained by
the differences in affinities of LT
and TNF to the p55 and p75
TNFRs.
As shown in Fig. 5A, TNF rapidly
dissociated from the p75 TNFR cells. In striking contrast to this,
LT appeared to have a relatively slow rate of dissociation from
this receptor, indicating that the on-off rate of the LT
p75
complex is much slower than the on-off rate of the TNF
p75
complex. The experiments shown were performed at room temperature and
indicate at least a 6-fold difference between TNF and LT
in the
rate of dissociation from the p75 TNFR at this temperature (t
5 min for TNF versus more than
30 min for LT
). The rate of dissociation for LT
and TNF from
the p55 TNFR appeared to be indistinguishable, and somewhat lower (t
> 60 min) than the rate of dissociation
for LT
from the p75 TNFR (Fig. 5B). These data
demonstrate that there are major differences between TNF and LT
in
their interaction with the p75 TNFR, whereas the experiments reveal no
differences in the interaction with the p55 TNFR.
Figure 5:
Dissociation of TNF and LT from the
p75 (A) and the p55 TNFR (B). In A were
SW480-
Gal cells incubated with saturating amounts of iodinated TNF
(
) and LT
(
) in the presence of 10 µg/ml htr-9 for
3 h. In B were Fs4 cells incubated with saturating
concentrations of iodinated TNF (
) and LT
(
) in the
presence of 10 µg/ml utr-1 as above. Cells were washed, and
unlabeled TNF and LT
were added to wells containing bound labeled
TNF and LT
, respectively. Cell-associated radioactivity was
measured at the indicated time points thereafter as described under
``Experimental Procedures.''
Figure 6:
Effect of soluble p55 and p75 TNFRs on
activity of TNF and LT. TNF (A) and LT
(B)
were added to WEHI clone 13 cells in the absence or presence of either
300 ng/ml soluble recombinant p75 TNFR (sp75; squares) or 300 ng/ml soluble recombinant p55 TNFR (sp55; triangles). After incubation for 24 h, cell
viability was determined according to the MTT
method.
Figure 7:
Effect of antagonistic p55 and p75
monoclonal antibodies on TNF and LT effects. SW480-
Gal and
KYM-1 cells were stimulated with TNF (A and C) and
LT
(B and D) either alone (open
circles) or in the presence of htr-5 (open triangles) and
utr-1 (closed triangles) (all antibodies were applied at a
concentration of 10 µg/ml) followed by measurement of
-galactosidase activity (A and B) or
cytotoxicity (C and D) as described under
``Experimental Procedures.''
Why TNF and LT show, depending on the cell type, either
similar or different biological effects remains one of the unresolved
questions in the cytokine field. The present paper demonstrates a
correlation of cell responsiveness to TNF and LT
with the
expression of the p75 TNFR. Thus, TNF was 100-1000-fold more potent
than LT
in cells expressing a high p75/p55 TNFR ratio, whereas
both cytokines showed equal efficacy in evoking biological responses of
cells expressing largely the p55 TNFR. The correlation of expression of
a high proportion of the p75 TNFR with the relative resistance of cells
to LT
is not restricted to the three cell lines described here.
Also the human myeloma cell line OH-2, which predominantly expresses
the p75 TNFR, is 100-100-fold more resistant to the growth
stimulatory action of LT
compared with TNF. (
)Furthermore, in U937 cells, which express about 80% p75
and 20% p55 TNFRs(41, 42) , LT
is required in at
least 10-fold higher concentrations than TNF to obtain a similar
cytotoxic effect (41) . (
)These data, together with
the results on the lack of homology in the receptor binding regions of
TNF and LT
(4) , their differences in trimer
formation(43) , and stability of trimers(44) , suggest
different modes of receptor interactions and/or receptor triggering
caused by TNF and LT
.
To verify this hypothesis, we first
studied the interaction of TNF and LT with the two TNFR by
measuring their affinities and on-off rates of dissociation. Comparable K
values have been detected for both cytokines
upon their binding to the two TNFRs. Together with previously published
data(41, 45) , it suggests that different efficacy of
TNF and LT
cannot be explained by their different affinities for
the TNFRs. Of importance, differences between TNF and LT
in the
dynamic interaction with the receptors have been observed, which are
not reflected in the measurements of affinity constants. Thus, TNF had
a much more rapid on-off rate in the interaction with the p75 TNFR than
with the p55 TNFR. In contrast, LT
showed only minor differences
in its rate of dissociation from the p75 TNFR compared with that from
the p55 TNFR and exhibited a significantly slower rate of dissociation
from the p75 TNFR than TNF.
Secondly, the aforementioned difference
between TNF and LT in the dynamic interaction with the p75 TNFR
has been confirmed by using soluble TNFRs. In accordance with the data
reported by other groups (17, 18) , the soluble p55
TNFR was considerably more potent than the soluble p75 TNFR in
inhibiting a TNF effect. Assuming that a rapid on-off rate of TNF in
the interaction with soluble receptors increases the probability of
interaction of TNF with a signal transducing cell surface receptor, the
difference reported here can be explained by the differences in the
rates of dissociation for TNF between the p55 and p75 TNFR. Also, in
the case of LT
, similar rates of dissociation in the interaction
with the two TNFRs are reflected in similar abilities of the two
soluble receptors in inhibiting the LT
cytotoxic effect.
Interestingly, however, the inhibition of the LT
effect by either
TNFR type was much less pronounced than the inhibition of the TNF
activity by the p55 TNFR. These results confirm the finding that
LT
has a lower potency to bind to the soluble form of the p55 TNFR
when compared to TNF(46) . Furthermore, in attempts to
attenuate cytokine action in vivo, the implication of these
results could be that in the case of TNF soluble p55 TNFR should be a
more potent antagonist than the p75 TNFR, whereas perhaps none of the
soluble receptors may be strong antagonists of LT
action in
vivo.
Thirdly, the application of antagonistic TNFR antibodies
in this study has revealed the different use of the p75 TNFR by TNF and
LT. Blocking of both the p55 and p75 TNFRs led to a marked
inhibition of the TNF effects in SW480-
Gal and KYM-1 cells. In
accordance with previously published
observations(6, 8, 36, 41) , it
indicates that TNF uses both TNFR types for signaling. In contrast, the
antagonistic p75 TNFR antibody utr-1 potentiated the ability of LT
to mediate CMV promoter activation in SW480-
Gal cells and to cause
cytotoxic effect in KYM-1 cells, whereas blocking of the p55 TNFR
inhibited the LT
effects. In line with this, only the p55 TNFR has
been reported to mediate the LT
cytotoxic effect in U937 cells (41) and its ability to up-regulate the expression of adhesion
molecules in human vascular endothelial cells (47) as well as
the proliferative response of human primary fibroblasts(44) ,
whereas both receptors were important for the TNF
effects(36, 41) . Thus, despite the ability of LT
to bind with high affinity to the p75 TNFR(28) , at least in
some cell lines, this cytokine does not seem to mediate signal
transduction through this receptor type.
The p75 TNFR has also been
proposed to play an accessory function by passing TNF to the p55 TNFR
due to its higher affinity, thereby providing more efficient signal
transduction by the p55 TNFR(28) . However, it is unlikely that
the p75 TNFR mediates interaction of LT with the p55 TNFR in
SW480-
Gal and KYM-1 cells, because in this case the on-off rate
apparently is much slower. On the opposite, it could attenuate LT
and prevent its binding to the p55 TNFR. Indeed, antibodies which block
the binding of LT
to the p75 TNFR without affecting the binding to
the p55 TNFR potentiated the LT
effects but inhibited the TNF
activities in SW480-
Gal and KYM-1 cells. Thus, whereas the p75
TNFR is capable of both directly propagating TNF signals and
``passing'' TNF to the p55 TNFR, it appears to attenuate
LT
and to serve as a ``decoy'' receptor for this
cytokine in SW480-
Gal and KYM-1 cells.
Under normal
physiological conditions, LT forms a heteromeric complex with
LT
on the cell surface(9) . It has been published that
LT
can be found only in a membrane-associated form(40) . A
novel LT
specific receptor was identified, which binds to
heterotrimers with the stoichiometry of LT
2/LT
1 but not to
those with the stoichiometry LT
1/LT
2(10) . Moreover,
LT
LT
complexes with different stoichiometries can be
distinguished by the LT
R and the p55 TNFR(40) . To the
best of our knowledge, no binding between the LT
R and LT
homotrimers has been reported. In addition, radiolabeled TNF can be
displaced with excess cold LT
and vice versa, and the combination
of the p55 and p75 TNFR antagonistic antibodies completely prevented
the binding of both cytokines to the cell lines. (
)Thus, the
binding data obtained in this study with soluble LT
give no
indication of the involvement of LT
or the LT
specific
receptor in the binding of homotrimeric LT
to the cell lines
examined.
Interestingly, TNF and LT exhibited comparable
abilities to inhibit proliferation of the human breast carcinoma cell
line BT-20, that had only low affinity receptors (suggesting the p55
TNFR), whereas in cell lines that had high affinity receptors
(suggesting the p75 TNFR), TNF was 20 to 320 time more potent than
LT
(45) . It is plausible that the lack of LT
signaling through the p75 TNFR and the ability of this receptor to
attenuate LT
, found in the present study, could be the reasons
explaining differential biological effects of these cytokines. In
contrast, LT
, but not TNF, acts as a growth factor for
Epstein-Barr virus-infected B cell lines which express mainly the p75
TNFR(26) . The reason for this discrepancy is not clear but
could be related to the importance of the two TNFRs in mediating
specific biological responses in various cell types. Further studies of
the role of the two TNFRs in TNF and LT
signaling, especially in
the cell types where the p75 TNFR is the main signal transducing
molecule, are needed to bring a better understanding of the mechanisms
behind differential or similar effects of these two cytokines.