(Received for publication, July 12, 1995; and in revised form, September 1, 1995)
From the
Fluorescence resonance energy transfer (FRET) was used to
investigate whether interleukin-1 (IL-1) causes the aggregation of IL-1
type I receptors (IL-1 RI) at the cell surface. For these experiments,
a noncompetitive anti-IL1 RI monoclonal antibody, M5, was labeled
separately with a donor probe, fluorescein isothiocyanate, or with an
acceptor carbocyanine probe, Cy3. Donor-labeled M5 and acceptor-labeled
M5 were simultaneously bound to transfected mouse IL-1 RI on either
C-127 mouse mammary carcinoma cells or on Chinese hamster ovary
(CHO)-K1 cells, and the ratio of acceptor emission at 590 nm to donor
emission at 525 nm (excitation at 488 and 514 nm) was monitored with
flow cytometry as an indicator of FRET. Addition of a saturating
concentration of human IL-1 at 22 °C causes a time-dependent
increase in FRET for both cell lines that indicates IL-1-dependent
self-association of IL-1 RI. Binding of the IL-1 receptor antagonist at
22 °C causes little or no FRET for both cell lines, indicating a
correlation between receptor aggregation and the ability of the ligand
to stimulate a functional response. When donor-labeled and
acceptor-labeled Fab fragments of M5 are used to monitor FRET,
IL-1
causes efficient energy transfer in the CHO-K1 cells at 22
°C, but not at 4 °C. In contrast, IL-1
causes much less
FRET at 22 °C in C-127 cells when the M5 Fab fragments are used
instead of the intact bivalent M5. In a striking parallel,
IL-1
-dependent activation of prostaglandin E
production depends on the bivalent M5 antibody in the C-127
cells, but is independent of this monoclonal antibody in the CHO-K1
cells. These results provide a strong correlation between the ability
of IL-1 to cause the aggregation of IL-1 RI and the stimulation of a
functional response.
IL-1 (
)and
are polypeptide cytokines
produced by a variety of cell types in response to injury and
infection, and they play central roles in immune and inflammatory
responses(1, 2, 3) . They share similar
biological activities and receptor binding properties, and their
three-dimensional structures are closely
related(4, 5) . A receptor antagonist polypeptide,
IL-1ra, competes with IL-1
and
for binding to receptors but
does not stimulate a functional response(6) . There are two
known receptors for IL-1. IL-1 RI binds IL-1
or IL-1
to
mediate the activation of T cells and hematopoietic cells, and to
regulate the synthesis and secretion of acute-phase proteins,
prostaglandins, and collagenase (7, 8, 9, 10) . This receptor is
found on almost all cell types and is often involved in immune and
inflammatory responses(11, 12) . IL-1 RII is found on
a more limited number of cell types, including B cells and fibroblasts,
and may act as a ``decoy'' to regulate IL-1 RI-mediated
responses(12, 13) .
IL-1 RI is a single polypeptide
chain with an extracellular segment composed of three
immunoglobulin-like domains that make up the IL-1 binding
site(14) . A single hydrophobic transmembrane sequence connects
the extracellular region to an intracellular segment of 219 residues
that has sequence homology to the Drosophila gene product Toll (15) but provides no clear prediction of the mechanism by which
transmembrane signaling is mediated. Recently, Greenfeder et al.(16) have cloned a 66-kDa polypeptide that interacts
with IL-1 RI and exhibits significant sequence homology to this
receptor polypeptide. Several recent studies have indicated that IL-1
activates a cascade of serine/threonine
phosphorylation(17, 18, 19) , but the
earliest events in signal transduction stimulated by this receptor are
still unknown.
A number of different cytokines and growth factor
polypeptides have been shown to cause aggregation of their receptors to
form dimers and sometimes higher order aggregates. One particular case
with structural similarities to IL-1/IL-1 RI is the fibroblast growth
factor (FGF) receptor system, in which the binding of FGF causes
transphosphorylation of FGF receptors (20) and phosphorylation
of downstream substrates on tyrosine residues(21) , resulting
in a cascade of signal transduction which leads to various
physiological responses (22) . FGF has a three-dimensional
structure that is related to IL-1 and
(23) , and it
binds to the external domain of the FGF receptor that contains three
immunoglobulin domains, in analogy to IL-1 RI(24) . The
receptor for stem cell factor, c-kit, contains five immunoglobulin-like
domains in its extracellular region, and recent studies have shown that
binding of stem cell factor to the first three domains cause
self-aggregation of the receptors that involves interactions dependent
on the fourth immunoglobulin-like domain (25) .
There are
only limited data to suggest that IL-1 may cause the association of
IL-1 RI with each other or with other
polypeptides(14, 16) . One observation that is
consistent with a functional role for ligand-induced receptor
aggregation is the ability of the overexpressed mutant IL-1 RI lacking
a cytoplasmic segment to inhibit IL-1-dependent activation of
PGE production by endogenous IL-1 RI in transfected CHO-K1
cells(26) . In an analogous situation, co-transfection of
excess inactive c-kit receptors with active receptors was shown to
inhibit stem cell factor-dependent signaling in a process involving the
co-dimerization of active and inactive receptors(27) .
The
present study used FRET monitored by flow cytometry to investigate
whether IL-1 causes the aggregation of IL-1 RI at the cell surface. For
these experiments, a noncompetitive, anti-IL1 RI mAb or its Fab
fragments were labeled separately with the donor and the acceptor
probes and bound simultaneously to transfected mouse IL-1 RI on either
C-127 mouse mammary carcinoma cells or on CHO-K1 cells. By monitoring
the ratio of acceptor emission to donor emission, we can readily detect
sensitized acceptor emission and donor quenching that occurs upon
addition of IL-1. Our results indicate that IL-1 binding leads to
time-dependent aggregation of IL-1 RI, and that this aggregation is
likely to play an important role in IL-1-dependent signal transduction.
The Cy3 derivative of M5 was prepared as
follows: M5 mAb (1 mg/ml) was dialyzed overnight in borate-buffered
saline (BBS: 0.16 M sodium chloride, 0.2 M sodium
borate, pH 9.1). One vial of the Cy3 dye was dissolved in 650 µl of
BBS, and 160 µl of this were added to 0.2 ml of the mAb solution
and incubated at room temperature in the dark for 45 min. Glycine was
added at a final concentration of 8 mM to quench the reaction,
and the sample was then microcentrifuged and exhaustively dialyzed in
PBS/EDTA, pH 7.4, to remove unconjugated dye. Molar ratios of coupling
were estimated to be 5.9 or 8.8 to 1 Cy3:M5 in two preparations as
based on extinction coefficients of 130,000 M cm
(552 nm) for Cy3,
6,500 M
cm
(280 nm) for
Cy3, and 210,000 M
cm
(280 nm) for M5 mAb.
Cells were harvested
and resuspended at 3 10
cells/ml in HBS as
described above, and equimolar donor-labeled and acceptor-labeled M5
mAb or M5 Fab fragments were combined and added to the cell suspension.
After 50 min at 22 °C, the mixture was divided into two or more
samples, and labeled cells were monitored in the flow cytometer as a
function of time. After monitoring the cell-associated fluorescence to
establish a baseline, either IL-1 or IL-1ra was added to one sample,
and no addition was made to a control sample. Other samples routinely
measured in parallel were cells incubated with donor- and
acceptor-labeled M5 (or Fab) after preincubation with 20-fold excess
unlabeled M5 (nonspecific control), and cells incubated with
donor-labeled M5 only.
where and
are the flow
cytometry measurements described above.
refers to
measurements with the dually labeled samples, and the
values refer to measurements with the nonspecific control
samples. The second term in corrects for FITC fluorescence
that is observed at the
wavelengths in the dually
labeled sample; in this equation the ratio
/
was
determined from the sample labeled with donor only. does
not require a similar correction because no significant Cy3
fluorescence is observed at the
wavelengths (data not
shown).
For most experiments the acceptor to donor fluorescence
ratio,
/
,
observed with the dually labeled sample was normalized by dividing this
value for the liganded (IL-1 or ILra) cells by the value for the
nonliganded cells at each parallel time point (see Fig. 1).
Figure 1:
IL-1-dependent FRET between donor
FITC-M5 and acceptor Cy3-M5 bound to IL-1 RI on the surface of CHO-mu1c
cells. A, a mixture of 5 nM FITC-M5 and 5 nM Cy3-M5 was incubated with CHO-mu1c cells (3
10
cells/ml) containing wild-type transfected receptors for 50 min
at 22 °C. IL-1
(
) or IL-1ra (
) was added at a final
concentration of 30 nM immediately after the time point at t = 0 min (arrow), and changes in the ratio of
Cy3-M5 fluorescence to FITC-M5 fluorescence were monitored over time.
Changes in this ratio were also monitored for the control sample to
which no ligand was added (
). B, normalized
fluorescence ratio for cells with added IL-1
(
) or IL-1ra
(
) calculated from data in A, as described under
``Experimental Procedures.''
The rat mAb M5 has been previously shown to bind to murine
IL-1 RI at a site in the extracellular region that is distinct from the
IL-1 binding site(29) . This mAb appears to cause the
dimerization of these receptors at the cell surface(32) , but
fails to cause any significant cellular response, and does not
interfere with the ability of IL-1 to stimulate these responses in
EL-4 cells(29) . In initial experiments using flow cytometry,
we established that the FITC and Cy3 derivatives of M5 mAb bind to
transfected murine IL-1 RI on CHO-K1 cells and on C-127 cells with the
properties expected from previous results with
I-derivative of M5 mAb (29) (data not shown). In
addition, fluorescence microscopy and steady state fluorescence
spectroscopy were used to look for endocytosis-mediated quenching due
to acidification in endosomes, and we established that >95% of IL-1
RI labeled by these derivatives remains at the surface for at least 1 h
at 37 °C in the presence or absence of IL-1 ligands (data not
shown). Because of these properties, we were able to bind donor- and
acceptor-labeled M5 mAb simultaneously to CHO-K1 cells to monitor
time-dependent changes in the ratio of acceptor emission to donor
emission
(
/
) as a
sensitive indicator of FRET at the cell surface. In this experiment,
IL-1-dependent aggregation of its receptors might be expected to bring
donor and acceptor probes into closer proximity that could be monitored
with FRET. This process is revealed by an increase in sensitized
acceptor emission and a concomitant decrease in donor
emission(33) .
As shown in Fig. 1A (),
addition of a saturating amount of IL-1
to CHO-mu1c cells
prelabeled with equimolar amounts of FITC-M5 and Cy3-M5 results in a
time-dependent increase in the ratio of
/
.
In a control sample, labeled cells monitored during the same time
period in the absence of IL-1
show a small increase in this ratio
over time that generally exhibits a constant slope. This upward drift
in acceptor/donor fluorescence ratio in the absence of added ligands is
seen to varying extents in different experiments (see below), and the
time-dependent changes due to ligand addition are therefore represented
as shown in Fig. 1B (
) as normalized
fluorescence ratios by dividing for each time point the measured ratio
for the samples with added ligands (
) by the corresponding ratio
for the control sample (
). As seen in Fig. 1B,
the time-dependent increase in the normalized ratio becomes maximal by
about 60 min following the addition of IL-1
, with a half-time of
about 20 min. As summarized in Table 1, line 1, similar results
were obtained in four different experiments with two different
preparations of labeled M5 mAb.
Also shown in Fig. 1(,
) is the effect of addition of IL-1ra to the same labeled cells.
In this case, a much smaller increase in the normalized fluorescence
ratio is observed. The difference between the fluorescence ratio for
IL-1ra-treated cells and control cells (Fig. 1A) is
probably not statistically significant at any particular time point,
but similar small increases over time were observed in three out of
three experiments with this ligand (Table 1, line 2). These
initial observations indicated that, in the presence of M5 mAb,
IL-1
causes a time-dependent increase in FRET, and that this
effect is much smaller with the functionally inactive IL-1ra. In
support of these conclusions, control experiments in which cells were
labeled singly with FITC-M5 or Cy3-M5 showed no consistent change in
either FITC emission or Cy3 emission in response to IL-1
(data not
shown). Thus, the time-dependent increase in the normalized
fluorescence ratio due to the addition of IL-1
(Fig. 1B) represents a significant amount of FRET that
indicates IL-1-dependent co-aggregation of donor and acceptor-labeled
IL-1 RI which correlates with the stimulatory capability of this ligand
compared to IL-1ra.
Because of the potential for receptor
dimerization by the M5 mAb, labeled Fab fragments of M5 were prepared
and used to investigate whether IL-1-dependent aggregation
detected by FRET was influenced by M5-mediated dimerization. Fig. 2(
) shows that the addition of IL-1
causes a
time-dependent increase in the normalized fluorescence ratio for
CHO-mu1c cells labeled with FITC-M5 Fab and Cy3-M5 Fab. Similar to the
results with M5-labeled cells in Fig. 1, the time course of the
increase in FRET is relatively slow, reaching a maximal value by 80
min, with half-time of
40 min. The value for the maximum increase
in the normalized ratio of acceptor emission/donor emission in this
experiment (
1.5) is greater than the value observed with the M5
derivatives in Fig. 1, suggesting that the extent of aggregation
observed with the M5-Fab fragments is at least as great as that
observed with the bivalent M5 labels. The maximal values observed for
the normalized fluorescence ratios due to IL-1
-dependent FRET were
generally found to be very reproducible for a particular set of donor-
and acceptor- labeled M5 derivatives, but some variation in this
maximal value is observed with different preparations of these
derivatives, and with different ratios of donor-labeled and
acceptor-labeled M5 derivatives (data not shown). In some experiments
we examined the IL-1
dose-dependence of FRET using donor and
acceptor-labeled Fab fragments of M5. A concentration of 0.5 nM IL-1
is sufficient to occupy
50% of the IL-1 RI (data
not shown), and this concentration results in a maximal value of FRET
that is
80-90% of that observed with saturating (30
nM) IL-1
. A lower dose of IL-1
(0.1 nM)
results in less binding and FRET than observed at 0.5 nM IL-1
(data not shown).
Figure 2:
IL-1 but not IL-1ra causes
aggregation between IL-1 RI-labeled with FITC and Cy3 Fab fragments of
M5 as detected by FRET. A mixture of 20 nM FITC-M5-Fab and 20
nM Cy3-M5-Fab was added to CHO-mu1c cells transfected with
wild-type receptors and incubated at 22 °C for 50 min. IL-1
(
) or IL-1ra (
) was added to a final concentration of 10
nM immediately after the time point at 0 min. Changes in the
normalized ratio of Cy3-M5 Fab fluorescence to FITC-M5 Fab fluorescence
were monitored over time at 22 °C, as described under
``Experimental Procedures.''
Also shown in Fig. 2is the
time course for the normalized fluorescence ratio of CHO-mu1c cells
labeled with M5 Fab, following the addition of IL-1ra () in the
same experiment. In this situation, no significant FRET was detected
due to IL-1ra addition, and similar observations were made in two other
experiments (Table 1, line 4). These results suggest that the
small amount of FRET in response to IL-1ra on CHO-K1 cells labeled with
bivalent M5 (Fig. 1) may depend on the ability of M5 to dimerize
IL-1 RI which could facilitate a small amount of further aggregation by
IL-1ra.
Monitoring the ratio of acceptor emission to donor emission
as shown in Fig. 1and Fig. 2using equimolar amounts of
donor and acceptor-labeled M5 or its Fab fragments provided the most
sensitive and reliable indication of FRET in these experiments, but
this method of analysis does not readily yield the efficiency of energy
transfer since the amount of sensitized acceptor emission is dependent
on several parameters that are difficult to measure directly in this
situation (33) . In attempts to quantify the energy transfer
efficiency, several experiments were carried out in which cells were
labeled with a mixture of FITC-M5 Fab and Cy3-M5 Fab in a molar ratio
of 0.6:1, to maximize detection of energy transfer by donor quenching.
With this ratio, the donor signal is reduced, but there is an increased
probability that the donor-labeled Fab will be adjacent to an
acceptor-labeled IL-1 RI during IL-1 dependent aggregation. In these
experiments, addition of IL-1 caused a maximum quenching of 10 and
11% of the donor emission in two separate experiments after 70 min of
incubation (data not shown). Using the simplest model of FRET between
single donor-acceptor pairs(34) , in one limit we assume that
all of the donor FITC probes bound to aggregated IL-1 RI are within 10
Å of a Cy3 acceptor probe in these measurements. In this case the
estimated R
value of 55 Å for the FITC/Cy3
donor-acceptor pair predicts that E
1.0 for these donors,
and thus at least 10% of these are co-aggregated with acceptor-labeled
IL-1 RI. In the other limit, all of the labeled IL-1 RI donor are
assumed to be adjacent to acceptor-labeled IL-1 RI due to complete
aggregation, so that the value of R
estimated and
the FRET efficiency measured lead to an average of
70 Å
between donors and acceptors bound to aggregated IL-1 RI.
In an
effort to understand the molecular basis for IL-1-dependent FRET, we
investigated the temperature dependence of this process. Fig. 3() shows an experiment in which CHO-K1 cells were
initially labeled with FITC-M5 Fab and Cy3-M5 Fab fragments at 4
°C, then the acceptor/donor fluorescence ratio was monitored before
and after the addition of IL-1
at 4 °C. Under these
conditions, no significant change in the normalized fluorescence ratio
was detected following addition of IL-1
for a period of 80 min. In
several other experiments, a small amount of FRET could be detected
following extended incubation at 4 °C, but this was always
substantially less than the FRET observed with a parallel sample at 22
°C (data not shown). Following the incubation with IL-1 at 4
°C, the temperature of the cells was raised to 22 °C and a
substantial increase in the normalized fluorescence ratio was observed (Fig. 3,
). The maximal value of this ratio occurred
after 50 min of incubation at 22 °C, and was similar in magnitude
to that obtained with a separate sample of the labeled cells that were
treated with IL-1
at 22 °C from the outset of the experiment (Fig. 3,
). These latter cells were cooled to 4 °C
after 80 min of incubation at 22 °C, and we continued to monitor
the fluorescence ratio for a additional 80 min. As seen in Fig. 3(
), only a small decline in the normalized
fluorescence ratio was observed during this extended time period at 4
°C. These results are representative of three separate experiments
and indicate that the IL-1-dependent aggregation process detected by
FRET is highly temperature dependent, but, once it has occurred, it
remains stable during incubation of the cells at the nonpermissive
temperature. In several other experiments, we found that
IL-1
-dependent FRET occurs at 37 °C to a similar extent as at
22 °C, and the time course for this process is similar (data not
shown).
Figure 3:
IL-1-dependent energy transfer between
IL-1 RI is temperature dependent. A mixture of 20 nM FITC-M5
Fab and 12 nM Cy3-M5 Fab was added to CHO-mu1c cells (3
10
cells per/ml) with transfected wild-type IL-1 RI
and preincubated at either 4 °C (
) or 22 °C (
) for
50 min. Immediately after the base-line data point at t = 0 min, IL-1
was added (arrow) at a final
concentration of 10 nM to both samples. Changes in the
normalized ratio of Cy3-M5 Fab fluorescence to FITC-M5 Fab fluorescence
was monitored over time at the corresponding preincubation temperature.
At t = 85 min, the temperature for sample (
)
was changed from 4 to 22 °C, and the temperature for sample (
)
was changed from 22 to 4 °C. Changes in the normalized fluorescence
ratio continued to be monitored until t = 180
min.
To determine whether interaction of the cytoplasmic segment
of IL-1 RI with each other or with other cellular components are
necessary for IL-1-dependent aggregation detected by FRET, we carried
out energy transfer experiments with a mutant that is lacking the
C-terminal 194 residues out of 219 in the cytoplasmic
segment(26) . CHO-extn cells containing these mutant receptors
were initially labeled with donor- and acceptor-labeled M5 mAb, but we
found that in this situation, there was a large time-dependent increase
in the ratio acceptor emission to donor emission even in the absence of
IL-1, suggesting that the bivalent M5 mAb might be causing
efficient aggregation of these mutant receptors (data not shown). When
labeled M5 Fab fragments were used instead of the intact M5 mAb, the
ratio of acceptor emission to donor emission was found to be nearly
constant in the absence of IL-1 (data not shown). Fig. 4(
) shows the results from an experiment in which
IL-1
caused a time-dependent increase in the normalized
fluorescence ratio that is somewhat less than that for the wild-type
receptor (
) measured in the same experiment. In four separate
experiments, we observed a wider variation in the amount of energy
transfer with the truncated receptor than with the wild-type receptor,
but in all four experiments some energy transfer was observed with this
mutant. These results suggest that the cytoplasmic segment of IL-1 RI
is not essential for IL-1-dependent aggregation, but that it may
regulate the aggregation process.
Figure 4:
IL-1-dependent FRET can be detected
between FITC-M5 Fab and Cy3-M5 Fab bound to the cytoplasmic
tail-deleted mutant IL-1 RI on CHO-extn cells. A mixture of 20 nM FITC-M5 Fab and 12 nM Cy3-M5 Fab was added to wild-type
transfected receptors on CHO-mu1c cells and incubated at 22 °C for
50 min (
). A mixture of 20 nM FITC-M5 Fab and 12
nM Cy3-M5 Fab was added to CHO-extn cells (cytoplasmic tail
deleted mutant IL-1 RI) and incubated at 22 °C for 50 min (
).
IL-1
was added to a final concentration of 20 nM at the
arrow, and changes in the normalized ratio of Cy3-M5 Fab fluorescence
to FITC-M5 Fab fluorescence were monitored over time at 22
°C.
To relate the FRET results to a
functional response mediated by IL-1 RI, we measured the production of
PGE by the CHO-mu1c cells in response to IL-1
. As
shown in Fig. 5A, IL-1
stimulates the production
of PGE
by about 10-fold over that in the unstimulated
cells, and neither M5 Fab fragments nor the bivalent M5 mAb had any
significant effect on PGE
secretion in the presence or
absence of IL-1
. These results are consistent with previous
measurements obtained with EL-4 cells in the presence and absence of M5
mAb(29) . We also examined the PGE
secretion
response in a separate IL-1 RI transfected cell line, C-127 mouse
mammary cells, which express substantially more receptors on their cell
surface than the CHO-mu1c cells (data not shown). As shown in Fig. 5B, these cells exhibit virtually no response to
IL-1
by itself or in the presence of the Fab fragment of M5, but
they show a significant response to IL-1
in the presence of the
bivalent M5 mAb. As with the CHO-K1 cells, M5 in the absence of
IL-1
did not stimulate PGE
production.
Figure 5:
Stimulation of PGE production
by IL-1
in the presence and absence of M5 mAb or M5 Fab in
CHO-mu1c cells (A) and in C-127 cells (B). Confluent
cultures in 6-well plates, each well containing equal numbers of cells
were incubated with HBS alone, HBS containing a saturating amount of M5
Fab (30 nM), or HBS containing a saturating amount of M5 mAb
(15 nM) for 1 h at 37 °C. IL-1
was then added (solid bars) or not added (hatched bars), and
incubation was continued for an additional 3 h. Aliquots of
supernatants were collected and assayed for PGE
release as
described under ``Experimental Procedures.'' The data shown
are the average values of PGE
release from eight wells per
sample in three separate experiments for CHO-mu1c cells and from 14
wells in five separate experiments for C-127 cells. The error bars represent the standard deviations of these
results.
These
results suggest that M5 mAb can facilitate a functional response to
IL-1 in the C-127 cells. In order to investigate whether this
facilitation is related to an effect on IL-1-dependent receptor
aggregation, we monitored FRET in the C127 cells as described above for
the CHO-K1 cells. As shown in Fig. 6A (
), C-127
cells labeled with M5 mAb exhibit a time-dependent increase in the
normalized fluorescence ratio in response to IL-1
that is
generally smaller in magnitude than that observed with the CHO-K1
cells, but which occurs on a somewhat faster time scale. As for the
CHO-mu1c cells, IL-1ra causes a much smaller amount of FRET under these
conditions (
), even though it binds and occupies most of the
receptors (data not shown). In a separate experiment, we compared the
ability of IL-1
to cause a time-dependent increase in FRET in
C-127 cells labeled with either bivalent M5 derivatives or with M5 Fab
fragments. As shown in Fig. 6B, IL-1
causes little or
no increase in the normalized fluorescence ratio for cells labeled with
the monovalent Fab fragments (
), even though the expected
amount of FRET is observed with the cells labeled with bivalent M5
(
). This clear difference was observed in two separate experiments
and is in striking contrast to the results with CHO-mu1c cells
described above, in which cells labeled with the Fab fragments showed
at least as much IL-1
-dependent FRET as the same cells labeled
with the bivalent M5 derivatives. These M5 valency-dependent
differences in IL-1-dependent FRET between the two cell lines parallel
the differential requirements of these cell lines for IL-1
stimulated PGE
production in the presence and absence of M5
mAb. The results, taken together, provide a strong correlation between
the ability to detect IL-1
-dependent aggregation of receptors by
FRET and the ability of IL-1
to stimulate a functional response.
Figure 6:
IL-1-dependent FRET between FITC-M5
and Cy3-M5 bound to IL-1 RI on C-127 cells depends on the bivalency of
M5 mAb. A, a mixture of 7.5 nM FITC-M5 and 7.5
nM Cy3-M5 was added to C-127 cells (3
10
cells/ml) containing
2 nM wild-type IL-1 RI, followed by
preincubation at 22 °C for 50 min. IL-1
(
) or IL-1ra
(
) was added at the arrow at a final concentration of 30
nM, and changes in the normalized ratio of Cy3-M5 fluorescence
to FITC-M5 fluorescence were monitored over time at 22 °C. B, IL-1
(30 nM) was added (arrow) to
C-127 cells labeled as in A with FITC-M5 and Cy3-M5 (
) or
to C-127 cells labeled with a mixture of 40 nM FITC-M5 Fab and
24 nM Cy3-M5 Fab for 50 min at 22 °C (
), and
changes in the normalized ratio of Cy3 fluorescence to FITC
fluorescence were monitored over time at 22
°C.
The molecular mechanism by which IL-1 binding to IL-1 RI
causes transmembrane signaling that leads to the activation of
pro-inflammatory cellular responses has been difficult to ascertain (14) . Recent studies indicate that IL-1, like tumor necrosis
factor-, activates a stress-sensitive cascade of mitogen-activated
protein-kinase-related enzymes(35, 36) , but the
earliest events that follow IL-1 binding are largely unknown. Other
recent results indicate that IL-1 binding stimulates serine/threonine
phosphorylation of a IL-1 RI-associated 65-kDa substrate(37) ,
and this phosphorylation may play an important role in the initiation
of the kinase cascade. Our present results indicate that binding of
IL-1
causes co-aggregation of IL-1 RI at the cell surface, and
that this aggregation process is highly correlated with the activation
of at least one functional response, the production of PGE
.
Using flow cytometric FRET we have established a sensitive and
straightforward method to detect IL-1-dependent aggregation of IL-1 RI
labeled with monoclonal anti-IL-1 RI or their Fab fragments. This
method can be applied to other cytokine or growth factor receptor
system, provided that a noncompetitive mAb specific for the receptor of
interest is available. As demonstrated by our results, this method can
readily detect ligand-dependent receptor aggregation when fewer than
10 receptors/cell are present, as for the transfected CHO
cells in some of our experiments (data not shown). These experiments
were carried out with a simple analytical flow cytometer that
simultaneously excites both donor (488 nm) and acceptor (514 nm, at
lower intensity), and the detection of changes in the ratio of
(sensitized) acceptor emission to (quenched) donor emission provides a
sensitive means of detecting small changes in FRET that are readily
observed upon addition of ligand.
The time course of FRET that we
detect in response to IL-1 binding is slow relative to the time
course of IL-1
binding. Under the conditions of our FRET
experiments, FITC-IL-1 saturates the IL-1 RI receptors and attains a
steady state within about one minute of addition (data not shown). In
contrast, the maximum amount of receptor aggregation detected by FRET
typically requires
60 min for the CHO-mu1c cells at 22 °C,
with a half-time of 20-30 min in most experiments. This indicates
that there is a rate-limiting step subsequent to binding that is
necessary for receptor aggregation to occur. In the presence of
bivalent M5, the IL-1-dependent aggregation process is significantly
faster in the C-127 cells, suggesting that the rate-limiting step is
sensitive to some difference between these two cell lines. This
rate-determining difference is unlikely to be lateral mobility, as IL-1
RI actually diffuses faster in CHO-K1 cells than in C-127 cells, as
measured by fluorescence photobleaching recovery using labeled M5. (
)It is possible that differences in the stoichiometry of
IL-1 RI and the newly discovered 66-kDa receptor-associated polypeptide (16) might affect the kinetics of IL-1 RI aggregation.
Our
measurements do not distinguish between the formation of receptor
dimers and larger aggregates due to IL-1 binding, but under
conditions of maximal FRET, IL-1-dependent formation of patches of
aggregated IL-1 RI are not detectable by confocal fluorescence
microscopy, suggesting that aggregation is limited to substantially
less than 1000 receptors per aggregate (data not shown). The strong
temperature dependence for aggregation that we observe suggests that
the rate-limiting step in the aggregation process has an activation
energy barrier with a large temperature coefficient. It is reminiscent
of the temperature dependencies reported for epidermal growth
factor-dependent epidermal growth factor receptor aggregation (38) and decreased rotational diffusion(39) , both
observed in plasma membrane preparations. Although the molecular basis
for this temperature dependence remains to be determined, it provides a
useful experimental strategy for investigating the relationship between
IL-1
-dependent aggregation and other changes in IL-1 RI that can
be monitored by physical and biochemical methods.
Our FRET
measurements provide direct evidence for IL-1-dependent
association of IL-1 RI with each other and allow us to examine whether
this process is related to the initiation of signaling by this ligand.
As summarized in Table 1, the ability to detect a functional
response as represented by PGE
secretion that is elicited
for a particular combination of cells, ligand, and mAb is highly
correlated with the ability to detect a substantial amount of FRET.
Minimal aggregation detected by FRET in response to IL-1ra or in
response to IL-1
on C-127 cells in the absence of bivalent M5 is
apparently insufficient to cause productive signaling, while larger
amounts of FRET (Table 1, ++
+++++) detectable at 22 °C correlate with
productive signaling at 37 °C. The ability to detect substantial
FRET with the mutant IL-1 RI that lacks most of the cytoplasmic segment
of this receptor indicates that aggregation is not a consequence of
signals generated by the liganded receptor, since this mutant fails to
cause detectable signaling(26) . This result further indicates
that receptor-receptor interactions, if they occur during aggregation
detected by FRET, are probably mediated by the extracellular or
transmembrane segments of IL-1 RI, although we cannot rule out the
possibility that interactions involving the cytoplasmic segment play
some role in the aggregation process with wild-type receptors. It is
also formally possible that receptor-bound IL-1
could directly
interact with each other to mediate the aggregation process, but there
is currently no evidence for self-aggregation of IL-1
.
It is
notable that aggregation as detected by FRET occurs as readily in
response to IL-1 at 22 °C as it does at 37 °C, although
stimulated PGE
production is detectable only at the higher
temperature (data not shown). This indicates that other biochemical
processes occurring subsequent to IL-1-dependent receptor aggregation
have a different temperature dependence and are also necessary for this
functional response. The difference in the magnitude of stimulated
PGE
production (and FRET) between the CHO-mu1c cells and
the C-127 cells further indicates that other biochemical events and/or
components are involved and can determine the extent of the functional
response. These results, taken together, indicate that IL-1-dependent
aggregation of IL-1 RI is probably a necessary but, by itself, an
insufficient step in the sequence of events leading to IL-1 RI-mediated
signaling. The molecular mechanism by which IL-1 mediates the
aggregation of its receptor remains to be determined, but the FRET
method we have developed should provide a means by which to further
understand the structural basis and biochemical consequences of this
process.