(Received for publication, September 11, 1995; and in revised form, November 27, 1995)
From the
The extracellular ``soluble'' domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130) form a hexameric ternary receptor complex together with IL-6, consisting of two molecules of each component. In this report we have investigated the interactions of the partial IL-6 antagonist (Q159E,T162P)IL-6 ((QT)IL-6), with the sIL-6R and sgp130. The kinetic rate constants of the binding of sIL-6R to immobilized monomeric (QT)IL-6 or IL-6 were obtained using an optical biosensor with analysis of the primary data by linear and nonlinear regression. Both methods of analysis showed that, due to a higher off-rate, sIL-6R has lower apparent affinity for (QT)IL-6 than IL-6. The lower affinity of (QT)IL-6 was further confirmed by equilibrium binding measurements at the sensor surface and in solution. Using the biosensor it was also shown that the (QT)IL-6 complex interacts with sgp130, supporting the notion that the biological activity of (QT)IL-6 is mediated via gp130. However, the IL-6 mutant, when incubated with sIL-6R and sgp130, failed to induce a stable hexameric receptor complex, as shown by narrowbore size exclusion chromatography.
Interleukin-6 (IL-6) ()is a cytokine that exhibits
pleiotropic effects on a wide range of target cells. It is involved in
the growth and differentiation of B lymphocytes, differentiation and/or
activation of T lymphocytes and macrophages, maturation of
megakaryocytes, and enhancement of multipotential hematopoietic colony
formation, expression of acute phase proteins, and bone metabolism.
IL-6 has also been implicated in a variety of disease states, including
lymphoid malignancies and autoimmune and inflammatory disorders such as
glomerulonephritis and rheumatoid arthritis (reviewed in (1) ).
The biological activities of IL-6 are mediated by the IL-6 receptor
(IL-6R), which binds IL-6 specifically and with low
affinity(2) , and gp130, which associates with the
IL-6IL-6R complex, resulting in high affinity binding and
activation of intracellular
signaling(3, 4, 5) . gp130 does not associate
with IL-6 in the absence of the IL-6R(3) . gp130 also forms
part of the receptor complexes of the cytokines IL-11, oncostatin M,
leukemia inhibitory factor, and ciliary neurotrophic factor (reviewed
in (6) ), and cardiotrophin-1(7) .
To obtain sufficient quantities of IL-6 for our structure-function studies we have expressed IL-6 in Escherichia coli using the pUC8 expression vector(8, 9) . We have recently demonstrated that this material, purified to apparent homogeneity from bacterial inclusion bodies, can be separated into stable monomeric and dimeric IL-6 by size exclusion chromatography (SEC)(10) . By SEC and analytical ultracentrifugation, using the extracellular domains of the IL-6R and gp130 (sIL-6R and sgp130, respectively), we have shown that one molecule of monomeric IL-6 binds one molecule of IL-6R in the binary complex, while the ternary receptor complex of IL-6 is a hexamer consisting of two molecules each of IL-6, IL-6R, and gp130(10) .
The present investigation compares the
affinities of IL-6 and the partial IL-6 antagonist (Q159E,T162P)IL-6 ()((QT)IL-6) for binding to sIL-6R and sgp130, using an
optical biosensor (BIAcore(TM)) employing surface plasmon resonance
(SPR) detection for monitoring the interactions in real
time(11) . (QT)IL-6 has been shown to act as an IL-6 antagonist
on human CESS and hepatoma HepG2 cell lines, apparently by preventing
the formation of the IL-6
IL-6R complex(12) . It was
concluded that the (QT)IL-6
IL-6R complex was defective in its
interaction with gp130. However, on HepG2 cells, at high concentrations
of (QT)IL-6, an agonist activity corresponding to 10-20% of the
maximal response of IL-6 was consistently observed. This activity was
tentatively explained by the presence on HepG2 cells of a third
receptor chain necessary for high affinity binding or by differences in
relative numbers of high and low affinity receptors on CESS and HepG2
cells(12) . It was later reported that the (QT)IL-6
sIL-6R
complex has residual affinity for sgp130(13) ; however, it
remained to be determined whether (QT)IL-6 is capable of inducing the
formation of the hexameric receptor complex.
For reliable kinetic
analysis using a biosensor it is important to use homogeneous binding
components, as heterogeneity of the material may affect the
interpretation of the results. For example, the interaction of a Fab
fragment of a monoclonal antibody with its peptide antigen yielded
biphasic dissociation kinetics prior to purification of the antigen but
linear first-order kinetics after purification(14) . Since the
monomeric, but not the dimeric, form of recombinant IL-6 appears to
reflect the interactions of ``wild type'' IL-6 with the IL-6R
and gp130, ()we have used monomeric (QT)IL-6 and IL-6 for
the studies detailed below.
In this report, we determine the
apparent affinity of (QT)IL-6 for the sIL-6R by linear and nonlinear
regression of the biosensor data (15, 16) as well as
by equilibrium binding and compare the values obtained with those of
IL-6. This rigorous analysis was undertaken to confirm the
appropriateness of the mathematical models used, since disparity
between methods has been reported recently(17, 18) .
Using the biosensor and narrowbore SEC we also investigated the
interactions of the (QT)IL-6sIL-6R complex with sgp130. The
formation and characterization of the ternary receptor complex in the
presence of (QT)IL-6 is discussed.
Soluble human IL-6 receptor and sgp130 were expressed in Chinese hamster ovary cells transfected with pECEdhfr344 and pECEdhfrsgp620, respectively(22, 23) , and purified as described(10, 24) .
To investigate whether rebinding of sIL-6R to the sensor surface derivatized with (QT)IL-6 was occurring during dissociation, excess (300 nM) soluble (QT)IL-6 was injected immediately after injection of sIL-6R (21.9 nM), from the second injector loop in the microfluidic cartridge.
A typical sensorgram consists of an association phase,
during which the analyte passes over the derivatized sensor surface,
and a dissociation phase where the analyte is replaced with buffer. The
association and dissociation rate constants (k and k
, respectively) of the interaction can be
obtained from analysis of the sensorgrams by linear regression analysis (15) or by nonlinear least squares analysis(16) .
Determination of the k
according to the former
method relies on analysis of the data from the association phases of
various concentrations of analyte and assumes a pseudo-first-order
(1:1) interaction between the immobilized protein and the analyte.
Linear regression of the data was performed using , where R is the detector
response (in RU) resulting from the interaction of analyte with
immobilized component, R is the maximal response
at a given concentration of analyte, k
and k
are the association and dissociation rate
constants (in M
s
and
s
, respectively), and C is the
concentration of analyte (in M). Using this nomenclature the
rate equation may be expressed in the following
way.
This may be rearranged as the following.
When dR/dt is plotted as a function of R, the slope, k (in
s
), is the
following.
Thus, the slope of a plot of k as a
function of C yields the k
, whereas the k
can be obtained from the intercept on the y axis.
Using linear regression analysis the k at each individual analyte concentration can also be obtained
directly from the dissociation phase of a sensorgram. After the
association phase, when buffer is flowing over the sensor chip, C = 0, and becomes the
following.
This can be rewritten as the following,
where R is the response at the arbitrary
starting time t
and R
is the response
at time t
. Hence, the slope of a plot of
ln(R
/R
) as a function of time equals k
.
The K for the
interaction between two components can also be measured directly from
analysis of the response at equilibrium (R
).
Equilibrium binding is obtained at dR/dt = 0.
Thus, R = R
, and becomes the
following.
Since k/k
= K
, this may be rearranged as
follows.
Assuming a univalent interaction, a plot of R/C as a function of R
is linear, and the slope of the line equals K
= 1/K
.
Nonlinear least squares
analysis of the primary data analyzes the sensorgrams using the
integrated form of the rate equation(16) . By contrast to
linear regression analysis, it is theoretically possible to calculate
both an apparent k and k
using nonlinear regression analysis of a single concentration of
analyte. However, in practice it is preferable to analyze the data from
several concentrations to ensure that there is no variation of the
kinetic rate constants with concentration(16) .
The integrated form of the rate equation can be written as follows,
where R is the signal (in RU) at the start
of the analysis. For the dissociation phase, it can be written as
follows,
where A is the amplitude of the dissociation (in RU)
and R is the signal at infinite time when
the dissociation is complete.
Data were analyzed using the linearized forms of the rate equation in the BIA Evaluation Kinetics package supplied by Pharmacia. Nonlinear least squares analysis was performed using and on sensorgrams imported into Origin (Microcal Software Ltd., Northampton, MA) or analyzed using the BIA Evaluation, version 2.1, software supplied by the manufacturer and adapted with the appropriate equations. The binding kinetics in solution and at equilibrium were also plotted using Origin.
The
biological activity of monomeric (QT)IL-6 was assayed on mouse
hybridoma 7TD1 cells and found to be approximately 20-fold less potent
than that of monomeric IL-6 (EC = 150 pg/ml and 7
pg/ml for (QT)IL-6 and IL-6, respectively). The activity of (QT)IL-6 on
7TD1 cells was thus in good agreement with results obtained using mouse
hybridoma B9 cells, where this mutant was 7-fold less active than IL-6 (12) .
We have shown previously, by SEC and analytical
ultracentrifugation, that the stoichiometry of binding of sIL-6R with
IL-6 fits a model whereby one molecule of IL-6 binds to one molecule of
sIL-6R(10) . Incubation of (QT)IL-6 with sIL-6R resulted in the
formation of a binary receptor complex that had similar characteristics
to the IL-6sIL-6R complex, as determined by SEC and sedimentation
equilibrium centrifugation, suggesting that (QT)IL-6 binds with a 1:1
stoichiometry to sIL-6R (data not shown).
Figure 1:
Interaction of sIL-6R with immobilized
(QT)IL-6 measured by a biosensor employing SPR detection. Various
concentrations of sIL-6R were injected over a sensor chip with
immobilized (QT)IL-6 monomer, as detailed under ``Experimental
Procedures.'' A, sensorgrams of (from below)
buffer control, 2.6, 4.4, 6.6, 8.7, 10.9, 13.1, 15.3, 17.5, 19.7, and
21.9 nM sIL-6R. B, plot of kas a function of sIL-6R concentration (see under ``Experimental
Procedures''). The data are obtained from the sensorgrams in Fig. 1A (between 150 and 740 s). C, analysis
of initial 60-s dissociation (1030-1090 s) of 21.9 nM sIL-6R from immobilized (QT)IL-6 ( and ).
Data are from the corresponding sensorgram in Fig. 1A.
It can be seen () that the k should be independent of concentration. The
dissociation rate constant for the initial 60 s was found to be
constant at concentrations above 13.1 nM sIL-6R. However, at
lower concentrations (10.9-2.6 nM) sIL-6R, k
was found to be dependent on concentration,
values between 2.8 and 1.7
10
s
being obtained, resulting in an apparent higher affinity of
interaction with immobilized (QT)IL-6. Additionally, at later time
points, the plot of ln(R
/R
)
against time was markedly curvilinear. Analysis of the later time
points (300-440 s) indicated an apparent k
= 0.8
10
s
.
Such a phenomenon has been noted previously by others (26, 27) and may be due to rebinding of the analyte to
the immobilized ligand during dissociation(28, 29) .
To investigate whether rebinding of sIL-6R to (QT)IL-6 was affecting
the k
, excess ligand was injected during the
dissociation phase, as detailed under ``Experimental
Procedures.'' By contrast to the system of Panayotou and
co-workers(28) , who found that a large excess of soluble
peptide injected immediately after the analyte was able to prevent
rebinding to an immobilized peptide, we observed no major differences
in the dissociation kinetics in the presence or absence of over 10-fold
molar excess of soluble (QT)IL-6 (data not shown). These results
suggest that the k
was not influenced by rebinding
of sIL-6R to immobilized (QT)IL-6.
From the initial 50 s of the dissociation phases of 4.4-21.9
nM sIL-6R (sensorgrams shown in Fig. 1A)
apparent k = 2.3-3.3
10
s
were obtained (Table 2). The dissociation phases fitted well to a single
exponential function, with S.E. between 2.7 and 1.1% (
between 1.8 and 3.9) (Table 2). Analysis of the association
phases, using the above determined values for the k
, yielded k
=
8.8-2.4
10
M
s
(Table 3). Again the values for S.E.
and
were low. It can be seen (Table 3), that
at concentrations above 10.9 nM the results show little
dependence on concentration, with apparent K
values ranging from 10 to 13 nM. However, as we had
observed when using the linear regression analysis, at low
concentrations there was a progressive reduction in the values
calculated for the k
, with a concomitant increase
in the apparent k
.
Thus, apparent k = 2.4
10
M
s
, k
= 3.2
10
s
, and K
= 13
nM were determined for the interaction of sIL-6R with
immobilized (QT)IL-6, using nonlinear regression analysis.
Figure 2:
Analysis of binding kinetics of sIL-6R
with (QT)IL-6 at equilibrium and in solution. A, plot of
response as a function of sIL-6R concentration (2.6-21.9
nM) from sensorgrams in Fig. 1A. B,
plot of R/C as a function of R
(data from Fig. 1A) (see and under ``Experimental
Procedures''). C, sensorgrams of sIL-6R (21.9
nM) preincubated with (QT)IL-6 (0-160 nM).
Buffer is shown as a control. The sensor chip with immobilized (QT)IL-6
was used to determine the concentration of uncomplexed sIL-6R, as
detailed under ``Results.'' D, Scatchard analysis of
data in Fig. 2C, using the standard curve in Fig. 2A, as detailed under ``Results.''
Sensorgrams of sIL-6R preincubated with IL-6 (20, 40, 60, and 80
nM) are not shown, but data for IL-6 (open circles)
are plotted for comparison with (QT)IL-6 (solid
squares).
In these experiments the biosensor was used to measure
the concentration of free sIL-6R following preincubation with various
concentrations of ligand. As shown by the sensorgrams in Fig. 2C, sIL-6R (21.9 nM) preincubated with
20, 40, 80, or 160 nM (QT)IL-6 yielded a lower response with
immobilized (QT)IL-6 than 21.9 nM sIL-6R alone, indicative of
the formation of binary complex in solution. As these values (in RU)
all fell within the range of the standard curve obtained between 2.6
and 21.9 nM sIL-6R (Fig. 2A), the
concentration of uncomplexed sIL-6R could be determined
accurately. Based on a 1:1 interaction of ligand with sIL-6R, from the
total sIL-6R concentration (21.9 nM) and the concentration of
uncomplexed sIL-6R, the concentration of sIL-6R in the binary complex
could be calculated. The latter equals the concentration of complexed
ligand, B. Ligand not bound to sIL-6R, F, equals
total ligand in solution minus complexed ligand. According to the
Scatchard relationship(30) , when B/F is
plotted as a function of B, the slope of the line yields K = 1/K
. Such a plot
yielded a K
of 20 nM for the interaction
of (QT)IL-6 monomer with sIL-6R in solution (Fig. 2D and Table 1).
The sIL-6R standard curve in Fig. 2A could also be used to determine the
concentration of free sIL-6R and hence calculate the K for the interaction of IL-6 monomer with sIL-6R in solution. From
results obtained by preincubation of 21.9 nM sIL-6R with 20,
40, 60, and 80 nM IL-6 (sensorgrams not shown), a K
of 9 nM was calculated (Fig. 2D and Table 1).
Figure 3:
Analysis of binding kinetics of sIL-6R
with IL-6 at equilibrium. Various concentrations of sIL-6R were
injected over a sensor chip with immobilized IL-6 monomer, as detailed
under ``Experimental Procedures.'' A, sensorgrams of
(from below) 21.9, 43.7, 65.6, 87.4, 109.3, and 131.2 nM sIL-6R. B, plot of R/C as
a function of R
(data from Fig. 3A) (see and under
``Experimental Procedures'').
A plot of k against C from the corresponding association phases (see ) yielded an apparent k
= 2.2
10
M
s
(slope;
coefficient of correlation > 0.99). Direct analysis of the
dissociation phases (see and ) gave an
apparent k
= 1.0
10
s
. The resultant K
was 5
nM. In a separate experiment, analysis of lower concentrations
of sIL-6R (2.6-21.9 nM) yielded an apparent k
= 2.1
10
M
s
(slope;
coefficient of correlation > 0.98) and k
= 1.2
10
s
(ordinate intercept) (data not shown). The k
= 1.2
10
s
obtained from the intercept of a plot of k
against C compared well with the k
= 2.0
10
s
(slope of a plot of
ln(R
/R
) against time;
coefficient of correlation > 0.99) calculated from analysis of the
initial 60 s of the dissociation phase of 21.9 nM sIL-6R. As
was observed with (QT)IL-6, at lower concentrations (13.1-2.6
nM) sIL-6R the dissociation over the initial 60 s was found to
be concentration-dependent, ranging between k
= 1.8-1.0
10
s
(data not shown). Taken together, using
linear regression analysis, apparent k
=
2.1
10
M
s
, k
= 2.0
10
s
and K
= 10 nM were determined for the interaction of
sIL-6R with immobilized IL-6 monomer (Table 1).
Nonlinear
least squares analysis of the initial 50 s of the dissociation phases
of 21.9-131.2 nM sIL-6R, using a single exponential
function (see ), yielded apparent k = 0.5-0.9
10
s
, with standard errors between 1.8 and 2.1%
(
values between 3.2 and 17.5) (Table 2). These
values were used to constrain the calculations of the k
, yielding apparent k
= 1.9-2.1
10
M
s
, and hence K
= 3-5 nM (Table 3).
It should be noted that for the IL-6 data an improved fit (
= 0.5-0.9) could be obtained using a second
exponential function (data not shown). This analysis suggested that a
small component of the dissociation (<3%) had a rapid off-rate (k
5
10
s
) but that the remainder was described by an
apparent k
= 0.5
10
s
, i.e. similar to that calculated
using the single exponential decay. In a separate experiment, using
4.4-21.9 nM sIL-6R, apparent k
= 1.4-2.1
10
s
, k
=
1.6-1.8
10
M
s
, and K
=
8-13 nM were determined using a single exponential
function (data not shown).
As for (QT)IL-6, the apparent K was calculated independently by analysis of the
equilibrium data (see and ). From the maximal
responses in the concentration range of sIL-6R shown in Fig. 3A, the apparent K
was
calculated to be 10 nM (Fig. 3B).
The
interaction in solution of sIL-6R with (QT)IL-6 and IL-6 was also
measured on a sensor chip derivatized with IL-6 (sensorgrams not
shown). From results obtained by preincubation of 21.9 nM sIL-6R with various concentrations of ligand (40, 60, 80, 120, and
160 nM (QT)IL-6 or 20, 40, 60, 80, and 100 nM IL-6) a K of 16 nM for (QT)IL-6 and 6 nM for IL-6 were calculated (Table 1).
Figure 4:
Analysis of binding of
(QT)IL-6sIL-6R complex to sgp130, as detected by a biosensor
employing SPR. Soluble IL-6R was incubated with (QT)IL-6 and/or IL-6
prior to assay with immobilized sgp130, as detailed under
``Experimental Procedures.'' Sensorgrams correspond to 40
nM sIL-6R + 40 nM IL-6 (A), 40 nM sIL-6R + 40 nM IL-6 + 40 nM (QT)IL-6 (B), 40 nM sIL-6R + 40 nM IL-6 +
1000 nM (QT)IL-6 (C), 40 nM sIL-6R +
1000 nM (QT)IL-6 (D), 40 nM sIL-6R + 40
nM (QT)IL-6 (E), and 1000 nM (QT)IL-6 (F).
On a sensor chip derivatized with
sgp130, a response of 697 RU was obtained upon injection of 40 nM IL-6 preincubated with 40 nM sIL-6R (curve A).
By comparison, injection of 40 nM (QT)IL-6 preincubated with
40 nM sIL-6R yielded a response of only 117 RU (curve
E). When a large molar excess of (QT)IL-6 (1000 nM) was
preincubated with 40 nM sIL-6R the response increased slightly
to 210 RU (curve D) but was still substantially reduced
compared with that of the binary receptor complex of IL-6 (curve
A). No significant binding was obtained with 1000 nM (QT)IL-6 (40 RU; curve F), IL-6, or sIL-6R alone (not
shown). These data thus suggest that the (QT)IL-6sIL-6R complex
can interact with sgp130, albeit at a much lower level than the
IL-6
sIL-6R complex.
Upon injection of 1000 nM (QT)IL-6 preincubated with 40 nM IL-6 and 40 nM sIL-6R a response of 278 RU was obtained (curve C). This
response was lower than that obtained by the IL-6sIL-6R complex
alone (697 RU; curve A), showing that (QT)IL-6 can compete
with IL-6 for binding to the sIL-6R, thereby reducing the interaction
of the IL-6
sIL-6R complex with sgp130. Similarly, when 40 nM (QT)IL-6 was preincubated with 40 nM IL-6 and 40 nM sIL-6R, the response obtained (645 RU; curve B) was lower
than that of the IL-6
sIL-6R complex alone.
Figure 5:
Narrowbore size exclusion chromatography
of receptor complexes of (QT)IL-6. Chromatography was performed on a
Superose 12 PC 3.2/30 column (300 3.2-mm inner diameter)
operated at 40 µl/min in phosphate-buffered saline at 25 °C.
The sample loads (identical between runs) and M
of
the proteins were as indicated. A, (QT)IL-6 (0.98 µg;
20 kDa); B, sIL-6R (0.4 µg;
53 kDa); C,
IL-6 (0.99 µg;
21 kDa) + sIL-6R + sgp130 (0.74
µg;
90K); D, sgp130; E, (QT)IL-6 +
sIL-6R; F, (QT)IL-6 + sIL-6R + sgp130. The elution
position of the hexameric receptor complex is indicated by
.
(QT)IL-6 monomer (panel A), sIL-6R (panel B), sgp130 (panel D), and IL-6 monomer (not shown) eluted essentially as single peaks, confirming the homogeneity of the purified proteins. As shown with IL-6(10) , incubation of ligand with sIL-6R prior to chromatography results in both an increased peak height and a slight shift in elution position compared with the sIL-6R, indicative of the formation of the binary IL-6 receptor complex. Similarly, preincubation of (QT)IL-6 with sIL-6R yielded a decreased peak height of the ligand as well as an increased peak height and a minimal shift in elution position of the sIL-6R, compared with (QT)IL-6 and sIL-6R alone, indicating the formation of a binary complex of (QT)IL-6 with sIL-6R (compare panel E with panels A and B).
Whereas incubation of IL-6 with sIL-6R and sgp130 resulted in the formation of the hexameric receptor complex (see peak indicated by arrow in panel C), no significant formation of a hexameric complex was detected when (QT)IL-6 was used as a ligand (panel F). In a separate experiment, when IL-6, sIL-6R, and sgp130 were incubated together with (QT)IL-6 (6-fold molar excess of (QT)IL-6 over IL-6), no significant decrease in the peak height of the hexameric IL-6 receptor complex was noticed (data not shown).
When I-(QT)IL-6 and
I-IL-6 were incubated with
the sIL-6R alone, both ligands formed a higher molecular weight complex
coeluting with the binary complex (Fig. 6A). When incubated
with both sIL-6R and sgp130,
I-IL-6 clearly formed an
additional complex that eluted in an equivalent position to the
hexameric complex (Fig. 6B; c.f.Fig. 5C). By comparison, although the
I-(QT)IL-6
sIL-6R complex was formed, only 186 cpm
was found to elute in the position corresponding to the hexameric
complex (Fig. 6B).
Figure 6:
Narrowbore size exclusion chromatography
of receptor complexes of I-labeled (QT)IL-6 and IL-6.
Chromatography was performed as described in the legend to Fig. 5, with a flow rate of 100 µl/min. (QT)IL-6 (0.98
µg) and IL-6 (0.99 µg), mixed with the corresponding
I-labeled ligand, were incubated with 0.4 µg sIL-6R
alone (A) or sIL-6R (0.4 µg) and sgp130 (0.74 µg) (B). Fractions were collected every 30 s, and the associated
counts were determined using a
counter (
I-(QT)IL-6, solid line;
I-IL-6, dashed line). The
elution positions of the binary and hexameric receptor complexes
obtained from the corresponding UV traces (data not shown) are
indicated.
Using a biosensor employing surface plasmon resonance detection and narrowbore SEC we have investigated the interactions of an IL-6 antagonist, (QT)IL-6, with the sIL-6R and sgp130, the extracellular domains of the subunits of the IL-6 receptor complex.
Affinity data for the interaction of monomeric (QT)IL-6 with sIL-6R
were obtained from the association and dissociation rate constants as
well as from equilibrium binding measurements at the sensor surface and
in solution. With (QT)IL-6 immobilized to the sensor chip these methods
of calculation yielded K values that were in good
agreement with each other and with the binding kinetics in solution as
well as with the corresponding values for immobilized monomeric IL-6.
The small, but consistent, difference between the apparent K
values of (QT)IL-6 and IL-6 was due to the
difference in the apparent dissociation rate constants. Linear and
nonlinear regression analysis yielded k
= 3.1
and 3.2 s
10
for (QT)IL-6
and k
= 2.0 and 0.9 s
10
for IL-6, respectively. Our data
using soluble IL-6R are in good agreement with the relative
affinity of (QT)IL-6 to that of IL-6 for the IL-6R on human CESS cells
(500 and 360 pM, respectively)(12) .
Similar to
(QT)IL-6, in the case of the interaction of sIL-6R with immobilized
IL-6 monomer, the equilibrium binding data yielded an apparent K = 10 nM, which is in excellent
agreement with the apparent K
= 10 nM calculated from linear regression analysis. The apparent
association rate constants for the interaction of sIL-6R with IL-6
obtained by linear and nonlinear regression analysis were
indistinguishable (k
= 2.1
10
M
s
). However, the
apparent k
= 0.9
10
s
obtained by nonlinear regression analysis
was slightly lower than the apparent k
=
2.0
10
s
calculated using
linear regression, resulting in an apparent K
= 5 nM. A similar phenomenon was also observed in
a study on the interaction of soluble CD4 with an immobilized antibody,
where the nonlinear regression yielded a similar apparent k
, but a 6-fold lower apparent k
than the linear regression analysis(16) .
To measure
the interaction between two proteins using SPR, it is preferential to
immobilize the smaller component, as the detector response will be
proportional to the mass of bound analyte(25) . In the case of
the interaction between IL-6 (21 kDa) and sIL-6R (
53 kDa) it
is possible to immobilize either protein. In the present investigation,
using linear regression analysis, we have determined apparent k
= 2.1
10
M
s
and k
= 0.002 s
(apparent K
= 10 nM) for the interaction of
sIL-6R with immobilized IL-6 monomer. Using the same method of
analysis, but with the assay performed in the opposite orientation, the
interaction of IL-6 monomer with immobilized sIL-6R yielded an
apparent k
= 3.8
10
M
s
and k
= 0.018 s
(apparent K
= 47 nM).
A similar
discrepancy was found in a biosensor study on the interaction of human
IL-5 with the human soluble IL-5 receptor(31) . In this study,
the K
was calculated to be 1.7 nM using
immobilized receptor with IL-5 as the analyte; however, when the assay
was performed in the opposite orientation, the apparent K
was extrapolated to be 5.5
nM(31) .
The difference in the apparent K (10 nM and 47 nM for
immobilized IL-6 monomer and immobilized sIL-6R, respectively) is
mainly due to the difference in the calculated off-rates for the
analytes. One possible explanation for the difference could be the
immobilization procedure used. The N-ethyl-N
-(3-diethylaminopropyl)/N-hydroxysuccinimide
coupling chemistry, which, as evidenced by the good correlation between
solution phase and solid phase binding, appeared not to interfere with
the ability of (immobilized) IL-6 to interact with sIL-6R, may have
affected the interaction of (immobilized) sIL-6R with IL-6.
Additionally, the conditions used to regenerate the sensor chip between
measurements (4 M MgCl
in 10 mM Tris-HCl,
pH 7.4)
may have altered the properties of the immobilized
sIL-6R. In this respect it is worth mentioning that the sIL-6R is
considerably less stable than IL-6 when immobilized on the sensor
surface, with an average life-span of days as compared to weeks for
IL-6.
In this report the comparative studies of the solution
binding of sIL-6R with (QT)IL-6 and IL-6 have been performed with
analysis of uncomplexed sIL-6R concentration, using sensor chips
derivatized with both monomeric (QT)IL-6 and monomeric IL-6. The K values observed (20 and 16 nM for
(QT)IL-6, 9 and 6 nM for IL-6; Table 1) were essentially
independent of whether (QT)IL-6 or IL-6 was used for analysis, implying
that the immobilization had not drastically altered the ability of
(immobilized) (QT)IL-6 to interact with sIL-6R. Our data on the
solution binding of monomeric IL-6 with sIL-6R compare well with
previous results, where the concentration of uncomplexed sIL-6R was
measured using a sensor chip derivatized with an essentially dimeric
preparation of IL-6. In the latter study, the solution binding constant (K
) of sIL-6R with monomeric and essentially
dimeric preparations of IL-6 was 5
10
M
(K
= 20
nM) for both(24) .
Interaction of the
IL-6IL-6R complex with gp130 induces high affinity
binding(4) . On the basis of detecting low but not high
affinity binding of (QT)IL-6 to CESS cells, Brakenhoff and co-workers (12) concluded that the (QT)IL-6
IL-6R complex did not
interact with gp130 and that the antagonistic activity of (QT)IL-6 on
human HepG2 and CESS cells was caused by interference with the
formation of the IL-6
IL-6R complex. However, because of the
apparent inability to associate with gp130, it was difficult to explain
the small agonist activity of this mutant on HepG2 cells. Furthermore,
(QT)IL-6 also had residual bioactivity on the human myeloma cell line
XG-1(13) . Using a sandwich ELISA, de Hon et al.(13) showed that (QT)IL-6, in the presence of sIL-6R, has
residual affinity for sgp130. Here, using biosensor technology, we have
shown that (QT)IL-6 interferes with the association of the
IL-6
sIL-6R complex with sgp130 (Fig. 4). We also found
that the (QT)IL-6
sIL-6R complex is capable of interacting with
sgp130, further suggesting that the biological activity of (QT)IL-6 is
mediated by gp130.
We (10) and others (32) have
recently reported that the ternary receptor complex of IL-6 is a
hexamer consisting of two molecules each of IL-6, IL-6R, and gp130
(2:2:2 complex). Our recent data suggest that the formation of the
2:2:2 complex may involve the dimerization of an intermediate complex
consisting of one molecule each of IL-6, IL-6R, and gp130.
The existence of such a trimer in the signaling pathway of IL-6 was
previously suggested by Davis et al. by analogy with the
sequential formation of the receptor complexes of LIF and
CNTF(33) . (QT)IL-6 apparently does not induce the formation of
significant amounts of a stable hexameric complex using the
extracellular domains of the IL-6R and gp130 ( Fig. 5and Fig. 6). However, the bioactivity of (QT)IL-6 may be explained
by residual ability of this molecule to induce a hexameric complex on
the cell surface. The reason why CESS cells are unresponsive to
(QT)IL-6 (12) remains to be determined.
Simultaneous
incubation of (QT)IL-6 with IL-6, sIL-6R, and sgp130 did not reduce the
formation of the hexameric complex, as determined by SEC (data not
shown). Since the affinity of (QT)IL-6 for the sIL-6R is only
approximately 2-fold lower than that of IL-6, the 6-fold molar excess
of (QT)IL-6 used in this experiment should be sufficient to compete
with IL-6 for binding to the sIL-6R. The apparent inability of (QT)IL-6
to interfere with the formation of the 2:2:2 complex suggests that the
hexameric complex is of higher affinity than the binary complex of IL-6
and sIL-6R. This conclusion using the extracellular domains of IL-6R
and gp130 is in agreement with previous results on cells showing that
association of gp130 with the IL-6IL-6R complex confers high
affinity binding(4) .
The three regions of IL-6 apparently
involved in the association of the IL-6IL-6R complex with gp130
are residues Gln
-His
(denoted the
1 site)(12) , residues Lys
-Ala
(
2 site) (34) and residues Tyr
,
Gly
, Ser
, and Val
(
3
site)(35) . Replacement of the
2 region with the
corresponding mouse IL-6 residues resulted in drastically reduced
bioactivity of the mutant protein without disrupting its affinity for
the IL-6R(34) . An IL-6 mutant with substitutions of residues
Gln
for Glu, Thr
for Pro, and the
2
region for the corresponding mouse IL-6 residues was inactive on XG-1
cells and weakly antagonized IL-6-bioactivity on these cells (13) . By introducing two additional substitutions the affinity
of this mutant for the IL-6R was enhanced 5-fold, yielding an
antagonist that completely inhibited the activity of IL-6 on XG-1
cells(13) .
In homology models of human and mouse IL-6 based
on the structure of G-CSF(8, 34) , the 1 and
2 sites localize to the same side of the IL-6 molecule, suggesting
that they form a single gp130-associating determinant. We show here
that disruption of the
1 site, as in (QT)IL-6, interferes with the
formation of the hexameric receptor complex but does not prevent the
association of the (QT)IL-6
sIL-6R complex with sgp130. In
essential agreement with our results, during the preparation of this
manuscript it was reported that (W157R,D160R)IL-6, a
1 site mutant
different from (QT)IL-6, can interact with sIL-6R and sgp130 to form a
ternary complex consisting of one molecule of each ligand, sIL-6R and
sgp130, but is severely attenuated in its ability (approximately 5% of
``wild type'' IL-6 activity) to induce the hexameric receptor
complex(32) . The residual biological activity of (QT)IL-6 may
thus be explained by the partial functionality of the putative
1/
2 site. It remains to be determined whether disruption of
the
2 site alone has a similar effect on ternary IL-6 receptor
complex formation.
The affinity of (QT)IL-6 for the sIL-6R is lower
than that of IL-6, due to the increased k. We have
shown that the binary receptor complex of (QT)IL-6 has some affinity
for sgp130 but that (QT)IL-6 is ineffective in the induction of a
hexameric receptor complex. Our data suggest that the soluble hexameric
IL-6 receptor complex is of higher affinity than the binary complex of
IL-6 and sIL-6R.