(Received for publication, November 8, 1994; and in revised form, January 26, 1995)
From the
The pleiotropic cytokine interleukin-6 (IL-6) interacts with the
specific ligand binding subunit (IL-6R) of the IL-6 receptor, and
this complex associates with the signal-transducing subunit gp130
(IL-6R
). Human IL-6 acts on human and murine cells, whereas murine
IL-6 is only active on murine cells. The construction of a set of
chimeric human/murine IL-6 proteins has recently allowed us to define a
region (residues 43-55) within the human IL-6 protein, which is
important for the interaction with gp130. Subdividing this region shows
that mainly residues 50-55 of the human IL-6 are necessary for
this interaction. Recently, another human IL-6 double mutant (Q159E and
T162P) showed reduced affinity to gp130 but residual activity on the
human myeloma cell line XG-1. Into this IL-6 mutant we introduced the
murine residues 43-49 or 50-55 together with two point
mutations, F170L and S176A, which had been reported to increase the
affinity of IL-6 to the IL-6R
. The resulting IL-6 molecule, which
contained the murine residues 50-55, was inactive on human
myeloma cells and in addition completely inhibited wild type IL-6
activity on these cells. Such an antagonist may be used as a specific
inhibitor of IL-6 activity in vivo.
The plasma level of the pleiotropic cytokine IL-6 ()is increased upon injury or infection. IL-6 is involved in
a spectrum of activities like immune defense, hematopoiesis, and acute
phase response(1) . An increased expression of IL-6 has been
reported for several diseases like plasmacytoma/myeloma, mesangial
proliferative glomerulonephritis, osteoporosis, autoimmune diseases,
and AIDS(1) .
IL-6 activates target cells via a
transmembrane receptor, which consists of two glycoproteins. First,
IL-6 binds with low affinity (K =
500 pM) to the specific ligand binding subunit gp80
(IL-6R
). This complex binds with high affinity (K
= 30-50 pM) the
signal transduction subunit gp130 (IL-6R
). IL-6 by itself has no
measurable affinity to gp130(2) . IL-6/IL-6R
mediated
homodimerization of two gp130 molecules via a disulfide bridge induces
the IL-6 signal(3) .
Interleukin-6 is a protein of 184 amino
acids(4) . Secondary structure predictions point out that IL-6
belongs to a hemopoietic cytokine family characterized by four
antiparallel -helices (A, B, C, and D) (5) . This family
also includes leukemia inhibitory factor, oncostatin M, ciliary
neurotrophic factor, growth hormone, prolactine, and granulocyte colony
stimulating factor(5, 6, 7) . Structure
function studies of human IL-6 showed that the COOH terminus and the
end of the A-B loop/beginning of the B-helix (region 2c, residues
Gly
-Glu
) are involved in the
interaction with the
IL-6R
(8, 9, 10, 11, 12, 13, 14, 15) .
These results are in good agreement with a recently published human
IL-6 model (15) where these two regions are in close proximity.
Point mutations in the middle of the D-helix (F170L, S176R) resulted in
a 2-3-fold increased affinity to the
IL-6R
(12, 16) .
Human IL-6 acts on human and
murine cells, whereas murine IL-6 is only active on murine cells (the
identity between human and murine IL-6 is 42%(14) ). Recently,
we have exchanged the human residues 43-55 (beginning of the A-B
loop, region 2a) against the corresponding murine amino
acids(15) . This chimeric protein showed an only 5-fold reduced
affinity to the human IL-6R but a highly reduced human
IL-6R
dependent interaction with gp130 and no bioactivity on the
human hepatoma cell line HepG2. But on the more sensitive human myeloma
cell line XG-1, this IL-6 mutant reached full bioactivity at a 100-fold
higher concentration than human IL-6.
Brakenhoff et al.(17) have shown that substitution of two residues at the
beginning of the D-helix (Q159E/T162P) also reduced the affinity of the
IL-6IL-6R
complex to gp130, whereas the affinity of IL-6 to
the IL-6R
was decreased only 3-4-fold. This IL-6 mutant
could be used as an IL-6 receptor antagonist on human HepG2 cells and
the human Epstein-Barr virus-transformed B cell line CESS, but it had
full activity on the more sensitive human XG-1 cells when applied at
sufficiently high concentrations. (
)
In the present study,
we subdivided the region 2a into two parts (2a1, residues 43-49;
2a2, residues 50-55) to analyze which part is the most important
for the interaction with gp130. We exchanged each part in the human
IL-6 against the corresponding murine sequence. In the next step, we
wanted to construct an IL-6 molecule that should bind to the
IL-6R, but which should not activate gp130. In addition, this IL-6
protein should block the IL-6R
and thereby inhibit human IL-6
activity on the highly sensitive human myeloma cell line XG-1. For this
reason, we combined the murine residues 43-49 or 50-55 with
the two point mutations (Q159E/T162P) in the beginning of the D-helix,
which also decrease the interaction of the IL-6
IL-6R
complex
with gp130 (16) . In addition, we introduced point mutations in
the end of the D-helix (F170L/S176R) that increase the affinity to the
IL-6R
(12, 16) . These changes resulted in an
effective IL-6 receptor antagonist.
Construction of pRSET 5d-huIL-6-2a1 was as follows. Since there was no common restriction site in the human or murine IL-6-cDNA, we introduced the unique restriction site SacI at analogous positions using synthetic oligonucleotides. These nucleotide exchanges did not result in changes of the amino acid sequence. Chimeric human and murine IL-6-cDNA fragments were then produced by PCR using pRSET 5d-huIL-6 or pRSET 5d-huIL-6-2a as template(14, 15) . PCR reaction conditions were described(15) . The oligonucleotides hybridizing to the pRSET 5d vector, the human IL-6 cDNA or the mutant 2a cDNA, were 5`-TAATACGACTCACTATAG-3` sense (vector), 5`-CTCAGCTTCCTTTCGG-3` antisense (vector), 5`-TGTGAGAGCTCCAAAGAGGCAC-3` SacI sense (human IL-6), and 5`-CATCGGAGCTCTCACAATCAGA-3` SacI antisense (mutant 2a).
To introduce the two point mutations, F170L and S176R, into the huIL-6-Q159E/T162P cDNA (short designation, huIL-6-EP), a XbaI-Bsu36I fragment encoding the F170L and S176R substitutions was created with PCR technology. pRSET 6d-huIL-6-EP was used as a template, and the resulting fragment was subcloned into XbaI-Bsu36I-digested pRSET 6d-huIL-6-EP. PCR was carried out with Pyrococcus furiosus polymerase (Stratagene) under the following conditions: denaturation, 90 s at 95 °C; annealing, 60 s at 55 °C; extension, 60 s at 72 °C, 30 cycles. The following primers were used: a sense primer carrying the XbaI site (5`-GAATCTAGATGCAATAACCACC-3`) and an antisense primer encoding the two substitutions and the Bsu36I site (5`-GAAGAGCCCTCAGGCTGCGCTGCAGGAACTCCTTAAGGCTGCGCAGAATG-3`). The pRSET 6d-huIL-6-EP-F170L/S176R was called pRSET 6d-huIL-6-EP-LR.
The vectors pRSET 6d-huIL-6-EP-2a1-LR and pRSET 6d-huIL-6-EP-2a2-LR were constructed by ligating an NcoI-XbaI cDNA fragment from pRSET 5d-huIL-6-2a1 or -2a2 into NcoI-XbaI-digested pRSET 6d-huIL-6-EP-LR.
The integrity of all constructs was verified by restriction fragment analysis and DNA sequencing(18) .
First, we measured displacement of human I-IL-6
binding to a soluble form of the IL-6R
protein(14, 15, 19, 20, 21, 22) by an
excess of IL-6 mutants. As shown in Fig. 1A, human IL-6
displaced
I-IL-6 binding to 50% when used in a
10-20-fold molar excess. In contrast, murine IL-6 failed to
compete with human
I-IL-6 for binding. Chimera 2a showed
half-maximal displacement at an excess of approximately 60-fold whereas
chimera 2a1 and 2a2 showed similar affinity to the IL-6R
as human
IL-6.
Figure 1:
Binding and bioactivity of the mutants
2a1 and 2a2. A, binding of the IL-6 variants to a soluble
human IL-6R. Recombinant human soluble IL-6R
was incubated
with human
I-IL-6. Binding was competed with increasing
amounts of IL-6 variants. Average values of three experiments are
shown. B, proliferation of human XG-1 cells in response to
IL-6 mutants. Cells were grown in the presence of increasing amounts of
IL-6 variants, and proliferation was measured using
[
H]thymidine incorporation. One representative of
three experiments is shown. C1, induction of haptoglobin mRNA
expression in human hepatoma cells by IL-6 variants. C2,
photograph of the ethidium bromide-stained RNA gel. D, complex
formation between soluble forms of IL-6R
and gp130 and the IL-6
variants was determined using metabolically labeled
S-soluble IL-6R
and a fusion protein of human sgp130
and human IgG1. Complexes were precipitated with protein A-Sepharose,
separated by SDS-polyacrylamide gel electrophoresis, and visualized by
fluorography.
Human and murine IL-6 and the mutants 2a, 2a1, and 2a2 stimulated the proliferation of murine B9 cells to a similar extent (data not shown), which demonstrated that their structures were intact. On human myeloma XG-1 cells, human IL-6 but not murine IL-6 induced proliferation (Fig. 1B). The specific activity of chimera 2a was reduced by a factor of approximately 300. Chimera 2a1 was 5-fold and chimera 2a2 30-fold less active than human IL-6. These results indicate that the properties of chimera 2a reside mainly in part 2a2.
A similar result was obtained when we examined the induction of the acute phase protein gene haptoglobin by 10 ng/ml of the IL-6 variants in human hepatoma cells (HepG2). Untreated cells and cells treated with murine IL-6 and chimera 2a showed no haptoglobin mRNA induction (Fig. 1C). Stimulation of cells with human IL-6 led to full stimulation of haptoglobin mRNA expression. Chimera 2a2 was less active than chimera 2a1.
One possible
explanation for the discrepancy between binding of chimeras 2a1 and 2a2
to the IL-6R and their reduced bioactivity on human cells could be
a reduced interaction of the IL-6
IL-6R
complex with gp130.
To measure ternary complex formation of the IL-6 mutants with soluble
forms of IL-6R
and
gp130(19, 23, 24, 25, 26) ,
we used metabolically labeled
S soluble human IL-6R
protein and soluble human gp130 with the constant region of human IgG1
fused to its COOH terminus(15) . The complexes were
precipitated with protein A-Sepharose.
No precipitation of
sIL-6R occurred without IL-6 (Fig. 1D). In the
presence of human but not of murine IL-6, the sIL-6R
subunit was
coprecipitated with soluble gp130. The 2a mutant did not induce
measurable complex formation between IL-6R
and gp130. Chimera 2a1
led to a slightly reduced and chimera 2a2 to a more reduced ternary
complex formation. Therefore, region 2a2 is more important for the
interaction of the IL-6
IL-6R
complex with the gp130 molecule
than region 2a1. Whether this region is part of a binding side for
gp130 or it is necessary for stabilizing other epitopes responsible for
gp130 interaction remains open.
So far, it is not clear why the sensitivity of IL-6-responsive cells varies by 2-3 orders of magnitude. Cells that proliferate in response to IL-6 show a half-maximal response at 1-20 pg/ml, whereas cells that differentiate or express acute phase proteins have a half-maximal response at 1-5 ng/ml(14, 15) . This, however, cannot be explained by different receptor numbers(1, 34) . It cannot be excluded that there are differences in signal transduction or that the presence of additional receptor subunits on cells that proliferate in response to IL-6 renders these cells more sensitive to the cytokine. This phenomenon of differential sensitivity of different cell types has of course important implications for the therapeutic application of IL-6 receptor antagonists.
Fig. 2shows a ribbon representation of the IL-6
model (15, 27) . In our model, the two point mutations
(EP) and the regions 2a1 and 2a2 are in close proximity. Recently, the
substitution of two amino acids in the A-helix (Y31D/G35F) (Fig. 2) also lead to a reduced affinity of the
IL-6IL-6R
complex to gp130(28) . There is evidence
that the ligand-induced receptor complex consists of two molecules of
each, IL-6, IL-6R
, and gp130(22, 29) . So far, it
is unclear whether the mentioned regions are all involved in the
interaction with one gp130 or whether one IL-6 protein touches two
gp130 proteins or two IL-6 proteins touch each other.
Figure 2:
Ribbon representation of the human IL-6
model. Representation of amino acids important for IL-6R binding
and IL-6R
-dependent gp130 interaction (15, 35) (helix A, 19-45; helix B, 80-101;
helix C, 106-132; helix D, 157-184). The amino acids
Phe
, Ser
, Gln
,
Thr
, Phe
, and Ser
(see text)
are shown. The regions 2a1 and 2a2 are marked by the amino acids
Thr
, Cys
, and Glu
(see text). C, COOH terminus; N, NH
terminus
(corresponds to residue 17 of
IL-6)(15) .
We introduced
the murine region 2a2 and 2a1, respectively, into the mutant IL-6-EP
together with the two point mutations (F170L/S176R; short designation,
LR), which increase the affinity of human IL-6 to the IL-6R. The
two resulting IL-6 mutant proteins were called IL-6-EP-2a1-LR and
IL-6-EP-2a2-LR. Both mutants showed similar affinity to the IL-6R
as human IL-6 (Fig. 3A). Mutant IL-6-EP-2a1-LR and
IL-6-EP-2a2-LR fully induced proliferation of IL-6-dependent B9 cells
albeit at 10-100-fold higher concentrations (data not shown). For
mutant IL-6-EP-2a2-LR, no bioactivity on human XG-1 (Fig. 3B) and HepG2 (Fig. 3C) cells and
no IL-6R
-dependent ternary complex formation with gp130 (Fig. 3D) was detected. Mutant IL-6-EP-2a1-LR showed
residual bioactivity on XG-1 cells. This was in line with the higher
specific activity of 2a1 compared with 2a2.
Figure 3:
Binding and bioactivity of the mutants
IL-6-EP-2a1-LR and IL-6-EP-2a2-LR. A, binding of the IL-6
variants to the soluble human IL-6R. Average values of three
experiments are shown. B, proliferation of human XG-1 cells in
response to IL-6 variants. One representative of three experiments is
shown. C1, induction of haptoglobin mRNA expression in human
hepatoma cells by IL-6 variants. C2, photography of the
ethidium bromide-stained RNA gel. D, induction of complex
formation between soluble forms of IL-6R
and gp130 and the IL-6
variants. See text and legend to Fig. 1for
details.
To test for antagonistic activity, we added IL-6-EP-2a1-LR and IL-6-EP-2a2-LR at increasing concentrations, respectively, to human myeloma cells (XG-1) in the presence of 100 pg/ml human IL-6 (Fig. 4A). Mutant IL-6-EP-2a2-LR completely inhibited the bioactivity of IL-6 at an 100,000-fold excess, whereas mutant IL-6-EP-2a1-LR at this concentration only led to 70% reduction of cell proliferation. By increasing the amounts of human IL-6 to XG-1 cells in the presence of 10 µg/ml IL-6-EP-2a1-LR or IL-6-EP-2a2-LR, we observed that high concentrations of human IL-6 could completely overcome the antagonistic effect of both IL-6 variants (Fig. 4B).
Figure 4: Antagonistic effect of IL-6-EP-2a1-LR and IL-6-EP-2a2-LR on the human IL-6-induced proliferation of XG-1 cells. A, the indicated concentrations of IL-6-EP-2a1-LR and IL-6-EP-2a2-LR were added in the presence of 100 pg/ml human IL-6. One representative of two experiments is shown. B, the indicated concentrations of human IL-6 were added alone or in the presence of 10 µg/ml IL-6-EP-2a1-LR or IL-6-EP-2a2-LR.
IL-6-EP-2a2-LR behaved as a potent IL-6 receptor antagonist on XG-1
cells. Introduction of additional point mutations into IL-6 may further
increase the affinity to IL-6R. This will reduce the amount of
this antagonist necessary to inhibit IL-6 activity.
IL-6 plays a
role in a variety of diseases, such as plasmacytoma/myeloma, mesangial
proliferative glomerulonephritis, osteoporosis, autoimmune diseases,
and AIDS(1) . The administration of IL-6-neutralizing
monoclonal antibodies to patients with rheumathoid arthritis and
multiple myeloma has highly improved the conditions of the patients for
several weeks(30, 31) . But then the symptoms returned
because the high stability of antibodies in plasma increased the level
of IL-6, which normally is cleared from the circulation within several
minutes(32) . No beneficial effect can be expected by using
neutralizing monoclonal antibodies against IL-6R(33) . A
natural soluble form of this receptor subunit together with IL-6 has
been shown to behave as an
agonist(19, 20, 21) . Neutralizing antibodies
could lead to an accumulation of the soluble IL-6R
in the plasma.
Neutralizing monoclonal antibodies against the signal transduction
protein gp130, which is also part of the receptor of leukemia
inhibitory factor, ciliary neurotrophic factor, oncostatin M, and
IL-11(34) , could provoke a lot of side effects in
vivo. We therefore believe that the construction of IL-6 receptor
antagonists is the most promising strategy to neutralize IL-6 activity in vivo. We have obtained such an IL-6 receptor antagonist,
which can completely inhibit human IL-6 activity on human myeloma
cells. The availability of such an antagonist may offer an approach to
specifically inhibit IL-6 activity in vivo.