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*
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany; and
Diaclone, Besançon, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit (IL-6R, gp80, or
CD126), which in its soluble (s) form (sIL-6R) acts agonistically by
making the ligand accessible to the second subunit, the signal
transducer gp130 (CD130). Soluble forms of both receptor subunits are
present in human blood. Gel filtration of iodinated IL-6 that had been
incubated with human serum revealed that IL-6 is partially trapped in
IL-6/sIL-6R/sgp130 ternary complexes. sgp130 from human plasma was
enriched by immunoaffinity chromatography and identified as a 100-kDa
protein. Functionally equivalent rsgp130 was produced in
baculovirus-infected insect cells to study its antagonistic potential
on four different cell types. It was found that in situations in which
cells lacking membrane-bound IL-6R were stimulated with IL-6/sIL-6R
complexes, sgp130 was a much more potent antagonist than it was
on IL-6R-positive cells stimulated with IL-6 alone. In the latter case,
the neutralizing activity of sgp130 could be markedly enhanced by
addition of sIL-6R. As a consequence of these findings, sIL-6R of human
plasma must be regarded as an antagonistic molecule that enhances the
inhibitory activity of sgp130. Furthermore, in combination with sIL-6R,
sgp130 is a promising candidate for the development of IL-6
antagonists. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-6 acts on target cells via a receptor consisting of two transmembrane glycoproteins. After binding of IL-6 to its specific receptor (gp80 or IL-6R), the complex of IL-6 and IL-6R triggers the dimerization of the signal-transducing receptor component gp130 (11). This leads to the activation of different signaling cascades, of which the Janus kinase (Jak)3/STAT pathway is the best understood at the moment; the cytoplasmic part of gp130 is associated with tyrosine kinases of the Jak family that are activated upon receptor dimerization (12). These kinases phosphorylate tyrosine residues in the membrane-distal cytoplasmic part of gp130, which in turn become binding sites for the Src homology 2 domain containing transcription factors STAT1 and STAT3/acute phase response factor (APRF) (12, 13). Recruited STATs are also tyrosine phosphorylated by the associated kinases, leading to the formation of STAT homo- and heterodimers. STAT dimers are translocated into the nucleus, where they bind to responsive DNA elements and induce target gene expression (14). Furthermore, activation of the ras/raf/mitogen-activated protein kinase pathway was observed in different cells upon IL-6 stimulation (15, 16), as well as activation of various tyrosine kinases different from Jak kinases (17, 18).
The soluble form of the IL-6R (sIL-6R), which lacks the transmembrane and cytoplasmic parts, when complexed with IL-6 triggers the dimerization of gp130 and is therefore an agonistically acting molecule (19). Whereas numerous in vitro studies showed that sIL-6R acts agonistically by enhancing the effects of IL-6, the antagonistic potential of sgp130 has been described only in a single study (20). Since soluble forms of both receptor components (sIL-6R (21) and sgp130 (20)) have been detected in plasma and several biologic fluids of humans and mice, the physiologic significance of their opposite biologic effects is of great importance. To further characterize its molecular properties, we enriched sgp130 from human plasma and identified it as a monomeric 100-kDa glycoprotein that is able to efficiently bind IL-6/sIL-6R complexes. To study its antagonistic potential in greater detail, rsgp130 was expressed in baculovirus-infected insect cells. The recombinant protein was purified from conditioned media and characterized with respect to its oligomerization state and its ability to form complexes with IL-6 and sIL-6R. Here we show that sgp130 is a potent IL-6 antagonist on various cell types and that its antagonistic activity is markedly enhanced in the presence of sIL-6R.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Enzymes were purchased from Boehringer Mannheim (Mannheim,
Germany), and protein A-Sepharose was purchased from Pharmacia
(Freiburg, Germany). DMEM, DMEM/F12, RPMI 1640, Sf-900 II medium, and
antibiotics were obtained from Life Technologies (Eggenstein, Germany),
and FCS was obtained from Seromed (Berlin, Germany).
Bolton-Hunter reagent and [-32P]dATP were purchased
from Amersham International (Little Chalfont, U.K.), and
Tran[35S] label metabolic labeling reagent was
purchased from ICN (Meckenheim, Germany). Recombinant human IL-6 was
expressed in Escherichia coli, refolded, and purified as
described by Arcone et al. (22). The specific activity was
108 units/mg of protein in the B9 cell proliferation assay
(23). sIL-6R was expressed in insect cells as previously described
(24). The monoclonal gp130-Abs B-T12, B-P4, and B-T2 were generated as
described elsewhere (25). All other Abs were purchased from DAKO
(Hamburg, Germany). The frequently used PBS buffer contained 200 mM
NaCl, 2.5 mM KCl, 8 mM Na2HPO4, and 1.5 mM
KH2PO4.
sgp130 ELISA
Ninety-six-well microtiter plates (F96 MaxiSorp immunoplate; Nunc, Naperville, IL) were coated overnight at room temperature with anti-sgp130 mAb B-P4 (0.5 µg/well in PBS). The plates were incubated with saturation buffer (0.1 M Tris; 20% sucrose; and 0.1% sodium acide, pH 7.7) for 2 h at room temperature. After three washes with PBS and 0.02% Tween-20, the samples or standards (in PBS and 1% BSA) and the secondary biotinylated mAb (B-T2, 50 ng/well) were incubated simultaneously for 2 h at 37°C. The standard curve was obtained by twofold serial dilutions of rsgp130. Subsequently, the plates were washed again, and streptavidin poly-horseradish peroxidase (100 ng/ml in PBS and 1% BSA) was added and incubated for 45 min at room temperature. After a final wash, substrate solution (100 µg/ml tetramethylbenzidine in 0.1 M sodium acetate, pH 5.5, and 0.003% H2O2) was added. After incubation for 30 min in the dark, the color reaction was stopped with 2 M H2SO4, and the absorbance at 450 nm was determined using an ELISA reader (SLT-Labinstruments, Grödig, Austria). Affinity-purified rsgp130 from baculovirus-infected insect cells was used as a standard.
Construction of the recombinant baculoviruses and expression of rsgp130
The AccII-EcoRI fragment encoding the gp130 extracellular domains (codons 1–606) was cut out from the vector pVL-gp130. A pair of hybridized oligonucleotides (5'-AA TTC GGA (CAT)5 CAC TAG-3' and 5'-G ATC CTA GTG (ATG)5 TCC G-3') encoding a glycine and six histidine residues followed by a stop codon and a BamHI 5'-overhang was linked to the EcoRI site of the cDNA for soluble human gp130 and inserted into the polyhedrin locus-based baculovirus transfer vector pVL1392 using the BglII (blunt end) and the BamHI sites. Sf158 cells were cotransfected with 0.5 µg of recombinant gp130-baculovirus transfer vector and 0.125 µg of BaculoGold virus DNA as outlined in the BaculoGold transfection kit manual (Dianova, Hamburg, Germany). Single virus clones were obtained by end point dilution. Several clones were screened for expression of sgp130 by Western blotting. The selected virus clone was then amplified by infecting Sf158 cells at a multiplicity of infection less than 1. Sf158 insect cells were grown at 27°C as monolayer cultures in serum-free Sf-900 II medium. For protein expression, exponentially growing cells were infected with the rsgp130 baculovirus in suspension cultures at a multiplicity of infection from 10 to 20. Seventy-two hours after infection, the cells and cellular debris were sedimented by centrifugation, and the culture supernatants were stored at -20°C.
Immunoaffinity purification of sgp130 and rsgp130
The sgp130 mAb B-T12 was coupled to 0.5 g of CNBr-activated Sepharose CL-4B according to the protocol of the supplier (Pharmacia). Three milliliters of 1 M CaCl2 was added to 300 ml of human plasma, and after incubation at 37°C for 90 min, 10 ml of 0.5 M EDTA was added. Coagulated proteins were sedimented by centrifugation for 20 min at 14,000 x g. The supernatant was applied to the immunoaffinity column at a flow rate of 20 ml/h. To purify rsgp130, supernatants of baculovirus-infected insect cells were loaded onto the column. After washing with 100 ml PBS/0.05% Tween 20, sgp130/rsgp130 was eluted with 0.2 M glycine buffer, pH 2.5, and immediately neutralized with 1 M Tris/HCl, pH 8.0. Pooled fractions were dialyzed against PBS, and the sgp130 concentration was determined by ELISA. Total protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA).
Gel filtration
Analyses of complexes formed by iodinated IL-6 in human serum were performed on a calibrated Superdex 200 16/60 gel filtration column (Pharmacia) at a flow rate of 0.85 ml/min. sIL-6R depletion of human serum was performed as described (26). 125I-labeled IL-6 (20 ng) with a specific activity of 4.7 x 104 cpm/ng was incubated with 1 ml of human serum (either normal or sIL-6R depleted) or 1 ml of a solution of BSA (50 mg/ml) in PBS for 12 h at 4°C followed by 2 h at 37°C. After centrifugation for 10 min at 10,000 x g, the supernatant was applied to the gel filtration column, and 2.6-ml fractions were collected. Immunoprecipitations were performed using B-P4-saturated protein A-Sepharose. 125I-labeled IL-6 was quantified using a gamma counter.
Formation of ternary complexes of IL-6, sIL-6R, and sgp130
Binding experiments were performed using IL-6 radiolabeled with 125I according to the procedure of Bolton and Hunter (27), affinity-purified sIL-6R, and rsgp130 or conditioned media from Sf158 cells expressing rsgp130. All incubations were conducted at 4°C. 125I-labeled IL-6 (5.5 ng) with a specific activity of 8.3 x 104 cpm/ng was preincubated overnight with 100 ng sIL-6R in 500 µl TNET buffer (20 mM Tris-HCl, pH 7.5; 140 mM NaCl; 5 mM Na2-EDTA; 1% Triton X-100; 2 mM methionine; and 0.01% NaN3). Increasing amounts of purified rsgp130 or conditioned medium containing rsgp130 were added, and the incubation was continued for 2 h. The reaction mixtures were subjected to immunoprecipitation with the monoclonal gp130 Ab B-T12. Coprecipitated 125I-labeled IL-6 was quantified using a gamma counter.
Real-time interaction analysis
For direct monitoring of ternary complex formation, the IAsys
(Fisons, Cambridge, U.K.) system was used. The biosensor surface was
activated using
N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
and subsequently incubated with gp130 mAb B-T12 (50 µg/ml in 10 mM
sodium acetate, pH 4.8) for 8 min. Unbound Abs were removed by several
washes with PBS containing 0.05% Tween 20 (PBS-T), and residual
activated groups were blocked with 1 M ethanolamine, pH 8.5.
Supernatants of baculovirus-infected insect cells expressing rsgp130
(40 µl) were diluted 5-fold with PBS-T and added to the cuvette.
Subsequently, the cuvette was incubated with IL-6 (10 µg/ml in
PBS-T), sIL-6R (10 µg/ml in PBS-T), and IL-6 plus sIL-6R (5 µg/ml
each in PBS-T) (time intervals indicated in Fig. 4
A). After
each exchange of protein solution, the cuvette was rinsed with PBS-T.
Binding events were monitored as an increase of the resonance angle
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Ba/F3-gp130 (28) cells were cultured in RPMI 1640 containing
10% FCS, plated on 96-well plates (20,000 cells/well), and stimulated
with either IL-6/sIL-6R or 5% (v/v) conditioned medium from X63Ag-653
BPV-mIL-3 myeloma cells (as a source of IL-3) in the presence of
rsgp130 (concentrations indicated in Fig. 5). After 60 h of
incubation, viable and metabolically active cells were quantified using
a colorimetric assay based on the Cell Proliferation Kit II sodium
3'-[A-(phenylaminocarbonyl)-3.2
4-tetratolium]-bris-(4-methoxy-6-nitro) benzene sulfonic acid hydrate
(XTT) assay (Boehringer Mannheim).
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COS-7 and MDCK cells were cultured in DMEM containing 10% FCS.
The cDNA of IL-6R was subcloned into the expression vector pCB6 (kindly
provided by A. Le Bivic, Laboratoire de Genetique et Physiologie de
Développement, Faculté des Sciences, Marseille,
France). Transfection of MDCK cells was performed by a
modification of the calcium phosphate precipitation procedure described
by Graham and van der Eb (29). Resistant cells growing in the presence
of 0.5 mg/ml G418 (Life Technologies) for 14 days were screened for
IL-6R expression by indirect immunofluorescence and binding of
radioiodinated IL-6. To induce IL-6R expression, the MDCK/IL-6R cells
were treated with 10 mM sodium butyrate (Sigma, St. Louis, MO) for
15 h, and COS-7, MDCK, or MDCK/IL-6R cells were incubated
at 37°C for 15 min in the presence of IL-6, rsgp130, and sIL-6R
(concentrations as indicated in Figs. 6 and 9). Preparation of nuclear
extracts and EMSAs were performed as described (30). A mutated
double-stranded oligonucleotide corresponding to the c-fos promoter
(m67SIE, 5'-GAT CCG GGA GGG ATT TAC GGG GAA ATG CTG-3'), which provides
STAT3 and STAT1 binding sites, was used as 32P-labeled
probe. Protein/DNA complexes were separated on a 4.5% polyacrylamide
gel containing 7.5% glycerol in 23 mM Tris/23 mM boric acid, pH 8.0,
and 0.5 mM EDTA at 20 V/cm for 4 h. Gels were fixed in 10% (v/v)
methanol, 10% (v/v) acetic acid, and 80% (v/v) water for 30 min;
dried; and analyzed by autoradiography.
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HepG2 cells were incubated in DMEM/F12 with IL-6, rsgp130, and
sIL-6R (concentrations as indicated in Fig. 7) for 18 h and
metabolically pulse labeled with [35S]methionine for
3 h. Induction of the newly synthesized acute-phase protein
1-antichymotrypsin was measured in cell culture supernatants by
immunoprecipitation using a rabbit anti-human
1-antichymotrypsin antiserum. Immunocomplexes were
precipitated with protein A-Sepharose, separated on 10%
SDS-polyacrylamide gels, and visualized by autoradiography.
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Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by a semidry blotting procedure (31). The membranes were incubated with the Ab mixtures as indicated in the figures and were processed for chemiluminescence detection as described in the enhanced chemiluminescence manual (Amersham International).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of sgp130 in the modulation of IL-6 responses in a quantitative manner, an sgp130 ELISA was established. Several mAbs (described in 25 directed against the ectodomain of gp130 were tested for their usefulness in a sandwich ELISA. The best results regarding sensitivity and dose response were obtained by the use of B-P4 for coating of the microtiter plate and biotinylated B-T2 for the detection of sgp130. Concentrations between 100 pg/ml and 2000 pg/ml were quantified using purified rsgp130 as a standard (not shown). Plasma levels of sgp130 determined with our ELISA (320 ± 22 ng/ml, n = 6) are in good agreement with those previously reported (390 ± 70 ng/ml; 20 . The ELISA can also be used for the detection of sgp130 complexed with IL-6/sIL-6R (see below).
IL-6 added to human serum forms a high molecular mass complex with sgp130 that depends on the presence of sIL-6R
To prove the possible functional role of sgp130 in the modulation
of IL-6 responses, 125I-labeled IL-6 was added to human
serum, and the protein complexes formed were analyzed by gel
filtration. A substantial portion of 125I-labeled IL-6
appeared in a peak covering a molecular mass range of 450–100
kDa (Fig. 1A, closed
circles, fractions 25–29). An additional smaller peak of
125I-labeled IL-6 eluted with the void volume of the
column. Both peaks were not detected when 125I-labeled IL-6
was incubated with physiologic concentration of serum albumin in PBS
and then subjected to gel filtration (Fig. 1A, closed
triangles). The high background levels seen between fractions 22 and 31
may be due to association of 125I-labeled IL-6 with
different oligomeric forms of BSA. Analysis of the serum fractions by
ELISA revealed that sgp130 comigrates with the 450–100-kDa
125I-labeled IL-6 peak (Fig. 1B, open circles).
Immunoprecipitation of proteins in fractions 25–28 containing sgp130
using the sgp130 mAb B-P4 led to the coprecipitation of
125I-labeled IL-6 (Fig. 1C, fractions 25–28,
solid bars) indicative of a physical association of sgp130 and
125I-labeled IL-6. The highest portion of radioactivity was
precipitated in the fractions corresponding to a molecular mass of
450–200 kDa (fractions 25–27). No precipitation of
125I-labeled IL-6 with the sgp130 mAb was observed in the
low molecular mass 125I-labeled IL-6 peak (Fig. 1C, fraction 36, solid bar).
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A,
closed squares). The amount of radioactivity in the 450–100-kDa peak
was reduced from 12.5% of total radioactivity in the presence of
sIL-6R to 5.3% in the absence of sIL-6R. Reduction of radioactivity
was most pronounced in the fractions corresponding to a molecular mass
of 450–200 kDa. The portion of radioactivity that was precipitated
with the sgp130 Ab was in the range of only 1% of total radioactivity
of the corresponding fraction (Fig. 1C, gray bars). However,
in the presence of sIL-6R, up to 6.6% of radioactivity was
precipitated (Fig. 1C, solid bars). In the absence of
sIL-6R, the sgp130 peak was moderately shifted to lower molecular mass
(Fig. 1B, open squares). We conclude from these experiments
that the sgp130 peak obtained after gel filtration of plasma proteins
in the presence of radiolabeled IL-6 in its high molecular mass part
(fractions 25–27) contains IL-6/sIL-6R/sgp130 ternary complexes and in
its low molecular mass part (fractions 27–30) consists predominantly
of sgp130. Characterization of sgp130 from human plasma
To characterize sgp130 from human plasma, the protein was enriched
by immunoaffinity chromatography. Clotting of human plasma was induced
by the addition of Ca2+ ions, and precipitated fibrin was
separated by centrifugation. No major loss of sgp130 was observed by
the coagulation process as determined by ELISA. The clear supernatant
was loaded onto a column of the anti-sgp130 mAb B-T12 immobilized
to Sepharose. As shown in Fig. 2A, the sgp130 concentration
was drastically reduced in the flowthrough. After a washing step,
sgp130 was eluted from the column with acidic glycine buffer. The
eluate of the column was immediately neutralized and used for further
studies. The concentrations of total protein and sgp130 in the eluate
were determined. sgp130 made up 2% of total protein, which corresponds
to a 5000-fold enrichment in a single chromatographic step.
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B, lane 1).
Immunoblot analysis using the sgp130 mAb B-P4 revealed that the band of
about 100 kDa corresponds to sgp130 (Fig. 2B, lane
2). This is the molecular mass expected for gp130 (130 kDa)
lacking the transmembrane and cytoplasmic parts (30 kDa). Since the
molecular mass calculated from the amino acid sequence of the gp130
ectodomain is 65 kDa, the remaining 35 kDa are due to glycosylation,
which also explains the low intensity of this band after silver
staining. Consequently, after deglycosylation with
peptide-N-glycosidase F, the 100-kDa band disappeared and
shifted to lower molecular mass (not shown). Expression of human sgp130 in baculovirus-infected insect cells and characterization of the recombinant protein
Since the available amount of sgp130 from human plasma was too
low, we produced human rsgp130 for further studies. For this purpose,
insect cells were infected with a recombinant baculovirus encoding the
human gp130 ectodomain (amino acids 1–606 followed by a polyhistidine
tag; for details see Materials and Methods). After 3 days,
the highest concentration of rsgp130 was observed: about 3 µg/ml as
determined by ELISA. This corresponds to about 1% of total protein
(250–300 µg/ml). Furthermore, the immunoblot revealed an apparent
molecular mass of about 65 kDa for rsgp130 (not shown), suggesting a
much lower extent of glycosylation compared with sgp130 from human
plasma. Supernatants collected 3 days postinfection were used for
affinity purification of rsgp130 by the same method as described for
sgp130 from human plasma. Analysis of the fractions by SDS-PAGE
revealed that rsgp130 eluted as a pure protein (Fig. 3, lane 2). Purification,
however, was accompanied by a marked loss of recombinant protein.
Freshly prepared rsgp130 eluted from the gel filtration column as a
monomer (not shown).
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A, a surface plasmon
resonance experiment is shown, in which binding events are measured in
real time as an increase of the resonance angle . The sgp130 mAb
B-T12 was covalently linked to the sensor surface and subsequently
incubated with insect cell supernatants containing rsgp130, resulting
in an increase of the resonance angle due to binding of sgp130 to the
Ab. Addition of IL-6 alone did not lead to any binding event, whereas
addition of sIL-6R resulted in a weak increase of the resonance angle,
possibly due to a very low affinity binding of sIL-6R to rsgp130.
Addition of a combination of IL-6 and sIL-6R led to the expected strong
binding event. Similar results were obtained by coimmunoprecipitation
of 125I-labeled IL-6 (Fig. 4B). Using the sgp130
mAb B-T12, IL-6 was not precipitated in the presence of rsgp130 or in
the presence of sIL-6R alone. Only the combination of rsgp130 and
sIL-6R led to the coprecipitation of IL-6. The amount of coprecipitated
IL-6 corresponded to the increasing amounts of sgp130 used. The binding
assay worked with freshly purified rsgp130, as well as with conditioned
insect cell medium containing rsgp130. rsgp130 efficiently inhibits IL-6/sIL-6R-induced responses on cells lacking membrane-bound IL-6R
Ba/F3 cells, which constitute a pre-B cell line often used for the
study of cytokine responses, grow in the presence of IL-3. After
transfection with gp130, these cells become IL-6/sIL-6R responsive, but
due to the lack of membrane-bound IL-6R, they do not respond to IL-6
alone. A previously established Ba/F3-gp130 cell line (28) was used to
analyze the antagonistic potential of rsgp130. Ba/F3 cells were
incubated with IL-3 or IL-6 and sIL-6R in the presence of increasing
amounts of rsgp130. The response of the Ba/F3-gp130 cells to IL-3 was
not affected by rsgp130 (Fig. 5,
circles), whereas the response to IL-6/sIL-6R was suppressed in a
dose-dependent manner (Fig. 5, squares). Proliferation of the cells
incubated with rsgp130 alone (Fig. 5, triangles) were indistinguishable
from unstimulated control cells (not shown). In the presence of 10
ng/ml IL-6 and 1 µg/ml sIL-6R, 1 µg/ml sgp130 was sufficient for
the complete inhibition of the IL-6 signal.
To test the influence of rsgp130 on downstream signaling events, STAT
activation in COS-7 cells was analyzed. Since COS-7 cells endogenously
express gp130 but no membrane-bound IL-6R, again stimulation with
IL-6/sIL-6R was required. The EMSA presented in Fig. 6 shows that incubation of COS-7 cells
with IL-6/sIL-6R (10 ng/ml and 1 µg/ml, respectively) resulted in a
strong STAT1 activation, which is typical for this cell type (Fig. 6, lane 1). Whereas the presence of control supernatant had no
significant effect on the signal intensity (Fig. 6, lane 2),
STAT activation was completely abolished by the addition of supernatant
containing rsgp130 (final concentration in the assay, 1 µg/ml; Fig. 6, lane 3). Thus, on cells lacking membrane-bound IL-6R, the
stimulation with IL-6/sIL-6R can efficiently be blocked by rsgp130.
In the presence of rsgp130, the sIL-6R acts antagonistically on cells expressing membrane-bound IL-6R by increasing the inhibitory effect of rsgp130
Next, we investigated the antagonistic activity of rsgp130 on
cells expressing membrane-bound IL-6R. These cells respond to IL-6
without the requirement of sIL-6R. HepG2 cells, a human hepatoma cell
line, respond to IL-6 with the synthesis of the acute-phase protein
1-antichymotrypsin, which, after metabolic labeling, can be
immunoprecipitated from cell supernatants and detected by
autoradiography (Fig. 7, lane
6). Here, we repeatedly observed that amounts of rsgp130 (1
µg/ml) that were sufficient to completely suppress the IL-6 signal on
Ba/F3-gp130 or COS-7 cells showed only a weak antagonistic effect on
HepG2 cells (Fig. 7, lane 9). Most surprisingly, sIL-6R,
which normally acts agonistically on HepG2 cells (Fig. 7, lane
7), in combination with rsgp130 increased the antagonistic effect
of rsgp130 (Fig. 7, lane 8). We repeated this experiment
with physiologic concentrations of IL-6, sIL-6R, and sgp130. Again, an
inhibitory effect of sIL-6R in the presence of sgp130 was observed
(Fig. 7, lanes 1–5).
Due to the lack of membrane-bound IL-6R, MDCK cells do not respond to
IL-6 (Fig. 8, lane 5). After
stable transfection with a cDNA encoding membrane-bound IL-6R, these
cells become IL-6 responsive, resulting in a strong activation of STAT3
and STAT1 upon IL-6 stimulation (Fig. 8, lane 1). This
response can be enhanced by the addition of sIL-6R (Fig. 8, lane
2). As observed on HepG2 cells, sgp130 has only a weak influence
on the IL-6 response (Fig. 8, lane 3). Again, the
combination of sIL-6R and sgp130 led to a drastic reduction of the IL-6
response (Fig. 8, lane 4), confirming the antagonistic
activity of sIL-6R in the presence of sgp130. Taken together, these
findings allow some important conclusions on the physiologic role of
the naturally occurring sIL-6R and sgp130 in human plasma, as will be
outlined in the discussion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After enrichment by immunoaffinity chromatography, sgp130 appears as a monomeric protein of 100 kDa. Whether sgp130 is generated by shedding of the membrane-bound receptor or by translation of an alternatively spliced mRNA is not clear. Phorbol ester induces shedding of the IL-6R (34). Shedding of gp130, however, could hardly be detected (35). While the sIL-6R generated by alternative splicing (36) has been identified in human plasma (26), detection of the protein encoded by the alternatively spliced gp130 mRNA (37) has not been reported so far.
To investigate its antagonistic activity, in vitro studies were performed with rsgp130 from baculovirus-infected insect cells. rsgp130 was purified to homogeneity in a single step by immunoaffinity chromatography. Compared with sgp130 from human plasma, the purified rsgp130 showed a markedly lower apparent molecular mass as determined by SDS-PAGE, as well as by gel exclusion chromatography. The difference in molecular mass is most probably due to a different degree of glycosylation. A similar discrepancy in glycosylation levels was observed for the sIL-6R from human plasma (70 kDa) (26) and recombinant sIL-6R from baculovirus-infected insect cells (45 kDa) (24). Since purified rsgp130 has a tendency to aggregate upon aging, the carbohydrate moiety of the soluble receptor from human blood may be required to stabilize the protein. With two different approaches, i.e., plasmon resonance and coimmunoprecipitation, we have shown that rsgp130 binds IL-6/sIL-6R complexes and is therefore suited for the study of its antagonistic activity.
Maximal proliferation of our stably transfected Ba/F3-gp130 cells was
achieved with 10 ng/ml (0.5 nM) IL-6 and 1 µg/ml (20 nM) sIL-6R. For
half-maximal inhibition of the proliferative response
(ID50), 100 ng/ml (1.4 nM) rsgp130 was sufficient (see Fig. 5). IL-6/sIL-6R-induced proliferation was completely blocked at a
concentration of 1 µg/ml (14 nM) rsgp130. This is a 28-fold excess
over the IL-6 concentration used in this experiment. The same
concentration of rsgp130 was sufficient to inhibit the rapid
IL-6/sIL-6R-induced activation of STAT1 in COS-7 cells (Fig. 6).
Presumably, rsgp130 is such a potent antagonist because it neutralizes
IL-6/sIL-6R complexes by forming high affinity ternary complexes. In
previous studies, IL-6 and sIL-6R variants were designed to neutralize
IL-6 via the low affinity IL-6/IL-6R interaction. A concentration of at
least 100 nM sIL-6R mutated in the predicted IL-6R/gp130 interface was
required to achieve a partial inhibition of the IL-6 response (38).
IL-6 mutated in the predicted gp130-binding sites must be applied at a
1000-fold or larger excess to efficiently inhibit IL-6 responses (39, 40). The fact that the antagonistic activity of IL-6 variants was
enhanced by additional mutations strengthening the IL-6/IL-6R
interaction is in line with the above arguments (40).
On cells expressing membrane-bound IL-6 receptor that were stimulated
with IL-6 alone, we found that rsgp130 was a much weaker antagonist. A
concentration of 1 µg/ml rsgp130 was insufficient both on HepG2 and
on MDCK/IL-6R cells to completely inhibit the IL-6 response, although a
significant reduction was observed in both cases. Addition of
sIL-6R, which in the absence of sgp130 acts as an IL-6 agonist, now
potentiates the antagonistic activity of sgp130. We provide a simple
explanation for this phenomenon. In cases in which HepG2 or MDCK/IL-6R
cells are stimulated with IL-6 alone, the cytokine first binds to the
cell surface IL-6R before it interacts either with sgp130 or with
membrane-bound gp130. Here, 1 µg/ml rsgp130 is not sufficient to
strongly inhibit the IL-6 response, because it has to compete with
membrane-bound gp130 present in a high local concentration. Moreover,
the IL-6/IL-6R complexes, due to their membrane location, have to find
membrane-bound gp130 only in a two-dimensional space. When sIL-6R is
added, IL-6/sIL-6R complexes can be trapped by sgp130 in the soluble
high affinity ternary complexes and are thereby efficiently neutralized
before they bind to the cell surface receptors. Narazaki et al. (20)
observed an only 50% reduction in IL-6 responses, even at an sgp130
concentration of 2 µg/ml. Possibly, this weak inhibition is due to
the low sIL-6R concentration (
75 ng/ml) that these authors have used
in their experiments.
What is the functional role of the naturally occurring sgp130 and
sIL-6R? Since the concentration of sgp130 in human plasma (about 300
ng/ml or 3 nM; Ref. 20 and our data) is considerably higher than that
of sIL-6R (about 50 ng/ml or 0.7 nM; Refs. 21 and 26), it is reasonable
to assume that this soluble receptor pair has evolved to inhibit
systemic IL-6 responses. Indeed, when HepG2 cells were stimulated with
IL-6 in the presence of sIL-6R and rsgp130 at physiologic
concentrations, sIL-6R enhanced the antagonistic effect of rsgp130
(Fig. 7). However, the effect was more pronounced at higher
concentrations of IL-6, sIL-6R, and sgp130. To assess the functional
role of the soluble receptor proteins more quantitatively, we performed
some simple calculations applying the law of mass action (detailed in
the legend to Fig. 9). In Fig. 9, the
outcome is depicted as a series of graphs showing the concentration of
bioactive IL-6 ([IL-6]i - [IL-6/sIL-6R/sgp130]) as a
function of the initial IL-6 concentration ([IL-6]i) in
the presence of physiologic concentrations of sgp130 and sIL-6R. If
KD1 has a value of 5000 pM (Fig. 9, circles) or
higher, the bioavailability of circulating IL-6 is not influenced by
sIL-6R and sgp130. At a KD1 of 500 pM (Fig. 9, triangles), the concentration of bioactive IL-6 is considerably reduced
to about 50% due to ternary complex formation unless the IL-6
concentration is in the range of or even exceeds the sIL-6R
concentration. A KD1 of 50 pM (squares) would
lead to a very strong inhibition of IL-6 responses, since more than
90% of IL-6 is trapped in ternary complexes at moderately elevated
IL-6 concentrations. Using recombinant sIL-6R from baculovirus-infected
insect cells, we measured a KD1 of 500 pM for
the binding of iodinated IL-6 (24). A 10-fold higher affinity was
measured for ternary complex formation on cells expressing IL-6R and
gp130 (KD2 = 50 pM (41, 42)). Applying these
dissociation constants to our mathematical model (Fig. 9, triangles)
suggests that in the presence of sIL-6R and sgp130 at physiologic
concentrations the systemic IL-6 response is modulated in such a way
that the bioavailability of IL-6 decreases due to soluble ternary
complex formation. Instead of being an agonist (19), sIL-6R of human
plasma should therefore be regarded as a protein that enables sgp130 to
efficiently trap IL-6 in a soluble ternary complex, thereby acting as a
buffer to modulate systemic IL-6 responses. Furthermore, using the
above dissociation constants, Equation 3 predicts that at the sgp130
and sIL-6R concentrations used in most of our experiments (1 µg/ml
each), the amount of bioavailable IL-6 is reduced 50-fold. This
corresponds to the observed strong antagonistic effects. It should be
taken into consideration that due to cell activation or certain
pathologic conditions, the local concentrations of sgp130 and sIL-6R
may be considerably different from the ones observed in human plasma,
thus modulating the inhibitory capacity of this pair of soluble
receptors.
Modulation of biologic responses by antagonizing proteins is a common principle in cytokine biology. For example, the bioactivity of IL-1 can be down-regulated by a naturally occurring IL-1 receptor antagonist (43). For other soluble cytokine receptors, it has been demonstrated that they indeed play a physiologic role in the down-regulation of the response to the corresponding mediator. Very recently, a circulating leptin-binding protein has been detected in mice that is up-regulated during pregnancy. This protein efficiently neutralizes leptin and was identified as a soluble form of the leptin receptor (44). A similar functional role during pregnancy has been assigned to the soluble growth hormone receptor (45). Both receptors belong to the same family of cytokine receptors as do IL-6R and gp130. Also, for other members of the cytokine receptor family, soluble forms have been described (reviewed in 46 . In each case, their physiologic roles need to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter C. Heinrich, Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany. E-mail:
3 Abbreviations used in this paper: Jak, Janus kinase; s, soluble; MDCK, Madin-Darby canine kidney; EMSA, electrophoretic mobility shift assay.
Received for publication February 9, 1998. Accepted for publication August 11, 1998.
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mutated in the predicted gp130-binding interface. J. Biol. Chem. 270:12242.This article has been cited by other articles:
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) (10 ng/ml and 1 µg/ml,
respectively) or without any cytokine (
). Proliferation of cells was
measured after 72 h using the colorimetric XTT assay.
IL-6/sIL-6R), the concentration of IL-6 bound to sIL-6R can be
calculated by solving Equation 1
, normal human serum; 
,
sIL-6R-depleted human serum). C, The fractions of the
gel filtration experiment described in (A) that
contained sgp130 (25–28) and fraction 36 as a control were subjected
to immunoprecipitation with the sgp130 mAb B-P4. Coprecipitated
radioactivity due to association of 125I-labeled IL-6 is
depicted as a percentage of total radioactivity of the corresponding
fraction (solid bars, normal human serum; gray bars, sIL-6R-depleted
human serum).
