gp130 transducing receptor is involved in the formation of high
affinity receptors for the members of the IL-6 (
)family(1) . In addition to IL-6, this cytokine
family is composed of interleukin-11 (IL-11)(2) , leukemia
inhibitory factor (LIF)(3, 4) , oncostatin M
(OSM)(5) , ciliary neurotrophic factor (CNTF)(6) , and
cardiotrophin-1 (CT-1)(7) . Many biological properties of the
IL-6-related molecules are shared, such as activation of hepatocyte
transcription(8) , activation of neural proliferation and
differentiation(9) , and regulation of hematopoiesis (10, 11) . Moreover LIF, CNTF, CT-1, and OSM display
biological effects in the early stages of embryonic development,
allowing the in vitro growth of embryonic stem cells in an
undifferentiated state (12, 13, 14) . IL-6
and IL-11 are also important modulators of the immune response by
regulating immunoglobulin secretion(15, 16) . Gene
inactivation experiments in the mouse have confirmed the implication of
these cytokines in vivo by showing that LIF was essential for
embryo implantation (17) and that CNTF/CNTF receptor
interaction was critical for motoneuron
development(18, 19) . In addition, mice lacking IL-6
displayed some deficiencies in bone remodeling, inflammatory response,
and immunoglobulin secretion(20, 21) .
The shared
use of gp130 receptor in their multimeric receptor explained in part
the redundancy of their biological properties. The recruitment of the
gp130 transduction pathway by IL-6 also implicates an IL-6-binding
protein known as IL-6 receptor(22) , which can be produced
either as a transmembrane protein or as a truncated soluble product (23) . Both forms of IL-6 receptor can associate IL-6 to induce
gp130 dimerization and downstream signaling
events(1, 24) . More recently, an IL-11 binding chain
was isolated and a similar activation process for gp130 is
suspected(25, 26) . Regarding to the receptors for
LIF, OSM, and CT-1, gp130 will associate to a second transducing
subunit referred to as gp190/LIF
receptor(14, 27, 28) . For CNTF a third
additional component or
CNTF receptor was identified and is
required to reinforce the interaction of CNTF with the transducing
receptor complex composed of gp130 and gp190/LIF
receptor(29, 30) .
Dimerization of the transducing
subunits initiates intracellular signaling by activating members of a
family of cytoplasmic receptor-associated tyrosine kinases, referred to
as Jak (Janus kinase) (for review, see (31) ). Both gp130 and
gp190/LIF receptor can associate Jak1, Jak2, and Tyk2(32) . The
information is next relayed by a family of transcription factors known
as STATs (signal transducers and activators of transcription), which
are activated in the cytoplasm before translocation to the
nucleus(31) . IL-6-related cytokines will preferentially
activate STAT1 and STAT3(33, 34, 35) .
Induction of gp130 dimerization or its heterodimerization with LIF
receptor was well analyzed in the case of IL-6 and CNTF/LIF receptors,
respectively(24, 30) . Studies regarding to the
downstream activation processes mediated through the gp130/gp130 or
gp130/gp190 pathways were also performed by constructing chimeric
receptors composed in their external parts of G-CSF receptor, epidermal
growth factor, or TRKc and by the intracellular domains of gp130 or
gp190(33, 36) . In the present study we have analyzed
the agonistic properties of the B-S12 antibody directed against gp130,
and we show that cross-linking of gp130 transducing subunit was
sufficient to elicit IL-6 type responses.
MATERIALS AND METHODS
Cells and Reagents
The SK-N-MC neuroblastoma
cell line (ATCC, Rockville, MD), and the HepG2 cell line (ATCC) were
routinely grown in RPMI culture medium supplemented with 10% fetal calf
serum. For the multifactor-dependent TF1 cell line (37) and the
XG1 myeloma cell line(38) , the culture medium was
supplemented, respectively, with 1 ng/ml GM-CSF and 1 ng/ml IL-6.
Purified human recombinant LIF (10
units/mg) produced in
Chinese hamster ovary cell line, Escherichia coli recombinant
GM-CSF (10
units/mg), and human soluble IL-6 receptor were
kindly donated by Drs. K. Turner and M. Stahl (Genetics Institute,
Boston, MA). IL-6 (10
units/mg) and OSM (10
units/mg) were purchased from Peprotech (Canton, MA). Soluble
human gp130 was bought from R& Systems (Minneapolis, MN). Purified
B-S12 monoclonal antibody (IgG1) obtained as described previously (
)was kindly provided by Dr. John Wijdenes from Diaclone
(Besançon, France). B-S12 and control IgG1 Fab
fragments were generated by using immobilized papain from Pierce,
following the manufacturer's instructions. Fab fragments were
then separated from Fc fragments on a protein A column.
Bioassays
Serial dilutions of antibodies or
cytokines were added to 15
10
TF1 or XG1 cells in
triplicate in the assay(39) . The cells were then incubated for
72 h before being pulsed with 0.5 µCi of
[
H]Tdr for the last 4 h of the culture. HepG2
hepatoma cell line was plated in triplicate in 96-well plates at a
concentration of 50
10
cells/well in 200 µl of
RPMI medium containing 10% fetal calf serum, 10
M dexamethasone, and serial dilutions of IL-6 or
antibodies (40) . After 48 h, the supernatants were harvested
and the haptoglobin content determined by ELISA as described
previously(41) .
Hematopoietic progenitor cells
were purified from bone marrow from informed donors by using the
CD34-positive cell selection system from Applied Immune Science (Menlo
Park, CA) and following the manufacturer's instructions. Purity
of the cells was controlled on a FACScan by using an anti-CD34
phycoerythrin-labeled antibody (Becton Dickinson, Mountain View, CA)
and was routinely >97% CD34-positive cells. One hundred
CD34-positive purified cells were seeded in 24-well plates in
triplicate in Iscove's medium containing 20% fetal calf serum,
0.8% methylcellulose, 5
10
M
-mercaptoethanol, and the indicated combination of cytokines
or antibodies. After a 14-day culture period, the colony number was
scored.
Soluble gp130 Symmetric ELISA
Detection of the
dimeric form of soluble gp130 was achieved by coating the ELISA plates
with the B-T12 monoclonal antibody
at a concentration of 10
µg/ml in 100 mM carbonate buffer, pH 8.6. After washing
and a saturation step, 2 nM soluble gp130 supplemented with 2
nM IL-6 plus 2 nM soluble IL-6 receptor, or 6 nM B-S12 anti-gp130 antibody were added to the wells for an overnight
incubation at 4 °C. Biotinylated B-T12 mAb was used as tracer
antibody at a final concentration of 1 µg/ml. After a 6-h
incubation at 37 °C, streptavidin peroxidase (Dako, Glostrup,
Denmark) was added at a 1/20,000 dilution for an additional 1-h
incubation step. ABTS was used as substrate and the reading performed
at 405 nM.
Tyrosine Phosphorylation Analysis
Cells were
stimulated with OSM or antibodies before being lysed in 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30
mM sodium pyrophosphate, 50 mM sodium fluoride, 1
mM sodium orthovanadate, 1% Triton X-100 and proteinase
inhibitors. After pelleting insoluble material and protein
standardization, the supernatants were immunoprecipitated in the
presence of B-T2 antibody raised against gp130
, anti-Jak1
antibody (Upstate Biotechnology, Inc. (UBI), Lake Placid, NY), or
anti-Jak2 antibody (UBI). The complexes were then isolated with beads
coupled to protein A, submitted to SDS-polyacrylamide gel
electrophoresis, and transferred onto an Immobilon membrane (Millipore,
Bedford, MA). The membranes were incubated in the presence of 4G10
anti-phosphotyrosine mAb, followed by an incubation with a goat
anti-mouse immunoglobulin polyclonal antibody labeled with peroxidase
(Tago, Camarillo, CA). The reaction was visualized on a x-ray film with
ECL reagent (Amersham, Les Ulis, France) following the
manufacturer's instructions.
RESULTS AND DISCUSSION
B-S12 Antibody Triggered the Proliferative Response of
Hematopoietic Cell Lines and Increased Protein Transcription in HepG2
Hepatoma Cell Line
We recently characterized a set of new
monoclonal antibodies raised against the gp130 transducing
protein.
During the time course of the antibody screening,
we did identify the B-S12 mAb, which, in contrast to most other
identified antibodies, displayed agonistic properties in several
bioassays. The stimulatory properties of the B-S12 antibody were first
analyzed by using two human hematopoietic cell lines, XG1 and TF1,
respectively, known to be sensitive to the cytokines activating the
gp130 transduction pathway and both gp130 and gp190/LIF receptor
systems(38, 39) . B-S12 mAb was found to strongly
trigger the proliferation of TF1 erythroleukemia cell line and XG1
multiple myeloma cell line, whereas a control IgG1 did not support the
proliferation of these cells (Fig. 1, A and B). This result indicates that the B-S12 antibody can bypass
the cytokine requirement to activate the gp130 pathway, and that
recruitment of an
binding component was not necessary to generate
a functional response. A series of reports had shown that the integrity
of two proximal motifs in the intracellular part of gp130, and known as
box 1 and 2, were sufficient to generate a proliferative
response(33, 36, 42) . On the other hand, it
has been demonstrated that regulation of gene transcription in hepatoma
or neuroblastoma cell lines was under the dependence of an additional
gp130 motif called box 3, and implicating the recruitment of STAT3
shuttle transducing protein(33, 42) . In line with
these observations, we have tried to further characterize the
functional properties of the B-S12 antibody in order to know, if in
addition to its ability to stimulate cell proliferation, the antibody
could also increase gene transcription. The experiments were performed
in HepG2 hepatoma cell line where the induction of haptoglobin
secretion was monitored. Fig. 1C shows that B-S12
antibody was able to up-regulate the induction of haptoglobin
secretion, whereas an IgG1 control antibody was without effect.
Nevertheless, B-S12 was about a thousandfold less potent than IL-6 used
as positive control. This result shows that gp130 activation pathway by
the B-S12 antibody led not only to a proliferative response, as
observed in the hematopoietic cell lines, but also to an increase gene
transcription in hepatic cells.
Figure 1:
B-S12 anti-gp130 antibody triggered the
proliferation of the TF1 and XG1 hematopoietic cell lines and induced
haptoglobin secretion in the HepG2 hepatoma cell line. Fifteen thousand
TF1 or XG1 cells were cultured in triplicate in the presence of IL-6
(concentration expressed in ng/ml) (hatched bars), B-S12 (black bars), or IgG1 control (white bars) antibodies
(concentration expressed in µg/ml). After a 72-h culture period, a
[
H]Tdr pulse was performed and the incorporated
radioactivity determined by using a
counter. In C,
50,000 HepG2 cells were cultured in triplicate in the presence of IL-6,
B-S12, or IgG1 control antibodies. After a 48-h culture period, the
culture supernatants were harvested and their haptoglobin content
determined by ELISA.
B-S12 Antibody Induced Both Proliferation and
Differentiation of CD34 Hematopoietic Progenitors
Several
IL-6-related cytokines were reported to display synergistic activities
on both proliferation and differentiation processes of hematopoietic
cells. To examine the potential role of the B-S12 anti-gp130 antibody
on the expansion of hematopoietic cell progenitors, its effect was
tested in combination with GM-CSF on purified human CD34-positive cells
by using a methylcellulose clonal assay. B-S12 antibody alone, like the
IgG1 isotype control did not induce colony formation (Table 1).
Addition of 1 ng/ml GM-CSF to the culture stimulated the generation of
11 ± 1.4 colonies after a 2-week culture period. To assess the
synergistic effect of IL-6 type cytokines in the culture system, LIF
was preferred to IL-6 because this latest cytokine only displayed weak
effects in human CD34 cell culture and the adjunction of soluble IL-6
receptor was usually required to observe a response(43) .
Addition of LIF to GM-CSF doubled the score of colonies in the cultures
in agreement with the published studies(10, 44) .
Similar results were obtained by introducing B-S12 antibody in
combination with GM-CSF in the CD34-positive cell cultures. Moreover,
the average size of the observed colonies generated in the presence of
B-S12 and GM-CSF was twice larger than those detected in the presence
of GM-CSF alone. (
)These experiments show that B-S12
antibody, like most of the cytokines belonging to the IL-6 family,
synergistically promoted potent expansion of human hematopoietic
progenitor cells in vitro.
B-S12 Antibody-induced Dimerization of the gp130
Transducing Protein
Fab fragments were generated from the B-S12
antibody and tested in the proliferative TF1 assay to determine whether
the observed agonistic properties of the antibody were dependent on the
presence of the two functional sites expressed by the immunoglobulin. Fig. 2A shows that B-S12-derived Fab fragments failed
to trigger the proliferation of the TF1 cell line. Cross-linking of the
Fab fragments by using a second antibody recognizing the mouse
immunoglobulins did not allow the restoration of a functional signal,
indicating that a very precise configuration of the antibody was likely
required to bring the proliferative information. In addition, coating
of Fab fragments to a plastic dish did not restore the signal (data not
shown). The lost of biological properties of the Fab fragments
indicates that B-S12 might induce a dimerization of gp130 transducing
protein to generate a biological response. To assess this hypothesis,
induction of gp130 dimerization was analyzed in a symmetric ELISA where
the same B-T12 anti-gp130 mAb was used as coating and tracer antibody.
In these conditions the detection of an increased optical density value
will be a reflection of the dimerization of the soluble form of gp130.
As shown in Fig. 2B, addition of 2 nM soluble
IL-6 receptor and IL-6 to 2 nM soluble gp130 conducted to an
increased signal in the symmetric gp130 ELISA. This result sustains the
notion that IL-6, IL-6 receptor and gp130 can reassociate together in
solution to generate a trimeric, or an hexameric complex as reported
before(45, 46) . In a similar manner introduction of
the B-S12 antibody, instead of IL-6 and its soluble receptor, led to a
dimerization of the soluble form of gp130 transducing protein.
Interestingly B-S12 antibody failed to inhibit IL-6-driven
proliferation of the TF1 cell line and did not interfere with the
binding of radiolabeled IL-6 to its high affinity receptor (data not
shown). These results indicate that the antibody and the cytokine
recruited gp130 through different functional sites.
Figure 2:
B-S12 anti-gp130 antibody-induced
dimerization of the receptor transducing component. In A,
15,000 TF1 cells were cultured in the presence 20 µg/ml B-S12 mAb (black bars), IgG1 control mAb (hatched bars), or 20
µg/ml Fab-derived fragments from B-S12 antibody and IgG1 isotype
control, or a combination Fab-derived fragments (20 µg/ml) plus 20
µg/ml goat anti-mouse polyclonal antibody (Gam). After a
72-h culture period, a [
H]Tdr pulse was performed
and the incorporated radioactivity determined by using a
counter.
In B, soluble gp130-induced dimerization was measured in a
symmetric ELISA using the same B-T12 anti-gp130 mAb as coating and
tracer antibody. After coating and plate saturation, 2 nM soluble gp130, 2 nM IL-6, 2 nM soluble IL-6
receptor, or 6 nM B-S12 mAb were added to the wells as
indicated for an overnight incubation step. Biotinylated B-T12 mAb was
used as second antibody and peroxidase-labeled avidin used for the
detection. Reading was carried out at 405
nm.
gp130 and Jak Family Members Were Tyrosine-phosphorylated
in Response to B-S12 Stimulation
Receptor activation by the IL-6
family of cytokines results in the tyrosine phosphorylation of the
transducing receptor subunits and downstream regulatory
proteins(32, 33) . Functional activation of gp130
transducer by B-S12 antibody was assessed by analyzing tyrosine
phosphorylation of the transducing protein. The experiments were
performed by using the SK-N-MC neuroblastoma cell line, since
IL-6-related cytokines displayed many effects in neuronal tissue and,
in addition, gp130 tyrosine phosphorylation was readily detectable in
this cell line. Fig. 3A shows that the recruitment of
gp130, and its tyrosine phosphorylation by the B-S12 antibody was
dose-dependent. After a 5-min interaction with the agonistic antibody
tyrosine phosphorylation of gp130 could already be detected (Fig. 3B). The observed signal peaked after a 20-min
contact, but remained weaker than the one observed by incubating the
SK-N-MC cell line in the presence of OSM. A control antibody added in
the culture for 20 min did not activate gp130 tyrosine phosphorylation
underlining the specificity of the detected signal. Jak1, Jak2, and
Tyk2 kinases have been shown to be implicated in the activation of
gp130 and gp190/LIF receptor(32, 33, 34) .
This led us to investigate the involvement of Jak-Tyk kinases in
signaling initiated by the B-S12 monoclonal antibody. Tyrosine
phosphorylation of Jak1 and Jak2 was observed after stimulation by the
agonistic B-S12 antibody (Fig. 3C). In contrast we did
not detect Tyk2 activation after treating the cells with B-S12
antibody, or OSM, even if Tyk2 protein could be detected by
immunobloting in the cell lysate (data not shown). A similar pattern of
activation was described previously for another neuronal derived cell
line, and some tissue variations in the Jak-Tyk recruitment by the IL-6
type of cytokines were reported(32) . These results show that
B-S12 antibody was able to activate gp130 pathway in neuronal cells as
well, by recruiting Jak1 and Jak2 kinases and inducing tyrosine
phosphorylation of the transducing protein.
Figure 3:
B-S12
mAb-induced tyrosine phosphorylation of Jak1, Jak2, and gp130 receptor
component. In panel A, SK-N-MC neuroblastoma cells were
incubated for 20 min in the presence of increasing concentrations of
B-S12 mAb or with 50 ng/ml OSM. After cell lysis, gp130 was
immunoprecipitated, run on SDS-polyacrylamide gel electrophoresis,
blotted on a membrane, and its tyrosine phosphorylation content
analyzed by using an anti-Tyr(P) mAb. In B, SK-N-MC cells were
stimulated for 10 min with 50 ng/ml OSM or with 50 µg/ml antibodies
for the indicated periods of time. After cell lysis, the samples were
treated as described in A. In C, resting or B-S12
activated SK-N-MC cells (50 µg/ml B-S12 mAb, for 20 min) were
lysed, and Jak1 (lanes 1) and Jak2 (lanes 2)
signaling proteins immunoprecipitated. Their tyrosine phosphorylation
level was analyzed as described in A.
CONCLUSION
Receptor-induced dimerization is a common mechanism required
for signaling in both cytokine receptors and receptor tyrosine kinases
(for review, see (47) ). Activation processes of cytokine
receptors leading to the formation of functional dimeric complexes were
analyzed by using different approaches, such as introduction of
disulfide bonds between transducing subunits or by generating chimeric
receptors composed of the intracellular parts of the
and
IL-2 receptor chains and the extracellular region of c-Kit, GM-CSF
receptor, or G-CSF
receptor(48, 49, 50, 51) .
Biological properties of the IL-6 cytokine family mediated through an
homodimerization of gp130 transducing protein or its heterodimerization
with LIF receptor were analyzed by using chimeric receptors substituted
in their external parts by G-CSF receptor, epidermal growth factor
receptor, or TRKc corresponding regions(24, 30) . The
obtained results have reinforced the notion that homo- or
heterodimerization of gp130 and gp190 lead to an activation of gene
transcription mediated through the Stat3/box 3 distal motif
interaction, but little is known about the regulation of the
proliferative signal provided by dimerization of the signaling receptor
proteins(42, 33) . In the present study we have
reported that dimerization of gp130 was sufficient to elicit IL-6 type
responses. These results sustain the idea that the major contribution
of the
IL-6 receptor subunit is to increase IL-6/gp130
interaction and gp130 dimerization at the cell surface. In addition,
neutralizing antibodies directed against IL-6 or IL-6 receptor were not
able to inhibit the responses mediated through B-S12 activation,
indicating a direct effect of the agonistic antibody on gp130
recruitment (results not shown). We also show that B-S12-induced
dimerization of gp130 triggered both cell proliferation and gene
transcription, and that functional responses could be observed in
hematopoietic, hepatic, and neural derived cell lines. Tyrosine
phosphorylation analysis of gp190/LIF receptor performed after B-S12
cell stimulation did not reveal a significant increased activation of
this receptor subunit, indicating that the observed responses were only
under the dependence of gp130 pathway (data not shown). Interestingly a
study performed by using bispecific chimeric antibodies able to
recognize two of three receptor subunits involved in the formation of
high affinity IL-2 receptor has shown that cross-linking of transducing
receptor subunits in these conditions could lead to a functional
response(52) . Transposition of this approach to the
gp130/gp190 system, by generating bispecific antibodies able to
recognize both transducing components, could be of interest in some
situations such as expansion of hematopoietic progenitors, where
activation of gp130 by B-S12 antibody already led to a efficient
response.