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Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3495-3504
REVIEW ARTICLE
Interleukin-6 and Soluble Interleukin-6 Receptor: Direct
Stimulation of gp130 and Hematopoiesis
By
Malte Peters,
Albrecht M. Müller, and
Stefan Rose-John
From I. Medizinische Klinik, Abteilung Pathophysiologie, Johannes
Gutenberg Universität Mainz, Mainz, Germany; and Max Planck
Institut für Immunbiologie, Freiburg, Germany.
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INTRODUCTION |
THE INTERLEUKIN-6 (IL-6) family of
cytokines acts via receptor complexes that contain at least one subunit
of the signal transducing protein gp130.1 The family
comprises IL-6, IL-11, ciliary neurotrophic factor (CNTF),
cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF), and
oncostatin M (OSM).1 IL-6, IL-11, and CNTF first
bind to specific receptors, and these complexes associate with a
homodimer of gp130 in the case of IL-6 and IL-11 or, alternatively,
with a heterodimer of gp130 and the related protein LIF receptor
(LIF-R) in the case of CNTF. OSM and LIF first bind directly to gp130
and LIF-R, respectively, and form heterodimers with LIF-R and gp130.
Recently, a gp130-related protein was described that can heterodimerize
with gp130 and that acts as an alternative OSM receptor.2
CT-1 binds directly to the LIF-R and induces gp130/LIF-R heterodimer
formation.3 Recently, the presence of a specific
glycosylphosphatidylinositol (GPI)-anchored CT-1 receptor
on neuronal cells was implicated.4
Cytokines of the IL-6 family are involved in various steps of
hematopoiesis and have been used for the ex vivo expansion of hematopoietic cells.5-7 Whereas recent reviews have
concentrated on soluble cytokine receptors in general,8 on
the mechanisms of generation of soluble receptors,9,10 and
on various aspects of the IL-6 cytokine family,11 this
review will mainly focus on the consequences of direct stimulation of
gp130 on hematopoietic cells, through the complex of IL-6 and a soluble
form of the IL-6R, in vivo and in vitro.
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GENERATION AND OCCURRENCE OF SOLUBLE RECEPTORS |
Many if not all transmembrane proteins occur also in a soluble form
that consists of the major part of the extracellular domain. This
phenomenon has been observed for type I and type II transmembrane proteins.9,10 Two independent mechanisms lead to the
generation of such soluble proteins.
Firstly, transmembrane proteins can be cleaved by a transmembrane
metalloproteinase that most likely is a protease distinct from
matrix-type metalloproteinases to yield the soluble extracellular domain of the proteins. This mechanism has been studied in detail for
the human IL-6R.12-16 Cleavage is controlled by protein
kinase C and occurs at a distinct site that is not strictly sequence specific.15 The generation of the soluble IL-6R can be
prevented by hydroxamic acid compounds16 that previously
have been shown to inhibit the processing of the membrane form of tumor
necrosis factor (TNF).17,18 A TNF processing
metalloproteinase has recently been cloned19,20 and shown
to belong to the family of disintegrin domains containing
metalloproteinases (ADAMs).21 It is unclear whether
different family members of the ADAMs are highly substrate specific or
whether one protease is able to cleave more than one protein.
Alternatively, the generation of soluble counterparts of transmembrane
proteins has been shown to occur via translation from alternatively
spliced mRNAs.9 In particular, a soluble form of the IL-6R
can be synthesized by various cells from a spliced mRNA yielding a
protein that differs at its COOH-terminus by 14 amino acid
residues,22,23 indicating that for one transmembrane protein both mechanisms of generation of a soluble protein may exist.
Soluble IL-6R protein has been detected in the blood of normal
individuals at concentrations of 50 to 80 ng/mL.24
Increased concentrations have been found during infections and
malignant disorders.24-26 Interestingly, bacterial proteins
massively induce the shedding of several membrane proteins via the
activation of metalloproteinases.27,28
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IL-6-TYPE CYTOKINES TRANSSIGNALING VIA SOLUBLE RECEPTORS |
Soluble receptor proteins bind their ligands with similar affinities as
the cognate transmembrane receptors.9 Most soluble receptors for cytokines and growth factors compete with their membrane
bound counterparts for the binding of the ligand and therefore are
antagonists.9 In contrast, the soluble receptors of the
IL-6 cytokine family, when complexed with their ligands, exhibit
agonistic biological activities. These complexes can directly recruit
and activate homodimers of gp130 (in the case of IL-6 and IL-11) and
heterodimers of gp130 and LIF-R (in the case of CNTF).29-32
Cells that do not express specific receptors for IL-6, IL-11, or CNTF
are not able to respond to these cytokines. The presence of soluble
receptors leads to responsiveness of these cells
(Fig 1). This process has been named
transsignaling.9 Of note, soluble forms of gp130 and LIF-R
exist in vivo and have been demonstrated to possess antagonistic
biological activity.33,34
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| Fig 1.
Transsignaling of soluble receptors of IL-6 family. An
IL-6R-expressing cell (left) releases a soluble receptor by shedding
or alternative splicing. This soluble receptor binds IL-6 and induces
homodimerization of gp130 on a target cell (right) that expresses gp130
but no IL-6R. In this model, the target cell in the absence of soluble
IL-6R is not responsive to IL-6. gp130, red; IL-6R black; IL-6, blue.
This transsignaling model holds also true for the IL-6 family cytokines
IL-11 and CNTF.
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BIOLOGICAL PROPERTIES OF SOLUBLE IL-6R |
Taga et al,35 using coimmunoprecipitation techniques, were
the first to demonstrate that a soluble form of the IL-6R in the
presence of IL-6 associates with gp130. Consequently, release of a
soluble IL-6R by human peripheral blood mononuclear cells (PBMC) was
demonstrated and it was shown that soluble IL-6R together with IL-6
partly suppressed the Con A-induced proliferative response of
PBMC.24 The in vivo biological activity of soluble IL-6R has been demonstrated using a murine tumor rejection
model.36 In this assay, highly tumorigenic murine melanoma
cells (B78) were used. B78 cells injected into syngeneic mice caused
the formation of tumors and metastases, whereas cells transfected with
a cDNA coding for IL-6 protected the animals. Surprisingly,
transfection of B78 cells with a cDNA coding for the murine soluble
IL-6R led to an even more effective protection of the animals,
indicating that the soluble IL-6R interacted with the endogenous murine
IL-6.36
To study the in vivo function of the soluble IL-6R, we have constructed
transgenic mice that express a human IL-6R cDNA into which a
translational stop codon had been introduced upstream of the
transmembrane region. Expression of this soluble receptor was under the
transcriptional control of the liver specific phosphoenolpyruvate carboxykinase (PEPCK) promoter.37 Human IL-6 stimulates
human and murine cells, whereas murine IL-6 only stimulates murine
cells.38 Because of this species specificity of IL-6, the
transgenic human soluble IL-6R did not bind the endogenous murine IL-6,
and consequently the transgenic animals showed no transgene specific
phenotype. Upon injection of human IL-6 into transgenic mice and
nontransgenic control mice, the IL-6-specific induction of hepatic
genes was analyzed.37 Measuring the IL-6-induced hepatic
haptoglobin mRNA expression, it turned out that, in the presence of the
soluble IL-6R, hepatocytes were significantly sensitized towards human IL-6 (Fig 2A, left panel). A similar extent
of acute phase response was obtained with a 100-fold lower
concentration of human IL-6 in mice expressing the soluble
IL-6R.37 Time course studies showed that the expression of
hepatic haptoglobin mRNA was markedly prolonged in the presence of the
soluble IL-6R (Fig 2A, right panel), which was most likely caused by a
prolongation of the plasma half-life of IL-6 in mice expressing the
soluble IL-6R (Fig 2B).
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| Fig 2.
Sensitization of target cells and increase of the plasma
half- life of IL-6 in mice transgenic for the human soluble IL-6R. (A)
Dose-response of the IL-6-induced hepatic acute phase protein
expression. Haptoglobin expression in the liver of control mice
( /) and heterozygous (/+) and homozygous (+/+) mice was
analyzed. Mice were injected intraperitoneally with various dosages of
human IL-6 as indicated. Mice were killed 4 hours after injection. (B)
Time course of the hepatic acute phase protein expression. Mice were
injected intraperitoneally with 8 µg of human IL-6 and killed after
the time periods indicated. Total RNA was prepared from the liver and
subjected to Northern blot analysis. Filters were hybridized with a
32P-labeled 0.9-kb HinfI restriction fragment of
human haptoglobin cDNA. (C) Serum levels of human IL-6 in control mice
or heterozygous (/+) or homozygous (+/+) transgenic mice
expressing the human soluble IL-6R were measured 4 hours after
injection with a human IL-6-specific ELISA.
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Recently, it was shown that human IL-6-dependent myeloma cells were
unable to grow in the presence of low IL-6 concentrations when the
medium was depleted of soluble IL-6R produced by these cells. This
result seems to indicate that the membrane-bound IL-6R was not
sufficient to mediate the growth-stimulating signal of IL-6 and might
point to a more general importance of the IL-6/soluble IL-6R complex
for gp130-mediated signaling.39
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DEFINITION OF NEW TARGET CELLS OF THE IL-6/SOLUBLE IL-6R COMPLEX |
The function of the soluble IL-6R can be deduced from a situation
depicted in Fig 3A. Cells that express
gp130 and the specific IL-6R can be stimulated with human IL-6 or
alternatively by IL-6 complexed to the soluble human IL-6R. Figure 3B
schematically shows a hypothetical target cell that would only be
responsive to the complex of IL-6 and the soluble IL-6R. To address the
question of whether such target cells exist in vivo, we compared the
phenotype of mice transgenic for IL-6 alone with the one of mice
transgenic for both human IL-6 and human soluble
IL-6R.40-42
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| Fig 3.
Target cells of IL-6 and the IL-6/soluble IL-6R complex.
(A) Cells that express gp130 (gray) and membrane bound IL-6R (black)
are responsive to IL-6 and are sensitized by the presence of the
soluble IL-6R. (B) Cells that only express gp130 but no membrane bound
IL-6R are unresponsive to IL-6 but can be stimulated by the complex of
IL-6 and soluble IL-6R.
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It turned out that one of the main differences between single- and
double-transgenic mice was a massive extramedullary hematopoiesis in
liver and spleen of the adult animals.40 As shown in
Fig 4, the livers and spleens of
IL-6/sIL-6R double-transgenic mice contained a highly elevated number
of Lin /Sca1+/c-kit+ cells,
which have been demonstrated to contain a very high percentage of
hematopoietic stem cells43,44 (Peters et al, manuscript in
preparation). Moreover, spleen and liver contained highly
elevated numbers of granulocytes, macrophages, Sca1+
hematopoietic progenitor cells, and B cells. The presence of hematopoietic progenitor cells in liver and spleen resulted in a
time-dependent massive increase of peripheral blood cell numbers in
IL-6/sIL-6R double-transgenic mice (Fig 5).
These effects were completely absent in single transgenic mice and in
nontransgenic control animals.40
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| Fig 4.
Frequency of cells with a hematopoietic stem cell
phenotype in control, IL-6, or IL-6/soluble IL-6R transgenic mice. The
presence of Lin/Sca1+/c-kit+
cells present in bone marrow, spleen, and liver of ( ) control mice,
( ) IL-6 transgenic mice, and ( ) IL-6/soluble IL-6R
double-transgenic mice was analyzed by FACS.
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| Fig 5.
Peripheral blood cells of control, IL-6, or IL-6/soluble
IL-6R transgenic mice. White blood cell (A), neutrophil (B), platelets
(C), red blood cells (D), and hemoglobin (E) values were analyzed from
six transgenic mice and nontransgenic littermates per group at the ages
indicated. Mean values with standard deviations are presented.
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In murine embryogenesis, the first hematopoietic cells are generated in
the yolk sac at day 7.5 (E 7.5) of gestation. The first intraembryonic
tissue with multilineage and long-term repopulating activity is the
splanchnopleuric mesoderm/aorta, genital ridge, mesonephros (AGM)
region between E 8.5 and 11 of gestation.45 Later,
hematopoietic progenitor and stem cells can be found in the fetal liver
and around birth in the spleen and bone marrow. The presence of
multipotent hematopoietic progenitor cells and cells with a stem cell
phenotype in IL-6/sIL-6R double-transgenic adult animals might point to
the fact that the adult liver retains a hematopoietic microenvironment
and hematopoietic stem cells from the fetal developmental stage
independent from other hematopoietic tissues such as spleen and bone
marrow. Bone marrow hematopoiesis in double-transgenic mice was
unaffected by the presence of IL-6 and IL-6R.40 This
suggests that both tissues are affected in a different manner by IL-6
and soluble IL-6R.
Several studies have described functional changes in the hematopoietic
system during development (reviewed in Bonifer et al46). Lansdorp et al47 and Vormoor et al48 have
recently reported that human fetal liver cells have a higher
proliferation and self-renewal rate as compared with cord blood cells
and that cord blood cells have a higher proliferative and self-renewal
capacity as compared with adult bone marrow cells. There is now growing
evidence that the functional difference of stem cells isolated from
different developmental stages is reflected by a developmental-specific cytokine/growth factor receptor expression pattern on the surface of
hematopoietic cells. Several lines of evidence support this notion:
whereas cells isolated from bone marrow are optimally expanded with a
combination of Flt-3 ligand, stem cell factor (SCF), and
IL-3,49,50 cord blood cells require Flt-3 ligand, IL-6, and
the soluble IL-6R for efficient expansion.50 A further example of a developmental-specific growth factor activity of an IL-6
family member is OSM. Mukouyama et al51 report that treatment with OSM leads to the expansion of AGM-derived multipotent hematopoietic progenitors, but no stimulation of colony formation was
detected with bone marrow-derived cells.
Taken together, the data from the single- and double-transgenic mice
indicate that expansion of early extramedullary hematopoietic progenitor cells could only be stimulated by IL-6 in the presence of
the soluble IL-6R. This finding is strongly supported by recent data
from Tajima et al,52 who found that CD34+ cells
can be subdivided into IL-6R-expressing and nonexpressing cells. Both
cell populations express gp130. It was demonstrated that
IL-6R-expressing cells can be stimulated to form
granulocyte-macrophage colonies, whereas IL-6R negative cells upon
stimulation with IL-6 and soluble IL-6R form various types of colonies
including erythroid bursts, granulocyte-macrophage colonies,
megakaryocytes, and mixed colonies.52 These findings are
further supported by data from McKinstry et al,53 who
demonstrate that the number of IL-6R on hematopoietic progenitor cells
increases significantly with maturation of these cells. The
CD34+ subpopulation that does not express IL-6R includes
most of the erythroid, megakaryocytic, and primitive human
hematopoietic progenitors. Such cells are target cells for IL-6/soluble
IL-6R but not for IL-6 alone.
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STIMULATION OF HEMATOPOIETIC PROGENITOR CELLS WITH THE IL-6/SOLUBLE
IL-6R COMPLEX IN VITRO |
Experimental strategies to expand hematopoietic cells often used
cytokines of the IL-6 family.5-7 Sui et al54
were the first to demonstrate that stimulation of gp130 by IL-6 and
soluble IL-6R resulted in superior ex vivo expansion of primitive human hematopoietic progenitor cells when compared with IL-6 alone. They
showed that human cord blood CD34+ cells were stimulated by
stem cell factor combined with IL-6 and soluble IL-6R
(Fig 6). These studies were extended by the same group52,55 and have meanwhile been confirmed by
several laboratories.50,56 The most interesting aspect of
these studies is that direct stimulation of gp130 seems not to be a
proliferative stimulus by itself. However, IL-6 in combination with
stem cell factor and IL-3 has been reported to attenuate
differentiation of hematopoietic cells.50,54,56
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| Fig 6.
Expansion of CD34+ human cord blood-derived
progenitor cells in the presence of IL-6 or IL-6/soluble IL-6R. Two
thousand CD34+ cord blood cells containing 684 progenitors were cultured in serum containing suspension culture medium
supplemented with the factors indicated. The expansion of hematopoietic
progenitor cells was tested after 2 weeks of liquid culture in
methylcellulose assays. Adapted and reprinted with permission from Sui
et al.54 Copyright 1995 National Academy of
Sciences, U.S.A.
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A DESIGNER CYTOKINE THAT DIRECTLY STIMULATES gp130 |
The effective concentration of IL-6 (50 ng/mL) and sIL-6R (>1,000
ng/mL)54 needed for the stimulation of human hematopoietic progenitor cells is high, considering a kd of approximately 1 nmol/L.57,58 Recently, it has been reported that the
ligand/receptor interaction is mainly determined by the
off-rate,59 suggesting that the average half-life of the
IL-6/sIL-6R complex might be shorter than the time needed to assemble
the IL-6/sIL-6R/gp130 complex. Accordingly, to lower the effective dose
needed for IL-6 bioactivity, IL-6 muteins with a lower off-rate have
been generated that render the complexes with IL-6R more
stable.60 As a novel approach, we postulated that the
formation of the IL-6/IL-6R complex could be enhanced by converting it
into an unimolecular protein by using a flexible polypeptide as a
linker (Fig 7).
The distance between the C-terminus of IL-6R and the N-terminus of IL-6
was calculated from our three-dimensional model of the complex to be in
the order of 40 Å.61 Consequently, we used the 16 N-terminal nonhelical and presumably flexible amino acid residues of
IL-6 together with a 13 residue sequence rich in glycine and serine to
connect IL-6 and the sIL-6R.56
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| Fig 7.
Hyper-IL-6: a highly active designer cytokine consisting
of IL-6 and soluble IL-6R. Molecular model of the fusion protein
consisting of IL-6 (gray) and sIL-6R (yellow) fused by a flexible
peptide linker (green). A, B, C, and D denote the four helices of IL-6;
D-II and D-III are the two cytokine-binding receptor domains of the
sIL-6R used for the construction of the fusion protein.
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On gp130-expressing cells, the fusion protein that we call Hyper-IL-6
turned out to be fully active at 100- to 1,000-fold lower
concentrations compared with the combination of unlinked IL-6 and
IL-6R. The fusion protein was therefore tested for its ability to
stimulate expansion of hematopoietic progenitor cells in vitro. It
turned out that stimulation with Hyper-IL-6 was at least as effective
as IL-6/soluble IL-6R at 100 times lower concentrations than those used
for unlinked IL-6 and IL-6R.56,62
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gp130 STIMULATION OF HEMATOPOIESIS |
Hematopoiesis is arranged in an descending hierachy: clonogenic
hematopoietic stem cells pass through several stages of differentiation and finally produce functionally mature blood cells, including erythrocytes, megakaryocytes, granulocytes, monocytes, macrophages, mast cells, and the different classes of lymphocytes.63 The ability of a cell to generate and sustain the production of both mature
myeloid and lymphoid cells for the whole lifetime of an hematologically
compromised animal after transplantation has now been widely accepted
as a useful and functional definition for its assignment to the stem
cell compartment.64-66
In the double-transgenic IL-6/soluble IL-6R mice, hematopoietic
progenitor cells in adult liver and spleen have been
detected.40 As discussed earlier, it cannot be decided
whether these hematopoietic progenitor cells originate from the fetal
developmental stages or whether they have been washed in via the
circulation. Presumably, these cells have expanded during several weeks
of hepatic and spleenic hematopoiesis, and therefore renewal of
hematopoietic progenitor cells must have occurred. In this respect, it
is noteworthy that in double-transgenic IL-6/soluble IL-6R mice, but
not in single-transgenic IL-6 mice, we find a massive upregulation of stem cell factor mRNA and cell surface protein expression in the liver
that might contribute to stimulation and homing of hematopoietic progenitor cells (M. Peters, unpublished results).
So far, it is not clear whether gp130 stimulation contributes to
hematopoiesis in vivo. From our transgenic mice data40 together with the data from Zandstra et al,50 it is likely
that gp130 stimulation is more important during fetal liver than during adult medullary hematopoiesis. Accordingly, gp130-deficient mice die
perinatally between day 12.5 and term and the mutant embryos have
reduced numbers of pluripotential and committed hematopoietic progenitors in the liver and reduced differentiated lineages such as T
cells in the thymus.67 In contrast, mice that lack the LIF-R have normal hematologic compartments.68 These data
argue that either the LIF-R is not important in hematopoiesis or that the biological activity of LIF-R can be substituted.
The case of OSM is more complicated, because human OSM can interact
with both gp130/LIF-R and gp130/OSM-R complexes.2 In contrast, murine OSM seems only to interact with the gp130/OSM-R complex.69 In transgenic mice that overexpress OSM, no
hematological abnormalities have been reported,70 except
for an accumulation of immature and mature T cells in lymph
nodes.71 However, it was recently reported that OSM is
expressed in the AGM region and that OSM stimulated expansion of
AGM-derived multipotential hematopoietic progenitor cells in
vitro.51
Functional gp130 is required for normal fetal liver hematopoiesis.
Among the cytokines of the IL-6 family, only IL-6 and IL-11, which use
gp130 homodimers for signaling, may play a role in hematopoiesis in
vivo. IL-11 has been demonstrated to possess thrombopoietic potential
and can induce serial repopulating ability of murine hematopoietic stem
cells.72 However, mice deficient for the IL-11 receptor do
not show hematological abnormalities.73 IL-6 is involved in
the regulation of stem cells and committed progenitor cells in vivo,
but hematopoiesis still occurs in IL-6-deficient mice.74 A
likely explanation for these findings is that IL-6 and IL-11, which
both interact with a gp130 homodimer, can complement each other.
However, it cannot be excluded that other, as yet unidentified
gp130-stimulating cytokines exist that possess hematopoietic activities.
The second question that arises from the reviewed data is whether
soluble receptors (eg, soluble IL-6R or soluble IL-11R) are involved in
gp130 stimulation during hematopoiesis. Because several reports
indicate that hematopoietic progenitor cells do not express
IL-6R,40,52,53 gp130 stimulation on these cells might
involve soluble receptors (Fig 8A) or
intercellular stimulation (Fig 8B). However, it is difficult to
discriminate between the two models. The only way to ultimately clarify
the biological role of the soluble IL-6R will be to generate mice
unable to produce a sIL-6R. However, the generation of such an animal
model is a complex undertaking, because generation of the sIL-6R by
shedding and splicing would have to be blocked. Therefore, a mouse
lacking the exon used for the alternatively spliced
sIL-6R23 would have to be constructed. This mutation then
would have to be combined with a mutation, resulting in the deletion of
the shedding sites of the IL-6R,14,27,28 or, alternatively,
with a mutation resulting in the deletion of the shedding protease.
Problems in this respect might arise from our finding that at least
three different cleavage sites can be used by the shedding
protease14,27,28 and that the IL-6R shedding protease has
not been molecularly defined.
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| Fig 8.
gp130 stimulation via soluble or membrane-bound IL-6R.
(A) A donor cell (bottom) releases the soluble IL-6R that, in the
presence of IL-6, stimulates the target cell (top) to dimerize gp130
(gray) and initiate signal transduction. (B) The contact between donor
cell (bottom) and target cell (top) is mediated by the membrane-bound
IL-6R of the donor cell, IL-6, and the two gp130 molecules of the
target cell leading to gp130 dimerization and signaling. For reasons of
simplicity, on donor cells, gp130 has been omitted.
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CELLULAR RESPONSIVENESS DEPENDS ON THE RATIO BETWEEN gp130 AND IL-6R |
The tissue expression of gp130 is believed to be ubiquitous and the
cellular gp130 expression seems not to be the subject to major
regulation.1,75 However, the IL-6R is only expressed on
certain cell types,1,57 including hepatocytes and B cells, and its expression is regulated by glucocorticoids.58 With
the number of gp130 signal transducers being rather constant on all cells of the body, the number of IL-6R molecules expressed on the
surface of target cells may vary from one cell type to another (Fig 9).
Cells that do not express any IL-6R on their surface can be stimulated
only by the IL-6/sIL-6R complex and are insensitive towards IL-6 alone
(Fig 9A). Examples for such cells are hematopoietic progenitor
cells,40 endothelial cells,76 neuronal
cells,77,78 and osteoclasts.79 Osteoclast
progenitor cell differentiation can be stimulated by IL-6 and the
membrane bound IL-6R on osteoblasts (see Fig 8B) or by the combination
of IL-6 and soluble IL-6R.79 It has also been demonstrated
recently that osteoblasts can be stimulated via gp130 activation to
express osteoclast differentiation factor that might bind to an as yet
unidentified osteoclast differentiation factor receptor expressed on
osteoclast progenitors and induce their differentiation into
osteoclasts.80
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| Fig 9.
Cellular responsiveness to IL-6 or IL-6/soluble IL-6R is
determined by the expression levels of IL-6R. The number of the
ubiquitously expressed gp130 protein (red) is believed to be rather
constant on all cells of the organism. The number of the IL-6R
molecules (black) varies on different cell types. See text for
explanations.
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Cells that express fewer IL-6R molecules on their surface than gp130
signal transducers respond towards IL-6 alone, and this response can be
enhanced by the presence of the sIL-6R (Fig 9B). Examples for such
cells are hepatocytes and plasmacytoma cells. Cells that show a
balanced expression of IL-6R and gp130 on their surface respond towards
IL-6, and this response is not altered by the sIL-6R (Fig 9C).
Theoretically, on cells that express more IL-6R molecules than gp130
proteins, the addition of the soluble IL-6R might inhibit the IL-6
response, because the formation of inactive complexes containing only
one gp130 molecule would be favored (Fig 9D). Using transfected cells,
such a situation has recently been mimicked using the IL-11 receptor
system. Whereas on gp130-expressing cells the complex of IL-11 and
soluble IL-11R was agonistic, on cells overexpressing the
membrane-bound IL-11R, increasing amounts of soluble IL-11R inhibited
the IL-11 response of the cells.81
Interestingly, Wang et al82 recently showed that the
stimulation of peripheral T cells with IL-6 results in a downregulation of gp130 molecules that might represent a rescue mechanism of the
cytokine receptor system to avoid hyperstimulation.
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gp130 STIMULATION AND INHIBITION OF DIFFERENTIATION |
As outlined above, the stimulation of gp130 on hematopoietic progenitor
cells might result in a differentiation-inhibiting activitiy. This is
reminiscent of the activity of LIF83 on embryonic stem
cells, which now are widely used to generate knock-out animals. Interestingly, LIF can also be replaced by other cytokines of the IL-6
family that interact with the gp130/LIR heterodimer, such as OSM and
CNTF.84 ES cell differentiation is also completely prevented when cells are treated with the combination of IL-6 and
sIL-6R.85 It is therefore tempting to speculate that the consequence of gp130 stimulation on early hematopoietic progenitor cells might be an inhibition of differentiation related to the effect
of LIF seen in embryonic stem cells. Further experiments are needed to
support this hypothesis.
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CONCLUSIONS |
The reviewed data indicate that gp130 stimulation is of importance for
hematopoiesis in vivo. Probably, fetal liver hematopoiesis and
medullary hematopoiesis require different stimulating cytokines, reflecting the ontogenic difference between the hematopoietic cells
involved. Cytokines that directly stimulate gp130 will be of use for in
vitro expansion of hematopoietic progenitor cells and should replace
IL-6, which is commonly used in such cytokine cocktails.
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ACKNOWLEDGMENT |
The authors are thankful to H. Geiger (Freiburg, Germany) for help with
the FACS analysis. We thank Dr Connie Eaves (Vancouver, British
Columbia, Canada) and Dr Christoph Huber (Mainz, Germany) for reading
the manuscript, help, and advice.
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FOOTNOTES |
Submitted May 5, 1998;
accepted July 10, 1998.
Supported by the Deutsche Forschungs-Gemeinschaft (Bonn, Germany), the
NMFZ (Mainz, Germany), and the Stiftung Rheinland Pfalz für
Innovation (Mainz, Germany).
Address reprint requests to Stefan Rose-John, PhD, I. Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg
Universität Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz,
Germany; e-mail: rosejohn{at}mail.uni-mainz.de.
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REFERENCES |
1.
Taga T, Kishimoto T:
gp130 and the interleukin-6 family of cytokines.
Annu Rev Immunol
15:797, 1997[Medline]
[Order article via Infotrieve]
2.
Mosley B, De Imus C, Friend D, Boiani N, Thoma B, Park LS, Cosman D:
Dual oncostatin M (OSM) receptors. Cloning and characterization of an alternative signaling subunit conferring OSM-specific receptor activation.
J Biol Chem
271:32635, 1996[Abstract/Free Full Text]
3.
Pennica D, Shaw KJ, Swanson TA, Moore MW, Shelton DL, Zioncheck KA, Rosenthal A, Taga T, Paoni NF, Wood WI:
Cardiotrophin-1. Biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex.
J Biol Chem
270:10915, 1995[Abstract/Free Full Text]
4.
Pennica D, Arce V, Swanson TA, Vejsada R, Pollock RA, Armanini M, Dudley K, Phillips HS, Rosenthal A, Kato AC, Henderson CE:
Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons.
Neuron
17:63, 1996[Medline]
[Order article via Infotrieve]
5.
Morrison SJ, Uchida N, Weissman IL:
The biology of hematopoietic stem cells.
Annu Rev Cell Dev Biol
11:35, 1995[Medline]
[Order article via Infotrieve]
6.
Ogawa M:
Differentiation and proliferation of heamtopoietic stem cells.
Blood
81:2844, 1993[Abstract/Free Full Text]
7.
Ikebuchi K, Wong GG, Clark SC, Ihle JM, Hirai Y, Ogawa M:
Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipotential hematopoietic progenitors.
Proc Natl Acad Sci USA
84:9035, 1987[Abstract/Free Full Text]
8.
Heaney ML, Golde DW:
Soluble cytokine receptors.
Blood
87:847, 1996[Free Full Text]
9.
Rose-John S, Heinrich PC:
Soluble receptors for cytokines and growth factors: Generation and biological function.
Biochem J
300:281, 1994
10.
Hooper NM, Karran EH, Turner AJ:
Membrane protein secretases.
Biochem J
321:265, 1997
11.
Kishimoto T, Akira S, Narazaki M, Taga T:
Interleukin-6 family of cytokines and gp130.
Blood
86:1243, 1995[Free Full Text]
12.
Müllberg J, Schooltink H, Stoyan T, Heinrich PC, Rose-John S:
Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor.
Biochem Biophys Res Commun
189:794, 1992[Medline]
[Order article via Infotrieve]
13.
Müllberg J, Dittrich E, Graeve L, Gerhartz C, Yasukawa K, Taga T, Kishimoto T, Heinrich PC, Rose-John S:
Differential shedding of the two subunits of the interleukin-6 receptor.
FEBS Lett
332:174, 1993[Medline]
[Order article via Infotrieve]
14.
Müllberg J, Schooltink H, Stoyan T, Gunther M, Graeve L, Buse G, Mackiewicz A, Heinrich PC, Rose-John S:
The soluble interleukin-6 receptor is generated by shedding.
Eur J Immunol
23:473, 1993[Medline]
[Order article via Infotrieve]
15.
Müllberg J, Oberthur W, Lottspeich F, Mehl E, Dittrich E, Graeve L, Heinrich PC, Rose-John S:
The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site.
J Immunol
152:4958, 1994[Abstract]
16.
Müllberg J, Durie FH, Otten Evans C, Alderson MR, Rose-John S, Cosman D, Black RA, Mohler KM:
A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor.
J Immunol
155:5198, 1995[Abstract]
17.
McGeehan GM, Becherer JD, Bast RC Jr, Boyer CM, Champion B, Connolly KM, Conway JG, Furdon P, Karp S, Kidao S, McElroy AB, Nichols J, Pryzwanszky M, Schoenen F, Sedut L, Truesdale A, Verghese M, Warner J, Ways JP:
Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor.
Nature
370:558, 1994[Medline]
[Order article via Infotrieve]
18.
Mohler KM, Sleath PR, Fitzner JN, Cerretti DP, Alderson M, Kerwar SS, Torrance DS, Otten Evans C, Greenstreet T, Weerawarna K, Kronheim SR, Petersen M, Gerhart M, Kozlosky CJ, March CJ, Black RA:
Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing.
Nature
370:218, 1994[Medline]
[Order article via Infotrieve]
19.
Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP:
A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.
Nature
385:729, 1997[Medline]
[Order article via Infotrieve]
20.
Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Warner J, Willard D, Becherer JD:
Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.
Nature
385:733, 1997[Medline]
[Order article via Infotrieve]
21.
Wolfsberg TG, White JM:
ADAMs in fertilization and development.
Dev Biol
180:389, 1996[Medline]
[Order article via Infotrieve]
22.
Lust JA, Jelinek DF, Donovan KA, Frederick LA, Huntley BK, Braaten JK, Maihle NJ:
Sequence, expression and function of an mRNA encoding a soluble form of the human interleukin-6 receptor (sIL-6R).
Curr Top Microbiol Immunol
194:199, 1995[Medline]
[Order article via Infotrieve]
23.
Lust JA, Donovan KA, Kline MP, Greipp PR, Kyle RA, Maihle NJ:
Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor.
Cytokine
4:96, 1992[Medline]
[Order article via Infotrieve]
24.
Honda M, Yamamoto S, Cheng M, Yasukawa K, Suzuki H, Saito T, Osugi Y, Tokunaga T, Kishimoto T:
Human soluble IL-6 receptor: Its detection and enhanced release by HIV infection.
J Immunol
148:2175, 1992[Abstract]
25.
Frieling JTM, van Deuren M, Wijdenes J, van der Meer JWM, Clement C, van der Linden CJ, Sauerwein RW:
Circulating interleukin-6 receptor in patients with sepsis syndrome.
J Infect Dis
171:469, 1995[Medline]
[Order article via Infotrieve]
26.
Lavabre Bertrand T, Exbrayat C, Liautard J, Gaillard JP, Baskevitch PP, Poujol N, Duperray C, Bourquard P, Brochier J:
Detection of membrane and soluble interleukin-6 receptor in lymphoid malignancies.
Br J Haematol
91:871, 1995[Medline]
[Order article via Infotrieve]
27.
Walev I, Vollmer P, Palmer M, Bhakdi S, Rose-John S:
Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide.
Proc Natl Acad Sci USA
93:7882, 1996[Abstract/Free Full Text]
28.
Vollmer P, Walev I, Rose-John S, Bhakdi S:
Novel pathogenic mechanism of microbial metalloproteinases: Liberation of membrane-anchored molecules in biologically active form exemplified by studies with the human interleukin-6 receptor.
Infect Immun
64:3646, 1996[Abstract]
29.
Taga T, Kishimoto T:
Immune and hematopoietic cell regulation: Cytokines and their receptors.
Curr Opin Cell Biol
2:174, 1990[Medline]
[Order article via Infotrieve]
30.
Mackiewicz A, Schooltink H, Heinrich PC, Rose-John S:
Complex of soluble human IL-6-receptor/IL-6 up-regulates expression of acute-phase proteins.
J Immunol
149:2021, 1992[Abstract]
31.
Davis S, Aldrich TH, Ip NY, Stahl N, Scherer S, Farruggella T, DiStefano PS, Curtis R, Panayotatos N, Gascan H, Chevalier S, Yancopulos GD:
Released form of CNTF receptor alpha component as a soluble mediator of CNTF responses.
Science
259:1736, 1993[Abstract/Free Full Text]
32.
Baumann H, Wang Y, Morella KK, Lai CF, Dams H, Hilton DJ, Hawley RG, Mackiewicz A:
Complex of the soluble IL-11 receptor and IL-11 acts as IL-6-type cytokine in hepatic and nonhepatic cells.
J Immunol
157:284, 1996[Abstract]
33.
Narazaki M, Yasukawa K, Saito T, Ohsugi Y, Fukui H, Koishihara Y, Yancopoulos GD, Taga T, Kishimoto T:
Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gp130.
Blood
82:1120, 1993[Abstract/Free Full Text]
34.
Layton MJ, Cross BA, Metcalf D, Ward LD, Simpson RJ, Nicola NA:
A major binding protein for leukemia inhibitory factor in normal mouse serum: Identification as a soluble form of the cellular receptor.
Proc Natl Acad Sci USA
89:8616, 1992[Abstract/Free Full Text]
35.
Taga T, Hibi M, Hirata Y, Yamasaki K, Yasukawa K, Matsuda T, Hirano T, Kishimoto T:
Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130.
Cell
58:573, 1989[Medline]
[Order article via Infotrieve]
36.
Mackiewicz A, Wiznerowicz M, Roeb E, Karczewska A, Nowak J, Heinrich PC, Rose-John S:
Soluble interleukin 6 receptor is biologically active in vivo.
Cytokine
7:142, 1995[Medline]
[Order article via Infotrieve]
37.
Peters M, Jacobs S, Ehlers M, Vollmer P, Müllberg J, Wolf E, Brem G, Meyer zum Büschenfelde KH, Rose-John S:
The function of the soluble interleukin 6 (IL-6) receptor in vivo: Sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6.
J Exp Med
183:1399, 1996[Abstract/Free Full Text]
38.
van Snick J:
Interleukin-6: An overview.
Annu Rev Immunol
8:253, 1990[Medline]
[Order article via Infotrieve]
39.
Gaillard J-P, Liautard J, Klein B, Brochier J:
Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines.
Eur J Immunol
27:3332, 1997[Medline]
[Order article via Infotrieve]
40.
Peters M, Schirmacher P, Goldschmitt J, Odenthal M, Peschel C, Dienes HP, Fattori E, Ciliberto G, Meyer zum Büschenfelde KH, Rose-John S:
Extramedullary expansion of hematopoietic progenitor cells in IL-6/sIL-6R double transgenic mice.
J Exp Med
185:755, 1997[Abstract/Free Full Text]
41.
Peters M, Odenthal M, Schirmacher P, Blessing M, Fattori E, Ciliberto G, Meyer zum Büschenfelde KH, Rose-John S:
Soluble IL-6 receptor leads to a paracrine modulation of the IL-6-induced hepatic acute phase response in double transgenic mice.
J Immunol
159:1474, 1997[Abstract]
42.
Schirmacher P, Peters M, Ciliberto G, Fattori E, Lotz J, Meyer zum Büschenfelde KH, Rose-John S:
Hepatocellular hyperplasia, plasmacytoma formation, and extracellular hematopoiesis in interleukin (IL)-6/soluble IL-6 receptor double-transgenic mice.
Am J Pathol
153:639, 1998[Abstract/Free Full Text]
43.
Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y, Suda T:
In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells.
Blood
80:3044, 1992[Abstract/Free Full Text]
44.
Osawa M, Nakamura K, Nishi N, Takahasi N, Tokuomoto Y, Inoue H, Nakauchi H:
In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/) hemopoietic stem cells.
J Immunol
156:3207, 1996[Abstract]
45.
Dzierzak E, Medvinsky A:
Mouse embryonic hematopoiesis.
Trends Genet
11:359, 1995[Medline]
[Order article via Infotrieve]
46.
Bonifer C, Faust N, Geiger H, Müller AM:
Developmental changes in the differentiation capacity of haematopoietic stem cells.
Immunol Today
19:236, 1998[Medline]
[Order article via Infotrieve]
47.
Lansdorp PM, Dragowska W, Mayani H:
Ontogeny-related changes in proliferative potential of human hematopoietic cells.
J Exp Med
178:787, 1993[Abstract/Free Full Text]
48.
Vormoor J, Lapidot T, Pflumio F, Risdon G, Patterson B, Broxmeyer HE, Dick JE:
Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.
Blood
83:2489, 1994[Abstract/Free Full Text]
49.
Petzer AL, Zandstra PW, Piret JM, Eaves CJ:
Differential cytokine effects on primitive (CD34+CD38) human hematopoietic cells: Novel responses to Flt3-ligand and thrombopoietin.
J Exp Med
183:2551, 1996[Abstract/Free Full Text]
50.
Zandstra PW, Conneally E, Piret JM, Eaves CJ:
Ontogeny-determined changes in the cytokine responses of primitive human hematopoietic cells.
Br J Haematol
101:770, 1998[Medline]
[Order article via Infotrieve]
51.
Mukouyama Y-S, Hara T, Xu M-J, Tamura K, Donovan PJ, Kim H-J, Kogo H, Tsuji K, Nakahata T, Miyajima A:
In vitro expansion of murine multipotential hematopoietic progenitors from the embryonic aorta-gonad-mesonephros region.
Immunity
8:105, 1998[Medline]
[Order article via Infotrieve]
52.
Tajima S, Tsuji K, Ebihara Y, Sui X, Tanaka R, Muraoka K, Yoshida M, Yamada K, Yasukawa K, Taga T, Kishimoto T, Nakahata T:
Analysis of interleukin-6 receptor and gp130 expressions and proliferative capability of human CD34+ cells.
J Exp Med
184:1357, 1996[Abstract/Free Full Text]
53.
McKinstry WJ, Li CL, Rasko JE, Nicola NA, Johnson GR, Metcalf D:
Cytokine receptor expression on hematopoietic stem and progenitor cells.
Blood
89:65, 1997[Abstract/Free Full Text]
54.
Sui X, Tsuji K, Tanaka R, Tajima S, Muraoka K, Ebihara Y, Ikebuchi K, Yasukawa K, Taga T, Kishimoto T, Nakahata T:
gp130 and c-Kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells.
Proc Natl Acad Sci USA
92:2859, 1995[Abstract/Free Full Text]
55.
Kimura T, Sakabe H, Tanimukai S, Abe T, Urata Y, Yasukawa K, Okano A, Taga T, Sugiyama H, Kishimoto T, Sonoda Y:
Simultaneous activation of signals through gp130, c-kit, and interleukin-3 receptor promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors.
Blood
90:4767, 1997[Abstract/Free Full Text]
56.
Fischer M, Goldschmitt J, Peschel C, Kallen KJ, Brakenhoff JPJ, Wollmer A, Grötzinger J, Rose-John S:
A designer cytokine with high activity on human hematopoietic progenitor cells.
Nat Biotech
15:142, 1997[Medline]
[Order article via Infotrieve]
57.
Yamasaki K, Taga T, Hirata Y, Yawata H, Kawanishi Y, Seed B, Taniguchi T, Hirano T, Kishimoto T:
Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor.
Science
241:825, 1988 |
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Introduction
Conclusions