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* Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, Wales, U.K.;
Department of Microbiology, Tokyo Medical and Dental University, Tokyo 113, Japan;
Institute of Nephrology, University of Wales College of Medicine, Cardiff CF14 4XN, Wales, U.K.; and
§ Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA
1Correspondence: Cardiff School of Biosciences, Biomedical Sciences Building, Cardiff University, Museum Ave. (P.O. Box 911), Cardiff CF10 3US, Wales, U.K. E-mail: JonesSA{at}cf.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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Key Words: cytokines • soluble receptors • interleukin 6 • inflammation • shedding
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INTERLEUKIN 6 AND INTERLEUKIN 6 RECEPTOR SIGNALING |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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; elevated IL-6 concentrations have been associated
with disease states (2
3
4
5
6
7
8)
. The functional properties of
IL-6 are extremely varied and this is reflected by the terminology
originally used to describe the activities of this cytokine
[interferon ß2 (IFN-ß2, hepatocyte-stimulating factor, cytotoxic T
cell differentiation factor, B cell differentiation factor, and B cell
stimulatory factor 2]. Indeed, IL-6 promotes inflammatory events
through the expansion and activation of T cells, differentiation of B
cells, and the induction of acute-phase reactants by hepatocytes. In
contrast, IL-6 also performs a protective role during disease and
counteracts the manifestation of certain inflammatory responses. In
septic shock, for example, IL-6 suppresses acute neutrophil
accumulation caused by intratracheal administration of endotoxin
(9
, 10)
. This is paralleled by studies showing that IL-6
down-regulates proinflammatory cytokine expression while simultaneously
inducing the expression of IL-1 receptor antagonist and the soluble p55
tumor necrosis factor 
(TNF-) receptor (11
12
13)
.
Thus, IL-6 acts not only as a pro- but also as an anti-inflammatory
cytokine, and as a result serves a pivotal role during disease.
The receptor complex mediating the biological activities of IL-6
consists of two distinct membrane-bound glycoproteins, an 80 kDa
cognate receptor subunit (IL-6R, CD126) and a 130 kDa
signal-transducing element (gp130, CD130). Expression of the
trans-membrane-spanning gp130 is found in almost all organs,
including heart, kidney, spleen, liver, lung, placenta, and brain
(14)
. In contrast, cellular distribution of the cognate
IL-6R is limited and its expression is predominantly confined to
hepatocytes and leukocyte subpopulations (monocytes, neutrophils, T
cells, and B cells). Although gp130 was initially identified as the
signal-transducing component of the IL-6 receptor, it is now apparent
that cognate receptors for interleukin 11 (IL-11), oncostatin-M (OSM),
ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), leukemia
inhibitory factor (LIF), and novel neurotrophin-1/B cell-stimulating
factor-3 all transmit activation signals via gp130
(15
16
17
18)
. As a consequence, each of these cytokines
possess overlapping activities, and the phenotypic characteristics of
mice lacking either IL-6, IL-11, LIF, or CNTF (19
20
21)
are
less severe than the apparent pleiotropic properties of these mediators
would suggest. In contrast, targeted disruption of the gp130 gene is
embryonically lethal (22)
.
Interleukin 6 signaling is facilitated through the homodimerization of
gp130 to the ligand–receptor complex. Intracellular signaling is
subsequently triggered via activation of gp130-associated cytoplasmic
tyrosine kinases (JAK1, JAK2, and TYK2) and phosphorylation of STAT1
and STAT3 (23
, 26)
. In contrast, the high-affinity
receptors of LIF, OSM, and CNTF activate cells by a heterodimerization
between gp130 and a gp130-related protein (the LIF receptor)
(27)
. Such homo- or heterodimers activate distinct but
overlapping patterns of tyrosine phosphorylation through the Jak-Tyk
family of cytoplasmic tyrosine kinases (28)
. This may
contribute to the different cellular responses associated with this
family of proteins. Interleukin 6 also activates the Ras-Raf signaling
cascade, which regulates phosphorylation of MAP-kinase and ultimately
activation of the transcription factors NF-IL-6 (a C/EBP family member)
and AP-1 (c-Jun and c-Fos) (29
30
31)
. Stimulation of this
Ras-dependent MAP kinase cascade has been suggested to perform an
important role in IL-6-mediated proliferation, since activation of this
pathway was associated only with cell types that proliferate in
response to IL-6 (31)
. Subsequent reports have also
confirmed the involvement of Sak, Hck, Fes, Btk, and Tec-kinases in
IL-6 signaling (32
33
34
35)
. However, the significance of
their activation remains to be determined. For a more comprehensive
review of signaling via gp130, the reader is directed elsewhere
(36)
.
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THE SOLUBLE IL-6 RECEPTOR |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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, 38)
. This soluble receptor binds IL-6 with an affinity similar
to that of the cognate receptor (0.5–2 nM) (39
, 40)
and
prolongs its plasma half-life (41)
. More important, the
[sIL-6R/IL-6] complex is capable of activating cells via interaction
with membrane-bound gp130 (Fig. 1
). This feature makes the [sIL-6R/IL-6] complex an agonist for cell
types that although they express gp130, would not inherently respond to
IL-6 alone. Hence, the sIL-6R has the ability to widen the repertoire
of cell types that are responsive to IL-6. This is in contrast to the
function of most soluble cytokine receptors that bind their ligand and
antagonize cellular signaling by preventing the interaction of the
cytokine with their respective plasma membrane-bound cognate receptor
(42)
. However, it of interest that genetically engineered
forms of soluble IL-11 and CNTF receptors (the LIF-receptor) can also
mimic the stimulatory properties of the [sIL-6R/IL-6] complex once
associated with their ligands (43
44
45)
.
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The importance of the sIL-6R as a regulator of IL-6 responses is
illustrated by the increasing number of studies that have now been
published describing the agonistic properties to this soluble receptor.
Consequently, the [sIL-6R/IL-6] complex should be thought of as being
an heterodimeric cytokine that exerts its action through gp130. In
terms of structure, this may be analogous to the heterodimeric IL-12,
whose p40 subunit shares extensive amino acid homology with the entire
extracellular domain of the IL-6R (46)
.
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ACTIVITIES MEDIATED THROUGH THE ACTION OF sIL-6R |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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48
49)
. It was later confirmed that
sIL-6R purified from human plasma also retain similar biological
activities (38
, 50)
; roles for sIL-6R in cellular
proliferation, differentiation, and the activation of inflammatory
responses have now been described.
Cellular proliferation and differentiation
Initials experiments observed that sIL-6R could bind IL-6 and
efficiently suppress the proliferation of peripheral blood mononuclear
cells activated with concanavalin A (38)
. Subsequently, it
has been reported that the [sIL-6R/IL-6] complex promotes the growth
of many cell types including Kaposi’s sarcoma cell lines
(51)
, hematopoietic progenitor cells (52)
,
and synovial fibroblasts (53)
. Indeed, the potential
effect of the sIL-6R on proliferative events was recently emphasized by
a study of hepatocyte proliferation in double-transgenic mice
expressing high levels of human IL-6 and sIL-6R (54
, 55)
.
These mice spontaneously develop nodules of hepatocellular hyperplasia
around periportal spaces, which is consistent with regenerative
hyperplasia events commonly associated with myeloproliferative and
immunological conditions (55)
. This implies that the
[sIL-6R/IL-6] complex may act as a primary stimulus for hepatocyte
proliferation and as a promoter of hepatocellular transformation.
The sIL-6R has also been implicated in cellular differentiation. For
example, IL-6 alone is unable to induce osteoclast formation in
cocultures of mouse bone marrow and osteoblastic cells, but when
combined with the sIL-6R, osteoclast formation is promoted
(56
57
58)
. Further evidence for sIL-6R acting as a mediator
of differentiation events has also been obtained by comparing the
phenotypic characteristics of transgenic mice expressing either human
IL-6 or sIL-6R alone with animals coexpressing both IL-6 and sIL-6R.
The main difference between these mice is the dramatic increase in
extramedullary hematopoiesis that occurs within the liver and spleen of
double-transgenic adult mice (59
, 60)
. This feature of
sIL-6R/IL-6 double-transgenic animals is highlighted by the elevated
numbers of granulocytes, macrophages, B cells, and hematopoietic
progenitor cells in the liver and spleen, and by a massive increase in
circulating leukocytes and red blood cells within these animals. For a
detailed review of sIL-6R and hematopoiesis events, see ref
60
.
Based on recent studies, it has been questioned whether IL-6 not only
acts as a cytokine, but also as a neurotrophic factor
(61)
. Trophic factors are broadly defined by their
capacity to promote neuronal survival and development. Although many
neuronal cells are capable of producing IL-6, they remain unresponsive
to stimulation by IL-6 itself. Differentiation and survival of neuronal
cells can, however, be mediated through the action of sIL-6R. For
example, sympathetic and sensory neurons from neonatal superior
cervical ganglia and embryonic dorsal root ganglia both show a marked
increase in survival and neurite outgrowth when stimulated by the
[sIL-6R/IL-6] complex (62
, 63)
. In addition, sIL-6R may
perform a role in axon growth from dorsal root ganglia and in the
development of Schwann cell progenitors, which express myelin basic
protein after activation with a combination of IL-6 and sIL-6R
(64)
. Thus, sIL-6R in conjunction with IL-6 may be
important in nerve regeneration through the promotion of remyelination
events.
In many cases, the cellular responses coordinated by the
[sIL-6R/IL-6] complex can also be controlled by the action of other
gp130-stimulating cytokines. For instance, stimulation of cardiomyocyte
proliferation and the development of pathological ventricular
hypertrophy in response to the [sIL-6R/IL-6] complex (65
, 66)
have been described as functional properties of CT-1
(67)
. Similarly, sIL-6R-mediated expression of
neuropeptides and transmitter biosynthetic enzymes by sympathetic
neuronal cells (63)
can also be mimicked by the action of
LIF, CNTF, OSM, and CT-1 (67
68
69)
, whereas osteoclast
formation is mediated by IL-11, LIF, and OSM alone or via sIL-6R in
conjunction with IL-6 (58
, 70)
. This raises the following
question: if a given cell type, which was naturally devoid of the
cognate IL-6R, were to express the IL-6R, would activation of these
cells by IL-6 alone be able to fulfill many of the functions assigned
to the [sIL-6R/IL-6] complex? Predicting the answer to this question
may not be as straightforward as initially thought, and is highlighted
by studies of the proliferative capability of human
CD34+ cells after stimulation by [sIL-6R/IL-6]
complex (71
, 72)
. Although CD34+
cells bear gp130, expression of IL-6R is limited to only 30–50% of
the total cell population (71)
. Subsequent analysis of
sorted CD34+ cells showed that
CD34+ IL-6R- cells expand
into a variety of hematopoietic progenitor and erythroid cells in the
presence of IL-6, sIL-6R, and stem cell factor (71)
. Under
identical experimental conditions, stimulation of
CD34+ IL-6R+ cells with
IL-6 alone promoted the expansion of granulocyte/macrophage colonies
(71)
. These data may provide initial evidence for
potential functional differences between the activity of IL-6 and the
[sIL-6R/IL-6] complex or, alternatively, that IL-6R expression
reflects a progenitor cell type at a different stage of differentiation
and is more committed to expansion into a specific cell lineage.
Regulation of inflammatory mediators
Examination of IL-6-deficient (IL-6-/-)
mice has recently suggested that sIL-6R regulates leukocyte recruitment
in a subcutaneous (s.c.) air pouch model of inflammation
(73)
. It was subsequently shown that although endothelial
cells lack the cognate IL-6R, they could be activated to phosphorylate
STAT3, produce chemokines (IL-8, MCP-1, and, to a lesser extent,
MCP-3), and up-regulate adhesion molecule (ICAM-1, VCAM-1) expression
in response to the [sIL-6R/IL-6] complex (73
, 74)
. This
activation likely occurs via direct interaction with the gp130
signal-transducing element. Thus, the [sIL-6R/IL-6] complex plays a
significant role in regulation of leukocyte recruitment and may serve a
positive role in the prothrombotic/proinflammatory activation of
endothelial cells. Consistent with this finding, it was recently
reported that the degree of leukocyte infiltration into arthritic
joints correlates with elevated sIL-6R levels in synovial fluid
(75)
. Furthermore, activation of skin fibroblasts
(76)
, smooth muscle cells (77)
, thyroid
follicular cells (78)
, and astrocytes (79)
by
the [sIL-6R/IL-6] complex has been shown to promote cytokine (IL-6)
and chemokine production. In contrast, the [sIL-6R/IL-6] complex does
not appear to stimulate chemokine production by astrocytes, but has the
capacity to block TNF--mediated expression of VCAM-1 by these cells
(80
, 81)
. In addition to enhanced chemokine and adhesion
molecule expression, the sIL-6R has also been shown to regulate
expression of certain proteases and protease inhibitors (82
, 83)
.
In vivo properties of the sIL-6R
Initial in vivo experiments designed to examine the
functional properties of sIL-6R were based on the observation that
intracerebroventricular administration of IL-6 caused an increase in
body temperature and a decrease in locomotory activity and food intake.
When these animals were treated with IL-6 and an accompanying injection
of sIL-6R, all of these central effects were enhanced and prolonged
(84)
, suggesting that sIL-6R not only heightens IL-6
responsiveness, but also extends the circulating half-life of IL-6.
Subsequent studies have now confirmed and extended these observations
through the use of transgenic mice overexpressing either human sIL-6R
alone or together with human IL-6 (54
, 59
, 60
, 85)
. In all
these cases, the model system exploits the species specificity of human
IL-6 and its receptor, which are capable of transmitting signals via
interaction with murine IL-6R and murine gp130, respectively. In
contrast, murine IL-6 is unable to bind the human IL-6R, allowing
sIL-6R bioactivity to be reconstituted only through the presence of
human IL-6. In general, double-transgenic mice (overexpressing sIL-6R
and IL-6) are smaller than their wild-type littermates and have reduced
body weight, whereas autopsy and histopathological analysis show that
these animals have markedly reduced fat deposits, enlarged spleen, and
shrunken disfigured livers (54
, 59
, 85)
. These latter
morphological characteristics may be accounted for by increases in
hepatic and splenic hematopoiesis, hepatocellular hyperplasia, and
plasma cell proliferation associated with the double-transgenic mice
(54
, 59)
. Consequently, the sIL-6R appears to represent a
major stimulator of cellular growth and hematopoietic cells in
vivo.
Although these studies emphasize the significance of sIL-6R in
vivo, detection of high circulating levels of human IL-6 (10–20
ng/ml) and sIL-6R (4–8 µg/ml) in these transgenic mice populations
(59)
infers that this murine model reflects the influence
of systemically elevated sIL-6R. Conversely, when considering the
involvement of sIL-6R in disease progression, it is also important to
appraise the effect of locally produced sIL-6R in eliciting cellular
events. To date, few studies have examined the biological consequence
of locally produced sIL-6R. Nevertheless, the significance of locally
produced sIL-6R has been demonstrated through the use of IL-6/sIL-6R
double-transgenic mice (85)
. In this instance, human IL-6
and sIL-6R were coexpressed in mice under the respective control of
metallothionein and PEPCK promoters. Although both promoters were
functional within the liver, their activities were confined to distinct
regions of the organ. Using in situ hybridization
techniques, the authors showed that IL-6-induced expression of the
acute-phase reactant haptoglobin was localized to areas of the liver
were sIL-6R was expressed (85)
. Thus, sIL-6R has the
potential to act as a paracrine mediator whose effects are elicited
close to its site of generation. In addition, sIL-6R release by
leukocyte subpopulations (86
87
88)
suggests that activation
of infiltrating leukocytes may also contribute to locally elevated
sIL-6R levels that are commonly associated with several inflammatory
disorders. However, a direct link between infiltrating leukocytes and
enhanced local concentrations of sIL-6R remains unsubstantiated.
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MECHANISMS OF SOLUBLE IL-6 RECEPTOR PRODUCTION |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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Soluble forms of transmembrane proteins that retain bioactivity have
now been identified in biological fluids. These include soluble
adhesion molecules and receptors for cytokines and growth factors. Two
independent cellular processes have been identified as controlling
their production (42)
. First, differential mRNA splicing
can lead to the expression of soluble factors that lack cytoplasmic and
membrane-spanning domains of the cell-associated protein. Examples
include receptors for IL-4, epidermal growth factor (EGF), and LIF
(42)
. The second mechanism involves proteolytic cleavage
of a membrane-anchored protein at a site close to the cell surface.
Regulatory proteins processed in this manner include soluble receptors
for IL-1, IL-2, TNF-, platelet-derived growth factor, and the
adhesion molecule L-selectin (CD62L). In the case of the sIL-6R, both
processes regulate release (Fig. 2
). Thus, two distinct isoforms of the sIL-6R contribute to the overall
properties of this soluble receptor. These isoforms will be referred to
here as either DS-sIL-6R or PC-sIL-6R to denote whether the receptor is
released as the product of differential mRNA splicing (DS) or shed
after proteolytic cleavage (PC). The information detailed below reviews
the evidence for sIL-6R release through both mechanisms and the manner
in which they are regulated.
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Differential mRNA splicing
Examination of IL-6R expression by monocytic and tumor cell lines
shows that these cells encode two distinct IL-6R mRNA transcripts
(89
90
91
92)
. The shorter of the two transcripts lacks 94 base
pairs [nucleotides 1504 (T) to 1597 (G)] encoding for the putative
transmembrane region of the cognate IL-6R (89
, 90)
. A role
for differential mRNA splicing in the construction of this shorter
transcript was indicated by the identification of consensus sequences
for splicing donor (nucleotides 1501–1503, CAG) and splicing acceptor
(nucleotide 1598, G) motifs that flank the deleted region
(90)
(Fig. 3
). Absence of this 94 base pair section predicts the introduction of a
reading frameshift and the subsequent incorporation of a novel 10 amino
acid (GSRRRGSCGL) sequence at the COOH-terminal tail of this sIL-6R
isoform (90)
(Fig. 3)
. Antibodies specifically raised
against this putative peptide sequence have been used to demonstrate
expression of DS-sIL-6R both in vitro (88
, 90)
and in vivo (92
, 93)
. Examination of sIL-6R
levels in plasma of healthy individuals shows that the predominant
circulating form of sIL-6R is the product of differential mRNA splicing
(94)
. More recently, we have established that plasma
levels of DS-sIL-6R decrease with age (93)
. For example,
mean circulating levels of DS-sIL-6R in individuals 21–30, 31–40, and
41–60 years of age were found to be 18.3 ng/ml, 7.2 ng/ml, and 3.1
ng/ml, respectively. Hence, regulation of DS-sIL-6R release appears to
be complex. To date, there is a lack of information pertaining to the
transcriptional regulation of the splicing mechanism, whereas very few
activators of this process have been described. In particular, T cells
release DS-sIL-6R upon stimulation with phytohemoagglutinin
(90)
, whereas IL-1 (94)
and oncostatin-M
(94
, 95)
promote DS-sIL-6R secretion by hepatocyte cell
lines.
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Proteolytic cleavage (shedding)
A second mechanism of sIL-6R generation involves shedding of the
cognate membrane-bound receptor from the cell surface after proteolytic
cleavage (86
, 96
97
98)
. Examination of COS cells stably
expressing full-length IL-6R showed that phorbol esters could activate
sIL-6R release (96)
. Although this indicated that protein
kinase C regulates generation of sIL-6R, it is unknown whether this
phorbol ester response represents an increase in the activity of the
cleavage protease, increased expression of the receptor, or increased
trafficking of IL-6R to the plasma membrane. Purification and
COOH-terminal amino acid analysis of the liberated sIL-6R identified
the proteolytic cleavage site as being immediately prior to the
putative transmembrane domain (residues
Gln357-Asn358
(98
; Fig. 3
).
Initial attempts to identify the class of protease responsible for the
liberation of PC-sIL-6R using known protease inhibitors were fruitless
(86
, 95)
and suggested the involvement of a novel
protease. Subsequent studies showed that hydroxamic acid-based
metalloprotease inhibitors, such as those known to block pro-TNF-
processing (e.g., the TNF- (processing) protease inhibitor or TAPI)
(99)
, could prevent the phorbol ester- and
ionomycin-induced shedding of IL-6R from monocytic cells (86
, 100)
and multiple myeloma cell lines (101)
.
Although hydroxamic acid-based metalloprotease inhibitors also block
shedding of the TNF- receptors (86
, 102)
, L-selectin
(103
, 104)
, CD14 (105)
, and the processing of
protransforming growth factor- (TGF-) (106)
and
pro-EGF (107)
, no conserved proteolytic cleavage site has
been identified within any of these proteins. In addition to the
inhibitory action of hydroxamic acid-based metalloprotease inhibitors,
TIMP-3 (101)
and high concentrations of
1,10-phenanthrolene (105
, 108)
have also been reported to
prevent sIL-6R shedding.
The cleavage enzyme responsible for liberating sIL-6R remains
unidentified. Based on the inhibitory properties of hydroxamic
acid-based metalloprotease inhibitors, it would follow that the IL-6R
cleavage enzyme is closely related to the recently cloned protease
responsible for the processing of pro-TNF-. This enzyme, termed the
TNF--cleavage enzyme (TACE, also known as ADAM-17), is a zinc
binding metalloprotease disintegrin and belongs to the adamalysin
family (109
, 110)
. Although TACE has been implicated in
the processing of pro-TGF- as well as L-selectin and p75
TNF--receptor shedding (111)
, a role for TACE in sIL-6R
shedding has not been proposed. A second metalloprotease disintegrin
(ADAM-10) has been reported to mediate processing of pro-TNF-, but
again this enzyme was unable to cleave peptides representing the
processing sites in various proteins, including IL-6R
(112)
. Although IL-6R shedding via an ADAM directed
proteolytic event remains to be fully substantiated, bacterial-derived
metalloproteases, which are sensitive to agents such as TAPI, have been
shown to release sIL-6R (108)
. The cleavage site where
these proteases act, however, is distinct from that used by the
endogenous cellular protease (98
, 108)
.
Since the initial observation that IL-6R shedding can be promoted by
stimulation with phorbol esters, very few physiologically relevant
activators of sIL-6R shedding have been identified. Bacterial-derived,
pore-forming toxins streptolysin-O and hemolysin-A have been shown to
promote shedding of functionally active sIL-6R from monocytic cells via
a TAPI-sensitive mechanism (105)
, while activation of
human neutrophils with f-Met-Leu-Phe causes a significant loss of
surface-bound IL-6R as detected by flow cytometry (74)
. In
a study of sIL-6R production by monocytic THP-1 cells, a series of
cytokines (IL-1ß, TNF-, IFN-
, IL-4, IL-6, IL-10), chemokines
(RANTES, MCP-1), and growth factors such as platelet-derived growth
factor (PDGF-AA, PDGF-BB) were tested for their ability to induce
sIL-6R production (100)
. However, none of these agents
significantly elevated sIL-6R production by these cells. Subsequent
studies revolved around the rationale that IL-6R shedding may be
activated by factors known to coordinate release of other soluble
receptors/adhesion molecules (e.g., soluble TNF- and soluble
L-selectin) or agents known to modulate IL-6 activity or to be
regulated by IL-6. Based on these criteria human neutrophils were found
to shed IL-6R after stimulation by the acute-phase reactant C-reactive
protein (CRP) (88)
. During the onset of inflammation or
tissue injury, plasma concentrations of CRP are dramatically elevated
from 
1 µg/ml in healthy individuals to as much as 500 µg/ml
during the acute-phase response (113)
. In vitro
studies show that control of this response is primarily regulated by
IL-6 (114)
. CRP plays a significant role in host defense
against pathogens and also binds specific receptors on human
neutrophils, causing neutrophil responses, such as chemotaxis
(115)
and the activation of superoxide generation and
degranulation by chemoattractants (116)
, to be diminished.
Recently, the major receptor for CRP on leukocytes has been identified
as Fc receptor-IIa (CD32) (117)
. CRP and biologically
relevant CRP-peptides have been shown to prevent neutrophil adhesion to
endothelial cells via induction of L-selectin shedding
(118)
, a process that is prevented by a hydroxamic acid
based metalloprotease inhibitor (118)
. Surprisingly, the
CRP-induced shedding of sIL-6R by neutrophils was largely unaffected by
the hydroxamic acid-based metalloprotease inhibitor TAPI
(88)
. Thus, an alternative shedding mechanism may be
responsible for the CRP-induced release of sIL-6R from human
neutrophils. Indeed, it has been suggested that certain
leukocyte-derived proteases may facilitate the release of a variety of
surface proteins (119
120
121)
, with cathepsin-G being
implicated in IL-6R shedding (122)
.
|
DIFFERENTIAL RELEASE OF SOLUBLE IL-6 RECEPTOR ISOFORMS |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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, 100
, 105)
. In contrast, secretion of DS-sIL-6R is relatively
slow, and enhanced concentrations of this isoform are typically seen
8–24 h after activation (95)
. The delay in DS-sIL-6R
release presumably occurs because of the necessity for de
novo synthesis, since cycloheximide substantially diminishes
production of this isoform (95)
. We infer from this that
sIL-6R derived via alternate mRNA splicing is not under the same
control as that released through proteolytic cleavage. Credence for
this statement is provided by both in vitro and in
vivo studies. Examination of human monocytic THP-1 cells, hepatoma
HepG2 cells, and myeloma cells has shown that these lines release both
PC- and DS-sIL-6R. (95
, 100
, 123)
. Indeed, it has been
shown that basal sIL-6R production by THP-1 cells occurs through
differential mRNA splicing, whereas phorbol ester and ionomycin
stimulated release of sIL-6R results from activation of proteolytic
shedding (100)
. A differential pattern of sIL-6R isoform
production is also apparent in vivo (92, Fig. 4
); however, the significance of this has not been considered fully.
|
Since PC- and DS-sIL-6R may independently contribute to the action of
sIL-6R, the relevance of both isoforms needs to be considered when
determining the overall properties of sIL-6R. Thus, it is necessary to
consider how production of these isoforms is coordinated and also the
potential biological significance of both forms. At present, there is
no antibody available for the specific detection of PC-sIL-6R.
Consequently, patterns of sIL-6R isoform production can only be
monitored by comparing levels of total sIL-6R (detected using
antibodies that recognize the extracellular portion of IL-6R and are
unable to distinguish between the isoforms) with sIL-6R concentrations
measured using antibodies specific for the unique COOH-terminal
sequence of DS-sIL-6R (88
, 92
, 93
, 100)
. Using such an
approach, it was initially shown that the predominant circulating
isoform in normal healthy individuals was derived from differential
mRNA splicing [93, Fig. 4A
]. However, it has now been
established that serum levels of this isoform substantially decrease
with age whereas total circulating concentrations of sIL-6R remain
largely unaltered (Fig. 4B
). This may reflect either a
switch from DS-sIL-6R secretion to PC-sIL-6R release or conceivably a
processing of the COOH-terminal sequence of DS-sIL-6R rendering the
sIL-6R undetectable with anti-DS-sIL-6R antibodies. Few studies have
examined the temporal production of sIL-6R isoforms during disease.
However, based on the limited information currently available, it is
clear that PC- and DS-sIL-6R release is differentially regulated during
specific disease processes. This is most apparent in conditions linked
with retroviral infections such as AIDS and adult T cell leukemia or
human T cell leukemia virus-1 (HTLV-1)-associated myelopathy. Although
circulating sIL-6R levels are significantly elevated in each of these
diseases (38
, 92)
, closer inspection of the isoform
composition reveals that the increased levels associated with HTLV-1
infection are derived from differential mRNA splicing whereas
concentrations of DS-sIL-6R in AIDS patients remain unaltered from
those observed in healthy volunteers (92)
. Similarly,
levels of DS-sIL-6R in rheumatoid arthritis patients do not account for
the increase in sIL-6R concentrations associated with this disease
(Fig. 4A
; 92
), suggesting that proteolytic
shedding may be accounting for the increases detected.
So far it is unclear why two distinct mechanisms are used for the
production of sIL-6R. By coupling the differential release of the two
sIL-6R isoforms with the presence of a unique COOH-terminal sequence in
DS-sIL-6R, it is tempting to question whether structurally distinct
forms of sIL-6R may differ in their capacity to activate cellular
events. The ability of the [sIL-6R/IL-6] complex to signal via gp130
highlights that the trans-membrane region of the IL-6R is
not required for the functional interaction of IL-6R with gp130.
Furthermore, the NH2-terminal domain of the IL-6R
is dispensable for ligand recognition/signaling, whereas proximal
cytokine receptor domains (termed D2 and D3) are required for IL-6
binding and receptor interaction with gp130 (124)
. It
remains to be determined whether the extended COOH-terminal sequence of
DS-sIL-6R influences in some way the binding properties of these
proximal cytokine receptor domains. An initial comparison of the
biological activities of the two isoforms revealed no apparent
difference in their capacity to signal (125)
. However, it
is unclear whether responses facilitated via PC- and DS-sIL-6R differ
with respect to their potency or efficacy.
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ANTAGONISM OF SIL-6R ACTIVITY |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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.
Relatively high circulating levels of sgp130 are detected in human
blood and sgp130 associates with the [sIL-6R/IL-6] complex to inhibit
signaling via membrane-bound gp130 (126
, 127)
. Indeed,
sgp130 has been shown to inhibit sIL-6R-mediated proliferation of
Kaposi’s sarcoma cells (51)
, STAT activation and the
expression of 1-antichymotrypsin (40)
. However, the
inhibitory capacity of sgp130 is not solely restricted to preventing
sIL-6R signaling, since sgp130 also inhibits the action of LIF and OSM
(128)
, which may in part account for the high levels of
sgp130 detected in the plasma of healthy and diseased individuals.
Recently it was hypothesized that sIL-6R acts as an antagonistic
molecule that enhances the inhibitory capacity of sgp130
(40)
. This conclusion was based on the observation that
although sgp130 could not block IL-6-induced activation of
IL-6R+ cells, inclusion of sIL-6R in the assay
setup enhanced the antagonistic properties of sgp130 by promoting
formation of an [IL-6-sIL-6R-sgp130] tertiary complex. Hence, sgp130
and sIL-6R may conspire to prevent IL-6 signaling through the cognate
receptor. Consequently, when considering the effect of sIL-6R in
vivo, it is also necessary to consider the time frame of sIL-6R
and sgp130 production during disease progression. In this respect,
examination of a recently developed SCID mouse model for multiple
myeloma revealed that release of sIL-6R levels preceded those of sgp130
(129)
.
Several studies have examined the mechanism of sgp130 release and, like
sIL-6R production, both differential mRNA splicing and proteolytic
cleavage have been implicated in sgp130 release (127
, 130
131
132)
. Although only a few physiological activators of
sgp130 release have been identified (127)
, the
[sIL-6R/IL-6] complex was recently shown to up-regulate gp130
expression in smooth muscle cells (77)
. However, it is
unclear whether enhanced expression of gp130 was also paralleled by an
increase in sgp130 concentrations.
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SOLUBLE IL-6 RECEPTOR AND DISEASE |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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), in most instances the biological significance of sIL-6R within these
pathologies remains unclear. We have selected a few disorders in which
currently available information from clinical studies and in
vitro and in vivo investigations highlights the
potential involvement of sIL-6R in disease progression.
|
Arthritic lesions
High IL-6 concentrations have been documented in the serum and
synovial fluids of rheumatoid arthritis and juvenile rheumatoid
arthritis patients (6
, 133
134
135)
, whereas a prominent role
for IL-6 participation in the clinical manifestation of rheumatoid
arthritis has been emphasized by several in vivo studies
(136
137
138
139)
. Indeed, administration of an anti-IL-6
monoclonal antibody to arthritic patients in a limited trial proved to
be clinically beneficial (140)
. Similarly, suppression of
arthritic bone destruction was associated with inhibition of IL-6
production after adenovirus transfer of the csk gene (which
blocks Src kinase activity) into rat ankle joints susceptible to
adjuvant-induced arthritis (141)
. Taken together, these
studies appear to contradict the anti-inflammatory properties assigned
by some to IL-6 (9
10
11
12
13)
and accentuate the necessity to
understand the regulation of IL-6-mediated events and the effect of
sIL-6R on arthritic lesions.
Several studies have shown that sIL-6R levels are elevated in both
rheumatoid arthritis and juvenile rheumatoid arthritis (57
, 142
, 143)
; although sIL-6R concentrations are raised in
osteoarthritic patients, increases in sIL-6R are more pronounced in
rheumatoid arthritis (57)
. Indeed, levels of sIL-6R and
IL-6 have been shown to be highest in the more progressive stages of
rheumatoid arthritis development (143)
. Through a series
of in vitro approaches, sIL-6R has been implicated in a
variety of cellular consequences typically associated with
arthritis—for instance, the severe destruction of cartilage and bone,
which is a characteristic hallmark of rheumatoid arthritis. Examination
of both IL-6 and sIL-6R concentrations in synovial fluid from arthritic
patients showed that the extent of joint destruction correlated with
the increased concentration of these mediators (57)
.
Furthermore, synovial fluids from rheumatoid arthritis patients
containing high levels of IL-6 and sIL-6R promoted osteoclast-like cell
formation when added to cocultures of osteoblastic cells and bone
marrow cells (57)
. Inclusion of a blocking anti-IL-6R
antibody in these experiments significantly reduced the cellular
differentiation, confirming an involvement of sIL-6R in this process. A
role for sIL-6R in bone matrix degradation and resorption is also
supported by the observation that rat osteoblasts release collagenase-3
after costimulation with IL-6 and sIL-6R (82)
. In addition
to bone remodeling, the [sIL-6R/IL-6] complex may partially account
for the loss of proteoglycan commonly associated with arthritic lesions
since its activity is associated with suppression of proteoglycan
synthesis (144)
. Together, these findings indicate that
sIL-6R contributes, at least in part, to joint destruction. In contrast
to these proinflammatory responses, it has been suggested that sIL-6R
performs a protective role in cartilage metabolism. Experiments using
isolated human articular synovial fibroblasts and chondrocytes revealed
that stimulation of these cells by the [sIL-6R/IL-6] complex induced
expression of TIMP-1 and prevented the collagenolytic activity of
conditioned medium from IL-1-induced synoviocytes (83)
.
The presence of elevated sIL-6R levels in arthritic episodes strongly
suggests that sIL-6R production is coordinated as part of the
inflammatory response. Through comparison of sIL-6R determinations in
serum and synovial fluid of patients with rheumatoid arthritis, it is
difficult to judge whether the elevated sIL-6R levels associated with
this disease are derived from systemic or local sources
(75)
. To date, the cellular origin of sIL-6R in arthritis
remains unknown, although structural cells of the joint (chondrocytes,
synoviocytes, fibroblasts, and endothelial cells) are unlikely to
contribute to the sIL-6R levels seen in this disease (75)
.
It is conceivable that sIL-6R may be released from activated
leukocytes. This may account for the observed correlation between
leukocyte influx into arthritic joints and the increased concentration
of sIL-6R in synovial fluid (75)
. In the event of such a
scenario, it is of interest to consider the capacity of the
[sIL-6R/IL-6] complex to induce chemokine production and enhance
adhesion molecule expression (73
, 74
, 77
, 78)
. Thus,
sIL-6R release by infiltrating leukocytes would promote further
leukocyte recruitment. Conversely, systemic sIL-6R production may also
contribute to the regulation of IL-6 responses in arthritis, since
sIL-6R levels are significantly elevated in systemic onset juvenile
chronic arthritis (142)
.
Multiple myeloma
Early studies into the action of IL-6 led investigators to name
this cytokine by a variety of functional synonyms, including B cell
differentiation factor and/or B cell-stimulating factor-2
(145)
. Consequently, it is not surprising to find that
IL-6 plays a major role in the proliferation and survival of clonal
malignant plasma cells in multiple myeloma (146
, 147)
.
Indeed, levels of IL-6 are significantly elevated in patients suffering
from this condition (148)
. An active role for the sIL-6R
in multiple myeloma was first presented by the observation that
physiological concentrations of sIL-6R increase the sensitivity of
myeloma cells to IL-6 (149)
. As a result, numerous studies
have now established that sIL-6R levels are significantly elevated in
serum from multiple myeloma patients (150
151
152
153)
, whereas
the extent of sIL-6R in circulation appears to correlate with the
severity/stage of multiple myeloma (151
152
153)
. Indeed,
circulating levels of sIL-6R may act as a useful prognosis marker,
since high serum sIL-6R concentrations were associated with patients
who died within 3 years of diagnosis (151)
.
Although IL-6 has been implicated in the proliferation and survival of
myeloma cells, a precise role for sIL-6R in these processes is far from
clear. In particular, no correlation could be made between sIL-6R and
IL-6 (as a myeloma cell growth factor) levels or thymidine kinase (as a
marker of cellular proliferation) activity within a population of 207
multiple myeloma patients (151)
. Even so, expansion of
myeloma cell lines has been reported to be promoted by a combination of
IL-6 and sIL-6R (154
, 155)
as well as by stimulatory
monoclonal anti-human gp130 antibodies (129)
. In this
latter study, the authors used these agonistic antibodies to develop a
SCID mouse model of human multiple myeloma. Human myeloma cells were
transplanted into the mouse peritoneum and tumor growth promoted by the
presence of anti-gp130 monoclonal antibodies (129)
. When
circulating levels of human sIL-6R were periodically tested within
these mice, elevated serum concentrations of sIL-6R were detected prior
to formation of a palpable tumor mass (129)
. In contrast,
elevated sgp130 levels were raised some 2–3 wk after the initial
observation of tumor expansion, suggesting that sIL-6R activity remains
unchecked during the early stages of tumor growth (129)
.
The significance of this finding remains to be determined.
Crohn’s disease
Crohn’s disease is a chronic inflammatory condition of the
gastrointestinal tract that typically manifests itself by the formation
of bowel strictures, ileus, and fistulas. Development of this condition
appears to be controlled by type-1 T-helper cells (Th1) and is
associated with elevated concentrations of Th1-polarizing cytokines
(e.g., IFN-). In addition, elevated concentrations of IL-6 and
sIL-6R have been described in both Crohn’s disease and ulcerative
colitis (156)
, suggesting a role for these mediators in
the pathogenesis of these conditions. Using a SCID mouse model of
colitis, it was recently demonstrated that treatment of mice with a
blocking anti-IL-6R monoclonal antibody significantly impaired disease
progression (157)
. Specifically, mice treated with an
anti-IL-6R antibody retained normal growth, whereas sham mice
administered with a nonimmune IgG control lost weight. This observation
was paralleled by suppression of colonic ICAM-1, VCAM-1, IFN-,
TNF-, and IL-1ß expression after anti-IL-6R antibody treatment
(157)
. These results point to a critical role for IL-6 in
the pathogenesis of colitis episodes and are supported by the finding
that sIL-6R in combination with IL-6 prevents T cell apoptosis in
experimental colitis (158)
. In particular, activity of the
[sIL-6R/IL-6] complex was associated with induced expression of the
anti-apoptotic genes bcl-2 and bcl-x1, while blockade of
sIL-6R-mediated signaling (using either anti-IL-6R monoclonal
antibodies or an inhibitory gp130-Fc fusion protein) promoted apoptosis
of lamina propria T cells (158)
. Furthermore, inhibition
of sIL-6R activity in several murine models of colitis suppressed the
clinical indices of disease progression (158)
. Thus,
therapeutic measures that target sIL-6R signaling may be of potential
importance in the treatment of Crohn’s disease.
|
QUESTIONS FOR THE FUTURE |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTIONS FOR THE FUTURECONCLUDING REMARKSREFERENCES |
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, the principle mechanism for coordinating
IL-6-mediated events would be through the release of sIL-6R.
Consequently, it is necessary for future studies to establish the
source of sIL-6R. It is essential to consider elevated sIL-6R levels in
terms of localized increases in sIL-6R concentrations as well as
systemic sIL-6R production. In cases of multiple myeloma, for instance,
it is apparent that systemically elevated sIL-6R levels have a
pronounced bearing on the outcome of the disease
(151
152
153)
. In contrast, sIL-6R levels in synovial fluid
from rheumatoid arthritis patients correlate with the extent of
leukocyte infiltration into the joint (75)
and with the
degree of bone destruction (57)
, suggesting that local
sIL-6R levels also contribute to disease progression. Thus, a better
appreciation of how sIL-6R concentrations are regulated is required. In
particular, determining the time frame of sIL-6R production during
disease progression is central to our understanding of when sIL-6R is
likely to participate in the disease process. In line with this, it is
necessary to consider not only temporal changes in the release of both
DS- and PC-sIL-6R, but also the effect sgp130 may have in the balance
between the agonistic properties of IL-6 and its soluble receptor and
the antagonistic function of sgp130.
Interleukin 6 has been proposed to act as both a pro- and
anti-inflammatory cytokine. For instance, examination of the regulatory
properties of IL-6 in local and systemic acute inflammation using
IL-6-/- mice reveals that the absence of IL-6
correlates with increased expression of TNF-, IFN-, GM-CSF, and
MIP-2 (the murine homologue of IL-8) and by significantly elevated
neutrophilia (11)
. Hence, IL-6 has the capacity to limit
neutrophil recruitment and suppress the activities of proinflammatory
mediators. In contrast, the degree of leukocyte infiltration in an s.c.
air pouch inflammation model has been reported to be mediated via
sIL-6R (73)
, whereas in vitro studies show that
the [sIL-6R/IL-6] complex has the capacity to enhance chemokine and
adhesion molecule expression (73
, 74)
. These findings
support a proinflammatory role for IL-6 and sIL-6R in disease
pathogenesis. Indeed, the detrimental consequences of their action have
now been documented in several in vivo models of arthritis
(136
137
138
139)
and chronic intestinal inflammation (157
, 158)
. Thus, it would appear that the assignment of inflammatory
properties to IL-6 depends on the clinical condition in which it acts.
As a result, it is essential that the biological processes controlled
by IL-6 alone be distinguished from those mediated via the sIL-6R.
|
CONCLUDING REMARKS |
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TOPABSTRACTINTERLEUKIN 6 AND INTERLEUKIN...THE SOLUBLE IL-6 RECEPTORACTIVITIES MEDIATED THROUGH THE...MECHANISMS OF SOLUBLE IL-6...DIFFERENTIAL RELEASE OF SOLUBLE...ANTAGONISM OF SIL-6R ACTIVITYSOLUBLE IL-6 RECEPTOR AND...QUESTI |
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