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*
Department of Cell Biology, University of Alabama, Birmingham, AL; and
Section of Pathophysiology, First Department of Medicine, University of Mainz, Mainz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, and
IFN-
. In this study, we investigated the influence of IL-6 on
astroglial adhesion molecule expression. IL-6, the soluble form of the
IL-6R (sIL-6R), or both IL-6 plus sIL-6R, had no effect on VCAM-1 or
ICAM-1 gene expression. Interestingly, the IL-6/sIL-6R complex
inhibited TNF--induced VCAM-1 gene expression but did not affect
TNF--induced ICAM-1 expression. The inhibitory effect of IL-6/sIL-6R
complex was reversed by the inclusion of anti-IL-6R and gp130 Abs,
demonstrating the specificity of the response. A highly active fusion
protein of sIL-6R and IL-6, covalently linked by a flexible peptide,
which is designated H-IL-6, also inhibited TNF--induced VCAM-1
expression. sIL-6R alone was an effective inhibitor of TNF--induced
VCAM-1 due to endogenous IL-6 production. These results indicate that
the IL-6 system has an unexpected negative effect on adhesion molecule
expression in glial cells and may function as an immunosuppressive
cytokine within the CNS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression (14, 15) and
inducing the expression of soluble TNF- receptors and the IL-1R
antagonist (16, 17). Studies have also documented an antiinflammatory
effect of IL-6 in animal models of endotoxemia, lung injury, T cell
activation-associated hepatic injury, and Theiler’s virus-induced
demyelination (18, 19, 20, 21).
Cells responsive to IL-6 express on their surface a low affinity
receptor (IL-6R) that does not have transducing activity. The
IL-6/IL-6R complex induces the homodimerization of a signal-transducing
component, gp130, leading to cytoplasmic signaling cascades that
activate components of the Janus Kinase (JAK)/STAT pathway,
particularly the activation of the transcription factor STAT-3 (for
review, see 1 . A soluble form of the IL-6R (sIL-6R) can be
generated by shedding of the membrane-bound receptor (22, 23) or by
mRNA alternative splicing (24). Since the transmembrane and cytoplasmic
regions of the IL-6R are not essential for signal transduction, sIL-6R
can form a complex with IL-6 in solution and associate with gp130,
thereby activating signal transduction. This ability of sIL-6R to
confer IL-6 responsiveness to cells devoid of the membrane-bound IL-6R,
but expressing gp130, has been referred to as
trans-signaling (for review, see 25 . The sIL-6R is
found in the serum, urine, synovial fluid, and cerebrospinal fluid
(CSF) of normal individuals (26), and augmented serum levels have been
documented in various autoimmune diseases (for review, see 25 .
There are numerous cellular sources of IL-6 within the CNS; these
include astrocytes, microglia, and neurons (27, 28, 29, 30, 31). However, the
function of IL-6 within the CNS, particularly on glial cells, has not
been well established. IL-6, in conjunction with the sIL-6R, can confer
IL-6 sensitivity to endothelial cells, resulting in the expression of
the adhesion molecules ICAM-1, VCAM-1, and E-selectin, as well as
chemokine production (10, 12). Astrocytes, the major glial cell type in
the CNS, can express ICAM-1 and VCAM-1 upon stimulation with the
proinflammatory cytokines TNF-, IL-1ß, and IFN- (32, 33, 34, 35).
Adhesion molecules such as ICAM-1 and VCAM-1 are increased in the CNS
particularly during times of inflammation and are thought to contribute
to extravasation of leukocytes across the blood-brain barrier (BBB) and
into CNS parenchyma (36, 37). In disease states, ICAM-1 and VCAM-1
expression have been detected on the endothelial cells comprising the
BBB, as well as astrocytes and microglia, which contribute to the
structural integrity of the BBB (37).
In this study, we have examined potential functional effects of IL-6 on
human astrocytes and human astroglioma cells, with an emphasis on
modulation of ICAM-1 and VCAM-1 expression by these cells. IL-6
treatment of astroglioma cells/astrocytes did not induce either ICAM-1
or VCAM-1 expression, and the inclusion of sIL-6R did not change this
response. However, the IL-6/sIL-6R complex did inhibit TNF--induced
VCAM-1 expression, while not affecting TNF--induced ICAM-1
expression. A comparable level of VCAM-1 inhibition was achieved using
a highly active fusion protein of sIL-6R and IL-6 that is designated
H-IL-6 (38). Furthermore, inclusion of sIL-6R alone to TNF--treated
astroglioma cells resulted in inhibition of VCAM-1 expression due to
endogenous IL-6 production by these cells. These findings indicate that
sIL-6R can modulate the responsiveness of astrocytes to IL-6.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U373-MG human astroglioma cells were maintained in MEM with 1 mM Earles BSS media with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS. For passage, monolayers were rinsed with PBS and then dislodged by trypsinization (0.25% trypsin, 0.02% EDTA). Human fetal astrocytes were prepared as previously described (39). Briefly, human fetal brain tissue between 8 and 15 wk gestation from legal curettage were minced and passed through a 19-gauge needle. Cells were plated at 1 x 106 cells/ml in Eagle’s MEM with 10% FBS, 2 mM glutamine, penicillin/streptomycin, and fungizone. Cells were passaged for 4 to 6 wk before use. Astrocyte purity was determined by staining for glial fibrillary acidic protein (GFAP); the astrocyte cultures were >92% GFAP positive (39). Biopsy material from patients undergoing surgery to treat intractable epilepsy were used to prepare human adult astrocyte cultures, as previously described (40). Astrocytes were obtained after 30 days in culture and were 87 to 93% GFAP positive (40). Comparable results were obtained with fetal or adult astrocyte cultures.
Reagents
Human recombinant TNF- (sp. act. 5.6 x 107
U/mg) was the generous gift of Genentech (South San Francisco, CA),
human recombinant sIL-6R was purchased from R&D Systems (Minneapolis,
MN), and human recombinant IL-6 was from Collaborative Biomedical
Products (Bedford, MA) and R&D Systems. Hybrid-IL-6 (H-IL-6) was
prepared as previously described (38). Neutralizing monoclonal
anti-human IL-6 Ab (clone 6708.111), neutralizing monoclonal
anti-human sIL-6R Ab (clone 17506.11), and monoclonal
anti-human gp130 neutralizing Ab (clone 28126.111) were from R&D
Systems. Anti-human VCAM-1 Abs (clone 1G11B1, IgG1 isotype) were
obtained from Serotec (Washington, DC). Monoclonal Ab to GFAP was
obtained from Boehringer Mannheim (Indianapolis, IN). Goat
anti-mouse H and L chain FITC-labeled Abs were from Southern
Biotechnology Associates (Birmingham, AL). Mouse anti-human ICAM-1
mAb (IgG1) conjugated to phycoerythrin (PE) was purchased from
PharMingen (San Diego, CA), and mouse IgG1 conjugated to phycoerythrin
was from Southern Biotechnology Associates. Polyclonal STAT-3 and
STAT-1 antisera were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA), and monoclonal antiphosphotyrosine Ab 4G10 was from Upstate
Biotechnology (Lake Placid, NY).
Immunoprecipitation and Western blot analysis
U373-MG cells/human astrocytes were untreated or treated with
IL-6, IL-6/sIL-6R, H-IL-6, or IFN- for 30 min. Cell lysates from
control or treated cells were prepared as previously described (41),
and 0.5 to 1 mg of protein was precleared with normal rabbit serum
before incubation with polyclonal antisera against STAT-3 or STAT-1
(5 µl). Protein G-agarose (50 µl) was added for 2 h at 4°C.
The immunoprecipitates were washed 3 to 5 times with lysis buffer,
eluted from the agarose beads by boiling in 2x SDS-sample buffer, and
subjected to 6% SDS-PAGE. Proteins were then transferred to
nitrocellulose and probed with monoclonal anti-phosphotyrosine Ab
4G10 (1 µg/ml), as previously described (41). Enhanced
chemiluminescence (ECL) was used for detection of bound Ab. Membranes
were stripped at 50°C in buffer containing 100 mM 2-ME, 2% SDS, 62.5
mM Tris-HCl (pH 6.7) with occasional shaking, and reprobed for either
STAT-3 or STAT-1 protein.
RNA isolation, riboprobes, and RNase protection assay (RPA)
Total cellular RNA was isolated from cell monolayers that were incubated for 12 h with the different cytokines, as previously described (42). Briefly, cells were washed once with PBS and lysed directly in the culture dish. RNA was extracted with guanidinium isothiocyanate and phenol and precipitated with ethanol. A pBluescript SK(±) vector (Stratagene, La Jolla, CA) containing a fragment of the human VCAM-1 cDNA (bp 1307–2811) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The vector was linearized with SpeI, which digests within the VCAM-1 cDNA insert. In vitro transcription of this linearized vector with T7 RNA polymerase results in a 449-bp antisense RNA probe, and the protected fragment is 427 nucleotides (35). A pAMP-1 vector (Life Technologies, Grand Island, NY) containing a fragment of the human GAPDH cDNA (bp 43–531) was linearized with NcoI, which digests within the GAPDH cDNA insert. In vitro transcription of this linearized vector with T7 RNA polymerase results in a 290-bp antisense RNA probe, and the protected fragment is 230 nucleotides. GAPDH mRNA was utilized as a "housekeeping gene" since its levels are not affected by cytokine treatment.
RNase protection assay (RPA) was conducted with an RPA kit according to the manufacturer’s instructions (Ambion, Austin, TX), as previously described (35). Briefly, 15 µg of total cellular RNA was hybridized with VCAM-1 (2.5 x 104 cpm) and GAPDH (2.0 x 104 cpm) riboprobes at 42°C overnight in 20 µl of 40 mM PIPES (pH 6.4), 80% deionized formamide, 400 mM NaOAc, and 1 mM EDTA. The hybridized mixture was then treated with RNase A/T1 (1:200 dilution in 200 µl of RNase digestion buffer) at room temperature for 1 h, and RNA was precipitated and analyzed by 5% denaturing (8 M urea) polyacrylamide gel electrophoresis. The gels were exposed to x-ray film, and quantitation of protected RNA fragments was performed by scanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Values for VCAM-1 were normalized to GAPDH mRNA levels for each experimental condition.
Analysis of VCAM-1 and ICAM-1 protein expression by immunofluorescence flow cytometry
Human U373-MG astroglioma cells (2 x 105/well)
or primary human astrocytes (5 x 105/well) were
plated in six-well (35-mm2) plates (Costar, Cambridge, MA)
and grown to 
80% confluency. At that time, the original medium was
aspirated off; cells were washed with PBS, and then fresh serum-free
media (1 ml) was added to wells. Duplicate wells were treated with
medium alone, cytokines, sIL-6R, or H-IL-6 for 48 h. Cells were
trypsinized, suspended in PBS containing 5% FBS and 0.02% azide,
stained with anti-human VCAM-1 (1:500 dilution), washed twice, and
then stained with FITC-labeled goat anti-mouse Abs (1:100
dilution). Cells were also stained for ICAM-1 as previously described
(43). After washing three times, cells were fixed in 1%
paraformaldehyde and analyzed on the FACStar (Becton Dickinson,
Mountain View, CA) for VCAM-1 and ICAM-1 expression. We have previously
determined that trypsinization does not affect cell surface expression
of VCAM-1 or ICAM-1 (35, 43). Negative controls were incubated with an
isotype-matched (IgG1) control mAb. Ten thousand cells were analyzed
for each sample. VCAM-1 is expressed as the percentage of positive
cells, and ICAM-1 expression is presented in arbitrary units and is
calculated as the percentage of positive cells x mean
fluorescence intensity (MFI).
Measurement of IL-6 activity
U373-MG cells were incubated with medium alone, TNF-, sIL-6R,
or TNF- plus sIL-6R for 24 h; then supernatants were collected.
IL-6 activity in culture supernatants was determined in a biologic
assay using the IL-6-dependent B cell hybridoma B9, as previously
described (30). Briefly, B9 cells (2 x 103
cells/well) were plated in 96-well microtiter plates; serial dilutions
of conditioned medium and recombinant human IL-6 (used as a standard)
were added and incubated at 37°C for 72 h. Triplicate cultures
were set up for each condition. After this incubation, B9 cell growth
was assessed using the MTT assay, as described previously (30).
Measurement of sVCAM-1 production
U373-MG cells were incubated with medium alone, TNF-, sIL-6R,
or TNF- plus sIL-6R for 72 h; then culture supernatants were
collected, centrifuged, and stored at -70°C until use. sVCAM-1 in
culture supernatants was quantitated using a dual-Ab solid phase ELISA
(R&D Systems), according to the manufacturer’s instructions. Briefly,
supernatants were diluted 1:2 in the dilution buffer provided with the
ELISA kit. The diluted supernatants and recombinant sVCAM-1 (as a
standard) were applied to the wells. Unbound protein was removed by
washing, and horseradish peroxidase-conjugated anti-VCAM-1 Ab was
added. After the color reaction with substrate, the optical density was
recorded at 450-nm wavelength with an automated ELISA reader. sVCAM-1
concentrations were determined in relation to the standard curve
generated with recombinant sVCAM-1 provided by the
manufacturer.
Statistical analysis
Levels of significance for comparisons between samples were determined using Student’s t test distribution.
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Results |
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As an initial measure of responsiveness to IL-6, we tested the
ability of IL-6, IL-6/sIL-6R, or H-IL-6 to induce tyrosine
phosphorylation of STAT-3 in the U373-MG astroglioma line and human
astrocytes. As shown in Figure 1
A, stimulation of U373-MG
cells with IL-6 alone has a minimal effect on STAT-3 phosphorylation
(lane 2). These results suggest that the astroglioma
cells constitutively express very low levels of the IL-6R, which was
confirmed by FACS analysis (data not shown). However, when sIL-6R was
used together with IL-6, a clear induction of STAT-3 phosphorylation
was observed (lane 3). As well, we tested H-IL-6,
which is a fusion protein of sIL-6R and IL-6, linked by a flexible
peptide chain (38). H-IL-6 has been determined to be fully active at a
100-fold lower concentration than the combination of unlinked IL-6 and
sIL-6R (38). H-IL-6 at 10 ng/ml also stimulates STAT-3 tyrosine
phosphorylation in U373-MG cells (lane 4), at a level
comparable to IL-6/sIL-6R (lane 3). The blot was
stripped and reprobed for STAT-3 to determine amounts of STAT-3 protein
present in each lane (Fig. 1A, bottom panel). In
some cell types, IL-6 is able to induce tyrosine phosphorylation of
STAT-1 (for review, see 1 . U373-MG cells were stimulated with
IFN- (100 U/ml) or IL-6/sIL-6R; then STAT-1 phosphorylation was
examined. IFN- stimulation results in STAT-1 tyrosine
phosphorylation (Fig. 1B, lane 2), while
IL-6/sIL-6R is without effect (lane 3). The blot was
stripped and reprobed for STAT-1 protein (Fig. 1B,
bottom panel). Similar results were obtained in primary
human astrocytes. IL-6 alone did not stimulate STAT-3 phosphorylation
(Fig. 1C, lane 2), while the combination of
IL-6/sIL-6R did (lane 3). Furthermore, IL-6 or
IL-6/sIL-6R did not activate STAT-1 (lanes 5 and
6). These results collectively indicate that IL-6/sIL-6R
complexes activate STAT-3, but not STAT-1, in astroglioma cells and
primary human astrocytes.
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, IL-1ß, and IFN- (35, 43, 44). In U373-MG
cells, IL-6 alone or IL-6 plus sIL-6R had no effect on either VCAM-1 or
ICAM-1 expression, compared with the positive control of TNF- (Table I). IL-6 in conjunction with TNF-
resulted in minimal inhibition of VCAM-1 and ICAM-1 expression (9%).
Interestingly, the combination of IL-6 plus sIL-6R significantly
inhibited TNF--induced VCAM-1 expression, while having no effect on
TNF--induced ICAM-1 (Table I). A representative histogram of the
FACS results is shown in Figure 2.
TNF- treatment enhances the percentage of VCAM-1-positive cells
(Fig. 2A, Profile 2), and inclusion of IL-6/sIL-6R inhibits
the number of VCAM-1 positive cells (Profile 3). ICAM-1 is
constitutively expressed on most cells (Fig. 2B, Profile 1),
and TNF- treatment enhances ICAM-1 expression (Profile 2).
IL-6/sIL-6R complexes do not modulate TNF--enhanced ICAM-1
expression (Profile 3). IL-6 was tested in the range of 0.5 to 100
ng/ml in the presence of sIL-6R at 100 ng/ml; optimal inhibition was
observed using IL-6 at 5 to 10 ng/ml (data not shown). These results
indicate that IL-6/sIL-6R complexes can activate signaling in
astroglioma cells through gp130, leading to a selective inhibition of
TNF--induced VCAM-1 expression. Comparable results were observed
using primary human astrocytes; TNF- (50 ng/ml) induces VCAM-1
expression on astrocytes (0.9% to 45.5 ± 7.8% positive), while
IL-6 (5 ng/ml)/sIL-6R (100 ng/ml) reduces TNF--induced VCAM-1
expression to 19.3 ± 2.4% positive (58% inhibition).
TNF--induced ICAM-1 expression was not affected by IL-6/sIL-6R (data
not shown).
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plus IL-6 (5 ng/ml)/sIL-6R
(100 ng/ml) for 48 h, or sIL-6R was incubated with anti-sIL-6R
Ab (10 µg/ml) for 30 min at room temperature, then added to the cells
in conjunction with TNF- plus IL-6 for 48 h. The inclusion of
Ab against the sIL-6R reversed the inhibitory effect of IL-6/sIL-6R on
TNF--induced VCAM-1 expression by 80%, while anti-gp130 Ab
at 1 µg/ml reversed the inhibition by 70%. The Abs alone had no
effect on VCAM-1 expression (data not shown). These results indicate
that prevention of signaling through the IL-6R and the
signal-transducing protein gp130 blocks IL-6/sIL-6R inhibition of
TNF--induced VCAM-1 expression. Inhibition of VCAM-1 expression by H-IL-6
The fusion protein H-IL-6 was utilized in our studies for effects
on VCAM-1 expression. U373-MG cells were incubated with increasing
concentrations of H-IL-6 (0.1–10 ng/ml) alone or in conjunction with
TNF-; then VCAM-1 expression was examined. As shown in Figure 3, H-IL-6 alone did not affect VCAM-1
expression but inhibited TNF--induced expression of VCAM-1 in a
dose-dependent manner. The extent of inhibition with 10 ng/ml of H-IL-6
(78%) was greater than that obtained with IL-6/sIL-6R (68%
inhibition). The inhibitory effect of H-IL-6 was abrogated when cells
were preincubated with 2 µg/ml of anti-gp130 Ab, then exposed to
H-IL-6 plus TNF- (Fig. 3). H-IL-6, similar to IL-6/sIL-6R, did not
inhibit TNF--induced ICAM-1 expression (data not shown). These
results indicate that the H-IL-6 fusion protein is fully functional for
inhibition of TNF--induced VCAM-1 expression.
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Our initial experiments tested the ability of either IL-6 or
IL-6/sIL-6R to inhibit VCAM-1 expression. We next wished to determine
whether sIL-6R alone influenced TNF--induced VCAM-1 expression. For
these experiments, cells were incubated with TNF- plus sIL-6R or
IL-6/sIL-6R; then VCAM-1 expression was assessed. The inclusion of
sIL-6R inhibited TNF--induced VCAM-1 expression by 65%, and
addition of IL-6 with sIL-6R minimally potentiated the ability of
sIL-6R to inhibit VCAM-1 (Table II). The
effect of sIL-6R was dose dependent, with maximal inhibition obtained
using 50 to 100 ng/ml of sIL-6R (data not shown). These results
indicate that sIL-6R alone is strongly inhibitory for TNF--induced
VCAM-1 expression. We have previously determined that astrocytes
produce IL-6 in response to TNF- stimulation (31). It was possible
that TNF- stimulation of U373-MG astroglioma cells resulted in IL-6
production and that the presence of sIL-6R then mediated the inhibitory
effect of IL-6. To determine whether this was the case, we included a
neutralizing Ab to human IL-6 (2 µg/ml) in the cultures containing
sIL-6R plus TNF-. The inclusion of the anti-IL-6 Ab completely
reversed the inhibitory effect of sIL-6R (Table II). These results
suggest that IL-6 is produced during incubation of cells with TNF-
and sIL-6R, and blocking the interaction of IL-6 with sIL-6R prevents
inhibition of VCAM-1 expression. To formally demonstrate this, IL-6
protein was measured from culture supernatants obtained from cells
stimulated with TNF-, sIL-6R, or both. TNF- induced IL-6 protein
production, while sIL-6R alone was without effect. However, a strong
synergistic effect of TNF- plus sIL-6R for enhanced IL-6 expression
was noted in U373-MG astroglioma cells (Table III).
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-induced release of sVCAM-1 is inhibited by sIL-6R
Another aspect of adhesion molecule expression is the generation
of soluble forms of these molecules, as has been described for sVCAM-1,
sICAM-1, and sE-selectin (45, 46, 47). We performed experiments to assess
whether TNF--induced release of sVCAM-1 could also be affected by
sIL-6R. U373-MG cells were stimulated in the presence of TNF-,
sIL-6R, or TNF- plus sIL-6R for 72 h; then supernatants were
collected and analyzed for levels of sVCAM-1. TNF- induced the
release of sVCAM-1, and sIL-6R inhibited this response by 42% (Fig. 4). Thus, sVCAM-1 that is generated in
the presence of TNF- can be partially inhibited by the inclusion of
sIL-6R.
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-induced VCAM-1 mRNA expression
We next examined whether IL-6/sIL-6R could modulate expression of
VCAM-1 mRNA. As illustrated in Figure 5,
U373-MG cells are constitutively negative for VCAM-1 mRNA
(lane 1), while TNF- induces expression
(lane 2). Inclusion of IL-6 alone has no effect on
TNF--induced VCAM-1 expression (lane 3), while
sIL-6R inhibits TNF--induced VCAM-1 mRNA levels by 40%
(lane 4). The addition of IL-6 with sIL-6R
(lane 5) slightly potentiates the inhibitory effect
of sIL-6R (45%). These findings at the mRNA level are comparable to
what was observed at the VCAM-1 protein level (Tables I and II).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and C). The
inclusion of the sIL-6R restored the ability of IL-6 to activate STAT-3
(Fig. 1, A and C), indicating that astroglioma
cells/astrocytes lack sufficient IL-6R subunits to transmit
IL-6-induced signals. This finding has also been demonstrated in human
astrocytes for induction of the 1-antichymotrypsin (ACT)
gene (48). IL-6 could not induce ACT expression in astrocytes, while
cotreatment of cells with sIL-6R and IL-6 resulted in potent
stimulation of ACT (48).
In this study, we demonstrate another function of sIL-6R and IL-6 on
human astrocytes, that being the regulation of VCAM-1 expression. Our
findings indicate that IL-6/sIL-6R complexes or the fusion protein
H-IL-6 do not induce either ICAM-1 or VCAM-1 expression; rather they
can inhibit TNF--induced VCAM-1 expression, while having no effect
on TNF--induced ICAM-1 expression (Table I; Figure 2). These results
were surprising given the literature that IL-6/sIL-6R complexes
up-regulate ICAM-1 and VCAM-1 expression in endothelial cells and HepG2
cells (10, 12, 13). IL-6 activates ICAM-1 transcription in HepG2 cells
by activating both STAT-1 and STAT-3 (13), while the mechanism for
VCAM-1 induction is unknown. Our results in Figure 1 demonstrate that
IL-6/sIL-6R complexes can activate STAT-3, but not STAT-1, in
astroglioma cells/astrocytes. This may explain why ICAM-1 gene
expression is not induced in these cells by IL-6/sIL-6R. Our findings
demonstrate a selective inhibitory effect of IL-6/sIL-6R on
TNF--induced VCAM-1 expression, with inhibition observed at both the
mRNA and protein level. The regulatory elements that mediate
TNF--induced ICAM-1 and VCAM-1 gene expression are distinct (for
review, see 51 . TNF- mediated transcriptional activation of
VCAM-1 requires two tandem binding sites for NF-
B located in the
VCAM-1 promoter at positions -73 and -58, as well as the binding site
for IFN regulatory factor-1 (IRF-1) (51, 52). ICAM-1 transcription
induced by TNF- involves activation of NF-B and
CCAAT/enhancer-binding protein (C/EBP) transcription factors
(for review, see 51 . While it is clear in human astrocytes that
the IL-6/sIL-6R signal is not sufficient to induce expression of either
VCAM-1 or ICAM-1, the IL-6/sIL-6R complex may selectively inhibit
VCAM-1 expression by inducing negative repressors or interfering with
the NF-B/IFN regulatory factor-1-mediated induction of VCAM-1.
Interestingly, the results obtained in this study contrast with our
previous findings in rat astrocytes (42). In those cells, IL-6 alone
was a potent inhibitor of TNF-, IL-1ß, or IFN--induced ICAM-1
expression (VCAM-1 expression was not tested). Thus, while human
astrocytes need the inclusion of sIL-6R to respond to IL-6 (Ref. 48 and
this study), IL-6 alone is able to induce signal transduction events in
rat astrocytes (14, 42). Furthermore, the VCAM-1 gene is the target of
IL-6-mediated inhibition in human astrocytes while ICAM-1 expression is
unaffected. These results collectively demonstrate that the
intracellular targets of the IL-6/sIL-6R/gp130 signaling pathways
differ between rat and human astrocytes.
Astrocytes are well documented to produce IL-6 upon different
immunogenic stimuli, including TNF- stimulation (30, 31, 53). Our
results indicate that TNF- treatment of astroglioma cells results in
the secretion of biologically active IL-6 (Table III) and that, upon
the addition of sIL-6R, these cells can utilize the endogenously
produced IL-6 for inhibition of VCAM-1 expression (Table II). As well,
IL-6 production increased when cells were incubated with TNF- plus
sIL-6R, suggesting that endogenously produced IL-6 can complex with
sIL-6R, leading to a positive feedback loop for increased IL-6
production. IL-6/sIL-6R induction of IL-6 has been documented in other
cell types, including neurons (27), endothelial cells (10, 12),
fibroblasts (54), and Kaposi’s sarcoma cells (55). These findings
suggest that, when sIL-6R is present, inducers of IL-6 such as TNF-
can initiate an autocrine amplification pathway of astrocyte
activation.
It appears that, for many cell types, including human astrocytes, the
limitation in mounting a biologic response to IL-6 is the lack of the
IL-6R subunit. sIL-6R has been detected in the CSF of normal
patients (0.8–1.67 ng/ml) (26, 56), and levels do not appear to
increase in disease states such as Alzheimer’s (48, 56). However,
within the normal CNS, IL-6 is not constitutively present but is
readily detected in the CSF of patients with multiple sclerosis (57, 58), traumatic brain injury (59, 60), and Alzheimer’s disease (61). It
should be noted that the literature regarding elevated IL-6 levels in
Alzheimer’s disease is controversial since other groups have not
observed an increase in CSF levels of IL-6 (56, 62). Thus, the limiting
factor for IL-6 responsiveness during various disease states may be
IL-6 itself, given that gp130 is constitutively expressed in the brain
(63, 64) and that sIL-6R levels are readily detectable in both normal
and disease states (26). sIL-6R, in conjunction with IL-6, may provide
the stimulus for astrocyte activation, leading to diverse responses,
such as astrogliosis, ACT production, IL-6 production, and inhibition
of VCAM-1 expression (Refs. 48 and 65, and this study). It will be of
importance to determine whether the IL-6/sIL-6R complex affects other
aspects of astrocyte function. As well, the source of sIL-6R within the
CNS is unknown, as is the mechanism by which sIL-6R is produced within
this site. Future studies will investigate potential cellular sources
of sIL-6R in the CNS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Department of Cell Biology, Room 350 MCLM, University of Alabama, Birmingham, Alabama 35294. E-mail address:
3 Abbreviations used in this paper: CNS, central nervous system; ACT, 1-antichymotrypsin; BBB, blood-brain barrier; CSF, cerebrospinal fluid; H-IL-6, hybrid IL-6; RPA, ribonuclease protection assay; sIL-6R, soluble form of IL-6R; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein.
Received for publication May 1, 1998. Accepted for publication June 25, 1998.
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References |
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involves binding of distinct factors to a palindromic response element. J. Biol. Chem. 269:21146. expression by the cytokines TGF-ß, IL-6 and IL-10. Int. J. Dev. Neurosci. 13:341.[Medline]
. J. Immunol. 152:841.[Abstract]
induces adhesion molecule expression on human fetal astrocytes. J. Exp. Med. 176:1631., interleukin-1ß, and interferon- in astrocytes. J. Neuroimmunol. 51:209.[Medline]
-induced class II MHC gene expression does not involve inhibition of phosphorylation of JAK1, JAK2, or STAT1 or modification of IFNEX expression. J. Immunol. 154:610.[Abstract]
-bearing lymphocyte clones. J. Immunol. 154:1069.[Abstract]
1-antichymotrypsin expression in human cortical astrocytes. J. Biol. Chem. 273:4112.B and cytokine-inducible enhancers. FASEB J. 9:899.[Abstract]
B as a transcriptional activator of vascular cell adhesion molecule 1. Mol. Cell. Biol. 15:2558.[Abstract]
induces IL-6 production by astrocytes but not by microglia. Brain Res. 583:296.[Medline]
) is a potent growth factor for AIDS-associated Kaposi’s sarcoma (KS) cells; the soluble form of gp130 is antagonistic for sIL-6R-induced AIDS-KS cell growth. Int. Immunol. 8:595.This article has been cited by other articles:
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  S. J. Lee, H. Qin, and E. N. Benveniste Simvastatin inhibits IFN-{gamma}-induced CD40 gene expression by suppressing STAT-1{alpha} J. Leukoc. Biol., August 1, 2007; 82(2): 436 - 447. [Abstract] [Full Text] [PDF]
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 S. A. Jones Directing Transition from Innate to Acquired Immunity: Defining a Role for IL-6 J. Immunol., September 15, 2005; 175(6): 3463 - 3468. [Abstract] [Full Text] [PDF]
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R. M. McLoughlin, S. M. Hurst, M. A. Nowell, D. A. Harris, S. Horiuchi, L. W. Morgan, T. S. Wilkinson, N. Yamamoto, N. Topley, and S. A. Jones Differential Regulation of Neutrophil-Activating Chemokines by IL-6 and Its Soluble Receptor Isoforms J. Immunol., May 1, 2004; 172(9): 5676 - 5683. [Abstract] [Full Text] [PDF]
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M. A. Nowell, P. J. Richards, S. Horiuchi, N. Yamamoto, S. Rose-John, N. Topley, A. S. Williams, and S. A. Jones Soluble IL-6 Receptor Governs IL-6 Activity in Experimental Arthritis: Blockade of Arthritis Severity by Soluble Glycoprotein 130 J. Immunol., September 15, 2003; 171(6): 3202 - 3209. [Abstract] [Full Text] [PDF]
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J. G. Bode, J. Schweigart, J. Kehrmann, C. Ehlting, F. Schaper, P. C. Heinrich, and D. Haussinger TNF-{alpha} Induces Tyrosine Phosphorylation and Recruitment of the Src Homology Protein-Tyrosine Phosphatase 2 to the gp130 Signal-Transducing Subunit of the IL-6 Receptor Complex J. Immunol., July 1, 2003; 171(1): 257 - 266. [Abstract] [Full Text] [PDF]
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D. Deon, S. Ahmed, K. Tai, N. Scaletta, C. Herrero, I.-H. Lee, A. Krause, and L. B. Ivashkiv Cross-Talk Between IL-1 and IL-6 Signaling Pathways in Rheumatoid Arthritis Synovial Fibroblasts J. Immunol., November 1, 2001; 167(9): 5395 - 5403. [Abstract] [Full Text] [PDF]
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V. Marin, F. A. Montero-Julian, S. Gres, V. Boulay, P. Bongrand, C. Farnarier, and G. Kaplanski The IL-6-Soluble IL-6R{alpha} Autocrine Loop of Endothelial Activation as an Intermediate Between Acute and Chronic Inflammation: an Experimental Model Involving Thrombin J. Immunol., September 15, 2001; 167(6): 3435 - 3442. [Abstract] [Full Text] [PDF]
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 K. Naruishi, S. Takashiba, F. Nishimura, H.-H. Chou, H. Arai, H. Yamada, and Y. Murayama Impairment of Gingival Fibroblast Adherence by IL-6/sIL-6R Journal of Dental Research, May 1, 2001; 80(5): 1421 - 1424. [Abstract] [PDF]
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 S. A. JONES, S. HORIUCHI, N. TOPLEY, N. YAMAMOTO, and G. M. FULLER The soluble interleukin 6 receptor: mechanisms of production and implications in disease FASEB J, January 1, 2001; 15(1): 43 - 58. [Abstract] [Full Text] [PDF]
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S. T. Ahmed and L. B. Ivashkiv Inhibition of IL-6 and IL-10 Signaling and Stat Activation by Inflammatory and Stress Pathways J. Immunol., November 1, 2000; 165(9): 5227 - 5237. [Abstract] [Full Text] [PDF]
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S. J. Lee, K. Drabik, N. J. Van Wagoner, S. Lee, C. Choi, Y. Dong, and E. N. Benveniste ICAM-1-Induced Expression of Proinflammatory Cytokines in Astrocytes: Involvement of Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways J. Immunol., October 15, 2000; 165(8): 4658 - 4666. [Abstract] [Full Text] [PDF]
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J. Wang, R. J. Homer, L. Hong, L. Cohn, C. G. Lee, S. Jung, and J. A. Elias IL-11 Selectively Inhibits Aeroallergen-Induced Pulmonary Eosinophilia and Th2 Cytokine Production J. Immunol., August 15, 2000; 165(4): 2222 - 2231. [Abstract] [Full Text] [PDF]
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 T. Kielian and W. F. Hickey Proinflammatory Cytokine, Chemokine, and Cellular Adhesion Molecule Expression during the Acute Phase of Experimental Brain Abscess Development Am. J. Pathol., August 1, 2000; 157(2): 647 - 658. [Abstract] [Full Text] [PDF]
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M. C.-J. Cheeran, S. Hu, G. Gekker, and J. R. Lokensgard Decreased Cytomegalovirus Expression Following Proinflammatory Cytokine Treatment of Primary Human Astrocytes J. Immunol., January 15, 2000; 164(2): 926 - 933. [Abstract] [Full Text] [PDF]
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 M. Molnick and T. Jaskoll Mouse Submandibular Gland Morphogenesis: a Paradigm for Embryonic Signal Processing Critical Reviews in Oral Biology & Medicine, January 1, 2000; 11(2): 199 - 215. [Abstract] [Full Text] [PDF]
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 N. J. Van Wagoner, J.-W. Oh, P. Repovic, and E. N. Benveniste Interleukin-6 (IL-6) Production by Astrocytes: Autocrine Regulation by IL-6 and the Soluble IL-6 Receptor J. Neurosci., July 1, 1999; 19(13): 5236 - 5244. [Abstract] [Full Text] [PDF]
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