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Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Serge Rivest, Laboratory of Molecular Endocrinology, CHUL Research Center, Department of Anatomy and Physiology, Laval University, 2705 boulevard Laurier, Québec, Canada G1V 4G2. E-mail: serge.rivest{at}crchul.ulaval.ca
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Introduction |
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2-macroglobulin, and
1-acid glycoprotein (2). In
addition to be produced by both systemic lymphoid and nonlymphoid
cells, IL-6 protein and messenger RNA (mRNA) have been found in
specific populations of cells in the central nervous system (CNS)
during different experimental conditions. One of these stimuli is the
ip or iv administration of the endotoxin lipopolysaccharide (LPS) that
caused a profound transcriptional activation of the gene encoding the
cytokine in the choroid plexus (chp) and the circumventricular organs
(CVOs), structures devoid of blood-brain barrier (4). This
phenomenon is of particular interest, as it provides evidence that IL-6
may be secreted in the cerebrovascular spinal fluid (CSF) and reach its
receptor subunits, which are widely distributed throughout the neural
tissue (4) to influence different neurophysiological
functions (for a review, see Ref. 5). The first step in the induction of the transduction signals by IL-6 is the binding of the ligand to its IL-6 receptor subunit (IL-6R), which is either located at the cell surface or present in a soluble form in the liquids of the organism. The association of these two molecules with the membrane subunit gp130 forms a high affinity complex that triggers specific transduction signals, namely the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Once activated, the STAT proteins may activate different genes in combining their SH2 domains and forming homodimers (6, 7). The JAK/STAT signaling events are inhibited by at least two intracellular systems to avoid exaggerated responses. The first is the internalization of the IL-6/IL-6R/gp130 complex and its degradation by specific enzymes (8, 9, 10). This process does not require activation of the JAK kinases and is transduced by a degradation of the receptor at the level of the cell surface. The second involves activation of the transduction signals and the de novo production of inhibitory proteins that prevent phosphorylation of the transcription factor STAT and activation of the mitogen-activated protein kinases by interacting with the catalytic domain of the JAK kinases (11). These cytokine-inducible proteins, suppressors of cytokine signaling (SOCS-1 to SOCS-7) and cytokine-inducible SH2 protein (CIS), are rapidly induced by IL-6 and other members sharing the gp130 receptor subunit (12, 13). The SOCS proteins are characterized by a highly conserved carboxyl-terminal SOCS box motif that is preceded by an SH2 domain (13). Although SOCS-1 and SOCS-3 have potent activity for the inhibition of IL-6 signaling, the other members of the SOCS family and CIS have little or no impact (for a review, see Ref. 14). Both SOCS-1 and SOCS-3 act by preventing cytokine-dependent activation of the JAK/STAT pathway, but the intracellular mechanisms involved in these effects are quite different. SOCS-1 inhibits the intrinsic kinase activity by interacting with the catalytic domain of the JAK kinases (especially the JAK2 kinase), whereas SOCS-3 prevents IL-6 signaling at steps distal to JAK activation, i.e. recruitment of STAT factors and/or via binding to the tyrosine-phosphorylated receptor (15). Indeed, recent studies have shown that SOCS-3 is unable to inhibit directly either JAK1 or JAK2 kinase activity (16).
Elegant studies have recently reported that circulating leptin has the ability to induce specific expression of SOCS-3 mRNA in hypothalamic nuclei and that this inhibitory protein acts as an endogenous blocker for the leptin receptor-mediated signal transduction in mammalian cell lines (17). Interestingly, the leptin receptor is a member of the gp130 family, the signal-transducing subunit of the IL-6 cytokine receptor family (18, 19). Expression of this inhibitory protein may therefore be a very useful tool to investigate the signaling events occurring in the brain during immunogenic stimuli that involve IL-6. The present study was designed to identify the fine distribution of the SOCS-3 mRNA throughout rat and mouse brains under basal conditions and after different systemic immunogenic treatments. Intraperitoneal injection of the bacterial endotoxin LPS and im turpentine insult were used as models of immune challenge capable of inducing the release of IL-6 in the circulation. On the other hand, IL-6-deficient mice and their wild-type littermates were used to determine whether SOCS-3 expression was dependent on IL-6 production in response to the bacterial endotoxin. We also tested the hypothesis that preinduction of IL-6 receptors during the acute phase response is a needed transient mechanism, allowing circulating IL-6 to trigger SOCS-3 transcription. We demonstrate here the fine distribution of the gene encoding SOCS-3 protein in specific cellular populations of the brain that express both IL-6 receptor subunits during systemic inflammation. Endogenous production of the cytokine appears to be an essential prerequisite, as LPS-induced SOCS-3 transcription is prevented in microvascular-associated elements in IL-6-deficient mice. Finally, we provide evidence that expression of this inhibitor of the JAK/STAT signaling molecules is following the necessary preinduction of IL-6 receptors during the time-related events that take place in the acute phase reaction.
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Materials and Methods |
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Intraperitoneal injection of the endotoxin LPS in rats
In the morning (at 0900 h), rats received an ip injection
of the bacterial LPS (250 µg/100 g BW dissolved in 300 µl 0.9%
sterile pyrogen-free saline; LPS from Escherichia coli,
serotype 055:B5, catalogue no. L-2880, lot no. 56H4096,
Sigma, Oakville, Canada) and were killed 1, 3, 6, 12, or
24 h thereafter. Four nontreated rats were killed at the beginning
of the protocol and used as controls (time zero). The dose and time
postinjection were selected on the basis of previous results showing a
strong induction of IL-6 mRNA in the rat brain after such treatment
(4). The animals were conscious and freely moving at all
times throughout the procedure, and no mortality occurred after the
injections. The animals were deeply anesthetized with an ip injection
(500 µl) of a mixture of ketamine hydrochloride (91 mg/ml) and
xylazine (9 mg/ml) and then rapidly perfused transcardially with 0.9%
saline, followed by 4% paraformaldehyde in 100
mM borax (pH 9.5 at 4 C). The brains were removed
from the skulls and placed in a solution of 4% paraformaldehyde-borax
buffer at 4 C for 2 to 5 days. They were then placed in the same
fixative solution containing 10% sucrose overnight at 4 C. The brains
were mounted onto a sliding microtome (SM2000R, Leica Corp., Nussloch, Germany), frozen with dry ice, and cut into
30-µm coronal sections from the olfactory bulb to the caudal medulla.
The slices were collected in a cold cryoprotectant solution [50
mM sodium phosphate buffer (pH 7.3), 30%
ethylene glycol, and 20% glycerol] and stored at -20 C.
Intravenous injection of LPS and IL-6 in rats
We have previously found that preinduction of IL-6 receptors
with a very low dose of LPS enhances the effects of the cytokine in the
brain, whereas a single bolus of a high dose of IL-6 has very little
influence on neuronal functions (20, 21). The same
experimental conditions were used here to trigger transcription of both
IL-6 receptor subunits in cells lining the cerebral blood vessels and
the CVOs. Rats were anesthetized with an ip injection (300 µl) of a
mixture of ketamine hydrochloride and xylazine, and a sterile catheter
filled with sterile pyrogen-free heparin-saline (5.0 U/ml) was
implanted into their right jugular vein. The catheter was made from a
piece of SILASTIC tubing (id, 0.500 mm; od, 0.940 mm; SILASTIC medical
grade tubing, Dow Corning Corp., Midland, MI) connected to
Intramedic polyethylene tubing (id, 0.580 mm; od, 0.965 mm; PE-50, Clay
Adams, Parsippany, NJ). The internal SILASTIC tip of the catheter was
positioned in the atrium, and the outlet was placed at an interscapular
position and sealed. After the surgery, the rats were housed
individually for a recuperation period of 5 days. On the day of the
experiment (0900 h), the outlet of the catheter was fastened to a
truncated 27-gauge needle attached to PE-50 tubing. This connector was
then fixed to a 1-ml syringe, and the rats were placed individually in
a quiet room for at least 2 h before experimentation. This
procedure was used to avoid disturbing the animals during the
injections.
Rats first received an iv injection of either LPS (5 µg
dissolved in 300 µl 0.9% sterile pyrogen-free saline) or vehicle
solution. The animals were conscious and freely moving during the
procedure. After a 6-h waiting period, they received a second iv
injection of either recombinant rat IL-6 (1.5 µg dissolved in 300
µl sterile pyrogen-free distilled water; bioactivity, 
1 x
107 U/mg; endotoxin, <0.1 ng/µl; catalogue no.
PRC0065, lot H032506; Biosource Technologies, Inc.,
Camarillo, CA) or vehicle solution. One hour later, animals were deeply
anesthetized with an iv injection of ketamine-xylazine and perfused
transcardially, and their brains were processed as described.
To ascertain the lack of effect of a single bolus of IL-6 on brain SOCS-3 transcription, another group of male rats received a higher dose of recombinant rat IL-6 from a different source (3.0 µg dissolved in 200 µl sterile pyrogen-free distilled water; purity, >97%; bioactivity: ED50 = 0.04–0.12 ng/ml; catalogue no. 506-RL-050, lot no. ANW01906B, R&D Systems, Minneapolis, MN). Animals (n = 26) were implanted with a chronic indwelling cannula into the jugular vein 6 days before receiving the single injection of the recombinant cytokine. They were conscious and freely moving during the procedure and were killed before (time zero) and 1, 3, 6, or 12 h after the single iv bolus of IL-6.
Intramuscular injection of turpentine in rats
Rats were injected into the left thigh muscles
with 50 µl/100 g BW turpentine (catalogue no. TU 109, lot no. CAS
8006–64-2, Spectrum Chemical Manufacturing Corp., Gardena, CA) or
0.9% sterile pyrogen-free saline and transcardially perfused at 1, 3,
6, and 12 h postinjection. This dose was determined on the basis
of previous studies showing evident swelling of the left hind limb and
a robust increase in the gene encoding inhibitory factor-
B
(IB) and cyclooxygenase 2 in the endothelium of the brain
capillaries (22, 23, 24). In the present case, we observed a
robust inflammation 6 and 12 h after im injection of turpentine,
whereas no visible change was observed in vehicle-administered rats.
This experimental model of sterile inflammation induces local tissue
damage that is responsible for the development of a systemic acute
phase response. This model also produces a more restricted cytokine
response, in particular IL-1ß and IL-6, to the acute localized
inflammatory insult (25).
Intraperitoneal injection of LPS in mice
In the morning (
0830 h), IL-6 knockout and wild-type mice
received an ip injection of LPS (25 µg dissolved in 100 µl 0.9%
sterile pyrogen-free saline) and were killed before (time zero) and 1,
4, and 8 h after injection of the bacterial endotoxin. They
received an ip injection of the ketamine-xylazine mixture (50 µl) and
were rapidly perfused transcardially with 0.9% saline, followed by 4%
paraformaldehyde in 100 mM borax (pH 9.5 at 4 C). The
brains were removed from the skulls and postfixed in a solution of 4%
paraformaldehyde-borax buffer at 4 C for 10–36 h before being placed
in the same fixative solution containing 10% sucrose overnight at 4 C.
The brains were mounted onto a sliding microtome, frozen with dry ice,
and cut into 20-µm coronal sections from the olfactory bulb to the
caudal medulla.
Riboprobe preparation and in situ hybridization histochemistry
The rat SOCS-3 complementary DNA (cDNA) fragment that was
initially inserted in a pEF-FLAG-I vector (provided by Dr. Doug Hilton,
The Walter and Eliza Hall Institute of Medical Research, Melbourne,
Australia) was extracted with XbaI and reinserted into a
pCRII vector (Invitrogen, Carlsbad, CA). The new
construction was then linearized with XhoI. The pBlueBac
plasmid containing rat gp130 cDNA (provided by Dr Gerald Fuller,
University of Alabama, Birmingham, AL) was digested with
HindIII. The inserts were then subcloned into pBluescript
SK- and linearized with XhoI. The
pBluescript plasmid containing the rat IL-6R cDNA fragment (no. 63081,
American Type Culture Collection, Manassas, VA) was
linearized with EcoRV. Radioactive antisense complementary
RNA copies were synthesized by incubating 250 ng linearized plasmid in
6 mM MgCl2, 40
mM Tris (pH 7.9), 2 mM
spermidine, 10 mM dithiothreitol, 0.2
mM ATP/GTP/CTP, 200 µCi
[-35S]UTP (NEG039H, NEN Life Science Products, Boston, MA), 40 U RNasin (Promega Corp.,
Madison, WI), and 20 U SP6 (SOCS-3), T7 (IL-6R), or T3 (gp130) RNA
polymerase for 60 min at 37 C. Unincorporated nucleotides were removed
by using the ammonium-acetate method; 100 µl deoxyribonuclease
solution (1 µl deoxyribonuclease, 5 µl 5 mg/ml transfer RNA, and 94
µl 10 mM Tris/10 mM
MgCl2) were added, and 10 min later an extraction
was accomplished using a phenol-chloroform solution. The complementary
RNA was precipitated with 80 µl 5 M ammonium
acetate and 500 µl 100% ethanol for 20 min on dry ice. After
centrifugation, the pellet was washed with 500 µl 70% ethanol,
dried, and resuspended in 100 µl 10 mM Tris/1
mM EDTA. A concentration of
107 cpm probe was mixed into 1 ml hybridization
solution [500 µl formamide, 60 µl 5 M NaCl,
10 µl 1 M Tris (pH 8.0), 2 µl 0.5
M EDTA (pH 8.0), 50 µl 20 x Denhart’s
solution, 200 µl 50% dextran sulfate, 50 µl 10 mg/ml transfer RNA,
and 10 µl 1 M dithiothreitol; 118 µl
diethylpyrocarbonate water - volume of probe used]. This
solution was mixed and heated for 5 min at 65 C before being spotted on
slides.
Hybridization histochemistry localization of each transcript (SOCS-3, IL-6R, and gp130 mRNAs) was carried out on every sixth section of the whole brain from the olfactory bulb to the end of the medulla as described previously (26). The sections were exposed at 4 C to x-ray films (Biomax, Eastman Kodak Co., Rochester, NY) for 24–48 h, defatted in xylene, dipped into NTB2 nuclear emulsion (Eastman Kodak Co.; diluted 1:1 in distilled water), and exposed for 10 days (SOCS-3 and gp130 mRNA) or 12 days (IL-6R). Adjacent sections were also hybridized with sense probes to ascertain the specificity of the positive signal obtained with the antisense probes.
Combination of immunocytochemistry with in situ
hybridization
Immunohistochemistry and in situ hybridization
techniques were combined to determine whether SOCS-3, IL-6R, and gp130
transcripts were expressed in endothelial cells of the cerebral blood
vessels. An antisera directed against von Willebrand factor (vWF) was
used to stain the endothelial cells of the brain microvasculature.
Every sixth brain section was processed using the avidin-biotin bridge
method with peroxidase as a substrate. Briefly, slices were washed in
sterile diethylpyrocarbonate-treated 50 mM
potassium PBS (KPBS) and incubated at room temperature with vWF
antibody mixed in sterile KPBS, 0.4% Triton X-100, 0.25% heparin
sodium salt USP (ICN Biomedicals, Inc., Aurora, OH), and
1% BSA (fraction V, Sigma, St. Louis, MO). vWF antibody
(sheep antirat, catalog no. CL20176A-R, lot no. AB22–74, Cedarlane
Laboratory, Hornby, Canada) was diluted 1:2500 in the solution
described above. Two hours after incubation with the primary antibody,
the brain slices were rinsed in sterile KPBS and incubated with a
mixture of sterile KPBS, Triton X-100, heparin, and biotinylated
secondary antibody (rabbit antisheep IgG; 1:1500 dilution; Vector Laboratories, Inc., Burlingame, CA) for 60 min. Sections were
then rinsed with KPBS and incubated at room temperature with an
avidin-biotin-peroxidase complex (Vectastain ABC elite
kit, Vector Laboratories, Inc.). After several rinses in
sterile KPBS, the brain slices were reacted in a mixture containing
sterile KPBS, the chromagen 3,3'-diaminobenzidine tetrahydrochloride
(0.05%), and 0.003% hydrogen peroxide
(H2O2).
Thereafter, tissues were rinsed in sterile KPBS, mounted onto gelatin- and poly-L-lysine-coated slides, desiccated under vacuum, fixed in 4% PFA for 30 min, and digested by proteinase K [10 µg/ml in 100 mM Tris HCl (pH 8.0) and 50 mM EDTA (pH 8.0) at 37 C for 25 min]. Prehybridization, hybridization, and posthybridization steps were performed as described previously (22). After being dried under vacuum, sections were exposed for 14 days, developed in D19 developer (Kodak) for 3.5 min at 15 C, and fixed in rapid fixer (Kodak) for 5 min. Thereafter, tissues were rinsed in running distilled water for 1 h, rapidly dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with distrene plasticizer xylene mounting medium (BDH, Poole, UK). The presence of SOCS-3, IL-6R, and gp130 transcripts was evident as silver grains clearly visible in perikarya, and vWF-immunoreactive cell bodies were stained in brown.
Quantitative analysis
Quantitative analyses of SOCS-3 hybridization signal were
carried out on x-ray films over four CVOs and along the ependymal
lining cells of the mouse right lateral ventricle. The selected
structures were the organum vasculosum of the lamina terminalis
(OVLT), subfornical organ (SFO), median eminence (ME), and area
postrema (AP). These regions were chosen to facilitate the analysis
among animals, although the hybridization signal was not limited to
these regions (see Results). Transmittance values (referred
to here as OD) of the hybridization signals were measured using a
Northern Light Desktop Illuminator (Imaging Research, Inc., St. Catherines, Canada) using a Sony camera video system
(Tokyo, Japan) attached to a MicroNikkor 55-mm Vivitar extension tube
set for Nikon lens (New Hyde Park, NY) and coupled to a
Macintosh computer (Power Macintosh 7100/66) and NIH Image
software version 1.61/ppc (written by W. Rasband at the NIH and
available on the internet by anonymous ftp from zippy.nih.gov). OD
values for each pixel were calculated using a known standard of
intensity and distance measurements from a logarithmic specter adapted
from Bioimage Visage 110s (Millipore Corp., Ann Arbor,
MI). Brain sections from all animals were digitized and subjected to
densitometric analysis, yielding measurements of mean density per area.
The OD of each region was then corrected for the average background
signal by subtracting the OD of areas surrounding the CVOs or the
cerebral ependyma. All measurements were performed in triplicate. Data
are reported as mean OD values (±SEM), and
statistical analysis was performed by ANOVA followed by a
Bonferroni/Dunn test procedure as post-hoc comparison, using
StatView software (version 4.01, Macintosh).
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Results |
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shows the distribution of SOCS-3 mRNA in the rat brain at different
times post-LPS administration. The arcuate nucleus (Arc) was the only
structure of the brain exhibiting constitutive SOCS-3 expression, and
the message increased as soon as 1 h after the ip injection with
the endotoxin (Figs. 1A and 2A). At that
time, a low hybridization signal was also detected in the OVLT, SFO,
chp, paraventricular nucleus (PVN) of the hypothalamus, supraoptic
nucleus (SON), ME, and AP. The expression level was maximum in these
regions at 3 and 6 h post-LPS administration (
Figs. 1–4). A
stimulation of SOCS-3 transcription was also found in
vascular-associated cells across the brain and leptomeninges as well as
in ependymal lining cells of the cerebroventricular system, a
phenomenon that was particularly intense 3 h after the single ip
bolus of LPS. Although the hybridization signal declined in cells
lining the ventricles, PVN, and SON, it remained high in the
endothelium of the brain capillaries and the circumventricular organs
(CVOs) at 6 h post-LPS treatment (Figs. 1B, 2, and 4).
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and 2).
As shown in Fig. 4, LPS activated SOCS-3 transcription in
the ependymal lining cells of the third ventricle, the parvo- and
magnocellular parts of the PVN, and along the endothelium of the
hypothalamic capillaries. Despite the clear agglomeration of silver
grains delineating positive SOCS-3 cells in vascular- and
ependymal-associated elements, the message over the PVN parenchyma was
more diffused. Dual labeling within different populations of neurons
was therefore not possible because of this homogenous distribution of
silver grains across the endocrine hypothalamus. This message was
nevertheless specific, as no positive signal was detected in adjacent
sections hybridized with the sense probe (data not shown).
SOCS-3 expression in cells of the blood-brain barrier (BBB)
A robust induction of the gene encoding SOCS-3 was found in
vascular-associated cells 3 and 6 h after the single ip LPS bolus
(Fig. 5). SOCS-3 transcription was also
stimulated in cells of the cerebral capillaries in response to a
localized and systemic inflammatory insult (data not shown). Indeed,
the hybridization signal was positive at 6 h and reached a
moderate level 12 h after the im turpentine injection. Of interest
is the fact that the increase in SOCS-3 transcription in brain vascular
elements paralleled the swelling in the left hind paw, i.e.
the site of turpentine injection. The hybridization signal was more
intense in some blood vessels than others, but the positive cells were
regularly lining the internal walls (Fig. 5, x250). This led us to
believe that the endothelium of the cerebral blood vessels and small
capillaries was probably the cell type expressing the transcript.
Figure 6 (top panels) shows
that SOCS-3-expressing cells were localized within the endothelial
marker vWF. To determine whether these cells contained the biosynthetic
machinery for the IL-6 receptors, dual labeling was performed in
adjacent brain sections of LPS-challenged rats. This approach confirmed
that the genes encoding IL-6R and gp130 were induced in the endothelium
of the cerebral microvasculature by systemic treatment with the
endotoxin (Fig. 6, middle and bottom panels).
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depicts the distribution of SOCS-3 mRNA in the brains of wild-type and
IL-6 knockout mice before (time zero, control) and at various times
after ip LPS injection. As in rats, positive SOCS-3 hybridization
signal was detected in the Arc of both wild-type and knockout mice
under basal conditions. A low, but positive, signal was also found in
the PVN of these animals. By 1 h post-LPS administration, the
OVLT, SFO, chp, ME/Arc, SON, and AP of IL-6-deficient mice and their
wild-type littermates expressed SOCS-3 mRNA (Fig. 7, 1 h). The
hybridization signal increased 4 h after the endotoxin challenge
in wild-type mice, but not in IL-6-deficient animals (Fig. 8A). Moreover, LPS caused a strong
transcriptional activation of SOCS-3 in the ependymal lining cells of
the cerebroventricular system and along the microvasculature in
wild-type mice (Fig. 7, 4 h). Such induction was absent in
IL-6-deficient mice that received the same dose of LPS and were killed
together with their wild-type littermates (Fig. 7, 4 h, and Fig. 8B). The expression of the gene encoding SOCS-3 returned to basal
levels at 8 h in the ependymal walls of the ventricles (Fig. 8B),
blood vessels, and PVN, although the message remained positive in all
CVOs and chp of both mouse strains (Fig. 7, 8 h, and Fig. 8A).
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, SOCS-3 is no longer expressed in the
inducible structures at that time with this low dose of LPS iv
(LPS+Veh), although IL-6R can be detected in the CVOs and cells of the
BBB (20). Injection of IL-6 in these presensitized animals
provoked a strong activation of SOCS-3 transcription in the CVOs and
blood vessels (Fig. 9, LPS+IL-6), whereas the cytokine failed to
activate this inhibitory molecule in the brains of rats that received
the vehicle solution instead of the endotoxin (Fig. 9, Veh+IL-6).
Semiquantitative analysis of Fig. 10
shows that IL-6 significantly increased CVO SOCS-3 expression only in
the presensitized rats. Consistent with our previous data, the Arc was
the unique nucleus that exhibited constitutive expression of SOCS-3
mRNA (Fig. 9, Veh+Veh).
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Discussion |
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, which can activate
the synthesis of IL-6 receptors in cells of the BBB (4).
The stimulation of IL-6R and gp130 in the endothelium of brain
capillaries and CVOs is probably part of the mechanisms that produce
the cellular response when IL-6 is available in the circulation. The
profound induction of SOCS-3 may, in turn, be a determinant
intracellular signal to inhibit IL-6-induced JAK/STAT signaling within
selective structures of the brain, namely the ventricle ependyma and
the endothelium of the cerebral blood vessels.
The rapid induction of SOCS-3 transcript in the CVOs, chp,
and microvessels is quite interesting, as these elements have
previously been shown to express IL-6 receptors during the acute phase
reaction (4). This indicates that IL-6 can trigger
JAK/STAT signaling in these groups of cells, which may allow
transcription of the target genes involved in the neural-immune
interface. The exact JAK and STAT molecules involved in these events
have yet to be determined, although investigating the transduction
pathways is technically limited in vivo. Nevertheless, such
an approach is essential to study the complex systems that interact
together and the key populations of cells involved during the acute
phase response. In this regard, the essential role of IL-6 in mediating
SOCS-3 expression in the endothelium and the ependymal lining cells of
the brain is quite intriguing, as the molecules of the SOCS family are
rather redundant and can be triggered by numerous other cytokines (for
a review, see Ref. 27). These data contrast with our
recent report that nuclear factor-B (NF-B) signaling in the brain
is independent of the release of proinflammatory cytokines during
endotoxemia (22). However, in the presence of the CD14
soluble form, circulating LPS has the ability to directly activate the
NF-B pathway in endothelial cells (28). The NF-B
pathway is actually the main transduction signal for the endotoxin, and
there is little convincing evidence that LPS can trigger, at least by
itself, JAK/STAT molecules in endothelial cells. Induction of this
signal transduction pathway must therefore be dependent on the
production of circulating inflammatory cytokines in response to LPS.
The late expression of SOCS-3 in the cerebral blood vessels and brain
ependymal cells supports this concept. This is quite different from the
rapid transcription of the inhibitor of NF-B, IB, which is
stimulated in the microvessels as early as 30 min after ip LPS
injection (22).
A single systemic injection of LPS caused a rapid increase in
TNF and IL-1ß in the bloodstream, followed by a gradual elevation
of plasma IL-6 (29). Despite the possible overlap with the
JAK/STAT/SOCS pathways, TNF and IL-1 are well recognized
NF-B-inducible cytokines (30, 31), and their
contributions may be minimal in regulating SOCS-3. Supporting this idea
is the fact that the inhibitory factor was no longer expressed in the
cerebral microvasculature and the ventricle lining walls of
IL-6-deficient mice during endotoxemia. Such a phenomenon, however, was
not generalized to all responsive structures, as the CVOs and chp of
these animals still exhibited a positive signal for SOCS-3 mRNA after
the LPS challenge. This interesting result suggests that IL-6 is a key
ligand for activating SOCS-3 transcription only in barrier-associated
groups of cells, not in the CVOs and chp. Other cytokines produced
locally within these organs are likely to be responsible for the
residual SOCS-3 production in the brains of IL-6-deficient mice.
Indeed, systemic LPS administration caused a rapid biosynthesis of
different proinflammatory cytokines in all CVOs and chp
(32, 33, 34), and these molecules are likely to induce
JAK/STAT signaling within the cells that bear the receptors for these
ligands. Besides, IL-6 remains a potential inducer of SOCS-3 expression
in the CVOs, because the hybridization signal for this transcript was
higher in wild-type mice than in IL-6-deficient animals 4 h after
LPS administration. Without being essential, this cytokine-dependent
gp130 receptor subunit may be an important player to intensify SOCS-3
activity in the CVOs and chp at key moments during endotoxemia. On the
other hand, the obligatory role of IL-6 in triggering the JAK/STAT
signaling pathways that lead to SOCS-3 transcription seems only
specific to two cell groups, namely the cerebral capillaries and the
ventricle ependyma.
Although circulating IL-6 is most likely able to stimulate the microvasculature, IL-6 of systemic origin may not contribute to SOCS-3 expression in cells lining the cerebroventricular system during endotoxemia. The cytokine has very limited access to the neural tissue, because of its high mol wt, which does not allow diffusion across the BBB. However, systemic LPS challenge is associated with a robust, but selective, transcriptional activation of the gene encoding IL-6 in all CVOs and chp of rats (4). The chp secretes CSF, and molecules produced by this organ circulate freely across the cerebroventricular system and thereafter through the brain parenchyma. The ependymal lining cells are the first physical barrier to be reached by the CSF and its secreted products, and they express both IL-6 receptor subunits during endotoxemia (4). Together these data suggest that IL-6 secreted by the chp may circulate in the CSF and target its receptors, which produce the JAK/STAT transduction pathways within the ependymal cells of the cerebroventricular walls. The subsequent increase in SOCS-3 may be the key mechanism to inhibit this signal when the ligand is no longer produced or to prevent an exaggerated response.
Despite the fact that SOCS-3 transcription is dependent on the
endogenous release of IL-6 in cells lining the BBB and the ventricles,
a single administration of the cytokine failed to activate the
signaling events leading to SOCS-3 production in vivo. This
results may be explained by the lack of constitutive expression of both
IL-6 receptors, at least in the endothelium of the brain microvessels
(4, 20). As shown here, systemic LPS challenge induced
both IL-6R and gp130 transcripts in vWF-ir cells, and this phenomenon
is a prerequisite for IL-6 signaling during the acute phase response.
Whether IL-6R and gp130 expression in the brain microvasculature
depends on the release of IL-1ß and/or TNF or is a direct action
of the endotoxin on the cerebral endothelium is an open question that
is currently under investigation. Nevertheless, IL-1ß is capable of
activating IL-6 receptors in cells that can be reached from the
systemic circulation (4), and IL-1ß precedes IL-6
release during systemic inflammation. This potential mechanism is quite
interesting, as it follows the time-related events that occur during
the acute phase reaction of an immune challenge. Further supporting
this concept is the late expression of SOCS-3 in the brain
microvasculature of animals that were injected with turpentine into the
left hind paw, used here an experimental model of sterile and localized
inflammatory insult associated with a specific induction of IL-1ß and
IL-6 (25).
It is therefore tempting to propose the following sequence
of events. 1) IL-1ß and TNF- are released early in the bloodstream
during the acute phase reaction. 2) These circulating cytokines have
the ability to reach the large arterioles, small capillaries, and
venules, which triggers NF-B nuclear translocation and stimulates
IL-6R and gp130 transcription in endothelial cells. 3) These receptors
may then respond when the ligand becomes available in the circulation
that activates selective JAK/STAT molecules. 4) Phosphorylated
homodimers of STATs (most likely STAT1 and/or STAT3) target SOCS-3
promoter and stimulate its transcription. 5) The increase in SOCS-3
protein inhibits this cytokine signaling and the proinflammatory signal
transduction pathways. Similar events may take place directly in the
brain, with the chp resident macrophages being the cellular source of
IL-1ß, TNF, and IL-6 and the ependymal as being the cells
responding to these ligands of central origin. Together these data
suggest that preinduction of IL-6 receptors is a prerequisite allowing
IL-6 to trigger the transducing events and then SOCS-3 production in
the cerebral endothelium and a specific group of supportive cells. The
cytokine, when present in the circulation, may then act as the
subsequent step to maintain the neuronal activity involved in the
adequate control of homeostatic balance during systemic inflammation.
Moreover, IL-6-induced SOCS-3 is likely to be part of the
antiinflammatory mechanisms that take place in a very well organized
manner within the CNS.
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Acknowledgments |
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Footnotes |
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2 Medical Research Council of Canada scientist.
Received March 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity and COX-2
transcription in cells of the blood-brain barrier in response to
systemic and localized inflammation, but not during endotoxemia. J
Neurosci 19:10923–10930B
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