From the Instituto de Parasitologia y Biomedicina Lopez-Neyra, Consejo Superior de Investigaciones Científicas, Granada 18001, Spain
Received for publication, March 28, 2003 , and in revised form, May 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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IFN- constitutes one of the most potent microglia-activating
factors. Binding of IFN-
to its receptor induces the assembly of an
active receptor complex and consequent transphosphorylation of the
receptor-associated Janus tyrosine kinases Jak1 and Jak2
(4,
5). The activation of these
kinases induces phosphorylation of the cytoplasmic tail of the receptor
itself, which lacks intrinsic kinase activity. The cytosolic protein signal
transducer and activator of transcription (STAT1) is then recruited to the
activated IFN-
-receptor complex and phosphorylated
(4). Upon phosphorylation,
STAT1 forms homodimers and translocate to the nucleus, where they bind to the
IFN-
-activated site (GAS), also termed STAT binding element (SBE),
found in the promoter of many IFN-
-induced genes including the IFN
regulatory factor-1 (IRF-1) and ICAM-1 genes
(69).
Many of the regulatory effects of IFN-
in microglia appear to be
mediated by IRF-1 and/or STAT1, which transactivate multiple effector genes
including IFN-
-inducible protein-10 (IP-10), CD40, IL-12, and the
inducible nitric-oxide synthase (iNOS)
(1020).
Vasoactive intestinal peptide (VIP) and the structurally related peptide,
the pituitary adenylate cyclase activating polypeptide (PACAP), are two
neuropeptides that elicit a broad spectrum of biological functions, including
actions on natural and acquired immunity (reviewed in Refs.
2125).
Although VIP and PACAP affect a variety of immune functions, their primary
immunomodulatory function is anti-inflammatory in nature. VIP has been shown
to inhibit cytokine production and proliferation in T cells, and several
macrophage functions, including phagocytosis, respiratory burst, and
chemotaxis (23), as well as
LPS-induced IL-6, TNF-, IL-12, NO, and chemokine production
(2125).
Similarly, we have recently demonstrated that VIP and PACAP act as potent
microglia-deactivating factors by inhibiting the production of
endotoxin-induced proinflammatory mediators in vitro
(26,
27). The inhibition of
proinflammatory mediators is responsible, at least partially, for the
protective effect of VIP/PACAP in vivo in murine models for septic
shock, inflammation-induced neurodegeneration, brain trauma, and Parkinson's
disease
(2830).
To further understand the molecular mechanisms through which VIP and PACAP
attenuate the inflammatory responses in the central nervous system, we
examined the effects of VIP/PACAP on IFN-
-induced Jak-STAT1 activation
and IRF-1 synthesis in murine microglial cells. Our results indicate that
VIP/PACAP inhibit Jak12/STAT1 phosphorylation, binding of STAT1 to the
GAS motif in the IRF-1 promoter, and subsequently, IRF-1 transcription and
synthesis. These effects are correlated with an inhibitory effect of VIP/PACAP
on IFN-
-induced CD40, IP-10, and iNOS expression, mediated through the
VIP receptor VPAC1 and the cAMP/PKA transduction pathway.
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EXPERIMENTAL PROCEDURES |
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Cell CulturesMicroglial cell cultures were prepared as previously described (31). Briefly, cerebral cortical cells from 1-day-old BALB/c mice were dissociated after a 30-min trypsinization (0.25%) and were plated in 75-cm2 Falcon culture flasks in Dulbecco's modified Eagle's medium high glucose formula (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), containing 10 mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessential amino acids, 2 mM glutamine, 50 mM 2-mercaptoethanol, 100 units/ml penicillin, and 10 µg/ml streptomycin (complete medium). The medium was replenished 1 and 4 days after plating, and on day 8 of culture, plates were shaken for 20 min at a speed of 200 rpm in an orbital shaker to remove oligodendrocytes. On day 12 of culture, plates were shaken again for 2 h at a speed of 180200 rpm. Harvested cells were filtered through a 20-µm nylon mesh, plated in a 60-mm Petri dish, and incubated for 15 min at 37 °C. After extensive washing with culture medium, adherent cells (microglia) were collected with a rubber policeman and centrifuged (1000 rpm, 10 min). Purified microglial cell cultures comprised a cell population in which >98% stained positively with MAC-1 antibodies (Roche Molecular Biochemicals) and <2% stained positively with antibodies specific to the astrocyte marker glial fibrillary acid protein (Sigma).
Microglia monolayers were incubated with complete medium and stimulated
with 50 units/ml IFN- in the presence or absence of VIP or PACAP38
(from 1012 to 106
M) at 37 °C in a humidified incubator with 5% CO2.
Cell-free supernatants were harvested at the designated time points and kept
frozen (20 °C) until IP-10 determination.
RNA Extraction and Northern Blot AnalysisNorthern blot
analysis was performed according to standard methods. Murine primary microglia
cells were cultured at a concentration of 2 x 106 cells/ml in
100-mm tissue culture dishes and stimulated with IFN- (50 units/ml) in
the presence or absence of VIP (108 M)
or PACAP (108 M) for different time
periods at 37 °C. Cells were collected, and total RNA was extracted by the
acid guanidinium-phenol-chloroform method, electrophoresed on 1.2%
agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher &
Schuell), and cross-linked to the nylon membrane using UV light.
The probes for murine IRF-1, GAPDH, and IP-10 were generated by reverse
transcription-PCR as previously described
(32,
33). Oligonucleotides were
end-labeled with [-32P]ATP (3000 Ci/mmol, Amersham
Biosciences) by using T4 polynucleotide kinase. The RNA-containing membranes
were prehybridized for 16 h at 42 °C and hybridized at 42 °C for 16 h
with the appropriate probes. The membranes were washed twice in 2x SSC
containing 0.1% SDS at room temperature (20 min each time), once at 37 °C
for 20 min, and once in 0.1x SSC containing 0.1% SDS at 50 °C (20
min). The prehybridization and hybridization buffers were purchased from 5
Prime
3 Prime Inc. (Boulder, CO). The membranes were exposed to x-ray
films (Eastman Kodak Co.). CD40 mRNA expression was determined by RNase
protection assay as described previously
(34).
Electrophoretic Mobility Shift Assay (EMSA)Nuclear extracts were prepared by the mini-extraction procedure of Schreiber et al. (35) with slight modifications. Briefly, microglia cells were cultured at a density of 107 cells in 6-well plates, stimulated as described above, washed twice with ice-cold PBS plus 0.1% bovine serum albumin, and harvested from the dishes. Incubation times for the different factors assayed were optimized empirically. The cell pellets were homogenized with 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After 15 min on ice, Nonidet P-40 was added to a final 0.5% concentration, the tubes were gently vortexed for 15 s, and nuclei were sedimented and separated from cytosol by centrifugation at 12,000 x g for 40 s. Pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A, and the soluble nuclear proteins were released by adding 0.1 ml of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After incubation for 30 min on ice, followed by centrifugation for 10 min at 14,000 rpm at 4 °C, the supernatants containing the nuclear proteins were harvested, the protein concentration was determined by the Bradford method, and aliquots were stored at 80 °C for later use in EMSAs.
Oligonucleotides corresponding to the IFN stimulus response element (ISRE)
(nucleotides 234 to 206) motif of the IP-10 promoter
(16,
17,
36), to the IRF-1 (nucleotides
933 to 906) motif of the iNOS promoter
(11,
12), to the mGAS (nucleotides
491 to 467) site of CD40 promoter
(19,
20), and to the GAS site of
IRF-1 promoter were synthesized (nucleotides 131 to 105)
(6):
5'-CTCACGCTTTGGAAAGTGAAACCTACCTC-3' (ISRE for IP-10),
5'-CACTGTCAATATTTCACTTTCATAATG-3' (IRF-1 for iNOS),
5'-GGAAACTCTTCCTTGAAACGCCTCC-3' (GAS for CD40), and
5'-GCCTGATTTCCCCGAAATGACGGC-3' (GAS for IRF-1). The
oligonucleotides were annealed after incubation for 5 min at 85 °C in 10
mM Tris-HCl, pH 8.0, 5 mM NaCl, 10 mM
MgCl2, and 1 mM DTT. Aliquots of 50 ng of the
double-stranded oligonucleotides were end-labeled with
[-32P]ATP by using T4 polynucleotide kinase. For EMSA we
used 20,00050,000 cpm of double-stranded oligonucleotides,
corresponding to
0.5 ng, per reaction. The binding reaction mixtures (15
µl) were set up containing: 0.51 ng of DNA probe, 5 µg of nuclear
extract, 2 µg of poly(dI-dC)·poly(dI-dC), and binding buffer (50
mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 5%
glycerol, and 10 mM Tris-HCl, pH 7.5). The mixtures were incubated
on ice for 15 min before adding the probe, followed by another 20 min at room
temperature. Samples were loaded onto 4% nondenaturing polyacrylamide gels and
electrophoresed in TGE buffer (50 mM Tris-HCl, pH 7.5, 0.38
M glycine, and 2 mM EDTA) at 100 V, followed by transfer
to Whatman paper, drying under vacuum at 80 °C, and autoradiography. In
competition and antibody supershift experiments, the nuclear extracts were
incubated for 15 min at room temperature with the specific antibody (1 µg)
or competing cold oligonucleotide (50-fold excess) before the addition of the
labeled probe.
Western Blot Analysis of iNOSFor detection of iNOS by Western blotting, microglia cells (107 cells) were lysed in cold 40 mM Tris-HCl buffer, pH 8.0, containing 0.1 mM PMSF, 5 µg/ml aprotinin, 50 µg/ml leupeptin, 1 µg/ml chymostatin, and 5 µg/ml pepstatin, and lysed by sonication. Thirty micrograms of protein extracts was separated by 7.5% SDS-PAGE gels under reducing conditions. After electrophoresis, the gel was electroblotted in Tris-glycine buffer (48 mM Tris, 39 mM glycine, pH 9.2) containing 40% methanol onto a reinforced nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked with TBS-T buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% milk powder for1hat room temperature, then incubated for an additional 6 h at 4 °C with a monoclonal antibody against iNOS (1:1000) in 5% nonfat milk/TBS-T solution. After washing three times in TBS-T, membranes were incubated at room temperature for 1 h with peroxidase-conjugated goat anti-mouse IgG at 1:2000 dilution. After washing three times in TBS-T for 5 min each, and once in Tris-buffered saline for 5 min, the membrane was drained briefly and subjected to the enhanced chemiluminescence detection system (ECL, Amersham Biosciences). The x-ray films were exposed for 520 min.
Analysis of STAT1 and Jak1/2 PhosphorylationThe
phosphorylation status of Jak1/Jak2 and STAT1 was assessed by
immunoprecipitation followed by immunoblot analysis as previously described
(38). Cells (107
cells) were lysed in ice-cold immunoprecipitation buffer (50 mM
Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA pH 7.4, 1
mM Na3VO4, 20 mM -glycerol
phosphate, 1 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 1
µg/ml pepstatin, 1% Nonidet P-40, 0.25% deoxycholate, and 0.1% SDS). STAT1
and Jak1/Jak2 molecules were immunoprecipitated by incubation of lysates with
polyclonal Abs to STAT1 (10 µg/ml) or Jak1 and Jak2 (10 µg/ml),
respectively. Ab-antigen complexes were captured on protein G-Sepharose beads
for 1 h at 4 °C. Precipitated proteins were released from the beads by
washing in immunoprecipitation buffer (three times) and boiling in SDS sample
buffer and then separated by SDS-PAGE on a 10% acrylamide resolving gel.
Immunoblot analysis using an anti-phosphorylated STAT1 Abs (0.5 µg/ml, for
Tyr701 and Ser727 STAT1 analysis) or an Ab specific for
phosphotyrosine residues (1 µg/ml, for Jak1 and Jak2 analysis) was done
essentially as described above. Following analysis of phosphorylated STAT1,
phosphorylated Jak1, and phosphorylated Jak2, the blots were stripped and
reprobed with Abs directed to STAT1, Jak1, or Jak2, respectively, as described
above.
IP-10 ELISAThe amounts of IP-10 present in culture supernatants were determined using a specific sandwich ELISA. Briefly, a capture monoclonal anti-murine IP-10 antibody (clone A 1026, 2.5 µg/ml) was used for coating, and a biotinylated rabbit anti-mouse IP-10 polyclonal antibody (4 µg/ml) was used for detection. The detection limit for the assay is 15 pg/ml IP-10.
Determination of NO Synthase ActivityThe NO synthase activity was measured as described (39). Briefly, primary microglia cells were cultured at a density of 107 cells in 6-well plates, stimulated as described above, washed twice with ice-cold PBS plus 0.1% bovine serum albumin. Cells were lysed by three cycles of freeze/thaw in 40 mM Tris-HCl, pH 8.0, 0.1 mM EGTA, 0.1 mM EDTA, 12 mM 2-mercaptoethanol, and protease inhibitors (0.1 mM PMSF, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml chymostatin, and 5 µg/ml pepstatin), and centrifuged at 10,000 x g for 60 min. For the NO synthase activity assay, microglia lysates were incubated with 2 mM L-arginine, 2 mM NADPH, 4 µM flavin-adenine dinucleotide, 4 µM tetrahydrobiopterin, 2 mM DTT, and1mM EGTA for2hat37 °C.The reaction was terminated by adding 15 units/ml L-lactic dehydrogenase and 83 mM sodium pyruvate and incubating for an additional 15 min. The amount of NO formed in the reaction mixture was estimated from the accumulation of the stable NO metabolite nitrite by the Griess assay. Equal volumes of culture supernatants and Griess reagents (1% sulfanilamide, 0.1% N-[naphthyl]ethylenediamine dihydrochloride in 2.5% H3PO4) were mixed, and the absorbance was measured at 550 nm. The amount of nitrite was calculated from a NaNO2 standard curve.
Determination of CD40 Protein Expression by Immunofluorescence Flow CytometryCD40 expression was determined by flow cytometry as previously described (20). Briefly, primary microglia were plated at 2 x 105 cells/well into 12-well plates (Costar) and stimulated as described above. The cells were scraped in ice-cold RPMI complete medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated fetal calf serum (wash buffer). Cells were incubated with 10 µg/ml rat IgG2a-k anti-mouse CD40 Ab (clone 3/23) at 4 °C for 45 min, then washed, and incubated with 10 µg/ml biotinylated anti-rat IgG2a, for 30 min at 4 °C. After washing, the cells were incubated with 10 µg/ml phycoerythrin-conjugated streptavidin for 30 min at 4 °C. Isotype-matched Abs were used as controls, and IgG block (Sigma) was used to block the nonspecific binding to Fc receptors. After extensive washing, the cells were fixed in 1% paraformaldehyde. Stained microglia cells, gated according to forward and side scatter characteristics, were analyzed on a FACScan flow cytometer (Becton Dickinson). Samples in which isotype-matched antibody was used instead of specific antibody were used as negative controls to determine the proper region or window setting. Fluorescence data were expressed as mean fluorescence intensity and as percentage (%) of positive cells after subtraction of background isotype-matched values.
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RESULTS AND DISCUSSION |
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VIP and PACAP Reduce IFN--induced Jak1 and Jak2
Phosphorylation in MicrogliaIFN-
has been studied
extensively as a transcriptional regulator, whose action is initiated by the
binding of IFN-
to its receptor resulting in the activation of Jak1/2
by transphosphorylation (4,
44). First, whether VIP and
PACAP regulate Jak1 and Jak2 activation was investigated. Cell extracts of
IFN-
-stimulated microglia, treated with or without VIP or PACAP, were
immunoprecipitated with anti-Jak1 or anti-Jak2 Ab, followed by Western
blotting with an anti-phosphotyrosine Ab. Both Jak1 and Jak2 were weakly
phosphorylated in unstimulated controls; the Jak1/Jak2 phosphorylation level
increased within 5 min following IFN-
stimulation
(Fig. 1). Treatment with VIP or
PACAP reduced Jak1 and Jak2 phosphorylation at all time points
(Fig. 1).
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VIP and PACAP Inhibit the IFN--induced Phosphorylation
of STAT1The activation of Jak1/2 by IFN-
induces
phosphorylation and activation of the cytoplasmic tail of the receptor itself,
which recruits the cytosolic protein STAT1 and phosphorylates it
(4). Tyrosine phosphorylation
of STAT1 protein induces its homodimerization and translocation to the nucleus
(4,
44,
45). To determine whether VIP
and PACAP affect the activation of STAT1, microglia cells were stimulated with
IFN-
for different times in the presence or absence of VIP or PACAP.
Cell extracts were immunoprecipitated with anti-STAT1 Ab, and the levels of
phosphorylated STAT1 were assessed by Western blot using a specific Ab against
Tyr701-phosphorylated STAT1
. IFN-
stimulated in a
time-dependent manner the phosphorylation of STAT1
(Fig. 2). Treatment with VIP or
PACAP significantly reduced the levels of phosphorylated STAT1 in activated
microglia (Fig. 2A).
In addition of phosphorylation in Tyr701, to act with maximal
efficiency as a transcription factor, STAT1 must also be phosphorylated on
Ser727 by a kinase not clearly defined
(46). As
Fig. 2B shows, VIP and
PACAP also decreased IFN-
-induced STAT1 phosphorylation on
Ser727, suggesting that both neuropeptides fully deactivate STAT1
pathway.
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The majority of IFN--responsive genes are induced through the
interaction of phosphorylated STAT1 homodimers and an inducible palindromic
enhancer termed GAS/SBE present in the promoter of many IFN-
-induced
genes, including IRF-1 and CD40
(4,
6,
7,
19,
20,
44). IFN-
also induces
transcription of a subset of genes, such as IP-10, in a GAS-independent
fashion through a motif termed the ISRE
(1618).
VIP and PACAP Inhibit STAT1 Binding to the GAS Site of the IRF-1
PromoterAlthough the IRF-1 promoter contains a complex array of
transactivating binding sites, GAS/SBE appears to be essential for maximal
IRF-1 gene transcription following IFN- stimulation
(4,
5). To investigate whether
VIP/PACAP affect STAT1 binding to the GAS motif, nuclear extracts from
microglia stimulated with IFN-
in the presence or absence of VIP or
PACAP were used in gel shift assays. IFN-
treatment led to increased
binding to an oligonucleotide containing the GAS motif, and treatment with VIP
and PACAP inhibited this binding (Fig.
3A). The GAS binding was competed by an excess of
unlabeled homologous oligonucleotide (GAS) but not by a non-homologous
oligonucleotide (NF
B) (data not shown). Antibody supershift experiments
indicate that the GAS-binding complexes in IFN-
-stimulated macrophages
contain STAT1 (Fig.
3A). The VIP/PACAP inhibition of STAT1-GAS DNA binding in
the IRF-1 promoter was correlated with an inhibitory effect on
IFN-
-induced IRF-1 mRNA expression
(Fig. 3B).
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VIP and PACAP Inhibit IFN--induced iNOS Expression by
Decreasing IRF-1 Binding to Its PromoterIFN-
may indirectly
affect many effector genes through IRF-1. For example, although the iNOS
promoter contains a complex array of trans-activating binding sites, IRF-1
appears to be essential for maximal iNOS transcription after IFN-
stimulation
(1113).
To investigate whether VIP and PACAP affect IRF-1 nuclear translocation and
DNA binding in microglia stimulated with IFN-
, IRF-1 binding to the
iNOS promoter was assessed by EMSA. Stimulation of microglia cells with
IFN-
led to an increase in IRF-1 binding, and treatment with VIP or
PACAP significantly inhibited this binding
(Fig. 4A). The
specificity of the IRF-1 binding was evident by the supershifted
IRF-1-specific band observed in IFN-
-stimulated cells
(Fig. 4A).
VIP/PACAP-mediated decrease in IRF-1 DNA binding to the iNOS promoter was
correlated with an inhibition of iNOS protein expression and activity in
IFN-
-activated microglia (Fig. 4,
B and C). Therefore, VIP and PACAP decrease
IFN-
-induced iNOS expression by inhibiting Jak1/2-STAT1-mediated IRF-1
expression. In contrast to neurons and endothelial cells that express a
constitutive NOS, microglia express an inducible type of NOS, iNOS,
responsible for the prolonged, high output production of nitric oxide. In
general, expression of iNOS follows a generalized or localized inflammatory
response resulting from infection or tissue injury. Despite its beneficial
role in host defense, sustained nitric oxide production can be deleterious for
the host, especially under inflammatory conditions in the central nervous
system, such as brain trauma and Parkinson's disease
(13,
4749).
Therefore, the selective inhibition of expression of iNOS represents an
important therapeutic goal. In this sense, we have previously shown that VIP
down-regulated in vivo expression of microglial iNOS during brain
inflammation, brain trauma, and Parkinson's disease
(26,
27,
29,
30), this effect being
accompanied by a neuroprotective action.
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VIP and PACAP Inhibit IP-10 Expression by Down-regulating STAT1 Binding
to the ISRE Motif on Its PromoterIP-10 (CXCL10) is a member of the
CXC chemokine gene family that is expressed constitutively in lymphoid organs,
and strongly induced by IFN- in a variety of cell types including
macrophages, dendritic cells, and microglia
(4954).
ISRE has been identified as the essential element in the IP-10 promoter
involved in its transcriptional activation following IFN-
stimulation
(1618,
36). To investigate whether
VIP and PACAP affect STAT1 binding to the ISRE motif of the IP-10 promoter,
EMSAs were performed. Stimulation of microglia with IFN-
led to strong
STAT1 DNA binding to the ISRE site of the IP-10 promoter in comparison with
unstimulated cells, and VIP and PACAP significantly inhibited this binding
(Fig. 5A). The binding
specificity was confirmed by the displacement with 50-fold excess of unlabeled
homologous (ISRE) but not non-homologous oligonucleotide (NF
B) (data
not shown). Antibody supershift experiments indicate the specific binding of
STAT1, but not STAT2, to the ISRE motif
(Fig. 5A, right
panel). VIP/PACAP-mediated decrease in STAT1 DNA binding to the ISRE site
of the IP-10 promoter was accompanied by an inhibition of IP-10 mRNA and
protein expression in IFN-
-activated microglia
(Fig. 5, B and
C). In recent reports, VIP has been shown to inhibit
IP-10 production by LPS-stimulated macrophages and dendritic cells
(55,
56); however, molecular
mechanisms involved in such effect were not addressed. The biological
significance of the VIP/PACAP inhibition of IP-10 production by stimulated
microglia is supported by in vivo experiments. IP-10 and its receptor
CXCR3 have been associated with Th1 dominant immune responses. CXCR3 is
expressed primarily on T lymphocytes of the Th1 phenotype, a finding
consistent with the preferential chemoattraction of activated Th1 cells by
IP-10 (57,
58). As expected, IP-10 is
expressed in high levels in several Th1-type diseases such as rheumatoid
arthritis, psoriasis, multiple sclerosis, inflammatory bowel disease, and type
I diabetes
(5963).
Thus, the inhibition of directed migration of Th1 cells is of particular
significance for multiple sclerosis, where IP-10 neutralization prevented
central nervous system recruitment of effector Th1 cells in a model of
experimental autoimmune encephalomyelitis (EAE)
(64). Therefore, VIP/PACAP
impairment of IP-10 production by activated microglia in an inflamed brain
parenchyma could be beneficial for the treatment of multiple sclerosis
avoiding the preferential recruitment of activated autoreactive Th1 effector
cells.
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VIP and PACAP Inhibit IFN--induced CD40 Expression by
Down-regulating STAT1-GAS BindingCD40, a member of the tumor
necrosis factor receptor family, is expressed on antigen-presenting cells such
as dendritic cells, monocytes, B cells and microglia, where after its ligation
with CD40 ligand, expressed transiently on activated T cells, produces
bidirectional signaling essential for both costimulation of T cell and
CD40-bearing cell activation (reviewed in Ref.
65). IFN-
is the most
potent inducer of CD40 expression by microglia
(19,
20,
66). IFN-
-induced CD40
expression is mainly mediated by the binding of STAT1 homodimers to the
GAS/SBE site of its promoter
(19,
20). Therefore, I next
investigated whether VIP and PACAP could affect Jak/STAT1-mediated CD40
expression. EMSA experiments showed that treatment of microglia cells with
IFN-
led to strong binding of nuclear extracts to the
CD40/GAS-containing probe compared with unstimulated cells, and treatment with
VIP or PACAP decreased this binding (Fig.
6A). Antibody supershift experiments were performed to
determine the composition of the CD40/GAS-binding factors. Addition of
anti-STAT1, but not anti-STAT2 Abs, to the binding reaction resulted in a
marked reduction in the intensity of the GAS band and led to the appearance of
a slow migrating band, indicating that the GAS-binding complex of the CD40
promoter in IFN-
-activated microglia is composed primarily of STAT1
homodimers (Fig. 6A).
Inhibition of STAT1 binding to the GAS motif of the CD40 promoter by VIP/PACAP
was correlated by a decreased IFN-
-induced CD40 expression, at both
mRNA and protein levels (Fig. 6, B
and C). A recent report showed that VIP and PACAP inhibit
CD40 expression in LPS-stimulated rat microglia
(67), an effect indirectly
mediated through the production of the anti-inflammatory cytokine IL-10.
Although IL-10 involvement cannot be totally excluded in our study, because of
the different timing in the expression of both CD40 and IL-10 following
microglia activation, a direct effect of VIP/PACAP on IFN-
-induced
signal transduction seems to be the most plausible mechanism mediating the
inhibition of CD40 expression by both neuropeptides. Ligation of microglial
CD40 plays a crucial role in the development of immune and inflammatory
responses in the central nervous system. This is particularly important in
vivo under pathological conditions. For example, ligation of microglial
CD40 is essential for the development of EAE
(68,
69), and its levels in the
central nervous system correlate with the severity of the disease
(70,
71), governing the
pathogenesis of EAE locally by controlling the recruitment/retention of
autorreactive T cells (72). In
addition, microglial CD40 ligation induces microglia maturation and the
production of proinflammatory neurotoxic cytokines
(7375).
Therefore, negative regulation of IFN-
-induced CD40 in microglia by
VIP/PACAP, is physiologically important, because it may be one of the
mechanisms by which these neuropeptides antagonizes the development of Th1
cells and dampens inflammatory responses.
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Involvement of VPAC1 and cAMP in the Inhibitory Effect of VIP on
IFN--induced Jak1-STAT1 Activation in
MacrophagesNext we investigated whether the inhibitory effect of
VIP/PACAP could be related to occupancy of specific receptors. VIP and PACAP
act through a family of receptors consisting of VPAC1, VPAC2, and PAC1
(76). Reverse
transcription-PCR analysis indicates that murine primary microglia express
both PAC1 and VPAC1 mRNA (26).
In contrast, VPAC2 mRNA was not expressed even following activation. A similar
pattern of VIP/PACAP receptor expression was observed in rat primary microglia
(77) and in the two murine
microglia cell lines, EOC13 and BV2
(26).
The majority of the effects of VIP and PACAP in LPS-activated microglia are
exerted through the VIP/PACAP-receptor VPAC1, and the subsequent activation of
the cAMP/PKA pathway (26,
27). Therefore, it was next
investigated whether the inhibitory effect of VIP on the Jak/STAT/IRF
signaling pathway could be related to occupancy of this specific receptor and
to activation of PKA. The VPAC1 is coupled primarily to the adenylate cyclase
system (76), and
IFN--induced Jak1-STAT1 activation and production of some of the
factors studied (i.e. IP-10, CD40, and iNOS) is indeed inhibited in
activated microglia by agents that increase intracellular cAMP levels
(33,
39,
7884).
We investigated the ability of a specific VPAC1 antagonist
(85) and of H89, a potent and
selective PKA inhibitor, to reverse the effects of VIP. Both the VPAC1
antagonist and the PKA inhibitor reversed the inhibitory effects of VIP on
IP-10, CD40, iNOS, and IRF-1 expression
(Fig. 7), on the binding of
STAT1 to GAS/IRF-1 (Fig. 7),
and on STAT1 and Jak1 phosphorylation (Fig.
7). In contrast, PACAP638, an antagonist
specific for the VIP/PACAP receptors PAC1 and VPAC2
(86), or calphostin C, a PKC
inhibitor, did not affect the inhibitory effects of VIP (data not shown).
These results suggest that the inhibition of Jak/STAT/IRF signaling pathway by
VIP is mediated through VPAC1 and the cAMP/PKA pathway. This is supported by
the fact that, whereas forskolin (a cAMP-inducing agent) and a VPAC1 agonist
(87) exert an effect similar
to VIP, the VPAC2 agonists Ro-25-1553 and helodermin
(76) and the VIP fragment
1028 did not show any significant effect on IFN-
-induced STAT1
phosphorylation, IRF-1 expression, and IP-10 production
(Fig. 7). The link between the
cAMP pathway and the Jak1/2 activation has not been elucidated. One of the
possibilities is the induction of some of the members of the family of the
suppressors of cytokine signaling (SOCS) by VIP/PACAP. The members of the SOCS
family act as inhibitors of cytokine signaling
(88), and SOCS1 and SOCS3
inhibit STAT1 activation by IFN-
(89). However, VIP and PACAP
did not induce the expression of SOCS1 and 3 in unstimulated microglia, and
did not alter SOCS1 and/or SOCS3 expression in cells treated with IFN-
(data not shown). Another possibility that should be addressed is that
VIP/PACAP activate tyrosine phosphatases that dephosphorylate Jaks and STAT1.
Results showed in Fig. 8 raise
against this possibility because the tyrosine phosphatase inhibitor sodium
orthovanadate did not reverse the VIP-dependent suppression of STAT1 Tyr
phosphorylation and GAS binding. In summary, the present work demonstrates
that, as in macrophages (37),
VIP and PACAP sequentially inhibit IFN-
-induced Jak1/Jak2 activation,
and the subsequent activation of the STAT1, in terms of phosphorylation,
nuclear translocation, and binding to the GAS motif in the IRF-1 and CD40
promoters and to the ISRE motif in the IP-10 promoter
(Fig. 9). Because IFN-
is one of the most important microglia stimulators in vivo
participating in inflammation and Th1 activation/differentiation, and through
its effect in the IFN-
-induced Jak/STAT1 pathway, VIP and PACAP are
able to control the gene expression of IP-10, CD40, and iNOS, three
microglia-derived mediators that plays an essential role in several
pathologies, i.e. inflammation and autoimmune disorders, VIP and
PACAP emerge as two attractive therapeutic factors of such disorders and could
represent a significant element in the regulation of the inflammatory response
in the central nervous system by endogenous neuropeptides.
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FOOTNOTES |
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To whom correspondence should be addressed: Instituto de Parasitologia y
Biomedicina Lopez-Neyra, Consejo Superior de Investigaciones
Científicas, Calle Ventanilla, 11, Granada 18001, Spain. Fax:
34-958-203323; E-mail:
mdelgado{at}ipb.csic.es.
1 The abbreviations used are: TNF-, tumor necrosis factor
;
GAS, interferon
-activated site; IFN, interferon; IRF-1, interferon
regulatory factor 1; NO, nitric oxide; iNOS, inducible nitric-oxide synthase;
IP-10, interferon-induced protein-10; ISRE, interferon-stimulated response
element; PAC1, pituitary adenylate cyclase activating polypeptide-preferring
receptor; PACAP, pituitary adenylate cyclase activating polypeptide; STAT1,
signal transducer and activator of transcription 1; VIP, vasoactive intestinal
peptide; VPAC1 and VPAC2, vasoactive intestinal peptide/pituitary adenylate
cyclase activating polypeptide receptors 1 and 2; IL, interleukin; Ab,
antibody; PBS, phosphate-buffered saline; SOCS, suppressors of cytokine
signaling; TBS-T, Tris-buffered saline with Tween 20; SBE, signal transducer
and activator of transcription binding element; LPS, lipopolysaccharide; DTT,
dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PKA, cAMP-dependent
protein kinase; EMSA, electrophoretic mobility shift assay; ELISA,
enzyme-linked immunosorbent assay; EAE, experimental autoimmune
encephalomyelitis.
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REFERENCES |
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