From the Department of Oral Biology, University of Nebraska Medical Center, Lincoln, Nebraska 68583 and the ¶ Department of Pharmacology, Mayo Clinic at Jacksonville, Jacksonville, Florida 32224
Received for publication, September 8, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Interleukin-12 (IL-12) is composed of two
different subunits, p40 and p35. Expression of p40 mRNA but not
that of p35 mRNA in excessive amount in the central nervous system
of patients with multiple sclerosis (MS) suggests that IL-12 p40
may have a role in the pathogenesis of the disease. However, the mode
of action of p40 is completely unknown. Because nitric oxide produced from the induction of nitric-oxide synthase (iNOS) also plays a vital
role in the pathophysiology of MS, the present study was undertaken to
explore the role of p40 in the induction of NO production and the
expression of iNOS in microglia. Both IL-12 and p402, the p40 homodimer, dose-dependently induced the production
of NO in BV-2 microglial cells. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Induction of
NO production by the expression of mouse p40 cDNA but not that of
the mouse p35 cDNA suggests that the p40 but not the p35 subunit of
IL-12 is involved in the expression of iNOS. In addition to BV-2 glial
cells, p402 also induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 and
p402 were unable to induce the production of NO in mouse
primary astrocytes. Because activation of NF- Nitric oxide (NO), derived in excessive amount from the activation
of inducible nitric-oxide synthase
(iNOS)1 in glial cells
(microglia and astrocytes), is assumed to contribute to oligodendrocyte
degeneration in demyelinating diseases and neuronal death during
neurodegenerative diseases (1-5). Evidence from several laboratories
emphasizes the involvement of NO in the pathophysiology of multiple
sclerosis (MS) and experimental allergic encephalomyelitis (EAE), the
animal model of MS (2, 6-7). Analysis of CSF from MS patients has
shown increased levels of nitrite and nitrate compared with normal
control (9). The reaction of NO with
O On the other hand, interleukin-12 (IL-12) plays a critical role in the
early inflammatory response to infection and in the generation of T
helper type 1 Th-1 cells, which favor cell-mediated immunity (14).
Recently, it has been found that overproduction of IL-12 can be
dangerous to the host as it is involved in the pathogenesis of a number
of autoimmune inflammatory diseases (e.g. multiple
sclerosis, arthritis, insulin-dependent diabetes, Refs. 15,
16). IL-12 consists of a heavy chain (p40) and a light chain (p35)
linked covalently by disulfide bonds to give rise to a heterodimeric
(p70) molecule (17, 18). It is known that the heterodimeric p70
molecule is the bioactive IL-12 cytokine, and both subunits must be
coexpressed in the same cell to generate the bioactive form (19).
However, the level of p40 is much higher than that of p35 in IL-12
producing cells (19). Again, several reports (15, 19-21) indicate that
the level of p40 mRNA in the central nervous system (CNS) of
patients with MS is much higher than the CNS of control subjects
whereas the level of p35 mRNA is about the same or decreases
compared with controls. Similarly, in mice with experimental allergic
encephalomyelitis (EAE), an animal model of MS, the expression of p40
mRNA but not that of p35 mRNA increases in brain and spinal
cord (22). However, the functional significance of marked
overexpression of IL-12 p40 subunit in neural tissues of MS patients
and EAE animals has not been delineated so far.
We herein report the first evidence that p402, the IL-12
p40 homodimer, markedly induces the production of NO and the expression of iNOS through the activation of NF- Reagents--
Fetal bovine serum, Hank's balanced salt solution
and DMEM/F-12 were from Life Technologies, Inc.
L-NG-Monomethylarginine
(L-NMMA) and
D-NG-monomethylarginine
(D-NMMA), NF- Isolation of Mouse Microglia and Astrocytes--
Astrocytes were
prepared from mouse cerebral tissue as described by McCarthy and
DeVellis (23). Cells were maintained in DMEM/F-12 medium containing
10% fetal bovine serum. After 10 days of culture, astrocytes were
separated from microglia and oligodendrocytes by shaking for 24 h
in an orbital shaker at 240 rpm. To ensure the removal of
oligodendrocytes and microglia, the shaking was repeated twice after a
gap of 1 or 2 days. Astrocyte cultures were >95% positive for glial
fibrillary acidic protein, a specific marker for astrocytes. Cells were
trypsinized, subcultured, and stimulated with IL-12 p70, IL-12
p402, and other cytokines in serum-free DMEM/F-12.
Microglial cells were isolated from mixed glial cultures according to
the procedure of Guilian and Baker (24). Briefly, on day 7-9, the
mixed glial cultures were washed three times with DMEM/F-12 and were
shaken at 240 rpm for 2 h at 37 °C on a rotary shaker. The
floating cells were washed and seeded onto plastic tissue culture
flasks and incubated at 37 °C for 2 h. The attached cells were
removed by trypsinization and seeded onto new plates for further
studies. Ninety to ninety-five percent of this preparation was found to
be positive for Mac-1 surface antigen. For the induction of NO
production, cells were stimulated with IL-12 p70, IL-12 p402, and other cytokines in serum-free DMEM/F-12.
Mouse BV-2 microglial cells (kind gift from Virginia Bocchini of the
University of Perugia) were also maintained and induced with different
stimuli as indicated above.
Isolation of Mouse Macrophages and Induction of NO
Production--
Resident macrophages were obtained from mouse by
peritoneal lavage with sterile RPMI 1640 medium containing 1% fetal
bovine serum and 100 µg/ml gentamicin (25). Cells were washed three times with RPMI 1640 at 4 °C and were maintained at 37 °C in a humidified incubator containing 5% CO2 in air. Macrophages
at a concentration of 2 × 106/ml in RPMI 1640 medium
containing L-glutamine and gentamicin were added in volumes
of 1 ml to a 35-mm plate. After 1 h, nonadherent cells were
removed by washing, and 1 ml of serum-free RPMI 1640 medium with
various stimuli was added to the adherent cells. After 24 h, the
culture supernatants were transferred to measure NO production.
Construction of Mouse p40 and p35 cDNA Expression
Constructs--
Recombinant plasmids containing cDNA for mouse p40
(ATCC 87595) and p35 (ATCC 87596) in pBluescript SK+ were obtained from the ATCC. The p35 and p40 cDNA were cut out of the plasmids
utilizing the restriction enzymes XhoI and NotI.
The enzyme reaction products were size fractionated on 0.8%
agarose/0.5× Tris-acetate-EDTA and visualized by ethidium
bromide fluorescence. Bands of ~750 base pairs and 1050 base pairs
corresponding to p35 and p40 cDNA, respectively, were isolated from
the gel using Qiagen gel miniprep kit according to the manufacturer's
specifications. The isolated cDNA was ligated into
XhoI/NotI-cut pCIneo mammalian expression vector
(Promega, Madison, WI) utilizing T4 DNA ligase according to the
manufacturer's specifications. The cloned cDNA was used to
transform competent E. coli JM109. Several transformed
clones were isolated and plasmids were prepared from each. The sequence of the inserted DNA in several plasmid constructs was confirmed at the
core facilities of the Beadle Center for Biotechnology, University of Nebraska.
Expression of Mouse p40 and p35 cDNAs in BV-2 Glial
Cells--
Cells at 50-60% confluence were transfected with 1 µg
each of p40 and p35 cDNAs by LipofectAMINE Plus (Life Technologies, Inc.) following the manufacturer's protocol (26, 27). Twenty-four hours after transfection, cells were incubated with serum-free media.
To exclude the influence of bioactive IL-12 p70 on this experiment, we
also added anti-IL-12 p70 (1 µg/ml) to the serum-free media. After
24 h of incubation, culture supernatants were transferred to
measure NO production.
Assay for NO Synthesis--
Synthesis of NO was determined by
assay of culture supernatants for nitrite, a stable reaction product of
NO with molecular oxygen. Briefly, 400 µl of culture supernatant was
allowed to react with 200 µl of Griess reagent (28-31) and incubated
at room temperature for 15 min. The optical density of the assay
samples was measured spectrophotometrically at 570 nm. Fresh culture
medium served as the blank in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of
NaNO2 in the assay. Protein was measured by the procedure
of Bradford (32).
Immunoblot Analysis for iNOS--
Immunoblot analysis for iNOS
was carried out as described earlier (28-30). Briefly, cells were
scraped off, washed with Hank's buffer, and homogenized in 50 mM Tris-HCl, pH 7.4 containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin). After electrophoresis, the proteins were transferred onto a nitrocellulose membrane, and the
iNOS band was visualized by immunoblotting with antibodies against
mouse macrophage iNOS and by chemiluminescence assay.
RNA Isolation and Northern Blot Analysis--
Cells were taken
out of the culture dishes directly by adding Ultraspec-II RNA reagent
(Biotecx Laboratories Inc.), and total RNA was isolated according to
the manufacturer's protocol. For Northern blot analyses, 20 µg of
total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose
gels, electrotransferred to Hybond nylon membrane (Amersham Pharmacia
Biotech) and hybridized at 68 °C with 32P-labeled
cDNA probe using Express Hyb hybridization solution (CLONTECH) as described by the manufacturer. The
cDNA probe was made by polymerase chain reaction amplification
using two primers (forward primer, 5'-CTCCTTCAAAGAGGCAAAAATA-3';
reverse primer, 5'-CACTTCCTCCAGGATGTTGT-3'; Refs. 28-30). After
hybridization, the filters were washed two or three times in solution I
(2× SSC, 0.05% SDS) for 1 h at room temperature followed by
solution II (0.1× SSC, 0.1% SDS) at 50 °C for another hour. The
membranes were then dried and exposed to x-ray films (Kodak). The same
amount of RNA was hybridized with probe for glyceraldehyde-3-phosphate dehydrogenase.
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay--
Nuclear extracts from p402-stimulated or
unstimulated cells (1 × 107 cells) were prepared
using the method of Dignam et al. (33) with slight
modifications. Cells were harvested, washed twice with ice-cold
phosphate-buffered saline, and lysed in 400 µl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM
MgCl2, 0.5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin
A, and 5 µg/ml leupeptin) containing 0.1% Nonidet P-40 for 15 min on
ice, vortexed vigorously for 15 s, and centrifuged at 14,000 rpm
for 30 s. The pelleted nuclei were resuspended in 40 µl of
buffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin). After 30 min on ice,
lysates were centrifuged at 14,000 rpm for 10 min. Supernatants
containing the nuclear proteins were diluted with 20 µl of modified
buffer C (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.05 M KCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride)
and stored at Assay of Transcriptional Activity of NF- IL-12 Induces the Production of NO and the Expression of iNOS in
BV-2 Microglial Cells--
IL-12 is a potent regulator of
cell-mediated immune responses (14, 19). To understand the role of
IL-12 in the induction of iNOS, we examined the effect of IL-12 p70 on
the production of NO in mouse BV-2 microglial cells. Results in Table
I show that mouse IL-12 markedly induced
the production of NO. The inhibition of NO production by arginase, an
enzyme that degrades the substrate (L-arginine) of NOS and
L-NMA, a competitive inhibitor of NOS, but not by
D-NMA, a negative control of L-NMA, suggest
that IL-12-induced NO production in BV-2 glial cells is dependent on
NOS-mediated arginine metabolism (Table I). To understand the mechanism
of NO production in IL-12-stimulated BV-2 cells, we examined the effect
of IL-12 on the protein level of iNOS. Western blot analysis with
antibodies against murine macrophage iNOS of IL-12-stimulated BV-2
cells clearly showed that IL-12 significantly induced the expression of
iNOS protein (Fig. 1B). To
understand the specificity of induction of iNOS, BV-2 glial cells were
stimulated with different cytokines and LPS. Among all the inducers
tested, LPS, IFN- IL-12 p40 Induces the Production of NO and the Expression of iNOS
in BV-2 Microglial Cells--
It is known that biologically active
IL-12 is a 70-kDa heterodimeric glycoprotein comprised of
disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits (17-19).
However, the p40 but not the p35 mRNA is expressed in excessive
amount in neural tissues of MS and EAE (15, 19-21). Therefore, we
examined the effect of p402 on the expression of iNOS. Fig.
2A shows that p402
dose-dependently induced the production of NO. About
18-20-fold induction of NO production was observed when
p402 was used at a concentration of 5 or 10 ng/ml (Fig.
2A). This recombinant p402 (Pharmingen) was
pure, and it showed a single 40-kDa protein band with
SDS-polyacrylamide gel electrophoresis(data not shown). The induction
of NO production by p402 was also inhibited by anti-mouse
p40 but not by anti-mouse p70 (data not shown) suggesting that
bioactive IL-12 p70 is not involved in p402-mediated
induction of NO production. Moreover, recombinant mouse
p402 obtained from a different source (R&D) also induced
the production of NO in BV-2 glial cells (data not shown). Taken
together, these observations clearly show that p402 is
capable of inducing the production of NO in BV-2 glial cells. To
understand the mechanism of induction of NO production, we examined the
effect of p402 on protein and mRNA levels of iNOS. Western blot analysis with antibodies against murine macrophage iNOS
and Northern blot analysis for iNOS mRNA of
p402-stimulated BV-2 glial cells clearly showed that
p402 induced the expression of iNOS protein (Fig.
2B) and iNOS mRNA (Fig. 2C). Under
physiological conditions, IL-12 p40 exists as both monomer and dimer
(19). To understand the role of p40 monomer in the induction of iNOS, we examined the effect of human p40 monomer (obtained from R&D) on the
induction of NO production in human THP1 monocytic cells. In contrast
to the effect of mouse p402 on NO production in mouse BV-2
glial cells, the human p40 monomer alone did not induce the production
of NO in THP1 cells. However, the human p40 monomer markedly stimulated
the production of NO in IFN- Expression of Mouse p40 cDNA Induces the Production of NO and
the Expression of iNOS in BV-2 Glial Cells--
To further confirm the
induction of iNOS by p40, we examined the effect of transient
expression of mouse p40 and p35 cDNAs on the production of NO and
the expression of iNOS in BV-2 glial cells. Similar to macrophages,
microglial cells are also known to produce IL-12 (34, 35). Mouse p40 or
p35 protein generated from transfected p40 or p35 cDNA may react
with endogenous p35 or p40 protein to produce the bioactive IL-12 p70
heterodimer, which in turn may influence the effect of transfected p40
or p35 cDNA. Therefore, to exclude the possible influence by IL-12
p70, we also added anti-mouse p70 to the serum-free media. Consistent to the induction of iNOS by p402, expression of p40
cDNA but not that of p35 cDNA markedly induced the production
of NO (Fig. 3A) and the
expression of iNOS protein (Fig. 3B) suggesting that p40 but
not the p35 subunit of IL-12 is involved in the induction of iNOS in
microglial cells and that p40 induces iNOS independent of the so-called
bioactive IL-12 p70.
IL-12 p70 and IL-12 p40 Induce the Production of NO in Mouse
Peritoneal Macrophages and Primary Microglia but Not in Primary
Astrocytes--
To understand whether p402 induces the
production of NO in primary cells, we examined the effect of
p402 on the production of NO in mouse primary glial cells
(astrocytes and microglia) and peritoneal macrophages (Table
II). Consistent to the induction of iNOS
in BV-2 glial cells, both IL-12 p70 and p402 markedly induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 p70 and p402 were unable
to induce the production of NO in mouse primary astrocytes (Table 2)
suggesting that p402 specifically induces iNOS in mouse
microglia and macrophages but not in astrocytes.
IL-12 p40 Induces the Expression of iNOS through the Activation of
NF-
The cytokines (TNF- IL-12, a heterodimeric cytokine, is most noted for its ability to
regulate the balance between type 1 and type 2 helper T cells (14, 19).
Neither IL-12 subunits (p35 or p40) alone was found to display
significant biological activity over a large range of concentrations
(17-19). However, several lines of evidence indicate that p40 is
expressed in excessive amount in the CNS of different demyelinating
diseases such as multiple sclerosis (MS), Guillain-Barre syndrome, and
animal models experimental autoimmune encephalomyelitis and neuritis
(15, 16, 19-21). On the other hand, the expression of p35 remains
almost constant or decreased to some extent in the CNS of these
demyelinating diseases compared with the CNS of control subjects
(19-21). However, the biological significance of this overexpression
of p40 in the CNS of patients with demyelinating diseases is not known.
Several lines of evidence presented in this manuscript clearly support
the conclusion that IL-12 p40 homodimer, p402, induces the
expression of iNOS in mouse microglia and macrophages. This conclusion
was based on the following observations. First, p402 induces the production of NO, which is inhibited by arginase, the
enzyme which degrades the substrate of NOS, and by L-NMA, an inhibitor of NOS. Second, p402-mediated production of NO
and expression of iNOS is inhibited by anti-mouse p40 but not by
anti-mouse p70. Third, the expression of mouse p40 cDNA but not the
mouse p35 cDNA induces the production of NO and the expression of
iNOS, suggesting that the p40 but not the p35 subunit of IL-12 is
involved in the induction of iNOS. Because NO produced from the
activation of iNOS in the CNS participates in the pathophysiology of MS
(4, 6-12), the overexpression of p40 mRNA in the CNS of MS
patients (15, 19-21) and the induction of iNOS by p40 suggest that p40 may participate in the pathophysiology of MS through the induction of
iNOS.
The level of p40 is much higher (5- to 500-fold) than that of the
heterodimeric p70 in IL-12-producing cells (19). This excess p40
produced either in vitro in activated cells or in
vivo in serum of endotoxin-treated mice exists as both dimer
(20-40%) and monomer (the remainder, Ref. 19). Although the
biological role of the monomeric as well as the dimeric form of p40 is
not known, it has been suggested that p402 may act as a
physiologic regulator of bioactive IL-12, because p402
possesses IL-12 antagonist activity (19, 39). Therefore, the induction
of iNOS by p402 suggests that p402 exhibits
IL-12 antagonist activity possibly through the activation of iNOS.
However, our observation that both p402 and the so-called
bioactive IL-12 (heterodimeric p70) induce the production of NO and the
expression of iNOS precludes this possibility. If iNOS-derived
NO mediates the IL-12 antagonist activity of p402 then
IL-12 itself can regulate its own function through the activation of
iNOS. Apart from the IL-12 antagonist activity of p40, experiments on
Listeria monocytogenes infection in p40- and p35-deficient
mice have shown that p40-deficient mice were susceptible to infection,
but p35-deficient mice were able to eliminate bacteria despite the
mouse's inability to produce biologically active heterodimeric IL-12
(19). Interestingly, it has also been found that the p35-deficient
mouse produces normal levels of p40 (19). Taken together, these
observations suggest that p40 alone may carry out some of the
biological functions of heterodimeric IL-12. Here we present the first
evidence that similar to IL-12, p402 can also induce the
expression of iNOS and that iNOS-derived NO may account for the
bacteria-eliminating property of both IL-12 and p40.
The signaling events in cytokine-mediated induction of iNOS are not
completely established so far. Proinflammatory cytokines (TNF- Our results have clearly shown that p402 induces the
expression of iNOS through the activation of NF- At present, it is unclear how p402 activates NF- NO, a diffusible free radical, plays many roles as a signaling and as a
effector molecule in diverse biological systems including neuronal
messenger, vasodilation, and antimicrobial and antitumor activities
(51, 52). In the nervous system the NO appears to have both neurotoxic
and neuroprotective effects and may have a role in the pathogenesis of
stroke and other neurodegenerative diseases and in demyelinating
conditions (e.g. multiple sclerosis, experimental allergic
encephalopathy, X-adrenoleukodystrophy) associated with infiltrating
macrophages and the production of proinflammatory cytokines (53). NO
and peroxynitrite (reaction product of NO and
O Because IL-12 p40 is overexpressed in the CNS of the neuroinflammatory
diseases, the induction of iNOS expression by IL-12 p40 in microglia
and macrophages suggests that expression of p40 may induce/potentiate
the neural injury in the inflamed CNS through the induction of NO production.
B is important for the
expression of iNOS, we investigated the effect of p402 on
the activation of NF-
B. Induction of the DNA binding as well as the
transcriptional activity of NF-
B by p402 and inhibition
of p402-induced expression of iNOS by SN50, a
cell-permeable peptide carrying the nuclear localization sequence of
p50 NF-
B, but not by SN50M, a nonfunctional peptide mutant, suggests
that p402 induces the expression of iNOS through the
activation of NF-
B. This study delineates a novel role of
IL-12 p40 in inducing the expression of iNOS in microglial cells, which
may participate in the pathogenesis of neuroinflammatory diseases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, a strong nitrosating agent capable of nitrosating
tyrosine residues of a protein to nitrotyrosine. Increased levels of
nitrotyrosine have been found in demyelinating lesions of MS brains as
well as in spinal cords of mice with EAE (10, 11). Subsequently, semiquantitative reverse transcriptase-polymerase chain reaction for
iNOS mRNA in MS brains also shows markedly higher expression of
iNOS mRNA in MS brains than in normal brains (12, 4).
B in mouse microglia.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B SN50, and NF-
B SN50 M were purchased from Biomol. LPS (Escherichia coli, serotype
0111:B4) and arginase were from Sigma. Antibodies against mouse
macrophage iNOS were obtained from CalBiochem. Recombinant mouse IL-12
and p40 homodimer were obtained from Pharmingen. Recombinant mouse IFN-
, TNF-
, and IL-1
were obtained from R&D.
70 °C until use. Nuclear extracts were used for the
electrophoretic mobility shift assay using the NF-
B DNA-binding
protein detection system kit (Life Technologies, Inc./Life
Technologies, Inc.) according to the manufacturer's protocol.
B--
To assay the
transcriptional activity of NF-
B, cells at 50-60% confluence were
transfected with pNF-
B-Luc, an NF-
B-dependent reporter construct (obtained from Stratagene), using the LipofectAMINE Plus method (Life Technologies, Inc.) (26, 27). All transfections included 50 ng/µg total DNA of pRL-TK (a plasmid encoding
Renilla luciferase, used as transfection efficiency control;
Promega). After 24 h of transfection, cells were treated with
different stimuli for 6 h. Firefly and Renilla
luciferase activities were obtained by analyzing total cell extract
according to standard instructions provided in the Dual Luciferase Kit
(Promega) in a TD-20/20 Luminometer (Turner Designs). Relative
luciferase activity of cell extracts was typically represented as
(firefly luciferase value/Renilla luciferase value) × 10
3.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and IL-12 efficiently induced the production of
NO and the expression of iNOS protein whereas IL-1
was less
efficient in inducing the expression of iNOS (Fig. 1). However, there
was no induction of iNOS by TNF-
and IL-6 (Fig. 1). Similarly,
TNF-
and IL-6 were also ineffective in inducing the production of NO
in mouse primary microglia (data not shown).
Induction of NO production by IL-12 in BV-2 glial cells
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Fig. 1.
Induction of NO production and expression of
iNOS by different cytokines in BV-2 glial cells. Cells were
cultured with different cytokines under serum-free conditions.
A, after 24 h, supernatants were used for nitrite assay
as described under "Materials and Methods." Data are mean ± S.D. of three different experiments. B, cell homogenates
were electrophoresed, transferred onto nitrocellulose membranes, and
immunoblotted with antibodies against mouse macrophage iNOS as
described under "Materials and Methods." Concentrations of
different stimuli were: LPS, 1.0 µg/ml; TNF- , 50 ng/ml; IL-1
,
10 ng/ml; IFN-
, 25 units/ml; IL-12, 5 ng/ml; IL-6, 20 ng/ml.
-treated THP1 cells.2
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Fig. 2.
Dose-dependent induction of NO
production and expression of iNOS by IL-12 p40 in BV-2 glial
cells. Cells were cultured with different p402
concentrations under serum-free conditions. A, after 24 h, supernatants were used for nitrite assay. Data are mean ± S.D.
of three different experiments. B, cell homogenates were
electrophoresed, transferred onto nitrocellulose membranes, and
immunoblotted with antibodies against mouse macrophage iNOS.
C, after a 6-h incubation, cells were taken out directly by
adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) to the
plates for isolation of total RNA, and Northern blot analysis for iNOS
mRNA was carried out as described under "Materials and
Methods."
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Fig. 3.
Expression of p40 cDNA but not that of
p35 cDNA induces the expression of iNOS in BV-2 glial cells.
Cells plated at 50-60% confluence in 6-well plates were transfected
with 1 µg each of p40 and p35 cDNAs by LipofectAMINE Plus (Life
Technologies, Inc.) as described under "Materials and Methods."
Twenty-four hours after transfection, cells were incubated with
serum-free medium in the presence of 1 µg/ml of anti-mouse IL-12 p70.
A, after 24 h, supernatants were used for nitrite
assay. Data are mean ± S.D. of three different experiments.
B, cell homogenates were electrophoresed, transferred onto
nitrocellulose membranes, and immunoblotted with antibodies against
mouse macrophage iNOS.
Induction of NO production by IL-12 p70 and IL-12 p402 in mouse
primary astrocytes, microglia, and macrophages
B in BV-2 Glial Cells--
Because activation of NF-
B is
necessary for induction of iNOS (26-30, 36, 37), to understand the
basis of expression of iNOS, we examined the effect of p402
on the activation of NF-
B in BV-2 glial cells. Activation of NF-
B
was monitored by both DNA binding and transcriptional activity of
NF-
B (26, 27, 31). DNA binding activity of NF-
B was evaluated by
the formation of a distinct and specific complex in a gel shift DNA
binding assay. Treatment of BV-2 glial cells with different
concentrations of p402 resulted in
dose-dependent induction of DNA binding activity of NF-
B
with the maximum induction observed at 5 ng/ml (Fig. 4A). This gel shift assay
detected a specific band in response to p402 that was
competed off by an unlabeled probe suggesting that p402
induces the DNA binding activity of NF-
B. We then tested the effect
of p402 on NF-
B-dependent transcription of
luciferase in BV-2 glial cells, using the expression of luciferase from
a reporter construct, pNF-
B-Luc (Stratagene), as an assay.
Consistent to the effect of p402 on the DNA binding
activity of NF-
B, p402 also induced
NF-
B-dependent transcription of luciferase in a dose-dependent manner (Fig. 4B). To further
confirm the activation of NF-
B by p402, we expressed
mouse p40 and p35 cDNAs in BV-2 glial cells and examined the
transcriptional activity of NF-
B. It is evident from Fig.
4C that expression of mouse p40 but not that of p35 cDNA
induced NF-
B-dependent expression of luciferase, suggesting that the p40 but not the p35 subunit of IL-12 is involved in
the activation of NF-
B. Because the activation of NF-
B is important for the induction of iNOS, these results also suggest that
unlike p40, p35 is unable to induce iNOS because of its inability to
induce the activation of NF-
B (Fig.
5). To investigate further that
p402-induced expression of iNOS in BV-2 microglial cells depends on the activation of NF-
B, we examined the effect of SN50 on
p402-mediated expression of iNOS. SN50 is a synthetic peptide containing signal sequences of Kaposi's fibroblast growth factor and the nuclear localization sequence of NF-
B p50 (38). It
has been reported to have the capacity to specifically block the
nuclear translocation of activated NF-
B (38). Inhibition of
p402-mediated activation of NF-
B (Fig.
6A), induction of NO production (Fig. 6B), and expression of iNOS mRNA (Fig.
6C) by SN50 but not by SN50M, a nonfunctional mutant of
SN50, suggests that activation of NF-
B is necessary for the
expression of iNOS in p402-stimulated BV-2 cells. However,
p402 was unable to activate NF-
B in mouse primary
astrocytes (data not shown), suggesting that p402 is unable
to induce iNOS in mouse astrocytes (Table 2) because of its inability
to induce the activation of NF-
B.
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[in a new window]
Fig. 4.
IL-12 p40 induces the activation of
NF- B in BV-2 glial cells. A,
cells incubated in serum-free DMEM/F-12 were treated with different
concentrations of p402. After 1 h of incubation, cells
were taken out to prepare nuclear extracts, and nuclear proteins were
used for the electrophoretic mobility shift assay as described under
"Materials and Methods." B, lanes 1-3 represent nuclear
extract of control cells, nuclear extract of p402-treated
cells, and nuclear extract of p402-treated cells incubated
with a 100-fold excess of unlabeled oligonucleotide. The concentration
of p402 used in this experiment was 5 ng/ml. The
upper arrow indicates the induced NF-
B band, and the
lower arrow indicates the unbound probe. C, cells
plated at 50-60% confluence in 6-well plates were cotransfected with
1 µg of pNF-
B-Luc (an NF-
B-dependent reporter
construct) and 50 ng of pRL-TK (a plasmid encoding Renilla
luciferase, used as transfection efficiency control) using
LipofectAMINE Plus as described under "Materials and Methods."
After 24 h of transfection, cells were stimulated with different
concentrations of p402 for 6 h under serum-free
conditions. Firefly and Renilla luciferase activities were
obtained by analyzing total cell extract as described under
"Materials and Methods."
View larger version (23K):
[in a new window]
Fig. 5.
Expression of p40 cDNA but not that p35
cDNA induces the activation of NF- B in
BV-2 glial cells. Cells plated at 50-60% confluence in 6-well
plates were cotransfected with 1 µg of pNF-
B-Luc (an
NF-
B-dependent reporter construct) and 1 µg of p40,
p35, or the empty vector using the LipofectAMINE Plus method. All
transfections included 50 ng/µg total DNA of pRL-TK. After 24 h
of transfection, cells were incubated with serum-free medium for
24 h in the presence of 1 µg/ml of anti-mouse IL-12 p70. Firefly
and Renilla luciferase activities were obtained by analyzing
total cell extract as described under "Materials and
Methods."
View larger version (27K):
[in a new window]
Fig. 6.
SN50, a specific cell-permeable peptide
inhibitor of NF- B nuclear translocation,
inhibits p402-mediated activation of
NF-
B and expression of iNOS in BV-2 glial
cells. A, cells plated at 50-60% confluence in 6-well
plates were cotransfected with 1 µg of pNF-
B-Luc and 50 ng of
pRL-TK using LipofectAMINE Plus. After 24 h of transfection, cells
were incubated with different concentrations of SN50 or SN50M (a
nonfunctional mutant of SN50) for 1 h followed by stimulation with
5 ng/ml of p402 for 6 h under serum-free conditions.
Firefly and Renilla luciferase activities were obtained by
analyzing total cell extract as described above. B, cells
preincubated with different concentrations of SN50 or SN50M for 1 h received 5 ng/ml of p402 under serum-free conditions.
After 24 h, supernatants were used for nitrite assay. Data are
mean ± S.D. of three different experiments. C, after
6 h of incubation, cells were taken out directly by adding
Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) to the plates for
isolation of total RNA, and Northern blot analysis for iNOS mRNA
was carried out as described above.
, IL-1
, IFN-
, IL-12 p70, IL-12 p40, IL-6)
or drugs (SN50 or SN50M) used under these experimental conditions had
no effect on the viability of BV-2 glial cells, measured by trypan blue
exclusion. Therefore, the conclusions drawn in this study are not due
to any change in viability of the cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
IL-1
, or IFN-
) bind to their respective receptors and induce the
expression of iNOS via NF-
B activation (26-30, 36, 37). The
presence of a consensus sequence in the promoter region of iNOS for the
binding of NF-
B (36) and the inhibition of iNOS expression with the
inhibition of NF-
B activation establishes an essential role of
NF-
B activation in the induction of iNOS (26-30, 36, 37).
Activation of NF-
B by various cellular stimuli involves the
proteolytic degradation of IkB, the inhibitory subunit of NF-
B
complex, and the concomitant nuclear translocation of the liberated
NF-
B heterodimer (40, 41). Although the biochemical mechanism
underlying the degradation of IkB remains unclear, it appears that
degradation of IkB induced by various mitogens and cytokines occurs in
association with the transient phosphorylation of IkB on serines 32 and
36 (42). Consistently, two closely related kinases (IKK
and IKK
)
that directly phosphorylate IkB
have also been described (43-45).
Upon phosphorylation, IkB that is still bound to NF-
B apparently
becomes a high affinity substrate for an ubiquitin-conjugating enzyme
(46). After phosphorylation-controlled ubiquitination, the IkB is
rapidly and completely degraded by the 20 S or 26 S proteosome, and the
NF-
B heterodimer enters into the nucleus (47) and binds to the
consensus DNA-binding site present in the promoter region of iNOS.
B. First,
p402 induces the DNA-binding as well as the transcriptional
activity of NF-
B. Second, expression of the mouse p40 cDNA but
not the mouse p35 cDNA induces the activation of NF-
B and the
expression of iNOS. Third, SN50, a cell-permeable peptide carrying the
nuclear localization sequence of p50 NF-
B, but not mutant SN50
(SN50M) inhibits p402-mediated activation of NF-
B and
expression of iNOS. It has been demonstrated that SN50 specifically
blocks the nuclear translocation of NF-
B, but does not affect the
activity of AP-1, SP-1 factor, and OCT-1 transcriptional factors (48)
suggesting that SN50 inhibits the expression of iNOS in
p402-stimulated microglial cells by inhibiting the
activation of NF-
B. In addition, these results also suggest that
IL-12 p402 is biologically active and that p402
alone can activate microglial cells.
B and
induces iNOS in microglial cells. IL-12 p402 has been shown
to antagonize bioactive IL-12 heterodimer by binding to the IL-12
receptor complex (19). The high affinity IL-12 receptor is composed of
a low affinity IL-12R
1 combined with a low affinity IL-12R
2,
which are responsible for Tyk2/Jak2 activation, respectively, and STAT4 activation (19, 49). It appears that p402 binds to
IL-12R
1 rather than IL-12R
2, whereas bioactive IL-12 binds the
receptor complex with high affinity (50). Therefore, it is possible
that p402 activates NF-
B and induces the expression of
iNOS through the IL-12R
1.
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FOOTNOTES |
---|
* This study was supported by Grants NS39940 from the National Institutes of Health, 0692 from Smokeless Tobacco Research Council and 00-09 from the UNMC College of Dentistry.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Dept. of Oral Biology,
University of Nebraska Medical Center, 40th and Holdrege, Lincoln, NE
68583-0740. Tel.: 402-472-1324; Fax: 402-472-2551; E-mail:
kpahan@unmc.edu.
§ Present address: Dept. of Biology, Walter Reed Army Inst. of Research, Washington, D. C.
Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M008262200
2 K. Pahan and X. Liu, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: iNOS, inducible nitric-oxide synthase; IL-12, interleukin 12; DMEM, Dulbecco's modified Eagle's medium; LPS, lipopolysaccharide.
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