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by IL-12/IL-18-Activated Macrophages Requires STAT4 Signaling and Is Inhibited by IL-41
*
Institute of Clinical Microbiology, Immunology, and Hygiene and
Department of Dermatology, University of Erlangen, Erlangen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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on combined stimulation with IL-12 and
IL-18, but the signaling requirements of this process and its
regulation by other cytokines are unknown. Here, we demonstrate that
STAT4 is indispensable for IL-12/IL-18-induced production of IFN- by
mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we
previously found to be a prerequisite for IL-12-induced IFN-
production in NK cells, was not required for IFN- production by
these macrophages. IL-12 alone already induced the expression of
IFN- mRNA, but nuclear translocation of STAT4, the release of
IFN- protein, and the subsequent production of NO was strictly
dependent on the simultaneous presence of IL-18. NF-
B, which
mediates IL-18 effects in T cells, was only weakly activated by IL-12
and/or IL-18 in macrophages. Known inhibitors of macrophage functions
(e.g., IL-4 and TGF-
) also suppressed macrophage IFN- production
and the subsequent production of NOS2-derived NO. The inhibitory effect
of IL-4 was paralleled by nuclear translocation of STAT6, which in
EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These
results further define the production of IFN- by macrophages and
point to a diversity in the signals required for IFN- production by
various cell types. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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, which is typically released by NK
cells, CD4+ type 1 Th cells, and several other
subsets of T cells (e.g., 
T cells, NKT cells,
CD8+ T cells), has been a hallmark of the immune
responses against intracellular pathogens and tumor cells as well as of
certain autoimmune reactions. IFN- activates macrophages to produce
cytokines, to express antimicrobial and tumoricidal effector pathways,
and to act as APCs (1, 2). During more recent years,
macrophages themselves were recognized also to be producers of IFN-
under certain conditions. IFN- mRNA and/or protein was detected in
various populations of mononuclear phagocytes, including human alveolar
macrophages (3), and resting peritoneal macrophages
(4, 5), peritoneal exudate macrophages
(6, 7, 8), bone marrow-derived macrophages (9),
splenic macrophages (10), and lung macrophages from mice
(11). Although a possible (minor) contamination with T,
NK, or NKT cells has not always been vigorously excluded, most of these
studies unequivocally demonstrate the production of IFN- by
monocytes/macrophages. The stimuli that were reported to induce IFN-
in monocytes/macrophages include type I IFNs (7), IFN-
itself (4), IL-12 (bioactive p70 homodimer) (3, 5, 10), LPS (6, 8) (which largely acts via induction
of endogenous IL-12), Mycobacterium tuberculosis
(3), Mycobacterium bovis bacillus
Calmette-Guérin plus IL-12 (11), and a combination
of IL-12 and IL-18 (9). In the latter case, the levels of
IFN- protein found in the cultures of bone marrow-derived
macrophages approached or even exceeded the amounts that are usually
released by T or NK cells. The activation of macrophages for the
secretion of IFN- by IL-12 and IL-18 is of particular interest,
because both IL-12 and IL-18 are known products of macrophages, which
suggests the possibility of autocrine stimulation. Cytokines that are
able to counteract this pathway have not yet been defined.
In T and NK cells, STAT4 is critical for the production of IFN- in
response to IL-12, which was shown by the analysis of STAT4-deficient
mice (12, 13, 14). In addition, NK cells, but not T cells,
required NO derived from type 2 NO synthase
(NOS2)3 for IL-12
signaling, i.e., for the activation of Tyk2 kinase, the tyrosine
phosphorylation of STAT4, and the production of IFN-
(15). Recently, evidence was provided that a
STAT4-independent pathway of IFN- production exists in
CD8+ T cells; however, it was only observed after
cross-linking of the TCR and not after stimulation with IL-12/IL-18
(16). A similar pathway was also observed in
CD4+ T cells lacking both STAT4 and STAT6
(14). In dendritic cells isolated from mouse spleens,
IL-12 signaling was reported to involve nuclear translocation of
NF-B rather than activation of members of the STAT family
(17). In contrast, in human blood monocyte-derived
dendritic cells stimulation with IL-12 led to tyrosine-phosphorylation
of Tyk2 and Jak2 kinase as well as of STAT3 and STAT4
(18). In LPS-activated human monocytes, STAT4 protein was
shown to be expressed and tyrosine-phosphorylated on stimulation with
IFN-
(19). However, whether NF-B, NOS2, and/or the
Jak/STAT pathway are actually required for the production of IFN- by
dendritic cells or macrophages is unknown to date. In the present
study, we show that STAT4 is essential for the production of IFN- by
inflammatory macrophages in response to IL-12/IL-18, whereas
NOS2-derived NO is dispensable. We also demonstrate that IL-4, IL-10,
IL-13, and TGF-1 inhibit IL-12/IL-18-induced IFN- production in
macrophages. The effect of IL-4 appears to be mediated by the
activation of STAT6, which in EMSA was able to bind to the same DNA
oligonucleotide as IL-12/IL-18-induced STAT4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Female CD1 mice (20–24 g; 8–12 wk old) and C57BL/6 mice
(16–18 g; 6–8 wk old) were purchased from Charles River Breeding
Laboratories (Sulzfeld, Germany). Breeding pairs of (129/SvEv x
C57BL/6) mice with a disrupted NOS2 gene
(NOS2-/-) (20) and wild-type
controls (NOS2+/+) were originally provided by
C. F. Nathan (New York, NY) and J. S. Mudgett (Merck, Rahway,
NJ). The NOS2+/+ and
NOS2-/- mice used here were obtained from
homozygous intercrosses in the F8 to
F9 generation (129/SvEv x C57BL/6). C57BL/6
mice deficient for the IFN- gene
(IFN--/-) were provided by M. Kopf (Basel
Institute for Immunology, Basel, Switzerland). Breeding pairs of FVB/NJ
mice deficient for the STAT4 gene (STAT4-/-)
and the respective wild-type controls (STAT4+/+;
Ref. 13) were kindly provided by Dr. J. N. Ihle
(Department of Biochemistry, St. Jude Children’s Research Hospital,
Memphis, TN). Breeding pairs of mice with a deletion of the J281
gene segment, which lack V14-positive NKT cells and were crossed
back on a C57BL/6 background for eight generations (21),
were kindly provided by Drs. M. Taniguchi and T. Nakayana (Chiba
University, Chiba, Japan). Breeding pairs of
Rag2-/- mice were obtained from Dr. Bernd
Arnold (University of Heidelberg, Heidelberg, Germany). All mice were
housed under specific pathogen-free conditions in our own animal
facilities.
Macrophages and cell culture
Four days after i.p. injection of 3 ml of 4% Brewer’s thioglycolate broth (Difco, Detroit, MI), peritoneal exudate cells were harvested from the above-mentioned mouse strains by flushing the peritoneal cavity twice with 10 ml of PBS. The cells were resuspended in RPMI 1640 culture medium supplemented with 2 mM glutamine, 10 mM HEPES, 13 mM NaHCO3, 50 µM 2-ME, 100 µg/ml penicillin, and 100 µg/ml streptomycin (all from Seromed-Biochrom, Berlin, Germany) plus 2.5% FBS (Sigma, Deisenhofen, Germany).
Macrophages were seeded into 96-well plates (2 x
105 cells/well in 100 µl), 24-well plates
(1 x 106 cells/well in 500 µl),
24-cm2 culture dishes (6 x
106 cells/dish in 3 ml), or
64-cm2 culture dishes (16 x
106 cells/dish in 8 ml) and were cultured at
37°C in 5% CO2/95% humidified air. After
90–120 min, nonadherent cells were washed off (three washes with warm
PBS). The adherent macrophage monolayers (containing >95% F4/80- or
Mac-1-positive cells and <<1% CD4+,
CD8+, or B220+ cells) were
further incubated in fresh medium with the given stimuli. Used stimuli
were recombinant murine (rm) IL-12 (BD PharMingen, San Diego, CA; R&D
Systems, Minneapolis, MN), recombinant human TGF-1, rmIL-4, rmIL-11,
rmIL-17, rmIL-18 (all from R&D Systems), IFN- (kindly provided from
Dr. G. Adolf at the Ernst Boehringer Institut, Vienna, Austria), and
LPS (O111:B4; Sigma). The concentrations of IL-12 and IL-18 were 0.1
and 10 ng/ml, unless stated differently.
The LPS content of the cytokine stock solutions and of the final culture medium was <10 pg/ml as determined by a colorimetric Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
Determination of nitrite accumulation and measurement of
IFN-
NO2- in culture
supernatants was determined by the Griess assay (22). The
capture ELISA for measuring IFN- was performed as described
(15) and had a detection limit of 39–78 pg/ml.
Preparation of total cell lysates for Western blot analysis and immunoprecipitation
For the detection of Stat4 and tyrosine-phosphorylated Stat4 protein, macrophage monolayers were lysed in 40 mM Tris buffer (pH 8.0) plus protease and phosphatase inhibitors as described (15). The lysates (60–80 µg protein/lane) were separated by 7.5% SDS-PAGE, transferred to reinforced nitrocellulose (0.2 µM; Schleicher & Schuell, Dassel, Germany), and analyzed by ECL-based Western blotting with affinity-purified rabbit-anti-mouse STAT4 IgG (sc-486 (C-20); Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phosphotyrosine mouse IgG (PY-99; Santa Cruz Biotechnology) as described (15, 23). Immunoprecipitations (with anti-STAT4 (sc-485 or sc-486) or anti-phosphotyrosine Ab (PY-99)) were performed as published previously (15).
RNA preparation, cDNA synthesis, and RT-PCR
Total RNA was isolated from macrophage monolayers by the
guanidinium isothiocyanate method, reverse transcribed (1 µg), and
analyzed by qualitative or quantitative (competitive) RT-PCR as
published (22). The competitor plasmids for the
different genes were as follows: 1) piNOSL1
(HincII 162 bp) for NOS2 (22); 2)
pMCQ for -actin and IFN- (24); and 3) pIL-12R1
and pIL-12R2 for IL-12 receptor 1 and IL-12 receptor 2,
respectively (15). The primer sequences for NOS2, IFN-,
-actin, and IL-12 receptor 1 and 2 were as published
previously (15, 25). The sequences of the IL-18 receptor
upstream and downstream primer used for qualitative PCR analysis were
5'-CGT GAC AAG CAG AGA TGT TG-3' and 5'-ATG TTG TCG TCT CCT TCC TG-3',
respectively. The annealing temperatures were 57°C (IL-18R), 58°C
(NOS2, IL-12R1, and IL-12R2), or 60°C (-actin and IFN-).
The number of PCR cycles was 35.
Oligonucleotide probes
Single-stranded oligonucleotides binding STAT4 (derived from the
IFN regulatory factor-1 gene promotor; Refs. 26 and
27), STAT6 (derived from the human secreted-type IL-1
receptor antagonist promotor; Ref. 28), or NF-B
(derived from the mouse NOS2 promotor; Ref. 29) were
obtained from MWG-Biotech (Ebersberg, Germany) and were annealed in 10
mM Tris buffer (pH 7.5; containing 50 mM NaCl, 10 mM
MgCl2, and 1 mM DTT) by incubation for 5 min at
85°C and subsequent slow cooling to room temperature: STAT4, 5'-CT
AGA GCC TGA TTT CCC CGA AAT GAT GAG-3', 3'-T CGG ACT AAA
GGG GCT TTA CTA CTC GAT C-5'; STAT4 mutant ("CCC" 
"TTT"),
5'-CT AGA GCC TGA TTT CTT TGA AAT GAT GAG-3', 3'-T CGG ACT
AAA GAA ACT TTA CTA CTC GAT C-5'; STAT6, 5'-GAT CGC TCT TCT TCC CAG GAA
CTC AAT-3', 3'-CG AGA AGA AGG GTC CTT GAG TTA CAG CT-5'; NF-B,
5'-GAA GCT TGG GGA CTC TCC CTT TG-3', 3'-ACC CCT GAG AGG GAA ACC
CTT-5'.
The double-stranded oligomers were filled in by the Klenow fragment of
DNA polymerase I with [-32P]dCTP (3000
Ci/mmol; Amersham Life Science, Braunschweig, Germany) to obtain
labeled probes. Unlabeled or mutant oligomers were used as competitors
for EMSA.
Preparation of nuclear extracts
Nuclear extracts were prepared as published previously with minor modifications (30). Briefly, after stimulation, macrophages were washed twice with ice-cold PBS (plus 100 µM sodium orthovanadate) and scraped into 400 µl of lysis buffer (20 mM Tris buffer, pH 8.0, containing 10 mM KCl; 5 mM MgCl2; 1 mM each of PMSF, EDTA, sodium orthovanadate, sodium pyrophosphate, and sodium fluoride; 0.5 mM each of EGTA and DTT; 0.1 mM sodium molybdate; 10 µM each of pepstatin A and aprotinin; and 5 µM each of leupeptin and chymostatin). The cells were allowed to swell on ice for 5 min before Nonidet P-40 was added to a final concentration of 0.5%, and the tube was vortexed for 10 s. The homogenate was centrifuged and the supernatant (cytosol) was harvested in a fresh tube. The nuclear pellets were washed once in lysis buffer without Nonidet P-40 and finally resuspended in 50–100 µl of nuclear extract buffer (lysis buffer plus 0.4 M KCl). After incubation on ice for 45 min, cell debris was removed by centrifugation (13,000 x g, 15 min, 4°C), and the supernatants (containing DNA binding proteins) were stored at -70°C.
EMSA
Binding reactions (20 µl total) were performed by incubating
the nuclear extracts (4–5 µg of protein) with 5x reaction buffer
(50 mM Tris buffer, pH 7.5; 500 mM KCl; 25 mM
MgCl2; 5 mM of DTT and EDTA; 25% glycerol) and 2
µg of poly(dI-dC)·poly(dI-dC) in the presence or absence of an
excess (100- to 1000-fold or as indicated) of cold competitor or mutant
oligomer for 5 min, followed by a 30-min incubation at room temperature
with the labeled dsDNA probe (0.1–1 ng; 
1 x
105 cpm). For supershift analysis, 2 µg of
rabbit anti-mouse NF-B p50 (sc-114X), rabbit anti-mouse
NF-B p65 (sc-372X), rabbit anti-STAT4 Ab (sc-485-X or sc-486-X),
or rabbit anti-STAT6 (sc-981-X; all from Santa Cruz Biotechnology)
were added to the reaction mixture and incubated at room temperature
for 30 min before the labeled DNA probe was added for another 15 min of
incubation. The formed DNA/protein complexes were separated on 4.5–6%
polyacrylamide gels in 0.5 x Tris-borate-EDTA buffer at 40 mA for
1.5 h. The gels were dried without fixation, and the DNA protein
complexes were visualized by autoradiography.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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protein in response
to IL-12/IL-18, which leads to the production of NO
In a previous study, mouse bone marrow-derived macrophages were
shown to produce IFN- in response to IL-12 and IL-18, whereas
commonly used macrophage cell lines failed to do so (9).
Here, we demonstrate that an entirely different population of
macrophages, i.e., thioglycolate-elicited peritoneal macrophages from
CD1, C57BL/6, FVB/NJ, as well as from Rag2-/-
mice (which lack functional T and B lymphocytes) and J281-deficient
mice (which lack V14/J281-positive NKT cells, high output
producers of IFN-), secreted IFN- after simultaneous exposure to
IL-12 and IL-18. IFN- was readily detectable at 24 h of
stimulation and reached its plateau at 48 h (Fig. 1
A and data not shown). At 10
ng/ml IL-18, 100 pg/ml IL-12 was sufficient to induce maximal
production of IFN- (9.7 ± 4.4 ng/ml, mean ± SEM of 27
experiments). After stimulation of the macrophages with either
cytokine alone, IFN- protein was still measurable in the culture
supernatants, but the concentrations were 5- to 10-fold lower (Fig. 1A).
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by IL-12/IL-18-stimulated peritoneal exudate
macrophages was paralleled by the accumulation of nitrite (Fig. 1
B). In accordance with previous data on bone marrow-derived
macrophages (9), no nitrite was detectable in the culture
medium of macrophages from IFN--deficient mice (data not shown).
Thus, the production of NO was not a direct effect of IL-12 and IL-18,
but mediated by the endogenous IFN-.
Cytokine regulation of macrophage IFN- production
Having seen that IL-12 and IL-18 are potent inducers of IFN-,
we next analyzed whether other known macrophage activators (LPS,
TNF-, or IL-17; Ref. 31) could replace IL-12 or IL-18.
We found that LPS (0.2–200 ng/ml), TNF- (0.1–50 ng/ml), or IL-17
(0.1–10 ng/ml) alone were unable to stimulate macrophages for the
production of IFN-. However, LPS in combination with IL-18 (but not
in combination with IL-12) led to a potent release of IFN- after
48 h (9.0 ± 0.7 ng/ml, mean ± SEM of 12 experiments).
TNF- and IL-17, in contrast, were ineffective when combined with
either IL-12 or IL-18 (data not shown).
A number of cytokines have been described to down-regulate various
macrophage functions, including IL-4, IL-10, IL-11, IL-13, or TGF-
(Refs. 32, 33, 34 and references therein). When added 2 h
before or simultaneously with IL-12 and IL-18 to the macrophage
cultures, IL-4 and TGF- inhibited the production of IFN-, whereas
IL-10, IL-11, and IL-13 (0.1–100 ng/ml) were only weakly suppressive
or completely inactive (Fig. 2, A–C and data not shown). The mean suppression achieved by
IL-4 and TGF- was 61.9 ± 7.1% and 70.8 ± 9.4%,
respectively (mean ± SEM of six experiments). TGF-, but none
of the other cytokines, was also active when added 2 h after
initiation of the stimulation with IL-12 plus IL-18 (Fig. 2D).
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, and NOS2
As a first approach to understanding the synergistic action of
IL-12 and IL-18 on IFN- protein production, we analyzed the
expression of their receptors and their effect on IFN- mRNA.
Unstimulated macrophage monolayers already constitutively expressed
IL-12R1, IL-122, and IL-18R mRNA. With PCR analysis, there was no
appreciable difference in the mRNA expression levels of the receptors
when the macrophages were stimulated with IL-12, IL-18, or IL-12 plus
IL-18 (data not shown). These data indicate that the two cytokines do
not act by up-regulating the mRNA of each other’s receptor. However, a
possible mutual influence on the surface expression of the receptor
proteins is not excluded.
IL-12, IL-18, or IL-12 plus IL-18 induced the expression of IFN-
mRNA, which was undetectable in resting macrophages. However, we did
not observe a synergistic action of IL-12 and IL-18 (Fig. 3A). In contrast, the
induction of NOS2 mRNA was most prominent under costimulation
conditions and delayed by 4–6 h relative to the expression of IFN-
mRNA (Fig. 3B). Together with the data in Fig. 1A
these results suggest that the synergistic action of IL-12 and IL-18
mainly occurs on the level of IFN- protein and that the expression
of IFN- precedes the production of NO.
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B
T cells and NK cells activate STAT4 on stimulation with IL-12
(12, 13, 35, 36). Furthermore, STAT4 protein was found in
human dendritic cells, monocytes, and macrophages (18, 19). Therefore, we envisaged a role of STAT4 in macrophage
activation by IL-12 plus IL-18. Unstimulated adherent macrophages
expressed STAT4 protein, which was readily detectable by Western
blotting of whole-cell lysates. The detected band was confirmed to
represent STAT4 by parallel analysis of macrophage lysates from
STAT4+/+ and STAT4-/-
mice (Fig. 4A).
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, B and
C). The required minimum period of stimulation was 90–120
min (Fig. 4B and data not shown). Stimulation of the
macrophages with IL-12 or IL-18 alone, in contrast, did not cause
translocation of STAT4 into the nucleus (Figs. 4B and
7A).
As NF-B was shown to be activated by IL-18 in T cells (37, 38) and by IL-12 in dendritic cells (17), we also
analyzed the nuclear translocation of NF-B in macrophages stimulated
with IL-12, IL-18, or IL-12 plus IL-18 for 15 min to 6 h. Our EMSA
(Fig. 5 and data not shown) revealed that
the combination of both cytokines (but neither cytokine alone) weakly
enhanced the DNA binding activity of nuclear extracts to an
oligonucleotide containing a NF-B consensus sequence. The activation
of NF-B might contribute to the stimulatory effect of IL-12 plus
IL-18 on macrophage IFN- production.
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production by IL-12 plus IL-18
To directly assess the role of STAT4 in macrophage IFN-
production, we used macrophages from STAT4-/-
mice. Whereas wild-type macrophages produced high amounts of IFN-
and NO in response to IL-12 plus IL-18, IFN- protein and NO were not
detectable in the culture supernatants of
STAT4-/- macrophages (Fig. 6, A and B). In
contrast, macrophages with a deletion of the NOS2 gene were fully
capable of producing IFN- after stimulation with IL-12 plus IL-18
(Fig. 6A). Thus, STAT4, but not NOS2-derived NO, is required
as a signaling molecule in macrophages for IL-12/IL-18-induced IFN-
production. Importantly, IFN- mRNA was still present in
IL-12/IL-18-stimulated STAT4-/- macrophages,
although its level of expression was clearly lower compared with
STAT4+/+ macrophages (Fig. 6C).
Therefore, STAT4 does not only regulate the expression of IFN- mRNA
but also the synthesis of IFN- protein. This conclusion is further
supported by the observation that in wild-type macrophages IFN- mRNA
was expressed after stimulation with IL-12, IL-18, or IL-12 plus IL-18
(Fig. 3A and Fig. 6C), whereas activation of
STAT4 and production of IFN- protein were only achieved after
combined stimulation with IL-12 and IL-18 (Figs. 1A and
4).
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production by IL-4 is paralleled
by the activation of STAT6 that binds comparably well to the same DNA
oligonucleotide as STAT4
The above experiments have demonstrated that the
IL-12/IL-18-induced production of IFN- by macrophages is dependent
on STAT4 and is inhibited by IL-4. Therefore, we performed EMSA
analysis to investigate whether IL-4 can antagonize the activation of
STAT4 by IL-12 plus IL-18. Costimulation of the macrophages with IL-4
led to the appearance of a second, slower-migrating DNA/protein complex
(II) when using the STAT4 probe, whereas the intensity of the
IL-12/IL-18-induced complex I (containing STAT4) decreased. No such
effect was seen when TGF- instead of IL-4 was used (Fig. 7, A and C,
lane 3 vs lane 7). Complex II was formed by STAT6
because it was completely supershifted by an anti-STAT6 Ab (Fig. 7B), and it was also observed when a DNA oligonucleotide
with a STAT6 instead of a STAT4 binding site was used (Fig. 7C, lane 7 vs lane 8). Importantly,
IL-4-activated STAT6 bound comparably well to the STAT4 and STAT6 probe
(Fig. 7C, lane 11 vs lane 12), whereas
the IL-12/IL-18-activated STAT4 only interacted with the STAT4
oligonucleotide, but not with the STAT6 probe (Fig. 7C,
lane 3 vs lane 4). The addition of unlabeled
STAT4 competitor to nuclear extracts from IL-4/IL-12/IL-18-stimulated
macrophages completely eliminated both complex I and complex II,
whereas complex I was maintained in the presence of nonradiolabeled
STAT6 competitor (Fig. 7C, lane 7 vs lanes
9 and 10).
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, A and B).
Unlabeled STAT6 oligonucleotide competed the binding of STAT6 protein
to the STAT4 DNA probe at 
10-fold lower concentrations than the
STAT4 oligonucleotide. In contrast, the unlabeled STAT6 oligonucleotide
was unable to antagonize the interaction between the STAT4 protein and
the STAT4 probe (Fig. 8). Thus, STAT4 and STAT6 protein have similar
affinities to the STAT4 DNA oligonucleotide used in these in vitro
assays, and the affinity of STAT6 protein to the STAT6 DNA
oligonucleotide is only 10-fold higher.
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(data not shown). The
possibility that the inhibitory effect of IL-4 results from the
activation of STAT6 and its ability to interfere with signaling
functions of STAT4 is supported by previous experiments with mouse
macrophages in which strong evidence for a competition between
IFN--induced STAT1 and IL-4-induced STAT6 for occupancy of the STAT
binding element in the promotor of the IFN regulatory factor-1 gene was
presented (39). However, it is important to emphasize that
data obtained by EMSA analysis are only suggestive of, but by no means
provide a reflection of, the real events that take place on a promotor
in its genomic context. Furthermore, the promotor elements that are
occupied by STAT4 and mediate the STAT4-dependent induction of IFN-
in macrophages (or T cells) are unknown. Their ability to bind STAT4 vs
STAT6 might be completely different from the properties of the DNA
oligonucleotides that were used in the current analysis. Concluding remarks
Macrophages share with other types of cells the ability to produce
IFN-. This is the first study to demonstrate that macrophages,
similar to NK and T cells, are strictly dependent on STAT4 for the
release of IFN- protein in response to IL-12 plus IL-18. However,
unlike NK cells (15), NOS2-derived NO was not required for
macrophage IFN- production. We obtained no evidence for a
significant activation of NF-B by IL-12 plus IL-18 in macrophages as
it was described for dendritic cells (17). The production
of IFN- by macrophages is likely to amplify their antimicrobial
activities and to serve as an autoregulatory circuit that helps to
expand Th1 responses. However, IL-4, the prototypic Th2 cytokine, might
antagonize such a process by inhibiting macrophage IFN- production,
presumably via induction of STAT6 that is able to occupy the STAT4 DNA
binding site.
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Footnotes |
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2 Address correspondence and reprints requests to Dr. Christian Bogdan, Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Universität Erlangen-Nürnberg, Wasserturmstrasse 3, D-91054 Erlangen, Germany.
3 Abbreviations used in this paper: NOS2 (iNOS), type 2 (or inducible) NO synthase; rm, recombinant murine.
Received for publication September 9, 2000. Accepted for publication December 20, 2000.
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I. Wittmann, M. Schonefeld, D. Aichele, G. Groer, A. Gessner, and M. Schnare Murine Bactericidal/Permeability-Increasing Protein Inhibits the Endotoxic Activity of Lipopolysaccharide and Gram-Negative Bacteria J. Immunol., June 1, 2008; 180(11): 7546 - 7552. [Abstract] [Full Text] [PDF]
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 P. Geraghty, C. M. Greene, M. O'Mahony, S. J. O'Neill, C. C. Taggart, and N. G. McElvaney Secretory Leucocyte Protease Inhibitor Inhibits Interferon-{gamma}-induced Cathepsin S Expression J. Biol. Chem., November 16, 2007; 282(46): 33389 - 33395. [Abstract] [Full Text] [PDF]
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 U. I. Chaudhry, S. C. Katz, T. P. Kingham, V. G. Pillarisetty, J. R. Raab, A. B. Shah, and R. P. DeMatteo In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells FASEB J, May 1, 2006; 20(7): 982 - 984. [Abstract] [Full Text] [PDF]
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I. Fricke, D. Mitchell, J. Mittelstadt, N. Lehan, H. Heine, T. Goldmann, A. Bohle, and S. Brandau Mycobacteria Induce IFN-{gamma} Production in Human Dendritic Cells via Triggering of TLR2 J. Immunol., May 1, 2006; 176(9): 5173 - 5182. [Abstract] [Full Text] [PDF]
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 R. Parihar, R. Trotta, J. M. Roda, A. K. Ferketich, S. Tridandapani, M. A. Caligiuri, and W. E. Carson III Src Homology 2-Containing Inositol 5'-Phosphatase 1 Negatively Regulates IFN-{gamma} Production by Natural Killer Cells Stimulated with Antibody-Coated Tumor Cells and Interleukin-12 Cancer Res., October 1, 2005; 65(19): 9099 - 9107. [Abstract] [Full Text] [PDF]
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 P. Puddu, M. Carollo, I. Pietraforte, F. Spadaro, M. Tombesi, C. Ramoni, F. Belardelli, and S. Gessani IL-2 induces expression and secretion of IFN-{gamma} in murine peritoneal macrophages J. Leukoc. Biol., September 1, 2005; 78(3): 686 - 695. [Abstract] [Full Text] [PDF]
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 J. Tang, K. Kozaki, A. G. Farr, P. J. Martin, P. Lindahl, C. Betsholtz, and E. W. Raines The Absence of Platelet-Derived Growth Factor-B in Circulating Cells Promotes Immune and Inflammatory Responses in Atherosclerosis-Prone ApoE-/- Mice Am. J. Pathol., September 1, 2005; 167(3): 901 - 912. [Abstract] [Full Text] [PDF]
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I. N. Buhtoiarov, H. Lum, G. Berke, D. M. Paulnock, P. M. Sondel, and A. L. Rakhmilevich CD40 Ligation Activates Murine Macrophages via an IFN-{gamma}-Dependent Mechanism Resulting in Tumor Cell Destruction In Vitro J. Immunol., May 15, 2005; 174(10): 6013 - 6022. [Abstract] [Full Text] [PDF]
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 U. Schleicher, A. Hesse, and C. Bogdan Minute numbers of contaminant CD8+ T cells or CD11b+CD11c+ NK cells are the source of IFN-{gamma} in IL-12/IL-18-stimulated mouse macrophage populations Blood, February 1, 2005; 105(3): 1319 - 1328. [Abstract] [Full Text] [PDF]
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A. Mavropoulos, G. Sully, A. P. Cope, and A. R. Clark Stabilization of IFN-{gamma} mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells Blood, January 1, 2005; 105(1): 282 - 288. [Abstract] [Full Text] [PDF]
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D. S. Vinay, B. K. Choi, J. S. Bae, W. Y. Kim, B. M. Gebhardt, and B. S. Kwon CD137-Deficient Mice Have Reduced NK/NKT Cell Numbers and Function, Are Resistant to Lipopolysaccharide-Induced Shock Syndromes, and Have Lower IL-4 Responses J. Immunol., September 15, 2004; 173(6): 4218 - 4229. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Maegawa, T. Kondo, A. Kimura, Y. Iwakura, S. Nakamura, and N. Mukaida Essential Involvement of IFN-{gamma} in Clostridium difficile Toxin A-Induced Enteritis J. Immunol., March 1, 2004; 172(5): 3018 - 3025. [Abstract] [Full Text] [PDF]
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A. G. Rothfuchs, M. R. Kreuger, H. Wigzell, and M. E. Rottenberg Macrophages, CD4+ or CD8+ Cells Are Each Sufficient for Protection against Chlamydia pneumoniae Infection through their Ability to Secrete IFN-{gamma} J. Immunol., February 15, 2004; 172(4): 2407 - 2415. [Abstract] [Full Text] [PDF]
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K. Schroder, P. J. Hertzog, T. Ravasi, and D. A. Hume Interferon-{gamma}: an overview of signals, mechanisms and functions J. Leukoc. Biol., February 1, 2004; 75(2): 163 - 189. [Abstract] [Full Text] [PDF]
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Y. Ishida, T. Kondo, T. Takayasu, Y. Iwakura, and N. Mukaida The Essential Involvement of Cross-Talk between IFN-{gamma} and TGF-{beta} in the Skin Wound-Healing Process J. Immunol., February 1, 2004; 172(3): 1848 - 1855. [Abstract] [Full Text] [PDF]
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D. Durali, M.-G. de Goer de Herve, J. Giron-Michel, B. Azzarone, J.-F. Delfraissy, and Y. Taoufik In human B cells, IL-12 triggers a cascade of molecular events similar to Th1 commitment Blood, December 1, 2003; 102(12): 4084 - 4089. [Abstract] [Full Text] [PDF]
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G.-X. Zhang, S. Yu, B. Gran, J. Li, I. Siglienti, X. Chen, D. Calida, E. Ventura, M. Kamoun, and A. Rostami Role of IL-12 Receptor {beta}1 in Regulation of T Cell Response by APC in Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2003; 171(9): 4485 - 4492. [Abstract] [Full Text] [PDF]
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J. H. Bream, R. E. Curiel, C.-R. Yu, C. E. Egwuagu, M. J. Grusby, T. M. Aune, and H. A. Young IL-4 synergistically enhances both IL-2- and IL-12-induced IFN-{gamma} expression in murine NK cells Blood, July 1, 2003; 102(1): 207 - 214. [Abstract] [Full Text] [PDF]
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A. Ito, A. Matejuk, C. Hopke, H. Drought, J. Dwyer, A. Zamora, S. Subramanian, A. A. Vandenbark, and H. Offner Transfer of Severe Experimental Autoimmune Encephalomyelitis by IL-12- and IL-18-Potentiated T Cells Is Estrogen Sensitive J. Immunol., May 1, 2003; 170(9): 4802 - 4809. [Abstract] [Full Text] [PDF]
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 A. M. Harandi, K. Eriksson, and J. Holmgren A Protective Role of Locally Administered Immunostimulatory CpG Oligodeoxynucleotide in a Mouse Model of Genital Herpes Infection J. Virol., December 20, 2002; 77(2): 953 - 962. [Abstract] [Full Text] [PDF]
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J. Pirhonen, S. Matikainen, and I. Julkunen Regulation of Virus-Induced IL-12 and IL-23 Expression in Human Macrophages J. Immunol., November 15, 2002; 169(10): 5673 - 5678. [Abstract] [Full Text] [PDF]
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H.-R. Jiang, E. Muckersie, M. Robertson, H. Xu, J. Liversidge, and J. V. Forrester Secretion of interleukin-10 or interleukin-12 by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture J. Leukoc. Biol., November 1, 2002; 72(5): 978 - 985. [Abstract] [Full Text] [PDF]
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C. Bovolenta, E. Pilotti, M. Mauri, B. Panzeri, M. Sassi, P. Dall'Aglio, U. Bertazzoni, G. Poli, and C. Casoli Retroviral Interference on STAT Activation in Individuals Coinfected with Human T Cell Leukemia Virus Type 2 and HIV-1 J. Immunol., October 15, 2002; 169(8): 4443 - 4449. [Abstract] [Full Text] [PDF]
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A. C. Kirby, U. Yrlid, and M. J. Wick The Innate Immune Response Differs in Primary and Secondary Salmonella Infection J. Immunol., October 15, 2002; 169(8): 4450 - 4459. [Abstract] [Full Text] [PDF]
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M. Strengell, T. Sareneva, D. Foster, I. Julkunen, and S. Matikainen IL-21 Up-Regulates the Expression of Genes Associated with Innate Immunity and Th1 Response J. Immunol., October 1, 2002; 169(7): 3600 - 3605. [Abstract] [Full Text] [PDF]
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A. Finnegan, M. J. Grusby, C. D. Kaplan, S. K. O'Neill, H. Eibel, T. Koreny, M. Czipri, K. Mikecz, and J. Zhang IL-4 and IL-12 Regulate Proteoglycan-Induced Arthritis Through Stat-Dependent Mechanisms J. Immunol., September 15, 2002; 169(6): 3345 - 3352. [Abstract] [Full Text] [PDF]
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J. C. M. Morel, C. C. Park, K. Zhu, P. Kumar, J. H. Ruth, and A. E. Koch Signal Transduction Pathways Involved in Rheumatoid Arthritis Synovial Fibroblast Interleukin-18-induced Vascular Cell Adhesion Molecule-1 Expression J. Biol. Chem., September 13, 2002; 277(38): 34679 - 34691. [Abstract] [Full Text] [PDF]
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 V. B. Mahajan, C. Wei, and P. J. McDonnell III Microarray Analysis of Corneal Fibroblast Gene Expression after Interleukin-1 Treatment Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2143 - 2151. [Abstract] [Full Text] [PDF]
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 R. Hontecillas, M. J. Wannemeulher, D. R. Zimmerman, D. L. Hutto, J. H. Wilson, D. U. Ahn, and J. Bassaganya-Riera Nutritional Regulation of Porcine Bacterial-Induced Colitis by Conjugated Linoleic Acid J. Nutr., July 1, 2002; 132(7): 2019 - 2027. [Abstract] [Full Text] [PDF]
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E. Kuroda, T. Kito, and U. Yamashita Reduced Expression of STAT4 and IFN-{gamma} in Macrophages from BALB/c Mice J. Immunol., June 1, 2002; 168(11): 5477 - 5482. [Abstract] [Full Text] [PDF]
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C. Parham, M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, R. Zhang, K. P. Singh, F. Vega, et al. A Receptor for the Heterodimeric Cytokine IL-23 Is Composed of IL-12R{beta}1 and a Novel Cytokine Receptor Subunit, IL-23R J. Immunol., June 1, 2002; 168(11): 5699 - 5708. [Abstract] [Full Text] [PDF]
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 A. A. Lighvani, D. M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, B. D. Hissong, B. V. Nguyen, M. Gadina, A. Sher, W. E. Paul, et al. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells PNAS, December 18, 2001; 98(26): 15137 - 15142. [Abstract] [Full Text] [PDF]
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A. Gigliotti Rothfuchs, D. Gigliotti, K. Palmblad, U. Andersson, H. Wigzell, and M. E. Rottenberg IFN-{alpha}{beta}-Dependent, IFN-{gamma} Secretion by Bone Marrow-Derived Macrophages Controls an Intracellular Bacterial Infection J. Immunol., December 1, 2001; 167(11): 6453 - 6461. [Abstract] [Full Text] [PDF]
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D. L. Laskin, B. Weinberger, and J. D. Laskin Functional heterogeneity in liver and lung macrophages J. Leukoc. Biol., August 1, 2001; 70(2): 163 - 170. [Abstract] [Full Text] [PDF]
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D. Stober, R. Schirmbeck, and J. Reimann IL-12/IL-18-Dependent IFN-{{gamma}} Release by Murine Dendritic Cells J. Immunol., July 15, 2001; 167(2): 957 - 965. [Abstract] [Full Text] [PDF]
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