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*
Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan; and
Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases and
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by APC as well as IFN- production
by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress
Stat4 induction in DC and macrophages when present during maturation
and activation, respectively, diminishing IFN- production. In
contrast, IL-4 has no effect on Stat4 levels in mature DC and actually
augments IFN- production by DC during Ag presentation, indicating
that IL-4 acts differently in a spatiotemporal manner. The functional
importance of Stat4 is evident in Stat4-/- DC and
macrophages, which fail to produce IFN-. Furthermore,
Stat4-/- macrophages are defective in NO production in
response to IL-12 and are susceptible to Toxoplasma.
Autocrine IL-12 signaling is required for high-level IFN- production
by APC at critical stages in both innate and adaptive immunity, and the
control of Stat4 expression is likely an important regulator of this
process. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
production by NK cells. At the same time, IL-12
specifically promotes the differentiation of CD4+
T cells to Th1 effector cells producing IFN- (5, 6).
IFN-, in turn, acts to increase NK activity and to enhance killing
of intracellular pathogens. Impaired NK responses and lower IFN-
production from CD4+ T cells have been observed
in IL-12-deficient mice, indicating the essential role of IL-12 in both
innate and acquired immunity (7).
It has long been assumed that the only cells responding to IL-12 are T,
B, NK, and NKT cells (5, 6). Recently, however, we and
others have demonstrated that DC and macrophages also respond to IL-12
by producing IFN- (8, 9, 10, 11). DC express a high-affinity
receptor for IL-12 and are capable of producing significant amounts of
IFN- in response to IL-12 (9, 10, 12). In fact, the
amounts of IFN- produced by DC are severalfold higher than those by
NK cells (9). Similar to DC, bone marrow-derived
macrophages produce IFN- when stimulated with IL-12, but high-level
production requires stimulation with IL-18 in combination with IL-12
(8). Taken together, these recent advances suggest the
possibility of autocrine regulation of macrophage and DC functions by
IL-12.
As with other type I cytokines, IL-12 activates a signal transduction
cascade consisting of specific Janus family tyrosine kinases (Jaks) and
Stats, families of signal transduction molecules that play pivotal
roles in cytokine-induced gene expression (13, 14, 15, 16, 17). Among
these, Stat4 is absolutely required for IL-12-dependent IFN-
production as shown by studies with Stat4-/-
mice, although it is by no means clear that Stat4 directly regulates
the IFN- gene (18, 19). Nonetheless, these observations
suggested that Stat4 is required in IL-12-dependent IFN- production
in APC as well.
In this study, we demonstrate that the expression of Stat4, but not
Jak2 or Tyk2, is regulated in a maturation-dependent manner in DC.
Stat4 is not expressed in immature DC but its expression is induced
upon maturation as well as in macrophages upon activation. Mature DC
and activated macrophages express high levels of Stat4, and IL-12
promotes phosphorylation and nuclear translocation of Stat4 in these
APC. We further show that the Th2 cytokines IL-4 and IL-10 suppress
Stat4 induction in DC and macrophages when present during maturation
and activation, respectively, diminishing IFN- production. In
contrast, IL-4 has no effect on Stat4 levels in mature DC and actually
augments IFN- production by DC during Ag presentation, indicating
that IL-4 acts differently in a spatiotemporal manner. This unique
regulatory mechanism of IFN- production in DC suggests a novel
Th1/Th2 driving system by DC. Moreover, the functional significance of
autocrine IL-12 signaling in APC is highlighted by defects in
Stat4-deficient macrophages. Normal macrophages produce IFN- and NO
in response to IL-12 and display microbicidal activity against
Toxoplasma gondii, whereas Stat4-deficient macrophages are
not capable of this function. Thus, we propose a model for cellular
immunity which includes autocrine stimulation of APC by IL-12, a
process involving dynamic regulation of Stat4 by various stimuli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from Sankyo Labo Service (Tokyo,
Japan). B10.D2-Rag-2-deficient mice, Rag-2-/-
mice that had been backcrossed to B10.D2/nSnJ for 10 generations, were
obtained from Taconic Farms (Germantown, NY). Breeding pairs of
BALB/c-Stat4-/-,
BALB/c-IFN--/-, and
C57BL/6-IFN--/- mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). All mice were maintained in
specific pathogen-free conditions in our animal facilities and used
between 6 and 12 wk of age. All experiments were performed in
accordance with our Institutional Guidelines.
Cytokines and reagents
Recombinant murine (m) IL-12 (mIL-12) was purchased from Sigma
(St. Louis, MO) or, where indicated, R&D Systems (Minneapolis,
MN). Recombinant mIL-10 was purchased from Pharma Biotechnologie
Hannover (Hannover, Germany) or R&D Systems. Recombinant mTNF- was
purchased from PeproTech (Rocky Hill, NJ). Purified recombinant mIL-4
and mGM-CSF were generous gifts from A. Miyajima (University of Tokyo,
Tokyo, Japan). LPS was obtained from Calbiochem (San Diego, CA). RPMI
1640 supplemented with 10% FCS (HyClone, Logan, UT), 2-ME (50 µM),
L-glutamine (2 mM), penicillin G (100 U/ml), streptomycin
(100 µg/ml), and sodium pyruvate (1 mM) was used as the culture
medium here.
Antibodies
The following mAbs were purchased from BD PharMingen (San Diego,
CA): HL3-FITC, -PE (anti-CD11c); M1/70-biotin (anti-Mac-1);
PO3-biotin (anti-CD86); 3/23-biotin (anti-CD40); 53-6.7-PE,
-biotin (anti-CD8); 25-9-17-PE, -biotin
(anti-I-Ab); AMS-32.1-biotin
(anti-I-Ad); and C15.1-biotin
(anti-IL-12). F4/80-FITC and streptavidin-Cy5 were obtained from
Caltag (Burlingame, CA). Affinity-purified rabbit anti-Stat4,
-Jak2, and -Tyk2 antisera were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Polyclonal Ab against phosphorylated Stat4 was
obtained from Zymed (San Francisco, CA). Anti-cystatin C antiserum was
purchased from Upstate Biotechnology (Lake Placid, NY).
Rhodamine-conjugated affinity-purified goat anti-rabbit IgG (H + L)
was purchased from Jackson ImmunoResearch (West Grove, PA). Rabbit
polyclonal anti-asialoGM1 (anti-ASGM1) Ab was purchased
from Wako Pure Chemical (Osaka, Japan).
DC preparation
Immature and mature DC were prepared as described in previous
studies (10, 11). Briefly, collagenase-digested spleen
cells were suspended in a dense BSA solution in PBS (
= 1.080;
Sigma), overlaid with 1 ml of FCS-free RPMI 1640 medium, and
centrifuged in a swing bucket rotor at 9500 x g for 15
min at 4°C. The cells in a low-density fraction at the interface were
collected and washed twice. In experiments requiring further
purification, the cells were incubated with anti-mouse Ig (H + L)
beads (Perspective Biosystems, Cambridge, MA), and contaminated B cells
were excluded by a MACS magnet (Miltenyi Biotec, Bergisch Gladbach,
Germany). DC were then positively selected using anti-CD11c (N418)
MicroBeads and MACS column or AutoMACS (Miltenyi Biotec) from the
negatively selected cells. All procedures except the collagenase
digestion step were performed on ice. Purified cells were routinely
>95% CD11c+ I-A+ and used
as immature DC. Freshly isolated DC were induced to mature by
cultivation in culture medium overnight in the presence or absence of
cytokines. In selected experiments, the cells in a low-density fraction
from the density gradient centrifugation of collagenase-digested cells
were stained with a mixture of the following biotinylated mAbs:
anti-CD3
, 145-2C11; anti-CD4, GK1.5; and anti-B220,
RA3-6B2. Cells were then incubated with streptavidin MicroBeads and
depleted by MACS or AutoMACS. An additional purification procedure was
done to separate DC into CD8+ and
CD8- subsets. The
CD8+ subset was positively selected with
anti-CD8 (Ly-2) MicroBeads (Miltenyi Biotec) and from the
negative fraction of this selection, the CD8-
subset was purified with anti-CD11c (N418) MicroBeads (Miltenyi
Biotec).
Macrophage preparation
Bone marrow-derived macrophages were prepared using standard
techniques (20). Briefly, bone marrow cells were filtered
through nylon mesh and erythrocytes were lysed using ACK (0.15 M
NH4Cl, 10 mM KHCO3, and 0.1 mM
Na2EDTA, pH 7.4) lysis buffer solution
(BioWhittaker, Walkersville, MD). The cells were then cultured
for 8–10 days on 150 x 15-mm tissue culture dishes in culture
medium and 25 ng/ml M-CSF (R&D Systems) or 10 ng/ml GM-CSF. The medium
was replaced every 3 days. After 8–10 days of culture, nonadherent
cells were removed and adherent cells were detached using 10 mM EDTA in
PBS. These cells were routinely >98% positive for Mac-1 and FcR as
determined by flow cytometry. The harvested macrophages were then
washed with PBS and enumerated before subsequent experiments.
Western blots
Cells were lysed in a lysis buffer solution consisting of 20 mM
Tris-HCl (pH 7.4), 250 mM NaCl, 20 mM 
-glycerophosphate, 1 mM sodium
orthovanadate, 5 mM p-nitrophenylphosphate, 2 mM DTT, 1%
aprotinin, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and
1% Nonidet P-40. Postnuclear supernatants were obtained by
centrifugation at 10,000 x g for 30 min. Cell lysates
were boiled for 3 min in a Laemmli sample buffer solution, fractionated
on SDS-PAGE, transferred to polyvinylidene difluoride (PVDF)
membranes, and blotted with the indicated Abs. The reactive bands were
visualized with HRP coupled to the appropriate secondary Abs with an
ECL Western blotting detection system (Amersham, Buckinghamshire,
U.K.).
Preparation of nuclear fractions
After a 1-h stimulation with IL-12 (10 ng/ml), DC were washed twice with ice-cold PBS, harvested, and resuspended in 300 µl of a hypotonic buffer solution A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, and 10 µg/ml of leupeptin, aprotinin, and pepstatin) for 10 min on ice. The cells were then lysed in 0.6% Nonidet P-40 by vortexing for 10 s. Nuclei were separated from cytosol by centrifugation at 12,000 x g for 30 s and washed with 300 µl of the above buffer solution A. They were resuspended in a buffer solution consisting of 20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin and briefly sonicated on ice. Nuclear extracts were obtained by centrifugation at 12,000 x g for 10 min.
Immunohistochemical analysis
Intracellular immunofluorescence staining was performed as described previously (9). Immunohistochemical analysis of spleen sections was performed as follows. Spleens were embedded in Tissue-Tek (Miles, Elkhart, IN) and frozen at -80°C. Cryosections (7-µm thick) were fixed in acetone for 10 min and incubated with the primary Abs for 30 min at room temperature. Immunofluorescence labeling was performed with rhodamine-conjugated secondary Ab or streptavidin-Cy5 for 30 min at room temperature. Incubation was terminated by washing the samples with PBS. Samples were sealed with Prolong reagent (Molecular Probes, Eugene, OR). Specimens were examined under a Zeiss LSM510 confocal laser scanning microscope (Zeiss, Thornwood, NY) or a Axiovert 100 fluorescence microscope (Zeiss) equipped with an IPLab Spectrum image analysis system (Signal Analytics, Vienna, VA).
Toxoplasma infections
Bone marrow-derived macrophages (2 x
105/well) were plated in 96-well plates and
treated as indicated with IL-12 (10 ng/ml), IL-18 (50 ng/ml), IFN-
(100 U/ml), and/or anti-IFN- (XMG6, rat IgG1, 20 µg/ml). LPS
(10 ng/ml) was added 18 h later. Two hours after LPS treatment,
the macrophages were infected with tachyzoites of the RH strain of
T. gondii at a multiplicity of infection of 0.1. Eighteen
hours after T. gondii infection, 50-µl aliquots of the
supernatants were used to determine NO production by the Griess
reaction. Plates were then pulsed at 48 h with 1 µCi of
[3H]uracil. Subsequently, macrophage cultures
were harvested on glass fiber filters and incorporated uracil was
measured by liquid scintillation. Net cpm was calculated by subtracting
background cpm in uninfected cultures from counts measured in infected
cultures.
Miscellaneous
Flow cytometric analysis was performed as follows. Cells were
stained with FITC-, PE-, or biotin-conjugated mAbs in PBS-2% FCS,
washed, and analyzed on a FACScan using the CellQuest program (Becton
Dickinson, San Jose, CA). Biotinylated mAbs were detected with
streptavidin Red670 (Life Technologies, Rockville, MD). Titers of
IFN- in the culture supernatants were determined by a Quantikine M
ELISA kit using the manufacturer’s protocol (R&D Systems).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fact that IL-12 induces IFN- production in DC
indicates the presence of functional signal transduction downstream of
the IL-12R. We therefore set out to understand the mechanism of
signaling in these cells. First, we examined the expression of Jak2,
Tyk2, and Stat4, key intermediates in IL-12 signaling
(21, 22, 23, 24). When purified mature splenic DC were lysed and
analyzed for the expression of relevant signaling molecules, we
detected high-level expression of these signaling molecules (Fig. 1
A). Maturation of DC leads to
dramatic changes in the functions of DC (25, 26, 27, 28); thus,
the possibility existed that signaling functions vary during the
maturation process of DC. Since it has been reported that TCR
stimulation induces the expression of Stat4 in T cells
(22), we considered the possibility that Stat4 was
inducible in DC as well.
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, B–E; Ref.
11). We then examined changes in their phenotypes
during maturation induced by overnight incubation. Intracellular
components containing MHC class II molecules were readily detected in
freshly isolated DC, and cell surface expression was induced after
overnight incubation (Fig. 1C). Moreover, while freshly
isolated DC expressed costimulatory molecules such as CD86 and CD40 at
low levels on their surface, their expression levels increased during
maturation (Fig. 1D). Furthermore, Western blotting
demonstrated high-level expression of cystatin C, an endogenous
inhibitor of cathepsin S, in freshly isolated DC and its reduction in
mature DC (Fig. 1E) (29). These results
collectively show that freshly isolated DC are at an early stage of
maturation (25, 26, 27, 28). We then compared expression of the
above signaling molecules between immature and mature DC. As shown in
Fig. 1F, expression levels of Jak2 and Tyk2 were unaltered
during maturation. In contrast, the expression of Stat4 was highly
up-regulated during maturation. Mature DC express substantial levels of
Stat4, whereas only a low level of Stat4 was detected in immature DC
(Fig. 1F). The level of Stat4 expression in purified mature
DC was equal to or higher than that in purified splenic NK cells
(>90% purity; data not shown). The expression of Stat4 was unchanged
for several hours after isolation of immature DC but increased after
overnight incubation (data not shown). Maturation signals thus
up-regulate Stat4, but not Jak2 or Tyk2, in DC.
Differential expression of Stat4 in immature CD8+
and CD8- DC
We next examined the expression patterns of the Jak-Stat
components of IL-12 signaling, comparing CD8+
DC and CD8- DC (9, 10, 30, 31).
CD8+ and CD8- DC in
both immature and mature stages were lysed, and total cell extracts
were subjected to Western blotting analysis. Again, the levels of Jak2
or Tyk2 were unaltered during maturation as measured by the ratio to
heat shock protein 90 (HSP90), although we noted that the level
of Tyk2 was slightly higher in CD8+ than in
CD8- DC (data not shown). In contrast, a more
dramatic difference among the two subsets was observed in the
expression level of Stat4 (Fig. 1G). Immature
CD8+ DC did not express Stat4, whereas
immature CD8- DC expressed Stat4 at a low
level. Nonetheless, maturation-induced up-regulation of Stat4 was
observed in both CD8+ DC and
CD8- DC, with mature cells expressing nearly
the same levels of Stat4. These results suggest that the expression of
Stat4 is more strictly regulated by maturation in
CD8+ DC than in
CD8- DC.
Activation induces Stat4 expression in macrophages
Macrophages along with DC are important APC, but they play
additional direct roles in host defense by killing intracellular
pathogens. Recently, it has been shown that mouse macrophages produce
IFN- in response to a combination of IL-12 and IL-18 (8, 9). We examined whether mouse macrophages express Stat4 in the
same manner as DC. To this end, we derived macrophages from mouse bone
marrow with GM-CSF. It is known that these cultured macrophages are
activated in response to IFN- and bacterial products such as LPS
(32). We thus examined Stat4 expression in cultured
macrophages with or without activation signals. As shown in Fig. 1H, Stat4 was expressed in cultured macrophages at a low
level, but the expression levels were dramatically augmented in
response to LPS. IFN- further enhanced LPS-induced Stat4
up-regulation. These results collectively indicate that Stat4 is
induced in murine DC and macrophages in maturation- and
activation-dependent manners, respectively, and are consistent with our
recent findings in humans (33).
LPS induces up-regulation of Stat4 in DC and macrophages in vivo
To examine whether the maturation-dependent Stat4 induction in APC
occurs in vivo, we injected mice with LPS, which induces maturation of
DC and activation of macrophages in the spleen (34, 35).
We used Rag-2-/- mice pretreated with -ASGM1
Ab, in which the majority of cells in the spleen are DC and macrophages
(9). We then analyzed the amount of Stat4 protein in the
splenocytes after injection of LPS or PBS. Administration of LPS
augmented the Stat4 expression level in the spleen as demonstrated by
Western blotting analysis (Fig. 2A). Immunohistochemical
analysis of the spleen sections showed that the
CD11c+ DC in the spleen from LPS-treated C57BL/6
mice were brightly positive for Stat4 expression, whereas DC in the
spleen of PBS-treated mice were negative for Stat4 expression (Fig. 2B, upper panels). Similarly
F4/80+ macrophages expressed Stat4 in the spleen
of LPS-treated animals at levels much higher than detected in
PBS-treated control animals (Fig. 2B, lower
panels). These results demonstrate that the Stat4 expression is
induced in DC and macrophages in vivo and regulated by the state of
activation of these cells.
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As shown above, Jak2, Tyk2, and Stat4 are highly expressed in
mature DC and activated macrophages, indicating the presence of central
components of the IL-12-induced Jak-Stat pathway in APC. Since
activation results in phosphorylation and nuclear localization of Stats
(13, 14, 15, 16, 17), we examined whether nuclear translocation of
Stat4 occurs in DC upon IL-12 stimulation. Although Stat4 was detected
in the cytoplasm of DC following maturation (Fig. 3A, a), IL-12
stimulation caused Stat4 to translocate to the nucleus (Fig. 3A, b). Furthermore, Stat4 was readily detectable
by Western blotting analysis in the nuclear fraction of DC treated with
IL-12, whereas only a trace amount of Stat4 was observed in that of
untreated DC (Fig. 3B). Similarly, phosphorylation of Stat4
was clearly observed in response to IL-12 in macrophages prepared from
wild-type but not Stat4-/- mice (Fig. 3C). Taken together, these results indicate that the
Jak-Stat pathway for IL-12 signaling is present and functional in
APC.
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Having determined that DC and macrophages respond to
IL-12 by phosphorylation and nuclear translocation of Stat4, we sought
to determine whether this signaling had physiological functions. The
one well-characterized gene that is induced by IL-12 in a
Stat4-dependent manner is the IFN- gene, although the exact role of
Stat4 in this process has not been elucidated. We therefore examined
the role of Stat4 in IL-12-dependent IFN- production in DC. As shown
in Fig. 4A, DC prepared from
wild-type mice produced high amounts of IFN- in response to IL-12 or
a combination of IL-12 and IL-18 as reported elsewhere (9, 10). In contrast, DC from Stat4-/- mice
failed to produce IFN- in response to these stimuli. Thus, one role
of Stat4 in DC is to allow IL-12-dependent IFN- production, a key
cytokine produced at levels that could clearly affect the relative
Th1/Th2 balance during Ag presentation. We have previously shown that
DC produce IFN- during Ag presentation in mixed cocultures with
allogeneic T cells (11). Importantly, cocultures of normal
splenic DC with allogeneic IFN--/- T cells
resulted in high levels of IFN- production (Fig. 5). These levels were equivalent to those
seen in allogeneic cocultures of IFN--/- DC
with wild-type T cells, indicating that the contribution of IFN- by
DC during Ag presentation is clearly substantial.
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production in
DC
Several key cytokines have important effects on functions and
maturation processes of DC (27). Therefore, we next
examined the effects of several cytokines on maturation-dependent Stat4
induction in DC. For example, GM-CSF and TNF- are well known as
growth and maturation factors for DC (27, 36). We first
examined whether these maturation factors alter the expression level of
Stat4 in DC when added during the maturation process. As shown in Fig. 4B, upper panel, neither GM-CSF nor TNF-
influenced the level of Stat4 expression in DC after maturation. In
contrast, IL-10, a Th2 cytokine (6, 37, 38, 39, 40), inhibited the
induction of Stat4 in maturing DC (Fig. 4B, lower
panel). IL-4, another Th2 cytokine, showed a similar effect,
although the activity was not as strong as that of IL-10. In addition,
when IL-4 or IL-10 was included in the overnight culture to generate
mature DC, the resultant cells had significantly decreased IFN-
production in response to IL-12 compared with DC that were cultured in
the absence of these cytokines (Fig. 4C). Neither of these
Th2 cytokines affected the expression levels of the high-affinity
IL-12R (Fig. 4D).
We next examined whether Th2 cytokines affect IFN- production during
Ag presentation. As shown previously (11), when DC
prepared from wild-type C57BL/6 were cultured with
CD4+ T cells derived from
IFN--/- mice on a BALB/c background, a
substantial amount of IFN- production was observed (Fig. 5). When DC
were matured in the presence of IL-4 or IL-10, levels of IFN-
production from DC in response to allogeneic
IFN--/- CD4+ T cells
were significantly reduced (Fig. 5A). This is clearly not
the effect of IL-4 on mature DC, as we have previously shown that IL-4
enhances IFN- production from DC when acting on mature DC
(10). Unlike in immature DC, IL-4 had no effect on the
expression level of Stat4 in mature DC (data not shown). Consequently,
when mature DC of the C57BL/6 background were cultured with allogeneic
CD4+ T cells derived from
IFN--/- mice of the BALB/c background in the
presence of various concentrations of IL-4, IFN- production by DC
was augmented by IL-4 in a dose-dependent manner (Fig. 5B).
In contrast, when DC prepared from IFN--/-
mice on a BALB/c background were cultured with
CD4+ T cells derived from wild-type C57BL/6 mice,
IFN- production by CD4+ T cells was suppressed
by IL-4 in a dose-dependent manner (Fig. 5B). No significant
difference in proliferation of allogeneic T cells was observed in these
cultures (data not shown). These results indicate that IL-4 shows
differing effects on IFN- production between T cells and mature DC
and between mature and immature DC, and these effects on DC correlate
with the effects on Stat4 levels.
Autocrine IL-12 signaling in macrophages requires Stat4
IFN- production by DC during Ag presentation can function to
alter Th1/Th2 differentiation in adaptive immunity and host defense.
However, we hypothesized that autocrine IL-12 signaling by APC might
have additional functional implications for innate immunity as well. In
addition to Ag-presenting functions, macrophages have direct actions in
eliminating pathogenic organisms, a process that is regulated by
IFN-. Therefore, we next examined the importance of Stat4 in
IL-12-dependent IFN- production by macrophages and its functional
implications. Similar to DC, bone marrow macrophages derived from
wild-type mice produced IFN- in response to IL-12 or a combination
of IL-12 and IL-18 as previously reported (Fig. 6A; Refs. 8, 9). In contrast, Stat4-deficient macrophages were unable to
produce IFN- in response to these stimuli (Fig. 6A).
These results demonstrate that Stat4 is also required for
IL-12-dependent IFN- production by macrophages.
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B, Th2
cytokines inhibited the induction of Stat4 in macrophages as they do in
murine DC and in human monocytes/macrophages (33). It was
therefore not surprising that IFN- production from activated
macrophages in response to a combination of IL-12 and IL-18 was greatly
reduced by Th2 cytokines (Fig. 6C). These results
collectively indicate that Th2 cytokines suppress Stat4 production from
macrophages when they act on or before maturation/activation, and this
correlates with levels of IFN-. Function of autocrine IL-12 signaling in pathogen elimination
To further elucidate the importance of Stat4-mediated IL-12
signaling in macrophage functions, we examined microbicidal activity of
macrophages by infection with T. gondii in vitro. As shown
in Fig. 6D, growth of T. gondii in infected
macrophages was dramatically inhibited by addition of IL-12 or a
combination of IL-12 and IL-18. To demonstrate that this effect was
dependent on endogenous IFN- production, we also assessed the effect
of anti-IFN- Ab and found that growth inhibition by IL-12 and
IL-18 was reversed. As expected, addition of exogenous IFN- resulted
in killing of the organisms. In contrast, T. gondii grew in
Stat4-/- macrophages in the presence of IL-12
or a combination of IL-12 and IL-18 (Fig. 6D). However, the
Stat4-/- macrophages were not intrinsically
incapable of killing this organism, as addition of exogenous IFN-
inhibited growth of the organism, supporting the hypothesis that
IL-12-induced IFN- is critical in the microbicidal activity of
macrophages. It has been shown that NO production in response to
IFN- is required for microbicidal activity of macrophages against
T. gondii (41). As shown in Fig. 6E,
NO production upon T. gondii infection was greatly enhanced
by IL-12, or a combination of IL-12 and IL-18 in wild-type macrophages
but was impaired in Stat4-/- macrophages.
Again, exogenous IFN- induced NO production in
Stat4-/- macrophages. Since the addition of
anti-IFN- Ab suppressed NO production in wild-type macrophages
and exogenous IFN- restored NO production in
Stat4-/- macrophages, we conclude that
endogenous IFN- production generated via autocrine IL-12 signaling
is critical for the NO production and subsequent microbicidal activity
against T. gondii infection. This is dependent upon Stat4,
which is dynamically regulated in macrophages and DC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, we have previously shown that DC
are capable of producing large amounts of IFN- in response to IL-12
and during Ag presentation to CD4+ T cells
(9, 10, 11). These results suggest the presence of a
functional signal transduction cascade downstream of IL-12R in DC. In
fact, recent studies have shown the expression of Jak2, Tyk2, and Stat4
in human DC and human monocytes/macrophages (33, 42). Our
present study shows that there is a unique regulatory mechanism of
Stat4 expression in DC and in macrophages also. Stat4 expression allows
IL-12-dependent IFN- production by both DC and macrophages. The
expression of Stat4, but not Jak2 or Tyk2, is not detectable in
unstimulated or immature APC, but is induced in DC in a
maturation-dependent manner and in macrophages upon activation.
Furthermore, an important difference exists in the basal expression
level of Stat4 between CD8+ and
CD8- DC in their immature stages. Stat4 was
detected in immature CD8- DC but not in
CD8+ DC. Considering that the
CD8+ DC are the major cells producing IFN-
in response to IL-12 (9, 10), such strict regulation of
Stat4 expression in CD8+ DC is likely
important in the control of IL-12-dependent IFN- production by DC.
The fact that Stat4-deficient DC and macrophages do not produce IFN-
in response to IL-12 unequivocally indicates the essential functional
role of Stat4 in IL-12 signaling in APC, although this does not mean
that it necessarily regulates the transcriptional activation of the
IFN- gene directly.
We present here evidence for the importance of Stat4 in both innate and
acquired immune responses. These data argue that DC and macrophages are
capable of producing IFN- in an autocrine manner by producing IL-12
which, in turn, induces further IFN- production by T and NK cells.
IFN- production by APC has functional significance, as demonstrated
by the essential role of Stat4 in microbicidal activity of macrophage
against T gondii. Macrophages are unable to produce NO in
response to IL-12 or a combination of IL-12 and IL-18 in the absence of
Stat4. They are unable to kill infected T gondii in response
to IL-12 due to their inability to make IFN-. Moreover, addition of
exogenous IFN- restores both NO production and microbicidal activity
of Stat4-deficient macrophages, whereas anti-IFN- abolishes NO
production and microbicidal activity in wild-type macrophages. Thus, we
propose that early in infection, IFN- production from APC provides a
rapid lymphoid-independent mechanism for growth inhibition of infected
pathogens. Our results also unequivocally indicate that Stat4 is
essential in the signaling pathway of IL-12 in macrophages during
innate immune responses.
DC constitute a highly efficient system for capturing Ags in the
periphery and delivering them to the T cell areas of lymphoid tissues
(27, 43). DC show dramatic changes in their functional
properties during maturation. In the periphery, DC are in an immature
state characterized by high endocytotic and weak Ag-presenting
capabilities. In response to maturation signals such as inflammatory
cytokines, bacterial products, or CD40 ligation, DC are induced to a
mature state with acquisition of potent Ag-presenting ability and loss
of endocytotic capacity (25, 26, 27, 28). It has also been
reported the critical role of APC, particularly DC, in the control of
Th1/Th2 balance (44, 45, 46). Our data indicating that DC
produce substantial levels of IFN- provide another straightforward
mechanism for this regulation. The importance of this role is clearly
demonstrated by the high levels of IFN- produced in cocultures of
normal DC and allogeneic IFN--/- T cells
(Ref. 11 ; Fig. 5). It has been known that ligation of CD40
on DC induces IL-12 production (2, 3). Thus, activated T
cells expressing CD40 ligand (CD154), such as the allogeneic T
cells in our coculture experiments, activate DC to produce IL-12, which
in turn acts on DC to produce IFN- in an autocrine manner. Thus,
IL-12 as well as IFN- would then induce efficient Th1
differentiation.
The inhibition of Stat4 up-regulation in DC and macrophages by Th2
cytokines is quite intriguing. Since it has been demonstrated that
IFN- along with IL-12 is required for the effective Th1 induction
(6), we propose that IFN- derived from DC is likely
important for Th1 induction. IL-4 and IL-10, however, suppress Stat4
production by DC and inhibit IFN- up-regulation when they are
present during the maturation of DC. These cytokines also suppress
Stat4 and IFN- production by macrophages when they are present
during macrophage activation. In contrast, IL-4 has no inhibitory
effect on Stat4 production by mature DC and, in fact, augments IFN-
production. Thus, Th2 cytokines act on DC in distinct manners depending
upon the maturation stages of DC; their negative effect on APC
production of IFN- correlates directly with their ability to inhibit
Stat4 expression.
During maturation and migration processes, cytokines in the surrounding
environment influence DC maturation and modulate their functions
(27, 36, 38, 39, 40). From our studies, the following picture
emerges: IL-4 and IL-10 suppress IFN- production by DC when they are
present in peripheral tissue, where DC are in the immature state. In
contrast, IL-4 augments IFN- production by DC when they are present
in lymphoid tissue where mature DC are localized. This regulation is
indeed novel, as IL-4 augments IFN- production by DC in response to
allogeneic T cells while at the same time it suppresses IFN-
production by T cells. As sources of IL-4, we propose that mast cells
and Th2 cells provide this cytokine to DC in the periphery and lymphoid
organs, respectively. Such spatiotemporal action of Th2 cytokines on
IFN- production by DC likely affects the Th1/Th2 balance. In this
regard, there have been several observations where IL-4 favors Th1
induction, which seemed contradictory to the general views that IL-4
suppresses IFN- production (47, 48, 49, 50). Such
seemingly contradictory observations would be explained by our findings
that Th2 cytokines regulate IFN- production by DC through differing
spatiotemporal action mechanisms.
In summary, we demonstrate that Stat4 levels are tightly and
dynamically regulated in DC and macrophages. IL-12 signals in DC and
macrophages, inducing Stat4 phosphorylation and IFN- production.
Proinflammatory and maturation signals up-regulate its expression,
whereas Th2 cytokines inhibit its expression. Since DC and macrophages
are themselves major producers of IL-12, these findings have several
functional implications. Autocrine IL-12 signaling allows IFN-
production by macrophages, inducing NO production and providing
sentinel protection during innate immune responses. Moreover, it
results in IFN- production by DC, altering the relative Th1/Th2
balance during Ag presentation in acquired immune responses. These
functional effects appear to be dependent upon the regulated expression
of Stat4 in DC and macrophages.
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Acknowledgments |
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Footnotes |
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2 T.F. and D.M.F. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Shigeo Koyasu, Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
4 Abbreviations used in this paper: DC, dendritic cell; Jak, Janus family tyrosine kinase; m, murine; ASGM1, asialoGM1; PVDF, polyvinylidene difluoride; HSP90, heat shock protein 90.
Received for publication August 31, 2000. Accepted for publication January 24, 2001.
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20% of those for immature
DCs. H, Macrophages were prepared from bone marrow by
cultivating with GM-CSF. Cells were then stimulated with the
indicated reagents overnight and examined for the expression of Stat4
by Western blotting (protein amount equivalent to 1 x
105 cells/lane). The concentrations of IFN-
) DC (1 x 105/well) were then incubated with
allogeneic IFN-