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The Journal of Immunology, 2001, 166: 4446-4455.
Copyright © 2001 by The American Association of Immunologists

Inducible Expression of Stat4 in Dendritic Cells and Macrophages and Its Critical Role in Innate and Adaptive Immune Responses1

Taro Fukao2,*, David M. Frucht2,{dagger}, George Yap{ddagger}, Massimo Gadina{dagger}, John J. O’Shea{dagger} and Shigeo Koyasu3,*

* Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan; and {dagger} Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases and {ddagger} Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-{gamma} production by APC as well as IFN-{gamma} 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-{gamma} production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-{gamma} 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-{gamma}. 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-{gamma} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell-mediated immunity is critical for host defenses against intracellular pathogens. APC play essential roles during cell-mediated immunity, by both presenting Ag and killing pathogens. One of the most important cytokines produced by mature dendritic cells (DC)4 and macrophages is IL-12 (1, 2, 3, 4), a key cytokine in the induction of cellular immune responses to intracellular pathogens. Additionally, in the innate immune response to microbial pathogens, IL-12 triggers IFN-{gamma} and TNF-{alpha} production by NK cells. At the same time, IL-12 specifically promotes the differentiation of CD4+ T cells to Th1 effector cells producing IFN-{gamma} (5, 6). IFN-{gamma}, in turn, acts to increase NK activity and to enhance killing of intracellular pathogens. Impaired NK responses and lower IFN-{gamma} 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-{gamma} (8, 9, 10, 11). DC express a high-affinity receptor for IL-12 and are capable of producing significant amounts of IFN-{gamma} in response to IL-12 (9, 10, 12). In fact, the amounts of IFN-{gamma} produced by DC are severalfold higher than those by NK cells (9). Similar to DC, bone marrow-derived macrophages produce IFN-{gamma} 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-{gamma} production as shown by studies with Stat4-/- mice, although it is by no means clear that Stat4 directly regulates the IFN-{gamma} gene (18, 19). Nonetheless, these observations suggested that Stat4 is required in IL-12-dependent IFN-{gamma} 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-{gamma} production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-{gamma} production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. This unique regulatory mechanism of IFN-{gamma} 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-{gamma} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

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-{gamma}-/-, and C57BL/6-IFN-{gamma}-/- 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-{alpha} 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{alpha}); 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 ({rho} = 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{epsilon}, 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{alpha}+ and CD8{alpha}- subsets. The CD8{alpha}+ subset was positively selected with anti-CD8{alpha} (Ly-2) MicroBeads (Miltenyi Biotec) and from the negative fraction of this selection, the CD8{alpha}- 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 Fc{gamma}R 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 {beta}-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-{gamma} (100 U/ml), and/or anti-IFN-{gamma} (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-{gamma} in the culture supernatants were determined by a Quantikine M ELISA kit using the manufacturer’s protocol (R&D Systems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stat4 is up-regulated in DC during maturation

The fact that IL-12 induces IFN-{gamma} 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. 1GoA). 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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  		FIGURE 1. Maturation- and activation-dependent Stat4 up-regulation in mouse APC. A, Total cell extracts (protein amount equivalent to 2 x 105 cells in the first lane) of mature DC were separated on 8% SDS-PAGE, transferred onto a PVDF membrane, and analyzed by Western blotting with Abs against indicated proteins. Blotting with anti-HSP90 was performed as a control. An extract of the Rag-2-/- splenocyte population containing 30% NK cells was used as a positive control. B, Freshly isolated immature DC were stained with PE-conjugated anti-CD11c mAb or anti-Leu4 mAb as a negative control (thin line) and analyzed on a FACScan. C, Immature (a) and mature (b) DC were mounted on coverslips, fixed, stained with anti-MHC II molecule Ab followed by staining with rhodamine-conjugated secondary Ab, and analyzed using a confocal laser scanning microscope. Bars, 10 µm. D, Immature (upper panels) and mature (lower panels) DC were stained with biotin-conjugated anti-CD86 (left panels) or anti-CD40 (right panels) mAbs followed by incubation with streptavidin-Red670 and analyzed on a FACScan. Negative controls (thin lines) were obtained by staining with biotin-conjugated anti-Leu4 mAb. E, Total cell lysates (protein amount equivalent to 2 x 105 cells/lane) of immature (IM) or mature (M) DC were separated on 15% SDS-PAGE, transferred onto a PVDF membrane, and blotted with anti-cystatin C serum (upper panel). Membranes were reblotted with anti-HSP90 Ab (lower panel). F, Total cell lysates (protein amount equivalent to 2 x 105 cells/lane) of immature or mature DC were separated on 8% SDS-PAGE, transferred onto a PVDF membrane, and blotted with the indicated antisera. Membranes were reblotted with anti-HSP90 Abs. G, Immature and mature DCs of both subsets (CD8{alpha}+, 93% CD8{alpha}+CD11c+I-A+ and CD8{alpha}-, 94% CD8{alpha}- CD11c+ I-A+) were prepared as described in Materials and Methods. Total cell extracts (protein amount equivalent to 2 x 105 cells on the lanes of immature DC) were subjected to Western blotting analysis with anti-Stat4 Abs. Membranes were reblotted with anti-HSP90 Ab. The amounts of cell extracts used for mature DCs were ~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-{gamma} and LPS were 10 ng/ml and 1 µg/ml, respectively. Membranes were reblotted with anti-{beta}-tubulin Ab.

 
To this end, a quick isolation procedure was established to obtain splenic DC in an immature state (Fig. 1Go, 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. 1GoC). 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. 1GoD). 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. 1GoE) (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. 1GoF, 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. 1GoF). 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{alpha}+ and CD8{alpha}- DC

We next examined the expression patterns of the Jak-Stat components of IL-12 signaling, comparing CD8{alpha}+ DC and CD8{alpha}- DC (9, 10, 30, 31). CD8{alpha}+ and CD8{alpha}- 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{alpha}+ than in CD8{alpha}- DC (data not shown). In contrast, a more dramatic difference among the two subsets was observed in the expression level of Stat4 (Fig. 1GoG). Immature CD8{alpha}+ DC did not express Stat4, whereas immature CD8{alpha}- DC expressed Stat4 at a low level. Nonetheless, maturation-induced up-regulation of Stat4 was observed in both CD8{alpha}+ DC and CD8{alpha}- 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{alpha}+ DC than in CD8{alpha}- 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-{gamma} 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-{gamma} and bacterial products such as LPS (32). We thus examined Stat4 expression in cultured macrophages with or without activation signals. As shown in Fig. 1GoH, Stat4 was expressed in cultured macrophages at a low level, but the expression levels were dramatically augmented in response to LPS. IFN-{gamma} 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 {alpha}-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. 2GoA). 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. 2GoB, 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. 2GoB, 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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  		FIGURE 2. Maturation- and activation-dependent Stat4 up-regulation in APC in vivo. A, B10.D2-Rag-2-/- mice were injected i.v. with 300 µg of anti-ASGM1 Ab to deplete NK cells. After 3 days, NK cell-depleted mice were injected i.v. with 25 µg of LPS in PBS or PBS alone. After 10 h, splenocytes were collected and total cell lysates were subjected to Western blotting (protein amount equivalent to 0.5 x 105 cells/lane) with anti-Stat4 Ab. Membranes were reblotted with anti-HSP90 Ab. B, C57BL/6 mice were injected i.v. with 25 µg of LPS in PBS or PBS alone. After 10 h, cryosections were prepared from spleens from each mouse and stained with rabbit anti-Stat4 Ab and biotinylated anti-CD11c Ab followed by rhodamine-conjugated goat anti-rabbit IgG Ab and streptavidin Cy5 (upper panels), or rabbit anti-Stat4 Ab and FITC-conjugated F4/80 followed by rhodamine-conjugated goat anti-rabbit IgG Ab (lower panels). The samples were analyzed using a confocal laser scanning microscope. Bars, 20 µm.

 
IL-12 signaling in APC

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. 3GoA, a), IL-12 stimulation caused Stat4 to translocate to the nucleus (Fig. 3GoA, 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. 3GoB). Similarly, phosphorylation of Stat4 was clearly observed in response to IL-12 in macrophages prepared from wild-type but not Stat4-/- mice (Fig. 3GoC). Taken together, these results indicate that the Jak-Stat pathway for IL-12 signaling is present and functional in APC.



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  		FIGURE 3. IL-12-dependent activation of Stat4 in DC and macrophages. A, Purified DC were stimulated with IL-12 (10 ng/ml) or left unstimulated for 1 h and analyzed under an immunofluorescence microscope with anti-Stat4 Ab. Bars, 10 µm. NC, Negative control using preimmune rabbit serum instead of anti-Stat4 Ab. B, Mature DC were stimulated with IL-12 (10 ng/ml) or left unstimulated for 1 h at 37°C. Nuclear fractions from each sample were then prepared, separated on 8% SDS-PAGE, and analyzed by Western blotting with anti-Stat4 Ab. Both preparations contained similar amounts of HDAC2, a well known nuclear protein, as revealed by reblotting the membrane with anti-HDAC2 Ab. In addition, no I{kappa}B{alpha} protein was detected in the nuclear fractions, confirming that there was no contamination of the cytoplasmic fraction (data not shown). C, Bone marrow-derived macrophages from wild-type (WT) and Stat4-/- mice were stimulated with IL-12 (10 ng/ml) or left unstimulated for 20 min at 37°C. Total cell extracts (protein amount equivalent to 2.5 x 106 cells/lane) were subjected to Western blotting analysis with anti-phosphorylated Stat4, anti-Stat4, and anti-Jak3 Abs.

 
Function of autocrine IL-12 signaling in DC

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-{gamma} 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-{gamma} production in DC. As shown in Fig. 4GoA, DC prepared from wild-type mice produced high amounts of IFN-{gamma} 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-{gamma} in response to these stimuli. Thus, one role of Stat4 in DC is to allow IL-12-dependent IFN-{gamma} 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-{gamma} during Ag presentation in mixed cocultures with allogeneic T cells (11). Importantly, cocultures of normal splenic DC with allogeneic IFN-{gamma}-/- T cells resulted in high levels of IFN-{gamma} production (Fig. 5Go). These levels were equivalent to those seen in allogeneic cocultures of IFN-{gamma}-/- DC with wild-type T cells, indicating that the contribution of IFN-{gamma} by DC during Ag presentation is clearly substantial.



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  		FIGURE 4. Function of autocrine IL-12 signaling in DC. A, A low-density fraction (see Materials and Methods) containing 40% DC from wild-type (WT) or Stat4-/- mice was incubated overnight and then cultured (1 x 105/well in 200 µl of culture medium) with or without IL-12 (10 ng/ml) and/or IL-18 (50 ng/ml) for 3 days. Titers of IFN-{gamma} in the culture supernatants were then determined by ELISA. Results are presented as the mean values ± SE. B, Immature DC were isolated and cultivated overnight in the presence or absence of GM-CSF (10 ng/ml), TNF-{alpha} (20 ng/ml), IL-10 (10 ng/ml), or IL-4 (10 ng/ml). Total cell extracts (protein amount equivalent to 2 x 105 cells/lane) were subjected to Western blotting analysis with anti-Stat4 Ab. Membranes were reblotted with anti-HSP90 Ab. C, Immature DC (5 x 104/well) were prepared and incubated in the presence or absence of IL-4 (10 ng/ml) or IL-10 (10 ng/ml) and then cultured with various concentrations of IL-12. After 3 days of culture, titers of IFN-{gamma} in the culture supernatants were measured by ELISA. Data are presented as the mean values ± SD. D, Immature DC (5 x 104/well) prepared and incubated in the presence or absence of IL-4 (10 ng/ml) or IL-10 (10 ng/ml) were incubated on ice for 1 h with IL-12 (1 µg/ml), washed, and further incubated with biotinylated anti-IL-12 mAb (10 µg/ml) for 30 min. Cells were then incubated with streptavidin-Red670 and analyzed on a FACScan. Negative controls (thin lines) were obtained by staining without IL-12.

 


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  		FIGURE 5. A, Immature DC (1 x 105/well) prepared from C57BL/6 spleen and then matured in the presence or absence of IL-4 (10 ng/ml) or IL-10 (20 ng/ml) were cultured with CD4+ T cells (2 x 105/well) prepared from IFN-{gamma}-/- mice of a BALB/c background. After 3 days, titers of IFN-{gamma} in the culture supernatants were determined by ELISA. Results are presented as the mean values ± SD. B, Mature splenic DC were prepared from wild-type C57BL/6 mice and IFN-{gamma}-/- mice of the BALB/c background in the absence of any exogenous cytokines. Splenic CD4+ T cells were also prepared from wild-type C57BL/6 and IFN-{gamma}-/- mice of the BALB/c background. Wild-type (•) or IFN-{gamma}-/- ({circ}) DC (1 x 105/well) were then incubated with allogeneic IFN-{gamma}-/- (•) or wild-type ({circ}) CD4+ T cells (2 x 105/well) for 3 days in the presence of indicated concentrations of IL-4. Titers of IFN-{gamma} produced by DC (•) or CD4+ T cells ({circ}) in the culture supernatants were then determined by ELISA. Results are presented as the mean values ± SE.

 
Th2 cytokines regulate Stat4 expression and IFN-{gamma} 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-{alpha} 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. 4GoB, upper panel, neither GM-CSF nor TNF-{alpha} 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. 4GoB, 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-{gamma} production in response to IL-12 compared with DC that were cultured in the absence of these cytokines (Fig. 4GoC). Neither of these Th2 cytokines affected the expression levels of the high-affinity IL-12R (Fig. 4GoD).

We next examined whether Th2 cytokines affect IFN-{gamma} 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-{gamma}-/- mice on a BALB/c background, a substantial amount of IFN-{gamma} production was observed (Fig. 5Go). When DC were matured in the presence of IL-4 or IL-10, levels of IFN-{gamma} production from DC in response to allogeneic IFN-{gamma}-/- CD4+ T cells were significantly reduced (Fig. 5GoA). This is clearly not the effect of IL-4 on mature DC, as we have previously shown that IL-4 enhances IFN-{gamma} 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-{gamma}-/- mice of the BALB/c background in the presence of various concentrations of IL-4, IFN-{gamma} production by DC was augmented by IL-4 in a dose-dependent manner (Fig. 5GoB). In contrast, when DC prepared from IFN-{gamma}-/- mice on a BALB/c background were cultured with CD4+ T cells derived from wild-type C57BL/6 mice, IFN-{gamma} production by CD4+ T cells was suppressed by IL-4 in a dose-dependent manner (Fig. 5GoB). 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-{gamma} 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-{gamma} 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-{gamma}. Therefore, we next examined the importance of Stat4 in IL-12-dependent IFN-{gamma} production by macrophages and its functional implications. Similar to DC, bone marrow macrophages derived from wild-type mice produced IFN-{gamma} in response to IL-12 or a combination of IL-12 and IL-18 as previously reported (Fig. 6GoA; Refs. 8, 9). In contrast, Stat4-deficient macrophages were unable to produce IFN-{gamma} in response to these stimuli (Fig. 6GoA). These results demonstrate that Stat4 is also required for IL-12-dependent IFN-{gamma} production by macrophages.



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  		FIGURE 6. Function of autocrine IL-12 signaling in macrophages. A, M-CSF cultured macrophages obtained from wild-type (WT) or Stat4-/- mice were cultured (1 x 105/well in 200 of µl culture medium) with or without IL-12 (10 ng/ml) and/or IL-18 (50 ng/ml) for 3 days. Titers of IFN-{gamma} in the culture supernatants were then determined by ELISA. Results are presented as the mean values ± SD. B and C, GM-CSF-induced macrophages prepared from bone marrow were incubated with or without IL-4 (10 ng/ml) or IL-10 (10 ng/ml) for 2 h and then stimulated with LPS (1 µg/ml) overnight. B, Total lysates (protein amount equivalent to 1 x 105 cells/lane) were then analyzed for the expression of Stat4 by Western blotting. C, Cells were then cultured (1 x 105/well in 200 µl of culture medium) with or without IL-12 (5 ng/ml) and/or IL-18 (5 ng/ml) for another 3 days. Titers of IFN-{gamma} in the culture supernatants were then determined by ELISA. Results are presented as the mean values ± SD. D and E, Macrophages prepared from wild-type and Stat4-/- mice were infected with T. gondii. As indicated, certain cultures were pretreated for 24 h with IL-12, IL-18, anti-IFN-{gamma} (negative control, 20 µg/ml), or IFN-{gamma} (positive control, 100 U/ml). Growth of T. gondii was determined by uptake of radioactive uracil (D). NO production was also determined after a 24-h incubation (E).

 
Since Stat4 is unequivocally required for autocrine IL-12 signaling in macrophages, we hypothesized that levels of Stat4 also correlate with macrophage responses to IL-12. As shown in Fig. 6GoB, 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-{gamma} production from activated macrophages in response to a combination of IL-12 and IL-18 was greatly reduced by Th2 cytokines (Fig. 6GoC). 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-{gamma}.

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. 6GoD, 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-{gamma} production, we also assessed the effect of anti-IFN-{gamma} Ab and found that growth inhibition by IL-12 and IL-18 was reversed. As expected, addition of exogenous IFN-{gamma} 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. 6GoD). However, the Stat4-/- macrophages were not intrinsically incapable of killing this organism, as addition of exogenous IFN-{gamma} inhibited growth of the organism, supporting the hypothesis that IL-12-induced IFN-{gamma} is critical in the microbicidal activity of macrophages. It has been shown that NO production in response to IFN-{gamma} is required for microbicidal activity of macrophages against T. gondii (41). As shown in Fig. 6GoE, 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-{gamma} induced NO production in Stat4-/- macrophages. Since the addition of anti-IFN-{gamma} Ab suppressed NO production in wild-type macrophages and exogenous IFN-{gamma} restored NO production in Stat4-/- macrophages, we conclude that endogenous IFN-{gamma} 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.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although conventionally it is assumed that T cells and NK cells are the major producers of IFN-{gamma}, we have previously shown that DC are capable of producing large amounts of IFN-{gamma} 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-{gamma} 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{alpha}+ and CD8{alpha}- DC in their immature stages. Stat4 was detected in immature CD8{alpha}- DC but not in CD8{alpha}+ DC. Considering that the CD8{alpha}+ DC are the major cells producing IFN-{gamma} in response to IL-12 (9, 10), such strict regulation of Stat4 expression in CD8{alpha}+ DC is likely important in the control of IL-12-dependent IFN-{gamma} production by DC. The fact that Stat4-deficient DC and macrophages do not produce IFN-{gamma} 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-{gamma} 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-{gamma} in an autocrine manner by producing IL-12 which, in turn, induces further IFN-{gamma} production by T and NK cells. IFN-{gamma} 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-{gamma}. Moreover, addition of exogenous IFN-{gamma} restores both NO production and microbicidal activity of Stat4-deficient macrophages, whereas anti-IFN-{gamma} abolishes NO production and microbicidal activity in wild-type macrophages. Thus, we propose that early in infection, IFN-{gamma} 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-{gamma} provide another straightforward mechanism for this regulation. The importance of this role is clearly demonstrated by the high levels of IFN-{gamma} produced in cocultures of normal DC and allogeneic IFN-{gamma}-/- T cells (Ref. 11 ; Fig. 5Go). 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-{gamma} in an autocrine manner. Thus, IL-12 as well as IFN-{gamma} 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-{gamma} along with IL-12 is required for the effective Th1 induction (6), we propose that IFN-{gamma} derived from DC is likely important for Th1 induction. IL-4 and IL-10, however, suppress Stat4 production by DC and inhibit IFN-{gamma} up-regulation when they are present during the maturation of DC. These cytokines also suppress Stat4 and IFN-{gamma} 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-{gamma} 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-{gamma} 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-{gamma} production by DC when they are present in peripheral tissue, where DC are in the immature state. In contrast, IL-4 augments IFN-{gamma} 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-{gamma} production by DC in response to allogeneic T cells while at the same time it suppresses IFN-{gamma} 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-{gamma} 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-{gamma} production (47, 48, 49, 50). Such seemingly contradictory observations would be explained by our findings that Th2 cytokines regulate IFN-{gamma} 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-{gamma} 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-{gamma} production by macrophages, inducing NO production and providing sentinel protection during innate immune responses. Moreover, it results in IFN-{gamma} 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.


   Acknowledgments
 
We are grateful to Dr. A. Miyajima for recombinant IL-4 and GM-CSF; Drs. S. Matsuda, M. Amagai, T. Yamada, T. Ohteki, E. Mansfield, C. Prussin, and A. Sher for helpful discussions; Dr. T. Ohta, Dr. S. Fan, M. Fujiwara, and M. Suzuki for help in some experiments, and A. Sakurai for excellent animal care.


   Footnotes
 
1 This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (Grant 10153261), a Keio University Special Grant-in-Aid for Innovative Collaborative Research Project, and a grant from the Japan Society for the Promotion of Science (Grant JSPS-RFTF 97L00701). Back

2 T.F. and D.M.F. contributed equally to this work. Back

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. Back

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. Back

Received for publication August 31, 2000. Accepted for publication January 24, 2001.


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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.
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