STAT4 Signaling and Is Inhibited by IL-4

The Production of IFN-γ by IL-12/IL-18-Activated Macrophages Requires STAT4 Signaling and Is Inhibited by IL-4

This information is current as Heike Schindler, Manfred B. Lutz, Martin Röllinghoff and of September 24, 2021. Christian Bogdan J Immunol 2001; 166:3075-3082; ; doi: 10.4049/jimmunol.166.5.3075 http://www.jimmunol.org/content/166/5/3075 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Production of IFN-␥ by IL-12/IL-18-Activated Macrophages Requires STAT4 Signaling and Is Inhibited by IL-41

Heike Schindler,* Manfred B. Lutz,† Martin Ro¨llinghoff,* and Christian Bogdan2*

Macrophages release IFN-␥ 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, Downloaded from 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. The Journal of Immunology, 2001, 166: 3075–3082. http://www.jimmunol.org/ he activation of macrophages by IFN-␥, which is typically protein found in the cultures of bone marrow-derived macrophages released by NK cells, CD4ϩ type 1 Th cells, and several approached or even exceeded the amounts that are usually released T other subsets of T cells (e.g., ␥␦ T cells, NKT cells, CD8ϩ by T or NK cells. The activation of macrophages for the secretion T cells), has been a hallmark of the immune responses against of IFN-␥ by IL-12 and IL-18 is of particular interest, because both intracellular pathogens and tumor cells as well as of certain auto- IL-12 and IL-18 are known products of macrophages, which sug- immune reactions. IFN-␥ activates macrophages to produce cyto- gests the possibility of autocrine stimulation. Cytokines that are kines, to express antimicrobial and tumoricidal effector pathways, able to counteract this pathway have not yet been deﬁned. and to act as APCs (1, 2). During more recent years, macrophages In T and NK cells, STAT4 is critical for the production of IFN-␥

themselves were recognized also to be producers of IFN-␥ under in response to IL-12, which was shown by the analysis of STAT4- by guest on September 24, 2021 certain conditions. IFN-␥ mRNA and/or protein was detected in deﬁcient mice (12–14). In addition, NK cells, but not T cells, re- various populations of mononuclear phagocytes, including human quired NO derived from type 2 NO synthase (NOS2)3 for IL-12 alveolar macrophages (3), and resting peritoneal macrophages (4, signaling, i.e., for the activation of Tyk2 kinase, the tyrosine phos- 5), peritoneal exudate macrophages (6–8), bone marrow-derived phorylation of STAT4, and the production of IFN-␥ (15). Re- macrophages (9), splenic macrophages (10), and lung macro- cently, evidence was provided that a STAT4-independent pathway phages from mice (11). Although a possible (minor) contamination of IFN-␥ production exists in CD8ϩ T cells; however, it was only with T, NK, or NKT cells has not always been vigorously ex- observed after cross-linking of the TCR and not after stimulation cluded, most of these studies unequivocally demonstrate the pro- with IL-12/IL-18 (16). A similar pathway was also observed in duction of IFN-␥ by monocytes/macrophages. The stimuli that CD4ϩ T cells lacking both STAT4 and STAT6 (14). In dendritic were reported to induce IFN-␥ in monocytes/macrophages include cells isolated from mouse spleens, IL-12 signaling was reported to type I IFNs (7), IFN-␥ itself (4), IL-12 (bioactive p70 homodimer) involve nuclear translocation of NF-␬B rather than activation of (3, 5, 10), LPS (6, 8) (which largely acts via induction of endog- members of the STAT family (17). In contrast, in human blood enous IL-12), Mycobacterium tuberculosis (3), Mycobacterium monocyte-derived dendritic cells stimulation with IL-12 led to ty- bovis bacillus Calmette-Gue´rin plus IL-12 (11), and a combination rosine-phosphorylation of Tyk2 and Jak2 kinase as well as of of IL-12 and IL-18 (9). In the latter case, the levels of IFN-␥ 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, *Institute of Clinical Microbiology, Immunology, and Hygiene and †Department of NOS2, and/or the Jak/STAT pathway are actually required for the Dermatology, University of Erlangen, Erlangen, Germany production of IFN-␥ by dendritic cells or macrophages is unknown Received for publication September 9, 2000. Accepted for publication December 20, to date. In the present study, we show that STAT4 is essential for 2000. the production of IFN-␥ by inﬂammatory macrophages in response The costs of publication of this article were defrayed in part by the payment of page to IL-12/IL-18, whereas NOS2-derived NO is dispensable. We charges. This article must therefore be hereby marked advertisement in accordance ␤ with 18 U.S.C. Section 1734 solely to indicate this fact. also demonstrate that IL-4, IL-10, IL-13, and TGF- 1 inhibit IL- ␥ 1 This work was supported by grants from the Deutsche Forschungsgemeinschaft 12/IL-18-induced IFN- production in macrophages. The effect of (Sonderforschungsbereich 263) to C.B. (Project A5) and M.B.L. (Project C13). IL-4 appears to be mediated by the activation of STAT6, which in 2 Address correspondence and reprints requests to Dr. Christian Bogdan, Institut fu¨r Klinische Mikrobiologie, Immunologie und Hygiene, Universita¨t Erlangen-Nu¨rnberg, Wasserturmstrasse 3, D-91054 Erlangen, Germany. E-mail address: christian. 3 Abbreviations used in this paper: NOS2 (iNOS), type 2 (or inducible) NO synthase; [email protected] rm, recombinant murine.

EMSA was able to bind to the same DNA oligonucleotide as IL- RNA preparation, cDNA synthesis, and RT-PCR 12/IL-18-induced STAT4. Total RNA was isolated from macrophage monolayers by the guanidinium isothiocyanate method, reverse transcribed (1 ␮g), and analyzed by qualitative or quantitative (competitive) RT-PCR as published (22). The Materials and Methods competitor plasmids for the different genes were as follows: 1) piNOSL1 Mice (HincII 162 bp) for NOS2 (22); 2) pMCQ for ␤-actin and IFN-␥ (24); and 3) pIL-12R␤1 and pIL-12R␤2 for IL-12 receptor ␤1 and IL-12 receptor ␤2, Female CD1 mice (20–24 g; 8–12 wk old) and C57BL/6 mice (16–18 g; respectively (15). The primer sequences for NOS2, IFN-␥, ␤-actin, and 6–8 wk old) were purchased from Charles River Breeding Laboratories IL-12 receptor ␤1 and ␤2 were as published previously (15, 25). The se- (Sulzfeld, Germany). Breeding pairs of (129/SvEv ϫ C57BL/6) mice with Ϫ Ϫ ϩ ϩ quences of the IL-18 receptor upstream and downstream primer used for a disrupted NOS2 gene (NOS2 / ) (20) and wild-type controls (NOS2 / ) qualitative PCR analysis were 5Ј-CGT GAC AAG CAG AGA TGT TG-3Ј were originally provided by C. F. Nathan (New York, NY) and J. S. ϩ ϩ Ϫ Ϫ and 5Ј-ATG TTG TCG TCT CCT TCC TG-3Ј, respectively. The annealing Mudgett (Merck, Rahway, NJ). The NOS2 / and NOS2 / mice used temperatures were 57°C (IL-18R), 58°C (NOS2, IL-12R␤1, and IL- here were obtained from homozygous intercrosses in the F8 to F9 genera- ␤ ␤ ␥ tion (129/SvEv ϫ C57BL/6). C57BL/6 mice deficient for the IFN-␥ gene 12R 2), or 60°C ( -actin and IFN- ). The number of PCR cycles was 35. (IFN-␥Ϫ/Ϫ) were provided by M. Kopf (Basel Institute for Immunology, Basel, Switzerland). Breeding pairs of FVB/NJ mice deficient for the STAT4 gene (STAT4Ϫ/Ϫ) and the respective wild-type controls Oligonucleotide probes (STAT4ϩ/ϩ; Ref. 13) were kindly provided by Dr. J. N. Ihle (Department Single-stranded oligonucleotides binding STAT4 (derived from the IFN of Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN). regulatory factor-1 gene promotor; Refs. 26 and 27), STAT6 (derived from Breeding pairs of mice with a deletion of the J␣281 gene segment, which ␣ the human secreted-type IL-1 receptor antagonist promotor; Ref. 28), or lack V 14-positive NKT cells and were crossed back on a C57BL/6 back- ␬ NF- B (derived from the mouse NOS2 promotor; Ref. 29) were obtained Downloaded from ground for eight generations (21), were kindly provided by Drs. M. Tan- from MWG-Biotech (Ebersberg, Germany) and were annealed in 10 mM iguchi and T. Nakayana (Chiba University, Chiba, Japan). Breeding pairs Ϫ Ϫ Tris buffer (pH 7.5; containing 50 mM NaCl, 10 mM MgCl ,and1mM of Rag2 / mice were obtained from Dr. Bernd Arnold (University of 2 DTT) by incubation for 5 min at 85°C and subsequent slow cooling to Heidelberg, Heidelberg, Germany). All mice were housed under specific Ј pathogen-free conditions in our own animal facilities. room temperature: STAT4, 5 -CT AGA GCC TGA TTT CCC CGA AAT GAT GAG-3Ј,3Ј-T CGG ACT AAA GGG GCT TTA CTA CTC GAT C-5Ј; STAT4 mutant (“CCC” 3 “TTT”), 5Ј-CT AGA GCC TGA TTT Macrophages and cell culture CTT TGA AAT GAT GAG-3Ј,3Ј-T CGG ACT AAA GAA ACT TTA CTA CTC GAT C-5Ј; STAT6, 5Ј-GAT CGC TCT TCT TCC CAG GAA http://www.jimmunol.org/ Four days after i.p. injection of 3 ml of 4% Brewer’s thioglycolate broth CTC AAT-3Ј,3Ј-CG AGA AGA AGG GTC CTT GAG TTA CAG CT-5Ј; (Difco, Detroit, MI), peritoneal exudate cells were harvested from the NF-␬B, 5Ј-GAA GCT TGG GGA CTC TCC CTT TG-3Ј,3Ј-ACC CCT above-mentioned mouse strains by flushing the peritoneal cavity twice with GAG AGG GAA ACC CTT-5Ј. 10 ml of PBS. The cells were resuspended in RPMI 1640 culture medium The double-stranded oligomers were filled in by the Klenow fragment supplemented with 2 mM glutamine, 10 mM HEPES, 13 mM NaHCO ,50 3 of DNA polymerase I with [␣-32P]dCTP (3000 Ci/mmol; Amersham Life ␮M 2-ME, 100 ␮g/ml penicillin, and 100 ␮g/ml streptomycin (all from Science, Braunschweig, Germany) to obtain labeled probes. Unlabeled or Seromed-Biochrom, Berlin, Germany) plus 2.5% FBS (Sigma, Deisen- hofen, Germany). mutant oligomers were used as competitors for EMSA. Macrophages were seeded into 96-well plates (2 ϫ 105 cells/well in 100 ␮l), 24-well plates (1 ϫ 106 cells/well in 500 ␮l), 24-cm2 culture dishes (6 ϫ 106 cells/dish in 3 ml), or 64-cm2 culture dishes (16 ϫ 106 cells/dish Preparation of nuclear extracts by guest on September 24, 2021 in 8 ml) and were cultured at 37°C in 5% CO /95% humidified air. After 2 Nuclear extracts were prepared as published previously with minor mod- 90–120 min, nonadherent cells were washed off (three washes with warm ifications (30). Briefly, after stimulation, macrophages were washed twice PBS). The adherent macrophage monolayers (containing Ͼ95% F4/80- or ϩ ϩ ϩ with ice-cold PBS (plus 100 ␮M sodium orthovanadate) and scraped into Mac-1-positive cells and ϽϽ1% CD4 , CD8 , or B220 cells) were fur- 400 ␮l of lysis buffer (20 mM Tris buffer, pH 8.0, containing 10 mM KCl; ther incubated in fresh medium with the given stimuli. Used stimuli were 5 mM MgCl ; 1 mM each of PMSF, EDTA, sodium orthovanadate, sodium recombinant murine (rm) IL-12 (BD PharMingen, San Diego, CA; R&D 2 pyrophosphate, and sodium fluoride; 0.5 mM each of EGTA and DTT; 0.1 Systems, Minneapolis, MN), recombinant human TGF-␤1, rmIL-4, rmIL- mM sodium molybdate; 10 ␮M each of pepstatin A and aprotinin; and 5 11, rmIL-17, rmIL-18 (all from R&D Systems), IFN-␥ (kindly provided ␮M each of leupeptin and chymostatin). The cells were allowed to swell on from Dr. G. Adolf at the Ernst Boehringer Institut, Vienna, Austria), and ice for 5 min before Nonidet P-40 was added to a final concentration of LPS (O111:B4; Sigma). The concentrations of IL-12 and IL-18 were 0.1 and 10 ng/ml, unless stated differently. 0.5%, and the tube was vortexed for 10 s. The homogenate was centrifuged The LPS content of the cytokine stock solutions and of the final culture and the supernatant (cytosol) was harvested in a fresh tube. The nuclear Ͻ pellets were washed once in lysis buffer without Nonidet P-40 and finally medium was 10 pg/ml as determined by a colorimetric Limulus amebo- ␮ cyte lysate assay (BioWhittaker, Walkersville, MD). resuspended in 50–100 l 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 ϫ g, 15 min, 4°C), and the supernatants (containing Determination of nitrite accumulation and measurement DNA binding proteins) were stored at Ϫ70°C. of IFN-␥ NO Ϫ in culture supernatants was determined by the Griess assay (22). The 2 EMSA capture ELISA for measuring IFN-␥ was performed as described (15) and had a detection limit of 39–78 pg/ml. Binding reactions (20 ␮l total) were performed by incubating the nuclear extracts (4–5 ␮g of protein) with 5ϫ reaction buffer (50 mM Tris buffer, Preparation of total cell lysates for Western blot analysis and pH 7.5; 500 mM KCl; 25 mM MgCl2; 5 mM of DTT and EDTA; 25% ␮ ⅐ immunoprecipitation glycerol) and 2 g 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 For the detection of Stat4 and tyrosine-phosphorylated Stat4 protein, mac- mutant oligomer for 5 min, followed by a 30-min incubation at room tem- rophage monolayers were lysed in 40 mM Tris buffer (pH 8.0) plus pro- perature with the labeled dsDNA probe (0.1–1 ng; Ϸ1 ϫ 105 cpm). For tease and phosphatase inhibitors as described (15). The lysates (60–80 ␮g supershift analysis, 2 ␮g of rabbit anti-mouse NF-␬B p50 (sc-114X), rabbit protein/lane) were separated by 7.5% SDS-PAGE, transferred to reinforced anti-mouse NF-␬B p65 (sc-372X), rabbit anti-STAT4 Ab (sc-485-X or nitrocellulose (0.2 ␮M; Schleicher & Schuell, Dassel, Germany), and an- sc-486-X), or rabbit anti-STAT6 (sc-981-X; all from Santa Cruz Biotech- alyzed by ECL-based Western blotting with affinity-purified rabbit-anti- nology) were added to the reaction mixture and incubated at room tem- mouse STAT4 IgG (sc-486 (C-20); Santa Cruz Biotechnology, Santa Cruz, perature for 30 min before the labeled DNA probe was added for another CA) and anti-phosphotyrosine mouse IgG (PY-99; Santa Cruz Biotechnol- 15 min of incubation. The formed DNA/protein complexes were separated ogy) as described (15, 23). Immunoprecipitations (with anti-STAT4 (sc- on 4.5–6% polyacrylamide gels in 0.5 ϫ Tris-borate-EDTA buffer at 40 485 or sc-486) or anti-phosphotyrosine Ab (PY-99)) were performed as mA for 1.5 h. The gels were dried without fixation, and the DNA protein published previously (15). complexes were visualized by autoradiography. The Journal of Immunology 3077

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 Rag2Ϫ/Ϫ mice (which lack functional T and B lymphocytes) and J␣281-deficient mice (which lack V␣14/J␣281-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. 1A 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 Ϯ 4.4 ng/ml, mean Ϯ 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). The release of IFN-␥ by IL-12/IL-18-stimulated peritoneal exudate macrophages was paralleled by the accumulation of nitrite (Fig. 1B). 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 Downloaded from shown). Thus, the production of NO was not a direct effect of IL-12 and IL-18, but mediated by the endogenous IFN-␥. FIGURE 1. IL-12 plus IL-18 stimulates peritoneal exudate macrophages for the production of IFN-␥ and NO. IFN-␥ (A) and nitrite content ␥ (B) of culture supernatants from macrophages stimulated for 48 h with Cytokine regulation of macrophage IFN- production http://www.jimmunol.org/ various concentrations of IL-12 with or without IL-18. The values repre- Having seen that IL-12 and IL-18 are potent inducers of IFN-␥,we Ϯ sent the mean ( SEM) of two experiments. Similar results (i.e., induction next analyzed whether other known macrophage activators (LPS, of IFN-␥ and NO Ϫ by optimal concentrations of IL-12 plus IL-18, but not 2 TNF-␣, or IL-17; Ref. 31) could replace IL-12 or IL-18. We found by IL-12 and IL-18 alone) were obtained in 20 additional experiments. 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 Results and Discussion production of IFN-␥. However, LPS in combination with IL-18 ␥ Inflammatory mouse macrophages release IFN- protein in (but not in combination with IL-12) led to a potent release of response to IL-12/IL-18, which leads to the production of NO IFN-␥ after 48 h (9.0 Ϯ 0.7 ng/ml, mean Ϯ SEM of 12 experi-

In a previous study, mouse bone marrow-derived macrophages ments). TNF-␣ and IL-17, in contrast, were ineffective when com- by guest on September 24, 2021 were shown to produce IFN-␥ in response to IL-12 and IL-18, bined with either IL-12 or IL-18 (data not shown).

FIGURE 2. Inhibition of macrophage IFN-␥ production by IL-4, IL-10, IL-13, and TGF-␤. Macro- phage monolayers were exposed to IL-4, IL-10, IL-11, IL-13 (10 ng/ml each or as mentioned), or TGF-␤ (5 ng/ml or as mentioned) simultaneously with (A and B), before (C), or after (D) activation by IL-12 plus IL-18. IFN-␥ in the culture supernatants was determined at 48 h of stimulation with IL-12/IL-18. Mean (ϮSEM) of seven (A), nine (C), and six (D) experiments. B shows one of three representative experiments. 3078 MACROPHAGE, IFN-␥, AND STAT4 Downloaded from http://www.jimmunol.org/

FIGURE 3. Induction of IFN-␥ mRNA and NOS2 mRNA by IL-12, IL-18, or IL-12 plus IL-18 as determined by competitive PCR analysis. A, Expression of IFN-␥ and NOS2 mRNA at 24 h of stimulation with IL-12, IL-18, or IL-12 plus IL-18. B, Expression of IFN-␥ and NOS2 mRNA after stimulation with IL-12 plus IL-18 for different periods of time. One of three experiments. by guest on September 24, 2021 A number of cytokines have been described to down-regulate various macrophage functions, including IL-4, IL-10, IL-11, IL- FIGURE 4. Expression and activation of STAT4 in macrophages on 13, or TGF-␤ (Refs. 32–34 and references therein). When added stimulation with IL-12 plus IL-18. A, Western blot analysis of STAT4 protein expression in total cell lysates (80 ␮g/lane) from wild-type and 2 h before or simultaneously with IL-12 and IL-18 to the macro- Ϫ/Ϫ ␤ ␥ STAT4 macrophages stimulated with IL-12 and IL-18 for 1 h. B,Nu- phage cultures, IL-4 and TGF- inhibited the production of IFN- , clear translocation of STAT4 in wild-type macrophages stimulated with whereas IL-10, IL-11, and IL-13 (0.1–100 ng/ml) were only IL-12 with or without IL-18. In the gel-shift reaction, nuclear extracts (4 weakly suppressive or completely inactive (Fig. 2, A–C and data ␮g/lane) were combined with a [32␣-P]dCTP-labeled DNA oligonucleotide not shown). The mean suppression achieved by IL-4 and TGF-␤ containing a STAT4 binding site in the absence or presence of a 100-fold was 61.9 Ϯ 7.1% and 70.8 Ϯ 9.4%, respectively (mean Ϯ SEM of excess of the unlabeled probe or with a labeled STAT4 mutant oligonu- six experiments). TGF-␤, but none of the other cytokines, was also cleotide. C, STAT4 EMSA with nuclear extracts from wild-type vs Ϫ/Ϫ active when added 2 h after initiation of the stimulation with IL-12 STAT4 macrophages stimulated with IL-12 plus IL-18 for 2 h. plus IL-18 (Fig. 2D). 4–6 h relative to the expression of IFN-␥ mRNA (Fig. 3B). To- ␥ mRNA expression of IL-12R, IL-18R, IFN- , and NOS2 gether with the data in Fig. 1A these results suggest that the syn- As a first approach to understanding the synergistic action of IL-12 ergistic action of IL-12 and IL-18 mainly occurs on the level of and IL-18 on IFN-␥ protein production, we analyzed the expres- IFN-␥ protein and that the expression of IFN-␥ precedes the pro- sion of their receptors and their effect on IFN-␥ mRNA. Unstimu- duction of NO. lated macrophage monolayers already constitutively expressed IL- 12R␤1, IL-12␤2, and IL-18R mRNA. With PCR analysis, there Macrophage stimulation with IL-12 plus IL-18 leads to strong was no appreciable difference in the mRNA expression levels of activation of STAT4, but only weakly stimulates the nuclear ␬ the receptors when the macrophages were stimulated with IL-12, translocation of NF- B IL-18, or IL-12 plus IL-18 (data not shown). These data indicate T cells and NK cells activate STAT4 on stimulation with IL-12 that the two cytokines do not act by up-regulating the mRNA of (12, 13, 35, 36). Furthermore, STAT4 protein was found in human each other’s receptor. However, a possible mutual influence on the dendritic cells, monocytes, and macrophages (18, 19). Therefore, surface expression of the receptor proteins is not excluded. we envisaged a role of STAT4 in macrophage activation by IL-12 IL-12, IL-18, or IL-12 plus IL-18 induced the expression of plus IL-18. Unstimulated adherent macrophages expressed STAT4 IFN-␥ mRNA, which was undetectable in resting macrophages. protein, which was readily detectable by Western blotting of However, we did not observe a synergistic action of IL-12 and whole-cell lysates. The detected band was confirmed to represent IL-18 (Fig. 3A). In contrast, the induction of NOS2 mRNA was STAT4 by parallel analysis of macrophage lysates from most prominent under costimulation conditions and delayed by STAT4ϩ/ϩ and STAT4Ϫ/Ϫ mice (Fig. 4A). The Journal of Immunology 3079

␬

FIGURE 5. Nuclear translocation of NF- B in unstimulated or stimu- Downloaded from lated macrophages (IL-12, IL-18, or IL-12 plus IL-18 for 4 h). As a positive control, nuclear extracts (5 ␮g/lane) were also prepared from macrophages activated with IFN-␥ plus LPS (20 ng/ml each). Supershift analyses with anti-NF-␬B p50 or p65 Abs identiﬁed the DNA/protein complexes as p50/50 homodimers and as p65/50 heterodimers. http://www.jimmunol.org/ Analysis of STAT4 activation by anti-STAT4 (or anti-phosphotyrosine) immunoprecipitation followed by anti-phosphotyrosine (or anti-STAT4) Western blotting was not possible because two different anti-STAT4 Abs (sc-485 (L-18) and sc-486 (C-20); Santa Cruz Biotechnology) and an anti-phosphotyrosine Ab (PY-99; Santa Cruz Biotechnology) failed to bind (tyrosine-phosphorylated) STAT4 in macrophage lysates under nondenaturing conditions (data not shown). Therefore, we performed EMSA to see whether FIGURE 6. STAT4 is required for macrophage IFN-␥ production. Mac- stimulation with IL-12, IL-18, or IL-12 plus IL-18 leads to acti- rophages from STAT4Ϫ/Ϫ or NOS2Ϫ/Ϫ mice or from the respective wild- by guest on September 24, 2021 vation (i.e., nuclear uptake) of STAT4. By using a STAT probe type controls were stimulated with IL-12 plus IL-18 for 48 h before the that can bind multiple STATs including STAT4 (27), we found culture supernatants were analyzed for their content of IFN-␥ (A) and Ϫ that IL-12 plus IL-18 induced a protein-DNA complex that was NO2 (B). Total RNA preparations from resting or IL-12/IL-18-stimulated Ϫ Ϫ formed by STAT4 because it was not observed with nuclear STAT4 / or wild-type macrophages were analyzed for their content of Ϫ Ϫ ␥ extracts from STAT4 / macrophages (Fig. 4, B and C). The re- IFN- mRNA by quantitative PCR (C). One of three (A) or two (B) quired minimum period of stimulation was 90–120 min (Fig. 4B experiments. 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). derived NO, is required as a signaling molecule in macrophages As NF-␬B was shown to be activated by IL-18 in T cells (37, for IL-12/IL-18-induced IFN-␥ production. Importantly, IFN-␥ 38) and by IL-12 in dendritic cells (17), we also analyzed the mRNA was still present in IL-12/IL-18-stimulated STAT4Ϫ/Ϫ nuclear translocation of NF-␬B in macrophages stimulated with macrophages, although its level of expression was clearly lower IL-12, IL-18, or IL-12 plus IL-18 for 15 min to 6 h. Our EMSA compared with STAT4ϩ/ϩ macrophages (Fig. 6C). Therefore, (Fig. 5 and data not shown) revealed that the combination of both STAT4 does not only regulate the expression of IFN-␥ mRNA but cytokines (but neither cytokine alone) weakly enhanced the DNA also the synthesis of IFN-␥ protein. This conclusion is further sup- binding activity of nuclear extracts to an oligonucleotide contain- ported by the observation that in wild-type macrophages IFN-␥ ing a NF-␬B consensus sequence. The activation of NF-␬B might mRNA was expressed after stimulation with IL-12, IL-18, or IL-12 contribute to the stimulatory effect of IL-12 plus IL-18 on macro- plus IL-18 (Fig. 3A and Fig. 6C), whereas activation of STAT4 and phage IFN-␥ production. production of IFN-␥ protein were only achieved after combined stimulation with IL-12 and IL-18 (Figs. 1A and 4). STAT4, but not NOS2, is required for the activation of macrophage IFN-␥ production by IL-12 plus IL-18 Inhibition of macrophage IFN-␥ production by IL-4 is To directly assess the role of STAT4 in macrophage IFN-␥ pro- paralleled by the activation of STAT6 that binds comparably duction, we used macrophages from STAT4Ϫ/Ϫ mice. Whereas well to the same DNA oligonucleotide as STAT4 wild-type macrophages produced high amounts of IFN-␥ and NO The above experiments have demonstrated that the IL-12/IL-18- in response to IL-12 plus IL-18, IFN-␥ protein and NO were not induced production of IFN-␥ by macrophages is dependent on detectable in the culture supernatants of STAT4Ϫ/Ϫ macrophages STAT4 and is inhibited by IL-4. Therefore, we performed EMSA (Fig. 6, A and B). In contrast, macrophages with a deletion of the analysis to investigate whether IL-4 can antagonize the activation NOS2 gene were fully capable of producing IFN-␥ after stimula- of STAT4 by IL-12 plus IL-18. Costimulation of the macrophages tion with IL-12 plus IL-18 (Fig. 6A). Thus, STAT4, but not NOS2- with IL-4 led to the appearance of a second, slower-migrating 3080 MACROPHAGE, IFN-␥, AND STAT4

DNA/protein complex (II) when using the STAT4 probe, whereas tween either STAT4 or STAT6 protein and the radiolabeled the intensity of the IL-12/IL-18-induced complex I (containing STAT4 DNA probe was very similar (Fig. 8, A and B). Unlabeled STAT4) decreased. No such effect was seen when TGF-␤ instead STAT6 oligonucleotide competed the binding of STAT6 protein to of IL-4 was used (Fig. 7, A and C, lane 3vslane 7). Complex II the STAT4 DNA probe at ϳ10-fold lower concentrations than the was formed by STAT6 because it was completely supershifted by STAT4 oligonucleotide. In contrast, the unlabeled STAT6 oligo- an anti-STAT6 Ab (Fig. 7B), and it was also observed when a nucleotide was unable to antagonize the interaction between the DNA oligonucleotide with a STAT6 instead of a STAT4 binding STAT4 protein and the STAT4 probe (Fig. 8). Thus, STAT4 and site was used (Fig. 7C, lane 7 vs lane 8). Importantly, IL-4-acti- STAT6 protein have similar affinities to the STAT4 DNA oligo- vated STAT6 bound comparably well to the STAT4 and STAT6 nucleotide used in these in vitro assays, and the affinity of STAT6 probe (Fig. 7C, lane 11 vs lane 12), whereas the IL-12/IL-18- protein to the STAT6 DNA oligonucleotide is only 10-fold higher. activated STAT4 only interacted with the STAT4 oligonucleotide, Together these findings suggest that IL-4 induces nuclear trans- but not with the STAT6 probe (Fig. 7C, lane 3 vs lane 4). The location of STAT6, which not only interacts with STAT6 DNA addition of unlabeled STAT4 competitor to nuclear extracts from binding sites, but might also compete the DNA binding of IL-12/ IL-4/IL-12/IL-18-stimulated macrophages completely eliminated IL-18-activated STAT4. STAT6, when bound to the STAT4 motif, both complex I and complex II, whereas complex I was maintained apparently does not exert transactivating functions, because stim- in the presence of nonradiolabeled STAT6 competitor (Fig. 7C, ulation of macrophages with IL-4 alone does not lead to the pro- lane 7 vs lanes 9 and 10). duction of IFN-␥ (data not shown). The possibility that the inhib- Finally, we performed EMSA analyses with nuclear extracts itory effect of IL-4 results from the activation of STAT6 and its from macrophages stimulated with IL-12/IL-18 plus IL-4 and with ability to interfere with signaling functions of STAT4 is supported Downloaded from titrated amounts of nonradiolabeled oligonucleotides to test the by previous experiments with mouse macrophages in which strong relative affinities of STAT4 and STAT6 protein to bind to STAT4 evidence for a competition between IFN-␥-induced STAT1 and or STAT6 oligonucleotides. The ability of nonradiolabeled STAT4 IL-4-induced STAT6 for occupancy of the STAT binding element oligonucleotide to compete for the formation of complexes be- in the promotor of the IFN regulatory factor-1 gene was presented http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. Effect of IL-4 on the nuclear translocation of STAT4 in macrophages stimulated with IL-12 plus IL-18. Nuclear extracts (4 ␮g/lane) were prepared from macrophages activated by IL-12 plus IL-18 for4hintheabsence or presence of IL-4 (10 ng/ml) or TGF-␤1 (5 ng/ml). EMSA analysis was performed with DNA oligonucleotides containing a STAT4 binding site (A–C) or a STAT6 binding site (C). The Journal of Immunology 3081

tion, presumably via induction of STAT6 that is able to occupy the STAT4 DNA binding site.

References 1. Farrar, M. A., and R. D. Schreiber. 1993. The molecular cell biology of interferon-␥ and its receptor. Annu. Rev. Immunol. 11:571. 2. Murray, H. W. 1994. Interferon-␥ and host antimicrobial defense: current and future clinical applications. Am. J. Med. 97:459. 3. Fenton, M., M. W. Vermeulen, S. Kim, M. Burdick, R. M. Strieter, and H. Kornﬁeld. 1997. Induction of ␥ interferon production in human alveolar macrophages by Mycobacterium tuberculosis. Infect. Immun. 65:5149. 4. Di Marzio, P., P. Puddu, L. Conti, F. Belardelli, and S. Gessani. 1994. Interfer- on-␥ upregulates its own gene expression in mouse peritoneal macrophages. J. Exp. Med. 179:1731. 5. Puddu, P., L. Fantuzzi, P. Borghi, B. Varano, G. Rainaldi, E. Guillemard, W. Malorni, P. Nicaise, S. F. Wolf, F. Belardelli, and S. Gessani. 1997. IL-12 induces IFN-␥ expression and secretion in mouse peritoneal macrophages. Infect. Immun. 159:3490. 6. Fultz, M. J., S. A. Barber, C. W. Dieffenbach, and S. N. Vogel. 1993. Induction of IFN-␥ in macrophages by lipopolysaccharide. Int. Immunol. 5:1383. 7. Fultz, M. J., and S. N. Vogel. 1995. Autoregulation by interferons provides an endogenous ‘priming’ signal for LPS-responsive macrophages. J. Endotoxin Res.

2:77. Downloaded from 8. Salkowski, C. A., K. Kopydlowski, J. Blanco, M. J. Cody, R. McNally, and S. N. Vogel. 1999. IL-12 is dysregulated in macrophages from IRF-1 and IRF-2 knockout mice. J. Immunol. 163:1529. 9. Munder, M., M. Mallo, K. Eichmann, and M. Modolell. 1998. Murine macrophages secrete interferon-␥ upon combined stimulation with IL-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. 187:2103. 10. Ohteki, T., T. Fukao, K. Suzue, C. Maki, M. Ito, M. Nakamura, and S. Koyasu. 1999. Interleukin-12-dependent interferon-␥ production by CD8␣ϩ lymphoid dendritic cells. J. Exp. Med. 189:1981. http://www.jimmunol.org/ 11. Wang, J., J. Wakeham, R. Harkness, and Z. Xing. 1999. Macrophages are a signiﬁcant source of type 1 cytokines during mycobacterial infection. J. Clin. Invest. 103:1023. FIGURE 8. Afﬁnity of STAT4 and STAT6 to DNA oligonucleotides 12. Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, and K. M. Murphy. 1995. Interleukin-12 signaling in T helper type containing a STAT4 or STAT6 binding site. A, EMSA analysis was per- 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator formed with a nuclear extract derived from macrophages stimulated with of transcription (Stat)3 and Stat4. J. Exp. Med. 181:1755. IL-12/IL-18 plus IL-4 (10 ng/ml) for3hinthepresence of a labeled 13. Thierfelder, W. E., J. M. van Deursen, K. Yamamoto, R. A. Tripp, S. R. Sarawar, STAT4 probe and increasing concentrations of unlabeled STAT4 or R. T. Carson, M. Y. Sangster, D. A. A. Vignali, P. C. Doherty, G. C. Grosveld, and J. N. Ihle. 1996. Requirement for Stat4 in interleukin-12-mediated responses STAT6 oligonucleotides (competitors). B, Densitometric evaluation of the

of natural killer and T cells. Nature 382:171. by guest on September 24, 2021 formed protein/DNA complexes (complex I ϭ STAT4/DNA, complex II ϭ 14. Kaplan, M. H., A. L. Wurster, and M. J. Grusby. 1998. A signal transducer and STAT6/DNA). Complex formation in the absence of competitor was taken activator of transcription (Stat)4-independent pathway for the development of T as 100%. Densitometric analysis was performed after scanning of the gels helper type 1 cells. J. Exp. Med. 188:1191. 15. Diefenbach, A., H. Schindler, M. Ro¨llinghoff, W. Yokoyama, and C. Bogdan. with the AIDA program (Ray Test, Straubenhardt, Germany). One of two 1999. Requirement for type 2 NO-synthase for IL-12 responsiveness in innate experiments. immunity. Science 284:951. 16. Carter, L. L., and K. M. Murphy. 1999. Lineage-specific requirement for signal transducer and activator of transcription (Stat) 4 in interferon-␥ production from CD4ϩ versus CD8ϩ T cells. J. Exp. Med. 189:1355. 17. Grohmann, U., M. L. Belladonna, R. Bianchi, C. Orabona, E. Ayroldi, (39). However, it is important to emphasize that data obtained by M. C. Fioretti, and P. Puccetti. 1998. IL-12 acts directly on DC to promote nuclear localization of NF-␬B and primes DC for IL-12 production. Immunity EMSA analysis are only suggestive of, but by no means provide a 9:315. reflection of, the real events that take place on a promotor in its 18. Nagayama, H., K. Sato, H. Kawasaki, M. Enomoto, C. Morimoto, K. Tadokoro, genomic context. Furthermore, the promotor elements that are oc- S. Asano, and T. A. Takahashi. 2000. IL-12 responsiveness and expression of IL-12 receptor in human peripheral blood monocyte-derived dendritic cells. cupied by STAT4 and mediate the STAT4-dependent induction of J. Immunol. 165:59. IFN-␥ in macrophages (or T cells) are unknown. Their ability to 19. Frucht, D. M., M. Aringer, J. Galon, C. Danning, M. Brown, S. Fan, M. Centola, bind STAT4 vs STAT6 might be completely different from the C.-Y. Wu, N. Yamada, H. El Gabalawy, and J. J. O’Shea. 2000. Stat4 is expressed in activated peripheral blood monocytes, dendritic cells, and macro- properties of the DNA oligonucleotides that were used in the cur- phages at sites of Th1-mediated inflammation. J. Immunol. 164:4659. rent analysis. 20. MacMicking, J. D., C. Nathan, G. Hom, N. Chartrain, D. S. Fletcher, M. Trumbauer, K. Stevens, Q.-w. Xie, K. Sokol, N. Hutchinson, H. Chen, and Concluding remarks J. S. Mudgett. 1995. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641. Macrophages share with other types of cells the ability to produce 21. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, ␥ M. Kanno, and M. Taniguchi. 1997. Requirement vor V␣14 NKT cells in IL- IFN- . This is the first study to demonstrate that macrophages, 12-mediated rejection of tumors. Science 278:1623. similar to NK and T cells, are strictly dependent on STAT4 for the 22. Stenger, S., H. Thu¨ring, M. Ro¨llinghoff, and C. Bogdan. 1994. Tissue expression release of IFN-␥ protein in response to IL-12 plus IL-18. However, of inducible nitric oxide synthase is closely associated with resistance to Leish- mania major. J. Exp. Med. 180:783. unlike NK cells (15), NOS2-derived NO was not required for mac- 23. Mattner, J., H. Schindler, A. Diefenbach, M. Ro¨llinghoff, I. Gresser, and rophage IFN-␥ production. We obtained no evidence for a signif- C. Bogdan. 2000. Regulation of type 2 NO synthase by type I interferons in icant activation of NF-␬B by IL-12 plus IL-18 in macrophages as macrophages infected with Leishmania major. Eur. J. Immunol. 30:2257. ␥ 24. Platzer, C., G. Richter, K. U¨ berla, W. Mu¨ller, H. Blo¨cker, T. Diamantstein, and it was described for dendritic cells (17). The production of IFN- T. Blankenstein. 1992. Analysis of cytokine mRNA levels in interleukin-4-trans- by macrophages is likely to amplify their antimicrobial activities genic mice by quantitative polymerase-chain-reaction. Eur. J. Immunol. 22:1179. and to serve as an autoregulatory circuit that helps to expand Th1 25. Diefenbach, A., H. Schindler, N. Donhauser, E. Lorenz, T. Laskay, J. MacMicking, M. Ro¨llinghoff, I. Gresser, and C. Bogdan. 1998. Type 1 inter- responses. However, IL-4, the prototypic Th2 cytokine, might an- feron (IFN-␣/␤) and type 2 nitric oxide synthase regulate the innate immune tagonize such a process by inhibiting macrophage IFN-␥ produc- response to a protozoan parasite. Immunity 8:77. 3082 MACROPHAGE, IFN-␥, AND STAT4

26. Yamamoto, K., O. Miura, S. Hirosawa, and N. Miyasaka. 1997. Binding se- macrophage effector function through the inhibition of nuclear factor-␬B. J. Im- quence of STAT4: STAT4 complex recognizes the IFN-␥ activation site (GAS)- munol. 159:5661. like sequence (T/A)TTCC(C/G)GGAA(T/A). Biochem. Biophys. Res. Commun. 34. Chomarat, P., and J. Banchereau. 1998. Interleukin-4 and interleukin-13: their 233:126. similarities and discrepancies. Int. Rev. Immunol. 17:1. 27. Wang, K. S., J. Ritz, and D. A. Frank. 1999. IL-2 induces STAT4 activation in 35. Yu, C.-R., J.-X. Lin, D. W. Fink, S. Akira, E. T. Bloom, and A. Yamauchi. 1996. primary NK cells and NK cell lines, but not in T cells. J. Immunol. 162:299. Differential utilization of Janus kinase-signal transducer and activator of tran- 28. Ohmori, Y., M. F. Smith, and T. A. Hamilton. 1996. IL-4-induced expression of scription signaling pathways in the stimulation of human natural killer cells by ␣ the IL-1 receptor antagonist gene is mediated by STAT6. J. Immunol. 157:2058. IL-2, IL-12, and IFN- . J. Immunol. 157:126. 29. Xie, Q.-W., Y. Kasshiwabara, and C. Nathan. 1994. Role of transcription factor 36. Kaplan, M. H., Y.-L. Sun, T. Hoey, and M. J. Grusby. 1996. Impaired IL-12 NF-␬B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4705. responses and enhanced development of Th2 cells in Stat4-deﬁcient mice. Sci- ence 382:174. 30. Schreiber, E., P. Matthias, M. M. Mu¨ller, and W. Schaffner. 1989. Rapid detection of octamer binding proteins with “mini-extracts”, prepared from a small 37. Robinson, D., K. Shibuya, A. Mui, F. Zonin, E. Murphy, T. Sana, S. B. Hartley, S. Menon, R. Kastelein, F. Bazan, and A. O’Garra. 1997. IGIF does not drive Th1 number of cells. Nucleic Acid Res. 17:6419. development but synergizes with IL-12 for interferon-␥ production and activates 31. Jovanovic, D. V., J. A. D. Battista, J. Martel-Pelletier, F. C. Jolicoeur, Y. He, IRAK and NF␬B. Immunity 7:571. M. Zhang, F. Mineau, and J.-P. Pelletier. 1998. IL-17 stimulates the production 38. Tsuji-Takayama, K., Y. Aizawa, I. Okamoto, H. Kojima, K. Koide, M. Takeuchi, and expression of proinﬂammatory cytokines, IL-1␤ and TNF-␣, by human mac- H. Ikegami, T. Ohta, and M. Kurimoto. 1999. Interleukin-18 induces interferon-␥ rophages. J. Immunol. 160:3513. production through NF-␬B and NFAT activation in murine T helper type 1 cells. 32. Bogdan, C., and C. Nathan. 1993. Modulation of macrophage function by trans- Cell. Immunol. 196:41. ␤ forming growth factor- , interleukin 4 and interleukin 10. Annu. NY Acad. Sci. 39. Ohmori, Y., and T. A. Hamilton. 1997. IL-4-induced STAT6 suppresses IFN-␥- 685:713. stimulated STAT1-dependent transcription in mouse macrophages. J. Immunol. 33. Trepicchio, W. L., L. Wang, M. Bozza, and A. J. Dorner. 1997. IL-11 regulates 159:5474. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021