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The Journal of Immunology, 2001, 166: 7082-7089.
Copyright © 2001 by The American Association of Immunologists

Suppressors of Cytokine Signaling (SOCS)-1 and SOCS-3 Are Induced by CpG-DNA and Modulate Cytokine Responses in APCs1

Alexander H. Dalpke, Sandra Opper, Stefan Zimmermann and Klaus Heeg2

Institute of Medical Microbiology and Hygiene, Philipps-University, Marburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During infection, the functional status of the innate immune system is tightly regulated. Although signals resulting in activation have been well characterized, counterregulative mechanisms are poorly understood. Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine-inducible negative regulators of Janus kinase/STAT signaling in cells of hemopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen-derived stimuli, we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines, bone marrow-derived dendritic cells, and peritoneal macrophages with CpG-DNA. In this study, we show that CpG-DNA, but not GpC-DNA, induces expression of mRNA for SOCS-1 and SOCS-3 in vitro and in vivo. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the mitogen-activated protein kinase pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA. CpG-DNA triggered synthesis of SOCS proteins that could be detected by Western blotting. SOCS proteins were functional because they inhibited IFN-{gamma} as well as IL-6- and GM-CSF-induced phosphorylation of STAT proteins. Furthermore, IFN-{gamma}-induced up-regulation of MHC class II molecules was also prevented. The same effects could be achieved by overexpression of SOCS-1. Hence, the results indicate a substantial cross-talk between signal pathways within cells. They provide evidence for regulative mechanisms of Janus kinase/STAT signaling after triggering Toll-like receptor signal pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During initial interaction of pathogens with the host’s immune system, pathogen-associated molecular pattern (PAMP)3 triggers cells of the innate immune system (1). Conserved bacterial components such as LPS, peptidoglycan, lipoteichoic acid, or bacterial CpG-DNA (2, 3) have been characterized as structural correlates of PAMP. PAMP are recognized by innate immune cells through pattern recognition receptors and cause activation and induction of effector functions (4). Toll-like receptors (TLR) play a pivotal role in recognition of PAMP (5). For LPS, peptidoglycan, lipoteichoic acid, and recently for CpG-DNA (6), the respective TLR have been identified. The intracellular TLR-triggered signaling cascade depends critically on the intracellular adapter protein MyD88 (7, 8), and converges with the IL-1R and CD40 signaling cascades. Finally, TLR signaling results in activation of mitogen-activated protein kinase (MAPK) pathways and translocation of NF-{kappa}B (9). The innate immune cell pool responding to PAMP is comprised mainly of dendritic cells (DC) and macrophages (1). Pathogen-derived stimuli act directly on these cells; however, responses are regulated and influenced by a cytokine milieu containing, for example, IFN-{gamma} (10).

Bacterial DNA activates cells of the innate immune system due to the relative abundance of unmethylated CpG motifs. Synthetic oligodeoxynucleotides (ODN) with immunostimulative CpG motifs mimic the effects of bacterial DNA and trigger macrophages and DC (2, 3). Inversion of the CpG dinucleotide to GpC completely prevents immunostimulation. As bacterial DNA, CpG-ODN trigger activation of MAPK cascades and NF-{kappa}B, resulting in transcription of cytokine mRNA such as TNF and IL-12 (11, 12, 13). CpG signaling critically depends on the presence of intracellular protein MyD88 and TRAF6 (14, 15). Recently, TLR-9 has been identified as the respective receptor for CpG-DNA, thus adding CpG-DNA to the family of TLR ligands (6).

After triggering with CpG-DNA, intracellular signaling pathways are activated within minutes. After hours, these cells respond with production and secretion of cytokines such as IL-12 or TNF and with differentiation and activation of effector functions (16). In vivo, cytokine release shows sharp kinetics: cytokine levels burst and peak after 1–2 h, followed by subsequent down-regulation (16). Thus, initial activation of DC and macrophages is followed by counterregulative events whose mechanisms have been characterized only incompletely to date. In addition, it has been shown that macrophages down-regulate MHC class II expression after initial activation by CpG-DNA (17).

Whereas initiation of signal transduction by cytokines or natural ligands is well characterized, little is known about inactivation of signal transduction pathways. In the last years, substantial progress has been achieved toward the understanding of molecular mechanisms of activation and down-regulation of the Janus kinase (JAK)/STAT signaling pathway, which is activated in response to a variety of cytokines and hormones (18). A new family of cytokine-induced inhibitors of JAK/STAT signals has been characterized (19, 20, 21). This family is comprised of at least eight members of SH2-domain-containing proteins, which are named suppressors of cytokine signaling (SOCS 1–7) (19), and cytokine-inducible SH2(C15)-containing proteins (22). These proteins can be induced in various tissues and cell lines (23), although SOCS-1 and SOCS-3 have been recognized to be most important for regulative effects in immune cells (24, 25, 26, 27, 28). The SOCS proteins act as negative regulators of the JAK/STAT pathway either by inhibiting JAK activity or by inhibiting binding of STAT factors to the cytoplasmatic domains of the respective receptors (23). SOCS-1 and SOCS-3 are tightly regulated within immune cells, and their induction depends on de novo transcription of mRNA (29). The respective promoter regions contain defined STAT binding sites (30). Induction may occur not only through cytokines, but also through other ligands that are associated with activation of JAK/STAT pathway (23, 31). Furthermore, there are indications that SOCS proteins even can be induced by STAT-independent signals (27), suggesting that multiple signal pathways are involved in regulation of SOCS.

Because signal pathways induced by CpG-DNA are quite well characterized, and because CpG-ODN with its internal negative control (GpC-ODN) represents a suitable and reliable tool to trigger TLR-related signal pathways (11, 12), we analyzed the role of CpG-DNA on the expression of SOCS proteins. We show induction of SOCS-1 and SOCS-3 mRNA, which was independent of protein synthesis and could be blocked partially by inhibitors of MAPK signaling. SOCS proteins were operative because they inhibited subsequent IFN-{gamma} as well as IL-6 and GM-CSF signaling pathways. Thus, the results indicate that TLR-related signal pathways not only activate functional SOCS proteins and might be responsible for down-regulative events after triggering with PAMP, but also point to a regulative cross-talk between distinct intracellular signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and reagents

J774 (American Type Culture Collection, Manassas, VA) and RAW 264.7 cells (a kind gift from R. Schumann, Universitätsklininkum Charite, Berlin, Germany) were cultured in Clicks/RPMI 1640 supplemented with 5% FCS, 50 µM 2-ME, and antibiotics (penicillin G (100 IU/ml of medium) and streptomycin sulfate (100 IU/ml of medium)). Phosphothioate-modified ODN were custom synthesized by TIB Molbiol (Berlin, Germany) and MWG Biotech (Munich, Germany). The following sequences were used (the bold letters indicate the CpG motifs): 1668, TCC ATG ACG TTC CTG ATG CT; SB-DG, TCC ATG ACG TTC CTG ATG CTA GAG A; 1668GC, TCC ATG AGC TTC CTG ATG CT (control ODN with inverted CpG motif); PZ-3, CTC CTA TTG GGG GTT TCC TAT (G-rich ODN). LPS and chloroquine were purchased from Sigma (Deisenhofen, Germany). Cycloheximide and the MAPK inhibitor SB203580 were delivered by Calbiochem (Schwalbach, Germany); UO126 came from Promega (Mannheim, Germany). IFN-{gamma} was obtained from E. Adolf (Vienna, Austria); IL-6 and GM-CSF were purchased from Tebu (Frankfurt, Germany). Stat-1, Stat-3, and Stat-5 Ab were a kind gift of T. Decker (Vienna, Austria). Abs to SOCS were purchased from Santa Cruz Biotechnology (Heidelberg, Germany); other Abs were obtained from New England Biolabs (Frankfurt, Germany). Recombinant IFN-{alpha}, IFN-{beta}, and neutralizing Abs against IFN-{alpha} and IFN-{beta} were obtained from PBL (New Brunswick, NJ).

Determination of cytokine secretion

Cytokine levels in culture supernatants were determined using a commercially available ELISA kit for TNF-{alpha}, according to the manufacturer’s instructions (BD Biosciences, Heidelberg, Germany). Each value represents mean of duplicate values.

Cell stimulation

Depending upon the experiments, 1 x 106 cells/well (RT-PCR) or 5 x 106 cells/well (Western blot) were plated in a 12-well culture plate and stimulated with different ODN in the indicated concentrations. When using inhibitors, these were preincubated for the indicated time before stimulation was performed. If not otherwise indicated, cells were stimulated for 4.5 h, then supernatants were removed and, where indicated, TNF-{alpha} was measured by ELISA.

TaqMan RT-PCR

Total RNA from the cells was isolated by using HighPure RNA kit (Roche, Mannheim, Germany), which included DNase I digestion. A total of 1 µg of total RNA preparation was reverse transcribed with cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) using oligo(dT)23. cDNA was diluted 1/4, and 2.5 µl was used as template in 25 µl TaqMan-PCR mix, according to the manufacturer’s protocol (PE Biosystems, Weiterstadt, Germany). MgCl2 was tested by titration and used with 5.5 mM. {beta}-actin primer (sense, CCC TGT GCT GCT CAC CGA; antisense, ACA GTG TGG GTG ACC CCG TC), TNF-{alpha} primer (sense, AAA ATT CGA GTG ACA AGC CTG TAG; antisense, CCC TTG AAG AGA ACC TGG GAG TAG), SOCS1 primer (sense, CAC CTT CTT GGT GCG CG; antisense, AAG CCA TCT TCA CGC TGA GC), and SOCS3 primer (sense, GCT CCA AAA GCG AGT ACC AGC; antisense, AGT AGA ATC CGC TCT CCT GCA G) were purchased from MWG (Munich, Germany). Fluorogenic probes (6-carboxyfluorescein) were: {beta}-actin, CCC CTG AAC CCT AAG GCC AAC CG; TNF-{alpha}, CAC GTC GTA GCA AAC CAC CAA GTG GA; SOCS1, TCG CCA ACG GAA CTG CTT CTT CG; SOCS3, TTG CGC ACG GCG TTC ACC AC. Specificity of RT-PCR was controlled by no template and no reverse-transcriptase controls.

Semiquantitative PCR results were obtained using the {Delta}{Delta}CT method (32). Because PCR efficiencies for all four reactions were similar (~0.95–0.98), threshold values were normalized to {beta}-actin and set in reference to unstimulated control cells. The calculated values represent thus the 2n-fold induction of mRNA.

Western blot

After stimulation, cells were lysed for 30 min on ice in 250 µl of lysis buffer (50 mM Tris-HCl, pH 7.4; 1% Igepal; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSA; 1 µg/ml each aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF). Lysates were cleared by centrifugation at 4°C for 10 min at 11,000 x g. Equal amounts of lysates were fractionated by 12% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked with TBS (pH 7.8)/5% nonfat dry milk/0.05% Tween 20, and were blotted with the indicated Abs. Detection was by ECL system (Amersham, Freiburg, Germany). Phosphospecific Abs to STATs and MAPK were used according to the manufacturer’s protocol. Where indicated, membranes were stripped with 0.2 M NaOH for 5 min and reprobed with Abs.

Bone marrow-derived DC (BMDDC) and peritoneal washout cells (PWC)

BMDDC from BALB/c mice were prepared as described (12). Briefly, femurs of mice were rinsed with cell culture medium using a syringe with a 27-gauge needle. Bone marrow cells (4 x 106) were seeded into a 80-cm2 tissue culture flask in culture medium with 200 U/ml GM-CSF. Nonadherent cells were used at day 9, when mature DC (CD11c+, GR-1-) represented >85% of the resulting cell population. PWC were prepared by peritoneal lavage of untreated BALB/c mice. Cells were plated in culture medium and washed 2 and 4 h after plating to remove nonadherent cells.

RNA preparation of spleen extracts

BALB/c mice were injected i.p. with 10 nmol ODN 1668 in 500 µl PBS. Mice were killed by cervical dislocation at the indicated time points. Spleens were prepared and flash-frozen in liquid nitrogen. Then spleens were homogenized on ice, and RNA was prepared using TRIzol (Life Technologies, Karlsruhe, Germany), according to the manufacturer’s protocol. To remove traces of DNA, an incubation with DNase I (Roche) was performed, and finally RNA was cleaned up in a second purification with RNeasy kit (Qiagen, Hilden, Germany). RNA was stored at -70°C before cDNA synthesis.

Flow cytometry analysis

Cells were washed in PBS/2% FCS. Fc-block was performed by incubating with anti-Fc{gamma}RII/III mAbs (clone 2.4G2; PharMingen, San Diego, CA) and 10% normal mouse serum for 15 min on ice. Then cells were stained with FITC-conjugated anti I-Ad/Ed mAbs (clone 2G9; PharMingen), FITC GR-1 (clone RB6-8C5; PharMingen), or PE-conjugated anti-CD11c Abs (clone HL3; PharMingen) for 45 min on ice. After staining, cells were washed and fixed in PBS/1% paraformaldehyde. Cells were analyzed on a Partec PAS flow cytometer (Dako, Hamburg, Germany).

Generation of stable RAW 264.7 transfectants

Mammalian expression vectors for SOCS-1 and SOCS-3 were described previously (19) and were obtained from D. Hilton (Victoria, Australia). pEF-Sem, which contains a neomycin resistance cassette, was a kind gift of H. Haecker (Munich, Germany). Stable transfections were established by cotransfection of either SOCS-1, SOCS-3, or empty vector expression plasmids, and pEF-Sem in a ratio of 10 to 1.5 x 106 RAW 264.7 cells were transfected by electroporation in 500 µl final volume (RPMI, 25% FCS) at 290 V, 1050 µF in an EasyjecT plus gene pulser (PeqLab, Erlangen, Germany). Cells were plated, and 24 h after transfection they were overlaid with soft agar containing 0.8 µg/ml G418 (Life Technologies). G418-resistant clones were picked, expanded, and tested for expression of SOCS mRNA by semiquantitative RT-PCR as well as SOCS protein expression by immunoprecipitation and Western blot with anti-Flag Abs (Sigma).

Clones were selected for equal SOCS-1 and SOCS-3 mRNA overexpression. They also showed equal protein levels, as determined by anti-Flag Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To analyze whether CpG-ODN might induce negative regulators of signaling, we set up semiquantitative mRNA determinations based on the TaqMan technology (33). We first determined the CpG-ODN-triggered induction of mRNA for TNF-{alpha}, known to be induced by CpG-DNA (11), and SOCS-1 and SOCS-3 in RAW 264.7 macrophage cell line (Fig. 1GoA). CpG-DNA induced TNF-{alpha} mRNA as well as SOCS-1 and SOCS-3 mRNA with similar dose-response curves. Time kinetics revealed that mRNA for TNF-{alpha} peaked at 1/2–1 h, then decreased slightly, but remained elevated for 24 h (Fig. 1GoB). SOCS-3 mRNA expression also peaked at 1–2 h, yet showed a second peak after 8 h. Again, sustained expression was observed even after 24 h. Significant mRNA induction of SOCS-1 was delayed in comparison with SOCS-3 and TNF. Expression started after 2 h and peaked at 4 h (Fig. 1GoB). After 48 h, mRNA expression for SOCS and TNF declined to background values (data not shown). Similar results were obtained with J774 macrophage cell line for TNF and SOCS-3 mRNA expression, while SOCS-1 mRNA was expressed at much lower levels (data not shown). mRNA expression of SOCS-3 resulted in subsequent translation of SOCS-3 proteins, as shown by Western blotting with extracts from J774 as well as RAW cells (Fig. 1GoC).



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  		FIGURE 1. CpG-ODN induce SOCS-1 and SOCS-3 expression in RAW 264.7 and J774 macrophages. A, RAW 264.7 macrophages were incubated with the indicated concentrations of ODN 1668 for 4 h. Subsequently, mRNA induction for TNF-{alpha}, SOCS-1, and SOCS-3 was quantified by TaqMan RT-PCR. Values give the fold mRNA induction compared with nonstimulated controls. B, RAW 264.7 cells were incubated with 1 µM ODN 1668 for the indicated time period, and then mRNA induction was determined. C, J774 and RAW 264.7 macrophages were stimulated with ODN 1668 (1 µM) for different time periods. Then SOCS protein expression was measured in the cell lysate by Western blot.

 
Next we tested whether the requirements for induction of SOCS mRNA fulfill the characteristics of immunostimulation by CpG-DNA (2, 3). Whereas canonical CpG-ODN (1668 or SB-DG) induced TNF-{alpha} as well as SOCS-1 and SOCS-3, GpC ODN failed to do so (Fig. 2GoA). ODN containing polyguanosine sequences but no CpG motifs (PZ-3) also were negative. In contrast, LPS induced TNF and SOCS expression almost as efficient as CpG-ODN. Thus, we concluded that TLR-dependent or related triggering of innate immune cells induces expression of negative regulators of cytokine signaling (SOCS). To exclude that SOCS mRNA is induced indirectly by CpG-ODN, we stimulated J774 cells in the presence of cycloheximide (Fig. 2GoB). Although production of TNF protein was completely abolished, mRNA expression of TNF-{alpha}, SOCS-1, and SOCS-3 was not significantly affected (Fig. 2GoB). Because it has been published that IFN-{alpha} and IFN-{beta} contributes to SOCS-1 induction mediated by LPS in murine bone marrow culture-derived macrophages in an autocrine fashion, we addressed this question by blocking experiments. IFN-{alpha} and IFN-{beta} were able to induce SOCS-1 mRNA, while SOCS-3 induction was only weak (Fig. 2GoC). Addition of a neutralizing Ab completely inhibited IFN-{alpha}- and IFN-{beta}-mediated SOCS induction. In contrast, the Ab did not inhibit either SOCS-1 or SOCS-3 induction by CpG-ODN, thus ruling out an autocrine mechanism via type I IFNs. Because the promotor region of SOCS-3 contains putative binding sites for NF-{kappa}B and AP-1, and because CpG-ODN trigger both transcription factors (11), yet do not induce tyrosine phosphorylation of STAT factors, we presume that CpG-ODN directly are capable of inducing expression of SOCS mRNA.



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  		FIGURE 2. CpG-ODN induce SOCS-1 and SOCS-3 mRNA directly and dependent on CpG motifs. A, RAW 264.7 cells were incubated with CpG-ODN (1668, SB-DG) or non-CpG ODN (1668GC, PZ-3) at 1 µM or LPS (1 µg/ml) for 5 h. Thereafter, induction of mRNA of TNF-{alpha}, SOCS-1, and SOCS-3 was determined. B, J774 macrophages were preincubated with either medium alone or medium containing cycloheximide at 50 µg/ml for 30 min. Cells were then stimulated with ODN 1668 (1 µM). After 4 h, induction of mRNA was measured. TNF-{alpha} production was determined by ELISA. C, RAW 264.7 cells were incubated with rIFN-{alpha} (300 U/ml), IFN-{beta} (100 U/ml), or ODN 1668 (1 µM) for 5 h. Where indicated, a neutralizing polyclonal Ab against IFN-{alpha} and IFN-{beta} (1000 neutralization U/ml) was added. mRNA induction of SOCS-1 and SOCS-3 was determined.

 
To further test the signal requirements for induction of SOCS by CpG-ODN, we analyzed the effects of different inhibitors of CpG signaling on expression of TNF and SOCS mRNA. Chloroquine inhibits CpG-DNA signaling, probably through inhibition of endosomal maturation (34), and therefore upstream in the signaling cascade. Addition of chloroquine inhibited in a dose-dependent manner the induction of mRNA of TNF-{alpha}, SOCS-1, SOCS-3 (Fig. 3GoA), as well as TNF protein secretion (data not shown). To analyze downstream signaling events, MAPK were blocked by specific inhibitors. SB203580, an inhibitor of the p38 MAPK, failed to impede expression of TNF and SOCS-1 mRNA, yet inhibited slightly SOCS-3 mRNA expression (Fig. 3GoB). Corroborating previous results (11), we found that SB203580 blocked almost completely TNF production, although mRNA expression for TNF was not inhibited. In contrast, UO126, which inhibits the extracellular signal-related kinase-1/2 MAPK, slightly suppressed expression of mRNA encoding for TNF and SOCS-1 (Fig. 3Go). SOCS-3 mRNA expression was more susceptible to blockade by UO126 (Fig. 3Go). From these experiments, we concluded that reagents known to block the CpG ODN-triggered signaling pathways also interfered with induction of SOCS-1 and SOCS-3 mRNA. Because we ruled out indirect mechanisms for induction of SOCS (Fig. 2GoB) and because inhibitors of CpG-ODN signaling block expression of SOCS mRNA, we conclude that SOCS-1 and SOCS-3 are direct target genes of CpG-ODN signaling.



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  		FIGURE 3. CpG-ODN-induced SOCS-1 and SOCS-3 mRNA expression can be blocked by inhibition of endosomal maturation or by inhibition of MAPK. J774 macrophages were preincubated with the inhibitors indicated for 45 min and then stimulated with ODN 1668 (0.1 µM) for 4.5 h. Thereafter, mRNA induction was measured by TaqMan RT-PCR, and TNF production was determined by ELISA. A, Inhibition by chloroquine. B, Inhibition of p38-MAPK pathway by SB203580 (8, 4, 2 µM) and of extracellular signal-related kinase-1/2 pathway by UO126 (5, 1, 0.2 µM).

 
Next we examined whether the results on macrophage cell lines also hold true for primary cells. We analyzed SOCS induction in BMDDC and peritoneal macrophages (PWC). In both cell types, stimulation with CpG-DNA resulted in induction of SOCS-1 and SOCS-3 mRNA (Fig. 4GoA). Although PWC showed high TNF-{alpha} induction, SOCS-1 and SOCS-3 induction was only moderate. In contrast, CpG-DNA induced higher amounts SOCS-1 and SOCS-3 mRNA in BMDDC, while TNF-{alpha} mRNA induction was lower. LPS induced similar mRNA expression in BMDDC (Fig. 4GoB), while PWC showed a higher SOCS induction as compared with CpG stimulation (Fig. 4GoB). Comparison of mRNA induction in J774 and RAW 264.7 cell lines further revealed cell type-specific differences (Fig. 4GoB). Again SOCS mRNA data were confirmed by Western blotting for SOCS protein expression (Fig. 4GoC). Taken together, primary cells as well as cell lines showed marked induction of SOCS-1 and SOCS-3 upon stimulation with LPS as well as with CpG-DNA.



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  		FIGURE 4. SOCS induction by CpG-DNA and LPS in primary APCs and macrophage cell lines. PWC and BMDDC were prepared as described in Materials and Methods. Cells were stimulated with A, 3 µM ODN 1668, or B, 1 µg/ml LPS for 4 h. TNF-{alpha} and SOCS mRNA induction was determined by TaqMan RT-PCR. C, Cells were incubated with either 3 µM ODN 1668 or 1 µg/ml LPS for 5 h, and subsequently SOCS-3 protein induction was determined by Western blot.

 
To determine SOCS mRNA induction in vivo, mice were injected i.p. with CpG-DNA, and mRNA was determined in spleen homogenates at different time points (Fig. 5Go). TNF-{alpha} mRNA peaked at 1 h after injection; SOCS-3 also was induced as early as after 1 h, peaking at 2 h. SOCS-1 induction was slightly delayed, nevertheless comparable with SOCS-3 mRNA induction, and peaked at 2 h. Thus, early kinetics of mRNA expression obtained showed similarities to in vitro results of RAW 264.7 cells (Fig. 1GoB). In contrast, mRNA expression for TNF-{alpha} and SOCS declined steadily in vivo toward basal rates at 23 h.



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  		FIGURE 5. SOCS mRNA induction in vivo. BALB/c mice were injected with 10 nmol ODN 1668 i.p. and stimulated for the indicated time period. Then spleen homogenates were prepared, and total RNA was extracted. TNF-{alpha} and SOCS mRNA induction was measured by TaqMan RT-PCR. Shown are the mean values of three experiments.

 
To assess whether SOCS proteins induced by CpG-ODN are functional, we analyzed whether ODN-induced SOCS could prevent tyrosine phosphorylation of STAT protein in response to different cytokines. Macrophage cell lines were incubated with CpG-ODN for various time intervals and then triggered with either IFN-{gamma}, IL-6, or GM-CSF. In control cells, tyrosine phosphorylation of STAT proteins could be readily detected upon stimulation (Fig. 6Go). Whereas preincubation with CpG-ODN for 1 h had no effect, prestimulation for 8 or 24 h blocked almost completely phosphorylation of the respective STAT proteins (Fig. 6Go). Inhibition of IFN-{gamma} as well as IL-6 and GM-CSF signaling correlated with the expression of SOCS proteins (compare Figs. 1Go and 6Go). These results were corroborated with RAW 264.7 cells overexpressing SOCS-1 and SOCS-3. SOCS-1, but not SOCS-3, overexpression prevented STAT-1 phosphorylation after stimulation with IFN-{gamma} (data not shown). Moreover, IL-6-induced STAT-3 phosphorylation was diminished in SOCS-1- as well as SOCS-3-overexpressing cells (data not shown). From these data, we conclude that CpG-ODN-induced SOCS proteins are functional in vitro and are capable of preventing signaling via the JAK/STAT signal pathway.



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  		FIGURE 6. Preincubation with CpG-ODN inhibits tyrosine phosphorylation of STAT factors induced by IFN-{gamma}, IL-6, and GM-CSF. RAW 264.7 macrophages were incubated with ODN 1668 (1 µM) for the time periods indicated. Thereafter, cells were stimulated with A, IFN-{gamma} (10 U/ml); B, IL-6 (50 ng/ml); and C, GM-CSF (50 ng/ml) for 15 min. Cell lysates were blotted and probed with phosphotyrosine-specific anti-STAT Abs, as indicated (upper line). Blots were then stripped and reprobed with anti-STAT Abs (lower line).

 
Finally, we analyzed whether preincubation with CpG-DNA would also result in loss of biological responses triggered by STAT-dependent signaling pathways. Therefore, we examined the effects of preincubation with CpG-DNA on IFN-{gamma}-induced up-regulation of MHC class II molecules by flow cytometry (Fig. 7Go). Treatment with CpG-DNA alone resulted in a moderate up-regulation of MHC II expression, while stimulation with IFN-{gamma} was much more effective (Fig. 7Go). However, in cells that were preincubated with CpG-DNA for 12 h, IFN-{gamma} failed to induce MHC class II up-regulation (Fig. 7Go). Thus, pretreatment with CpG-DNA diminishes subsequent biological effects triggered by IFN-{gamma} in macrophages. This effect correlated with induction of SOCS proteins by CpG-DNA. To further strengthen this correlation, we examined RAW 264.7 cells overexpressing either SOCS-1 or SOCS-3. In SOCS-1-expressing cells, IFN-{gamma} failed to induce up-regulation of MHC class II molecules, while expression of SOCS-3 had only minor inhibitory effects. (Fig. 8Go). Thus, SOCS-1 rather than SOCS-3 seems to play the predominant role for suppressing IFN-{gamma} signaling, which corroborates previous reports (35, 36).



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  		FIGURE 7. Effect of CpG-DNA on IFN-{gamma}-induced MHC-II expression. RAW 264.7 cells were stimulated with 0.5 µM ODN 1668 for 12 h and left without further treatment for 48 h (left panel). Further cells were treated with 5 U/ml IFN-{gamma} for 48 h (middle panel) and with IFN-{gamma} after 12-h preincubation with ODN 1668 (right panel). MHC-II protein expression was determined by flow cytometry. MHC-II expression on unstimulated cells is shown as control (light gray line).

 


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  		FIGURE 8. Effect of SOCS overexpression on IFN-{gamma} induced MHC-II expression. RAW264.7 cells stable transfected with either SOCS-1, SOCS-3, or empty vector expression plasmids were treated with IFN-{gamma} in the indicated concentration for 48 h. Subsequently MHC-II protein expression was measured by flow cytometry, and MHC-II+ cells were determined. SOCS protein expression levels were determined by immunoprecipitation and Western blot with anti-Flag Ab of equal numbers of transfected cells. Arrows indicate the migration of Flag-tagged SOCS proteins. Ig light and heavy chains are marked (lc, hc).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To date, induction of SOCS expression has been almost exclusively attributed to JAK/STAT-dependent signaling pathways (29). However, it has been reported that IL-10 induces SOCS-3 expression independent of STAT proteins (27). We show in this study that CpG-ODN can directly induce SOCS expression that is independent of protein synthesis. Because CpG-ODN signal transduction is not reported to involve JAK/STAT activation (11, 13) (own unpublished observations) and because inhibition of MAPK results in reduction of CpG-ODN-induced SOCS expression (Fig. 3Go), our data strongly suggest that pathways distinct from JAK/STAT also trigger SOCS induction. Consistently, chloroquine, which inhibits upstream events in CpG-ODN signaling, also completely blocks SOCS induction by CpG-DNA. These observations fit well with recent reports showing that endotoxin (LPS) or TNF-{alpha} induces SOCS expression in murine macrophages (37, 38). Furthermore, it has been shown that PMA triggers SOCS expression via MAPK activation (39). Thus, CpG-DNA induce SOCS proteins in a pathway independent of JAK/STAT. Because LPS (37) as well as CpG-DNA induced SOCS expression and because both stimuli trigger TLR-dependent signaling cascades (14, 15, 40), we concluded that expression of negative regulators of cytokine signaling can be induced by TLR-dependent signals in innate immune cells.

Recent reports on SOCS induction by TNF or LPS showed an almost exclusive induction of SOCS-3. We report in this study induction of SOCS-3 as well as SOCS-1 mRNA triggered by CpG-DNA or LPS. In addition, we provide evidence for cell type-specific SOCS induction (Fig. 4Go), which in turn may explain the reported results. It has been suggested that IFN-{alpha} and IFN-{beta} may contribute to SOCS-1 induction mediated by LPS in murine bone marrow culture-derived macrophages in an autocrine fashion (41). Although CpG-DNA induces efficiently production of type I IFNs (42, 43), our results do not support an autocrine mechanism because induction of SOCS was independent of protein synthesis and could not be inhibited by neutralizing type I IFN Abs (Fig. 2Go). Moreover, time kinetics of SOCS induction was similar to direct target genes of TLR pathways (e.g., TNF-{alpha}). However, an autocrine pathway of SOCS induction cannot be ruled out completely because CpG-triggered secretion of preformed cytokines might induce SOCS transcription. Clarifying experiments using distinct promotor/reporter construct will be needed.

SOCS-1 is important for inhibiting IFN-{gamma} signaling (44), while the role of SOCS-3 is not entirely clear. We show in this study that STAT phosphorylations induced by multiple cytokines (IFN-{gamma}, IL-6, GM-CSF) are reduced after preincubation with CpG-DNA. We further strengthen the importance of SOCS-1 for IFN-{gamma} signaling by showing impaired responses to IFN-{gamma} in SOCS-1-overexpressing cells (Fig. 7Go). It will be intriguing to analyze whether other JAK/STAT-dependent signal pathways will also be influenced by CpG-ODN. Furthermore, it will be interesting to examine whether SOCS also interacts with signal molecules distinct from the JAK/STAT pathway. In this context, SOCS-1 has been reported to inhibit Tec protein kinase (45), and SOCS-2 interferes with insulin-like growth factor-1 signaling (46). In addition, SOCS-1 interacts with other tyrosine kinases in yeast (47), indicating that the spectrum of signal cascades regulated by SOCS could be much broader.

Subsequent inhibition of IFN-{gamma}-induced STAT-1 tyrosine phosphorylation as well as MHC-II up-regulation after pretreatment with CpG-ODN indicates that perception of infectious danger via PAMP not only leads to initiation and activation of effector functions of the innate immune system, but also simultaneously engenders intrinsic feedback mechanisms. Although concomitant IFN-{gamma}- and TLR-dependent signals act synergistically on macrophages, we now provide evidence that stepwise activation may result in down-regulative mechanisms. In this context, it recently has been reported that infection of macrophages with Listeria monocytogenes resulted in an early enhancement of IFN-{gamma} signaling, while at later time points an inhibition was observed. This was discussed as a consequence of SOCS induction (48). Thus, timing of different signals is of decisive importance for distinct outcomes concerning macrophage activation and function. Down-regulation of IFN-{gamma} signaling after CpG-ODN pretreatment could play a physiological role as protective mechanism to excessive stimulation of innate immune cells. Furthermore, it indicates an important intracellular cross-talk between distinct signaling pathways that regulate the cells’ reception of extracellular signals as well as their immune function. Collectively, our data show that perception of infectious danger by innate immune cells not only activates effector mechanisms, but also leads to the induction of the negative regulatory elements SOCS, which link TLR and TLR-related signals to the JAK/STAT signaling pathway.


   Acknowledgments
 
We appreciate the excellent technical help of Claudia Trier. We thank Dr. T. Decker, Dr. D. Hilton, and Dr. H. Haecker for helpful discussions and generous support.


   Footnotes
 
1 This work was supported by grants from the Sonderforschungsbereich (SFB) 297 (C6) and the Deutsche Forschungsgemeinschaft (DFG) (He 1452/2). Back

2 Address correspondence and reprint requests to Dr. Klaus Heeg, Institute of Medical Microbiology and Hygiene, Philipps-University Marburg, Pilgrimstein 2, 35037 Marburg, Germany. E-mail address: heeg{at}post.med.uni-marburg.de Back

3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; DC, dendritic cell(s); BMDDC, bone marrow-derived DC; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide(s); PWC, peritoneal washout cell(s); SOCS, suppressors of cytokine signaling; TLR, Toll-like receptor(s). Back

Received for publication November 2, 2000. Accepted for publication April 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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