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Originally published In Press as doi:10.1074/jbc.M410992200 on October 18, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54708-54715, December 24, 2004
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Suppressor of Cytokine Signaling (SOCS) Proteins Indirectly Regulate Toll-like Receptor Signaling in Innate Immune Cells*{diamondsuit}

Andrea Baetz{ddagger}§, Markus Frey¶§, Klaus Heeg¶, and Alexander H. Dalpke{ddagger}||

From the {ddagger}Institute of Medical Microbiology and Hygiene, Philipps-University Marburg, 35037 Marburg, Germany and the Department of Hygiene and Medical Microbiology, University of Heidelberg, 69120 Heidelberg, Germany

Received for publication, September 24, 2004 , and in revised form, October 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-{alpha}. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-{beta} (IFN-{beta}), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF{kappa}B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF{kappa}B pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Suppressor of cytokine signaling (SOCS)1 proteins have been identified as negative feedback inhibitors for various cytokines signaling via the JAK/STAT pathway. Eight members, SOCS-1 to SOCS-7 and CIS, have been identified in this protein family (1). In general, SOCS are induced upon cytokine receptor activation through STAT-dependent elements in the respective promoters (2). In turn, SOCS proteins are able to limit further receptor signaling. To that end, members of the SOCS family use different mechanisms. SOCS-1 binds to JAKs, and by means of its SH2 domain and a proximal kinase inhibitory region, it is able to directly inhibit kinase activity. SOCS-3 is recruited by phosphotyrosine residues of the intracellular domain of the cytokine receptor and also inhibits JAK activity, while CIS is thought to compete for STAT recruitment. Besides the importance of the central SH2 domain for the activity of SOCS, these proteins contain yet another domain at the C terminus, which has been termed SOCS box (3). The SOCS box mediates interactions with elongins B and C, which are part of the E3 ubiquitin ligase complex (4). It has been suggested that SOCS can promote ubiquitinylation and proteosomal degradation of JAK2, thereby enhancing its inhibitory effects (5, 6).

Although, in vitro, SOCS proteins can be induced by a wide variety of different cytokines, in vivo studies with knock-out models have suggested that members of the SOCS family have specific actions. For example, SOCS-1-deficient mice have severely impaired IFN-{gamma} signaling and succumb to perinatal death with a multi-inflammatory syndrome (7, 8). Backcrossing to Rag or IFN-{gamma} knock-out mice rescues mice from early death (9). Thus, SOCS-1 has been deduced to play primarily a role in IFN-{gamma}-dominated adaptive immune responses.

It has been shown that SOCS-1, SOCS-3, and CIS can also be induced in innate immune cells, including macrophages and dendritic cells. Triggering of TLRs and stimulation with TNF-{alpha} induce SOCS proteins in these cells (10-14). In the case of SOCS-1 the mode of induction has been a matter of controversy as to the role of paracrine type I IFN signaling (11, 13). SOCS-1 regulates the sensitivity of macrophages to IFN-{gamma} (11, 15), whereas SOCS-3 has been implicated to dampen IL-6 sensitivity (16-18). Besides this mode of action, which has to be referred to as crosstalk inhibition, further reports suggested that SOCS proteins may have a broader inhibitory role which involves JAK/STAT-independent signaling pathways, namely, TNF-{alpha}, insulin, and vav (19-21). This led to the suggestion that SOCS proteins could also act as direct inhibitors of TLR signaling, thus, revealing a further role in innate immunity.

Toll-like receptors mediate recognition of conserved microbial structures, so called pathogen-associated molecular patterns. TLRs have been identified to be crucial receptors for the recognition of microbial encounter in innate immunity and mutations in TLRs result in impaired defense against infections (22). Upon TLR stimulation, at least some of the receptors dimerize. In turn, adaptor molecules are recruited to the receptor complex via the TIR domain that is shared with the interleukin-1 receptor. Subsequently, further signaling molecules among which are IRAKs and TRAF-6 are recruited and activate MAP kinase signaling, as well as NF{kappa}B. MyD88 was the first adaptor molecule to be identified, and MyD88-deficient mice show a loss of proinflammatory cytokine induction upon TLR stimulation (23). However, some signaling events are independent of MyD88, and further adaptors have been identified. Thus, a complicated picture has emerged in which two main signaling streams can be observed (24). First, there are direct target genes, which are induced via MAP kinases and NF{kappa}B, including the prototypical proinflammtory cytokine TNF-{alpha}. Some of these genes, however, are induced upon intermediate activation of I{kappa}B{zeta}, as shown recently (25). This pathway is dependent on MyD88; however, in the case of TLR-2 and TLR-4, an additional adaptor Mal/TIRAP can be used (26, 27). Second, at least some TLRs activate IRF-3, resulting in IFN-{beta} induction (24). In turn, IFN-{beta} becomes secreted and can act in an autocrine and paracrine manner to activate JAK/STAT signaling. This broadens the set of induced genes. IP-10 and iNOS are thought to be activated in this way, and also the upregulation of CD40 has been shown to be IFN-{beta}-dependent (28, 29). In the case of TLR-3 and TLR-4, two further adaptors, TRIF/TICAM-1 and TRAM/TICAM-2, are used (30-33). This pathway has been neglected for TLR-2 stimulation (34).

There is indirect evidence linking SOCS to TLR signaling, since it has been reported that SOCS-1-deficient mice exhibit increased LPS sensitivity, associated with elevated TNF-{alpha} secretion, and lack endotoxin tolerance (35, 36). These effects could be observed in macrophages and were also retained in SOCS-1/IFN-{gamma} double knock-out mice, thus, ruling out the possibility of a pre-activation of macrophages by increased IFN-{gamma} sensitivity. Direct effects of SOCS-1 on NF{kappa}B signaling have been proposed on the basis of transfection experiments. However, the precise mechanism and role of SOCS-1 for TLR signaling are still elusive.

Here we analyzed the MyD88 and paracrine type I IFN dependence of SOCS-1, SOCS-3, and CIS induction following TLR stimulation. Moreover, we made use of an overexpression system of SOCS in macrophages with careful control of expression levels. We were not able to observe inhibitory effects of SOCS on TLR-induced TNF-{alpha} and nitrite secretion. However, we found that all TLR ligands induced IFN-{beta} and subsequent IFN-{beta}-triggered STAT-1-dependent signaling. This secondary signaling pathway, as exemplified by IP-10 induction, was sensitive to inhibition by SOCS-1 and to a lesser extent by SOCS-3 and CIS. These data clearly show that the effects of SOCS-1 on TLR signal transduction are limited to the inhibition of secondary type I IFN-dependent amplification circuits.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Completely phosphorothioate-modified CpG-oligodeoxynucleotide (ODN) 1668 (TCC ATG ACG TTC CTG ATG CT) was purchased from TIB Molbiol (Berlin). Lipoteichoic acid from Staphylococcus aureus was a kind gift from S. Morath (Konstanz, Germany) and was shown to be TLR-2-dependent (37). Highly purified lipopolysaccharide from Salmonella minnesota was kindly provided by U. Seydel (Borstel, Germany). Poly(dI-dC) was purchased from Sigma (Schnelldorf, Germany). Pam3CSK4 was obtained from EMC Microcollections (Tübingen, Germany). Antibodies specific for phosphorylated MAP kinases, phosphorylated STAT-1, I{kappa}B{alpha}, and c-Myc were from Cell Signaling Technology (Frankfurt, Germany). Anti-FLAG M2 antibody was obtained from Sigma. Recombinant IFN-{gamma} and GM-CSF were purchased from Tebu (Frankfurt, Germany).

Mice—MyD88-deficient mice were obtained from A. Gessner, Erlangen, Germany. IFNAR-I-deficient mice were delivered from B&K Universal. Further mice were purchased from Harlan-Winkelmann, Borchen, Germany.

Cells and Culture Conditions—Cells were cultured in Clicks/RPMI 1640 supplemented with 5-10% FCS, 50 µM {beta}-mercaptoethanol, and antibiotics (penicillin G and streptomycin). Bone marrow-derived dendritic cells were isolated from Balb/c mice as described previously (38), albeit with slight modifications. Briefly, bone marrow cells (4 x 106) were seeded into an 80-cm2 tissue culture flask, containing culture medium and 20 ng/ml GM-CSF. Non-adherent cells were used at day 9 when immature dendritic cells (CD11c+, GR-1-) represented >85% of the resulting cell population. Peritoneal macrophages were obtained by thioglycollate injection and adherence purification. RAW 264.7 cells were a kind gift from R. Schumann (Berlin, Germany).

Generation of Stable RAW 264.7 Transfectants—Mammalian expression vectors for SOCS-1, -2, and -3 were obtained from D. Hilton (Victoria, Australia). pEF-Sem, which contains a neomycin resistance cassette, was a kind gift from H. Haecker (Munich, Germany). Stable transfections were established by cotransfection of either SOCS or empty vector expression plasmids and pEF-Sem in a ratio of 10 to 1. RAW 264.7 cells (5 x 106) were transfected by electroporation in 500 µl final volume at 290 V, 1050 µF in an EasyjecT plus gene pulser (PeqLab, Erlangen, Germany). Cells were plated, and multiple G418-resistant clones were picked, expanded, and tested both for expression of SOCS mRNA by quantitative RT-PCR, as well as for expression of SOCS protein by Western blot with anti-FLAG or anti-c-Myc antibodies.

Determination of Cytokine and Nitric Oxide Secretion—1.5 x 105 cells were stimulated in 96-well plates in 300 µl of medium as indicated in duplicates. Supernatants were harvested and analyzed for cytokines by commercially available ELISA kits (TNF-{alpha} and IL12p40 (BD Biosciences, Heidelberg, Germany) and IP-10 (R&D Systems, Wiesbaden, Germany). Nitric oxide accumulation was measured photometrically (550 nm) by mixing equal parts of supernatant and Griess reagent (1:1 mixture of 1% sulfanilamide, 5% H3PO4, and 0.1% naphthylethylenediamine dihydrochloride).

Quantitative RT-PCR—Total RNA from 1 x 106 cells was isolated using a HighPureTM RNA kit (Roche Applied Science, Mannheim, Germany) which included DNase I digestion. Total RNA (1 µg) was reverse-transcribed with a cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany). cDNA, diluted 1:4, was used as template in the quantitative PCR mix according to the manufacturer's standard protocol (Eurogentec, Seraign, Belgium) (ABI Prism 7700, Applied Biosystems). The primer sequences have been previously published (11) and are available on request. Quantifications were made using either fluorogenic probes (6-carboxyfluorescein/6-carboxytetramethylrhodamine) or by means of SYBR Green. Specificity of RT-PCR was controlled by no template and no RT controls. PCR efficiencies for all reactions were determined and were similar (0.94-1.0). Threshold values were normalized to expression of {beta}-actin. Quantitative PCR results are expressed relative to the induction of the housekeeping gene {beta}-actin (1/2{Delta}Ct).

Western Blot—2-5 x 106 cells were stimulated and subsequently 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 phenylmethylsulfonyl fluoride, 1 µg/ml each aprotinin, leupeptin, pepstatin, 1 mM Na3VO4, 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. Membranes were stained with the indicated antibodies, and proteins were detected using an enhanced chemiluminescence system (Amersham Biosciences, Freiburg, Germany). Where indicated, membranes were stripped and re-probed.

Reporter Gene Experiments—For reporter gene experiments, the NF{kappa}B-dependent ELAM promoter (obtained from H. Heine, Borstel, Germany), as well as the STAT-1-dependent IFP-53 promoter (from T. Decker, Vienna, Austria), were used coupled to a luciferase reporter gene. Transient transfections in RAW 264.7 cells were performed with 0.8 µg of reporter gene and 2.0 µg of SOCS expression plasmid using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany). Luciferase induction was measured with a LucLit kit (Packard) in a TopCountNXT.

NF{kappa}B Activation—Nuclear translocation of activated NF{kappa}B was measured using the TransAMTM kit (Activemotif, Rixensart, Belgium) according to the manufacturer's protocol after stimulation of cells for 45 min with 100 ng/ml LPS.

Flow Cytometry—Cells were washed in PBS, 2% FCS. Fc block was done with anti-Fc{gamma}RII/III monoclonal antibodies (clone 2.4G2, Pharmingen) and 10% normal mouse serum. Cells were stained with fluorescein isothiocyanate-conjugated anti-CD40 (clone HM40-3, Pharmingen) or phycoerythrin (PE)-CD86 (clone GL1, Pharmingen). For the analysis of phosphorylated STAT-1, cells were fixed with 2% paraformaldehyde/PBS for 30 min at room temperature. Subsequently, cells were further fixed and permeabilized in 90% methanol at -20 °C overnight. Cells were incubated with 1 µl of phosphotyrosine-specific STAT-1 antibody (clone 4a, Pharmingen) in 100 µl of volume together with Fc block for 2 h at room temperature. Finally cells were stained with PE-labeled secondary antibody (Dako, Hamburg, Germany). Cells were analyzed on a Partec PAS flow cytometer (Dako).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TLR-mediated SOCS Induction Differs with Regard to MyD88 Dependence—TLR activation induces various SOCS proteins; however, the precise mechanisms are still a matter of controversy. We first used MyD88-deficient mice to further gain insight into TLR-mediated SOCS induction (Fig. 1a). MyD88 has been shown to be used as an adaptor by most of the TLRs. Stimulation of peritoneal macrophages with prototypic ligands for different TLRs revealed that induction of SOCS-3 and CIS was completely dependent upon MyD88 in response to all TLR ligands tested. Moreover, peritoneal macrophages showed similar induction of SOCS-3 in response to the different ligands, whereas CIS induction was mainly achieved by LPS and LTA. Interestingly, SOCS-1 induction completely depended on MyD88 in the case of CpG-DNA and LTA stimulation, but induction was mostly preserved in MyD88-deficient mice stimulated with LPS or poly(dI-dC). Thus, a given TLR-dependent gene can be induced by disparate pathways which use distinct adaptor proteins.



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  		FIG. 1.
TLR ligands induce SOCS in a direct manner with differing needs of MyD88. a, peritoneal macrophages from wild type or MyD88-/- mice were stimulated with 10 µg/ml LTA, 5 µg/ml poly(dI-dC), 100 ng/ml LPS, or 300 nM CpG-DNA for 5 h. Expression levels for SOCS-1, SOCS-3, and CIS mRNA were determined by quantitative RT-PCR (n = 2, mean ± S.D.). b, peritoneal macrophages from C3H/HeJ and C3H/HeN mice were stimulated in a transwell system with either LPS or CpG as above. Expression levels of SOCS-1, TNF-{alpha}, and IP-10 were determined by quantitative RT-PCR (n = 3, mean ± S.D.). c, dendritic cells from IFNAR-1-/- or wild type mice were stimulated with either LTA, LPS, CpG-DNA, or 30 units/ml IFN-{gamma} and examined as above (n = 2, mean ± S.D.).

 
TLR Triggering Induces SOCS in a Direct and Type I IFN-independent Manner—Next we analyzed a possible role of paracrine acting type I IFN in TLR-mediated induction of SOCS, in particular, SOCS-1. Therefore, we used a transwell system with LPS-responsive C3H/HeN macrophages and LPS non-responsive C3H/HeJ cells in the respective chambers (Fig. 1b). Upon stimulation with LPS, induction of mRNA for the direct target gene TNF-{alpha} was only observed in the C3H/HeN macrophages. In contrast, IP-10, which is induced indirectly via secreted type I IFN, showed increased expression in both macrophage populations. SOCS-1 behaved like TNF-{alpha}, thus, ruling out a significant contribution of paracrine mediators. CpG-DNA as control showed induction of the different genes in all cells. In addition, we stimulated type I IFN receptor-deficient mice (Fig. 1c). All TLR stimuli induced SOCS-1, SOCS-3, and CIS in dendritic cells. SOCS-3 and CIS induction showed no differences in IFNAR-I-/- mice as was the case for TNF-{alpha}. SOCS-1 induction was not affected in the case of the control stimulus IFN-{gamma}, yet showed a slight, but not significant, decrease in response to LPS and CpG-DNA. In addition, no major role for type I IFN in TLR-mediated SOCS induction could be identified in dendritic cells.

TLR-1, -2, -3, -4, and -9 Ligands Induce Secondary Type I IFN-dependent Signaling—To further study the significance of SOCS for TLR signaling, we first assessed whether the concept of direct and indirect, type I IFN-dependent TLR signaling (see Fig. 7) holds true for different TLR ligands, a matter that is controversial in the literature. First, we examined induction of transcripts for IFN-{beta} as well as IFN-{beta}-dependent IP-10 in comparison with the direct target gene TNF-{alpha} (Fig. 2a). In general, all of the tested TLR ligands (which have been shown to be devoid of contaminants earlier) were able to induce IFN-{beta} and IP-10. The concentrations of the respective stimuli were chosen to give nearly equal TNF-{alpha} induction. However, poly(dI-dC) was more efficient in IFN-{beta} induction, and LPS, as well as poly(dI-dC), also induced more IP-10 transcription (in the range of 15-fold more than with CpG-DNA). The results were confirmed by Western blot with phosphotyrosine-specific STAT-1 detection (Fig. 2b). Upon prolonged TLR stimulation, STAT-1 activation could be observed with LPS, CpG-DNA, or LTA in C57Bl/6 macrophages. Again, LPS was more efficient than LTA or CpG-DNA. Results were corroborated with C3H/HeN mice; however, in this case CpG-DNA was much more active, arguing for strain-specific differences in type I IFN signaling. C3H/HeJ mice confirmed that indeed STAT-1 activation by lipoteichoic acid was not due to LPS contaminants. Moreover, STAT-1 activation differed with respect to MyD88 dependence, whereas CpG-DNA and LTA were completely dependent on MyD88. Finally, transwell experiments confirmed that STAT-1 activation is due to the paracrine action of a secreted factor, most probably type I IFNs (Fig. 2c).



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  		FIG. 7.
Model of SOCS action in TLR signaling.

 



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  		FIG. 2.
Activation of indirect type I IFN signaling in response to TLR stimulation. Peritoneal macrophages from C57Bl/6 mice were stimulated with 300 nM CpG-DNA, 100 ng/ml LPS, 5 µg/ml poly(dI-dC), 10 µg/ml LTA, or 10 µg/ml Pam3CSK4 for 6 h. a, expression of mRNA for IFN-{beta}, IP-10, and TNF-{alpha} was determined relative to {beta}-actin (n = 3, mean ± S.D.). b, lysates were prepared and equal amounts were blotted with phosphospecific STAT-1 Ab. c, peritoneal macrophages were placed in a transwell system (0.4 µm) and stimulated and analyzed as above.

 
Generation of Macrophages with Stable Overexpression of SOCS—To evaluate a possible role of SOCS in TLR signal transduction we elected to use an overexpression system. To that end, we generated RAW 264.7 macrophage clones with stable overexpression of the respective SOCS molecules. Overexpression at the mRNA level was carefully monitored in different clones (Fig. 3a), and clones with specific and high overexpression were selected for further experiments. Moreover, protein overexpression was also controlled with either FLAG or c-Myc antibodies (data not shown). Untransfected macrophages showed expression of SOCS-1, SOCS-3, and CIS but not of SOCS-2 in response to LPS (Fig. 3b). However, expression levels of SOCS-1 and SOCS-3 were lower than with the stably transfected cells, while CIS levels were comparable. Thus, the stable transfectants had SOCS expression levels at least equal to or higher than LPS activated cells. SOCS proteins at the selected expression levels were functional, since they inhibited prototypical JAK/STAT-dependent cytokine signaling, as shown for SOCS-1-mediated IFN-{gamma} inhibition (Fig. 3c and data not shown).



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  		FIG. 3.
Generation of RAW 264.7 cells stably overexpressing SOCS. RAW 264.7 macrophages were transfected with either SOCS-1, SOCS-2, SOCS-3 or CIS expression plasmids. Cells were selected for stable expression and twice subcloned. a, overexpression was controlled by quantitative RT-PCR for each cell clone. Shown are the RT-PCR for one typical clone out of four. nd, not detected. b, RAW macrophages were stimulated with LPS for 5 h and subsequently expression of SOCS-1, -2, and -3 and CIS were determined by quantitative RT-PCR (n = 2, mean ± S.D.). c, RAW macrophages stably overexpressing SOCS proteins were stimulated with 30 units/ml IFN-{gamma} for 26 h, and nitrite was measured in the supernatant (n = 3, mean ± S.D.).

 
SOCS Inhibit Indirectly, but Not Directly, Induced Genes upon TLR Stimulation—Using RAW 264.7 macrophages with stable overexpression of the various SOCS proteins, we next analyzed the responsiveness toward TLR stimulation. Neither LPS- nor CpG-DNA-mediated TNF-{alpha} secretion were significantly inhibited by expression of the various SOCS proteins (Fig. 4, a and c). Although single clones existed with slightly differing TLR sensitivity, no significant overall differences became obvious upon repeated experiments. The same results were also observed for lipoteichoic acid and Pam3CSK4 (data not shown). Although iNOS has been described to be partly dependent on paracrine type I IFN induction, we were not able to observe inhibitory effects of SOCS on TLR-mediated nitrite secretion (Fig. 4, b and d). However, when we analyzed the induction of the indirect, IFN-{beta}-dependent gene IP-10, we found that secretion of this chemokine upon LPS or CpG-DNA stimulation was severely inhibited by overexpression of either SOCS-1 or SOCS-3 (Fig. 4e). CIS had also a slightly lower inhibitory potential.



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  		FIG. 4.
SOCS negatively influence indirect but not direct TLR signaling. RAW macrophages stably overexpressing SOCS proteins were analyzed for the induction of proinflammatory mediators. Cells were stimulated with indicated amount of either LPS (a, b) or CpG-DNA (c, d) for 24 h. Subsequently TNF-{alpha} (a, c) and nitrite (b, d) were measured in the supernatant. Displayed is one out of five typical experiments. e, cells were stimulated with either 100 ng/ml LPS or 100 nM CpG-DNA for 24 h, and IP-10 secretion was determined by ELISA (n = 2, mean ± S.D.).

 
SOCS-1 Inhibits Induction of IP-10 mRNA upon TLR Stimulation—To corroborate the results obtained at the protein level, we also examined induction of TNF-{alpha} and IP-10 at the mRNA level (Fig. 5). Again, LPS and CpG-DNA were equally active in inducing TNF-{alpha} transcription, and this was not influenced by SOCS protein overexpression. However, in accord with the above results, SOCS-1 overexpression severely and significantly diminished IP-10 induction in response to TLR-4 and -9 triggering. SOCS-3 and CIS were less effective, yet a slight decrease in induction of IP-10 could also be observed.



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  		FIG. 5.
SOCS-1 inhibits IP-10 mRNA induction upon TLR activation. RAW macrophages stably overexpressing SOCS proteins were stimulated for 5 h with either 100 ng/ml LPS or 1 µM CpG-DNA. Expression of TNF-{alpha} and IP-10 mRNA was determined by RT-PCR and normalized to {beta}-actin (*, p < 0.05; n = 2, mean ± S.D.).

 
SOCS-1 Inhibits TLR-mediated STAT-1 Activation—Finally, we analyzed the effects of SOCS overexpression on TLR signaling pathways. In line with the above results, no significant effects of SOCS on LPS or CpG-DNA stimulation were observed for the rapid activation of direct signaling events including phosphorylation of p38, JNK, and ERK as well as degradation of I{kappa}B{alpha} (Fig. 6a). Again, different cell clones showed equal results in Western blotting. However, when cells were stimulated for prolonged time periods with TLR ligands, STAT-1 activation as measured by tyrosine phosphorylation became observable. SOCS-1 and to a lesser extent SOCS-3 and CIS impaired or abolished TLR-mediated STAT-1 activation (Fig. 6b). The same pattern could be observed for the inhibition of IFN-{beta} signaling upon direct stimulation. As conflicting reports have been published we confirmed the signaling data with different methods. Neither NF{kappa}B reporter gene assays (Fig. 6c) nor NF{kappa}B translocation assays (Fig. 6d) were able to show inhibitory effects of SOCS on TLR-induced NF{kappa}B activation. In controls, SOCS expression (SOCS-1 and -3) abolished IFN-{gamma} signaling at the chosen concentrations as expected. Using phosphotyrosine-specific antibodies in flow cytometry, we were again able to observe a negative regulatory effect of SOCS on LPS-mediated STAT-1 activation, thus, corroborating the above results (Fig. 6e). Again, SOCS-3 and CIS were only partly inhibitory.



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  		FIG. 6.
SOCS effects on TLR signaling pathways. a, RAW macrophages with stable overexpression of SOCS were stimulated with 100 ng/ml LPS or 1 µM CpG-DNA for 30 min. Equal amounts of protein lysates were blotted and probed with phosphospecific abs for ERK, JNK, and p38 MAP kinase as well as I{kappa}B{alpha}. b, cells were stimulated with 100 ng/ml LPS, 0.3 µM CpG-ODN for 4 h or with 10 units/ml IFN-{beta} for 15 min. Lysates were probed with phosphospecific STAT-1 Ab and subsequently reprobed with STAT-1 Ab recognizing total amount of STAT-1. c, RAW 264.7 macrophages were transfected with either the NF{kappa}B-dependent ELAM or the STAT-1-dependent IFP53 reporter together with SOCS expression plasmids. Cells were stimulated with either 100 ng/ml LPS or 30 units/ml IFN-{gamma} for 6 h. Induction of the luciferase reporter gene was measured. Experiments were normalized to mock transfected controls (n = 3, mean ± S.D.). d, macrophages with stable overexpression of SOCS were activated with 100 ng/ml LPS for 45 min. Activation of NF{kappa}B was measured by an ELISA-based method in nuclear extracts (shown is one of two experiments). e, SOCS overexpressing cells were stimulated for 4.5 h with 100 ng/ml LPS. Cells were stained with a phosphospecific STAT-1 Ab, and mean flourescence intensity was measured in a flow cytometer. The given stimulation index is the n-fold induction toward non-stimulated control cells (n = 3, mean ± S.D.; *, p < 0.05).

 
SOCS-1 Inhibits TLR-mediated CD40 Up-regulation—Indirect type I IFN signaling has been suggested to participate in regulation of costimulatory molecules on antigen presenting cells. Thus, we analyzed CD40 expression in SOCS overexpressing macrophages. While LPS induced CD40 expression in control cells, these effects were diminished in SOCS-1- and CIS-expressing cells (Table I). No significant effects were observed for CD86 (data not shown).


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  		TABLE I
SOCS-1 inhibits TLR-mediated CD40 up-regulation RAW macrophages with stable overexpression of SOCS were stimulated with 100 ng/ml LPS or 30 units/ml IFN-{gamma} for 48 h. Subsequently up-regulation of CD40 was measured by flow cytometry. Shown are the results of one out of two experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Based on the presented experiments, a clear picture of the role of SOCS in TLR signaling can be portrayed (Fig. 7) and delineated as follows. TLR-dependent stimuli are able to induce SOCS proteins in a manner that is independent of paracrine-secreted factors and, especially, independent of type I IFN. Moreover, SOCS induction depends on MyD88 with the exception that TLR-3 and TLR-4 are able to use a second pathway for SOCS-1 induction. In turn, SOCS proteins do not inhibit direct MAP kinase- and NF{kappa}B-dependent signaling, yet these proteins are able to inhibit autocrine, secondary signaling via IFN-{beta}. Thus, TLR stimulation negatively influences STAT-1 activation.

Concerning the mode of induction of SOCS after TLR stimulation, we did not find evidence for an exclusive role of secreted type I IFN as reported by another group (13), and thus, we confirm earlier results (10, 11). Moreover, experiments using a transwell approach suggested that secreted factors do not play a dominant role at all. However, while it is conceivable that regulation occurs at the transcriptional level (2, 10, 11, 36), the precise mechanism of SOCS induction still remains elusive, since to date no promoter studies in response to TLR stimulation have been performed. We now extend our previous findings, showing that at least for TLR-2 and TLR-9 induction of SOCS-1, SOCS-3, and CIS is completely dependent on MyD88. However, in the case of LPS and poly(dI-dC), an additional pathway seems to be operative. One of the candidates could be TIRAP, since this mediator is reported to mediate additionally activation by LPS (26, 27). However, no role for TIRAP has been found in TLR-3 signaling (39). Thus, it seems more probable that TRIF/TICAM-1 is involved in SOCS-1 induction by LPS and poly(dI-dC) (30, 31). This signaling pathway is also responsible for IFN-{beta} induction by the latter ligands. Interestingly, we now show that TLR-2 and TLR-9 stimulation also results in IFN-{beta} induction and this is completely MyD88-dependent. Thus, this particular gene (IFN-{beta}) can be activated by different TLR stimuli using different adaptors. This is not only true for the partial overlap of MyD88 and TIRAP but also seems to be the case for TRIF and MyD88. These findings are in line with results from the Akira group showing similar differences in MyD88 dependence for TLR-4 and -9 ligands with regard to IFN-{beta} induction and up-regulation of CD40 (40). Moreover, our results also demonstrate that TLR-2 does not lack the ability for IFN-{beta} induction, since lipoteichoic acid was able to activate this signaling branch. This contrasts with earlier findings (34); however, the lipoteichoic acid used was highly purified and characterized to be strictly TLR-2-dependent (37). In addition, lipoteichoic acid-mediated IFN-{beta} and STAT-1 activation was preserved in C3H/HeJ mice. It has to be stated that, possibly, TLR stimuli show different capacities to activate this signaling pathway yet there is no doubt that TLR-2 signaling itself is able to induce STAT-1 activation. The results are in line with a model proposed by O'Neill and Hamilton (24), which reconciled the role of IFNs in TLR signaling. Also, this confirms earlier results showing that cycloheximide is able to inhibit LPS-mediated iNOS induction while having no effects on IFN-{beta} (29).

Here we used IP-10 as a model gene that is induced by the secondary IFN-{beta}-dependent TLR pathway, a fact that has been studied in IFNAR-1-deficient mice earlier (40). Similarly, CD40 regulation in dendritic cells has been reported to be dependent on IFN-{beta} (28, 40). We here report that SOCS-1 only inhibits indirect IFN-{beta} signaling in response to TLR stimulation but has no effects on NF{kappa}B. Since up-regulation of CD40 was also diminished in SOCS-1 overexpressing cells, this indirectly argues for IFN-{beta} dependence thereby corroborating the cited results. In contrast, we did not find significant effects on CD86 regulation while others have reported that macrophages from TRIF- or IFNAR-1-deficient mice do not display CD86 regulation anymore (28). Whether this is due to cell type-specific differences has not yet been examined.

Regarding the role of SOCS proteins in TLR signaling, we were not able to find evidence for an inhibitory role of SOCS for direct target genes, in contrast to other groups (35, 36). We chose to use an in vitro overexpression system. Overexpression levels were carefully controlled, and SOCS proteins were functional as they inhibited the expected cytokines (e.g. IFN-{gamma}). Using this approach, neither TNF-{alpha} nor IL-6 (data not shown) nor nitrite were inhibited by the different SOCS proteins. However, LPS induced IP-10 as a maker of secondary IFN-{beta} action was diminished by SOCS-1, SOCS-3, and CIS expression. Analyzing the respective signaling pathways, we did not find conflicting results but confirmed that while STAT-1 tyrosine phosphorylation was affected by SOCS, no effects could be observed for MAP kinase and NF{kappa}B activation. This contrasts the reported finding using NF{kappa}B reporter gene assays. The results are difficult to bring together. However, there is indirect evidence that IFN-{beta} signaling has amplificatory effects on direct genes in a mode of positive feedback regulation (41, 42). Such positive feedback regulation could be of differing intensity among various cell types or depending on culture conditions. Indeed, we have preliminary results showing that some macrophage cell lines exhibit reduced TNF-{alpha} induction upon addition of neutralizing type I IFN antiserum. Thus, it could be that the experimental conditions between the groups differed with respect to actions of IFN-{beta}. Indeed, it has been shown that Tyk2 knock-out mice, which have a defect in IFN-{beta} signaling, show a decreased LPS sensitivity in a LPS shock model (43). This argues for a much more important role of auto/paracrine type I IFN signaling in vivo. This could also explain the differences between our results using in vitro overexpression and the observed effects with SOCS-1-deficient animals. Furthermore, it has been reported that SOCS-1/STAT-1 double-deficient mice show a marked increase in LPS shock resistance as compared with SOCS-1 knock-out mice (36). Although it was stated that these mice still were more susceptible than wild type mice, the differences were only minor suggesting that SOCS-1 effects, independent of STAT-1 inhibition, also play only a minor role in vivo.

Additional controversy resulted from the fact that we did not find a role of SOCS for TLR induced nitrite induction. Others, using substitution or depletion of IFN-{beta} (34, 41), have found that iNOS induction depends at least partly on the IFN-{beta} pathway in TLR signaling. However, as this pathway is markedly diminished in SOCS-1 expressing macrophages, one would assume that iNOS induction upon TLR triggering should be diminished. Nevertheless, we failed to observe effects of SOCS-1 on TLR-induced nitrite at an early time point of 24 h. This is in line with observations that IFN-{beta} null mice have equal levels of nitrite in response to LPS as compared with wild type mice in vivo (43). Based on these findings we suggest that early nitrite generation is induced in a direct manner but can be enhanced at later time points by secondary IFN-{beta} action, similar to other genes as described previously (42).

Finally, if one argues for an existing role of SOCS-1 in LPS signaling, clear mechanisms for NF{kappa}B affection should be identified. So far only one group was able to show a weak SH2-dependent interaction of SOCS-1 with IRAK in an overexpression situation. Recently, an interesting finding has been reported by Ryo et al. (44). These authors found that SOCS-1 can interact with p65 and mediate ubiquitinylation and proteosomal degradation, thus, contributing to NF{kappa}B inhibition. However, it is also known that proinflammtory genes differ in their need for duration of NF{kappa}B signaling (45, 46). While some genes are immediately induced and possibly require only a short pulse of transcription factor activity, others have to remodel their chromatin structure before being activated, a process that will take some time. Thus, it could be that additional direct target genes have to be examined and some of them might be partly affected by SOCS-1 inhibiting the duration of NF{kappa}B signaling. Regarding SOCS-3 and NF{kappa}B no evidence for a modulation of LPS-induced NF{kappa}B activation was reported by others (47).

Taken together, our results indicate that SOCS can mediate two effects within innate immune cells upon TLR activation. First, they mediate cross-talk inhibition thus, diminishing the sensitivity of the cell to further subsequent cytokine signals. This would guarantee the undisturbed induction of an inflammatory program upon initial activation. Second, SOCS-1 is able to inhibit signal amplification circuits by autocrine type I IFN signaling, thus, avoiding overshooting reactions. However, IFN-{beta} can act in a paracrine manner on otherwise still naive cells without prior microbial contact, thus, exerting priming activities.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grants Da 592/1 and He 1452/4. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{diamondsuit} This article was selected as a Paper of the Week. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 49-6421-286-6497; Fax: 49-6421-286-6420; E-mail: dalpke{at}mailer.uni-marburg.de.

1 The abbreviations used are: SOCS, suppressor of cytokine signaling; SH2, Src homology 2; CIS, cytokine-inducible SH2-containing protein; GM-CSF, granulocyte macrophage-colony-stimulating factor; IFN, interferon; IFNAR, interferon-{alpha} receptor; IRAK, interleukin-1 receptor-associated kinase; JAK, Janus kinase; LPS, lipopolysaccharide; LTA, lipoteichoic acid; Mal, MyD88-adaptor like; PBS, phosphate-buffered saline; STAT, signal transducer and activator of transcripition; TICAM, TIR-containing adaptor molecule; TIR, Toll-interleukin-1 receptor domain; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRAM, TRIF-related adaptor molecule; TRAF, TNF receptor-associated factor; TRIF, TIR-containing adaptor-inducing interferon-{beta}; MAP, mitogen-activated protein; iNOS, inducible nitric-oxide synthase; ODN, oligodeoxynucleotide; FCS, fetal calf serum; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; PE, phycoerythrin; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; Ab, antibody. Back


   ACKNOWLEDGMENTS
 
We thank Helene Bykow and Christine Barett for excellent technical support as well as Peter Murray for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alexander, W. S., and Hilton, D. J. (2003) Annu. Rev. Immunol. 22, 503-529[CrossRef]
  2. Auernhammer, C. J., Bousquet, C., and Melmed, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6964-6969[Abstract/Free Full Text]
  3. Hilton, D. J., Richardson, R. T., Alexander, W. S., Viney, E. M., Willson, T. A., Sprigg, N. S., Starr, R., Nicholson, S. E., Metcalf, D., and Nicola, N. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 114-119[Abstract/Free Full Text]
  4. Kamura, T., Sato S, Haque, D., Liu, L., Kaelin, W. G. J., Conaway, R. C., and Conaway, J. W. (1998) Genes Dev. 12, 3872-3881[Abstract/Free Full Text]
  5. Frantsve, J., Schwaller, J., Sternberg, D. W., Kutok, J., and Gilliland, D. G. (2001) Mol. Cell. Biol. 21, 3547-3557[Abstract/Free Full Text]
  6. Kamizono, S., Hanada, T., Yasukawa, H., Minoguchi, S., Kato, R., Minoguchi, M., Hattori, K., Hatakeyama, S., Yada, M., Morita, S., Kitamura, T., Kato, H., Nakayama, K., and Yoshimura, A. (2001) J. Biol. Chem. 276, 12530-12538[Abstract/Free Full Text]
  7. Starr, R., Metcalf, D., Elefanty, A. G., Brysha, M., Willson, T. A., Nicola, N. A., Hilton, D. J., and Alexander, W. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14395-14399[Abstract/Free Full Text]
  8. Naka, T., Matsumoto, T., Narazaki, M., Fujimoto, M., Morita, Y., Ohsawa, Y., Saito, H., Nagasawa, T., Uchiyama, Y., and Kishimoto, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15577-15582[Abstract/Free Full Text]
  9. Alexander, W. S., Starr, R., Fenner, J. E., Scott, C. L., Handman, E., Sprigg, N. S., Corbin, J. E., Cornish, A. L., Darwiche, R., Owczarek, C. M., Kay, T. W., Nicola, N. A., Hertzog, P. J., Metcalf, D., and Hilton, D. J. (1999) Cell 98, 597-608[CrossRef][Medline] [Order article via Infotrieve]
  10. Stoiber, D., Kovarik, P., Cohney, S., Johnston, J. A., Steinlein, P., and Decker, T. (1999) J. Immunol. 163, 2640-2647[Abstract/Free Full Text]
  11. Dalpke, A. H., Opper, S., Zimmermann, S., and Heeg, K. (2001) J. Immunol. 166, 7082-7089[Abstract/Free Full Text]
  12. Dalpke, A. H., Eckerle, S., Frey, M., and Heeg, K. (2003) Eur. J. Immunol. 33, 1776-1787[CrossRef][Medline] [Order article via Infotrieve]
  13. Crespo, A., Filla, M. B., Russell, S. W., and Murphy, W. J. (2000) Biochem. J. 349, 99-104[CrossRef][Medline] [Order article via Infotrieve]
  14. Bode, J. G., Nimmesgern, A., Schmitz, J., Schaper, F., Schmitt, M., Frisch, W., Haussinger, D., Heinrich, P. C., and Graeve, L. (1999) FEBS Lett. 463, 365-370[CrossRef][Medline] [Order article via Infotrieve]
  15. Crespo, A., Filla, M., and Murphy, W. (2002) Eur. J. Immunol. 32, 710-719[CrossRef][Medline] [Order article via Infotrieve]
  16. Lang, R., Pauleau, A. L., Parganas, E., Takahashi, Y., Mages, J., Ihle, J. N., Rutschman, R., and Murray, P. J. (2003) Nat. Immunol. 4, 546-550[CrossRef][Medline] [Order article via Infotrieve]
  17. Yasukawa, H., Ohishi, M., Mori, H., Murakami, M., Chinen, T., Aki, D., Hanada, T., Takeda, K., Akira, S., Hoshijima, M., Hirano, T., Chien, K. R., and Yoshimura, A. (2003) Nat. Immunol. 4, 551-556[CrossRef][Medline] [Order article via Infotrieve]
  18. Croker, B. A., Krebs, D. L., Zhang, J. G., Wormald, S., Willson, T. A., Stanley, E. G., Robb, L., Greenhalgh, C. J., Forster, I., Clausen, B. E., Nicola, N. A., Metcalf, D., Hilton, D. J., Roberts, A. W., and Alexander, W. S. (2003) Nat. Immunol. 4, 540-545[CrossRef][Medline] [Order article via Infotrieve]
  19. Morita, Y., Naka, T., Kawazoe, Y., Fujimoto, M., Narazaki, M., Nakagawa, R., Fukuyama, H., Nagata, S., and Kishimoto, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5405-5410[Abstract/Free Full Text]
  20. Kawazoe, Y., Naka, T., Fujimoto, M., Kohzaki, H., Morita, Y., Narazaki, M., Okumura, K., Saitoh, H., Nakagawa, R., Uchiyama, Y., Akira, S., and Kishimoto, T. (2001) J. Exp. Med. 193, 263-269[Abstract/Free Full Text]
  21. De Sepulveda, P., Okkenhaug, K., Rose, J. L., Hawley, R. G., Dubreuil, P., and Rottapel, R. (1999) EMBO J. 18, 904-915[CrossRef][Medline] [Order article via Infotrieve]
  22. Akira, S., and Takeda, K. (2004) Nat. Rev. Immunol. 4, 499-511[CrossRef][Medline] [Order article via Infotrieve]
  23. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Immunity 11, 115-122[CrossRef][Medline] [Order article via Infotrieve]
  24. Hertzog, P. J., O'Neill, L. A., and Hamilton, J. A. (2003) Trends. Immunol. 24, 534-539[CrossRef][Medline] [Order article via Infotrieve]
  25. Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Kuwata, H., Takeuchi, O., Takeshige, K., Saitoh, T., Yamaoka, S., Yamamoto, N., Yamamoto, S., Muta, T., Takeda, K., and Akira, S. (2004) Nature 430, 218-222[CrossRef][Medline] [Order article via Infotrieve]
  26. Horng, T., Barton, G. M., and Medzhitov, R. (2001) Nat. Immunol. 2, 835-841[CrossRef][Medline] [Order article via Infotrieve]
  27. Fitzgerald, K. A., Palsson-McDermott, E. M., Bowie, A. G., Jefferies, C. A., Mansell, A. S., Brady, G., Brint, E., Dunne, A., Gray, P., Harte, M. T., McMurray, D., Smith, D. E., Sims, J. E., Bird, T. A., and O'Neill, L. A. (2001) Nature 413, 78-83[CrossRef][Medline] [Order article via Infotrieve]
  28. Hoebe, K., Janssen, E. M., Kim, S. O., Alexopoulou, L., Flavell, R. A., Han, J., and Beutler, B. (2003) Nat. Immunol. 4, 1223-1229[CrossRef][Medline] [Order article via Infotrieve]
  29. Hattori, Y., Akimoto, K., Matsumura, M., Tseng, C. C., Kasai, K., and Shimoda, S. (1996) Biochem. Mol. Biol. Int. 40, 889-896[Medline] [Order article via Infotrieve]
  30. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., and Seya, T. (2003) Nat. Immunol. 4, 161-167[CrossRef][Medline] [Order article via Infotrieve]
  31. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., Sugiyama, M., Okabe, M., Takeda, K., and Akira, S. (2003) Science 301, 640-643[Abstract/Free Full Text]
  32. Oshiumi, H., Sasai, M., Shida, K., Fujita, T., Matsumoto, M., and Seya, T. (2003) J. Biol. Chem. 278, 49751-49762[Abstract/Free Full Text]
  33. Yamamoto, M., Sato, S., Hemmi, H., Uematsu, S., Hoshino, K., Kaisho, T., Takeuchi, O., Takeda, K., and Akira, S. (2003) Nat. Immunol. 4, 1144-1150[CrossRef][Medline] [Order article via Infotrieve]
  34. Toshchakov, V., Jones, B. W., Perera, P. Y., Thomas, K., Cody, M. J., Zhang, S., Williams, B. R., Major, J., Hamilton, T. A., Fenton, M. J., and Vogel, S. N. (2002) Nat. Immunol. 3, 392-398[CrossRef][Medline] [Order article via Infotrieve]
  35. Kinjyo, I., Hanada, T., Inagaki-Ohara, K., Mori, H., Aki, D., Ohishi, M., Yoshida, H., Kubo, M., and Yoshimura, A. (2002) Immunity 17, 583-591[CrossRef][Medline] [Order article via Infotrieve]
  36. Nakagawa, R., Naka, T., Tsutsui, H., Fujimoto, M., Kimura, A., Abe, T., Seki, E., Sato, S., Takeuchi, O., Takeda, K., Akira, S., Yamanishi, K., Kawase, I., Nakanishi, K., and Kishimoto, T. (2002) Immunity 17, 677-687[CrossRef][Medline] [Order article via Infotrieve]
  37. Lehner, M. D., Morath, S., Michelsen, K. S., Schumann, R. R., and Hartung, T. (2001) J. Immunol. 166, 5161-5167[Abstract/Free Full Text]
  38. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., and Steinman, R. M. (1992) J. Exp. Med. 176, 1693-1702[Abstract/Free Full Text]
  39. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Nature 420, 324-329[CrossRef][Medline] [Order article via Infotrieve]
  40. Hoshino, K., Kaisho, T., Iwabe, T., Takeuchi, O., and Akira, S. (2002) Int. Immunol. 14, 1225-1231[Abstract/Free Full Text]
  41. Jacobs, A. T., and Ignarro, L. J. (2001) J. Biol. Chem. 276, 47950-47957[Abstract/Free Full Text]
  42. Doyle, S. E., O'Connell, R., Vaidya, S. A., Chow, E. K., Yee, K., and Cheng, G. (2003) J. Immunol. 170, 3565-3571[Abstract/Free Full Text]
  43. Karaghiosoff, M., Steinborn, R., Kovarik, P., Kriegshauser, G., Baccarini, M., Donabauer, B., Reichart, U., Kolbe, T., Bogdan, C., Leanderson, T., Levy, D., Decker, T., and Muller, M. (2003) Nat. Immunol. 4, 471-477[CrossRef][Medline] [Order article via Infotrieve]
  44. Ryo, A., Suizu, F., Yoshida, Y., Perrem, K., Liou, Y. C., Wulf, G., Rottapel, R., Yamaoka, S., and Lu, K. P. (2003) Mol. Cell. 12, 1413-1426[CrossRef][Medline] [Order article via Infotrieve]
  45. Saccani, S., Pantano, S., and Natoli, G. (2001) J. Exp. Med. 193, 1351-1360[Abstract/Free Full Text]
  46. Hoffmann, A., Levchenko, A., Scott, M. L., and Baltimore, D. (2002) Science 298, 1241-1245[Abstract/Free Full Text]
  47. Park, S. H., Kim, K. E., Hwang, H. Y., and Kim, T. Y. (2003) DNA Cell Biol. 22, 131-139[CrossRef][Medline] [Order article via Infotrieve]

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Home page
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N. Vazquez, T. Greenwell-Wild, S. Rekka, J. M. Orenstein, and S. M. Wahl
Mycobacterium avium-induced SOCS contributes to resistance to IFN-{gamma}-mediated mycobactericidal activity in human macrophages
J. Leukoc. Biol., November 1, 2006; 80(5): 1136 - 1144.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
V. A. Dennis, A. Jefferson, S. R. Singh, F. Ganapamo, and M. T. Philipp
Interleukin-10 Anti-Inflammatory Response to Borrelia burgdorferi, the Agent of Lyme Disease: a Possible Role for Suppressors of Cytokine Signaling 1 and 3.
Infect. Immun., October 1, 2006; 74(10): 5780 - 5789.
[Abstract] [Full Text] [PDF]

Home page
 Innate ImmunityHome page
M. C. Gonzalez-Leon, A. Soares-Schanoski, C. del Fresno, A. Cimadevila, V. Gomez-Pina, E. Mendoza-Barbera, F. Garcia, E. Marin, F. Arnalich, P. Fuentes-Prior, et al.
Nitric oxide induces SOCS-1 expression in human monocytes in a TNF-{alpha}-dependent manner
Innate Immunity, October 1, 2006; 12(5): 296 - 306.
[Abstract] [PDF]

Home page
 J. Immunol.Home page
X. Zhu, M. S. Chang, R. C. Hsueh, R. Taussig, K. D. Smith, M. I. Simon, and S. Choi
Dual Ligand Stimulation of RAW 264.7 Cells Uncovers Feedback Mechanisms That Regulate TLR-Mediated Gene Expression
J. Immunol., October 1, 2006; 177(7): 4299 - 4310.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
G. M. Anderson, P. Beijer, A. S. Bang, M. A. Fenwick, S. J. Bunn, and D. R. Grattan
Suppression of Prolactin-Induced Signal Transducer and Activator of Transcription 5b Signaling and Induction of Suppressors of Cytokine Signaling Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat during Late Pregnancy and Lactation
Endocrinology, October 1, 2006; 147(10): 4996 - 5005.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
H. Frobose, S. Groth Ronn, P. E. Heding, H. Mendoza, P. Cohen, T. Mandrup-Poulsen, and N. Billestrup
Suppressor of Cytokine Signaling-3 Inhibits Interleukin-1 Signaling by Targeting the TRAF-6/TAK1 Complex
Mol. Endocrinol., July 1, 2006; 20(7): 1587 - 1596.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
H. Weighardt, J. Mages, G. Jusek, S. Kaiser-Moore, R. Lang, and B. Holzmann
Organ-Specific Role of MyD88 for Gene Regulation during Polymicrobial Peritonitis.
Infect. Immun., June 1, 2006; 74(6): 3618 - 3632.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Qasimi, A. Ming-Lum, A. Ghanipour, C. J. Ong, M. E. Cox, J. Ihle, N. Cacalano, A. Yoshimura, and A. L-F. Mui
Divergent Mechanisms Utilized by SOCS3 to Mediate Interleukin-10 Inhibition of Tumor Necrosis Factor {alpha} and Nitric Oxide Production by Macrophages
J. Biol. Chem., March 10, 2006; 281(10): 6316 - 6324.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Zimmermann, P. J. Murray, K. Heeg, and A. H. Dalpke
Induction of Suppressor of Cytokine Signaling-1 by Toxoplasma gondii Contributes to Immune Evasion in Macrophages by Blocking IFN-{gamma} Signaling
J. Immunol., February 1, 2006; 176(3): 1840 - 1847.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. Qiao, M. V. Andrade, F. A. Lisboa, K. Morgan, and M. A. Beaven
Fc{epsilon}R1 and toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells
Blood, January 15, 2006; 107(2): 610 - 618.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Kimura, T. Naka, T. Muta, O. Takeuchi, S. Akira, I. Kawase, and T. Kishimoto
Suppressor of cytokine signaling-1 selectively inhibits LPS-induced IL-6 production by regulating JAK-STAT
PNAS, November 22, 2005; 102(47): 17089 - 17094.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
P. Ekchariyawat, S. Pudla, K. Limposuwan, S. Arjcharoen, S. Sirisinha, and P. Utaisincharoen
Burkholderia pseudomallei-Induced Expression of Suppressor of Cytokine Signaling 3 and Cytokine-Inducible Src Homology 2-Containing Protein in Mouse Macrophages: a Possible Mechanism for Suppression of the Response to Gamma Interferon Stimulation
Infect. Immun., November 1, 2005; 73(11): 7332 - 7339.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. G. Mujtaba, L. O. Flowers, C. B. Patel, R. A. Patel, M. I. Haider, and H. M. Johnson
Treatment of Mice with the Suppressor of Cytokine Signaling-1 Mimetic Peptide, Tyrosine Kinase Inhibitor Peptide, Prevents Development of the Acute Form of Experimental Allergic Encephalomyelitis and Induces Stable Remission in the Chronic Relapsing/Remitting Form
J. Immunol., October 15, 2005; 175(8): 5077 - 5086.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Kaufmann, B. Musset, S. H. Limberg, V. Renigunta, R. Sus, A. H. Dalpke, K. M. Heeg, B. Robaye, and P. J. Hanley
"Host Tissue Damage" Signal ATP Promotes Non-directional Migration and Negatively Regulates Toll-like Receptor Signaling in Human Monocytes
J. Biol. Chem., September 16, 2005; 280(37): 32459 - 32467.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Kamimura, C. Viedt, A. Dalpke, M. E. Rosenfeld, N. Mackman, D. M. Cohen, E. Blessing, M. Preusch, C. M. Weber, J. Kreuzer, et al.
Interleukin-10 Suppresses Tissue Factor Expression in Lipopolysaccharide-Stimulated Macrophages via Inhibition of Egr-1 and a Serum Response Element/MEK-ERK1/2 Pathway
Circ. Res., August 19, 2005; 97(4): 305 - 313.
[Abstract] [Full Text] [PDF]

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