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Published as doi: 10.1096/fj.05-5493fje.
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(The FASEB Journal. 2006;20:985-987.)
© 2006 FASEB

IFN-ß-induced SOCS-1 negatively regulates CD40 gene expression in macrophages and microglia

Hongwei Qin, Cynthia A. Wilson, Sun Jung Lee and Etty N. Benveniste1

Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA

1Correspondence: Department of Cell Biology, 1918 University Blvd., MCLM 395, University of Alabama at Birmingham, Birmingham, AL 35294–0005, USA. E-mail: tika{at}uab.edu

ABSTRACT

Costimulation between T cells and antigen-presenting cells is required for adaptive immune responses. CD40, a costimulatory molecule, is expressed in macrophages and microglia. The aberrant expression of CD40 is involved in human diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer’s disease. CD40 expression is induced by a variety of stimuli, including IFN-{gamma} and lipopolysaccharide (LPS). In this study, we describe the molecular basis by which IFN-ß, a cytokine with immunomodulatory properties, regulates CD40 gene expression. IFN-ß induces CD40 expression in macrophages and microglia at the transcriptional level, and GAS elements in the CD40 promoter are required for IFN-ß-induced CD40 promoter activity. The critical role of signal transducers and activators of transcription-1{alpha} (STAT-1{alpha}) in this response was confirmed by utilizing primary microglia from STAT-1{alpha} deficient mice. IFN-ß induces suppressor of cytokine signaling-1 (SOCS-1) gene expression, which inhibits cytokine signaling by inhibiting activation of STAT proteins. The ectopic expression of SOCS-1 abrogates IFN-ß-mediated STAT-1{alpha} activation and inhibits IFN-ß-induced CD40 expression. IFN-ß-induced recruitment of STAT-1{alpha} and RNA Pol II and permissive histone modifications on the CD40 promoter are also inhibited by SOCS-1 overexpression. These novel results indicate that IFN-ß-induced SOCS-1 plays an important role in the negative regulation of IFN-ß-induced CD40 gene expression.—Qin H., Wilson C. A., Lee S. J., Benveniste E. N. IFN-ß-induced SOCS-1 negatively regulates CD40 gene expression in macrophages and microglia.


Key Words: macrophages • microglia

TYPE I AND II IFNs play important roles in the regulation of numerous physiological responses in humans (1 2 3) . Upon interaction with their respective receptors on target cells, the IFNs elicit antiviral, antiproliferative, and immunomodulatory responses (4) . The type 1 IFNs are comprised of the products of multiple (up to 13) IFN-{alpha} genes and a single IFN-ß gene (4) . The major pathway of intracellular signaling used by IFN-{alpha}/ß and their receptors involves the tyrosine kinases Janus-activated kinase (JAK)1 and TYK2, leading to the activation of signal transducers and activators of transcription-1{alpha} (STAT-1{alpha}) and STAT-2 to form a STAT-1{alpha}/STAT-2 heterodimer, which associates with the p48 protein, also known as IFN responsive factor-9 (IRF-9) to form the IFN-stimulated gene factor-3 (ISGF-3) complex. ISGF-3 recognizes the IFN-responsive sequence element (ISRE) in promoters of IFN-responsive genes (4) . Less frequently, a STAT-1{alpha} homodimer is formed in response to IFN-{alpha}/ß, which binds to IFN-{gamma} activation site (GAS) elements to induce gene expression (4) .

CD40, a costimulatory molecule for antigen presentation, is expressed by a wide variety of cells such as B cells, macrophages, microglia, dendritic cells, keratinocytes, endothelial cells, thymic epithelial cells, fibroblasts, and tumor cells. CD40 has been implicated in participating in many human diseases, particularly autoimmune diseases (5 , 6) . Blocking the interaction between CD40 and its ligand, CD40L (CD154), with anti-CD154 or anti-CD40 antibody (Ab) has been found to be beneficial in several animal models of autoimmune diseases (6) . These findings illustrate the importance of CD40-CD154 interactions for homeostasis of immune responses. We have previously shown that macrophages and microglia, the endogenous macrophage of the brain, constitutively express CD40 at a low level and that expression is strongly enhanced by IFN-{gamma} (7) . Examination of the CD40 promoter indicated that at least two GAS sites (dGAS and mGAS) and two Ets elements (EtsA and EtsB) in the CD40 promoter contribute to IFN-{gamma} induction of CD40 transcription. The transcription factors PU.1 and/or Spi-B constitutively bind to the EtsA element, and PU.1 binds to the EtsB element. IFN-{gamma}-activated STAT-1{alpha} homodimers bind to the mGAS and dGAS elements of the CD40 promoter (7) . Furthermore, optimal expression of CD40 in response to IFN-{gamma} requires endogenously produced TNF-{alpha}, and IFN-{gamma}-induced TNF-{alpha} activates NF-{kappa}B to bind three NF-{kappa}B elements within the CD40 promoter (8) . Collectively, these transcription factors interact with coactivators, likely CBP and/or p300, as well as the basal transcription machinery, to mediate CD40 gene transcription. We have recently shown that LPS functions as an inducer of CD40 gene expression (9) . In this case, LPS-induced CD40 gene expression is controlled by LPS activation of NF-{kappa}B and subsequent endogenous production of IFN-ß that induces STAT-1{alpha} activation. Activated NF-{kappa}B binds to NF-{kappa}B elements, and STAT-1{alpha} binds to GAS elements in the CD40 promoter, which coordinately control CD40 gene expression (9) .

Suppressor of cytokine signaling-1 (SOCS-1) is one of eight cytokine-inducible inhibitors of the JAK-STAT signaling pathway (10 11 12) . SOCS-1 interacts with phosphorylated JAK tyrosine kinases and inhibits IFN-{gamma} signaling (13) . SOCS proteins are defined by a conserved C-terminal motif, known as the SOCS box, and a central phosphotyrosine binding SH2 domain (14 , 15) . A number of studies have implicated SOCS-1 involvement in the inhibition of various signaling pathways (16 17 18 19 20) . Recently, it has been shown that SOCS-1 is rapidly induced by LPS and negatively regulates innate immune responses triggered by LPS (19 , 21) . Furthermore, SOCS-1 overexpression suppresses LPS-induced I{kappa}B phosphorylation and NF-{kappa}B transcriptional activity (19 , 21) . SOCS-1 expression is induced by numerous cytokines including interleukin (IL)-6, IL-2, IL-8, IL-4, IL-13, growth hormone, or IL-10 through the activation of various STAT proteins in T cells and macrophages, and overexpression of SOCS-1 can inhibit responses to several of these cytokines (17 , 20 , 22 23 24) .

We recently demonstrated that LPS-induced endogenous production of IFN-ß and the subsequent activation of STAT-1{alpha} are critical for optimal expression of CD40 in response to LPS (9) . The molecular mechanism of CD40 induction by exogenous IFN-ß is not known and was the subject of investigation in this study. We demonstrate that IFN-ß induces CD40 gene expression in macrophages and microglia and that induction is regulated at the transcriptional level and involves activation of STAT-1{alpha}. The critical role of STAT-1{alpha} in this response was shown utilizing primary microglia from STAT-1{alpha} deficient mice, which were unresponsive to IFN-ß induction of CD40 expression. Chromatin immunoprecipitation (ChIP) assays demonstrate that IFN-ß induces binding of STAT-1{alpha} and RNA Pol II to the CD40 promoter in vivo and also induces permissive modifications of histones H3 and H4 on the CD40 promoter. We also demonstrate that IFN-ß induces SOCS-1 gene expression in macrophages and microglia. A murine macrophage cell line ectopically expressing SOCS-1 was used to determine its effect on IFN-ß-mediated CD40 induction. Ectopic SOCS-1 expression abrogates IFN-ß-induced STAT-1{alpha} activation, which correlates with inhibition of IFN-ß-induced CD40 gene expression. SOCS-1 overexpression also inhibits IFN-ß-mediated STAT-1{alpha} and RNA Pol II recruitment to the CD40 promoter, as well as IFN-ß-mediated H3 and H4 acetylation on the CD40 promoter. These data indicate that SOCS-1 negatively regulates IFN-ß-induced CD40 gene expression, which may play an important role in the negative regulation of pathological inflammatory events.

MATERIALS AND METHODS

Recombinant proteins and reagents
Recombinant murine IFN-ß was purchased from Biosource International (Camarillo, CA), and recombinant human IFN-ß was purchased from R&D Systems (Minneapolis, MN). Rat-anti-imouse PE-conjugated CD40 Ab (clone 3/23), rat-PE-conjugated IgG isotype control, mouse-anti-human FITC-conjugated CD40 Ab (clone 5C3), and mouse-FITC-conjugated IgG isotype control were purchased from PharMingen (San Diego, CA). Antibodies against phospho-STAT-1{alpha}Tyr701, phospho-STAT-1{alpha}Ser727, and phospho-STAT-2Tyr690 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against STAT-1{alpha}, STAT-2, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Ab against SOCS-1 was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Antibodies against AcH3 and AcH4 were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies against Pol II and Pol IISer-5 were from Covance (Princeton, NJ). Expression vectors encoding wild-type (WT) and dominant-negative forms of IKK-{alpha} and IKK-ß were kindly provided by Dr. Randolph Noelle (Dartmouth Medical School, Lebanon, NH).

Cells
Primary microglia from C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME), WT mice, and STAT-1{alpha} deficient mice on the 129S6/SvEv background (Taconic, Germantown, NY) were prepared as described previously (25 , 26) . Primary human macrophages were generated by plating 10 x 106 peripheral blood mononuclear cells/well in a six-well plate and culturing the cells in Dulbecco’s modified Eagle’s medium as described previously (27) . The purity of the macrophage cultures is generally 95% or higher, as determined by staining for CD14. The murine microglia cell line EOC13 and the murine macrophage cell line RAW264.7 were maintained as described previously (8) .

Immunofluorescence flow cytometry
Murine cells were plated at 2 x 105 cells/well into 12-well plates, treated with medium or IFN-ß for up to 48 h, and then incubated with 100 µl of 2.4G2 hybridoma supernatant (which contains rat anti-mouse Fc{gamma}R Ab) for blocking Fc{gamma}R. Cells were then incubated with 10 µg/ml of PE-conjugated anti-mouse CD40 Ab and analyzed on the FACStar (Becton-Dickinson, Mountain View, CA), as described previously (7) . Human macrophages were incubated with 10 µg/ml of FITC-conjugated anti-human CD40 Ab. Negative controls were incubated with IgG isotype-matched Ab. Fold induction of CD40 expression was calculated by dividing the value of mean fluorescence intensity of IFN-ß treated samples by the value of untreated samples.

RNA isolation, riboprobes, and ribonuclease protection assay
Total cellular RNA was isolated from unstimulated or IFN-ß treated cells. The riboprobes for murine CD40, IRF-1, SOCS-1, RANTES and GAPDH, and human CD40 and GAPDH were prepared as described previously (7 , 28) . Twenty micrograms of total RNA were hybridized with the riboprobes at 42°C overnight. The hybridized mixture was treated with RNase A/T1 (1:200) and then analyzed by 5% denaturing (8 M urea) PAGE. Values for CD40, IRF-1, and SOCS-1 mRNA expression were normalized to GAPDH mRNA levels for each experimental condition.

CD40 and SOCS-1 promoter constructs
The characterization of the 758 bp human CD40 promoter construct (hCD40p0.7) has been described previously (7) . Site-directed mutant constructs of the three GAS elements were generated from the hCD40p0.7 plasmid backbone as described previously (7 , 8) . The 1480 bp (–1380 to +98) murine SOCS-1 promoter was amplified from genomic DNA, using the forward primer: 5'-TTGCCTGGGCTGGAAGCACAG-3' and the reverse primer: 5'-TTGGCTCACCTGGCGGCAGGA-3', and cloned into the pGL3 basic vector. The promoter sequence was confirmed by automatic sequencing.

Transient transfection and luciferase assays
CD40 or SOCS-1 promoter constructs (0.2 µg) were transiently transfected into 5 x 105 RAW264.7 cells in six-well plates using the LipofectAMINE Plus method as described previously (7) . Transfected cells were treated with IFN-ß for 12 h (CD40 promoter) or 8 h (SOCS-1 promoter), and luciferase activity of each sample was normalized to the total protein concentration of each well. Luciferase activity from the untreated sample was arbitrarily set at 1 for calculation of fold induction. For transfection with the IKK-{alpha} and IKK-ß expression constructs, 0.1 µg of the specific constructs were used, and differences in the amount of DNA were adjusted with appropriate empty vector.

Immunoblotting
Fifty micrograms of cell lysate was separated on 10% SDS-PAGE and probed with specific antibodies as described previously (29) . Membranes were stripped at 50°C in buffer containing 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) with occasional shaking and reprobed for total actin, STAT-1{alpha}, and/or STAT-2. SOCS-1 protein expression was analyzed using 15% SDS-PAGE gel and PDVF membranes.

RNA interference
DharmaFECTTM 1 siRNA transfection reagent, SMARTpool small interfering RNA (siRNAs) specific for murine STAT-2, and siCONTROL nontargeting siRNA were purchased from Dharmacon (Lafayette, CO); 5 x 105 RAW264.7 cells in six-well plates were transfected with 100 nM of siCONTROL or STAT-2 specific siRNAs using the DharmaFECTTM 1 following the manufacturer’s recommendations. After 72 h of transfection, RAW264.7 cells were treated with IFN-ß for 4 or 24 h to analyze CD40 mRNA and CD40 protein expression, respectively. Fifty micrograms of cell lysate were subjected to immunoblotting for STAT-2 detection as described above.

ChIP assays
ChIP analysis was done following a protocol provided by Upstate Biotechnology with modifications as described previously (29 , 30) . RAW264.7 cells were stimulated with IFN-ß for up to 8 h and fixed with 1% formaldehyde for 15 min at room temperature, and nuclei were isolated. Chromatin was sheared by sonication, and samples were precleared for 2 h at 4°C with salmon sperm DNA-saturated protein A/G-Sepharose. Chromatin solutions were precipitated overnight at 4°C with 5 µg of antibodies or isotype-matched control IgG. Input and immunoprecipitated chromatin were incubated at 65°C overnight to reverse crosslinks. After proteinase K digestion, DNA was extracted with the Qiagen Miniprep Kit. Purified DNA was analyzed by PCR with Taq polymerase. The primer pair 5'-CTACAGCCTCTGGATGGAGC-3' and 5'-TGCAGAACCGAAAGCGTCTC-3' was used to amplify a 250 bp region in the mouse CD40 promoter containing functional NF-{kappa}B and GAS elements. Densitometry was used to quantify the PCR results, and all results were normalized by the respective input values.

RESULTS

IFN-ß-induced STAT-1{alpha} activation and GAS elements in the CD40 promoter are critical for IFN-ß-induced CD40 gene expression
We have recently demonstrated that optimal induction of CD40 gene expression in response to LPS requires endogenous IFN-ß production, which subsequently activates STAT-1{alpha} in both macrophages and microglia (9) . The molecular basis of IFN-ß-induced CD40 gene expression has yet to be addressed. We initiated experiments to examine the kinetics of exogenous IFN-ß-induced CD40 gene transcription in macrophages (RAW264.7 cells). These cells were incubated with medium or IFN-ß for up to 12 h, and then CD40 mRNA expression was analyzed. In RAW264.7 cells, CD40 mRNA was detected 1 h after addition of IFN-ß, peaked at 4 h (7.6-fold induction), and then returned to basal levels at 12 h (Fig. 1 A). CD40 protein expression was analyzed by FACS. IFN-ß induced CD40 protein expression in a time-dependent manner in the RAW264.7 cells (Fig. 1B ).


Figure 1
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  		Figure 1. IFN-ß-induced STAT-1{alpha} activation and GAS elements in CD40 promoter are critical for IFN-ß-induced CD40 gene expression. A) RAW264.7 cells were treated with medium or IFN-ß (100 U/ml) for up to 12 h, and then total RNA was isolated and analyzed by RNase protection assay (RPA) for CD40 and GAPDH mRNA. Basal concentration of the untreated sample (UN) was set at 1.0, and fold induction on IFN-ß treatment was compared to that. Representative of 3 experiments. B) RAW264.7 cells were treated in the absence or presence of IFN-ß for up to 72 h and then stained with either PE-conjugated anti-CD40 or PE-conjugated isotype-matched control antibody (Ab). Cells were subjected to FACS analysis. Samples were analyzed by measuring mean fluorescence intensity (MFI). Fold induction of CD40 protein was calculated and is mean ± SD of 3 experiments. C) RAW264.7 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 4 h. Protein lysates were prepared and subjected to immunoblotting with antiphospho-STAT-1{alpha}Tyr701, antiphospho-STAT-1{alpha}Ser727, antiphospho-STAT-2Tyr690, stripped, and reprobed with anti-STAT-1{alpha}, STAT-2, and anti-actin as loading controls. D) WT and GAS mutant constructs of the human CD40 promoter. E) RAW264.7 cells were transiently transfected with 0.2 µg of indicated constructs, allowed to recover for 4 h, treated with medium or IFN-ß (100 U/ml) for 12 h, and then analyzed for luciferase activity. Values were normalized to total protein, and fold induction was calculated by dividing the IFN-ß treatment values by untreated levels. Data are presented as mean ± SD of 3 experiments. F) WT or STAT-1{alpha}-/- primary murine microglia were treated with IFN-ß for 4 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA expression. Representative of 3 experiments.

To investigate the activation of STATs by IFN-ß, RAW264.7 cells were treated in the absence or presence of IFN-ß for up to 4 h, and then protein lysates were subjected to SDS-PAGE analysis to detect the phosphorylation status of STAT-1{alpha} and STAT-2 (Fig. 1C ). Expression levels of total STAT-1{alpha}, STAT-2, and actin were used as loading controls. STAT-1{alpha}Tyr701 was strongly phosphorylated after 0.5–2 h of IFN-ß treatment, and the intensity of the band decreased at 4 h. Phosphorylation of STAT-1{alpha}Ser727 was detected at 0.5 h of IFN-ß treatment, peaked between 1 and 2 h, and then diminished at 4 h. Phosphorylation of STAT-2Tyr690 was detected at 0.5 h after IFN-ß treatment and persisted until 4 h. These results indicate that IFN-ß activates the STAT-1/STAT-2 signaling pathway in RAW264.7 cells.

We have previously described that GAS and NF-{kappa}B regulatory elements are conserved between the human and mouse CD40 promoters, as is the spacing between these sites (7 , 8) . Within the CD40 promoter, there are three GAS elements designated as dGAS, mGAS, and pGAS, and four NF-{kappa}B elements designated as d{kappa}B, m{kappa}B, m2{kappa}B, and p{kappa}B (7 , 8) . To define the cis-acting elements necessary for IFN-ß-induced CD40 promoter activity, GAS mutant constructs of the human CD40 promoter (Fig. 1D ) were tested in the RAW264.7 cells. Mutation of the dGAS and mGAS elements leads to a ~65% inhibition of CD40 promoter activity, while mutation of the pGAS element did not inhibit IFN-ß activation of the CD40 promoter (Fig. 1E ). Mutation of three of the four NF-{kappa}B sites (d{kappa}B, m2{kappa}B, and p{kappa}B) had no influence on IFN-ß-induced CD40 promoter activity (data not shown). Mutation of the m{kappa}B element had a slight inhibitory effect on IFN-ß-induced CD40 promoter activity (data not shown), which may be due to the fact that the mGAS (–494 bp) and the m{kappa}B (–483 bp) elements are very close to each other, and mutation of m{kappa}B affects the function of mGAS. These results suggest that two of the three GAS elements (dGAS and mGAS), but not the NF-{kappa}B elements, play an important role in IFN-ß-induced CD40 promoter activity.

We have previously demonstrated that STAT-1{alpha} binds to the dGAS and mGAS elements in the CD40 promoter (8) . To determine the importance of STAT-1{alpha} activation in IFN-ß-induced CD40 gene expression, primary microglia from STAT-1{alpha} deficient mice were examined. In these cells, IFN-ß-induced CD40 mRNA expression was attenuated compared to WT microglia (Fig. 1F ). These experiments indicate that STAT-1{alpha} plays a critical role in IFN-ß-induced CD40 gene expression.

STAT-2 and NF-{kappa}B are not involved in IFN-ß induction of CD40 gene expression and promoter activity
The major pathway of intracellular signaling used by IFN-ß is the STAT-1{alpha}/STAT-2/IRF-9 heterotrimer, known as the ISGF-3 complex, which recognizes ISRE elements in promoters of IFN-ß-responsive genes (4) . As previously described, there are no ISRE elements in the CD40 promoter (7 , 8) . To determine the potential role of STAT-2 in IFN-ß-induced CD40 gene expression, CD40 mRNA and protein expression were evaluated in RAW264.7 cells with reduced STAT-2 expression levels. siRNA was used to suppress STAT-2 protein expression. There was an ~85% reduction in STAT-2 protein levels comparing unstimulated siCONTROL nontargeting siRNA (lane 1) vs. unstimulated STAT-2 specific siRNA (lane 3) and an ~78% reduction comparing IFN-ß stimulated siCONTROL (lane 2) with IFN-ß stimulated STAT-2 specific siRNA (lane 4) after 72 h of transfection (Fig. 2 A). The RANTES promoter contains an ISRE element (31) , and RANTES mRNA is inducible by IFN-ß in RAW264.7 cells (Fig. 2B ). Reduced levels of RANTES mRNA (~55%) were observedin the presence of STAT-2 siRNA compared with siCONTROL (Fig. 2B ). In contrast, IFN-ß-induced expression of CD40 mRNA and protein were not affected in the presence of STAT-2 siRNA compared with siCONTROL (Fig. 2C and D ). These results indicate that STAT-2 is not involved in IFN-ß-induced CD40 gene expression.


Figure 2
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  		Figure 2. STAT-2 and NF-{kappa}B are not involved in IFN-ß induction of CD40 gene expression and promoter activity. A) RAW264.7 cells were transfected with 100 nM of siCONTROL nontargeting siRNA or STAT-2-specific siRNA for 72 h in the absence or presence of IFN-ß. Protein lysates were prepared and subjected to immunoblotting with anti-STAT-2 and anti-actin as a loading control. B) siCONTROL and STAT-2 siRNA transfected RAW264.7 cells were treated with medium or IFN-ß (100 U/ml) for 4 h, and then total RNA was isolated and analyzed by RPA for RANTES and GAPDH mRNA. C) siCONTROL and STAT-2 siRNA transfected RAW264.7 cells were treated with medium or IFN-ß (100 U/ml) for 4 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA. D) siCONTROL and STAT-2 siRNA transfected RAW264.7 cells were treated with medium or IFN-ß (100 U/ml) for 24 h and then stained with either PE-conjugated anti-CD40 or PE-conjugated isotype-matched control Ab. Cells were subjected to FACS analysis. Fold induction of CD40 protein was calculated and is mean ± SD of 3 experiments. E) RAW264.7 cells were transiently cotransfected with WT CD40 promoter construct (0.2 µg) and expression vectors (0.1 µg) containing DN IKK-{alpha} or IKK-ß cDNA and allowed to recover for 4 h. Cells were then treated with IFN-ß (100 U/ml) for 12 h and analyzed for luciferase activity. Differences in amount of DNA were adjusted with the empty vector pcDNA3. Data are mean ± SD of 3 experiments.

To exclude the involvement of NF-{kappa}B in IFN-ß-induced CD40 gene transcription, we first tested whether IFN-ß activated the NF-{kappa}B signaling pathway. There was no detectable phosphorylation of IKK{alpha}/ß, I{kappa}B{alpha}, or NF-{kappa}B on IFN-ß stimulation for up to 4 h, and there was no degradation of I{kappa}B{alpha} (data not shown). Next, dominant-negative (DN) expression constructs of IKK-{alpha} or IKK-ß that contain substitutions of alanine for an essential lysine in the ATP-binding site were utilized. We have previously shown that these DN constructs inhibit both IFN-{gamma} and LPS induction of CD40 gene expression (8 , 9) . The DN constructs were cotransfected with the WT CD40 promoter construct into RAW264.7 cells, and then the cells were treated with medium or IFN-ß. The IKK-{alpha} DN, IKK-ß DN, or both constructs did not significantly suppress IFN-ß-induced CD40 promoter activity (Fig. 2E ). These data indicate that activation of NF-{kappa}B is not critical for the induction of CD40 promoter activity by IFN-ß.

IFN-ß induces CD40 expression in primary macrophages and microglia
To examine whether CD40 expression was regulated by IFN-ß in primary cultures of macrophages and microglia, the following experiments were performed. In primary human macrophage cultures, low constitutive expression of CD40 mRNA and protein was observed, which was enhanced by IFN-ß at 4–8 h at the mRNA level and at 36 h at the protein level (Fig. 3 A and B). In primary murine microglia, CD40 mRNA was strongly inducible by IFN-ß in a time-dependent manner, followed by expression of the CD40 protein at 36 h (Fig. 3C and D ). We also examined a murine microglial cell line, EOC13, for IFN-ß-induced CD40 gene expression. CD40 mRNA was first detected 2 h after addition of IFN-ß, peaked at 4 h (6.7-fold induction), and then declined at 12 h in EOC13 cells (Fig. 3E ). IFN-ß induced CD40 protein expression in a time-dependent manner in the EOC13 cells (Fig. 3F ). These data demonstrate that IFN-ß induction of CD40 expression occurs in primary macrophages and microglia.


Figure 3
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  		Figure 3. IFN-ß induces CD40 gene expression in primary macrophages, and microglial cells. A) Human primary macrophages were treated with IFN-ß for 4 and 8 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA expression. B) Primary human macrophages were treated with IFN-ß (100 U/ml) for 36 h, and then cells were subjected to FACS analysis for CD40 protein expression. C) Murine primary microglia were treated with IFN-ß for up to 12 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA expression. D) Murine primary microglia were treated with IFN-ß (100 U/ml) for 36 h, and then cells were subjected to FACS analysis for CD40 protein expression. E) EOC13 cells were treated with medium or IFN-ß (100 U/ml) for up to 12 h, and then total RNA was isolated and analyzed by RPA for CD40 and GAPDH mRNA. F) EOC13 cells were treated in the absence or presence of IFN-ß for up to 72 h and then stained with either PE-conjugated anti-CD40 or PE-conjugated isotype-matched control Ab. Cells were subjected to FACS analysis. Data are mean ± SD of 3 experiments.

IFN-ß induces SOCS-1 expression in macrophages, and ectopic expression of SOCS-1 inhibits recruitment of STAT-1{alpha} and RNA Pol II to the CD40 promoter and modification of H3 and H4 histones in response to IFN-ß
Negative regulation of signal transduction pathways is necessary for an appropriate cellular and physiological response to cytokine stimulation (32) . SOCS-1 is one of eight cytokine-inducible inhibitors of cytokine signaling (12 , 15 , 33) . SOCS-1 expression is induced by stimuli such as IFN-{gamma} and LPS, and overexpression of SOCS-1 inhibits subsequent responses to IFN-{gamma} or LPS (17 , 20 , 22 23 24) . To test whether SOCS-1 gene expression was regulated upon IFN-ß treatment in macrophages, RAW264.7 cells were incubated with medium or IFN-ß for up to 12 h, and then SOCS-1 mRNA expression was analyzed. Constitutive expression of SOCS-1 mRNA was undetectable; however, SOCS-1 mRNA was induced at 1–2 h after addition of IFN-ß (Fig. 4 A), and SOCS-1 protein expression was detected between 2–8 h of IFN-ß treatment (Fig. 4B ). Activation of the murine SOCS-1 promoter (–1380 bp to +98 bp) was tested in RAW264.7 cells by transient transfection. IFN-ß induced SOCS-1 promoter activity by ~6.8-fold (Fig. 4C ). These results indicate that IFN-ß transcriptionally induces SOCS-1 gene expression.


Figure 4
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  		Figure 4. IFN-ß induces SOCS-1 expression in macrophages and ectopic expression of SOCS-1 inhibits recruitment of STAT-1{alpha} and RNA Pol II to the CD40 promoter and modification of H3 and H4 histones in response to IFN-ß. A) RAW264.7 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 12 h, and then total RNA was isolated and analyzed by RPA for SOCS-1 and GAPDH mRNA. Basal concentration of untreated sample was set at 1.0, and fold induction upon IFN-ß treatment was compared to that. B) RAW264.7 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 24 h. Protein lysates were prepared and subjected to immunoblotting with anti-SOCS-1 Ab, stripped, and reprobed with anti-actin as a loading control. C) RAW264.7 cells were transiently transfected with 0.2 µg of murine SOCS-1 promoter construct, allowed to recover for 4 h, treated with medium or IFN-ß (100 U/ml) for 8 h, and analyzed for luciferase activity. Values were normalized to total protein, and fold induction was calculated by dividing IFN-ß treatment values by untreated levels. Data are mean ± SD of 3 experiments. D) Protein lysates were prepared from RAW264.7 and RAW-SOCS-1 cells and subjected to immunoblotting with anti-SOCS-1 Ab for confirmation of ectopic SOCS-1 expression, stripped, and reprobed with anti-actin as a loading control. E) RAW264.7 and RAW-SOCS-1 cells were treated with IFN-ß (100 U/ml) for up to 24 h, and CD40, IRF-1, and GAPDH mRNA was analyzed. Fold induction- IFN-ß treatment was determined by comparison to untreated controls. F) RAW264.7 and RAW-SOCS-1 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 8 h, and then cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against STAT-1{alpha}, histone acetylation (Ac-H3, Ac-H4), RNA Pol II, phospho-Pol IISer-5, or normal rabbit IgG. Basal concentration of the untreated sample was set at 1.0, and fold induction upon IFN-ß treatment was compared to that. Representative of 3 experiments.

To study the possible negative regulation of IFN-ß-induced CD40 gene expression by SOCS-1, RAW264.7 cells overexpressing SOCS-1 (RAW-SOCS-1) were utilized (28) . SOCS-1 protein expression was confirmed by immunoblotting (Fig. 4D ). As shown in Fig. 4E , RAW and RAW-SOCS-1 cells were incubated with medium or IFN-ß for up to 24 h, and then CD40 mRNA expression was analyzed. IFN-ß-induced CD40 mRNA expression was suppressed at all time points in RAW-SOCS-1 cells compared with WT RAW264.7 cells. Similar results were observed for IFN-ß-induced IRF-1 mRNA expression (Fig. 4E ).

Gene transcription in eukaryotic cells is controlled by protein complexes including transcription factors, coregulators, chromatin-remodeling complexes, and complexes responsible for signal-specific histone modifications on promoters (34) . We investigated the kinetics of the recruitment of transcription factors to the endogenous CD40 promoter in vivo and how they are coupled to CD40 gene transcription upon exogenous IFN-ß stimulation. To monitor transcription factor binding in vivo, RAW264.7 cells were incubated in the absence or presence of IFN-ß for up to 8 h, and ChIP assays were performed utilizing antibodies against STAT-1{alpha} or rabbit IgG (as a negative control). PCR analysis of the positive control (input) indicates that the soluble chromatin samples obtained from each time point had equal amounts of chromatin fragments containing the CD40 promoter (Fig. 4F ). STAT-1{alpha} was weakly associated with the CD40 promoter in untreated cells, and increased recruitment of STAT-1{alpha} was observed 0.5–1 h after IFN-ß treatment (Fig. 4F ). The recruitment of STAT-1{alpha} to the CD40 promoter decreased over time and returned to basal levels after 4–8 h of IFN-ß treatment (Fig. 4F ). There was no association of NF-{kappa}B p65 or STAT-2 with the CD40 promoter in untreated or IFN-ß treated RAW264.7 cells (data not shown). IFN-ß-induced STAT-1{alpha} recruitment was repressed in RAW-SOCS-1 cells compared with WT RAW264.7 cells (Fig. 4F ). These results indicate that the STAT-1{alpha} transcription factor is recruited to the CD40 promoter in a time-dependent manner upon IFN-ß treatment, and this recruitment is inhibited by SOCS-1 overexpression.

Covalent histone modifications are indicators of the recruitment of histone modifying complexes, such as histone acetyltransferases, histone deacetylases, and histone methyltransferases, and also have functional roles in gene transcriptional regulation (35 , 36) . To study histone modifications during IFN-ß-induced CD40 gene transcription, ChIP assays were performed. The acetylation of histone H3 increased 1 h after IFN-ß treatment, peaked at 4 h and was maintained at 8 h (Fig. 4F ). Enhanced acetylation of histone H4 was observed at 0.5 h after IFN-ß treatment, peaked at 4 h, and then decreased at 8 h (Fig. 4F ). These permissive IFN-ß-induced histone modifications were inhibited by SOCS-1 overexpression in RAW-SOCS-1 cells (Fig. 4F ).

ChIP assays were performed for the binding of RNA Pol II to the CD40 promoter after IFN-ß treatment. Pol II was weakly associated with the CD40 promoter in untreated RAW264.7 cells. Increased recruitment of Pol II was observed at 0.5 h after IFN-ß addition, peaked at 1 h, and then decreased by 8 h of IFN-ß treatment (Fig. 4F ). Ab against phospho-Ser-5 of the Pol II C-terminal domain (CTD) was used to examine changes in phosphorylation during CD40 gene transcription. Ser-5 phosphorylation of Pol II CTD was increased 0.5 h after IFN-ß stimulation, reached maximal levels at 2 h, and then diminished over time (Fig. 4F ). In contrast, recruitment of Pol II and phosphorylation of Pol IISer-5 were inhibited in RAW-SOCS-1 cells compared to WT RAW264.7 cells (Fig. 4F ). These results suggest that IFN-ß-induced recruitment of Pol II and phosphorylation of Pol IISer-5 on the CD40 promoter are concurrent with activation of the CD40 gene and that overexpression of SOCS-1 blocks the IFN-ß-induced general gene transcription machinery.

IFN-ß induces SOCS-1 gene expression in microglia
Because of the importance of SOCS-1 in negative regulation of IFN-ß-induced signaling and the essential role of microglia in inflammatory diseases of the central nervous system (CNS), the induction of SOCS-1 expression was investigated. Murine primary microglia and EOC13 microglial cells were utilized. Cells were incubated with medium or IFN-ß for up to 12 h, and then SOCS-1 mRNA expression was analyzed. Constitutive expression of SOCS-1 mRNA was undetectable, but SOCS-1 mRNA was induced at 1–4 h after addition of IFN-ß (Fig. 5 A and C). SOCS-1 protein expression was analyzed by immunoblotting. Again, no constitutive SOCS-1 protein expression was detected, whereas expression of SOCS-1 protein was detected between 1–8 h of IFN-ß treatment in murine primary microglia (Fig. 5B ) and between 1–12 h of IFN-ß treatment in the EOC13 cells (Fig. 5D ). These results indicate that IFN-ß induces SOCS-1 gene expression in microglia.


Figure 5
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  		Figure 5. IFN-ß induces SOCS-1 gene expression in microglia. Murine primary microglia (A) or EOC13 cells (C) were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 12 h, and then total RNA was isolated and analyzed by RPA for SOCS-1 and GAPDH mRNA. Murine primary microglia (B) or EOC13 cells (D) were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 24 h. Protein lysates were prepared and subjected to immunoblotting with anti-SOCS-1 Ab, stripped, and reprobed with anti-actin as a loading control. Representative of 3 experiments.

Ectopic expression of SOCS-1 inhibits IFN-ß-stimulated STAT activation and CD40 gene expression
To study the effects of SOCS-1 on IFN-ß-induced signaling activation, STAT activation was investigated in the RAW-SOCS-1 cells. Cells were incubated with medium or IFN-ß for up to 6 h, and protein lysates were subjected to SDS-PAGE analysis to detect the phosphorylation status of STAT-1{alpha} and STAT-2. IFN-ß-induced STAT-1{alpha} and STAT-2 tyrosine phosphorylation was strongly inhibited at all time points in RAW-SOCS-1 cells compared to WT RAW264.7 cells (Fig. 6 A). Total STAT-1{alpha} and STAT-2 expression levels were tested in these cells. Interestingly, the constitutive level of STAT-1{alpha} protein was enhanced with IFN-ß treatment (2–6 h) in RAW264.7 cells, and this enhancement was suppressed by SOCS-1 overexpression (Fig. 6A ). IFN-ß treatment had no effect on the expression of the STAT-2 protein in WT RAW264.7 cells and RAW-SOCS-1 cells (Fig. 6A ). Further studies showed that ectopic SOCS-1 expression inhibited IFN-ß-induced CD40 protein expression and promoter activation (Fig. 6B and C). These results indicate that SOCS-1 negatively regulates IFN-ß-induced CD40 gene expression.


Figure 6
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  		Figure 6. Ectopic expression of SOCS-1 inhibits IFN-ß-induced CD40 gene expression and promoter activity. A) RAW264.7 and RAW-SOCS-1 cells were treated with IFN-ß (100 U/ml) for up to 6 h. Protein lysates were prepared and subjected to immunoblotting with antiphospho-STAT-1{alpha}Tyr701 and antiphospho-STAT-2Tyr690, stripped, and reprobed with anti-STAT-1{alpha}, anti-STAT-2, and anti-actin antibodies. B) RAW264.7 and RAW-SOCS-1 cells were treated with IFN-ß (100 U/ml) for 24 h and CD40 protein surface expression was analyzed by FACS. Fold induction of CD40 protein was calculated and is mean ± SD of 3 experiments. C) RAW264.7 and RAW-SOCS-1 cells were transiently transfected with 0.2 µg of the CD40 promoter construct, allowed to recover for 4 h, treated with medium or IFN-ß (100 U/ml) for 12 h, and analyzed for luciferase activity. Values were normalized to total protein, and fold induction was calculated by dividing IFN-ß treatment values by untreated levels. Data are mean ± SD of 3 experiments.

DISCUSSION

IFN-ß is a pleiotropic cytokine with numerous immunoregulatory effects on cells of the innate and adaptive immune systems. Many effects of IFN-ß are immunosuppressive in nature, such as inhibition of class II major histocompatibility complex (MHC) expression on a variety of cell types (37) ; suppression of matrix metalloproteinases (MMP)-9 expression (38) ; inhibition of IL-12, which inhibits Th1 development; and induction of IL-10 (39) . In this study, we demonstrate that IFN-ß induces the expression of the costimulatory molecule CD40, which is critical for efficient antigen presentation to T cells, thereby leading to T cell activation. However, IFN-ß also induces expression of the SOCS-1 protein, which functions in a negative regulatory feedback loop to inhibit IFN-ß signaling and ultimately CD40 expression. We investigated the molecular mechanisms underlying IFN-ß-induced activation of the CD40 gene. IFN-ß induction of CD40 occurs at the transcriptional concentration and involves recruitment of the transcription factor STAT-1{alpha} as well as RNA Pol II to the CD40 promoter in vivo in a stepwise and coordinated order. IFN-ß also induced permissive modifications of histones H3 and H4 that were concurrent with activation of the CD40 gene. These findings illustrate that the coordination of cell signaling, histone modifications, and recruitment of transcription regulators is important for IFN-ß induction of CD40 in response to IFN-ß. The STAT-1{alpha} transcription factor is critical for IFN-ß induction of CD40 as STAT-1{alpha}-deficient microglia do not express CD40 in response to IFN-ß. We also showed that the STAT-2 transcription factor is not involved in IFN-ß-induced CD40 expression, as reduction of STAT-2 protein by siRNA techniques had no influence on IFN-ß induction of CD40 mRNA and protein expression. Although the CD40 promoter contains four NF-{kappa}B elements, IFN-ß induction of CD40 does not involve activation of NF-{kappa}B. In murine macrophages and microglia, IFN-ß does not activate the NF-{kappa}B signal transduction pathway (data not shown), in contrast to what has been reported for human fetal microglia (3) . Thus, activation of the JAK/STAT-1{alpha} pathway is the mechanism by which IFN-ß induces CD40 gene expression.

SOCS-1 is a critical regulator of the JAK-STAT signaling pathway (40) . In this study, we have presented data that IFN-ß induces SOCS-1 mRNA expression between 1–4 h, and protein expression between 2–12 h. The kinetics of endogenous SOCS-1 protein expression is correlated with the decrease in IFN-ß-induced CD40 mRNA expression at 12 h and subsequent diminution of CD40 protein at 24–72 h, depending on the cell type studied. Our results also demonstrate that ectopic expression of SOCS-1 negatively regulates IFN-ß-induced CD40 gene expression in macrophages, attenuating IFN-ß-induced CD40 mRNA and protein expression as well as promoter activity. Additionally, IFN-ß-induced STAT-1{alpha} phosphorylation and recruitment to the CD40 promoter was abolished by SOCS-1 overexpression, as was RNA Pol II and phospho-Pol IISer-5 recruitment and histone H3 and H4 permissive modifications. Interestingly, we also observed that SOCS-1 overexpression inhibited IFN-ß enhancement of STAT-1{alpha} protein expression, suggesting another aspect of SOCS-1 inhibition of IFN-ß signaling. We conclude that SOCS-1 inhibits IFN-ß-induced signaling and gene expression, including CD40 and STAT-1{alpha} expression in macrophages and microglia.

SOCS-1 is up-regulated by many cytokines, most notably IFN-{gamma} (32 , 41) . IFN-{gamma} induction of IRF-1 leads to IRF-1 binding to the AANNGAAA repeat sequence within the SOCS-1 promoter, which is required for IFN-{gamma}-induced SOCS-1 gene expression (42) . IFN-{gamma} induction of IRF-1 occurs in a STAT-1{alpha}-dependent manner (42) . In addition to IFN-{gamma}, LPS also induces SOCS-1 gene expression. The transcription factor Egr-1 is responsible for LPS-induced SOCS-1 gene expression by binding to the GC-rich sequence 5'-GCGGGGGCG-3' within the SOCS-1 promoter (43) . The transcription factors STAT-6 and Ets cooperate to mediate IL-4-induced SOCS-1 gene expression through a functional IL-4-responsive element located –684/–570 of the SOCS-1 promoter (44 , 45) . Recently, it has been reported that STAT-5 is implicated in erythropoietin-induced SOCS-1 gene expression (46) . Results from our study indicate that IFN-ß is a potent inducer of SOCS-1 expression and promoter activity. As IFN-ß is a strong inducer of IRF-1 (4) , this may be a mechanism for induction of SOCS-1 promoter activity by IFN-ß, similar to what has been described for IFN-{gamma} induction of SOCS-1 (42) . The element(s) critical for IFN-ß induced SOCS-1 promoter activity are under investigation in our laboratory.

IFN-ß is currently used for therapeutic treatment of patients with MS, although efficacy is attenuated over time (47) . The beneficial effects of IFN-ß are thought to be mediated by induction of IL-10 expression, suppression of MMP-9 expression, inhibition of vascular cell adhesion molecule-1 expression, improving the integrity of the blood-brain-barrier, and dampening T cell and macrophage inflammatory responses, all functions considered to be "anti-inflammatory" in nature (39 , 48 49 50) . The involvement of IFN-ß in experimental allergic encephalomyelitis was recently shown in a study utilizing IFN-ß deficient mice (51) . The lack of endogenous IFN-ß led to sustained inflammation and tissue damage due to augmented microglial activation and increased TNF-{alpha}, IFN-{gamma}, and IL-4 cytokine production (51) . SOCS-1 induction by IFN-ß will lead to suppression of IFN-ß signaling, which in the context of MS may contribute to the loss of effectiveness of IFN-ß in this particular disease. However, our study documents that IFN-ß also has "pro-inflammatory" actions such as induction of CD40 expression, which is then attenuated by SOCS-1 expression. Thus, the role of IFN-ß and SOCS proteins may be multifaceted in autoimmune diseases such as MS, with both beneficial and detrimental effects, depending on the gene under investigation. A more detailed analysis of the effect of SOCS-1 on other IFN-ß regulated genes in T cells, endothelial cells, astrocytes, and macrophages/microglia will provide important information on the role of SOCS-1 in cells involved in CNS inflammatory responses.

ACKNOWLEDGMENTS

This work was supported by NIH Grants NS-45290 and NS-36765 to E. N. Benveniste and a Pilot and Feasibility Grant from AR-48311 to H. Qin. We acknowledge the support of the University of Alabama at Birmingham Flow Cytometry Core Facility (AM20614). We thank Dr. Akihiko Yoshimura (Kurume University, Kurume-shi, Japan) for the generous gift of the mouse SOCS-1 cDNA, Dr. Randolph Noelle (Dartmouth Medical School, Lebanon, NH) for providing dominant-negative constructs of IKK-{alpha} and IKK-ß, Dr. Vincent Nguyen (University of Texas, Galveston) for generation of the CD40 promoter constructs, and Dr. Olaf Kutsch (University of Alabama at Birmingham, Birmingham, AL) for providing primary human macrophages cultures. We thank Drs. Max Cooper and Duane Wesemann (University of Alabama at Birmingham, Birmingham, AL) for providing the STAT-1{alpha}-deficient mice.

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