Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0803375 on December 23, 2003

Published online before print December 23, 2003
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(Journal of Leukocyte Biology. 2004;75:560-568.)
© 2004 by Society for Leukocyte Biology

p38 activation through Toll-like receptors modulates IFN-{gamma}-induced expression of the Tap-1 gene only in macrophages

Alicia A. Cecil1 and Michael J. Klemsz2

Department of Microbiology and Immunology, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis

2 Correspondance: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Dr., MS5010, Indianapolis, IN 46202. E-mail: mklemsz{at}iupui.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although interferon-{gamma} (IFN-{gamma}) induces the transporter associated with antigen processing (Tap)-1 expression in macrophages, cooperation with lipopolysaccharide signaling through Toll-like receptor 4 (TLR4) accelerates the kinetics and increases the overall levels of this gene. In this report, we show that peptidoglycan signaling through TLR2 and bacterial CpG DNA signaling through TLR9 are functionally equivalent at synergizing with IFN-{gamma} in regulating Tap-1 expression in macrophages. Activation of the p38 mitogen-activated protein kinase is necessary for this response, which correlates with increased phosphorylation of signal transducer and activator of transcription-1 on serine 727. Activation of p38, however, is not sufficient, as this signaling event does not affect the response to IFN-{gamma} in HeLa cells. The cooperation between these different signaling pathways also requires membrane fluidity. These data suggest that macrophages possess an ability to coordinate the signaling between the IFN-{gamma} and TLR receptors.

Key Words: gene regulation • transcription factor • macrophages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A cellular immune response is initiated when an antigen is presented to a naïve CD8+ T cell in the context of major histocompatibility complex class I (MHC I) proteins by a macrophage or dendritic cell (DC). For macrophages, the first step of antigen presentation is activation by interferon-{gamma} (IFN-{gamma}) or other cytokines, which modulate the expression of several critical proteins. One of these is the transporter associated with antigen processing (Tap)-1 gene, which helps pump peptides into the endoplasmic reticulum (ER), where they associate with the MHC I molecules [1 , 2 ]. Cells without Tap-1 express very low levels of surface MHC I as well as an inability to present antigens to T cells [3 ].

IFN-{gamma} alone can modulate Tap-1 gene expression in macrophages, resulting in maximal expression within 24 h [4 ]. This is followed at 48 h with a maximal increase in MHC I expression. It is interesting that activation of macrophages with IFN-{gamma} in conjunction with the bacterial product lipopolysaccharide (LPS) can accelerate the kinetics of Tap-1 and MHC expression as well as the overall levels of these proteins [4 ]. This combination of two signals, resulting in maximal Tap-1 gene expression within 4–8 h, would allow a macrophage to present antigens to T cells at a much earlier time during an immune response. Studies with the human macrophage cell line THP-1 have shown that the response to IFN-{gamma} and LPS is controlled at the transcriptional level, and these two signaling events modulate Tap-1 gene expression via a signal transducer and activator of transcription-1 (STAT1)-dependent mechanism [5 ].

STAT1 is a member of a family of transcription factors that allows rapid responses to cytokine stimulation [6 ]. This is because members of this family are already present in the cytoplasm of cells, and following cytokine stimulation, they become phosphorylated by receptor-associated kinases, dimerize, and move to the nucleus where they activate gene expression. In macrophages, the Janus tyrosine kinase (TK; JAK)1 and JAK2 are associated with the IFN-{gamma} receptor (IFN-{gamma}R) [7 ]. Upon signaling, these kinases phosphorylate STAT1 monomers on tyrosine 701. The monomers associate via Src homology 2 domain interactions to form a transcriptionally active STAT1 homodimer, which can gain access to the nucleus where it binds to IFN-{gamma} activation site (GAS) elements in STAT1-dependent promoters. Although tyrosine phosphorylation is important for activating STAT1, it has also been shown that maximal transcriptional activity of STAT1 is dependent on phosphorylation of serine residue 727 [8 ]. The phosphorylation of S727 can be seen in macrophages in response to many different stimuli, including IFN-{gamma} itself, as well as UV light, tumor necrosis factor {alpha} (TNF-{alpha}), and LPS [9 , 10 ]. This serine residue lies within a mitogen-activated protein kinase (MAPK) motif, and its phosphorylation correlates with the activation of the p38 MAPK by UV, TNF, and LPS. In contrast, IFN-{gamma} appears to stimulate serine 727 phosphorylation independently of p38 activation, potentially through the activation of a Ca2+/calmodulin-dependent kinase [11 ].

LPS and other bacterial products signal through members of the Toll-like receptor (TLR) family, which are characterized by their intracellular homology to the Drosophila Toll receptor [12 ]. The function of the TLRs is to recognize the characteristic repeating patterns found in microbial products such as LPS [13 ]. This family of 10 receptors recognizes a wide range of microbial products. For example, TLR2 recognizes peptidoglycan (PG) from gram-positive bacteria, [14 , 15 ], TLR4 recognizes LPS from gram-negative bacteria [16 17 18 ], and TLR9 recognizes unmethylated CpG DNA from bacteria [19 , 20 ]. Most studies on signaling through TLRs have focused on the similarities between the Toll receptors, the interleukin-1 receptor (IL-1R), and the Drosophila Toll protein [21 , 22 ]. This is a result of the homology between these proteins in their cytoplasmic domains and the ability of these pathways to activate nuclear factor (NF)-{kappa}B-regulated gene expression in cells. These studies have shown that TLRs, similarly to the IL-1R, activate gene expression through a MyD88-dependent mechanism [23 ]. Engagement of the TLR with the appropriate ligand results in the recruitment of MyD88 to their translation initiation region (TIR) domain. This triggers the dissociation of IL-1R-associated kinase (IRAK) from Toll-inhibitory protein, allowing IRAK to interact with MyD88 and phosphorylate itself as well as additional, unknown proteins [24 , 25 ]. Activated IRAK is released from the receptor complex, and it associates with TNF receptor-associated factor 6. The result is the activation of inhibitor of {kappa}B kinase complexes and the induction of the NF-{kappa}B pathway. Studies with MyD88 knockout mice have suggested that LPS interaction with TLR4 can also activate a MyD88-independent signaling cascade resulting in NF-{kappa}B activation and the expression of many of the same genes [23 , 26 ]. This pathway appears to have delayed kinetics and may involve the interaction of TIR domain-containing adaptor protein/MyD88 adaptor-like protein with the TIR domain of TLR4 [27 ]. The MyD88-independent signaling pathway, however, has not been shown for other TLRs. In addition to NF-{kappa}B, signaling through TLRs can activate multiple MAPK pathways, including c-jun NH2-terminal kinase, extracellular regulated kinase, and p38 [28 , 29 ].

In this study, we addressed whether ligands for TLR2 and TLR9 can modulate IFN-{gamma} signaling to further increase Tap-1 transcription in macrophages, as we have previously shown for LPS and TLR4. Our results show that PG and bacterial CpG DNA can synergize with IFN-{gamma} to up-regulate Tap-1 gene expression in the human macrophage cell line, THP-1. The synergy with IFN-{gamma} was also seen using the inflammatory cytokine TNF-{alpha}, and our results suggested that the activation of p38 by each of these was important for the induction of Tap-1 gene expression. In contrast, activation of p38 in the human epithelial cell line HeLa was not sufficient to phosphorylate STAT1 on serine 727 nor synergize with IFN-{gamma} in regulating Tap-1 expression. These results suggest that macrophages possess a unique ability to coordinate activating signals that will increase antigen processing and presentation for MHC I and thus enhance the immune system’s ability to mount a cellular immune response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
The human macrophage cell line, THP-1, was cultured in RPMI-1640 (BioWhittaker, Walkersville, MD) media, supplemented with 10% Fetalclone I (HyClone Laboratories, Logan, UT) at 37°C with 7.5% CO2. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker), supplemented with 5% Fetalclone I at 37°C with 7.5% CO2. Human IFN-{gamma} was purchased from Roche Molecular Biochemicals (Indianapolis, IN). LPS (Escherichia coli serotype 055:B5) was purchased from Sigma Chemical Co. (St. Louis, MO). PG from Neisseria gonorrhea was prepared and shown to be free of endotoxin as described [30 ] and was a gift from Dr. Raoul Rosenthal (Indiana University School of Medicine, Indianapolis). DNA (Type VIII from E. coli Strain B) was purchased from Sigma Chemical Co. TNF-{alpha} was a gift from Dr. David Donner (Indiana University School of Medicine). SB203580 and LY294002 were purchased from Calbiochem (La Jolla, CA). Methyl-ß-cyclodextrin (MßCD) was purchased from Sigma Chemical Co.

Reporter plasmids and transfections
For transfections, pLTP, containing the human Tap-1 promoter, and p(I/G)3Luc were used [5 ]. THP-1 cells were transfected using diethylaminoethyl Dextran as described [5 ]. Cells were allowed to recover from the transfection for ~18 h. Drugs were added at the indicated concentrations 30 min before the cells were stimulated with IFN-{gamma}, LPS, PG, DNA, or TNF-{alpha} for 4 h. Following stimulation, the cells were harvested, washed with cold phosphate-buffered saline (PBS), and lysed in a buffer containing 100 mM K2PO4 (pH 7.8) and 0.2% Triton-X 100. Luciferase activity was meausured on a Lumat luminometer. HeLa cells were transfected using Geneporter I, according to the manufacturer’s instructions (Gene Therapy Systems, San Diego, CA).

Northern blot analysis
Total RNA was prepared from the cells using Tri Reagent (Molecular Research Center, Cincinnati, OH). For Northern blots, 12 µg RNA was electophoresed on a 1.1% denaturing agarose gel containing formaldehyde. The RNA was transferred to a nylon membrane and probed as described previously [4 ]. Radioactive probes were prepared using High Prime (Roche Molecular Biochemicals). Blots were hybridized with the probes for 18 h, washed, and exposed to X-ray film.

Western blot analysis
Cell lysates were prepared using standard procedures. Briefly, cells were stimulated, pelleted by centrifugation, and washed with cold PBS. The cells were lysed (25 mM Tris HCl, pH 7.4, 0.05 mM EDTA, 75 mM NaCl with protease and phosphatase inhibitors at 1:100 from Sigma Chemical Co.) and incubated on ice for 15 min. Cellular debris was pelleted by centrifugation at 13K rpm for 15 min. Supernatants were collected, and protein concentration was determined using the DC assay kit from Bio-Rad (Hercules, CA). Equal amounts of total protein were boiled in 5x sample buffer [50% glycerol, 2.5% sodium dodecyl sulfate, 62.5 mM Tris, pH 6.8, and 0.1% bromphenol blue] and were electrophoresed on 10% Tris-glycine gels (Gradipore, Bountiful, UT). Separated proteins were electroblotted onto nitrocellulose (Schleicher and Schuell, Keene, NH). The membranes were blocked with 5% milk or 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST). Primary antibodies were used at the recommended dilutions in milk or BSA for 1 h. STAT1, phospho-STAT1 (Ser 727), and phospho-STAT1 (Tyr 701) were from Upstate Biotechnology (Lake Placid, NY). Phospho-p38 and p38 antibodies were from Cell Signaling Technology (Beverly, MA). Following primary antibody incubation, the blots were washed three times for 5 min in TBST. Secondary antibodies conjugated to alkaline phosphatase (AP; Bio-Rad) or horseradish peroxidase (HRP; Jackson Immunolabs, West Grove, PA) were then added at the recommended dilutions. To visualize proteins, the blots were incubated with Immune Star for AP-conjugated antibodies or enhanced chemiluminescence for HRP-conjugated antibodies and were then exposed to film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Multiple bacterial products synergize with IFN-{gamma} to increase Tap-1 promoter activity through a STAT1-dependent mechanism
We have previously shown that LPS signaling through TLR4 synergizes with IFN-{gamma} to increase Tap-1 gene expression in the human macrophage cell line THP-1. This synergistic increase was seen at the transcription level and was shown to be a result of STAT1 binding to the Tap-1 promoter. With the recent characterization of additional TLRs that are bound by other bacterial products, we investigated whether PG binding to TLR2 or bacterial CpG DNA binding to TLR9 would also synergistically increase Tap-1 transcription in THP-1 cells. Transfection of the Tap-1 reporter plasmid pLTP into THP-1 cells showed the characteristic, synergistic increase in reporter gene activity following stimulation with IFN-{gamma} and LPS, as compared with IFN-{gamma} alone (Fig. 1A ). Using PG or unmethylated, bacterial CpG DNA in combination with IFN-{gamma}, we saw similar increases above the levels seen for IFN-{gamma} alone (Fig. 1A) . This suggested that bacterial products signaling through multiple TLRs have a common ability to modulate IFN-{gamma}-induced Tap-1 gene expression.



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  		Figure 1. Multiple bacterial products synergize with IFN-{gamma} to increase Tap-1 promoter activity through a STAT1-dependent mechanism. (A) The full-length human Tap-1 promoter luciferase plasmid pLTP was transfected into THP-1 cells. Luciferase activity was measured following stimulation for 4 h with 100 U/ml IFN-{gamma}, 100 ng/ml LPS, 1 mg/ml PG, or 5 µg/ml bacterial CpG DNA alone or the combination of IFN-{gamma} and each of the bacterial components. (B) The p(I/G)3Luc plasmid, containing a trimer of the IFN-sensitive response element (ISRE)/GAS site from the human Tap-1 promoter, was transfected into THP-1 cells, followed by stimulation as described for A. Each plasmid and condition was repeated a minimum of four times, and a representative experiment is shown. RLU, Relative light units.

 
Our studies about LPS have shown that the synergistic induction of Tap-1 by IFN-{gamma} and LPS is mediated by STAT1. We next asked if TLR2 and TLR9 also synergize with IFN-{gamma} through a STAT1-dependent mechanism. To answer this question, we performed transfection studies in THP-1 cells using a luciferase reporter plasmid, p(I/G)3Luc, which contains only a minimal TK promoter and a trimerized ISRE/GAS site from the Tap-1 promoter. The results from these experiments showed that all three bacterial products in combination with IFN-{gamma} increased STAT1-dependent reporter activity, significantly above the level seen for IFN-{gamma} alone (Fig. 1B) . These results suggest that STAT1 is important for the synergistic induction of Tap-1 by IFN-{gamma} in combination with LPS, PG, or CpG DNA.

Activation of the p38 MAPK is necessary for synergy between IFN-{gamma} and TLR signaling
It has been suggested that phosphorylation of STAT1 on serine 727 by the p38 MAPK is a point of convergence between the IFN-{gamma} and LPS signaling pathways in murine macrophage cell lines [9 , 10 ]. As our transfection studies indicated that STAT1 was mediating the synergistic induction of Tap-1 when THP-1 cells were stimulated with IFN-{gamma} plus three different bacterial products, we asked if p38 was involved in this response for all three TLR ligands studied. To address this question, we used the specific p38 inhibitor SB203580 in our transfections to block p38 activation. THP-1 cells transfected with the p(I/G)3Luc plasmid were pretreated with SB203580 before the addition of IFN-{gamma} and bacterial product. These experiments showed that SB203580 blocks the ability of LPS, PG, or CpG DNA to synergize with IFN-{gamma} by reducing the fold induction in these samples to levels seen with IFN-{gamma} alone (Fig. 2 ). The drug did not affect the fold induction of samples stimulated with only IFN-{gamma}. In contrast, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 or the MAPK (non-p38) inhibitor PD98059 had no effect on the synergy (data not shown). Thus, p38 appears to play a critical role in the integration of signals from TLR2, -4, and -9 and the IFN-{gamma}R on STAT1-regulated Tap-1 gene expression.



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  		Figure 2. Activation of the p38 MAPK is necessary for synergy between IFN-{gamma} and TLR signaling. The p(I/G)3Luc plasmid containing a trimer of the ISRE/GAS site from the human Tap-1 promoter was transfected into THP-1 cells. Cells were stimulated with IFN-{gamma} alone or in combination with three different bacterial products, as described for Figure 1 . In addition, transfected cells were incubated with the p38 MAPK inhibitor SB203580 (10 µM) before stimulation. Each plasmid and condition was repeated a minimum of four times, and a representative experiment is shown. DMSO, Dimethyl sulfoxide.

 
TNF-{alpha} signaling synergizes with IFN-{gamma} through the activation of p38
These studies extended our understanding of a role for p38 activation via several TLRs on Tap-1 gene expression. We next wanted to know if the synergy with IFN-{gamma} signaling was specific to TLRs or if other cytokines that activate p38, such as TNF-{alpha}, could also modulate IFN-{gamma}-induced Tap-1 gene expression. To address this question, we transfected pLTP or p(I/G)3 Luc into THP-1 cells and stimulated them with TNF-{alpha} or LPS alone or with IFN-{gamma}. The results from these studies showed TNF-{alpha} in combination with IFN-{gamma} induced either reporter plasmid to similar levels seen with LPS, and these levels were significantly higher than IFN-{gamma} alone (Fig. 3A ). In addition, pretreatment of the transfected cells with SB203580 blocked the synergy between TNF-{alpha} and IFN-{gamma}, which suggests that the synergy was a result of the activation of p38 (Fig. 3B) . In additional experiments, we saw similar results using IL-1, which also activates p38 (data not shown). These data suggest that activation of p38 through a wide variety of receptors can modulate Tap-1 promoter activity through its STAT1-binding site.



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  		Figure 3. TNF-{alpha} signaling synergizes with IFN-{gamma} through the activation of p38. (A) The Tap-1 promoter luciferase plasmid pLTP or the p(I/G)3Luc plasmid was transfected into THP-1 cells. Luciferase activity was measured following stimulation for 4 h with 100 U/ml IFN-{gamma}, 100 ng/ml LPS, or 1 nM TNF-{alpha} alone or the combination of IFN-{gamma} and LPS or TNF-{alpha}. (B) The p(I/G)3 Luc plasmid was transfected into THP-1 cells, as described above with or without incubation with the p38 MAPK inhibitor SB203580. Fold induction is shown. Each plasmid and condition was repeated a minimum of four times, and a representative experiment is shown.

 
Multiple bacterial products and TNF-{alpha} synergize with IFN-{gamma} to increase Tap-1 mRNA levels in THP-1 cells
From our transfection studies, we have shown that multiple inflammatory agents will synergize with IFN-{gamma} through STAT1 to up-regulate reporter gene expression. To show that these agents increase the level of Tap-1 mRNA expression, we performed Northern blot analysis on THP-1 cells treated for 24 h with IFN-{gamma} alone or in combination with LPS, bacterial CpG DNA, or TNF-{alpha}. Consistent with the transfection data, the results showed that LPS, DNA, or TNF-{alpha} plus IFN-{gamma} increased the level of Tap-1 mRNA expression significantly more than seen for IFN-{gamma} alone (Fig. 4 ). Together, these results indicate that multiple TLRs and other receptors that activate p38 can cooperate with IFN-{gamma} signaling pathways to synergistically increase Tap-1 expression in macrophages.



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  		Figure 4. Multiple bacterial products and TNF-{alpha} synergize with IFN-{gamma} to increase Tap-1 mRNA levels in THP-1 cells. Northern blot analysis was used to assess changes in Tap-1 mRNA expression in THP-1 cells stimulated with 100 U/ml IFN-{gamma} alone or in combination with 100 ng/ml LPS, 5 µg/ml bacterial CpG DNA, or 1 nM TNF-{alpha}. The blot was reprobed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control.

 
Bacterial CpG DNA, LPS, and TNF-{alpha} activate p38 and increase STAT1 serine 727 phosphorylation
PG and bacterial CpG DNA, as well as TNF-{alpha}, are functionally equivalent to LPS in their ability to cooperate with IFN-{gamma} signaling and up-regulate Tap-1 promoter activity and Tap-1 mRNA expression. Our use of the p38 inhibitor suggested a role for this MAPK in the synergistic response, possibly through modulating STAT1 phosphorylation. To determine whether in THP-1 cells the different TLRs and TNF-{alpha} can activate p38 and induce serine 727 phosphorylation of STAT1, we used DNA as a representative bacterial product in comparison with LPS and TNF-{alpha} in a series of Western blot analyses. Extracts were prepared from THP-1 cells stimulated for 20 min with IFN-{gamma}, LPS, DNA, TNF-{alpha} alone, or each of these stimuli in combination with IFN-{gamma}. As seen, stimulation with LPS, DNA, and TNF-{alpha} activated p38, as seen by its increased phosphorylation (Fig. 5A ). Stimulation with IFN-{gamma} alone had no effect on p38 phosphorylation nor did it alter the levels seen with any of the other stimuli. We next asked whether the activation of THP-1 cells with LPS, CpG DNA, or TNF-{alpha} alone or in combination with IFN-{gamma} resulted in increased STAT1 phosphorylation. As seen, stimulation with LPS, CpG DNA, or TNF-{alpha} alone resulted in increased STAT1 serine 727 but not tyrosine 701 phosphorylation (Fig. 5B and 5C) . IFN-{gamma} was also able to induce a similar increase in serine 727 phosphorylation through a p38-independent mechanism and the characteristic induction of tyrosine 701 phosphorylation. The combination of LPS, CpG DNA, or TNF-{alpha} with IFN-{gamma} resulted in higher levels of STAT1 serine 727 phosphorylation than seen for any stimuli alone. These data suggest that synergistic increases in Tap-1 gene expression following stimulation with IFN-{gamma} and bacterial products or TNF-{alpha} are a result of activation of p38 and increased phosphorylation of STAT1 on serine 727.



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  		Figure 5. Bacterial CpG DNA, LPS, and TNF-{alpha} activate p38 and increase STAT1 serine 727 phosphorylation (pS727). Western blot analysis was performed to assess changes in p38 MAPK activation and STAT1 serine 727 phosphorylation following the activation of THP-1 cells. Cells were stimulated for 20 min with the indicated agents. (A) Activation of p38 MAPK (ppP38) was determined by assaying its phosphorylation status with phosphospecific antisera. The blot was reprobed for total p38 protein. (B and C) STAT1 serine and tyrosine phosphorylation was determined with phosphospecific antisera. The blots were reprobed for total STAT1 protein. pY701, Tyrosine 701 phosphorylation.

 
Removal of cholesterol blocks synergy between IFN-{gamma} and LPS
It has been hypothesized that cell membranes have distinct domains based on lipid content that define regions involved in signaling [31 ]. These domains have high concentrations of sphingolipids and cholesterol, which allow the domains to move through the lipid bilayer. Our results suggest that movement of the IFN-{gamma} and TLRs and their associated signaling molecules within the membrane may be important for the induction of Tap-1 expression in macrophages. To test this, we used MßCD, which has been shown to deplete cholesterol from plasma membranes and disrupt trafficking of proteins [32 ]. Treatment of THP-1 cells with MßCD following transfection with the STAT1-responsive p(I/G)3Luc reporter plasmid showed that depletion of cholesterol blocked the synergy between IFN-{gamma} and LPS (Fig. 6A ). The addition of this drug up to 30 min after stimulation with IFN-{gamma} and LPS reduced the levels of luc to those seen with IFN-{gamma} alone. In contrast, addition of MßCD after 2 h had less ability to disrupt the synergy, suggesting that membrane fluidity was necessary for combining proximal signaling events between the IFN-{gamma} and TLRs.



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  		Figure 6. Removal of cholesterol blocks synergy between IFN-{gamma} and LPS. To study membrane fluidity and cooperation between IFN-{gamma}R and TLR signaling pathways, the cholesterol-depleting drug MßCD was used in transfection and Western blot analyses. (A) Following transfection of the p(I/G)3Luc plasmid, THP-1 cells were treated with MßCD (10 µM) at the indicated times in relation to stimulation with IFN-{gamma}, LPS, or both agents. Luciferase activity was measured and is reported as fold increases. This experiment was repeated three times, and a representative experiment is shown. (B) Western blot analysis was used to determine if loss of cholesterol and membrane fluidity could disrupt STAT1 phosphorylation. As shown, cells were left alone or pretreated with MßCD (10 µM) for 30 min followed by stimulation with the indicated agents for 20 min. STAT1 serine 727 (pS727) and tyrosine 701 phosphorylation (pY701) was determined using phosphospecific antisera. The blot was reprobed for total STAT1 protein levels.

 
To determine how MßCD blocked synergy between IFN-{gamma} and TLRs, we asked whether removal of cholesterol blocked phosphorylation of STAT1 on serine 727. Addition of MßCD to THP-1 cells before treatment with IFN-{gamma} and LPS decreased the levels of phosphorylation on serine 727 but had no effect on phosphorylation of tyrosine 701 (Fig. 6B) . Densitometric analysis showed serine phosphorylation on STAT1 was reduced by ~70%. These results suggest that cooperation between IFN-{gamma} and TLR signaling requires the movement of membrane domains containing cholesterol.

p38 activation alone is not enough to modulate STAT1 phosphorylation or activate Tap-1 gene expression
Our previous studies have shown that the activation of nonprofessional antigen-presenting cells (APCs) such as HeLa cells with IFN-{gamma} and LPS, does not result in a synergistic increase in Tap-1 gene expression. One possible reason could be that signaling through a TLR does not activate p38 in HeLa cells. To address this, extracts were prepared from HeLa cells stimulated with LPS or TNF-{alpha} alone or in combination with IFN-{gamma}. Western blot analysis showed that LPS and TNF-{alpha} activated p38 in HeLa cells (Fig. 7A ). It is also interesting to note the higher levels of p38 activation seen following stimulation with TNF-{alpha}, as compared with LPS. Thus, these data show that two different stimuli can activate p38 in HeLa cells. We next asked whether the activation of p38 resulted in increased levels of STAT1 serine 727 phosphorylation in HeLa cells. As seen, only IFN-{gamma} stimulation resulted in phosphorylation of STAT1 on serine 727 or tyrosine 701 (Fig. 7B) . In contrast, stimulation with LPS or TNF-{alpha} alone or in combination with IFN-{gamma} had no effect on serine 727 phosphorylation. These data show that despite high levels of p38 activation following stimulation with LPS or TNF-{alpha}, this does not translate into an effect on STAT1. To support these data, we transfected the p(I/G)3Luc plasmid into HeLa cells and stimulated with IFN-{gamma} alone or in combination with LPS or TNF-{alpha}. The data showed that the addition of LPS or TNF-{alpha} to IFN-{gamma} had no effect on the regulation of this STAT1-regulated reporter plasmid. These data suggest that the reason no synergy is seen in HeLa cells is a result of the inability to translate the activation of p38 into the phosphorylation of serine 727 on STAT1.



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  		Figure 7. p38 activation alone is not enough to modulate STAT1 phosphorylation or activate Tap-1 gene expression. Western blot analysis was performed to assess changes in p38 MAPK activation and STAT1 serine 727 phosphorylation following the activation of HeLa cells. Cells were stimulated for 20 min with the indicated agents. (A) Activation of p38 MAPK (ppP38) was determined by assaying its phosphorylation status with phosphospecific antisera. The blot was reprobed for total p38 protein. (B and C) STAT1 serine 727 (pS727) and tyrosine 701 phosphorylation (pY701) was determined with phosphospecific antisera. The blots were reprobed for total STAT1 protein. PDGF, Platelet-derived growth factor. (D) Transfection of the p(I/G)3Luc plasmid into HeLa cells, followed by stimulation for 4 h with the indicated agents. Fold induction of luciferase activity is shown for a representative experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The induction of Tap-1 gene expression in macrophages is critical for the transport of peptides into the ER and the presentation of antigens by MHC I molecules. We have previously shown that LPS will synergize with IFN-{gamma} to induce expression of the Tap-1 gene in macrophages. In this study, we investigated whether other bacterial products would also synergize with IFN-{gamma} to modulate Tap-1 expression. As shown by Northern blot analysis, bacterial CpG DNA and TNF-{alpha} in combination with IFN-{gamma} were able to significantly induce Tap-1 mRNA expression above the levels seen for stimulation with IFN-{gamma} alone. Transfection of the human macrophage cell line THP-1 with a Tap-promoter luciferase reporter plasmid showed that IFN-{gamma} would also synergize with PG, bacterial CpG DNA, as well as TNF-{alpha}, and inhibitor studies suggested that activation of the p38 MAPK was necessary for this response. Using the isolated STAT1-binding element, reporter plasmid p(I/G)3Luc showed that this response was controlled by STAT1. Western blot analysis showed that bacterial CpG DNA and TNF-{alpha} induced phosphorylation of STAT1 on serine 727 in THP-1 cells. In the nonmacrophage line HeLa, however, activation of p38 by LPS or TNF-{alpha} had no effect on STAT1 serine phosphorylation or luciferase reporter activity. This suggested that activation of p38 is not sufficient for cooperation between the IFN-{gamma} and TLRs. Finally, studies using MßCD to deplete cholesterol from plasma membranes showed that membrane fluidity was important for the phosphorylation of STAT1 on serine 727 and the synergistic signaling between the IFN-{gamma} and TLRs. These findings indicate that professional APCs, such as macrophages, have the unique capacity to combine signals from bacterial products and IFN-{gamma} to up-regulate expression of genes used for the presentation of antigens.

As STAT1 can be phosphorylated on tyrosine and serine residues, which results in its maximal transcriptional activity, it has emerged as an important protein, where IFN-{gamma} and other signaling pathways could combine in macrophages. In murine macrophages, it has been shown that activation of the p38 MAPK by LPS correlates with STAT1 serine phosphorylation [9 , 10 ]. When this signal was combined with IFN-{gamma}, higher levels of reporter gene expression were seen. Additional studies have shown that stimulation of macrophages and other cells with a wide variety of stimuli, including UV light, TNF-{alpha}, and types I and II IFNs, can result in increased serine 727 phosphorylation on STAT1 [9 , 10 , 33 , 34 ]. Thus, modulating IFN-{gamma}-induced gene expression could be controlled by how signals from different stimuli activate serine phosphorylation. In this report, we add PG, bacterial CpG DNA, as well as TNF-{alpha} as stimuli that cannot only activate phosphorylation on STAT1 serine 727 but will also synergize with IFN-{gamma} to regulate the expression of the Tap-1 gene in the human macrophage cell line THP-1. For signaling through TLRs, these studies suggest that TLR2 and TLR9 are functionally equivalent to TLR4 in their ability to activate p38 and cooperate with IFN-{gamma} during the activation of macrophages.

In contrast, our studies using an epithelial cell line suggest that activation of p38 is not sufficient for activating serine 727 phosphorylation on STAT1 and modulating IFN-{gamma}-induced gene expression. Despite high levels of p38 activation by LPS or TNF-{alpha}, neither stimuli increased the level of serine 727 phosphorylation in HeLa cells. This suggests that cooperation between these signaling pathways is specific for defined cell lineages. One possibility is that macrophages are able to uniquely coordinate signaling through IFN-{gamma} and TLR receptors. In support of this, addition of MßCD blocked the synergy between IFN-{gamma} and TLR4 and the increased phosphorylation of STAT1 on serine 727, normally seen in the macrophage line THP-1. As this drug removes cholesterol from plasma membranes [32 ], our data suggest that membrane fluidity is important for the transfer of a TLR signal to p38 activation and STAT1 serine phosphorylation. It is interesting that we also observed an enhanced level of tyrosine phosphorylation on STAT1 when we stimulated THP-1 cells in the presence of MßCD with IFN-{gamma} alone. Our transfection results suggested that an equivalent amount of STAT1 is translocating to the nucleus with or without MßCD. It has been reported that the IFN-{gamma}R-1 (IFNGR-1) subunit normally translocates away from the plasma membrane following stimulation with IFN-{gamma} [35 ]. Treatment of cells with filipin, which binds cholesterol and blocks the function of lipid domains that contain caveolae, blocked the movement of IFNGR-1. This suggests that blocking membrane fluidity may have the effect of keeping components of the IFN-{gamma}R signaling complex in close proximity at the plasma membrane.

Within the plasma membrane, lipid microdomains or lipid rafts are emerging as an important platform for concentrating receptors and associated signaling complexes [31 ]. These structures contain sphingolipids and cholesterol, which allow for the inclusion or exclusion of specific proteins as well as move as a coordinated unit throughout the membrane bilayer. Components of the IFN-{gamma}R complex including STAT1 have been found in these lipid domains [35 , 36 ]. It has also been reported that molecules involved in LPS signaling, including TLR4 and CD14, are found in lipid rafts [37 ]. Although our studies using MßCD suggest that raft movement is important for coordinating signaling between the IFN-{gamma} and TLR4 receptor complexes, we have not detected any movement of either of these receptor complexes into or out of a lipid raft fraction isolated using sucrose gradients (A. A. Cecil and M. J. Klemsz, unpublished observation).

In this report, we show that TLR2 and TLR9 are functionally equivalent to TLR4 in their ability to synergize with IFN-{gamma} in regulating Tap-1 gene expression in macrophages. Activation of the p38 MAPK is necessary but not sufficient for the enhanced expression of this gene, as increased phosphorylation of STAT1 on serine 727 was seen only in the macrophage line THP-1. These results and others show how signals normally associated with innate immunity can enhance the expression of genes required for the ability of macrophages to present antigens via MHC I molecules and initiate the acquired immune response.


   ACKNOWLEDGEMENTS
 
United States Public Health Service Grant CA71384 supported this work.


   FOOTNOTES
 
1 Current address: Department of Applied Biology, Rose-Hulman Institute of Technology, 5500 Wabash Ave., Terre Haute, IN 47803. Back

Received August 11, 2003; revised October 21, 2003; accepted November 13, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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