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Inhibition of Tumor Necrosis Factor--Inducible Inflammatory Genes by Interferon- Is Associated with Altered Nuclear Factor-B Transactivation and Enhanced Histone Deacetylase Activity

Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania

Received August 24, 2006; accepted November 15, 2006

	Abstract

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Airway smooth muscle (ASM) cells can act as effector cells in the initiation and/or perpetuation of airway inflammation in asthma by producing various inflammatory chemokines or cytokines. Previous studies from our laboratory and others showed that the combination of tumor necrosis factor- (TNF) and interferon- (IFN) or endogenous IFN results in a synergistic induction of various pro-inflammatory genes, including CD38 and regulated upon activation normal T-cell expressed and secreted (RANTES), in ASM cells. In contrast to these studies, we found that IFN (1000 U/ml) markedly inhibited TNF-induced expression of interleukin (IL)-6, IL-8, and eotaxin by 66.29 ± 3.33, 43.86 ± 7.11, and 63.25 ± 6.46%, respectively. These genes were also found to be NF-B-dependent in that TNF-induced expression of IL-6, IL-8, and eotaxin was dose-dependently inhibited by the selective IKK inhibitor 4-(2'-aminoethyl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline (BMS-345541) (1-30 µM). Using a luciferase reporter construct containing B sites, we found that IFN (10-1000 U/ml) inhibits NF-B-dependent gene transcription in a dose-dependent manner. Moreover, IFN failed to affect TNF-induced IK phosphorylation or IB degradation as well as nuclear NF-B/DNA interaction. It is noteworthy that IFN decreases TNF-induced histone acetyl transferase (HAT) and increases histone deacetylase (HDAC) activities. Finally, trichostatin A, an HDAC inhibitor, prevents IFN inhibitory action on TNF-induced gene expression. Together, our data indicate that IFN is a potent inhibitor of specific TNF-inducible inflammatory genes by acting on NF-B transactivation via the modulation of HDAC function.

Several lines of evidence demonstrate that TNF could play a role in the pathogenesis of asthma through a direct immuno-modulatory action on ASM cells (Halayko and Amrani, 2003). In cultured ASM cells, TNF, alone or in combination with other cytokines, such as IL-1 or IL-13, has been shown to stimulate the expression of various "pro-asthmatic" mediators, including cytokines (IL-6), chemokines (eotaxin, Rantes, IL-8) as well as adhesion molecules (ICAM-1, VCAM-1) (Halayko and Amrani, 2003). In addition, TNF also cooperates with type II IFN to synergistically induce different pro-inflammatory proteins such as RANTES (John et al., 1997), fractalkine (Sukkar et al., 2004), or CD38 (Tliba et al., 2006), although the underlying mechanisms of such cooperation have not been elucidated. In that regard, we made the interesting observation that induction of defined genes including RANTES as well as CD38 by TNF occurred, at least in part, via the secretion and autocrine action of endogenous IFN (Tliba et al., 2003, 2004). We were surprised to find that endogenous IFN acted as a negative regulator of IL-6 production induced by TNF (Tliba et al., 2003). This suggests that, in addition to exerting cooperative effects, IFNs also induce antagonistic effects on TNF-inducible genes. This hypothesis is supported by other reports in ASM showing that exogenous IFN can suppress TNF-inducible inflammatory genes including vascular endothelial growth factor (Wen et al., 2003), IL-17 receptor expression (Lajoie-Kadoch et al., 2006), and CD38 (Tliba et al., 2006). This is an important finding because a better understanding of the inhibitory mechanisms exerted by IFN on TNF-inducible inflammatory genes may offer new insight into the design of alternative approaches for the treatment of airway inflammation in asthma.

The purpose of the present study was to assess the suppressive mechanisms of IFN on TNF-inducible pro-asthmatic genes in human ASM cells. We found that IFN acts as a potent inhibitor of TNF-inducible, NF-B-sensitive genes, including IL-6, IL-8, and eotaxin. TNF-induced NF-B acetylation, transactivation, and HAT function were impaired by IFN, and HDAC activity was significantly enhanced. TSA, a specific HDAC inhibitor, was able to reverse IFN inhibitory effects on TNF-inducible genes. We therefore propose that IFN could negatively regulate expression of pro-inflammatory genes induced by TNF by impairing NF-B function through transcriptional repression exerted by an increased HDAC activity.

	Materials and Methods

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Smooth Muscle Cell Culture and Characterization. Human ASM cell culture was performed as described previously (Tliba et al., 2003). Human trachea was obtained from lung transplant donors in accordance with the procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings.

Western Blotting. Immunoblot analysis for total NF-B p65, total IKK, phosphorylated IKK and IKB, and anti-acetyl-lysine was performed as described previously (Amrani et al., 1999). In brief, cells were lysed in buffer containing 10 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 0.1% deoxycholate, 10 µg/ml leupeptin, 100 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 mM EDTA, 10 mM NaF, and 2 mM Na₃VO₄ for 20 min at 4°C. Postnuclear extracts were obtained by centrifugation of lysates at 14,000g for 10 min. Antibodies against total p65, total IKK, IB (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated IKK (Cell Signaling Technology, Danvers, MA) or anti-acetyl lysine antibody (2 µg/ml, clone 4G12; Upstate Biotechnology, Chicago, IL) were used as indicated by the manufacturer's instructions. Immunoprecipitation using NF-B p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was performed as indicated by the manufacturer's instructions. Equal amounts of protein were analyzed by 4 to 12% SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The membranes were blocked in 5% milk or 5% bovine serum albumin (anti-phospho-protein antibodies) in Tris-buffered saline for 1 h and then incubated overnight with the primary antibody of interest at 4°C. After incubation with the appropriate peroxidaseconjugated secondary antibody (Roche Applied Science, Indianapolis, IN), the bands were visualized by the enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and autoradiographed.

Transfection of ASM Cells. Because most standard transfection methods yield poor transfection efficiencies for ASM cells (10%), here we have optimized a high-efficiency transfection technique (Tliba et al., 2006). This technique is an extension of electroporation, using the Nucleofector kit for primary smooth muscle (Amaxa Biosystems, Cologne, Germany), in which plasmid DNA is transfected directly into the cell nucleus. Transfection was performed according to the manufacturer's instructions, and the program used was U-25. Sixteen to eighteen hours after transfection, the media were changed with serum-free media for the next 24 h. This method, using green fluorescence protein-pmax control vector (Amaxa Biosystems), enabled us to reach a transfection efficiency of 70%.

SEAP and -Galactosidase Assays. To monitor NF-B-dependent gene expression, ASM cells were cotransfected with B-secreted alkaline phosphatase (SEAP) reporter vector and with pSV--galactosidase vector used to normalize transfection efficiencies (Promega, Madison, WI). The activities of SEAP and -galactosidase were evaluated using Great Escape SEAP detection kit (Clontech, Mountain View, CA) and -galactosidase detection kit (Promega), respectively, according to the manufacturer's instructions.

RNA Isolation and Reverse Transcriptase PCR Analysis. Human ASM cells were serum-deprived in medium containing 0.1% fetal bovine serum for 24 h and exposed to 10 ng/ml TNF to 1000 U/ml IFN and TNF/IFN in combination for 24 h. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Reverse transcriptase PCR analysis of IL-6, IL-8, and eotaxin was then performed as reported previously (Amrani et al., 2000). Primers used for IL-6: forward, 5'-CCAGCTATGAACTCCTTCTCCACAAGC-3'; reverse, 5'-GCTGGACTGCAGGAACTCCTTAAAGC-3'; IL-8: forward, 5'-ATGACT TCCAAGCTGGCCGTGGCT-3'; reverse, 5'-TCTCAGCCCTCTTCAAAAACT TCTC-3' (Zhu et al., 2003); eotaxin: forward, 5'-GGATCCAACATGAAGGTCTCCG-3', and reverse, 5'-GAATTCTTATGGCTTTGGAGTTGGAG-3'(Nie et al., 2005). The semiquantitative PCR approach was performed in parallel by investigating human GAPDH mRNA levels with the following primers: forward, 5'-ATGGATGATGATATCGCCGC-3'; reverse, 5'-TTAATGTCACGCACGATTTC-3'.

PCR was performed for 30 cycles at 94°C denaturation, 60°C annealing, and 72°C extension using Taq DNA polymerase (Promega). Reaction products were confirmed on 1% agarose (Fisher Biotech, Fair Lawn, NJ) gels with size markers (New England Biolabs, Ipswich, MA) and stained with ethidium bromide. The intensity of density area was analyzed using a Gel-Pro Analyzer (Silver Spring, MD). The final PCR product was expressed as the ratio to Actin used for scanning analysis.

NF-B p65/DNA Interaction. Nuclear extraction was performed as described previously (Tliba et al., 2003, 2006). Five micrograms of nuclear extract wastested for p65-DNA binding activity by using TransAM NF-B p65 kit, using a wild type sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and a specific NF-B p65 antibody, according to the manufacturer's instructions (Active Motif, Carlsbad, CA). The results (optical density measured at 450 nm) were expressed as percentage increase over basal (untreated cells).

Total Acetylation and Deacetylation Analyses. Nuclear extracts derived from cytokines treated or control cells were prepared as described previously (Tliba et al., 2003, 2006). Fifty micrograms of nuclear extracts were used for assessing HAT and HDAC activities according to the manufacturer's instructions [Oxford Biomedical (Oxford, MI) and Upstate Biotechnology, respectively].

Materials and Reagents. Tissue culture reagents and primers used for PCR were obtained from Invitrogen (Carlsbad, CA). Human rTNF was provided by Roche Applied Science (Indianapolis, IN). rIFN, was purchased from R&D Systems. IKK inhibitor (BMS-345541) and HDAC inhibitor trichostatin A (TSA) were purchased from Calbiochem (San Diego, CA). Cytokine concentrations in the culture media were determined by enzyme-linked immunosorbent assay (Tliba et al., 2003).

Statistical Analysis. To compare differences between treatment means (expressed as mean ± S.E.), all data were subjected to one- or two-way analysis of variance when experiments were of a factorial design. After analysis of variance, Fisher's method of protected least significant differences was used as a multiple comparison test. Comparison of two populations was made with Student's t test. Values of P < 0.05 were sufficient to reject the null hypothesis for all analyses.

View larger version (28K): [in this window] [in a new window]   Fig. 1. IFN inhibited TNF-induced IL-6, IL-8, and eotaxin expression. ASM cells were incubated with 10 ng/ml TNF, with 1000 U/ml IFN and with both cytokines at indicated concentrations of IFN for 24 h. IL-6 (A), IL-8 (B), and eotaxin (C) gene expression were measured. The results are expressed in picograms per milliliter ± S.E.M. of three separate experiments. #, P < 0.001 compared with basal condition; *, P < 0.05 compared with cells treated with TNF alone; **, P < 0.001 compared with cells treated with TNF alone. D, cells were lysed, total mRNA was extracted, and reverse transcription-PCR was performed using specific IL-6, IL-8, and eotaxin primers, as described under Materials and Methods, under the same conditions (basal, TNF, IFN, TNF/IFN). The results show representative gels of three experiments for each gene.

	Results

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IKK Inhibitor BMS-345541 Suppressed TNF-Induced Expression of Inflammatory Genes. Although multiple NF-B binding sites are present in the promoters of different genes (Baldwin, 1996), few reports have examined the putative role of NF-B pathways in the regulation of inflammatory genes in human ASM cells. Using the selective inhibitor of IKK BMS-345541 (IC₅₀ = 0.3 µM) (Burke et al., 2003), we found that TNF-induced expression of IL-6, IL-8, and eotaxin was suppressed in a dose-dependent manner (1-30 µM) reaching the basal level (Figs. 2 A-C; P < 0.001). The specificity of BMS-345541 concentrations was confirmed using a NF-B reporter plasmid. As shown in Fig. 5A, TNF induced a time-dependent (6-24 h) increase in NF-B reporter activity that was completely abrogated in cells pretreated with the IKK inhibitor BMS-345541. We were also interested to find that IFN inhibited TNF-induced NF-B reporter activity in a concentration-dependent manner (Fig. 3B). Together, these results show that IKK plays an important role in mediating TNF-induced gene expression and raises the hypothesis that IFN interferes with TNF-induced gene expression by suppressing the NF-B pathway.

View larger version (20K): [in this window] [in a new window]   Fig. 2. The IKK inhibitor suppressed TNF-induced IL-6, IL-8, and eotaxin expression. A, ASM cells were treated with TNF (10 ng/ml) in the presence or absence of IKK inhibitor (BMS-345541) added alone at 30 µM or 1 h before TNF stimulation at the indicated concentrations (ranging from 1 to 30 µM). IL-6 (A), IL-8 (B), and eotaxin (C) expression was assessed by enzyme-linked immunosorbent assay as described under Materials and Methods. The results are expressed in picograms per milliliter of three separate experiments. #, P < 0.001 compared with basal condition; *, P < 0.05 compared with cells treated with TNF alone; **, P < 0.001 compared with cells treated with TNF alone; NS, nonsignificant compared with untreated cells.

View larger version (23K): [in this window] [in a new window]   Fig. 5. IFN inhibited TNF-induced histone acetyltransferase activity (A) and NF-B p65 acetylation (B). A, cells were stimulated with TNF (10 ng/ml), IFN, or the combination at the indicated time and lysed; nuclear extracts were prepared for HAT activity as described in Materials and Methods. B, cells stimulated with TNF, IFN, or the combination for 2 h were lysed, immunoprecipitated with anti-p65 antibody, and assayed for anti-acetyl lysine by immunoblot analysis (gradient 4-12% SDS-PAGE). The results are expressed as increase over basal. #, P < 0.005 compared with untreated cells (basal); ##, P < 0.001 compared with untreated cells (basal); **, P < 0.005 compared with cells treated with TNF alone.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Both IK inhibitor and IFN suppressed TNF-induced NF-B-dependent gene expression. ASM cells were stimulated with TNF (10 ng/ml), and the supernatant was taken for 6 and 24 h. Cells were transfected with SEAP reporter construct driven by a B site as described under Materials and Methods to assess NF-B gene expression. A, cells were treated with IKK inhibitor (BMS-345541, 30 µM) 1 h before TNF stimulation. B, cells were treated with 10 ng/ml TNF alone or in combination with IFN (10 and 1000 U/ml). Data are representative of three separate experiments. #, P < 0.01 compared with basal condition; ##, P < 0.001 compared with basal condition; *, P < 0.05 compared with cells treated with TNF alone; **, P < 0.01 compared with cells treated with TNF alone; ***, P < 0.001 compared with cells treated with TNF alone.

IFN Treatment Had No Effect on Either TNF-Induced IKK Phosphorylation or IB Degradation. To further study the mechanisms by which IFNs suppressed TNF-induced NF-B-dependent gene expression, we examined the effect of IFN on NF-B upstream activating cascades, including activation of IKK, degradation of IB, and NF-B nuclear translocation and DNA binding capabilities using the TransAM technology (Active Motif, Carlsbad, CA). As shown in Fig. 4, IFN pretreatment failed to prevent TNF-induced IKK phosphorylation (Fig. 4A), IB degradation (Fig. 4B), NF-B DNA binding activity assessed at 2 and 24 h (Fig. 4C), and TNF-induced p65 accumulation in the nucleus (Fig. 4D). IFN alone failed to activate any of these signaling pathways. These results show that the mechanisms underlying the inhibitory effect of IFN on cytokine-induced NF-B-dependent gene expression occurs at the nucleus, most likely on the transcriptional machinery.

View larger version (40K): [in this window] [in a new window]   Fig. 4. IFN failed to prevent TNF-induced IKK phosphorylation (A), IB degradation (B), NF-B p65/DNA interaction (C), or NF-B p65 nuclear translocation (D). Cells were stimulated for 24 h with TNF (10 ng/ml), IFN, or the combination at the indicated concentrations of IFN and lysed, and total cell lysates were prepared for either IKK phosphorylation or IB degradation by immunoblot analysis as described under Materials and Methods. Top, scanning of a representative gel; bottom, densitometry analysis with each value normalized over the mean density of the corresponding control (either total IKK or actin). C, cells were stimulated with TNF (10 ng/ml), IFN, or the combination at the indicated time (2 or 24 h) and lysed, and nuclear extracts were prepared for NF-B p65/DNA interaction using TransAM kit as described under Materials and Methods. The results are expressed as -fold increase over basal. D, immunoblot analysis was performed using p65 antibody on nuclear extracts of cells stimulated with TNF or TNF/IFN for 2 h as described under Materials and Methods. #, P < 0.001, compared with untreated cells (basal); NS, nonsignificant compared with cells treated with TNF alone.

HDAC Activity Was Synergistically Induced by TNF/IFN Combination. Because IFN inhibits HAT activity induced by TNF, we next examined whether IFN modulated HDAC activity. Our results demonstrated that, whereas TNF or IFN separately modestly activated HDAC activity, the combination of both cytokines leads to a synergistic induction of HDAC activity (Fig. 6). It is noteworthy that the inhibitory effects exerted by IFN on TNF-induced NF-B-dependent gene expression (Fig. 7A), as well as IL-6 and IL-8 (Fig. 7, B and C), could be reversed by pretreating cells with TSA, a well characterized HDAC inhibitor (Barnes et al., 2005). These results suggest that modulation of HDAC function by both IFN/TNF is playing a key role in the regulation of NF-B-dependent gene expression.

View larger version (17K): [in this window] [in a new window]   Fig. 6. IFN increased TNF-induced HDAC activity. Cells were stimulated with TNF (10 ng/ml), IFN, or the combination at the indicated time (2 or 24 h) and lysed; nuclear extracts were prepared for HDAC activity using a kit as described under Materials and Methods. The results are expressed as NF-B reporter activity increase over values for untreated cells. #, P < 0.005 compared with untreated cells (basal); **, P < 0.005 compared with cells treated with TNF alone.

View larger version (17K): [in this window] [in a new window]   Fig. 7. HDAC inhibitor (TSA) restored the inhibitory effect of IFN on TNF-induced NF-B (A), IL-6 (B), and IL-8 (C) expression. Cells were stimulated with 400 nM TSA 1 h before TNF (10 ng/ml), IFN (1000 U/ml), or the combination for 6 h. TSA completely prevented the inhibitory effect of IFN on TNF-induced NF-B (A), IL-6 (B), and IL-8 (C). ##, P < 0.001 compared with untreated cells (basal); , P < 0.05 compared with cells treated with TNF alone; , P < 0.01 compared with cells treated with TNF alone; NS, not significant.

	Discussion

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In ASM cells, several reports showed that IFN/TNF was an effective combination to synergistically induce different inflammatory genes including RANTES (John et al., 1997), COX-2 (Singer et al., 2003), CXCL10 (Hardaker et al., 2004), Fractalkine (Sukkar et al., 2004), or CD38 (Tliba et al., 2006). It is noteworthy that we found here that IFN can also act as a potent inhibitor of TNF-induced expression of other genes including IL-6, IL-8, and eotaxin (Fig. 1). Thus, our study demonstrates that IFN/TNF combination could result in either a cooperative or antagonistic expression of inflammatory genes in ASM cells. This IFN/TNF antagonism in ASM cells has been described previously by two reports showing that IFN can also suppress other TNF-inducible inflammatory genes, including vascular endothelial growth factor (Wen et al., 2003) and IL-17 receptor expression (Lajoie-Kadoch et al., 2006). Although studies in other cell types, including airway epithelial cells (Matsukura et al., 2003), dermal (Miyamasu et al., 1999) and corneal fibroblasts (Fukuda et al., 2002) demonstrated that IFN/TNF combination leads to similar effects (i.e., reduced cytokine production), others found opposite results in which IFN treatment enhanced IL-6 and IL-8 expression in response to TNF (van Wissen et al., 2002). Surprisingly little is known about the molecular mechanisms mediating IFN differential effects on gene expression in ASM as well as in other cell types, but our study demonstrates the IFN/TNF antagonism probably occurs at the transcriptional level via the modulation of NF-B pathways.

The NF-B transcription factor is a vital regulator of cellular processes involved in immune response, cellular proliferation, differentiation, and apoptosis (Baldwin, 1996). Recent evidence using newly developed pharmacological inhibitors identified the necessary NF-B-activating kinase, IKK, as a potential new target for treatment of inflammatory responses in asthma (Birrell et al., 2005). We and others showed that NF-B is activated by TNF or IL-1 in ASM cells and represents an essential transcription factor involved in the regulation of VCAM-1 (Issa et al., 2006), ICAM-1 (Amrani et al., 1999), CXC chemokine growth-related oncogene protein- (Issa et al., 2006), and granulocyte macrophage-colony-stimulating factor (Lalor et al., 2004). Through the use of BMS-345541, a potent IKK inhibitor (Burke et al., 2003), we demonstrated here that NF-B also played a central role in IFN-sensitive, TNF-inducible genes IL-6, IL-8, and eotaxin. Using a reporter plasmid containing multiple B enhancer elements (Fig. 3), we found that IFN almost completely abrogated TNF-induced NF-B-dependent gene expression. Combined together, these observations strongly suggest that IFN suppressed TNF-associated genes by impairing NF-B transactivation. It is noteworthy that some reports, but not all, showed the ability of type I or II IFNs (IFN or IFN) to inhibit TNF-induced NF-B pathways in different cell types, including 2fTGH fibroblasts (Ganster et al., 2005), Ewin's sarcoma EW-7 cells (Sanceau et al., 2002), and Jurkat T cells (Manna et al., 2000). We found that the inhibition of cytokine-induced NF-B transactivation by IFN most likely occurs at the transcriptional level because IFN failed to block upstream signaling events such as phosphorylation of IKK, degradation of the cytosolic inhibitor IB, as well as NF-B nuclear translocation and NF-B-DNA binding interaction (Fig. 4). Only one study performed in ME-180 cervical cancer cells demonstrated that IFN sensitized cells to apoptosis induced by TNF. This occurs via the suppression of NF-B-dependent gene transcription, an effect that was mimicked by overexpressing IRF-1, another member of IFN signaling (Suk et al., 2001). Our findings differ from previous observations made in other cell types that IFN blocks of NF-B pathways induced by TNF through multiple mechanisms, including 1) inhibition of NF-B-DNA interaction (Sanceau et al., 2002), 2) prevention of IB degradation (Manna et al., 2000), or 3) tight regulation of TNF receptor 1 activity induced by direct interaction with STAT1 (Wang et al., 2000; Wesemann and Benveniste, 2003). The molecular mechanisms by which IFN/TNF combination leads to the suppression of NF-B-inducible genes in ASM cells seem to be highly complex and distinct from other cell types. The present report presents the novel hypothesis that IFN/TNF-induced change in protein acetylation plays an important role in the regulation of inflammatory genes in ASM cells.

Transcription of eukaryotic genes is complex and depends on different coactivators, such as p300, pCAF, and cAMP response element-binding protein-binding protein, as well as HATs and HDACs. HATs are responsible mainly for destabilizing the chromatin structure to allow accessibility of different transcription factors, including NF-B, to the transcriptional site in the DNA. In contrast, HDACs serve as corepressors of gene transcription by restoring the condensation of DNA in the chromatin (Barnes and Karin, 1997). As reported in A549 cells (Rahman et al., 2004), we found that HAT activity is increased in ASM cells after TNF treatment, an effect that was sustained up to 24 h (Fig. 5A). It is logical to assume that this prolonged HAT activity would be required to ensure the time-dependent induction of the inflammatory genes IL-6, IL-8, and eotaxin previously reported in ASM cells (Pang and Knox, 2001; Ammit et al., 2002). In agreement with our observation, Nie et al. (2005) reported that TNF did induce histone H4 acetylation and the recruitment of NF-B at the eotaxin promoter in human ASM cells. Studies using trichostatin A, a specific inhibitor of HDACs (Barnes et al., 2005), confirmed the current hypothesis that histone acetylation is an essential event for mediating TNF-associated NF-B-dependent genes (Ashburner et al., 2001; Adam et al., 2003). This assumption is further supported in our study by two unexpected observations: 1) HDAC activity was significantly increased in cells treated with IFN/TNF combination and 2) TSA prevented the inhibitory actions of IFN on TNF responses (NF-B activation and IL-6 and IL-8 expression). Our results showed that p65 acetylation, which is important for the NF-B-dependent gene transcription (Hoberg et al., 2006), was also enhanced by TNF.We found that IFN decreases p65 acetylation induced by TNF (Fig. 5B). Thus, the p65 acetylation level, because of the HAT/HDAC activities, is probably a part of the mechanisms by which IFN inhibits TNF-inducible genes. Together, these data support the novel concept that changes in histone acetylation through the modulation of HDAC function may play an important role in the anti-inflammatory actions of IFNs. Our report is the first to show that HDAC function can be modulated by TNF/IFNs combination, although the nature of HDAC involved as well as the deacetylated proteins involved are unknown. In contrast to the unexplored role of HDAC in TNF signaling, HDAC1 activity has been shown to be required for the complete transcription of IFN-inducible genes (Nusinzon and Horvath, 2003). Whether HDAC1 plays any role in the "selective" anti-inflammatory effects of IFNs remains to be determined. The essential question that should be raised is the nature of the synergistic signals leading to aberrant HDAC activity and suppression of NF-B function.

In conclusion, in such diseases as asthma, an unbalance between HAT and HDAC activities could play an important role in the regulation of pro-inflammatory genes in ASM cells. It is noteworthy that subjects with severe asthma have reduced HDAC activity and increased HAT activity (Ito et al., 2002). Thus, a better understanding of the IFN mechanisms leading to increased HDAC function in the airways could provide new insight into the treatment of airway inflammatory diseases.

	Acknowledgements

 
We thank Mary McNichol for her assistance in the preparation of the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health grant HL064063, the University of Pennsylvania Research Foundation, and American Lung Association grant CI-21948-N (to Y.A.).

ABBREVIATIONS: TNF, tumor necrosis factor ; IB, inhibitor of B; IK, I kinase; IFN, interferon; NF-B, nuclear factor B; HAT, histone acetyltransferase; HDAC, histone deacetylase; IL, interleukin; ASM, airway smooth muscle; RANTES, regulated upon activation normal T-cell expressed and secreted; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; TSA, trichostatin A; PCR, polymerase chain reaction; SEAP, secreted alkaline phosphatase; r, recombinant; BMS 345541, 4-(2'-aminoethyl)amino-1,8-dimethylimidazo[1,2-a]quinoxaline.

Address correspondence to: Dr. Yassine Amrani, Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, University of Pennsylvania School of Medicine, Translational Research Laboratories Building, Suite 1200, 125 South 31st Street, Philadelphia PA 19104-3403. E-mail: amrani{at}mail.med.upenn.edu

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