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J. Biol. Chem., Vol. 278, Issue 50, 50615-50623, December 12, 2003

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Tumor Necrosis Factor Modulates Airway Smooth Muscle Function via the Autocrine Action of Interferon ^*

Omar Tliba, Samira Tliba, Chien Da Huang, Rebecca K. Hoffman, Peter DeLong, Reynold A. Panettieri, Jr., and Yassine Amrani

From the Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Received for publication, April 9, 2003 , and in revised form, August 18, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Current evidence suggests that tumor necrosis factor (TNF) and the family of interferons (IFNs) synergistically regulate many cellular responses that are believed to be critical in chronic inflammatory diseases, although the underlying mechanisms of such interaction are complex, cell-specific, and not completely understood. In this study, TNF in a time-dependent manner activated both janus tyrosine kinase 1 and Tyk2 tyrosine kinase and increased the nuclear translocation of interferon-regulatory factor-1, STAT1, and STAT2 in human airway smooth muscle cells. In cells transfected with a luciferase reporter, TNF stimulated -activated site-dependent gene transcription in a time- and concentration-dependent manner. Using neutralizing antibodies to IFN and TNF receptor 1, we show that TNF-induced secretion of IFN mediated -activated site-dependent gene expression via activation of TNF receptor 1. In addition, neutralizing antibody to IFN also completely abrogated the activation of interferon stimulation response element-dependent gene transcription induced by TNF. Secreted IFN acted as a negative regulator of TNF-induced interleukin-6 expression, while IFN augmented TNF-induced RANTES (regulated on activation normal T cell expressed and secreted) secretion but had little effect on TNF-induced intercellular adhesion molecule-1 expression. Furthermore TNF, a modest airway smooth muscle mitogen, markedly induced DNA synthesis when cells were treated with neutralizing anti-IFN. Together these data show that TNF, via the autocrine action of IFN, differentially regulates the expression of proinflammatory genes and DNA synthesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TNF¹ is now considered to be one of the most pleiotropic cytokines in mediating inflammatory and immune responses in chronic lung diseases. In vivo studies using selective inhibitors of TNF activity demonstrate that TNF plays a major role in antigen-induced airway inflammation (leukocyte infiltration) and airway hyper-responsiveness in animal models of asthma (1, 2). Others who used receptor knock-out mice confirmed the importance of both TNF receptors (TNFRs), TNFR1 and TNFR2, in the abnormal airway changes induced by allergen challenge in sensitized animals (3-5). A potential site for TNF deleterious action in the lungs is airway smooth muscle (ASM), a primary effector tissue thought to only regulate bronchomotor tone (6). In human cultured ASM cells that retain physiological responsiveness and express both TNF receptors (7), TNF alters proinflammatory gene expression that in turn may play an important role in the pathogenesis of allergic asthma. In previous reports, we showed that TNF increased expression of ICAM-1, IL-6, and RANTES by selectively activating TNFR1, although TNFR2 was also involved in TNF-induced RANTES secretion (8-10). TNFR1-associated gene expression has been involved in the development of bronchial hyper-responsiveness (6, 11, 12).

TNF also cooperates with other cytokines such as interferon (IFN) to regulate the expression of cytokines (IL-1 and IL-5) and chemokines (IL-8, eotaxin, and RANTES) as well as adhesion molecules such as ICAM-1, vascular cell adhesion molecule-1, and CD40 (for a review, see Ref. 10). Because ASM expresses receptors for a variety of cytokines and chemokines, investigators suggest that many secreted cytokines such as IL-1 and IL-5, in an autocrine manner, may modulate ASM function to elicit a "proasthmatic phenotype" (13, 14). Whether Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (STAT)-dependent signaling pathways mediate the cooperative interaction between TNF and IFN in human ASM cells remains unknown.

JAKs and STATs are the central components of IFN receptor signaling. Ligands stimulate IFN-receptor complexes (types I and II) and activate the receptor-associated tyrosine kinase JAK, specifically JAK1 and Tyk2 by IFN/ (type I) or JAK1 and JAK2 by IFN (type II) (15). JAKs then phosphorylate STAT proteins that assemble in dimeric or oligomeric forms, translocate to the nucleus, and regulate gene expression. STAT proteins play an important function in regulating immunological and inflammatory responses (16). The role of the JAK/STAT-dependent pathways in promoting airway diseases is supported by evidence that STAT proteins regulate a number of inflammatory responses associated with allergic diseases such as Th1 and Th2 differentiation, IgE regulation, and cytokine expression (for a review, see Ref. 17). Increased levels of STAT1 and STAT1-dependent genes such as IFN-regulatory factor IRF-1 or ICAM-1 in bronchial epithelium from subjects with asthma correlate with an accumulation of T cells in the airways, a defining feature of asthma (18). Although the factors responsible for STAT activation in asthma still remain unknown, these data suggest that altered STAT-dependent gene expression may be a key factor driving airway inflammation. In the present study, we investigated whether the modulatory effect of TNF on gene expression in human ASM cells involves activation of the JAK/STAT signaling pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Smooth Muscle Cell Culture and Characterization—Human ASM cell culture was performed as described previously (7). Human trachea was obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions, and the trachealis muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl₂, 640 units/ml collagenase, 1 mg/ml soybean trypsin inhibitor, and 10 units/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37 °C. The cell suspension was filtered through 105-µm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F-12 medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT). Aliquots of the cell suspension were plated at a density of 1.0 x 10⁴ cells/cm². The cells were cultured in Ham's F-12 medium supplemented with 10% FBS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 µg/ml amphotericin B, and this was replaced every 72 h. Human ASM cells in subculture during the second through fifth cell passages were used since the cells retain native contractile protein expression as demonstrated by immunocytochemical staining for smooth muscle actin and myosin.

Thymidine Incorporation Assays—DNA synthesis was evaluated by measuring thymidine incorporation as described previously (19). Cells were growth-arrested by incubating the cultures in serum-free medium consisting of Ham's F-12 medium with 0.1% bovine serum albumin. After 48 h in serum-free medium, the cells were stimulated with 1 unit/ml thrombin in the presence or absence of 10 ng/ml TNF with or without neutralizing anti-IFN. After 16-18 h of stimulation, human ASM cells were labeled with 3 µCi/ml [methyl-³H]thymidine (40-60 Ci/mmol, Amersham Biosciences) for 24 h. The cells were then scraped and lysed, and the protein and DNA were precipitated with 10% trichloroacetic acid. The precipitant was aspirated onto glass filters, extensively washed, dried, and counted.

Flow Cytometry Analysis—Flow cytometry was performed as described previously (20). Antibodies used for IFN receptor expression (anti-IFNAR1 and anti-IFNAR2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-conjugated secondary antibodies were bought from Jackson ImmunoResearch Laboratories (West Grove, PA).

Enzyme-linked Immunosorbent Assay—Cytokines in supernatants were measured using antibodies obtained from R&D Systems (Minneapolis, MN) (IL-6 and RANTES) and BD Pharmingen (IFN) as indicated by the manufacturers' instructions (9). As a positive control for IFN enzyme-linked immunosorbent assays, the renal carcinoma cell line (REN) was infected with either adenovirus encoding for IFN and -galactosidase (Ad-IFN) or a control adenovirus encoding for -galactosidase alone (Ad) (21). Levels of IFN were measured using a commercially available human IFN enzyme-linked immunosorbent assay kit (catalogue number 41400-1, R&D Systems). The range of detection using IFN standard was found to be between 125 and 10 000 pg/ml (R² = 0.9911).

Immunoblot Analysis—Immunoblot analysis for cyclin D1 and p27^Kip1 was performed as described previously (20, 22). 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 14,000 x g for 10 min. Immunoprecipitations using the Tyk2, JAK1, and STAT2 antibodies (Santa Cruz Biotechnology) were performed as indicated by the manufacturer's instructions. Equal amounts of proteins were analyzed by 4-12% SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The membranes were blocked in 5% milk or 5% bovine serum albumin (anti-phosphoprotein antibodies) in Tris-buffered saline and then incubated with either of the following antibodies: anti-phospho-STAT1 (Tyr-701), anti-STAT1, anti-Tyk2, and anti-JAK1 (Santa Cruz Biotechnology); anti-phospho-Tyk2 (Tyr-1054/1055) and anti-phosphotyrosine Tyr(P) (Cell Signaling, Beverly, MA); and anti-phospho-JAK1 (Tyr-1022/1023) (BioSource International, Camarillo, CA). Detection of IRF-1 proteins (Santa Cruz Biotechnology) was performed on nuclear extracts. After incubation with the appropriate peroxidaseconjugated secondary antibody (Roche Applied Science), the bands were visualized by the enhanced chemiluminescence system (Amersham Biosciences) and autoradiographed.

Immunocytochemistry of STAT1, STAT2, and IRF-1—Immunostaining for nuclear translocation experiments was performed as described previously (8) with the exception of the following antibodies: anti-phospho-STAT1 (Tyr-701), anti-STAT2, and anti-IRF-1 (Santa Cruz Biotechnology). Isotype-matched antibodies (rabbit and mouse IgG from R&D Systems) were used as negative controls. After staining, the glass coverslips were mounted onto glass slides, examined under epifluorescence microscopy (Nikon, Tokyo, Japan), and photographed using Olympus 1X70 (Hitech Instruments, Inc., Edgemont, PA).

Transfection of Human ASM Cells—Transfection of human ASM cells was performed as reported previously (9). In brief, human ASM cells were transfected with 10 µg of pGAS-Luc or 10 µg of pISRE-Luc to monitor the transcriptional activities of STAT1 and STAT2, respectively (Stratagene, La Jolla, CA), and 5 µg of a pSV--galactosidase control vector was used to normalize transfection efficiencies (Promega, Madison, WI). Forty-eight hours after transfection, the cells were rendered quiescent in medium containing 0.1% fetal bovine serum for 24 h and exposed to 10 ng/ml TNF or 500 units/ml IFN. In neutralizing experiments, human ASM cells were preincubated with anti-TNFR1 or anti-TNFR2 (20 µg/ml, 60 min) anti-IFN or isotype-matched IgG (same concentration). Cells were then harvested, and luciferase and -galactosidase activities were assessed with a Promega kit 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 at different times (0, 0.5, 1, 2, 3, and 4 h). Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcriptase PCR analysis of IFN, IFN, IFN, platelet-derived growth factor A (PDGF)-A, PDGF-B, and glyceraldehyde-3-phosphate dehydrogenase expression was then performed as reported previously (6). Consensus primers (upstream primer, 5'-TGATGGCAACCAGTTCCAGAAGGCTCAAG-3'; downstream primer, 5'-ACAACCTCCCAGGCACAAGGGCTGTATTT-3' (23)) were used to detect multiple human IFN subtypes (GenBank^TM accession numbers, based on the BLAST analyses): NM_024013 [GenBank] , Homo sapiens interferon 1 (IFNA1) mRNA; AF439447 [GenBank] , H. sapiens interferon 1b gene, partial cds; Y11834 [GenBank] , H. sapiens IFNA2 gene; AY255838 [GenBank] , H. sapiens interferon 2b mRNA, complete cds; NM_021068 [GenBank] , H. sapiens interferon 4 (IFNA4) mRNA; X02955 [GenBank] , human interferon gene IFN 4b; X02956 [GenBank] , human interferon gene IFN 5; X02958 [GenBank] , human interferon gene IFN 6; X02960 [GenBank] , human interferon gene IFN 7; X03125 [GenBank] , human interferon gene IFN 8; NM_002171 [GenBank] , H. sapiens interferon 10 (IFNA10) mRNA; X00803 [GenBank] , human interferon gene IFN 13; X02959 [GenBank] , human interferon gene IFN 14; X02957 [GenBank] , human interferon gene IFN 16; NM_021268 [GenBank] , H. sapiens interferon 17 (IFNA17) mRNA; NM_002175 [GenBank] , H. sapiens interferon 21 (IFNA21) mRNA; K01900 [GenBank] , human lymphocyte interferon type B mRNA, complete cds; V00532 [GenBank] , human gene for leukocyte () interferon C; J00210 [GenBank] , human leukocyte interferon (IFN) -d gene, complete cds; X00145 [GenBank] , human mRNA for interferon -F; V00533 [GenBank] , human gene for leukocyte () interferon H; V00531 [GenBank] , human interferon genes LeIF-L and LeIF-J; M34913 [GenBank] , human interferon -J1 (IFN-J1) mRNA, complete cds; M27318 [GenBank] , human interferon (IFN-M1) mRNA, complete cds; X00140 [GenBank] , human mRNA for interferon -N; K02055 [GenBank] , human interferon -WA gene, complete cds, clone -85; M28585 [GenBank] , human leukocyte interferon mRNA, complete cds. Other primers for IFN, IFN, PDGF-A, PDGF-B, and glyceraldehyde-3-phosphate dehydrogenase detection were identical to those reported previously (23, 24) and were designed to amplify at least one intron in the genes to exclude contamination of cDNA with genomic DNA. PCR products were separated on 1% agarose gels and stained with ethidium bromide.

Materials and Reagents—Tissue culture reagents and primers used for PCR were obtained from Invitrogen. Human rTNF was provided by Roche Applied Science. rIFN, rIFN, anti-TNFR1 neutralizing antibody, anti-IFN neutralizing antibody (sheep polyclonal antibody, catalogue number 31400-1, 5 µg/ml, 15 min), isotype-matched goat or mouse IgG were purchased from R&D Systems. Cycloheximide and thrombin were purchased from Sigma and Calbiochem, respectively. The anti-TNFR2 neutralizing antibody was obtained from Cell Sciences Inc. (Norwood, MA).

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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
TNF Stimulates STAT1 Activation and -Activated Site (GAS)-dependent Gene Expression in Human ASM Cells—In human ASM cells, TNF and IFNs synergize to regulate the expression of a number of proinflammatory genes (10). Here we tested the hypothesis that TNF modulates IFN-associated signaling pathways. We found that TNF stimulates STAT1 activation at 3 and 4 h as shown by the increased STAT1 phosphorylation at tyrosine residue 701 in cytokine-treated nuclear extracts. In comparison, STAT1 phosphorylation by IFN was swifter and more robust occurring at 30 min (Fig. 1A). Fig. 1B shows that TNF treatment for 3 h also stimulated the nuclear translocation of activated STAT1 in human ASM cells, an effect that was completely abrogated by cycloheximide, a protein synthesis inhibitor (data not shown). To address the functional consequences of TNF-induced STAT1 activation, ASM cells were transfected with a reporter construct containing GAS motifs that bind activated STAT1 (15). As shown in Fig. 1C, TNF stimulated luciferase activity in a time-dependent manner, although a slight decrease in GAS reporter activity was observed at 2 h (45%) when compared with untreated cells. Interestingly the effect of TNF on GAS-dependent gene expression temporally correlated with the increase in the phosphorylation of STAT1 induced by TNF (Fig. 1A). The effect of TNF on GAS-mediated gene expression was concentration-dependent (Fig. 2A) with significant increases at 1, 10, and 30 ng/ml with a 3.49 ± 0.6-, 5.2 ± 0.54-, and 13 ± 0.37-fold increase over basal, respectively. In contrast to TNF, IFN differentially regulates GAS-dependent gene expression, characterized by a swifter time course as early as 2 h with a 5 ± 0.7-fold increase over basal and more robust activation of reporter activity reaching an 18 ± 1.3-fold increase over basal at 4 h (Fig. 1D), suggesting that both cytokines activate GAS-dependent gene expression via potentially disparate pathways. Using antagonistic antibodies against TNFR receptors (9), we showed that neutralizing anti-TNFR1, but not anti-TNFR2, abrogated TNF-induced GAS-dependent transcription (Fig. 2B), while an isotype-matched IgG had no effect on TNF-induced STAT1 activation (data not shown). Together these results show that TNF, via TNFR1, induces a delayed activation of GAS-mediated gene expression in human ASM cells.

View larger version (40K): [in this window] [in a new window]   FIG. 1. TNF activates phosphorylation and nuclear translocation of STAT1 and STAT1-dependent gene expression. A, cells were stimulated with TNF (10 ng/ml) or IFN (100 units/ml) for the indicated times and lysed, and total cell lysates were prepared and assayed for either phosphorylated STAT1 (Tyr-701) (P-STAT1) or STAT1 by immunoblot analysis (15% SDS-PAGE). B, cells stimulated with TNF (10 ng/ml) for 3 h were fixed, permeabilized, and incubated with mouse anti-phosphorylated STAT1 (Tyr-701) followed by a fluorescein isothiocyanateconjugated anti-mouse antibody. This result is representative of three separate experiments. C and D, cells transfected with a luciferase reporter construct containing GAS motifs were then stimulated with TNF (10 ng/ml, C) or IFN (100 units/ml, D) for the indicated times. Cells were lysed, and the luciferase activity in cell extracts was normalized for -galactosidase activity as described under "Experimental Procedures." Data are representative of three separate experiments. *, p < 0.05 compared with untreated cells.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Concentration-dependent effects of TNF on GAS-dependent gene expression: role of TNFR1. Cells transfected with a luciferase reporter construct containing GAS enhancer elements were stimulated with the indicated concentration of TNF for 4 h (A) or were first preincubated with neutralizing anti-TNFR1 or anti-TNFR2 (20 µg/ml, 60 min) antibodies before adding 10 ng/ml TNF for 4 h (B). Cells were lysed, and the luciferase activity in cell extracts was normalized for -galactosidase activity as described under "Experimental Procedures." Data are representative of three separate experiments. *, p < 0.05 compared with untreated cells. #, p < 0.05 when compared with cells treated with TNF alone. NS, non-significant.

View larger version (28K): [in this window] [in a new window]   FIG. 3. TNF, via the autocrine action of secreted IFN, activates STAT1-dependent gene expression and IFN receptor-associated proteins. A, ASM cells were stimulated with TNF for the indicated times. Total mRNA (2 µg) was subjected to reverse transcriptase PCR with the primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and IFN. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. Data are representative of mRNA obtained from three different experiments. B, cells were stimulated with 10 ng/ml TNF for the indicated time, lysed, immunoprecipitated with anti-JAK1 or anti-Tyk2, and assayed for JAK1, phospho-JAK1 (P-JAK1), Tyk2, or phospho-Tyk2 (P-Tyk2) by immunoblot analysis (gradient 4-12% SDS-PAGE). Results are representative of three separate experiments. C, ASM cells were transfected with luciferase reporter construct containing GAS enhancer elements and stimulated for 4 h with TNF (10 ng/ml) or IFN (500 units/ml) in the presence or absence of neutralizing anti-IFN (5 µg/ml, 15 min). Cells were lysed, and luciferase activity in cell extracts was normalized for -galactosidase activity as described under "Experimental Procedures." Data are representative of four separate experiments. *, p < 0.05 when compared with control cells. #, p < 0.05; ##, p < 0.05 when compared with cells treated with IFN or TNF, respectively.

View larger version (36K): [in this window] [in a new window]   FIG. 4. TNF stimulates the nuclear translocation of STAT2 and induces ISRE-dependent gene expression. A, cells stimulated with TNF for the indicated time were lysed, immunoprecipitated with anti-STAT2 antibody, and assayed for phosphotyrosine (pTyr) or STAT2 by immunoblot analysis (gradient 4-12% SDS-PAGE). B, cells stimulated with TNF (10 ng/ml) for 3 h in the presence or absence of neutralizing anti-IFN (5 µg/ml, 15 min) were fixed, permeabilized, and incubated with mouse anti-STAT2 followed by a secondary fluorescein isothiocyanateconjugated anti-IgG antibody. This result is representative of three separate experiments. C and D, ASM cells were transfected with luciferase reporter construct containing ISRE enhancer elements and stimulated for the indicated time with TNF (10 ng/ml) or IFN (500 units/ml) in the presence or absence of neutralizing anti-IFN (5 µg/ml, 15 min). Cells were lysed, and luciferase activity in cell extracts was normalized for -galactosidase activity as described under "Experimental Procedures." Data are representative of three separate experiments. *, p < 0.05 when compared with control cells. #, p < 0.05; ##, p < 0.05 when compared with cells treated with TNF or IFN, respectively.

View larger version (60K): [in this window] [in a new window]   FIG. 5. TNF stimulates the nuclear translocation of IRF-1. A, cells stimulated with TNF for the indicated time were lysed, and nuclear extracts were prepared and assayed for IRF-1 by immunoblot analysis (15% SDS-PAGE). B, cells stimulated with TNF (10 ng/ml) for 3 h in the presence or absence of neutralizing anti-IFN (5 µg/ml, 15 min) were fixed, permeabilized, and incubated with mouse or anti-IRF-1 followed by a secondary fluorescein isothiocyanate-conjugated anti-IgG antibody. This result is representative of three separate experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 6. TNF does not affect expression of IFN receptor subunits IFNAR1 and IFNAR2. Cells were stimulated with TNF (10 ng/ml) for 3 h, and levels of IFNAR1 (A) and IFNAR2 (B) were assessed by flow cytrometry as described under "Experimental Procedures." The results shown are representative of three independent experiments.

View larger version (16K): [in this window] [in a new window]   FIG. 7. TNF, via the secretion of IFN, differentially regulates ICAM-1, IL-6, and RANTES expression. Cells were stimulated for 24 h with TNF (10 ng/ml) in the presence or absence of neutralizing anti-IFN (5 µg/ml, 15 min). Secretion of IL-6 (A) and RANTES (B) or expression of ICAM-1 (C) levels were assessed as described under "Experimental Procedures." Values shown are mean ± S.E. and are significantly different from controls. *, p < 0.05 when compared with basal. #, p < 0.05 when compared with cells treated with TNF alone.

View larger version (15K): [in this window] [in a new window]   FIG. 8. TNF, via the secretion of IFN, suppresses ASM cell proliferation and cell cycle regulators cyclin D1 and p27Kip1. Cells were stimulated for 40 h with thrombin (Thr) (1 unit/ml) and/or TNF (10 ng/ml) in the presence or absence of neutralizing anti-IFN (5 µg/ml, 15 min). A, [3H]thymidine incorporation was performed as described under "Experimental Procedures." The experiment represents the mean ± S.E. from three replicates per condition performed in three separate experiments. #, p < 0.05; *, p < 0.05 when compared with cells treated with diluent or thrombin alone, respectively (determined by analysis of variance with Bonferroni-Dunn correction). B, cells were lysed, and immunoblot analysis was performed with monoclonal antibodies specific to cyclin D1, p27Kip1, and -actin (lower panel) for equal amounts of protein loading. These data are representative of two experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous reports from our laboratory revealed that TNFR1-associated gene expression in ASM cells may play a central role in two features of asthma, i.e. airway inflammation and airway hyper-responsiveness (6-9, 11, 33). Although TNFR1 initiates signal transduction cascades by recruiting the adapter protein TRADD (TNF receptor-associated death domain) that in turn activates the signaling proteins TRAF2 and FADD (Fas-associated death domain) (for a review, see Ref. 33), our findings now suggest that IFN signal transducers represent active components in some but not all cellular responses induced by TNF. We now show that TNF, via activation of TNFR1, not only induces gene and protein expression of IFN but also activates IFN receptor-associated signaling molecules JAK1 and Tyk2 tyrosine kinases in human ASM cells. The molecular mechanism by which TNF stimulates IFN expression in ASM cells remains unknown, but investigators showed that transcription factors IRF-1 and NF-B, both activated in ASM cells (present study and Refs. 9 and 20), play a major role in the transcriptional activation of the IFN promoter (34). This raises the possibility that both NF-B and IRF-1 may induce IFN expression by TNF, although evidence suggests that IRF-1 could also act as a competitive inhibitor of NF-B-dependent gene expression (35). The effect of TNF on IFN-dependent signaling molecules, however, was not associated with changes in the expression of IFN receptor subunits, IFNAR1 or IFNAR2. Others reported that TNF increased expression of IFNAR2 in hepatocellular cell lines (29). In a variety of cell lines, activation of TNFR1 leads to a rapid activation of JAK/STAT pathway unlike that observed in ASM cells. In adipocytes and B cells, TNFR1 physically recruits both JAK1 and JAK2 within 5 and 15 min after stimulation (36, 37), while in HeLa cells both STAT1 and JAK2 are constitutively associated with both TNFR1 and TNFR2 in basal conditions, and this association was further increased by TNF at 15-30 min (36, 38). In ASM cells, however, we found that activation of JAK1 and Tyk2 by TNF was delayed and was associated with activation of both STAT1 and STAT2 at 2 h, while STAT3 was not activated by TNF (data not shown) in contrast to that reported in human B cells (37). Another major difference in our study was that nuclear translocated STAT1 and STAT2 induced by TNF were transcriptionally active as evidenced by the ability of TNF to activate GAS- and ISRE-dependent gene expression. In HeLa cells as well as in 3T3-L1 adipocytes, there was no DNA binding activity attributable to activated STAT1 in response to TNF (36, 38). The authors suggested that STAT1 may be acting as a negative regulator of TNF-induced NF-B activation via a blockade of TNFR1-associated signaling molecule TRAF2 (38). Another interesting finding was the inhibitory effect of TNF on GAS-dependent gene expression noticed at 2 h (but not observed at 3 and 4 h), while the ISRE-dependent transcription was induced by TNF. The reasons for the differential effect of TNF on gene expression controlled by GAS but not by ISRE binding elements at early time points remain unclear, and further studies are required to determine whether physical and/or functional interaction between members of TNF signal transduction and JAK/STAT pathways as shown in HeLa cells is involved (38). A recent study using STAT1-deficient cells U3A and STAT1 stably transfected cells U3APSG91 showed that STAT1 played a minor role in TNF-induced cell cytotoxicity (39), suggesting that the role and the type of STAT proteins activated by TNF are complex and highly cell-specific.

Although TNF increased IFN mRNA (40) or protein (41) in vascular and bronchial smooth muscle cells alone or in cells infected with Chlamydia pneumonia, the physiological consequence of IFN secretion by TNF was not examined. In our study, IFN, in an autocrine manner, promoted TNF-induced expression of RANTES while acting as a negative regulator of IL-6 expression. Previous reports showed that IFN induced RANTES expression in T cell lymphoma cell lines and in human macrophages (42) but not in endothelial cells (43), while in human fibroblasts IFN also requires the presence of TNF to induce RANTES expression (44). Interestingly secreted IFN does not affect TNF-induced ICAM-1 expression, although investigators using dominant negative proteins or reporter plasmids and their 5' deletion derivatives demonstrated the importance of STAT1 binding elements in the transcriptional activation of the ICAM-1 promoter (45). Others also showed that IFN inhibits TNF-induced ICAM-1 expression in brain endothelial cells (46). Our study shows that IFN negatively regulates cytokine-induced IL-6 gene expression, suggesting that the regulation of gene expression by IFN appears to be cell-specific and modulated by other cytokines. Further studies will provide essential information regarding the transcription factors involved in the differential effect of IFN on cytokine-induced gene expression. Because TNF regulates a variety of cytokines and/or chemokines in human ASM cells such as IL-8, eotaxin (47), or MCP-1 (48), IFN may also serve as an important regulator of other TNF-induced proinflammatory genes.

Finally we showed that secreted IFN inhibited ASM cell proliferation supporting our recent findings that exogenous IFNs are potent suppressors of ASM cell mitogenesis (32). Previous reports also showed that TNF inhibits thrombin-induced ASM cell proliferation, although the underlying mechanisms remained unclear (31). Blockade of IFN revealed that TNF exerts a growth-suppressive effect on both basal and agonist-induced proliferative responses via the autocrine action of IFN. These data suggest that IFN is a novel suppressor of ASM cell proliferation, and the alterations in the TNF-IFN pathways may play an important role in regulating changes in ASM mass seen in asthma (49). Our findings also raise the possibility that, in addition to IFNs, TNF represents a potential cytokine involved in the activation of STAT1 and IRF-1 transcription factors previously described in the airways of patients with asthma (18).

In summary, we showed that human ASM cells treated with TNF are a novel source of active IFN in the airways. Secreted IFN, in an autocrine manner, modulates cell mitogenesis as well as TNF-induced expression of proinflammatory genes RANTES and IL-6 that have been associated with chronic inflammatory diseases. Further studies are needed to determine the molecular mechanisms by which TNF promotes secretion of IFN as well as the pathophysiologic consequences of IFN secretion by ASM in asthma where TNF is thought to play a major role.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants 2R01-HL55301 (to R. A. P.) and 1P50 [PDB] -HL67663 (to R. A. P.) and by American Lung Association Grant RG-062-N (to Y. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Parker B. Francis fellow in pulmonary research. To whom correspondence should be addressed: Pulmonary, Allergy and Critical Care Division, University of Pennsylvania Medical Center, 421 Curie Blvd., 848 BRB II/III, Philadelphia, PA 19104-6160. Tel.: 215-573-9851; Fax: 215-573-4469; E-mail: amrani{at}mail.med.upenn.edu.

¹ The abbreviations used are: TNF, tumor necrosis factor ; TNFR, TNF receptor; ASM, airway smooth muscle; JAK, janus tyrosine kinase; IFN, interferon; STAT, signal transducers and activators of transcription; IRF, interferon-regulatory factor; GAS, -activated site; ISRE, interferon stimulation response element; PDGF, platelet-derived growth factor; IFNAR, IFN receptor; RANTES, regulated on activation normal T cell expressed and secreted; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; r (prefix), recombinant; TRAF2, tumor necrosis factor receptor-associated factor 2; cds, coding sequence.

	ACKNOWLEDGMENTS

 
We acknowledge Laura Kester for excellent technical assistance in immunoblot analyses and Mary McNichol for assistance in preparing the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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