Tumor Necrosis Factor Receptor (TNFR) 1, but Not TNFR2, Mediates Tumor Necrosis Factor- –Induced Interleukin-6 and RANTES in Human Airway Smooth Muscle Cells: Role of p38 and p42/44 Mitogen-Activated Protein Kinases

Little information is available regarding the mechanisms involved in cytokine-induced synthetic function of human airway smooth muscle (ASM) cells. Here, we report that tumor necrosis factor receptor (TNFR) 1-induced p38 and p42/44 mitogenactivated protein kinase (MAPK) activation modulates tumor necrosis factor(TNF )-mediated synthetic responses: expression of intercellular adhesion molecule-1 (ICAM-1) and secretion of interleukin (IL)-6 and the regulated-on-activation, normal T-cell expressed and secreted (RANTES) chemokine in human ASM cells. Pretreatment of ASM cells with SB203580, a p38 MAPK inhibitor, slightly enhanced TNF -induced ICAM-1 expression in a dose-dependent manner but partially inhibited secretion of RANTES and IL-6. In contrast, PD98059, a p42/44 inhibitor, reduced ICAM-1 expression by 50% but had no effect on TNF -induced RANTES or IL-6 secretion. SB203580 and PD98059 had little effect on TNF -induced nuclear factorB (NFB) activation as determined in cells transfected with a NFB–luciferase reporter construct. We also found that agonistic antibodies specific for either TNFR1 or TNFR2 stimulated IL-6 and RANTES secretion and activated p38 and p42/44 MAPKs. In addition, both antibodies induced NFB-mediated gene transcription. Using receptor-specific blocking antibodies, we found that TNFR1 primarily regulates TNF -induced IL-6 and RANTES secretion and activation of p38 and p42/44 MAPK pathways. Interestingly, we found that TNFR1 and TNFR2 are expressed differently on the cell surface of ASM cells. Our data suggest that despite the presence of functional TNFR2, TNFR1 associated with MAPK-dependent and -independent pathways is the primary signaling pathway involved in TNF -induced synthetic functions in ASM cells. Airway smooth muscle (ASM) is an important effector cell in asthma. Recent evidence suggests that cytokine-induced changes in the airway smooth muscle phenotype may modulate bronchial hyperresponsiveness and airway inflammation (Amrani et al., 2000a; Chung, 2000). Therefore, characterizing the cellular and molecular mechanisms that regulate ASM function will probably lead to new therapeutic approaches in the management of asthma. Using cultured ASM cells that retain their physiological responsiveness to agonist (Panettieri et al., 1989), investigators have shown that TNF , a cytokine present in high levels in the bronchoalveolar lavage fluid of asthmatic patients, stimulates ASM to express and/or secrete many proinflammatory mediators such as cytokines, chemokines, growth factors, and adhesion molecules known to be involved in asthma (Amrani et al., 2000a; Chung, 2000). The mechanisms underlying TNF -induced synthetic responses of ASM have not been fully elucidated. TNF initiates its pleiotropic action by binding to two receptors designated as p55 (TNFR1) and p75 (TNFR2) according to their apparent molecular mass. These receptors are coexpressed on the surface of most cells (for review, see Tartaglia and Goeddel, 1992). Although both TNFR1 and TNFR2 were found to be coexpressed on ASM cells and in native tissues (Amrani et al., 1996, 2000b), the majority of TNF effects on ASM are mediated by TNFR1 (for review, see Amrani et al., 2000a). TNFR1 was shown to regulate TNF -induced potentiation of agonist-evoked calcium signals, ASM cell proliferation (Amrani et al., 1996), and expression of adhesion molecules (Amrani et al., 2000b). Whether TNFR1 and/or TNFR2 activation This work was supported by Grants R01-HL64063 and R01-HL55301 from the National Institutes of Health. A.J.A. was supported by a C. J. Martin Fellowship (977301) from the National Health and Medical Research Council (Australia). ABBREVIATIONS: ASM, airway smooth muscle; TNF , tumor necrosis factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; IL, interleukin; PDGF, platelet-derived growth factor; ICAM-1, intercellular adhesion molecule-1; RANTES, regulated on activation, normal T-cell expressed and secreted; NFB, nuclear factorB; FBS, fetal bovine serum; BSA, bovine serum albumin; COX, cyclooxygenase. 0026-895X/01/6004-646–655$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 4 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 892/930958 Mol Pharmacol 60:646–655, 2001 Printed in U.S.A. 646 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from coordinately regulates ASM synthetic function remains unclear. Furthermore, the downstream signaling that mediates TNF -induced ASM synthetic function is not clearly understood. Mitogen-activated protein kinases (MAPKs), a family of serine/threonine kinases, consist of at least three distinct members: extracellular signal-regulated kinase (ERK, also called p42/p44 MAPK), p38 MAPK, and c-Jun NH2-terminal kinase (Davis, 1994). MAPKs regulate a variety of cellular responses, including inflammation, cell cycle progression, proliferation, and differentiation (for review, see Cowley et al., 1994). Recently, we and others have shown that p38 and p42/44 MAPKs are activated by a variety of proinflammatory agents such as cytokines (TNF and IL-1 ), growth factors (epidermal growth factor and PDGF), or contractile agonists (histamine and thrombin) (Orsini et al., 1999; Page et al., 1999). Collectively, these studies support the notion that MAPKs play an essential role in modulating contractile, proliferative, or synthetic responses in ASM cells. To better define the role of MAPK activation in modulating ASMinduced synthetic responses, we examined the role of p38 and p42/44 in TNF -induced ICAM-1 expression and IL-6 and RANTES secretion. In separate experiments, we also asked whether p38 and p42/44 modulate TNF -induced NFB activation because NFB seems to be critically important in regulating cytokine-induced ICAM-1 and IL-6 secretion in ASM cells and other cell types (Roebuck et al., 1995; Sanceau et al., 1995; Amrani et al., 1999). In addition, we investigated the contribution of TNF receptor subtypes in these responses. We report that TNFR1 and TNFR2 initiate similar cellular responses when activated individually using specific agonistic antibodies. However, we found that TNF -induced ICAM-1 expression, RANTES, and IL-6 secretion are mediated primarily via TNFR1. P38 and p42/44 pathways differentially regulate these cellular responses. In addition, although TNF activated p38 and p42/44, inhibition of p42/44 had little effect on TNF -induced IL-6 and RANTES secretion. Inhibition of p38 only partially affected these responses. Our data support the concept that TNF -induced synthetic responses are mediated primarily via the actions of TNFR1 and occur via MAPK-independent and -dependent pathways. Materials and Methods Human Airway Smooth Muscle Cell Culture. 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 isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor, and 10 U/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 105m Nytex mesh, and the filtrate was washed with equal volumes of cold Ham’s F-12 medium supplemented with 10% FBS (Hyclone Laboratories, Logan, UT). Aliquots of the cell suspension were plated at a density of 1.0 10 cells/cm. The cells were cultured in Ham’s F-12 medium supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 g/ml amphotericin B, and this was replaced every 72 h. Human ASM cells in subculture during the second through fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin. Flow Cytometry. Flow cytometric analysis was performed as described previously (Amrani et al., 1999). Human ASM cells were stained using either a fluorescein isothiocyanate-conjugated monoclonal antibody specific for ICAM-1 or an isotype-matched control (R & D Systems, Minneapolis, MN). Samples were then analyzed using an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA). ICAM-1 expression was expressed as the increase in mean fluorescence intensity over background. Measurement of IL-6 and RANTES Secretion by ASM Cells. Near-confluent, growth-arrested human ASM cells were pretreated with SB203580, SB202474 (negative congener), or with diluent for 30 min, before stimulation with TNF . In parallel experiments, cells were exposed to TNFR1, TNFR2 agonistic antibody (R & D Systems and Cell Sciences, Inc., Norwood, MA, respectively), or isotypematched goat or mouse IgG (R & D Systems). In experiments with receptor-blocking antibodies, cells were first preincubated with either anti-TNFR1 or anti-TNFR2 (R & D Systems) for 30 min before addition of TNF . After 24 h, cell supernatants were harvested and IL-6 or RANTES measured by an enzyme-linked immunosorbent assay according to the manufacturer’s instructions (R & D Systems). SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis. Immunoblot analysis for p38 and p42/44 was performed as described previously (Amrani et al., 1999): Briefly, ASM cells were washed with cold phosphate-buffered saline and resuspended in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 g/ml aprotinin and leupeptin. Proteins were analyzed on a 12.5% SDS-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The membranes were blocked in 3% BSA in Tris-buffered saline then incubated with a rabbit monoclonal IgG against the phosphorylated form of p38 or p42/44 (Cell Signaling, Beverly, MA). After incubation with the appropriate peroxidase-conjugated secondary antibody (Roche Molecular Biochemicals, Minneapolis, MN), the bands were visualized by the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ) and autoradiographed. Transfection of Human ASM Cells. Transfection of human ASM cells was performed as described previously (Amrani et al., 1999). Briefly, 4 10 cells were harvested and resuspended in 5 ml of Dulbecco’s modified Eagle’s medium [containing 200 g of DEAEdextran, 3 10 plaque-forming units of Ad5-GPT, and 10 g of pNFB-Luc designed for monitoring activation of NFB (CLONTECH, Palo Alto, CA)] and 2 g of pSV-b-galactosidase control vector to normalize transfection efficiencies (Promega, Madison, WI). The mixture was added to cells grown on 10-cm tissue culture plates and incubated for 2 h at 37°C. The media were then removed and the cells were washed for 1 min with 10% dimethyl sulfoxide in calciumand magnesium-free phosphate-buffered saline and incubated with Ham’s F-12 medium for 48 h. Cells were then rendered quiescent in medium containing 0.2% FBS for 16 h and exposed to TNF for 4 h in the absence or the presence of inhibitors or receptor-blocking antibodies. Cells were then harvested, and luciferase and -galactosidase activities were assessed using a Promega kit according to the manufacturer’s instructions. Immunostaining of TNF Receptors on Human ASM Cells. ASM cells were washed with HEPES buffer containing 137.5 mM NaCl, 1.25 mM CaCl2, 1.25 mM MgCl2, 0.4 mM NaH2PO4, 6 mM KCl, 5.6 mM glucose, 10 mM HEPES, and 0.1% BSA. The cells were fixed with 4% paraformaldehyde solution for 30 min at 4°C and then blocked in HEPES buffer (supplemented with 0.1% BSA) for 30 min at room temperature. ASM cells were then incubated with either anti-TNFR1 (htr-9) or anti-TNFR2 (utr-1) antibodies (Bachem Biosciences, King of Prussia, PA) for 120 min at 37°C. Negative controls included cells incubated with a mouse isotype IgG1 control (R & D Systems). After three washings, cells were incubated with a goat anti-mouse Alexa 594 (Molecular Probes, Eugene, OR). To stain the TNF Stimulates IL-6 and RANTES in Human Airway Smooth Muscle 647 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from nucleus, cells were then exposed to 1/5000 dilution of 4,6-diamidino2-phenylindole (2 mg/ml). After washing, the glass coverslips were mounted onto glass slides, examined under epifluorescence microscopy (Nikon, Tokyo, Japan), and photographed. Statistical Analysis. Statistical analysis was calculated using the Student’s t test for paired values (two-tailed test). Values were considered statistically significant if the probability (P) of chance alone causing the effect was less than 5%.