Tumor Necrosis Factor - ATS Journals

Tumor Necrosis Factor-a Stimulates Mucin Secretion and Cyclic GMP Production by Guinea Pig Tracheal Epithelial Cells In Vitro Bernard M. Fischer, Lori G. Rochelle, Judith A. Voynow, Nancy J. Akley, and Kenneth B. Adler Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh; Cystic Fibrosis/Pulmonary Research Center, University of North Carolina, Chapel Hill; and Division of Pediatric Pulmonary Diseases, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina

Tumor necrosis factor (TNF)-a, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-a–induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air–liquid interface culture. Exogenously applied TNF-a (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-a increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 mM); and mucin secretion in response to TNF-a or to the cGMP analogue dibutyryl cGMP (100 and 500 mM) was attenuated by the specific PKG inhibitor KT5823 (1 mM). Increased mucin secretion and increased cGMP production in response to TNF-a both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 mg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 mM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 mM). Collectively, the results suggest that TNF-a stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG. Fischer, B. M., L. G. Rochelle, J. A. Voynow, N. J. Akley, and K. B. Adler. 1999. Tumor necrosis factor-a stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro. Am. J. Respir. Cell Mol. Biol. 20:413–422.

(Received in original form April 6, 1998) Address correspondence to: Kenneth B. Adler, Ph.D., Dept. of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606. E-mail: [email protected] Abbreviations: Calphostin C, Calph. C; cyclic guanosine monophosphate, cGMP; diacylglycerol, DAG; dibutyryl cGMP, DBcGMP; dimethyl sulfoxide, DMSO; NG-monomethyl-D-arginine, DNMA; epidermal growth factor, EGF; enzyme-linked immunosorbent assay, ELISA; fetal bovine serum, FBS; femtomole(s), fmol; guanylate cyclase, GC-S; guinea pig tracheal epithelial, GPTE; 3-isobutyl-1-methylxanthine, IBMX; lactate dehydrogenase, LDH; NG-monomethyl-L-arginine, LNMA; nitric oxide, NO; nitric oxide synthase, NOS; nitrite and nitrate, NO x; optical density, OD; phosphate-buffered saline, PBS; phosphatidylcholine-specific phospholipase C, PC-PLC; PLC that hydrolyzes phosphatidylinositol, PI-PLC; protein kinase C, PKC; cGMP-dependent protein kinases, PKG; phospholipase C, PLC; tumor necrosis factor- a, TNF-a; 60-kD TNF-a receptor, TNFR-I. Am. J. Respir. Cell Mol. Biol. Vol. 20, pp. 413–422, 1999 Internet address: www.atsjournals.org

Tumor necrosis factor (TNF)-a is a proinflammatory cytokine implicated in the pathogenesis of airway inflammation (1), asthma (2), acute respiratory distress syndrome (ARDS) (3), and other respiratory/pulmonary disorders. In the airways, TNF-a can elicit many potentially deleterious effects, such as enhanced expression of adhesion molecules, bronchoconstriction, pulmonary edema, and stimulated production of cytokines and lipid mediators (2). In addition, we (4) and others (5) have shown that TNF-a can provoke secretion of mucin by airway epithelium, and we have demonstrated preliminarily that TNF-a induces mucin secretion via an intracellular pathway that appears to involve endogenously produced nitric oxide (NO) (4). NO has been recognized as a potential mediator of inflammatory lung disease. Activation of nitric oxide synthase (NOS) and elevated levels of exhaled NO have been demonstrated in asthmatics (6, 7) and in patients with other inflammatory airway diseases, such as bronchiectasis and chronic obstructive pulmonary disease (8, 9). NO also

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can affect mucociliary clearance in airways by altering both mucin secretion (4, 10) and ciliary beating (11). However, the cellular mechanisms remain unknown. One potential pathway associated with NO signaling in cells is via activation of the soluble form of guanylate cyclase (GC-S), resulting in production of cyclic guanosine monophosphate (cGMP). cGMP can exert a number of effects similar to those of NO, including stimulation of mucin secretion in the gut (12, 13) and alterations in ciliary beat frequency in airway epithelium (14), reflecting its role as an NO-regulated signaling molecule. cGMP also can affect other cellular functions, including regulation of ion channels, regulation of phosphodiesterases (PDEs), and activation of cGMP-dependent protein kinases (PKG) (15). In this study we examined effects of TNF-a on mucin secretion and cGMP production in guinea pig tracheal epithelial (GPTE) cells in vitro, a well-characterized model of differentiated airway epithelium (16). The results suggest that TNF-a provokes mucin secretion via a mechanism involving activation of the NOS →GC-S→cGMP→PKG pathway. In addition, protein kinase C (PKC) and a phosphatidylcholine-specific phospholipase C (PC-PLC) appear to be involved in mediating the secretory response to TNF-a, presumably upstream of activation of NOS.

Materials and Methods Sources of Reagents Healthy, viral antibody–free Hartley guinea pigs of either sex (150 to 250 g) were purchased from Charles River (Stone Ridge, NY) and were housed for at least 5 d in the laboratory animal facility at North Carolina State University, College of Veterinary Medicine, before isolation of GPTE cells. GPTE cells were cultured on 24-mm-diameter, 4.7-cm2, 0.45-mM-pore-size, collagen-treated Transwell-COL inserts purchased from Corning Costar (Cambridge, MA). Dulbecco’s modified Eagle’s medium-F12 (with 15 mM N-2-hydroxyethylpiperazine-N9-ethane sulfonic acid [DMEM/F12]), Hanks’ balanced salt solution (with and without calcium and magnesium supplementation [HBSS]), and phosphate-buffered saline (PBS) were purchased from the University of North Carolina Tissue Culture Facility, Lineberger Comprehensive Cancer Center (Chapel Hill, NC). HL-1 supplement for use with DMEM/F12 was purchased from BioWhittaker (Walkersville, MD). Human recombinant epidermal growth factor (hrEGF) and low-endotoxin bovine serum albumin (BSA) were purchased from Intergen (Purchase, NY). Gentamicin and L-glutamine were obtained from GIBCO (Grand Island, NY). Amphotericin B (Fungizone) was from Squibb (Princeton, NJ), and low-endotoxin fetal bovine serum (FBS) was from Hyclone (Logan, UT). Protein assay reagent was obtained from Bio-Rad (Melville, NY) and Calphostin C (Calph. C) from Biomol (Plymouth Meeting, PA). Human recombinant TNF-a was purchased from R&D Systems (Minneapolis, MN) and D609 (tricyclodecan-9-ylxanthogenate potassium salt) from Kamiya Biochemical (Thousand Oaks, CA). Calbiochem (San Diego, CA) was the source for bisindolylmaleimide (GF 109203X, Go 6850), LY83583 (6-anilino-5,8-quinolinequinone), KT5823, NG-

monomethyl-L-arginine (LNMA), and NG-monomethyl(DNMA). Cytotox 96 kits for assessment of cytotoxicity were purchased from Promega (Madison, WI). cGMP enzyme immunoassay (EIA) kits were obtained from Amersham (Arlington Heights, IL). The 96-well tissue culture treated plates and 96-well polystyrene high-protein binding enzyme-linked immunosorbent assay (ELISA) plates were procured from Corning Costar. Ro31-8220 was a gift from Dr. Keith Yagaloff (Hoffmann La Roche, Inc., Nutley, NJ). National Welders (Raleigh, NC) was the source for prepurified/medical-grade gases, including oxygen, helium, carbon dioxide, and nitrogen. 2,29-Azinobis-3-ethylbenzthiazolinesulfonic acid was obtained from Kirkegaard & Perry Labs (Gaithersburg, MD). The 100% (absolute) ethanol was purchased from North Carolina State University Central Warehouse (Raleigh, NC). Hydrochloric acid, sodium hydroxide (NaOH), ferrous ammonium sulfate, sodium bicarbonate, sodium carbonate, and sodium chloride were purchased from Fisher Scientific (Raleigh, NC). Sigma Chemical Co. (St. Louis, MO) was the source of all other reagents and chemicals including 3-isobutyl-1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO), ammonium molybdate, sodium nitrite, potassium nitrate, sulfuric acid, retinal acetate, nystatin, N2,29-O-dibutyryl-guanosine 39,59-cyclic monophosphate, (dibutyryl cGMP, [DBcGMP]), polyoxyethylenesorbitan monolaurate (Tween 20), protease XIV (pronase), DLdithiothreitol, and Trizma base (tris-[hydroxymethyl]aminomethane). D-arginine

Cell Culture Primary cultures of GPTE cells were established using the air–liquid interface procedure developed in this laboratory (16). Briefly, proteolytically dissociated tracheal epithelial cells were seeded on the apical surface of Transwell-COL inserts in six-well plates at a density of 50,000 to 60,000 cells/cm2 (250,000 to 300,000 cells/well) in 1 ml DMEM/ F12 (supplemented with 5% FBS, 4 mM L-glutamine, 25 ng/ ml hrEGF, 5 3 1028 M retinal acetate, 1% HL-1, 100 mg/ ml gentamicin, 40 U/ml nystatin, and 0.5 mg/ml amphotericin B) and 2 ml of supplemented medium, basally. Media were changed every other day until the cells were 70 to 80% confluent (4 to 6 d), at which time the FBS was removed from the media and the apical surface of the cells was exposed to humidified 97% air–3% CO2 at 378C (air– liquid interface culture method). For the next 4 to 6 d cultures were fed with serum-free media, basally only. Agonists and Inhibitors The specific agonists and inhibitors used are listed in Table 1 with the concentrations tested and their appropriate solubilization vehicles. For those inhibitors solubilized in DMSO, the final DMSO concentration was 0.1% unless otherwise indicated. All inhibitors tested were pre- and coincubated with the cells in the presence of the agonist or under control conditions for the designated time period. Measurement of Mucin Secretion Mucin secretion was measured as described previously (18). Briefly, mucin was collected from apical surfaces of the cells after an 8-h period (BASELINE) representing con-

Fischer, Rochelle, Voynow, et al.: TNF-a–Induced Mucin Secretion Depends on PLC, PKC, NO, and cGMP

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TABLE 1

Agonists and inhibitors used Compound

Agonists Human recombinant TNF-a DBcGMP (12) Inhibitor [and target] Calph. C. [PKC] (19) Bisindolylmaleimide [PKC] (25) Ro31-8220 [PKC] (25) LY83583 [GC-S] (22) KT5823 [PKG] (23) D609 [PC-PLC] (24) LNMA [NOS] (4) DNMA (4) IBMX [PDE] (17)

Concentration(s) Used

Solubilization Vehicle

0.1–150 ng/ml* 100 and 500 mM

PBS 1 0.1% BSA Sterile double-distilled water

0.1–0.5 mM 20 nM 10 mM 10 and 50 mM 1 mM 5–20 mg/ml 1 mM 1 mM 0.5 mM

DMSO DMSO DMSO DMSO DMSO Sterile double-distilled water HBSS HBSS Sterile double-distilled water

* TNF-a–specific activity: 2.86 3 107 U/mg protein; 10 ng/ml 5 286 U/ml; 15 ng/ml 5 429 U/ml.

stitutive secretion, and compared with mucin secreted in response to test agents or controls after an additional 8-h EXPERIMENTAL period. In all studies, cells were preincubated with the specific inhibitory agent, and then coincubated with the inhibitor plus TNF-a (or DBcGMP). Mucin secreted was expressed as the ratio of EXPERIMENTAL/ BASELINE for each well. By this method, each well could serve as its own control. An aliquot of each test-agent solution (agonist 6 inhibitor) not exposed to GPTE cells was used as an additional control. Mucin secretion was measured by a specific ELISA developed in this laboratory using two mouse anti–guinea pig tracheal mucin monoclonal antibodies in a sandwichstyle assay described previously (18). Two positive controls for the mucin ELISA were used: (1) conditioned medium (DMEM 1 20% FBS) containing mucin released from guinea pig tracheal explants and (2) mucin collected from GPTE cell cultures for 24 to 48 h before starting the BASELINE period. The negative control for the mucin ELISA was supplemented medium never exposed to tracheal explants or GPTE cells in culture. In addition, test-agent solutions (agonist 6 inhibitor) were tested for possible interference in the mucin ELISA. BASELINE and EXPERIMENTAL mucin samples for each culture well were assayed in duplicate on the same 96-well ELISA plate to minimize interplate variability. Optical density (OD) was measured from an ELISA reader at 405 nm, with 450 serving as a reference wavelength. Results were calculated by dividing the OD reading corresponding to mucin secreted for the EXPERIMENTAL period by the OD reading corresponding to mucin secreted during the BASELINE period. Results were expressed as percent of control. Measurement of cGMP Production IBMX, a nonspecific cellular PDE inhibitor was used to prevent breakdown of cGMP during the exposure period and to enhance the detectable levels of cGMP. GPTE cells were preincubated with 0.5 mM IBMX for 2 h at 37 8C. During the last 15 to 30 min of the IBMX preincubation period, any other inhibitors to be tested (Table 1) were also added. To start the stimulation period, TNF-a (15 ng/ ml) was added for 8 h. At the conclusion of the test period,

cells were rinsed with ice-cold PBS and frozen at 2208C for later extraction. To extract cGMP, cells were thawed and suspended in ice-cold 65% ethanol, vortexed vigorously, and then centrifuged at 2,000 3 g for 15 min at 48C. Supernatants and pellets were separated, and supernatants (containing extracted cGMP) were dried in a vacuum evaporator at 608C and then stored at 2208C until assayed for cGMP (19). Cellular pellets were resuspended in 1 N NaOH and further diluted 1:5 or 1:10 for protein analysis with the Bio-Rad protein assay system. cGMP was quantified utilizing a commercially available EIA. Briefly, the procedure was as follows: the 96-well plate in the kit was precoated with a donkey antirabbit immunoglobulin G antibody, and a rabbit anti-cGMP antibody was added to all wells except the blank and nonspecific-binding wells. Samples and standards were acetylated and added to the plate and incubated for 2 h at 4 8C. At that time, cGMP conjugated with horseradish peroxidase was added to all wells except the blank and the plate and incubated for an additional hour at 4 8C. After washing, tetramethylbenzidine substrate was added to all wells, the wells were incubated for 30 min at room temperature, and OD was measured in an ELISA reader at 450 nm. Results were compared with a standard curve (0 to 512 femtomoles [fmol]/well) and expressed as fmol cGMP/mg protein. Measurement of NO Production For assessment of GPTE cell NO production, total nitrite (N O22) and nitrate (N O23) (NOx) were measured utilizing an Antek Model 7020 NO detector (Antek Instruments, Inc., Houston, TX), a chemiluminescence detection system adapted from the method of Cox (20). Aliquots (100 ml each) of cell lysates from GPTE cells exposed to TNF-a in medium (similar to the mucin secretion studies) and the corresponding controls were individually injected into the reaction vessel containing a solution of 50% (vol/vol) sulfuric acid with 0.4% (wt/vol) ammonium molybdate and 0.4% (wt/vol) ferrous ammonium sulfate at 808C to reduce NOx to NO. Under vacuum, the vapor containing NO plus helium carrier gas was passed through a chiller and watervapor trap, then through an acid trap to neutralize any acid vapors, and finally into the detector. NOx values were

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compared with standards of potassium nitrate (KNO3) and sodium nitrite (NaNO2). Cell lysates were assayed for protein content with the Bio-Rad protein assay system. Results were expressed as nmol NOx/mg protein. Cytotoxicity Assessment The cytotoxicity of each agonist and inhibitor not previously tested in GPTE cells was assessed using a commercially available colorimetric assay for lactate dehydrogenase (LDH). GPTE cells were exposed to agonists and inhibitors separately as well as to TNF-a plus inhibitors. Both supernatants and cells lysates were collected and assessed for LDH content. As a positive control for cytotoxicity, additional untreated cells were incubated with distilled water and these supernatants and cell lysates were also assessed for LDH content. Percent of LDH release was calculated as follows: LDH in supernatant -------------------------------------------------------------------------------------------------- × 100 LDH in supernatant + LDH in cell lysate Statistical Analyses All data are presented as means 6 SEM unless otherwise indicated. Control and TNF-a–treated NO production were compared via an unpaired Student’s t test. All other results were analyzed by one-way analysis of variance with Bonferroni post-test correction for multiple comparisons (21). Data were considered significant at P , 0.05. Because primary cultures tend to vary from day to day, results were expressed as percent of control, as described previously (18).

Results Cytotoxicity None of the inhibitors or solvents used in these studies (Table 1) affected either mucin secretion or cGMP production in GPTE cells when added by themselves. In addition, each of the inhibitors and agonists, at the concentrations used, were determined by LDH release assays to be noncytotoxic, with the largest percent release in response to any of the agents tested being 5.67 6 0.61% in response to 0.5 mM IBMX after a 26-h exposure period, as compared with 4.51 6 0.69% in the corresponding control cells. Mucin Secretion Effect of TNF-a on mucin secretion. TNF-a provoked mucin secretion in both a concentration- and time-dependent manner. TNF-a exposure increased mucin secretion, with a peak response at TNF-a concentrations of 10 to 15 ng/ml (286 to 429 U/ml) and a decline to baseline levels at 100 ng/ml (Figure 1A). Mucin secretion was maximal at 8 h of TNF-a incubation (Figure 1B). Thus, TNF-a treatment conditions of 10 to 15 ng/ml and 8 h incubation time were used for all subsequent pharmacologic studies. Effect of NOS or GC-S inhibition on TNF-a–stimulated mucin secretion. To determine the role of NO and cGMP in mediating TNF-a–stimulated mucin secretion, enzymes that catalyze production of NO or cGMP were inhibited pharmacologically during TNF-a stimulation. NOSs, which catalyze NO production, were inhibited by the arginine

Figure 1. TNF-a–induced mucin secretion by GPTE cells. (A) Dose response. GPTE cells were continuously exposed to varying doses of TNF-a in medium for 5 to 8 h. TNF-a provoked maximal stimulation at 10 to 15 ng/ml (286 to 429 U/ml). Secretion is expressed as percentage of unpaired controls (medium alone). *Significantly different from control (P , 0.05). Values are means 6 SEM for n 5 6. Dotted line indicates control level of secretion. (B) Time course. GPTE cells were continuously exposed to TNF-a (15 ng/ml) in medium for 1 to 24 h. Results are expressed as sample or EXPERIMENTAL period OD/BASELINE period OD. *Significantly different from corresponding control (medium alone, P , 0.05). Values are means 6 SEM for n 5 5. Error bars not visible are indicative of SEM , 0.02.


derivative LNMA. LNMA treatment (1 mM) significantly inhibited TNF-a–stimulated mucin secretion, whereas its inactive stereoisomer, DNMA, had no effect (Figure 2A). TNF-a (15 ng/ml) significantly increased intracellular NOx levels (TNF-a: 2.19 6 0.29 versus control: 0.39 6 0.03 nmol/mg, P , 0.05, n 5 5; equivalent to 0.72 mM and 0.52 mM NOx in TNF-a–treated and control cell lysates,

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respectively), suggesting that TNF-a exposure activated NOS. NO activates GC-S, resulting in production of cGMP and subsequent activation of the downstream signal, PKG. Therefore, the role of cGMP in mediating TNF-a–stimulated mucin secretion was investigated. A specific GC-S inhibitor, LY83583 (10 to 50 mM) (22), inhibited mucin se-

Figure 2. Effect of NOS, GC-S, and PKG inhibition on TNF-a– induced mucin secretion by GPTE cells. (A) NOS inhibition by LNMA; GC-S inhibition by LY83583. GPTE cells were exposed to TNF-a (15 ng/ml) in medium for 8 h with or without pre- 1 coincubation with 1 mM LNMA, its inactive stereoisomer DNMA, or LY83583 (10 and 50 mM). LNMA and 50 mM LY83583, but not DNMA, inhibited TNF-a–induced mucin secretion. All agents in the absence of TNF-a had no significant effect on mucin secretion (LNMA, 99.8 6 4.8%; DNMA, 119 6 5.9%; 50 mM LY83583, 93.5 6 6.1%). Secretion is expressed as percentage of unpaired controls (medium alone). *Significantly different from control; †significantly different from TNF-a–treated cells; §significantly different from TNF-a 1 DNMA (P , 0.05). Values are means 6 SEM for n 5 6. Dotted line indicates control level of secretion. (B) PKG inhibition by KT5823. GPTE cells were exposed to TNF-a (15 ng/ml) for 8 h with or without pre- 1 coincubation with 1 mM KT5823. KT5823, 1 mM, inhibited TNF-a–induced mucin secretion. KT5823 in the absence of TNF-a had no effect on mucin secretion (not shown). Secretion is expressed as percentage of unpaired controls (medium alone). *Significantly different from control; †significantly different from TNF-a– treated cells (P , 0.05). Values are means 6 SEM for n 5 7. Dotted line indicates control level of secretion. ( C) DBcGMPinduced mucin secretion: effect of PKG inhibition by KT5823. GPTE cells were exposed to DBcGMP (100 or 500 mM) for 8 h with or without pre- 1 coincubation with 1 mM KT5823. DBcGMP stimulated secretion of mucin; 1 mM KT5823 inhibited DBcGMPinduced mucin secretion. Secretion is expressed as percentage of unpaired controls (medium alone). *Significantly different from control; †significantly different from 500 mM DBcGMP-treated cells (P , 0.05). Values are means 6 SEM for n 5 6. Dotted line indicates control level of secretion.

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cretion in response to TNF-a in a concentration-dependent manner (Figure 2A). In addition, the cell-permeable cGMP analogue DBcGMP (100 and 500 mM) (12) stimulated mucin secretion (Figure 2C), supporting the role of cGMP in mediating mucin secretion. To determine whether or not PKG is also required as an intracellular signal for TNF-a– stimulated mucin secretion, KT5823, a specific PKG inhibitor (23), was evaluated as an inhibitor of mucin secretion. KT5823 (1 mM) significantly attenuated TNF-a–induced mucin release (Figure 2B). KT5823 also attenuated the effect of DBcGMP on mucin secretion (Figure 2C), suggesting that PKG activation follows downstream from cGMP activation in the secretory pathway. To demonstrate that cGMP and downstream signals did mediate the effect of TNF-a on mucin secretion, we examined the effect of TNF-a stimulation on cGMP production. TNF-a (10 to 15 ng/ml) stimulated cGMP production at 8 h (Figure 3). TNF-a–induced upregulation of cGMP was inhibited by LNMA but not by DNMA (Figure 4). Neither LNMA nor DNMA affected control levels of cGMP pro-

duction in the absence of TNF-a. The GC-S inhibitor LY83583 also blocked induction of cGMP by TNF-a (Figure 4), suggesting that both NO and GC-S are required for TNF-a–stimulated production of cGMP in GPTE cells. Effect of inhibition of PC-PLC or PKC on TNF- a– induced mucin secretion. Because TNF-a receptor activation is known to trigger the downstream signaling molecules PC-PLC and PKC in many cell types, we investigated the putative role of these enzymes in mediating TNF-a–induced mucin secretion and cGMP production. D609, a specific inhibitor of PC-PLC (24) completely blocked TNF-a–induced mucin secretion (Figure 5A). Calph. C is a specific PKC inhibitor that binds to the diacylglycerol (DAG) binding site of the enzyme to block its activity (19). As illustrated in Figure 5B, Calph. C, in a concentration-dependent manner, significantly inhibited TNF-a–induced mucin secretion. Ro31-8220 and bisindolylmaleimide, two other PKC inhibitors structurally similar to straurosporine but more specific for PKC, and which interact with the adenosine triphosphate (ATP) binding site on PKC (25), also inhibited TNF-a–induced mucin secretion at concentrations of 10 mM and 20 nM, respectively (data not shown). In addition, both Calph. C and D609 completely inhibited increased cGMP production in response to TNF-a (Figure 6), supporting their roles as intracellular signals upstream

Figure 3. TNF-a–induced cGMP production in GPTE cells. Dose response. GPTE cells were exposed to varying doses of TNF-a (0.15 to 150 ng/ml) for 8 h in the presence of IBMX as described in MATERIALS AND METHODS. A concentration of 15 ng/ml TNF-a stimulated cGMP production. TNF-a (15 ng/ml) that was inactivated by boiling for 90 min before addition (heat inactivated) did not stimulate cGMP production. cGMP production is expressed as femtomoles cGMP/per milligram of protein. *Significantly different from control (medium); †significantly different from 15 ng/ ml TNF-a–treated cells (P , 0.05). Values are means 6 SEM for n 5 6.

Figure 4. Effect of NOS and GC-S inhibition on TNF-a–induced cGMP production in GPTE cells. GPTE cells were exposed to TNF-a (15 ng/ml) for 8 h with or without pre- 1 coincubation with 1 mM LNMA, 1 mM DNMA, or 50 mM LY83583 (GC-S inhibitor). LNMA and LY83583, but not DNMA, inhibited TNFa–induced cGMP production. In the absence of TNF-a, none of the agents affected cGMP production (not shown). Results are expressed as femtomoles cGMP per milligram of protein. *Significantly different from control (medium); †significantly different from TNF-a–treated cells; §significantly different from TNF-a 1 DNMA (P , 0.05). Values are means 6 SEM for n 5 7.


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Figure 6. Effect of PC-PLC and PKC inhibition on TNF-a– induced cGMP production in GPTE cells. GPTE cells were exposed to TNF-a (15 ng/ml) for 8 h with or without pre- 1 coincubation with 0.3 mM Calph. C (PKC inhibitor) or 5 to 20 mg/ml D609 (PC-PLC inhibitor). Calph. C and D609 each inhibited TNF-a–induced cGMP production. Either agent in the absence of TNF-a did not affect cGMP production (not shown). Results are expressed as femtomoles cGMP per milligram of protein. *Significantly different from control (medium); †significantly different from TNF-a–treated cells; §significantly different from TNF-a 1 5 mg/ml D609 (P , 0.05). Values are means 6 SEM for n 5 6.

of GC-S. Calph. C, Ro31-8220, and bisindolylmaleimide were all solubilized in DMSO. Concentrations of up to 0.5% DMSO did not significantly alter control or TNF-a– stimulated levels of mucin secretion or cGMP production (Figure 5B).

Discussion A variety of inflammatory mediators have been shown to enhance release of mucin from airway epithelial cells. To study intracellular signaling involved in secretion of mucin in response to such stimulation, we have focused on the

Figure 5. Effect of PC-PLC and PKC inhibition on TNF-a– induced mucin secretion by GPTE cells. (A) PC-PLC inhibition by D609. GPTE cells were exposed to TNF-a (15 ng/ml) for 8 h with or without pre- 1 coincubation with varying concentrations of D609 (5 to 20 mg/ml). TNF-a–stimulated secretion of mucin was attenuated by D609 at 10 and 20 mg/ml. D609 (20 mg/ml) in the absence of TNF-a had no effect on control levels of mucin secretion. Secretion is expressed as percentage of unpaired controls (medium alone). *Significantly different from control; †significantly different from TNF-a–treated cells (P , 0.05). Values are means 6 SEM for n 5 7. Dotted line indicates control level of secretion. (B) PKC inhibition by Calph. C. GPTE cells were exposed to TNF-a (15 ng/ml) in medium for 8 h with or without

pre- 1 coincubation with Calph. C (0.1 and 0.5 mM). Calph. C (0.5 mM) inhibited TNF-a–induced mucin secretion. Calph. C (0.1 to 0.5 mM) in the absence of TNF-a had no effect on control levels of mucin secretion (not shown). Secretion is expressed as percentage of unpaired controls (medium alone). DMSO, the vehicle for Calph. C, had no effect on control levels (not shown) or on TNF-a–induced mucin secretion. Ro31-8220 (10 mM) and bisindolylmaleimide (20 nM) also inhibited TNF-a–induced mucin secretion (not shown). *Significantly different from control; †significantly different from TNF-a–treated cells (P , 0.05). Values are means 6 SEM for n 5 6. Dotted line indicates control level of secretion.

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mechanism of TNF-a–induced mucin secretion because TNF-a is a major inflammatory mediator in airway disease. TNF-a (10 to 15 ng/ml) treatment for 8 h resulted in maximal mucin secretion from GPTE cells. These treatment conditions were similar to those used by Levine and colleagues (5) and Nettesheim and Bader (26) to stimulate mucin secretion by human airway epithelium in vitro. In addition, the levels of TNF-a used in these studies (10 to 15 ng/ml) are in the range of those reported in bronchoalveolar lavage fluid in patients with ARDS (10 to 15 ng/ml) (3) or those produced in vitro by leukocytes recovered by bronchoalveolar lavage from asthmatic patients (250 to 2,000 U/ml) (27). Thus, this model of TNF-a–induced mucin secretion uses clinically relevant concentrations of the cytokine and has been confirmed experimentally by other investigators. In addition, mucin secretion and related enzymatic activations did not appear related to nonspecific toxic responses to TNF-a because all concentrations used were not toxic, based on results of LDH release/retention assays. The response also could not be attributed to endotoxin contamination of the TNF-a preparation because boiling the TNF-a completely abolished the response, which would not be the case if sufficient endotoxin were present. Many of the studies reported here are based on use of well-established pharmacologic inhibitors such as LNMA, LY83583, KT5823, D609, Calph. C, and others, the specificity and efficacy of which have been reported extensively (Table 1). Additional studies were performed to ascertain these agents indeed were acting as inhibitors of specific enzyme systems in GPTE cells. For example, inhibitory effects of LNMA at 1 mM could be overcome with excess L-arginine; and LY83583 at the concentrations used in this report (50 mM) blocked production of cGMP in response to TNF-a (Figure 4), indicating inhibition of GC-S. In other studies, D609 completely inhibited TNF-a–induced activation of PC-PLC by GPTE and human airway epithelial cells (28). Relatedly, even when used at only a single concentration (e.g., KT5823) the concentration chosen was the same as reported effective in other systems (Table 1). However, it is possible that other effects of some of these agents may have been involved in their inhibitory actions on mucin secretion and/or cGMP generation. Interestingly, high concentrations of TNF-a (100 ng/ml [2,860 U/ml] or above) did not affect mucin secretion or cGMP production. This lack of response to higher concentrations of TNF-a might be attributable to shedding or downregulation of TNF-a receptors. A recent report (29) demonstrated downregulation of messenger RNA (mRNA) levels for the 60-kD TNF-a receptor (TNFR-I) by 50% within 2 h of stimulation of rat airway epithelial cells with 1,000 U/ml of TNF-a and a decrease in detectable protein for TNFR-I within 24 h. These authors reported that with 1,000 U/ml of TNF-a there was shedding of the extracellular domain of the receptor (soluble TNFR-I) within 30 min of stimulation. Soluble TNFR-I can function as an endogenous TNF-a inhibitor by binding to TNF-a and preventing it from binding to cell surface receptors (30). Thus, the TNFR-I may either up- or downregulate interactions of TNF-a in epithelial cells. Little is known about intracellular signaling mecha-

nisms that could mediate responses of airway epithelial cells to TNF-a. Levine and associates demonstrated that TNF-a–enhanced expression of mucin genes was mediated by PKC and tyrosine kinase (5), whereas Nettesheim and Bader implicated prostaglandins as mediators important in TNF-a–induced mucin secretion (26). Recent work from our laboratory demonstrated that NO was a common intermediate required for mucin secretion provoked by different inflammatory mediators, including TNF-a, histamine, oxidant stress, and platelet-activating factor (4). NO is also involved in mucin secretion by intestinal epithelium (12, 13). In the present study we explored signals both upstream and downstream of NO to further define the signaling pathways leading to mucin secretion by TNF-a. The results indicate that TNF-a provoked mucin release from GPTE cells via a mechanism that involved NO-induced GC-S, cGMP, and PKGs. Activation of PC-PLC and PKC were also involved in the response. In several cell types, TNF-a activates enzymes that hydrolyze membrane phospholipids, including phospholipase C (PLC) and phospholipase A2. PLC encompasses a group of enzymes that hydrolyze a variety of membrane phospholipids. The PLC that hydrolyzes phosphatidylinositol (PI-PLC) generates inositol-1,4,5-trisphosphate, which releases calcium from intracellular storage sites, and 1,2DAG, which activates PKC. The PLC that hydrolyzes PCPLC generates the products phosphocholine and DAG. TNF-a can activate PI-PLC or PC-PLC in different cell types. For example, TNF-a activates PC-PLC in monocytes (24, 31) but activates PI-PLC in osteoblasts (32). We assessed both PC-PLC and PI-PLC activity in GPTE cells in response to TNF-a. Surprisingly, TNF-a did not activate PI-PLC, as was shown previously for other mucin secretagogues such as histamine (33), oxidant stress (18), or ATP (34). However, in GPTE cells, TNF-a activated PC-PLC as demonstrated by two lines of evidence. First, both mucin secretion and cGMP production induced by TNF-a were significantly attenuated by D609, a specific inhibitor of PC-PLC (24). Second, recent thin-layer chromatography studies from our laboratory demonstrated that TNF-a activates PC-PLC in GPTE and human airway epithelial cells, a response that could be inhibited by D609 (28). These results are consistent with other models in which PC-PLC is a component of the signaling cascades of several cytokines and growth factors, including transforming growth factor-b (35), EGF (36), and interferon-g (IFN-g) (37). Thus, a potential signaling cascade for TNFa–induced mucin secretion is: TNF-α ⇒ PC-PLC ⇒ mucin hypersecretion. The products of PC-PLC–mediated hydrolysis of phosphatidylcholine are DAG and phosphocholine. In turn, DAG serves as an important component for activation of PKC. TNF-a has been shown to stimulate PKC activity in airway epithelium (38), and PKC has been shown to mediate many effects of TNF-a in the lung and respiratory tract, such as generation of pulmonary edema (39), activation of pulmonary artery endothelial cells (39, 40), stimulation of the respiratory burst of neutrophils (41), and induction of adhesion molecule expression in airway epithelium (42). PKC also has been implicated as a mediator


of airway mucin secretion (43–45). In addition to activating PKC, PC-PLC also has been shown to activate NOS and increase NO production in response to IFN-g 1 lipopolysaccharide (37). PKC also upregulates inducible NOS (iNOS) gene expression and activity (46–48), and overexpression of the PKCε isoform results in enhanced NO production and upregulation of iNOS mRNA expression (49) in macrophages. TNF-a has also been shown to enhance iNOS expression in lung epithelial cells (50). Therefore, both PC-PLC and PKC may increase NO production in GPTE cells, and a potential mechanism could be: TNF-α ⇒ PC-PLC → DAG → PKC → NOS → NO ⇒ mucin hypersecretion. Many effects of NO are mediated by activation of GC-S and subsequent cGMP production. In this study the role of cGMP in TNF-a–induced intracellular signaling was demonstrated by the following: (1) inhibition of NOS by LNMA resulted in decreased cGMP production and decreased mucin secretion; (2) inhibition of GC-S by LY83583 resulted in decreased cGMP production and decreased mucin secretion; and (3) DBcGMP stimulated mucin secretion. Therefore, cGMP is likely activated by NO in the TNF-a–stimulated signaling pathway: TNF-α ⇒ PC-PLC → DAG → PKC → NOS → NO → GC-S → cGMP ⇒ mucin hypersecretion.

3.

4.

5.

6. 7. 8. 9. 10. 11. 12. 13.

A potential pathway by which cGMP could enhance mucin secretion involves activation of PKG. PKG are serine kinases that have been implicated in a variety of biologic responses, including calcium signaling, neutrophil degranulation, potassium and chloride channel functioning, and secretory activity (51–53). As illustrated in Figure 2, both TNF-a- and DBcGMP-induced mucin secretion were significantly reduced by the PKG inhibitor KT5823, suggesting that PKG could be mediating the secretory response to TNF-a and cGMP. Thus, the complete pathway suggested by this study follows:

14. 15. 16.

17.

18.

TNF-α ⇒ PC-PLC → DAG → PKC → NOS → NO → GC-S → cGMP → PKG ⇒ mucin hypersecretion. Clearly, exposure of airway epithelial cells to the cytokine TNF-a appears to activate each of these intracellular signaling enzymes and second messenger molecules. These enzymes and second messengers may provide new therapeutic targets for potential intervention to prevent mucin hypersecretion in inflammatory airway disease. Acknowledgments: This work was supported by grants from the National Institutes of Health (RO1 HL36982 and F32 HL09063) and Glaxo Wellcome, Inc., Research Triangle Park, NC. Portions of this work were presented at the annual meetings of the American Lung Association/American Thoracic Society (Seattle, WA, May 1995; New Orleans, LA, May 1996) and at the 37th and 38th Aspen Lung Conferences (June 1995 and June 1996). One author (B.M.F.) completed portions of this work as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

19.

20. 21. 22.

23. 24.

25.

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