Introduction

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In industrialized countries, inflammatory airway diseases are frequent disorders with increasing incidence. The pathogenesis of these diseases is complex and poorly understood. Recent studies, however, suggest that in the broad array of cells involved in orchestrating airway inflammation (e.g., lymphocytes, mast cells, eosinophils, granulocytes), the airway epithelium itself plays a prominent and active role. Indeed, epithelial cell-derived mediators such as interleukin (IL)-8, 15-hydroxyeicosatetraenoic acid (15-HETE), tumor necrosis factor- (TNF-), granulocyte macrophage colony-stimulating factor, nitric oxide (NO), and prostaglandin E₂ (PGE₂) promote or reduce airway inflammation. In addition, bronchial epithelial cells are also targets of a variety of mediators released by neighboring cells (NO, TNF-, etc.) (1).

Currently available therapies for inflammatory and obstructive airway disease such as ₂-adrenoceptor agonists or glucocorticoids are nonspecific and not without side effects. One group of reagents with powerful anti-inflammatory activity consists of inhibitors of cyclic nucleotide phosphodiesterases (PDE). Thus far, seven PDE gene families encoding multiple PDE proteins have been identified (5). The PDE classification is based on substrate specificity and regulatory characteristics. The PDE isoenzyme pattern differs among tissues and cells. Our recent analysis in endothelial cells showed high activities of PDE2, PDE3, and PDE4 (8). These cells, however, lacked PDE5, the major cyclic guanosine monophosphate (cGMP)-degrading PDE. Airway epithelial cells (AEC) probably have an efficient cGMP-metabolizing capacity because they are continuously exposed to NO in the inhaled and exhaled air (11). The cyclic adenosine monophosphate (cAMP)-specific PDE4 has been demonstrated in many proinflammatory cell types, and PDE4 inhibition is a conceivable useful anti-inflammatory approach (12). Smooth-muscle cells possess PDE1-5, and inhibition of the cGMP-specific PDE5 regulates the tone of human peripheral airways (13).

The first objective of the present study, therefore, was to identify the PDE isoenzymes in AEC. This appears to be essential if a specific and site-directed therapy with both anti-inflammatory and antiobstructive properties is to be developed. Thus, we established AEC of porcine and human origin and also used the human bronchial epithelial cell line BEAS-2B.

The second objective of the present study was to test the concept that PDE inhibition alters the secretion of mediators and cytokines in AEC. It is well established that in the presence of enhanced intracellular levels of cyclic nucleotides, monocytes, T lymphocytes, eosinophils, and granulocytes demonstrate a decreased respiratory burst and cytokine generation (12, 14). Similarly, in endothelial cell monolayers, elevated cyclic nucleotides block endothelial hyperpermeability (8). Collectively, cAMP and cGMP appear to be important regulators of the inflammatory reaction. To test this concept, we focused on epithelial generation of IL-8 and of the lipid mediators PGE₂ and 15-HETE.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Materials

Tissue-culture plasticware was obtained from Becton-Dickinson, Heidelberg, Germany. Medium 199, serum-free keratinocyte medium, Dulbecco's modified Eagle's medium (DMEM)-F12, fetal calf serum (FCS), Hanks' balanced salt solution (HBSS), phosphate-buffered saline (PBS), N-2-hydroxypiperazine-N'-ethanesulfonic acid (Hepes), epidermal growth factor (EGF), L-glutamine, and bovine pituitary extract (BPE) were from GIBCO, Karlsruhe, Germany. Deoxyribonuclease type I (DNase), trypan blue, and antibiotics were from Boehringer Mannheim GmbH, Mannheim, Germany. Anticytokeratin (clone MNF 116) and avidin-biotin-horseradish peroxidase (HRP) were supplied from Dakopatts GmbH, Hamburg, Germany. Antivimentin (clone V9) was obtained from Dianova-Immunotech, Hamburg, Germany. Biotinylated horse antimouse antibody, Vectastain alkaline phosphatase kit, p-nitrophenylphosphate, and avidin-biotin blocking reagent were from Vector Laboratories, Inc., Burlingame, CA. The monoclonal antibody directed against PGE₂ was generously provided by K. Brune and J. Mollenhauer, Institute of Pharmacology, University of Erlangen, Germany. Recombinant human TNF- (2 × 10⁷ U/mg) was obtained from R&D Systems, Wiesbaden, Germany. Gelatin from porcine skin type I, protease XIV, insulin, bovine apotransferrin, forskolin, 3-isobutyl-1-methyl-xanthine (IBMX), Crotalus atrox snake venom, dipyridamole, sodium nitroprusside (SNP), bovine serum albumin (BSA), methyl formate, soybean trypsin inhibitor (SBTI), leupeptin, pepstatin, benzamidine, dithiothreitol (DTT), calmodulin, and W7 were purchased from Sigma Chemical Co., Munich, Germany. Erythro-9-(2- hydroxy-3-nonyl)-adenine (EHNA) was provided by Dr. Podzuweit, Max Planck Institute of Experimental Cardiology, Bad Nauheim, Germany. 4-(3'-cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone (rolipram) was kindly provided by Schering AG, Berlin, Germany; and 4, 5-dihydro-6- (4-[1H-imidazol-1-yl]-2-thienyl)-5-methyl-3-(2H)-pyridazinone (motapizone) was from Rhone-Poulenc Rorer GmbH, Cologne, Germany. Enoximone was supplied by Dr. H. D. Görlich, Marion Merrell Dow Pharmaceuticals, Berlin, Germany. 6-(difluormethoxy-3-methoxyphenyl)-3-(2H)-pyridazinone (zardaverine) was provided by Dr. Schudt, Byk Gulden GmbH, Konstanz, Germany. Vinpocetine, 8-methoxy-methyl-IBMX, trifluoperazine, and zaprinast were obtained from Calbiochem, Bad Soden, Germany, Tritiated cAMP and cGMP, IL-8 enzyme-linked immunosorbent assay (ELISA), and the ¹²⁵I-cAMP and ¹²⁵I-cGMP assay systems were from Amersham Buchler, Braunschweig, Germany. The 15-HETE assay system was purchased from Paesel and Lorei GmbH, Frankfurt, Germany; and C₁₈ columns (100-mg bed size) were from ICT, Bad Homburg, Germany. Retinoic acid was provided by Biofluids Inc., Rockville, MD. QAE-Sephadex A-25 columns were from Bio-Rad, Richmond, CA. All other chemicals used were of analytical grade.

Isolation and Culture of Porcine AEC

Tracheae and central bronchi were obtained from freshly slaughtered pigs and kept on ice during transportation. Cells were isolated according to Wu and Smith, with minor modifications (15). Specimens were rinsed with ice-cold HBSS, filled with protease solution (M199 containing 0.1% protease XIV, 0.001% DNase, 200 U/ml penicillin, 0.2 mg/ml streptomycin, and 10 µg/ml amphotericin B) and incubated for 16 h at 4°C. The protease solution was removed, and the cells were harvested by gentle agitation, washed twice in serum containing medium, and resuspended in DMEM/F12 supplemented with 1% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 5 µg/ml amphotericin B, 0.1 mg/ml L-glutamine, 5 µg/ml BPE, 5 ng/ml EGF, 5 µg/ml transferrin, 5 µg/ml insulin, 0.1 pg/ml retinoic acid, and 20 ng/ml T3. The number of isolated cells was determined in a Neubauer chamber, and viability was assessed by trypan blue (0.4%) exclusion. About 10⁶ viable cells/cm² were plated on collagen (10 µg/ml)-coated culture dishes, well plates, or chamber slides. The AEC were

grown in a 5% CO₂ atmosphere at 37°C and reached confluence in 7 to 10 d. Confluent primary cultures of porcine AEC were then used.

Isolation and Culture of Human AEC

Human tracheae and central bronchi were obtained from the Department of Pathology, University of Giessen. Preparations originated from patients who had died of a nonpulmonary disease and who had not been intubated. Human AEC were isolated according to the protocol described above, with the exception that exposure to protease solution was extended to 20 h. Only primary cultures of confluent AEC were used for the studies.

Culture of BEAS-2B Cells

A generous gift of Dr. Curtis Harris, National Institutes of Health, Bethesda, MD (16), BEAS-2B cells were grown on a matrix composed of 50 ng/ml fibronectin, 50 ng/ml vitronectin, and 0.01% BSA (fatty acid-free) in serum-free keratinocyte medium containing 0.1 pg/ml retinoic acid, 5 µg/ml BPE, 5 ng/ml EGF, 0.5 µg/ml epinephrine, 0.1 mg/ml L-glutamine, 100 U/ml penicillin, 0.1 µg/ml streptomycin, and 5 µg/ml amphotericin B. Cells were incubated in a 5% CO₂ atmosphere at 37°C and reached confluence within 14 d. For cell passage, monolayers in T75 flasks were exposed to trypsin-ethylenediamenetetraacetic acid solution (80 µg/ml trypsin) for 5 to 10 min at 37°C. After addition of 8 ml serum-free keratinocyte medium, the cell suspension was centrifuged at 800 rpm at 4°C for 5 min. Medium was removed and cell pellets were incubated in 200 µl SBTI solution (30 mg/ml) for 2 min, resuspended in complete medium, and passaged with a 1:3 split.

A549 cells were cultured according to Kwon and colleagues (17).

Characterization of Isolated and Cultured AEC by Immunohistochemistry

Isolated epithelial cells were characterized by morphologic and immunocytochemical criteria. AEC were grown on chamber slides and used for immunohistochemical examination after 8 d in culture. Cells were washed, permeabilized, and fixed with cold (20°C) acetone/methanol (1:1). Preparations were then saturated with avidin-biotin blocking reagent and with PBS containing 0.1% horse serum to avoid nonspecific binding. Thereafter, slides were incubated with mouse anticytokeratin or mouse antivimentin antibody. After careful removal of excess primary antibodies, a biotinylated horse antimouse antibody was added, followed by alkaline phosphatase ABC solution. After visualization with p-nitrophenylphosphate (2 mg/ml), slides were washed extensively, embedded, and photographed.

Assay of PDE

To determine PDE activity in AEC, cyclic nucleotide hydrolyzing activities were determined in cell lysates as described previously (9). Medium of T75 flasks was removed and cells were scraped in HBSS and washed twice by centrifugation. Cell pellets were resuspended in 200 µl ice-cold homogenization buffer (PBS containing 10 mM Hepes, 1 mM ethyleneglycol-bis-[-aminoethyl ether]-N,N'-tetraacetic acid, 1 mM MgCl₂, 5 mM DTT, 5 µM pepstatin, 10 µM leupeptin, 10 µM trypsin inhibitor, and 2 mM benzamidine) and disrupted by sonification (ten pulses, 1 s each, on ice at a power output of 60 W). Protein concentration was measured using a commercially available protein reagent (Bio-Rad, Munich, Germany), using BSA as the standard. Cell lysates were kept on ice and PDE assays were performed within 5 min. PDE activity was determined as previously described (8, 9). A standard reaction mixture containing 60 mM Tris (pH 7.4), 5 mM MgCl₂, 1.25 mM CaCl₂, 100 µM calmodulin, and 0.5 µM cyclic nucleotide/³H-labeled cyclic nucleotide (about 30,000 counts per minute) was used in a total volume of 200 µl. The reaction was initiated by addition of cell lysates (40 to 50 µg protein) and carried out at 37°C for 15 min. The reaction was stopped by adding 50 µl 0.2 N HCl and immediate cooling on ice for 10 min. Following incubation with 5'-nucleotidase (C. atrox snake venom, 50 µl, 2 mg/ml in 400 mM Tris, pH 8.5) at 37°C for 15 min, 200-µl aliquots of the assay volume were loaded on QAE-Sephadex A-25 columns (1-ml bed volume). The columns were eluted with 2 ml 30 mM ammonium formate (pH 6.0) directly into scintillation vials. Results were corrected for blanks using denatured protein.

Determination of PDE Isoenzyme Activities

With exception of PDE7, PDE isoenzyme activities in AEC lysates were determined by quantifying the inhibition of PDE activity in the presence of 1 µm motapizone (PDE3), 10 µM rolipram (PDE4), 10 µM zaprinast (PDE5), 100 µM EHNA (PDE2), or 100 µM vinpocetin or 8-methoxy- methyl-IBMX (PDE1). Overall PDE activity was blocked by 100 µM IBMX. The presence of PDE7 was suggested by demonstration of messenger RNA (mRNA) encoding for this PDE isoenzyme (see REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION section, below, for details). Because of the lack of a specific PDE7 inhibitor, the activity of this isoenzyme was calculated as the difference between PDE4-related and IBMX-insensitive cAMP-hydrolysis (see Table 1).

Determination of Cellular Cyclic Nucleotides

Cellular cyclic nucleotide content was measured by radioimmunoassays (RIAs) using commercially available ¹²⁵I-assay systems as described (8, 9, 18, 19). Confluent epithelial cells in 24-well plates (about 600,000 cells/well) were incubated with specific PDE inhibitors in HBSS 15 min before and throughout the experimental period. Cells were then extracted twice with 500 µl ice-cold 65% ethanol. Extracts were pooled, evaporated under a stream of nitrogen by 56°C, and dissolved in assay buffer. Aliquots of the extracts and of the standards were acetylated by addition of acetic anhydride and triethylamine (1:2) to enhance the sensitivity of cyclic nucleotide detection.

Determination of 15-HETE by Reversed-Phase High Pressure Liquid Chromatography (RP-HPLC) and ³H-RIA

Eicosanoids were extracted from cell supernatants by octadecylsilyl (ODS) solid-phase extraction columns (Bond Elute LRC solid phase C₁₈ cartridges, 100 mg sorbent). Cartridges were conditioned with 10 ml 100% methanol, 10 ml methanol/water/acetic acid (72.6/27.3/0.1 vol/vol/vol, pH 5.1), and 10 ml distilled water. Samples were then applied to the cartridges, washed with 10 ml distilled water, and eluted with 600 µl of methanol, followed by 2× 600 µl of formic acid methyl ester. Eluates were vacuum-evaporated and redissolved in 30 µl 100% methanol. The RP-HPLC was carried out on ODS columns (250 × 2 mm; Hypersil ODS 3-µm particles) at a constant flow of 200 µl/ min (Gynkotek pump, model 480) using methanol/water/ acetic acid (72.6/27.3/0.1 vol/vol/vol, pH 5.1) as mobile phase. Online detection and quantification of the eluted compounds was carried out using a UVIS VARIO 2 photometer/Spectra Physics integrator at 237 nm, with commercially available 15-HETE as standard reagent. The recovery determined under the conditions outlined above using different quantities of 15-HETE was 72 ± 3% (n = 10). Additionally, 15-HETE synthesis was measured using a commercially available ³H-RIA system. Supernatants of stimulated and control AEC were extracted as described for the RP-HPLC procedure. Samples were resolved in RIA buffer and assayed according to the manufacturer's instructions. All 15-HETE data obtained were corrected for their respective recovery of the overall analytical procedure and are expressed as nanograms of 15-HETE per 10⁶ AEC.

Determination of PGE₂

PGE₂ in AEC supernatants was examined using an avidin-biotin-HRP-based ELISA system as described (20). Conjugates of PGE₂ with BSA were prepared by carbodiimide coupling and were then separated from uncoupled prostanoid and free BSA by gel filtration on a Pharmacia G20 column. ELISA plates were coated with 200 µl of diluted PGE₂ conjugate solution (1 µl/ml in 46 mM Na₂CO₃/ NaHCO₃, pH 9.6) and incubated overnight at 4°C. After washing, a 100-µl sample volume was applied to each well, followed by 100 µl anti-PGE₂-antibody solution (5 µg/ml in PBS containing 2% BSA) for 16 h. ELISA was completed by adding biotinylated antimouse antibodies and biotin-HRP as described (20). Results are expressed as nanograms of PGE₂ per 10⁶ AEC.

IL-8 ELISA

IL-8 determination was performed using a commercially available IL-8 ELISA system (Amersham). Aliquots (100 µl) of supernatant of resting and TNF--stimulated cells were applied to a 96-well plate, and the ELISA was performed according to the manufacturer's instructions. Results are expressed as nanograms of IL-8 per 10⁶ AEC.

Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from approximately 1 × 10⁷ AEC as described by Chomczynski and Sacchi (21). One microgram of RNA was reverse-transcribed into complementary DNA (cDNA) using AMV reverse transcriptase according to the manufacturer's instructions (Promega, Heidelberg, Germany). The reverse transcription (RT)- generated cDNA encoding PDE4 and PDE7 genes was amplified by polymerase chain reaction (PCR) using specific primers designed from the reported primary sequences deposited with the Genbank data base (6). The primers used for PDE amplification, cycle numbers, annealing temperatures, and expected product sizes appear in Table 2. The amplification was performed with 0.5 U Taq-polymerase (InViTek, Berlin, Germany) in a Hybaid thermal cycler (MWG, Ebersberg, Germany). RT-PCR of the glyceraldehyde-3-phosphate dehydrogenase gene was routinely performed to confirm the integrity of epithelial cell RNA and equal loading of sample.

The PCR products were size-fractionated on 1.5% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light before Southern blotting to nylon membranes. The blots were hybridized with the cloned and sequenced cDNA for human PDE7 to confirm identity with the PCR product (6, 14). To control for possible genomic contamination, RNA was processed in parallel with the reverse-transcribed sample in the absence of reverse transcriptase. In addition, water blanks were subjected to PCR in parallel with test samples. None of these controls produced a detectable band on ethidium bromide-stained agarose gels or after Southern hybridization.

PDE7A1 PCR products were cloned into the pGEM-T-Vector (Promega) and sequenced using the Sequenase 2.0-system (Amersham). Six clones of the porcine cDNA were compared with the human sequence.

Statistical Methods

A one-way analysis of variance was used for data in Figures 1, 6, 7, and 8 and Table 1. Main effects were then compared by an F probability test. P < 0.05 was considered significant (22).

View larger version (16K): [in this window] [in a new window]   Figure 1.   PDE isoenzymes in AEC of different origins. The PDE isoenzyme patterns in porcine AEC (top), BEAS-2B (middle), and human AEC (bottom) are shown. All cell types express activities of PDE4, PDE5, and PDE7. Primary cultures of human AEC additionally possess the calcium/calmodulin- stimulated PDE1. PDE7 activity was calculated as the difference in cAMP hydrolysis between IBMX- and rolipram-treated samples as described in MATERIALS AND METHODS. Data are given as means ± SEM of at least five independent experiments.

View larger version (22K): [in this window] [in a new window]   Figure 6.   Cyclic nucleotide content in BEAS-2B and porcine AEC. The stimulated increases of cGMP (10 µM SNP) and cAMP (1 µM forskolin) in intact cells in the presence or absence (N) of PDE isoenzyme-specific inhibitors are shown. In BEAS-2B (top left) and porcine AEC (top right), cGMP levels increased significantly in the presence of the PDE5 inhibitor zaprinast only, whereas inhibition of all other PDE isoenzymes was without effect. As to cAMP, only inhibition of PDE4 with rolipram resulted in a significant accumulation of this cyclic nucleotide in BEAS-2B (bottom left) and porcine AEC (bottom right). Basal levels of cAMP and cGMP are shown as unfilled bars (U). Data represent means ± SEM of eight to 11 independent experiments.

View larger version (24K): [in this window] [in a new window]   Figure 7.   Cyclic nucleotide content in human AEC. Stimulated increases of cAMP (1 µM forskolin) and cGMP (10 µM SNP) in intact cells in the presence or absence (N) of PDE isoenzyme-specific inhibitors are shown. cAMP levels (left) increased significantly in the presence of rolipram. Inhibition of PDE5 (zaprinast) and PDE1 (vinpocetine or 8-methoxymethyl-IBMX) resulted in a significant accumulation of cGMP (right). Basal levels of cAMP and cGMP are shown as unfilled bars (U). Data represent means ± SEM of three independent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 8.   Effect of elevated cyclic nucleotides on AA/A23-induced generation of PGE2 and 15-HETE in porcine AEC. Stimulation of 15-HETE and PGE2 generation of porcine AEC was accomplished by exposure of cells to AA/A23 for 30 min. Two experimental sets of cells were pretreated with reagents prior to stimulation to increase cyclic nucleotides. cAMP was elevated by exposure of AEC to 1 µM forskolin/10 µM rolipram (F/R), and cGMP by exposure to 10 µM SNP/10 µM zaprinast (S/Z) for 10 min. Data indicate that AA/A23-stimulated PGE2-synthesis was significantly enhanced in AEC with elevated cAMP levels and reduced in AEC with elevated cGMP levels. Stimulated 15-HETE formation in AEC was not influenced by enhanced cyclic nucleotides. Data appear as means ± SEM of four to seven independent determinations.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Cell Characterization

Three bronchial epithelial cell types were used for the identification of the PDE enzyme pattern. Besides the well-established SV-40/adenovirus-transformed human bronchial epithelial cell line BEAS-2B, we studied confluent primary cultures of AEC of porcine and human origin. The purity of the cells used was > 97%, as indicated by morphologic and immunocytochemical criteria. These cells were cytokeratin-positive and vimentin-negative. Porcine cultured pulmonary artery endothelial cells (8, 9, 18, 23) were used as vimentin-positive controls. The major cyclooxygenase and lipoxygenase products in porcine AEC studied were PGE₂ and 15-HETE (24).

PDE Isoenzyme Activities in AEC

All three cell types studied hydrolyzed both cyclic nucleotides with different efficiencies (Table 1). BEAS-2B displayed the highest total cAMP- and cGMP-metabolizing capacities, followed by porcine and human AEC. Allosteric regulators, specific inhibitors, and mRNA analysis were applied to study the PDE isoenzyme pattern in lysates of AEC. cAMP hydrolysis was reduced by 50 to 80% in the presence of the specific PDE4-inhibitor rolipram, whereas other specific inhibitors had no effect (Table 1, Figure 1), suggesting that PDE4 is the major cAMP-degrading enzyme in AEC. The remaining cAMP hydrolysis might be attributable to PDE7, which by definition is a rolipram- insensitive, cAMP-hydrolyzing enzyme (6, 7). Selective inhibitors for PDE7 are unknown and no specific antibodies were available to us to confirm this notion. We therefore evaluated the presence of mRNA for PDE7 expressed in AEC by RT-PCR using primers designed to recognize a unique sequence in the human gene (Tables 2 and 3). Figure 2 shows an ethidium bromide-stained agarose gel of a representative experiment demonstrating amplified cDNA fragments derived from human AEC corresponding to the predicted size of human PDE7 (285 base pairs [bp], Table 3). This was subsequently confirmed by cloning of the PCR products of all three airway epithelial cell types studied into pGEM-T-Vector, followed by double-stranded sequencing (100 and 93.4% homology for human and porcine AEC, respectively; Table 4). Southern blot analysis of the PCR products derived from BEAS-2B and porcine AEC with the human PDE7-cDNA probe also confirmed the presence of PDE7 in these cells (Figure 3). In these studies, RT-PCR products of mRNA extracted from A549 and human lung tissue were used as positive controls (Figure 3).

View larger version (48K): [in this window] [in a new window]   Figure 2.   Expression of PDE7 mRNA in human AEC. An ethidium bromide-stained agarose gel of the RT-PCR products derived from human mRNA is shown. In the presence of reverse transcriptase, both the extracted RNA of human AEC (lane 1) and human lung (lane 4, positive control) show a single band with the appropriate size (285 bp). In the absence of reverse transcriptase (lane 2) or RNA (lane 3), no RT-PCR products were synthesized.

View larger version (39K): [in this window] [in a new window]   Figure 3.   Expression of PDE7 mRNA in airway epithelial cells. Shown is the Southern analysis of cDNA of A549 cells derived by RT-PCR, BEAS-2B, porcine AEC (one of four samples analyzed), and human lung (lane 8, positive control). The cDNA fragments of all cell types studied hybridized with the cloned and sequenced cDNA for human PDE7 (lanes 1, 3, and 5). In the absence of reverse transcriptase (RT, lanes 2, 4, and 6) or RNA (lane 7) no PCR products were synthesized, indicating specific amplification of the 285-bp fragment corresponding to PDE7.

PCR analysis of human AEC indicated the expression of mRNA encoding for the PDE4 variants 4A5, 4C1, 4D2, and 4D3 (Figure 4). mRNA extracted from human lung tissue, A549 cells, and BEAS-2B cells served as controls. Primers used for amplification of PDE4 cDNA fragments and their corresponding PCR products were cloned and sequenced as previously described (14).

View larger version (116K): [in this window] [in a new window]   Figure 4.   Expression of PDE4 mRNA of AEC. An ethidium bromide-stained agarose gel of the RT-PCR products derived from mRNA of primary cultured human AEC (lane A), A549 cells (lane B), BEAS-2B cells (lane C), and human lung (lane D) is shown. Human AEC expressed PDE4 variants 4A5, 4C1, 4D2, and 4D3. In the absence of reverse transcriptase (lane E), no RT- PCR products were synthesized. Human lung tissue expressed all PDE4 isoenzymes studies (lane D).

In the presence of the specific PDE5-inhibitor zaprinast, cGMP hydrolysis was inhibited by 80 to 100% (Table 1, Figure 1) whereas inhibitors of PDE2 and PDE3 (EHNA and motapizone) had no effect (Figure 1), suggesting that PDE5 is the major cGMP-degrading enzyme in AEC.

The addition of vinpocetine or 8-methoxy-methyl-IBMX to lysates of BEAS-2B and porcine AEC (in the presence of calmodulin and Ca²⁺) did not inhibit cGMP hydrolysis, indicating that PDE1 does not play a dominant role in cyclic nucleotide degradation in these cells (Table 1, Figure 1). In primary cultures of human AEC, however, vinpocetine was effective and reduced cGMP hydrolysis by approximately 20%, thereby suggesting the presence of a PDE1 (Table 1, Figure 1).

Cyclic Nucleotide Content in Intact AEC

PDE isoenzyme activities in cell lysates were correlated with cyclic nucleotide levels in intact cells. Increasing concentrations of rolipram induced a concentration-dependent accumulation of cAMP in intact porcine AEC stimulated with 1 µM forskolin with an IC₅₀ of 0.34 µM (Figure 5). Similarly, zaprinast induced a concentration-dependent accumulation of intracellular cGMP in intact AEC stimulated with SNP (IC₅₀ = 1 µM) (Figure 5). Both inhibitors were used at 10 µM for subsequent studies.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Concentration-dependent increase of cyclic nucleotides in porcine AEC. The accumulation of cAMP (open squares, right) and cGMP (filled squares, left) in the presence of increasing concentrations of zaprinast and rolipram is shown. Both inhibitors concentration-dependently increased cGMP or cAMP with IC50 values of 1 or 0.34 µM, respectively. Data are given as means ± SEM of four to seven independent experiments.

Forskolin-stimulated porcine AEC and BEAS-2B cells showed a substantial cAMP accumulation in the presence of rolipram. Suppression of all other PDE isoenzymes did not enhance cAMP concentration, indicating that PDE4 is the major cAMP-degrading enzyme in AEC (Figure 6). Because of the lack of specific inhibitors, it is currently not possible to assess the contributions made by PDE7 to overall cAMP metabolism in intact cells.

With respect to cGMP hydrolysis, BEAS-2B and porcine AEC were stimulated with SNP. cGMP concentrations increased in presence of zaprinast (Figure 6) only, indicating the predominant role of PDE5 for cGMP metabolism. Data for human AEC were very similar, with the exception that cGMP also increased in the presence of a PDE1 inhibitor (Figure 7). Overall, these cyclic nucleotide results in intact AEC are consistent with the PDE isoenzyme pattern determined in cell lysates.

Effect of Cyclic Nucleotide Levels on the Generation of PGE₂, 15-HETE, and IL-8 in AEC

To correlate AEC function with cyclic nucleotide levels, we focused on the ability of epithelial cells to generate PGE₂, 15-HETE, and IL-8 (Figures 8 and 9). A 7-fold increase in PGE₂ formation was noted after exposure of porcine AEC to 50 µM arachidonic acid/10 µM A23187 (AA/ A23) for 30 min (Figure 8, bottom). When cAMP was elevated first in AEC with forskolin/rolipram, there was a 4-fold increase in the effectiveness of the stimulus, resulting in an overall 28-fold increase in PGE₂ synthesis above baseline. Interestingly, preincubation of AEC with SNP/ zaprinast to increase maximally cGMP content reduced the effectiveness of the stimulus for PGE₂ production by 40% (Figure 8, bottom). Forskolin and rolipram are highly specific reagents; on the other hand, SNP and zaprinast in high concentrations may have effects unrelated to increased cGMP levels.

View larger version (16K): [in this window] [in a new window]   Figure 9.   Effect of elevated cyclic nucleotides on TNF-induced generation of IL-8 in BEAS-2B cells. IL-8 synthesis was stimulated in BEAS-2B cells by 10 ng/ml TNF- for 8 h. Two experimental sets of cells were pretreated with reagents prior to stimulation to increase cyclic nucleotides. cAMP was elevated by exposure of AEC to 1 µM forskolin/10 µM rolipram (F/R), and cGMP by exposure to 10 µM SNP/10 µM zaprinast (S/Z) for 10 min. Data indicate that TNF-stimulated IL-8 synthesis remained unchanged even in the presence of enhanced cyclic nucleotide levels in AEC. Data appear as means ± SEM of four independent determinations.

15-HETE formation in porcine AEC was increased 7-fold within 10 min by AA/A23 (Figure 8, top). Maneuvers taken to elevate cyclic nucleotide levels did not modify the stimulated synthesis of this lipoxygenase product. In control experiments we verified that the stimuli themselves (AA/ A23) did not increase cyclic nucleotides in epithelial cells (data not shown).

Stimulation of BEAS-2B cells with 10 ng/ml TNF- for 8 h increased IL-8 synthesis 9-fold (Figure 9). When cAMP or cGMP levels were increased first, TNF--induced IL-8 formation remained unchanged, suggesting that under the experimental conditions used, this epithelial cell mediator is not regulated by increased cyclic nucleotides.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

Three bronchial epithelial cell types were used for the identification of the PDE enzyme pattern. Besides the well- established human bronchial epithelial cell line BEAS-2B, emphasis was placed on the analysis of confluent primary cultures of AEC derived from human and porcine tissue (15). Morphologic and immunocytochemical criteria were applied to identify cells and to verify purity of the cultures. All epithelial cells were cytokeratin-positive and vimentin-negative; in the same set cultured pulmonary artery endothelial cells were used as vimentin-positive and cytokeratin-negative controls. The major cyclooxygenase and lipoxygenase products of epithelial cells were PGE₂ and 15-HETE; this arachidonic acid metabolite profile is similar to that described for human, bovine, and canine bronchial epithelial cells (3, 24).

Using a combination of biochemical, pharmacologic, and molecular techniques, we have demonstrated that primary cultures of human AEC express PDE1, PDE4, PDE5, and PDE7. We confirmed these observations also for primary cultures of porcine AEC and for the cell line BEAS-2B, both of which contained PDE isoenzymes 4, 5, and 7. Interestingly, these cells did not express PDE1, which may be due to species differences or to special circumstances introduced by virus transformation. Similar data for primary cultures of human AEC and for the alveolar epithelial cell line A549 were reported in abstract form by Rabe and colleagues (28) and Robichaud and associates (29), although no information was provided with repect to PDE7 and PDE4 variants. We applied RT-PCR using primers specific for the four PDE4 genes and detected PCR products that correspond to PDE4A5, 4C1, 4D1, 4D2, and 4D3. Rousseau and coworkers (30) used biochemical and pharmacologic methods to identify PDE isoenzymes in bovine tracheal epithelial cells. In that study only one broad peak of activity was resolved by diethylaminoethyl anion exchange chromatography; that peak was characterized using isoenzyme-selective inhibitors and allosteric effectors. The authors reported high activities of PDE1 in their study (30). As to PDE1, species differences appear to prevail because this isoenzyme is present in human and bovine but not in porcine AEC.

The absence of PDE3 in AEC contrasts with data published by Kelley and associates, who described the functional effects of PDE3 inhibitors on chloride fluxes in Calu-3, 16HBE, and transformed nasal polyp cells (31, 32). However, in that study no direct evidence for PDE3 was provided. Because these authors focused on specialized epithelial cell mutants with different degrees of cystic fibrosis transmembrane conductance regulator activities, their data with respect to PDE3 probably cannot be generalized. Using in situ hybridization, Reinhardt and coworkers (33) studied PDE3B expression during embryonic and postnatal development. Although PDE3B mRNA was demonstrated in rat bronchial epithelial cells, this enzyme was not documented at the protein level. Thus, like PDE1, species differences also appear to exist for PDE3.

Considering all this data together, it appears that in AEC PDE4 (4A5, 4C1, 4D1, 4D2, and 4D3) is the major cAMP-degrading pathway, whereas PDE5 is primarily responsible for the metabolism of cGMP. Regarding primary cultures of human AEC, PDE1 accounts for at least 20% of the total cGMP hydrolysis, and PDE5 the remainder.

In addition to the large amount of rolipram-sensitive cAMP hydrolysis (PDE4), there was also consistently a substantial fraction of rolipram-insensitive cAMP degradation (25 to 40% of total cAMP hydrolysis), suggesting the presence of a PDE7 which, by definition, is a high-affinity, cAMP-specific, rolipram-insensitive PDE (5). Because of the lack of selective inhibitors, it is not possible to attribute unequivocally the rolipram-resistant cAMP-PDE activity to PDE7. Moreover, PDE7 activity will probably be underestimated because IBMX does not block all PDE7 activity (34) and because high concentrations of rolipram candespite definitionreduce PDE7 by about 20% (6). Compelling evidence, however, to support the expression of PDE7 was provided by the unambiguous identification of PDE7 mRNA in AEC. To our knowledge this is the first demonstration of PDE7 in AEC.

Homogenates derived from human airways displayed activities for PDE1 to PDE5, with highest activities for PDE4 and PDE5, whereas PDE7 was not analyzed (13, 35). Previous studies using human airway smooth-muscle strips also revealed the presence of all PDE isoenzymes (36). The fact that AEC lack PDE2 and PDE3 suggests that airway smooth-muscle cells substantially contribute to the PDE isoenzyme pattern of airway homogenates. However, additional studies are required for an exact mapping of PDE isoenzymes in specific cell types of the airways.

On the basis of the PDE isoenzyme pattern analyzed, we tried to correlate AEC function with cyclic nucleotide levels and focused on the generation of PGE₂, 15-HETE, and IL-8. AA/A23 increased PGE₂ synthesis 7-fold in AEC. When cAMP was elevated first in AEC with forskolin/rolipram, there was a 4-fold increase in the effectiveness of the stimulus resulting in an overall 28-fold increase in PGE₂ synthesis above baseline and suggesting that PDE4 inhibition may enhance the anti-inflammatory and antiobstructive effects of endogenous PGE₂. Interestingly, increased cGMP levels reduced the effectiveness of the stimulus for PGE₂ production. Interpretation of these data in terms of airway physiology is complex. One possible explanation relates to the prevention of an unopposed bronchodilatation by simultaneous action of cGMP- and cAMP-dependent mechanisms. If increased cGMP levels decrease PGE₂ with a subsequent decrease in cAMP, this mechanism will substitute for a cGMP-stimulated, cAMP-degrading activity (37). This imitation of a PDE2-like activity would allow crosstalk between the two cyclic nucleotide species and compensate for the absence of a PDE2 in AEC.

Next we studied the interrelationship between cyclic nucleotides and the major epithelial cell lipoxygenase product 15-HETE. The AA/A23-related synthesis of 15-HETE was unaffected in SNP/zaprinast- or forskolin/rolipram-pretreated AEC, suggesting that increased nucleotides act on the cyclooxygenase but not on the lipoxygenase pathway in AEC.

IL-8 is an important proinflammatory cytokine that is released from AEC during airway inflammation upon exposure to TNF-, IL-1, and neutrophil elastase (4, 17, 38). Elevated cyclic nucleotide levels are known to downregulate the expression of cytokines (40), so we tested this hypothesis in BEAS-2B cells. However, under the experimental conditions used, TNF--induced IL-8 generation was unaffected by SNP/zaprinast or forskolin/ rolipram pretreatment. Similar data were reported for human mesangial cells which, upon stimulation with IL-1, secreted less IL-6 but undiminished amounts of IL-8 (39, 41). The regulation of IL-8 production in different airway epithelial cell types may vary. For example, Levine and colleagues could not demonstrate an IL-8 reduction in dexamethasone-pretreated, TNF--stimulated BEAS-2B cells (40), whereas corticosteroids were active in TNF--activated A549 and primary human epithelial cells (17). It is clear that several stimuli and AEC types must be studied to resolve the relationship between cyclic nucleotides and epithelial cell cytokine secretion.

An appreciation of the PDE isoenzyme pattern in AEC is of obvious importance for the design of new therapeutic strategies. For example, all pulmonary and inflammatory cell types studied possess a PDE4, and therefore PDE4 inhibition will result in a broad cAMP accumulation and exert a generalized anti-inflammatory and bronchodilative effect. As for cGMP, it may be of interest that inhibition of PDE5 would affect AEC and airway smooth-muscle cells but not pulmonary endothelial cells because their cGMP degradation is governed by PDE2 (8, 9, 18). Differences in the PDE isoenzyme spectrum among pulmonary cell types therefore will allow a better targeting of cyclic nucleotide- based therapies.

The interpretation of our study is limited for several reasons: (1) The data show differences between the different types of AEC examined (porcine cells, human cells, BEAS-2B cell line). Primary cultures of human AEC are the cell type of major interest and therefore these cells were included in the present study. (2) Human airway epithelial cells were grown submerged in the medium; it is well known that this can affect cell differentiation. The relevance of our in vitro findings to airway epithelium in vivo therefore remains to be established. Growth of AEC at conditions of liquid-air interphase for prolonged time periods will probably result in a better model of airway epithelium. (3) An airway epithelial cell line and primary cultures of large airway epithelium were used. For an exact analysis of airway epithelial cell dysfunction in clinical disorders, it would be desirable to study the contributions made by epithelial cells of small airways of human origin. Isolation and culture of these cells, however, is difficult, and therefore the applicability of the data presented to human airway disease is not clear.

In summary, human AEC express PDE isoenzymes 1, 4 (variants 4A5, 4C1, 4D1, 4D2, and 4D3), 5, and 7, whereas BEAS-2B cells and porcine AEC show similar profiles but lack PDE1. Enhanced cAMP levels substantially increased and enhanced cGMP level reduced epithelial PGE₂ generation, whereas IL-8 and 15-HETE synthesis were not affected. Currently available therapies for inflammatory airway disease are nonspecific and not free of side effects. We suggest that cyclic nucleotide-based treatments that rely on differences in the PDE-isoenzyme spectrum among pulmonary cell types could increase selectivity and reduce the side-effect profile of existing therapies. The identification of the PDE-isoenzyme pattern in AEC may contribute to this aim.