Phosphodiesterase expression in human epithelial cells

Lyndon C. Wright, Joachim Seybold, Annette Robichaud, Ian M. Adcock, and Peter J. Barnes

Department of Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London SW3 6LY, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Epithelial cells play a critical role in airway inflammation and have the capacity to produce many inflammatory mediators, including bioactive lipids and proinflammatory cytokines. Intracellular levels of cAMP and cGMP are important in the control of inflammatory cell function. These cyclic nucleotides are inactivated via a family of phosphodiesterase (PDE) enzymes, providing a possible site for drug intervention in chronic inflammatory conditions. We studied the expression of PDE activity in an epithelial cell line (A549) and in primary human airway epithelial cells (HAECs). We measured PDE function using specific inhibitors to identify the PDE families present and used RT-PCR to elucidate the expression of PDE isogenes. Both A549 cells and HAECs predominantly expressed PDE4 activity, with lesser PDE1, PDE3, and PDE5 activity. RT-PCR identified HSPDE4A5 and HSPDE4D3 together with HSPDE7. Inhibition of PDE4 and PDE3 reduced secretion by these cells. Epithelial PDE may be an important target for PDE4 inhibitors in the development of the control of asthmatic inflammation, particularly when delivered via the inhaled route.

phosphodiesterase type 3; phosphodiesterase type 4; inflammation; granulocyte-macrophage colony-stimulating factor; airway epithelial cells

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CYCLIC NUCLEOTIDES play a key role in controlling epithelial cell functions such as electrolyte transport, ciliary motility, and cytokine production (12, 30). The rate of breakdown of cAMP and cGMP is controlled by a specialized superfamily of hydrolytic enzymes called cyclic nucleotide phosphodiesterases (PDEs). Several families of PDE enzyme have now been identified and are classified according to differing substrate specificity, regulatory characteristics, and sensitivities to selective inhibitors (4). There are at least seven recognized families of PDE, and most of these are known to contain at least two different genes. Additionally, for many of these genes, more than one splice variant is expressed (7). The availability of specific inhibitors has meant that the most widely studied families are PDE3, PDE4, and PDE5 (5, 20). There has been particular interest in PDE4 because it is predominant in inflammatory cells such as mast cells (2), monocytes (34), macrophages (31), eosinophils (11), and T cells (27). This suggests that PDE4 inhibitors may be useful in the treatment of several inflammatory and allergic diseases including atopic dermatitis (24), arthritis (25), multiple sclerosis (28), and bronchial asthma (13). Several PDE4 inhibitors have been shown to attenuate cytokine release from inflammatory cells and inhibit activated inflammatory cells (11, 13, 14).

It has become increasingly apparent that airway epithelial cells play a key role in the initiation and maintenance of the airway inflammatory response. Epithelial cells are not only passive barrier-target cells but also play an integral role in the pathophysiology of airway diseases through the release of multiple inflammatory mediators, including prostaglandins and 15-hydroxyeicosatetraenoic acid (HETE) (23), and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) (10), interleukin (IL)-1 (1), IL-8 (16), and regulated on activation normal T cells expressed and secreted (RANTES) (6). Because PDE4 inhibitors block the release of proinflammatory cytokines in inflammatory cells, they may also have an effect on epithelial cells.

One of the problems encountered in clinical studies with PDE4 inhibitors is the relatively high incidence of side effects such as nausea, vomiting, and headaches (3). Delivery of a PDE4 inhibitor topically by inhalation may avert the systemic side effects. However, little is known about the expression of PDE on airway epithelial cells. We have therefore investigated the expression of PDEs in cultured epithelial cells and in a human epithelial cell line, A549. Because at least four human PDE4 genes have now been identified (4, 20), we have also studied which genes are expressed in these cells.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Poole, UK).

Primary cells. Human epithelial cells were obtained from normal human lung donors and cultured as previously described (16). The sections of tracheal tissue were carefully cleaned and stored overnight on ice bathed in saline containing antibiotic penicillin (1 U/ml), streptomycin (0.1 mg/ml), and the antifungal agent amphotericin B (5 µg/ml). The bathing solution also contained pronase (1%) to increase epithelial cell shedding from the basement membrane. The following day, sections were lavaged for 60 min to dissociate the epithelial cells. To increase the yield of cells, the trachea could then be left in fresh bathing solution for a further 12 h before being washed. Cells were filtered through a sterile muslin gauze to remove mucus and debris and were seeded (0.5 × 106 cells) into six-well plates (Costar) for culture at 37°C and 5% CO2 in a humidified incubator in supplemented Ham's F-12 medium (16).

A549 cells. The human epithelial-like (type II pneumocyte) cell line A549 (ATCC, Rockville, MD) was cultured in DMEM supplemented with 10% FCS, penicillin (1 U/ml), streptomycin (0.1 mg/ml), L-glutamine (2 mM), and amphotericin B (5 µg/ml) in a humidified incubator (37°C, 5% CO2) as a stock culture in T-75 flasks (Costar). Confluent cultures were split and seeded into six-well plates at a density of 1 × 105 cells/well and cultured to confluence before use.

PDE assay. The complement of PDE isoenzymes was determined for each cell type by assaying PDE activity and utilizing specific inhibitors and reaction conditions known to distinguish specific isoenzymes. The activity assay measures the breakdown of [3H]cAMP or [3H]cGMP (Amersham, Little Chalfont, UK) to the corresponding labeled monophosphate by PDE and the subsequent dephosphorylation by alkaline phosphatase with a modification of the method of Thompson and Appleman (32). Reactions were performed at 37°C in an assay cocktail including 1 µM cAMP (including ~280,000 dpm [8-3H]cAMP; Amersham), [8-14C]adenosine (~6,000 dpm; Amersham), 0.25 U alkaline phosphatase, 20 mM triethanolamine (pH 8.0), 1 mM EGTA, 5 mM magnesium acetate, 5 mM dithiothreitol (DTT), 500 µg/ml of BSA and, where appropriate, any drug being used. To a 270-µl cocktail, 30 µl of enzyme were added to initiate the reaction. After a period of time predetermined by the number of cells required to utilize no more than 25% of the substrate, the reaction was stopped by the addition of 1 ml of a solution of Dowex AG 1-X8 anion exchanger-methanol-water (1:2:1). The preparation was then vortex mixed for 30 min at 4°C before centrifugation at 12,000 g, 4°C for 5 min. Aliquots of 700 µl were taken from each tube and counted (~60% efficiency) in 2 ml of aqueous counting scintillant (ACS II, Amersham). Assays for cGMP hydrolysis were performed by substituting for cold and labeled cAMP in the assay cocktail.

Assays were performed on soluble (crude cytosolic) and particulate (crude microsomal) cellular fractions prepared by hypotonic lysis of cells at 4°C in lysis buffer (10 mM MOPS, pH 7.4, 1 mM EGTA, 2 mM magnesium acetate, and 5 mM DTT), and proteinase inhibitors (100 µM leupeptin, 100 µg/ml of bacitracin, 2 mM benzamidine, 100 µM phenylmethylsulfonyl fluoride, and 20 µg/ml of soybean trypsin inhibitor). After 60 min, the cell lysate was centrifuged at 4°C and 45,000 g for 30 min. The resulting supernatant was decanted and diluted in lysis buffer to the required cell equivalent per milliliter and used as a source of soluble enzyme. The pellet was resuspended at the required cell equivalent per milliliter and used as a source of membrane-associated enzyme.

Determination of PDE isoenzyme activities. Activities for individual PDE isoenzymes in A549 cells and human airway epithelial cells (HAECs) were determined by comparing total lysate hydrolytic activity in assay buffer alone with that seen in the presence of isoenzyme-specific inhibitors or allosteric modulators. Hydrolysis of cAMP and cGMP by soluble and particulate PDEs was assessed in the presence of 2 mM Ca2+ + 50 U calmodulin to unmask PDE1 activity. EGTA [1 mM; to eliminate residual PDE1 activity induced by Ca2+ in lysates (5 µM cGMP with or without 30 µM ORG-9935; Organon, Edinburgh, UK)] was used to unmask PDE2 and PDE3 activity (19); 1 mM EGTA with or without 50 µM rolipram (Alexis, Nottingham, UK) was used to inhibit PDE4 activity; 1 mM EGTA (with or without 30 µM zaprinast) was used to inhibit PDE5 activity; and 500 µM 3-isobutyl-1-methylxanthine (IBMX) was used for inhibition of all PDE activity except PDE7 (17).

RNA isolation and RT-PCR. We designed forward and reverse PCR primers according to primary sequence data relating to the isoforms of PDE4 and PDE7 found in the GenBank database. The following primer sequences were constructed, with annealing temperature and product size in parentheses: HSPDE4A5 sense 5'-AAG AGG AGG AAG AAA TAT CAA TGG-3' and antisense 5'-TTA CAG CAA CCA CGA ATT CCT CCC-3' (67°C, 272 bp); HSPDE4B sense 5'-AGG GCA GCC TGA GGT ATT AAA-3' and antisense 5'-CAC TCC TGG CTT ACA GTT GTA-3' (62°C, 363 bp); HSPDE4C sense 5'-GGA GTT GCC TGA CAC TGA ACT-3' and antisense 5'-AGA AAG ACA CCA GGG CAT CGT-3' (71°C, 335 bp); and HSPDE4D5 sense 5'-GCA AGA TCG AGC ACC TGG CAA-3' and antisense 5'-CGG TTA CCA GAC AAC TCT GCT-3' (68°C, 515 bp).

For HSPDE7 the primer sequences used were sense 5'-GGA CGT GGG AAT TAA GCA AGC-3' and antisense 5'-TCC TCA CTG CTC GAC TGT TCT-3' (59°C, 285 bp).

Extraction of RNA was performed after the modified guanidinium acid-phenol method of Chomczynski and Sacchi (9). Genomic DNA contamination was reduced by 1:1 phenol-chloroform extraction and ethanol precipitation. Human lung total RNA extracted from 100 mg of donor tissue was used as a positive control. Sample RNA was quantified by spectrophotometry (absorbency 260 nm), and 1 µg was reverse transcribed to cDNA in PCR buffer with 8 U avian myeloblastosis virus reverse transcriptase (Promega, Southampton, UK) and 0.2 µg of random hexamers (Pharmacia, Uppsala, Sweden) in a final volume of 20 µl. Then, the reaction mixture containing the cDNA was diluted to 100 µl and stored at -70°C. PCR reactions were performed on 5 µl of the cDNA with a Hybaid Omnigene thermal cycler (Hybaid, Middlesex, UK) in a final reaction volume of 25 µl in the presence of 0.5 U of Taq DNA polymerase. Twenty-six to forty cycles were used, with a denaturing step at 90°C for 30 s, followed by the specific primer annealing temperature and an extension step at 72°C for 1 min. The PCR products were then separated according to size on a 1.5% agarose gel containing ethidium bromide and visualized against a 1-kb ladder (GIBCO, Paisley, UK) over an ultraviolet light source.

To check that the desired target gene was being amplified, the product of each primer pair was cloned into pGEM5z (Promega), and double-stranded sequencing was performed with the Sequenase II kit (Amersham) to verify product identity.

GM-CSF Assay. Immunoreactive GM-CSF release from A549 cells and HAECs into culture medium (CM) was measured with an ELISA (Pharmingen, San Diego, CA), giving a lower detection limit of 15 pg/ml. The standard curves utilized for quantification were constructed with human recombinant GM-CSF (Genzyme, Cambridge, MA). Cells were grown in monolayers on six-well plates. When confluence was attained, the monolayers were washed to remove debris and nonadherent cells. Monolayers were then maintained for 24 h with fresh CM (1.0 ml) containing either drug or a volume equivalent of vehicle. The effect of PDE inhibitors (50 µM rolipram, 30 µM ORG-9935, and 500 µM IBMX) on GM-CSF production was assessed with control cultures and cultures in which GM-CSF production was stimulated by 1 ng/ml of human recombinant IL-1beta (R&D Systems, Oxford, UK). PDE inhibitors were added to the CM 1 h before IL-1beta stimulation. After 24 h, CM was aspirated and assayed immediately for GM-CSF. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability test was then performed on the monolayers to control for potential PDE inhibitor-mediated cytotoxicity (8).

Analysis of data and statistics. All data are presented as means ± SE of n independent experiments. Statistical analysis was performed on nontransformed data with analysis of variance, followed by a Bonferroni adjustment when multiple comparisons were made against a single mean. P < 0.05 was considered significant, and the null hypothesis was rejected.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification of functional PDEs. Figures 1 and 2 show the amount of cAMP and cGMP hydrolyzed by PDEs in primary HAECs and the type II pneumocyte adenocarcinoma epithelial-like A549 cells. Hydrolytic activity was measured under conditions selected to unmask the proportion of hydrolysis attributable to the different members of the PDE superfamily present in the cytosolic (Fig. 1) and crude microsomal (Fig. 2) fractions.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Cyclic nucleotide hydrolysis by airway epithelial cell cytosolic phosphodiesterase (PDE). Hydrolysis in cytosolic lysate from human airway epithelial cells (HAECs; A) and A549 cells (B) in presence of selective inhibitors and allosteric modulators. Data are means ± SE of 5 separate determinations. 3-Isobutyl-1-methylxanthine (IBMX) inhibits all PDEs except PDE7; control group shows mainly activity of PDE3 and PDE4 (cAMP substrate) or mainly PDE5 (cGMP substrate); Ca2+ (2 mM) + calmodulin (CaM; 50 U) stimulates PDE1 activity; cGMP (5 µM) stimulates PDE2 and inhibits PDE3; ORG-9935 (30 µM) inhibits PDE3; rolipram (50 µM) inhibits PDE4; and zaprinast (30 µM) inhibits PDE5. Significantly different from basal PDE activity, * P < 0.05.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   Cyclic nucleotide hydrolysis by airway epithelial cell microsmal PDE. Hydrolysis in microsomal lysate from HAECs (A) and A549 cells (B) in presence of selective inhibitors and allosteric modulators. Data are means ± SE of 5 separate determinations. IBMX inhibits all PDEs except PDE7; EGTA (1 mM) shows mainly activity of PDE3 and PDE4 (cAMP substrate) or mainly PDE5 (cGMP substrate); Ca2+ (2 mM) + CaM (50 U) stimulates PDE1 activity; cGMP (5 µM) stimulates PDE2 and inhibits PDE3; ORG-9935 (30 µM) inhibits PDE3; rolipram (50 µM) inhibits PDE4; and zaprinast (30 µM) inhibits PDE5. Significantly different from basal PDE activity, * P < 0.05.

The nonselective PDE inhibitor IBMX significantly suppressed cAMP and cGMP hydrolytic activity in the microsomal fractions and almost all of that associated with the cytosolic fractions, suggesting that the PDEs are the unique means of cyclic nucleotide catabolism in both cell types. However, a percentage of activity, albeit small, that was not blocked by IBMX remained in both HAECs and A549 cells. We reasoned that this could suggest the presence of small amounts of PDE7 in these cells, a speculation further supported by the RT-PCR data obtained (see Fig. 5). In the presence of the allosteric modulator calmodulin and an excess of Ca2+, cAMP hydrolysis increased above that seen in the presence of the chelating agent EGTA alone by 39.1 ± 10.3% and by 7.0 ± 5.6% for cytosolic and microsomal HAEC PDEs. Similarly, in A549 cells, the increase was 36.4 ± 9.4% for cytosolic PDE and 6.2 ± 1.4% for microsomal PDE. This indicates the presence of cytosolic PDE1 in both primary cells and A549 cells. The addition of cGMP in the presence of EGTA caused no significant difference in cAMP hydrolysis in HAECs, which, taken alone, suggests that PDE2 is absent. In the presence of the selective PDE3 inhibitor ORG-9935, cytosolic HAEC cAMP hydrolysis was inhibited significantly (23.8 ± 4.9%), whereas microsomal hydrolysis was not significantly inhibited. Taken together, these data suggest the presence of cytosolic but not membrane-associated PDE3. Likewise, in the A549 cell line, cGMP failed to induce activity in either subcellular fraction, suggesting that PDE2 is absent. ORG-9935 caused a significant (30.0 ± 2.6%) inhibition in the cytosol, suggesting the presence of PDE3 activity. Rolipram caused a substantial reduction in cAMP hydrolyzing activity in HAECs, with a 75.0 ± 6.2 and 62.1 ± 4.1% fall in cytosolic and microsomal activity, respectively. Likewise, in the A549 cells, cAMP hydrolysis was reduced in the cytosolic and microsomal fractions by 67.6 ± 7.0 and 73.1 ± 4.3%, respectively. This suggests that PDE4 was present in all subcellular fractions tested. In HAECs, cGMP was hydrolyzed by both cytosolic and microsomal cell fractions. This activity was inhibited by 30.2 ± 5.5 (cytosolic) and 38 ± 15.7% (microsomal) in the presence of 50 µM zaprinast, although this was not significant. This suggests that PDE5 was not the only source of cGMP metabolism.

By pooling these results, we are able to produce the PDE isoenzyme activity profile for HAECs and A549 cells shown in Fig. 3.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   PDE isoenzyme activities resolved in airway epithelial cells. According to assay criteria applied, HAECs (A) and A549 cells (B) displayed activities that may be attributed to PDEs 1, 3, and 4, whereas only HAECs displayed activity, suggesting presence of PDE5. No significant hydrolytic activity indicative of PDE2 in either cell type was found.

RT-PCR. Figure 4 is a representative photograph from three separate experiments of an ethidium bromide-stained gel showing the results of the RT-PCR amplification, the specific sequences corresponding to HSPDE4A, HSPDE4B, HSPDE4C, and HSPDE4D3 isoforms of PDE4. The primary HAECs and the A549 cells showed the same PDE4 profile, both cell types expressing PCR products corresponding to the message for the HSPDE4A and HSPDE4D isoforms but not for HSPDE4B or HSPDE4C. To complete the profile of known PDEs for these cell types, PCR was performed to check for HSPDE7 mRNA (Fig. 5; representative of 3 experiments), and both cell types expressed a strong signal.


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 4.   Expression of multiple HSPDE4 isoform mRNAs in airway epithelial cells. A representative ethidium bromide-stained agarose gel (n = 3 experiments) of HSPDE4 PCR products amplified from HAEC and A549 cell cDNA was obtained by reverse transcribing 1 µg of total cell RNA and subjecting this to PCR of 24-40 cycles in the presence of specific primers. Amplified product was separated by electrophoresis and visualized by ethidium bromide staining. Genomic contamination was controlled for by processing sample RNA in absence of reverse transcriptase (no RT), and total lung cDNA was used as a positive control.


View larger version (75K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of HSPDE7 mRNA in airway epithelial cells. A representative ethidium bromide-stained agarose gel (n = 3 experiments) of HSPDE7 PCR products amplified from HAEC and A549 cell cDNA was obtained by reverse transcribing 1 µg of total cell RNA and subjecting this to PCR of 24-40 cycles in the presence of specific primers. Amplified product was separated by electrophoresis and visualized by ethidium bromide staining. Genomic contamination was controlled for by processing sample RNA in presence (+RT) or absence (-RT) of reverse transcriptase or and total lung cDNA was used as a positive control.

GM-CSF ELISA. Figure 6 shows the release of GM-CSF from HAECs and A549 cells after culture for 24 h. PDE inhibitors failed to palliate this basal GM-CSF output. The presence of IL-1beta at 1 ng/ml causes a significant increase in the release of immunoreactive GM-CSF by both cell types. Treatment of stimulated cells with ORG-9935 or rolipram caused a significant decrease in GM-CSF output. ORG-9935 reduced GM-CSF release to baseline levels. IBMX, which gives inhibition of all PDE activity in these cells, obliterated IL-beta -mediated GM-CSF release. The MTT assay revealed that PDE inhibitor-mediated cytotoxicity cannot account for the reduction in GM-CSF secretion.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Selective PDE inhibitors partially abrogate granulocyte-macrophage colony-stimulating factor (GM-CSF) release from airway epithelial cells [HAECs (A) and A549 cells (B)]. Data are means ± SE of 5 separate experiments. Control cells were untreated, and PDE inhibitors had no effect on basal GM-CSF secretion. All other cultures were treated with interleukin (IL)-1beta (1 ng/ml) after 1-h pretreatment with either 30 µM ORG-9935, 50 µM rolipram, or 500 µM IBMX. GM-CSF release is significantly less than that seen with IL-1beta alone, * P < 0.05.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study characterized the PDE isoforms found in primary HAECs and the epithelial cell line A549. Previous studies characterized PDEs in bovine tracheal epithelial cells (22) and in human airways (21), but this is the first characterization of PDE in primary HAECs. Our results show the major cytosolic PDE activity in primary HAECs to be PDE4, with lesser activities of PDE3 and PDE1. A similar profile, but of lower activity, was seen for the enzyme associated with the membrane subcellular fraction. The A549 cell line presented activities for PDE4, PDE3, and PDE1, with a similar quantitative subcellular distribution but with activity ~10-fold higher than that seen in primary HAECs. This increased activity may be a consequence of the transformed nature of the A549 cells and suggests that these cells may not be suitable as a model for studying PDE induction patterns in airway epithelial cells. We have shown that PDE4 is the main enzyme responsible for cAMP hydrolysis in HAECs, and this is in broad agreement with findings in bovine tracheal epithelial cells (23) and human airway (21), although Rabe et al. (21) showed that the activity is associated with the cellular milieu in peripheral airways. This mixture of cell types may account for the discovery of significant amounts of PDE5 activity in whole peripheral airways, in contrast to our results in which PDE5 activity, as defined by zaprinast-mediated inhibition, did not attain significance. PDE4 has also been reported to be present as a major hydrolytic activity in both the cytosolic and membrane fractions of several cell types, including eosinophils (14), T lymphocytes (12), monocytes (26), and cells in the brain (18), myocardium (15), and trachea (33). PDE2 was absent from airway epithelial cells, and this suggests no role for this isoenzyme in regulating cellular function in the basal state. The small degree of hydrolytic activity found in all fractions that was not inhibited by IBMX may be due to a low background level of the methylxanthine-resistant PDE7 (17). Although this was supported by the identification of HSPDE7 mRNA in these cells, the negligible amount of activity suggests that this PDE does not perform a significant hydrolytic function in the basal state in these cell types. Message corresponding to PDE7 is also present in other inflammatory cells, including T cells (12), and is present in abundance in other tissues, but elucidation of the functional role, if any, played by this increasingly ubiquitous isoenzyme must await the development of specific inhibitors.

Using RT-PCR, we demonstrated that HSPDE4A5 and HSPDE4D3 were expressed in both primary HAECs and A549 cells, whereas HSPDE4B and HSPDE4C were absent in the basal state. To briefly summarize the data currently available concerning PDE4 mRNA subtype distribution in mammalian cells and tissues, PDE4A, PDE4B, and PDE4D appear to be fairly ubiquitous, with only PDE4C showing a more discrete peripheral distribution (20). The pattern of expression we have found in HAECs is similar to that observed in the T lymphocyte cell line Jurkat 30 and lung carcinomas (20). The significance of differential PDE subtype expression remains unknown, and a more discrete cell and tissue distribution of PDE may become apparent with elucidation of the expression of the spliced variants of PDE subtypes.

We have shown that IL-1beta promotes GM-CSF release from airway epithelial cells and that inhibition of PDE significantly reduces the liberation of this cytokine from these cells into their surrounding media. Interestingly, whereas hydrolysis experiments show that PDE3 is present in less significant amounts than PDE4 in both cell types, the PDE3-selective inhibitor ORG-9935 abrogates all GM-CSF release to baseline levels in these cells. Indeed, nonspecific inhibition of all major PDE activity by IBMX reduced release of this cytokine by no more than that achieved by inhibition of PDE3 alone. This suggests a key role for discrete PDE isoenzymes in control of cytokine release from airway epithelial cells, and the palliating effect of PDE inhibitors on GM-CSF release from these cells indicates that these drugs may be useful in asthma.

The demonstration that PDE4 predominates in airway epithelial cells suggests that these cells may be an important target for PDE4 inhibitors in the treatment of asthma and other inflammatory diseases of the airways. There is increasing evidence that airway epithelial cells in asthma produce and express cytokines such as GM-CSF (10), IL-1beta (1), RANTES (6), and monocyte chemoattractant-1 (29). PDE4 inhibitors may inhibit the release of cytokines from these cells through an increase in cAMP concentrations. This may be important in the clinical development of these drugs for asthma therapy because a major problem appears to be systemic side effects such as nausea and headache. Delivering these drugs via inhalation may make it possible to suppress inflammation and minimize these side effects. In the future, more selective PDE4 inhibitors may be developed (20), and possibly these will have fewer side effects than existing nonselective drugs. The identification of HSPDE4A5 and HSPDE4D3 in airway epithelial cells suggests that more selective inhibition might be possible.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the help and advice given by Drs. Mark A. Giembycz and Robert Newton in the preparation of this paper and the skilled technical support of Hilary Lewis and Koremu Meja.

    FOOTNOTES

This study was supported by Novartis (Basel, Switzerland) and the Medical Research Council, United Kingdom.

J. Seybold was a Deutsche Forschungsmeinschaft Research Fellow.

Address for reprint requests: P. J. Barnes, Dept. of Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse St., London SW3 6LY, UK.

Received 11 February 1997; accepted in final form 28 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abdelaziz, M. M., J. L. Devalia, O. A. Khair, M. Calderon, R. J. Sapsford, and R. J. Davies. The effect of conditioned medium from cultured human bronchial epithelial cells on eosinophil and neutrophil chemotaxis and adherence in vitro. Am. J. Respir. Cell Mol. Biol. 13: 728-737, 1995[Abstract].

2.   Alfonso, A., M. Esteves, M. C. Louzao, M. R. Vieytes, and L. M. Botana. Determination of phosphodiesterase activity in rat mast cells using the fluorescent cAMP analogue anthraniloyl cAMP. Cell. Signal. 7: 513-518, 1995[Medline].

3.   Barnes, P. J. Molecular mechanisms of anti-asthma therapy. Ann. Med. 27: 531-535, 1995[Medline].

4.   Beavo, J. A., M. Conti, and R. J. Heaslip. Multiple cyclic nucleotide phosphodiesterases. Mol. Pharmacol. 46: 399-405, 1994[Abstract].

5.   Beavo, J. A., and D. H. Reifsnyder. Primary sequences of cyclic nucleotide phosphodiesterase enzymes and the design of selective inhibitors. Trends Pharmacol. Sci. 11: 150-155, 1990[Medline].

6.   Berkman, N., A. Robichaud, V. L. Krishnan, G. Roesems, R. Robbins, P. J. Jose, P. J. Barnes, and K. F. Chung. Expression of RANTES in human airway epithelial cells: effect of corticosteroids and interleukin-4, -10 and -13. Immunology 87: 599-603, 1996[Medline].

7.   Bolger, G. B., I. McPhee, and M. D. Housley. Alternative splicing of cAMP specific phosphodiesterase mRNA transcripts. Characterization of a novel tissue specific isoform, RNPDE4A8. J. Biol. Chem. 271: 1065-1071, 1996[Abstract/Free Full Text].

8.   Carmichael, J., W. G. DeGraff, A. F. Gazdar, J. D. Minna, and J. B. Mitchell. Evaluation of tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47: 936-942, 1987[Abstract].

9.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

10.   Churchill, L., B. Friedman, R. P. Schleimer, and D. Proud. Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells. Immunology 75: 189-195, 1991.

11.   Dent, G., M. A. Giembycz, K. F. Rabe, and P. J. Barnes. Inhibition of eosinophil cyclic nucleotide PDE activity and opsonised zymosan-stimulated respiratory burst by `type IV'-selective PDE inhibitors. Br. J. Pharmacol. 103: 1339-1346, 1991[Abstract].

12.   Essayan, D. M., S. K. Huang, A. Kagey-Sobotka, and S. M. Lichenstein. Effects of nonselective and isoenzyme selective cyclic AMP phosphodiesterase 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2. Br. J. Pharmacol. 118: 1945-1958, 1995[Abstract].

13.   Giembycz, M. A., and G. Dent. Prospects for selective nucleotide phosphodiesterase inhibitors in the treatment of bronchial asthma. Clin. Exp. Allergy 22: 337-344, 1992[Medline].

14.   Hatzelmann, A., H. Tenor, and C. Schudt. Differential effects of non-selective and selective phosphodiesterase inhibitors on human eosinophil functions. Br. J. Pharmacol. 114: 821-831, 1995[Abstract].

15.   Kithas, P. A., M. Artman, W. J. Thompson, and S. J. Strada. Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activities in rabbit ventricular myocardium: relations to post-natal maturation. J. Mol. Cell. Cardiol. 21: 507-517, 1989[Medline].

16.   Kwon, O. J., B. T. Au, P. D. Collins, J. N. Baraniuk, I. M. Adcock, K. F. Chung, and P. J. Barnes. Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells. Immunology 81: 389-394, 1994[Medline].

17.   Lavan, B. E., T. Lakey, and M. D. Housley. Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes from liver and hepatocytes identified a novel IBMX insensitive form. Biochem. Pharmacol. 58: 4123-4126, 1989.

18.   Lobban, M., Y. Shakur, J. Beattie, and M. D. Housley. Identification of two splice variant forms of type IV-B cAMP phosphodiesterase, cytosolic compartments and differential expression in various brain regions. Biochem. J. 304: 399-406, 1994[Medline].

19.   Manganellio, V. C., M. Taira, E. Degerman, and P. Belfrage. Type III cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE3 gene family). Cell. Signal. 7: 445-455, 1995[Medline].

20.   Müller, T., P. Engels, and J. R. Fozard. Subtypes of the type 4 phosphodiesterases: structure, regulation and selective inhibition. Trends Pharmacol. Sci. 17: 294-298, 1996[Medline].

21.   Rabe, K. F., H. Tenor, G. Dent, C. Schudt, S. Liebig, and H. Magnussen. Phosphodiesterase isoenzymes modulating inherent tone in human airways: identification and characterization. Am. J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L458-L464, 1993[Abstract/Free Full Text].

22.   Rousseau, E., J. Gagnon, and C. Lugnier. Biochemical and pharmacological characterization of cyclic nucleotide phosphodiesterase in airway epithelium. Mol. Cell. Biochem. 140: 171-175, 1994[Medline].

23.   Salari, H., and M. Chan-Yeung. Release of 15-hydroxyeicosatetraenoic acid (15-HETE) and prostaglandin E2 (PGE2) by cultured human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 1: 245-250, 1989[Medline].

24.   Sawai, T., K. Ikai, and M. Uehara. Elevated cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leukocytes from children with atopic dermatitis. Br. J. Dermatol. 132: 22-24, 1995[Medline].

25.   Sekut, L., D. Yarnall, S. A. Stimpson, L. S. Noel, R. Bateman-Fite, R. L. Clark, M. F. Brackeen, J. A. Menius, Jr., and K. M. Connolly. Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation. Clin. Exp. Immunol. 100: 126-132, 1995[Medline].

26.   Seldon, P. M., P. J. Barnes, K. M. Meja, and M. A. Giembycz. Suppression of lipopolysaccharide-induced tumour necrosis factor-alpha generation from human peripheral blood monocytes by inhibitors of phosphodiesterase 4: interaction with stimulants of adenylyl cyclase. Mol. Pharmacol. 48: 747-757, 1995[Abstract].

27.   Seybold, J., R. Newton, L. Wright, P. A. Finney, N. Suttorp, P. J. Barnes, I. M. Adcock, and M. A. Giembycz. Induction of phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T-lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists. Potential role in beta2-adrenoreceptor desensitization. J. Biol. Chem. 273: 20575-20588, 1998[Abstract/Free Full Text].

28.   Sommer, N., P. A. Loschmann, G. H. Northoff, M. Weller, A. Steinbrecher, R. Meyermann, A. Reithmuller, and A. Fontana. The antidepressant rolipram suppresses cytokine production and prevents autoimmune encephalomyelitis. Nat. Med. 1: 244-248, 1995[Medline].

29.   Standiford, T. J., S. L. Kunkel, S. H. Phan, B. J. Rollins, and R. M. Strieter. Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells. J. Biol. Chem. 266: 9912-9918, 1991[Abstract/Free Full Text].

30.   Tamaoki, J., M. Kondo, and T. Takizawa. Effect of cAMP on ciliary function in rabbit tracheal epithelial cells. J. Appl. Physiol. 66: 1035-1039, 1989[Abstract/Free Full Text].

31.   Tenor, H., A. Hatzelmann, R. Kupferschmidt, L. Stanciu, R. Djukanovitc, C. Schudt, A. Wendel, M. K. Church, and J. K. Shute. Cyclic nucleotide phosphodiesterase isoenzyme activities in human alveolar macrophages. Clin. Exp. Allergy 25: 625-633, 1995[Medline].

32.   Thompson, W. J., and M. M. Appleman. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry 10: 311-316, 1971[Medline].

33.   Torphy, T. J., and L. B. Cieslinsky. Characterization and selective inhibition of cyclic nucleotide phosphodiesterase isozymes in canine tracheal smooth muscle. Mol. Pharmacol. 37: 206-214, 1990[Abstract].

34.   Verghese, M. W., R. T. McConnell, J. M. Lenhard, L. Hamacher, and S. L. Jin. Regulation of distinct cyclic AMP-specific phosphodiesterase (phosphodiesterase type 4) isozymes in human monocytic cells. Mol. Pharmacol. 47: 1164-1171, 1995[Abstract].


Am J Physiol Lung Cell Mol Physiol 275(4):L694-L700
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society