Selectivity of Cyclo-Oxygenase Inhibitors in Human Pulmonary Epithelial and Smooth Muscle Cells
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Copyright #ERS Journals Ltd 2000 Eur Respir J 2000; 15: 751±756 European Respiratory Journal Printed in UK ± all rights reserved ISSN 0903-1936 Selectivity of cyclo-oxygenase inhibitors in human pulmonary epithelial and smooth muscle cells S.P. Range, L. Pang, E. Holland, A.J. Knox Selectivity of cyclo-oxygenase inhibitors in human pulmonary epithelial and smooth muscle Respiratory Medicine Unit, City Hospital, cells. S.P. Range, L. Pang, E. Holland, A.J. Knox. #ERS Journals Ltd 2000. Hucknall Road, Nottingham, NG5 1PB, ABSTRACT: Cyclo-oxygenase (COX) inhibitors may have a role in reducing in- UK flammation in asthma and other pulmonary diseases. COX inhibitors have different Correspondence: S.P. Range selectivities for the two COX isoenzymes (COX 1 and COX 2) which vary between Dept of Respiratory Medicine purified enzyme and intact cell preparations. The relative selectivity of COX inhi- University Hospital bitors has not been studied in human airway cells. Queen's Medical Centre A number of COX inhibitors in cultured human airway cells were compared which Nottingham NG7 2UH exclusively express either COX 1 (primary degree cultured human airway smooth UK muscle (HASM) cells) or COX 2 (A549 pulmonary epithelial cell-line) as measured by Fax: 44 1159424554 Western blotting. COX activity was assayed by prostaglandin (PG)E2 production following 30 min incubation with 5 mM arachidonic acid. Keywords: A549 cells . -1 airway smooth muscle COX activity in both cell types was similar; HASM cells 92.212.1 ng PGE2 mg . -1 asthma protein, A549 cells 87.724.4 ng PGE2 mg protein. In HASM cells the median -5 -6 cyclo-oxygenase inhibitory concentration (IC50) was >10 M for nimesulide, 3.2610 M for N-(2- cyclo- cyclo-oxygenase inhibitor -8 hexyloxy-4-nitrophenyl)-methanesulphonamide (NS398), 1.8610 M for flurbiprofen, prostaglandin E2 6.7610-9 M for indomethacin and >10-5 M for aspirin. In A549 cells the IC50 was 1.86 10-9 M fornimesulide,4.1610-9 Mfor NS398,6.2610-10 Mfor flurbiprofen, 1.3610-8 M Received: January 11 1999 for indomethacin and >10-5 M for aspirin. Sodium valerate had no effect in either HASM Accepted after revision January 23 2000 or A549 cells. The COX 2:COX 1 selectivity ratio (COX 2 IC50/COX 1 IC50) was <0.0001 for nimesulide, 0.001 for NS398, 0.03 for flurbiprofen and 1.9 for indomethacin. L. Pang was supported by the National Asthma Campaign (UK). In conclusion the present study has shown that cyclo-oxygenase inhibitors have a range of selectivities for cyclo-oxygenase 1 and cyclo-oxygenase 2 in intact human air- way cells. The relative cyclo-oxygenase 2 selectivity of N-(2-cyclohexyloxy-4-nitro- phenyl)-methanesulphonamide and nimesulide may have implications for the treatment of asthma and other inflammatory pulmonary diseases. Eur Respir J 2000; 15: 751±756. Recent studies have suggested that cyclo-oxygenase muscle cells in vitro have suggested that COX products (COX) products may be involved in the pathophysiology can also regulate adenylate cyclase function and the pro- of several pulmonary diseases: COX products have been duction of the chemokine interleukin (IL)-8 [14±16]. implicated in the inflammation found in asthmatic airways, COXproductshavealsobeenimplicatedinotherpul- with different COX products exerting differential effects in monary diseases: lung fibroblasts from patients with idio- different situations in vivo [1±3]. Inhaled indomethacin pathic pulmonary fibrosis have been shown to have a and aspirin protect against indirect bronchoconstrictor diminished capacity to synthesize PGE2 [17]. Tracheal challenges in asthma [4, 5] suggesting that release of epithelial cells containing the DF508 cystic fibrosis (CF) constrictor prostaglandins such as prostaglandin (PG)D2 mutation exhibit enhanced PG synthesis compared to wild and PGF2a may contribute to the mechanisms of action of type cells [18]. Further evidence that pro-inflammatory these stimuli. Under different circumstances COX pro- COX products may be involved in the pathophysiology ducts may have a protective role. PGE2 has been shown to of CF has been provided by a study showing that the have a potential bronchoprotective role both in vitro [6, 7] COX inhibitor ibuprofen can slow the rate of pulmonary and in vivo [8, 9]. Studies showing that oral indomethacin function decline in these patients [19]. In order to gain inhibits refractoriness to repeated bronchoconstriction greater insight in to the possible role of COX inhibitors in challenge suggest that endogenous PGE2 may be involv- the modification of these and other pulmonary disease pro- ed in this protective response [10, 11]. A small number of cesses, further knowledge of the selectivity of COX inhi- patients with asthma exhibit aspirin sensitivity, whereby bitors against COX isoforms in airway cells is required. oral aspirin and other COX inhibitors produce broncho- COX exists in two isoforms COX 1 and COX 2. COX 1 choconstriction. This bronchoconstriction can be pre- is constitutively expressed in many tissues [20], whereas vented by inhalation of PGE2 [12, 13]. Collectively these COX 2 is an inducible form which has been shown to be studies implicate COX products in several aspects of induced in airway cells by stimuli present in inflammatory asthma pathophysiology, but it is clear that the responses pulmonary diseases [21±23]. Airway cells from patients to COX inhibition depend on the inhibitor studied, the with asthma have been shown to express COX 2 in great- route of administration, the circumstances of use and the er quantities than those from nonasthmatic subjects [24]. type of patients studied. Studies in cultured airway smooth COX inhibitors are available with a range of selectivities 752 S.P. RANGE ET AL. for COX 1 and COX 2. The selectivity of these agents air/5% CO2 at 378C. Culture media was replaced on alter- varies, however, between purified enzyme, broken cell nate days until the cells were confluent. Cells were growth and intact cell preparations [25]. A greater understanding arrested for 24 h in media without FCS prior to all experi- of the effects of these agents in the lung requires study of ments. their selectivity in whole cell preparations of airway cells. COX isoforms are expressed in several airway cells. The Cyclo-oxygenase activity authors and others have previously shown that COX 1 is the sole isoform expressed under resting conditions in Prior to each experiment, the culture medium was re- human airway smooth muscle (HASM), and that COX 2 placed with fresh culture medium without FCS. The COX can be induced by cytokines, particularly IL-1b [22, 26, inhibitor to be studied dissolved in DMSO (final concen- 27]. In contrast the epithelial cell line A549 expresses only tration 1% volume/volume) or DMSO control was then COX 2 both under resting [28] and cytokine-stimulated added, and the cells were incubated at 378C for 15 min. conditions [21, 28]. Similar findings have been shown in Exogenous arachidonic acid (AA; final concentration 5 primary cultures of airway epithelial cells [21, 28, 29]. mM) was then added and the cells were incubated for a Unstimulated HASM and A549 cells therefore represent further 30 min at 378C. Media was removed and imme- good whole cell models for studies of COX 1 and COX 2 diately frozen and stored at -208C until assayed for PGE2. respectively. Therefore, the effects of a number of COX Six COX inhibitors were studied based on their selectivity inhibitors were studied in these airway cells. The COX 1 in other systems: valeryl salicylate, a COX 1 selective inhi- selectivity of the drugs in unstimulated HASM cells that bitor; nimesulide and N-(2-cyclohexyloxy-4-nitrophenyl)- constitutively express COX 1 was examined along with the methanesulphonamide (NS 398), both COX 2 selective; COX 2 selectivity in unstimulated A549 epithelial cells and the nonselective COX inhibitors aspirin, indometha- that constitutively express COX 2. cin, and flurbiprofen. Materials and methods Prostaglandin E2 assay Cell culture PGE2 concentration was determined by radioimmuno- assay as previously described [31]. The authors have pre- Human airway smooth muscle cells. Primaryculturesof viously validated this assay [22, 23, 31] and have shown adult HASM cells were prepared from explants of airway it to have a low cross-reactivity with other metabolites of smooth muscle according to methods previously reported AA [22, 23]. Each sample analysed for PGE2 was assayed [22, 23, 30]. Briefly, human trachea was obtained from two in duplicate. post mortem individuals (one male aged 44 yrs and one female aged 52 yrs, with no evidence of airway disease) Protein assay within 12 h of death. The trachealis muscle was then dis- sected free of epithelium and connective tissue under sterile Cell protein was assayed in cell cultures used for conditions. Small (262 mm) explants of airway smooth Western blot analysis and relative COX activity. Following muscle were then excised and ~10 explants placed in one removal of the cell media for PGE2 estimation, extractable small petri dish. The explants were incubated in 10% foetal protein was removed from the cells by incubation with an calf serum (FCS)-Dulbecco'smodifiedEagle'smedium extraction buffer (0.9% NaCl, 20mM tris-HCl, pH 7.6, (DMEM) in humidified 5% CO2/95% air at 378C and the 0.1% triton6100, 1 mM phenylmethylsulforyl fluoride, medium was changed every 3 days. Smooth muscle cells 0.01% leupeptin) with gentle shaking. Extracted protein were usually seen ~7 days later. Once confluent, cells were concentration was determined using the Bio-Rad protein trypsinized and plated into 175 cm2 tissue culture flasks, assay (Bio-Rad Laboratories Ltd, Hemel Hempstead, Hert- grown to confluence again, trypsinized and cryopreserved fordshire, UK). at 16106 cells.mL-1. HASM cells out of cryopreservation were plated at a density of 26104 cells.well-1 in 12-well Western blot analysis of cyclo-oxygenase isoenzymes culture plates, cultured to confluence in 10% FCS-DMEM and growth-arrested in FCS-free DMEM for 24 h before Thirty micrograms of extracted protein was mixed 1:1 experiments.