Effect of interleukin‐1β, tumour necrosis factor‐α and interferon‐γ on the induction of cyclo‐oxygenase‐2 in cultured human airway smooth muscle cells

Abstract

1 Increased levels of several pro-in¯ammatory cytokines including interleukin-1b (IL-1b) and tumour necrosis factor-a (TNFa) have been found in bronchoalveolar lavage¯uid from symptomatic asthmatic patients. are known to stimulate a number of cells to produce in¯ammatory mediators such as prostaglandins. Although airway smooth muscle (ASM) is known to be a rich source of prostaglandins, the regulation of cyclo-oxygenase (COX) isoforms and prostanoid production by proin¯ammatory cytokines have not been studied in human airway smooth muscle. 2 We studied the e ects of IL-1b, TNFa and IFNg on the induction of two isoforms of cyclooxygenase and its relation to prostaglandin E 2 (PGE 2 ) release and COX activity (re¯ected by PGE 2 synthesis from exogenous arachidonic acid) in human cultured airway smooth muscle cells. 3 IL-1b, but not TNFa or IFNg, caused a time-and concentration-dependent enhancement in PGE 2 and other prostanoid (6-keto-PGF 1a , PGF 2a , thromboxane B 2 (TXB 2 ) and PGD 2 ) production, with PGE 2 and 6-keto-PGF 1a as the principal products. This stimulation was accompanied by a corresponding increase in COX activity. 4 COX-2 protein measured by Western blot analysis was not detectable in untreated cells, but was increased in a time-and concentration-dependent manner by IL-1b, but not TNFa or IFNg. In contrast, no variation in the expression of COX-1 protein was observed. 5 Pretreatment with the conventional non-steroidal anti-in¯ammatory drugs (NSAIDs), indomethacin and ibuprofen, and the selective COX-2 inhibitors, NS-398 and nimesulide, completely blocked IL-1b-induced PGE 2 release and COX activity. The glucocorticosteroid dexamethasone and protein synthesis inhibitors, cycloheximide and actinomycin D, not only markedly inhibited IL-1b-stimulated PGE 2 release and COX activity but also suppressed IL-1b-induced COX-2 induction. 6 This study demonstrates that human cultured ASM cells release prostanoids in response to IL-1b stimulation and that the response is mostly mediated by the induction of COX-2 rather than COX-1 isoenzyme, implying that airway smooth muscle may be an important source of prostaglandins in human airways and that COX-2 may play an important role in the regulation of the in¯ammatory process in asthma.

Inhibition of polyadenylation reduces inflammatory gene induction

“…This was not affected by cordycepin treatment. Because TNF does not induce the important inflammatory gene PTGS2 (COX2) in these cells (Pang and Knox 1997), we treated ASM cells with interleukin 1b after a 30-min preincubation with different doses of cordycepin, and found that PTGS2 mRNA induction is also very sensitive to cordycepin treatment (Fig. 3B).…”

“…HeLa and A549 lung epithelial carcinoma cells were cultured in DMEM with 10% fetal bovine serum with 4.5 g/L of glucose and 4 mM glutamine. Primary cultures from human airway smooth muscle cells (ASM) were prepared from explants as described previously (Pang and Knox 1997). The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 0.025 mg/mL amphotericin B, and 4 mM L-glutamine.…”

Selectivity of cyclo-oxygenase inhibitors in human pulmonary epithelial and smooth muscle cells

“…Cells at passage 3 were used for all experiments. The authors have previously shown that cells grown in this manner show the immunohistochemical and light microscopic characteristics of pure HASM cells [22].…”

“…Primary cultures of adult HASM cells were prepared from explants of airway smooth muscle according to methods previously reported [22,23,30]. Briefly, human trachea was obtained from two post mortem individuals (one male aged 44 yrs and one female aged 52 yrs, with no evidence of airway disease) within 12 h of death.…”

“…The authors and others have previously shown that COX 1 is the sole isoform expressed under resting conditions in human airway smooth muscle (HASM), and that COX 2 can be induced by cytokines, particularly IL-1b [22,26,27]. In contrast the epithelial cell line A549 expresses only COX 2 both under resting [28] and cytokine-stimulated conditions [21,28].…”