Induction of cyclo‐oxygenase‐2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type

Abstract

Cyclo‐oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H₂ (PGH₂) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX‐1 is expressed constitutively, whereas COX‐2 is expressed in response to pro‐inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined. COX‐1 and COX‐2 protein were measured by Western blot analysis with specific antibodies for COX‐1 and COX‐2. COX‐2 mRNA levels were assessed by Northern blot analysis by use of a COX‐2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE₂, thromboxane B₂ or 6‐ketoPGF_1α by radioimmunoassay. Under control culture conditions HASM cells expressed COX‐1, but not COX‐2, protein. However, a mixture of cytokines (interleukin‐1β (IL‐1β), tumour necrosis factor α (TNFα) and interferon γ (IFNγ) each at 10 ng ml⁻¹) induced COX‐2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 μm; added 30 min before the cytokines). Furthermore, COX‐2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 μm) and cyclohexamide (10 μg ml⁻¹; added 30 min before the cytokines). Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL‐1β, TNFα, IFNγ each at 10 ng ml⁻¹ for 24 h) caused the accumulation of PGE₂ and 6‐keto‐PGF_1α. In experiments where COX‐2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL‐1β in combination with TNFα caused a similar release of PGE₂ to that when the three cytokines were given in combination. In other experiments designed to measure COX‐2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 μm for 15 min) after which PGE₂ was measured. IL‐1β and TNFα increased COX‐2 activity and an additional small increase was produced by the three cytokines in combination. These findings suggest that the increased expression of COX‐2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro‐inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma. British Journal of Pharmacology (1997) 120, 910–916; doi:10.1038/sj.bjp.0700963