Induction of cyclo‐oxygenase‐2 by cytokines in human pulmonary epithelial cells: regulation by dexamethasone
Authors: Mitchell Jane A. , Belvisi Maria G. , Akarasereenont Pravit , Robbins Richard A. , Kwon O.‐Jung , Croxtall Jamie J. , Barnes Peter J. , Vane John R. 
Published: July 2012

Cyclo‐oxygenase metabolizes arachidonic acid to prostaglandin H2 (PGH2) and exists in at least two isoforms. Cyclo‐oxygenase‐1 (COX‐1) is expressed constitutively whereas COX‐2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo. Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX‐1 or COX‐2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549).

COX‐1 or COX‐2 protein was measured by western blot analysis using specific antibodies to COX‐2 and selective antibodies to COX‐1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE2, PGF2α, thromboxane B2 or 6‐oxo PGF1α measured by radioimmunoassay. Thus, COX‐1 or COX‐2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12 h before fresh culture medium was added containing exogenous arachidonic acid (30 μm) for 15min after which COX metabolites were measured.

Untreated primary cells or A549 cells contained low amounts of COX‐1 or COX‐2 protein. Bacterial LPS (1 μg ml−1 for 24 h) induced COX‐2 protein in the primary cells, a process which was enhanced by interferon‐γ, with no further increase in the presence of a mixture of cytokines (interleukin‐1β, tumour necrosis factor‐α and interferon‐γ, 10 ng ml−1 for all). In contrast, A549 cells contained only low levels of COX‐2 protein after exposure to LPS or LPS plus interferon‐γ, but contained large amounts of COX‐2 protein after exposure to the mixture of cytokines.

Untreated human pulmonary primary cells or A549 cells released low levels of all COX metabolites measured over a 24 h incubation period. This release was enhanced by treatment of either cell type with the mixture of cytokines (interleukin‐10, tumour necrosis factor‐α and interferon‐γ, 10ng ml−1 for all). PGE2 was the principal COX metabolite released by cytokine‐activated epithelial cells. The release of PGE2 induced by cytokines occurred after a lag period of more than 6 h.

The glucocorticosteroid, dexamethasone (1 μm; 30 min prior to cytokines) completely suppressed the cytokine‐induced expression of COX‐2 protein and activity in both primary cells and A549 cells.

In experiments where COX‐2 activity was supported by endogenous stores of arachidonic acid, treatment of A549 cells with interleukin‐10 but not tumour necrosis factor‐α or interferon‐γ alone caused a similar release of PGE2 to that seen when the cytokines were given in combination. However, both interleukin‐10 and necrosis factor‐α alone produced similar increases in COX‐2 activity (measured in the presence of exogenous arachidonic acid) as seen when the mixture of interleukin‐1β, tumour necrosis factor‐α and interferon‐γ were used to stimulate the cells.

These findings show that COX‐2 expression correlates with the exaggerated release of prostaglandins from cytokine‐activated human pulmonary epithelial cells and that the induction of the enzyme is suppressed by a glucocorticosteroid. These findings may be relevant to inflammatory diseases of the lung, such as asthma.