Assay of cyclooxygenase-1 and 2 in human monocytes

Objective and Design: There is frequently poor correlation between in vitro methods for calculated cyclooxygenase (COX)-1/COX-2 selectivities of infla mmatory agents. Therefore, we have examined the use of a single stimulus in a single cell type containing both COX isoforms, for determining the selec tivities of COX-inhibitory agents. Methods: Fresh human monocytes were stimulated with arachidonic acid (AA; 1 0 muM) for 15 min and prostaglandin E-2 (PGE(2)) and thromboxane B-2 (TXB2) production were used as a measure of COX-1 activity. To measure COX-2 acti vity, cells were transiently pre-treated with aspirin to irreversibly inhib it constitutive COX-1, treated with lipopolysaccharide (LPS) to induce COX- 2 and then stimulated with AA. Results: Eicosanoid production in resting monocytes was predominantly COX-1 derived since it was not inhibited by NS-398 and also, COX-2 was not detec table. In LPS treated monocytes pre-treated transiently with aspirin, neith er the level of induced COX-2 nor the activity was affected. Using the mean of the results for PGE(2) and TXB2 inhibition, the COX-1/COX-2 ratios of t he IC(5)0 values for aspirin and NS-398 are < 0.1 and > 130, respectively. Conclusions: This study has provided a system for investigating inhibition of COX isotypes without the potentially confounding effects of using differ ent cell types with different stimuli for each isotype as seen in other pub lished systems. Dose responses to aspirin and NS-398 which are COX-1 and CO X-2 selective inhibitors respectively, confirmed the utility of this system .