Molecular targets for glucocorticoids in macrophages: cytosolic phospholipase A2 and cytokine formation

Abstract: The aim of this investigation has been to study mechanisms by which glucocorticoids inhibit inflammatory processes in vitro. Type IV cytosolic 85 kDa phospholipase A2 (cPLA2) is an important modulator of inflammatory responses that is present in macrophages as well as in other cell types. In macrophages, the synthetic glucocorticoid dexamethasone (dex) was found to reduce the expression of cPLA2 and further to inhibit the activation of cPLA2. Stimulation with phorbol 12-myristate 13-acetate (PMA), zymosan or the protein phosphatase inhibitor okadaic acid increased the cPLA2 activity. After treatment with dex, okadaic acid, but not PMA or zymosan, was able to up-regulate cPLA2 activity. These results indicate that the effect of okadaic acid was exerted at, or downstream of, the dex-sensitive step(s). Phosphorylation of cPLA2 occurred on multiple sites. Upon stimulation with PMA or bacteria, the most C-terminal fragment (residues 698-749) of cPLA2 was the most heavily phosphorylated. We did not find any evidence for down-regulation of protein kinase C isoforms after dex treatment. Neither was the expression, nor the activation of, the mitogen-activated protein kinases (MAP kinases), extracellular signal- regulated kinase-2 (ERK-2) or p38, affected to the same extent as the cPLA2 activity. Although a minor inhibition of the zymosan-induced activation of the MAP kinases was observed, our results suggest that dex-mediated inhibition of cPLA2 activation is exerted down-stream of the MAP kinases. The effect of dex on the formation of two other inflammatory mediators in macrophages, the cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) was also investigated. Treatment with dex inhibited bacteria- induced IL-1beta expression primarily at the transcriptional level whereas TNF-alpha expression was only partially inhibited at the level of transcription but further inhibited at the translational level. The translational inhibition after dex treatment was overcome by okadaic acid. Also the post-translational processing of TNF-alpha was modified by okadaic acid. TNF-alpha was found both in the cleaved and uncleaved form after okadaic acid treatment. The different TNF-alpha forms exhibited different sensitivity to dex treatment. Based on these data, we suggest that the effect of dex on TNF-alpha translation is mediated by an okadaic acid-sensitive protein phosphatase. In conclusion, dex inhibits the synthesis of cPLA2. Studies with okadaic acid revealed protein phosphatases to be likely targets for dex both in the inhibition of cPLA2 activation and in inhibition of TNF-alpha translation.