Inflammatory cells such as monocytes can be activated by bacteria leading to the release of pro-inflammatory cytokines, particularly tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1β) which sequentially induce prostaglandin (PG) production. It has been shown that PGs, especially PGE2 and PGD2, can potently suppress the production of cytokines when added exogenously or produced endogenously by negative-feedback regulation. There are multiple receptor subtypes for these PGs and it is uncertain which subtypes are specifically involved in the suppression of cytokine production. Therefore, the aim of the present study was to ascertain the expression profile of these receptors following activation of monocytic cells in the presence of lipopolysaccharide (LPS) and the PGs, particularly PGE2. Monocytes were isolated from whole human blood and compared to the human monocytic cell line THP-1 which was cultured continuously with RPMI 1640 containing 10% fetal calf serum (FCS). Levels of TNF-α and IL-1β (in plasma or cell supernatants) were measured by ELISA. The expression of prostaglandin receptor genes for PGE2 and PGD2 (PTGER2, PTGER4, PTGDR1 and PTGDR2) were determined by quantitative real-time PCR. THP-1 cells were transfected with either siRNA duplexes for knock-down of the EP4 receptors or exogenous EP4 constructs in overexpression experiments. LPS increased TNF-α and IL-1β levels in blood, monocytes and THP-1 cells. The LPS-stimulated increase in levels of cytokines were suppressed by PGE2 and PGD2. Both EP2 and EP4 receptor agonists (butaprost and L-902, 688) inhibited the production of TNF-α but EP1/3 receptor agonist (sulprostone) did not alter TNF-α levels. Expression of the PTGER2, PTGER4, PTGDR1 and PTGDR2 genes were enhanced by LPS and inhibited by PGE2 and PGD2 in both monocytes and THP-1 cells.The EP4 receptor agonist (L-902, 688) inhibited both PTGER2 and PTGER4 genes expression but there was no obvious effect on PTGER2 expression using an EP2 agonist (butaprost). In contrast, the EP1/3 agonist (sulprostone) did not alter PTGER2 expression levels. Knock-down of PTGER4 resulted in a significant increase in TNF-α production and following exogenous transfection with an EP4 receptor construct, the TNF-α level was decreased. This implies that prostaglandins such as PGE2 and PGD2 can limit the level of cytokines released in response to LPS. It appears that there is cross regulation between EP2 and EP4 receptors because PTGER2 and PTGER4 were suppressed by both PGE2 and L-902, 688. This suggests that EP4 receptors play a crucial central role in the inhibition of TNF-α production.
|Date of Award||1 Feb 2015|
- University Of Strathclyde
|Supervisor||Dino Rotondo (Supervisor) & Alexander Gray (Supervisor)|