Regulation of prostanoid effects in whole blood : immediate-early anti-inflammatory effects of prostaglandin E2

Abstract

To understand the regulation of the pathophysiological processes, such as inflammation, thrombosis and atherosclerosis, it is very important to characterize the interactions between circulating cells and which molecules that contributes to and promote these interactions. As a part of this, the role of eicosanoids in cell-cell interactions in whole blood ex vivo and isolated blood cells are investigated. In this study, various inhibitors were used to regulate the amount of prostaglandins (and leukotrienes) and how these eicosanoids affect the activity- and expression level of tissue factor (TF), cytokines, enzymes, and receptors involved in the intercellular communication. Before the inhibition study, time course experiments revealed that the incubation times between 1.5 h and 2 h was sufficient for further study of the parameters under investigation. In the inhibition study, whole blood samples were preincubated with different eicosanoid inhibitors, and then stimulated with LPS for ninety minutes prior to TF activity measurement and real-time PCR analysis of cytokine gene expressions. Aspirin did not significantly enhance the lipopolysaccharide (LPS)-induced TF activity in whole blood, however a trend for enhance induction was indicated.

Prostaglandin inhibitors enhanced the LPS-induced TF activity compared to a vehicle control, with significant effect for the microsomal prostaglandin E synthase (mPGES) inhibitor MF63. A dual cyclooxygenase-2/ 5-lipoxygenase (COX-2/ 5-LOX) inhibitor was used to investigate what happened if the leukotriene pathways were blocked in parallel with COX-2. And although not significant, an inhibitory effect of monocyte TF activity was seen for the weakest and strongest dose.To measure the mRNA expression of the cytokines tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), IL-8 and monocyte chemotactic protein-1 (MCP-1), real-time PCR analysis was performed. Aspirin was found to generally increase the gene expression of these cytokines. Moreover, significant enhancement of the IL-8 mRNA expression was found for all doses of aspirin, varying from 67% to 78% enhancement for 20 μM and 200 μM, respectively. For IL-1β and TNF-α mRNA expression was increased by 86% (2 μM) and 36% (200 μM), respectively. Like aspirin, the selective cyclooxygenase-1 (COX-1) inhibitor SC-560, increased mRNA expression of all cytokines. The most pronounced effects were observed for IL-8 and IL-1β. The MCP-1 mRNA expression level was greatly enhanced, however due to testing a low number of individuals for this cytokine, the effect was not significant. The enhancement of IL-8 expression was also seen after addition of the selective COX-2 inhibitor, CAY10404. With the exception of IL-8, the highest dose of COX-2 inhibitor caused an insignificant reduction in mRNA expression for all cytokines. The lowest dose of prostaglandin synthesis inhibitor CAY10526 resulted into most pronounced mRNA expression for TNF-α, IL-1β and IL-8. However, the inhibitor did not significantly enhance the LPS-induced TF mRNA expression. Samples with mPGES-1 inhibitor (MF63) showed a bi-phased expression for nearly all the genes. However, the middle dose of the inhibitor induced gene expression at a higher level than controls and other doses. Flow cytometric analysis was carried out to investigate the platelet-leukocyte heteroconjugates in whole blood stimulated with LPS and LPS in combination with platelet activating factor (PAF). Whole blood samples were stimulated for 2 h. LPS-stimulated whole blood was found to increase platelet interactions with monocyte with approximately 50% (not significant). Addition of both LPS and PAF resulted in a 3-fold significant enhancement of the conjugate formation. The binding of platelets to granulocytes decreased when LPS was added but the aggregates were observed to slightly increase when stimulated with LPS and PAF in combination.

In conclusion, these experiments have demonstrated that inhibition of the PGE2 synthesis enhanced LPS-induced TF activity in whole blood monocytes and expression of the chosen proinflammatory cytokines. In platelet-monocyte interactions, platelets bound easily to monocytes, while binding to granulocytes seemed to require stronger stimuli.