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Abstract
The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells. To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells. Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation. Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.
Highlights
PGD2 is the major eicosanoid released from activated mast cells.[1,2] It has potent biological effects including bronchoconstriction via the TP receptor,[3,4] vasodilation via the DP1 receptor,[5] induction of eosinophilic lung inflammation[6] as well as activation and recruitment of group 2 innate lymphoid cells (ILC-2) and Th2 cells via the DP2 receptor,[7] called CRTH2 receptor.8-10 It is a current interest in targeting PGD2 for treatment of asthma and allergic diseases.[8]
First we examined the initial step in the PGD2 biosynthesis, that is, the dependence on the COX isozymes, in primary cultured mast cells from cord blood (CBMC) and peripheral blood (PBMC) as well as the LAD2 cell line (Figure 2A)
The study used pharmacologic inhibitors and a sensitive and selective mass spectrometry-based method to explore the metabolic effects of inhibiting the hematopoietic prostaglandin D synthase (hPGDS) enzyme that catalyses formation of PGD2 in four human mast cell models
Summary
PGD2 is the major eicosanoid released from activated mast cells.[1,2] It has potent biological effects including bronchoconstriction via the TP receptor,[3,4] vasodilation via the DP1 receptor,[5] induction of eosinophilic lung inflammation[6] as well as activation and recruitment of group 2 innate lymphoid cells (ILC-2) and Th2 cells via the DP2 receptor,[7] called CRTH2 receptor.8-10 It is a current interest in targeting PGD2 for treatment of asthma and allergic diseases.[8]. We hypothesised that inhibition of the biosynthesis of PGD2 might represent an attractive alternative interventional strategy requiring only one drug. We performed this in-depth study of the mechanistic consequences of selective inhibition of the biosynthesis of PGD2 in human lung mast cells (HLMC), as well as in three other human mast cell models. It has been speculated that inhibition of one enzyme distal of COX might increase the biosynthesis of other prostanoids, with such shunting of the intermediate into other pathways possibly leading to unwanted consequences.[14] There is no published study of whether or not such shunting occurs in human mast cells. The compound PPCA (2-Phenyl-pyrim idine-5-carboxylic acid (2,3-dihydro-indol-1-yl)-amide),[16] which belongs to the group of pyrimidine hydrazides,[17] showed more potent activity than HQL-799 and was selected for this study
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