Human LTD4 (Leukotriene D4) ELISA Kit

In alveolar macrophages, leukotriene (IT) B-4 and cysteinyl LTs (LTC4, LTD4 and LTE4) both enhance Fc gamma receptor (Fc gamma R)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gamma R-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB4 and LTD4 both enhanced PKC-delta and -alpha phosphorylation during Fc gamma R engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB4 effects were dependent on both PKC-alpha and -delta, while LTD4 effects were exclusively due to PKC-delta activation. Although both exogenous LTB4 and LTD4 enhanced p38 and ERK 1/2 activation, LTB4 required only ERK 1/2, while LTD4 required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB4 contributed to Fc gamma R-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB4 and LTD4 to enhance Fc gamma R-mediated phagocytosis reflect their differential activation of specific kinase programs. (C) 2008 Elsevier Ltd. All rights reserved.

The cysteinyl leukotrienes (cys-LTs), leukotriene C-4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA(4), in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 mu M and the corresponding 95% confidence interval was from 1.7 to 2.2 mu M. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner.