leukotriene-b4 and resiquimod

There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling.. The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo.. Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 α inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4).. These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.

Topics: Animals; Anti-Inflammatory Agents; Benzopyrans; Carboxylic Acids; Cytokines; Female; Fibronectins; Humans; Imidazoles; Leukotriene B4; Leukotrienes; Lipopeptides; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Monocytes; Neutrophils; Protein Isoforms; Pyrrolidines; Recombinant Proteins; Sulfonamides; Toll-Like Receptor 4; Transendothelial and Transepithelial Migration

Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8, the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself, it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP, platelet-activating factor, and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation, as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2alpha, promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1, an inhibitor of endosomal acidification, efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections.

Topics: Dinoprostone; Humans; Imidazoles; Inflammation Mediators; Leukotriene B4; Neutrophils; Platelet Activating Factor; RNA Viruses; Signal Transduction; Toll-Like Receptor 7; Toll-Like Receptor 8