Extra Domain A of Fibronectin Primes Leukotriene Biosynthesis and Stimulates Neutrophil Migration Through Activation of Toll-like Receptor 4

Objective. 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 B(4) (LTB(4)) 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. Methods. 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. Results. Recombinant human ED-A efficiently primed the biosynthesis of LTB(4) 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, Pam(2)CSK(4)) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB(4) receptor 1 (BLT(1)) antagonist CP105,696 or the cytosolic phospholipase A(2)alpha 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). Conclusion. These results demonstrate the ability of rhED-A to promote LTB(4) biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB(4) biosynthesis and PMN infiltration in inflammatory joint diseases.