Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis-derived secretory products

We previously reported that leukotriene B4 4 (LTB4) 4 ) contained in Trichomonas vaginalis-de--de- rived secretory products (TvSP) play an essential role in interleukin-8 (IL-8) production in human mast cell line (HMC-1 cells) via LTB4 4 receptor (BLT)-mediated Nuclear Factor- kappa B (NF-& kcy;B) & kcy; B) activation. Dynamin, a GTPase, has been known to be involved in endocytosis of receptors for signaling of production of cytokine or chemokines. In the present study, we investigated the role of dynamin-mediated BLT1 endocytosis in TvSPinduced IL-8 production. When HMC-1 cells were transfected with BLT1 or BLT2 siRNA, TvSP-induced IL-8 production was significantly inhibited compared with that in cells transfected with control siRNA. In addition, pretreatment of HMC-1 cells with a dynamin inhibitor (Dynasore) reduced IL-8 production induced by TvSP or LTB4. 4 . TvSPor LTB4- 4- induced phosphorylation of NF-& kcy;B & kcy; B was also attenuated by pretreatment with Dynasore. After exposing HMC-1 cells to TvSP or LTB4, 4 , BLT1 was translocated from the intracellular compartments to the plasma membrane within 30 min. At 60 min after stimulation with TvSP or LTB4, 4 , BLT1 remigrated from the cell surface to intracellular areas. Pretreatment of HMC-1 cells with dynamin-2 siRNA blocked internalization of BLT1 induced by TvSP or LTB4. 4 . Co-immunoprecipitation experiments revealed that dynamin-2 strongly interacted with BLT1 60 min after stimulation with TvSP or LTB4. 4 . These results suggest that T. vaginalis-secreted LTB4 4 induces IL-8 production in HMC-1 cells via dynamin 2-mediated endocytosis of BLT1 and phosphorylation of NF-& kcy;B. & kcy; B.