However blockade of receptor G2A did not reverse the LPC-mediated inhibition of cell death following LPS exposure

However blockade of receptor G2A did not reverse the LPC-mediated inhibition of cell death following LPS exposure. In contrast to earlier studies that concentrated on LPC action in the cell membrane, our work suggests that a mechanism of action for LPC is situated within the cytosol, where LPC binds directly to caspase-11 and thereby blocks its activation and oligomerization in response to LPS. LPC in experimental sepsis and endotoxemia. LPS (clone2D7/1) came from Abcam (USA). FITC-labeled LPS was purchased from Sigma. The liposomal transfection reagent Dotap was from Roche. Mouse IL-1 (MLA00) and IL-1 (MLB00C) Elisa Kit were from R&D. Macrophage Tianeptine preparation and activation Mouse peritoneal macrophages were isolated and cultured as explained (14). Briefly, C57BL/6 mice (8C10weeks older) were injected intraperitoneally with 2 mL of sterile 4% thioglycollate Tianeptine broth to elicit peritoneal macrophages. At 48C72h later on, cells were collected by lavage of the peritoneal cavity with 5 mL of sterile 11.6% sucrose. Harvested cells were washed, then re-suspended in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (Gibco). Peritoneal macrophages were plated in 12-well plates (106/well) and primed for 6 h with ultra-pure LPS (100ng/ml) or Pam3CSK4 (1g/ml). Primed cells were washed with phosphate-buffered saline (PBS) and treated for 2 h with stearoyl LPC, which had been prepared as before (13). Cells were washed again with PBS and treated with the combination of LPS (1g/ml) and a mixture of cholera toxin subunit B (CTB, 20g/ml) or Dotap, which had been prepared by combining CTB and Dotap in Optim (Gibco) Tianeptine for 20 min at space temp. At 16 h after treatment, cells were harvested and total lysates were prepared for analysis using Western blotting an assay for lactate dehydrogenase (LDH). In some experiments, macrophages were treated with LPC without priming. In additional experiments, macrophages were treated with anti-G2A antibody (1g/ml) only or with the combination of LPC and anti-G2A antibody (1g/ml). European blotting Total cell lysates were prepared and micro-centrifuged. Proteins in the supernatant were extracted using methanol/chloroform, fractionated using 12% SDS-PAGE, and transferred onto PVDF membranes (Millipore). Membranes were blotted with antibodies against caspase-11 (1:500)or Gsdmd (1:500). Band denseness was normalized to levels of -actin or GAPDH on the same blots (1:5000; Cell Signaling Technology, USA). Cell death assay Tradition press were harvested and centrifuged to sediment cells; the producing supernatant was subjected to the LDH Cytotoxicity Assay (Beyotime Biotechnology, China). Limulus amebocyte lysate (LAL) assay Isolation of cytosol portion from mouse peritoneal macrophages was performed as previously explained (15). Briefly, 5106 cells were treated with Stearoyl LPC for 2h, and then for 2h Tianeptine or 6h with LPS (1g/ml) plus CTB (20g/ml) as indicated. After treatment, cells were washed with sterile Dulbeccos Phosphate Buffered Saline (DPBS) then digested with 0.25% trypsin. Digestion was halted by addition of RPMI 1640 supplemented with 10% FBS. Cells were washed 3 times by Mouse monoclonal to Transferrin DPBS then treated with 300l of 0.005% digitonin extraction buffer for 20min on ice and the supernatant containing cytosol was collected after centrifugation and transferred to a new tube and stored at ?80C until analysis using a commercial LAL assay (Xiamen Bio Endo Technology, China). Circulation cytometry Stearoyl LPC was labeled with an Alexa Fluor 488-conjugated antibody using the Alexa Fluor 488 Labeling Kit (Life Systems, USA) according to the manufacturers instructions. Cells were incubated for 1 or 2 2 h with the producing stearoyl LPC-488(20M), washed three times with PBS, and then treated as previously (16) to quench extracellular fluorescence. After washing again with PBS, cells were digested with 0.25% trypsin and digestion was halted by adding RPMI 1640 supplemented with 10%FBS. Cell suspensions were centrifuged at 300for 5min, the supernatant was eliminated. Cell pellets were re-suspended in PBS supplemented with 2% BSA. Re-suspended cells were then analyzed by circulation cytometry on a BD FACS Cantoll. Fluorescence intensity assay Peritoneal macrophages were plated in 96-well fluorescence plates (105/well), primed and treated as explained above. Cells were incubated for 1 or 2h with LPC-488 (20M), washed three times with PBS, and treated as previously explained to quench extracellular fluorescence. After washing again with PBS, the intracellular fluorescence intensity was measured using a fluorescence micro.