For determination of the effects of C5a (Sino Biological), LPS (Sigma-Aldrich), or C5a/LPS, macrophages were incubated with each stimulator for 5 h, and then phagocytosis assay was performed. The percentage of killing was calculated as 100 × (1−CFUs after 2-h incubation/CFUs before 30-min incubation). For measurement of bactericidal activity, cells were incubated for 2 h after 30-min incubation for bacterial uptake. coli were plated onto Lysogeny broth agar plates, incubated overnight at 37☌, and the numbers of CFUs were counted. coli number, the plate was lysed with 0.1% Triton X-100 in PBS after 30 min of E. coli were washed out twice with PBS containing gentamicin (12.5 μg/ml) and another twice with PBS. After 30 min of incubation for the uptake of E. Macrophages were then incubated with 10 7 E. Thioglycolate-induced macrophages (1 × 10 6/ml) were seeded on a 24-well plate in a humidified incubator (37☌, 5% CO 2). These results show inappropriate activation of G2A −/− Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.īactericidal activity was determined as described previously ( 13, 15).
Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A +/+ but not from G2A −/− mice. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A +/+ peritoneal macrophages but not G2A −/− cells, which showed more PGE 2/nitrite release and intracellular reactive oxygen species levels.
IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Indomethacin effectively blocked both the increased i.p. Anti–IL-10 Ab reversed the impaired bacterial clearance in G2A −/− mice. G2A −/− mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl 3, an inhibitor of Kupffer cells. G2A is a GPCR abundantly expressed in immune cells.
0 kommentar(er)
