Secretion of TNF-α/IL-6 of human leukocytes
Human umbilical vein endothelial cell lines (HUVECs, RRID:CVCL_2959) were cultured in DMEM/F12 medium containing 10% FBS at 37°C until confluent. They were then detached by trypsinization and resuspended to obtain a cell density of 1×105 cells/ml. 100ul of the resuspended cells were added to each well of a 96-well plate and incubated at 37°C for 24 hours. On the day of the experiment, freshly extracted human WBCs could be added to the monolayer HUVEC culture in 96-well plates in order to establish three models of cellular interaction: one with (i) only WBCs, (ii) between WBCs and HUVECs, and (iii) between WBCs, RBCs, and HUVECs (WBC: RBC= 1: 5). After the experiment was completed, a different sample of WBCs was freshly extracted from a another individual. The experiment was repeated to ensure that the experimental results were reproducible. Incremental amounts of recIAP (5, 10, 20 U ml-1) were either pre-incubated with LPS for 3 hours in advance, or recIAP was added to 0.5 ng ml-1 LPS directly into the cell culture to stimulate WBCs. After cultured at 37°C for 24 hours, the supernatant was collected to detect TNF-α. Similarly, in an effort to examine the minimum required dosage of recIAP to elicit a response, incremental amounts of recIAP (0.5, 1.0, 2.0, 4.0U ml-1) and 0.5ng ml-1 LPS were added to the WBCs to stimulate TNF-α secretion. The levels of TNF-α and IL-6 were measured after 24 hours of culture. In another set of experiments, 0.5ng ml-1 of LPS was added to either 5 U ml-1 of recIAP or bovine intestinal alkaline phosphatase (bIAP) (P6774-2KU, Sigma) to stimulate WBCs, and the resulting culture medium was collected after 24 hours in order to detect TNF-α and IL-6 levels. Low concentrations (0.10, 0.25, 0.50, 1.00 μM) of ATP, ADP, AMP, and adenosine were also added to the cellular interaction model containing WBCs and HUVECs. The culture medium was subsequently collected after 24 hours to determine TNF-α levels. TNF-α and IL-6 in cell culture medium were measured by use of an enzyme-linked immunoassay kit (TNF-α Elisa kit, R & D system DY210; IL-6 Elisa kit, Huamei CSB -E04638h). For specific method and procedure, refer to the manufacturer’s instructions.