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.