recIAP inhibits the secretion of TNF-ɑ and IL-6 in the absence of LPS
For each experiment, we examined three conditions: (i) when recIAP alone was added to the cell culture (ii) when recIAP and LPS were added simultaneously to the cell culture, and (iii) when LPS and recIAP were pre-incubated together during a separate procedure for 3 hours before being added to the cell culture (Figure 3). Our results demonstrate that recIAP has an inhibitory effect on TNF-α generated by freshly extracted human leukocytes which occurs in a dose-dependent manner. Specifically when the WBCs were subject to conditions (ii) or (iii), the results showed that relatively similar TNF-α levels were secreted regardless of whether recIAP was added to the WBCs at the same time as LPS (Figure 3A black bar), or recIAP was pre-incubated with LPS for 3 hours separately before being added to the WBCs (Figure 3A gray bar). Because we expect that in condition (iii), the recIAP will neutralize the effect of LPS when they are pre-incubated together in a separate solution, this key result suggests that recIAP is able to inhibit leukocyte secretion of TNF-α independent of LPS presence. Additionally,the rest of the results seen in Figure 3ABC further support the finding that recIAP directly inhibits leukocyte secretion of TNF-α and IL-6 in the absence of LPS.
To determine the minimum effective dose of recIAP, we found that recIAP (2U ml-1) inhibits the secretion of TNF-α by leukocytes both in the presence of LPS (Figure 3B black bar) and in the absence of LPS(Figure 3B white bar). Furthermore, recIAP (2U ml-1) also inhibits the secretion of IL-6 by leukocytes in the presence of LPS (Figure 3C black bar) and in the absence of LPS (Figure 3C white bar,), indicating that recIAP also significantly reduces IL-6 secretion both in the presence or absence of LPS.