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.