Figure 5

Exogenous 'IL-32α induced' TNFα production by RAW 264.7 cells through activation of NF-κB and ERK1/2 MAPK. Two different concentrations of rIL-32α or LPS were added to RAW 264.7 cells in culture, followed by incubation for 24 hours. The level of TNFα in culture media was measured by a specific enzyme-linked immunosorbent assay (a). IL-32α alone as well as LPS was capable of inducing TNFα secretion into culture media by RAW 264.7 cells. Values are expressed as ± standard deviation (SD) of four determinations. RAW 264.7 cells were cultured with or without rIL-32α for 24 hours in combination with inhibitors for IκB and MAPKs, including DHMEQ, U0126, SB203580, and SP600125 (b). IL-32α-induced TNFα production was inhibited by DHMEQ or U0126 but not by SB203580 or SP600125 (*P < 0.05, **P < 0.01). Values are expressed as ± SD of four determinations. Phosphorylations of IκB and MAPKs in RAW 264.7 cells were determined by Western blotting by using anti-phospho-IκB, -ERK1/2, -p38, or -JNK antibodies after treatment with rIL-32α (100 ng/mL) (c). rIL-32α stimulated phosphorylation of IκB and ERK1/2, starting at 30 minutes and peaking at 90 (ERK1/2) or 120 (IκB) minutes, whereas significant phosphorylation was not observed in p38 or JNK. Degradation of IκB was also observed with a peak at 90 minutes. These data represent one of three independent experiments. DHMEQ, dehydroxymethylepoxyquinomicin; DMSO, dimethyl sulfoxide; IκB, inhibitor kappa B; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; rIL-32α, recombinant human interleukin-32α protein; TNFα, tumor necrosis factor-alpha.