Figure 4
High-mobility group box 1 (HMGB1) increases interleukin (IL)-8 secretion in normal human nasal epithelium (NHNE) cells grown under hypoxic conditions. (a) NHNE cells were incubated under hypoxic condition with/without N-acetyl cysteine (NAC) pretreatment. The amount of interleukin (IL)-8 secreted into apical culture medium was quantified by enzyme-linked immnunosorbent assay (ELISA). (b) NHNE cells were incubated with mammalian recombinant HMGB1 (rHMGB1) protein (4 μg ml−1) for 24 h. Amounts of IL-8 in the apical supernatants produced by NHNE cells were determined by ELISA assay. Flagellin (100 ng ml−1) was used as a positive inducer of IL-8. (c) NHNE cells were incubated under hypoxic conditions for 8 h and then the apical culture medium was collected. HMGB1 protein that had been secreted into the apical culture medium was precipitated and depleted using anti-HMGB1 antibody. HMGB1-precipitated culture supernatants were separated and a western blot assay was performed to confirm the precipitation of secreted HMGB1 protein. (d) NHNE cells were incubated under hypoxic conditions for 8 h and apical culture medium, which might contain secreted HMGB1 protein, was collected. This culture supernatant was precleared using protein G-Sepharose or serially precleared and precipitated using anti-HMGB1 antibody, to remove secreted HMGB1 protein. Supernatants that were only precleared or that had had HMGB1 removed were applied into NHNE cells for 24 h. Next, IL-8 produced in the apical supernatants by NHNE cells was determined by ELISA assay. (e) NHNE cells were incubated under hypoxic conditions with or without anti-HMGB1 blocking antibody for 24 h. Apical culture medium was collected and IL-8 produced by NHNE cells was determined using ELISA assay. For all panels, results are shown as mean±s.e.m. (N=3). *P<0.05.
