FIGURE 5:

HMGB1 induces TLR4-mediated TNF-α and IL-1β production. MTE cells were preincubated (2 h at 37°C) with an anti-TLR4 monoclonal antibody (20 mg/ml) or an isotype control antibody and then stimulated with human rHMGB1 (100 ng/ml) for 24 h. After stimulation, the culture supernatant and cells were collected. (A, B) TNF-α and IL-1β mRNA expression levels were determined by quantitative real-time PCR, and (C) concentrations of TNF-α and IL-1β in the supernatant were determined using a multiplex cytokine assay. Wild-type mice and TLR4-KO mice received an intravenous injection of 200 μg/kg rHMGB1 or vehicle (PBS) via the tail vein each day (n = 5 for each group) during acute (3 d) exposure to CS. (D) The BAL fluid was obtained and used to measure the differences in the proinflammatory cytokine concentrations. (E) Double immunohistochemical staining for TLR4 and TNF-α or IL-1β in the lung tissues from wild-type mice and TLR4-KO mice. The results are displayed as means ± SD from three independent experiments. *p < 0.05.