Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice were washed 3 times with serum-free medium and then cultured overnight with HMGB1 at different concentrations alone or with mouse hemoglobin (30 μg/ml) (A, B), or HMGB1 at 4 μg/ml with mouse hemoglobin at different concentrations (C,D). Concentrations of TNF (A, C) or IL-6 (B, D) in the supernatants were determined by ELISA. The results represent mean ± s.e. and are representative of more than three independent experiments. * P < .05, ** P < .01, compared between cells treated with and without hemoglobin.