High Mobility Group Box 1 (HMGB1) is Central to the Cytomix-Induced Pro-Inflammatory Response in Human Pulmonary Microvascular Endothelial Cells.

Endothelial cell death is an early event in acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). We have previously shown that cytomix (lipopolysaccharide (LPS), interleukin (IL)-1β, interferon-γ, and tumor necrosis factor-α), but not LPS alone, results in robust cell death in human pulmonary microvascular endothelial cells (hPMVEC). We hypothesized that the inflammatory cascade associated with cytomix treatment in hPMVEC would result in the release of HMGB1, a damage-associated molecular pattern (DAMP), which would enhance the pro-inflammatory response. Treatment of hPMVEC with cytomix for 24 hours resulted in a robust (25-fold) induction of HMGB1 protein levels in the media compared to controls. Cytomix treatment of hPMVEC resulted in nearly 5-fold greater cleaved caspase-1 protein levels and nearly 60-fold greater cleaved IL-1β protein levels than in control hPMVEC. Viable hPMVEC numbers were significantly reduced after cytomix treatment. To assess the role of TLR4 in the inflammatory response of hPMVEC following cytomix treatment, we utilized TAK-242, a TLR4 antagonist. The cytomix-induced increase in media HMGB1 levels was significantly attenuated by TAK-242. TAK-242 also significantly attenuated the cytomix-induced increases in cleaved caspase-1 and cleaved IL-1β. To assess the role of caspase 1 in the inflammatory response of hPMVEC following cytomix treatment, we utilized Ac-YVAD-CMK, a caspase-1 antagonist. Treatment with Ac-YVAD-CMK attenuated the cytomix-induced increase in media HMGB1 and cleaved IL-1β; and partially restored viable cell numbers following cytomix treatment. To further understand the role of HMGB1 in the pro-inflammatory process we treated hPMVEC with an HMGB1 antibody; and found that the HMGB1 antibody significantly attenuated the increases in HMGB1 and cleaved IL-1β release, as well as endothelial cell death caused by cytomix treatment. Our results demonstrate that treatment with cytomix results in release of HMGB1, which activates caspase 1 leading to greater HMGB1 production. Furthermore, HMGB1 activation of caspase-1 leads to cleaved IL-1β production as well as hPMVEC death. Preventing the interaction of HMGB1 and TLR4 attenuated HMGB1 and cleaved IL-1β release, and attenuated cytomix-induced reductions in viable cell numbers. We speculate that the release of damage-associated molecular patterns (DAMPs), such as HMGB1, may be crucial in the innate immune response of hPMVECs. These findings may have implications for the pathogenesis of ALI in inflammatory lung diseases.