Mechanisms of Toll‐like Receptor (TLR) 4 Mediated Pro‐inflammatory Cytokine Production in Human Pulmonary Microvascular Endothelial Cells

Abstract

Endothelial cell apoptosis is an early event in acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). An early pathogenic feature of ALI is inflammation‐induced endothelial cell death. 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) that interacts with TLR4, and thereby enhances TLR4 mediated production of pro‐inflammatory cytokines. Treatment of hPMVEC with cytomix for 24 hours resulted in a robust induction (~80‐fold, p<0.005) of HMGB1 protein levels in the media compared to controls. While, there was only a small and statistically non‐significant increase in the HMGB1 protein levels in the cells following cytomix treatment compared with controls. To assess the role of TLR4 in the inflammatory response of hPMVEC following cytomix treatment, we utilized TAK‐242, a TLR4 antagonist. Cytomix treatment of hPMVEC resulted in nearly 5‐fold greater cleaved caspase‐1 protein levels than in control hPMVEC. This cytomix‐induced increase in cleaved caspase‐1 was prevented by the addition of TAK‐242 to the media. Cytomix treatment of hPMVEC also resulted in a robust induction of both pro‐IL‐1β and cleaved IL‐1β protein levels compared to controls. However, the cytomix‐induced pro‐IL‐1β and cleaved IL‐1β protein levels were significantly attenuated when the hPMVEC were also treated with TAK‐242. Our results demonstrate that treatment with cytomix results in substantial release of HMGB1. Furthermore, cytomix treatment results in robust production of the TLR4‐mediated pro‐inflammatory cytokine IL‐1β. 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.