Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome

Abstract

Background: During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS.

Methods: ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera.

Results: The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating neutrophils. Both inhibiting the endothelial activation with an anti-toll like receptor (TLR) antibody and inhibiting the interaction of the endothelium with neutrophil using an anti-P-selectin antibody decreased the degree of neutrophil activation.

Conclusions: Extracellular histones are pro-inflammatory mediators in LPS-induced ARDS in mice. In addition to direct action to neutrophils, extracellular histones promote neutrophil adhesion and subsequent activation by first activating the pulmonary endothelium via TLR signaling. Thus, endothelial activation is important for extracellular histone-induced inflammatory injury.

Keywords: Acute respiratory distress syndrome; Endothelium; Extracellular histones; Inflammation; Neutrophil.

Fig. 1

Role of extracellular histones in endothelial and neutrophil activation in mice with ARDS. Mice were challenged with intravenous LPS (10 mg/kg, 24 h) or CTH (40 mg/kg, 6 h). Anti-H4 antibody (20 mg/kg) or non-specific mouse IgG (20 mg/kg) was injected intravenously once 30 min prior to LPS injection. The levels of circulating vWF and sTM were measured by ELISA (a, b). The translocation of P-selectin was measured by immunohistochemical detection (c, d). Neutrophil infiltration in the lungs was confirmed by immunohistochemical analysis of the specific marker Ly6G and neutrophil activation was examined by MPO activity (e, f). Data are presented as mean ± SD (n = 6). The immunohistochemical results are representative of three similar experiments. *p < 0.05 vs. the control group, ** p < 0.01 vs. the control group; ^# p < 0.05 vs. the LPS group, ^## p < 0.01 vs. the LPS group

Fig. 2

Effect of extracellular histones on endothelial activation in vitro. The MLVECs were challenged with LPS and then histone H4 in supernatant was measured by Western blot (a). After 10 min of incubation the binding of extracellular histones to the unchallenged endothelial cell membrane was examined by confocal laser microscopy (b). The MLVECs were treated with extracellular histones (1 h) and then P-selectin on endothelium was quantified by cell surface ELISA (c). The vWF in the supernatant was measured by ELISA (d). MLVECs were exposed to CTH and treated concurrently with anti-TLR2 or anti-TLR4 antibody. The inhibitory effect on endothelial activation was measured by P-selectin translocation (e) and release of vWF (f). Data are presented as mean ± SD (n = 6). The results of Western blot and confocal laser microscopy are representative of three similar experiments. *p < 0.05 vs. the control group, ** p < 0.01 vs. the control group; ^# p < 0.05 vs. the histones (40 mg/L) group, ^## p < 0.01 vs. the histones (40 mg/L) group

Fig. 3

Effect of histone-induced endothelial activation on neutrophil activation. Neutrophils were challenged by CTH (40 mg/L, 1 h), then exposed to unchallenged MLVECs. The anti-H4 antibody or non-specific IgG was given concurrently with CTH. The relative percentage of neutrophil adhesion to MLVECs was determined by a cell surface adhesion assay under static conditions (a) and the MPO activity in the supernatant was measured by ELISA (b). MLVECs were challenged by CTH (40 mg/L, 1 h), then exposed to unchallenged Neutrophils. The relative percentage of neutrophil adhesion to MLVECs (c) and the MPO activity in the supernatant (d) were measured. Both MLVECs and neutrophils were challenged by CTH (40 mg/L, 1 h), then exposed to each other. The relative percentage of neutrophil adhesion to MLVECs (e) and the MPO activity in the supernatant (f) were measured. Data are presented as mean ± SD (n = 6). *P < 0.05 vs. the control group, **P < 0.01 vs. the control group; ^# p < 0.05 vs. the injury group, ^## p < 0.01 vs. the injury group

Fig. 4

Roles of TLR signaling and P-selectin in endothelium-mediated neutrophil activation. MLVECs and neutrophils were challenged by CTH (40 mg/L, 1 h) and treated concurrently with anti-TLR2, anti-TLR4, anti-P-selectin antibody or non-specific IgG. The relative percentage of neutrophil adhesion to MLVECs (a, c) and the MPO activity in the cell supernatant (b, d) were measured. Data are mean ± SD of at least 6 experiments. *P < 0.05 vs. the control group, **P < 0.01 vs. the control group; ^# p < 0.05 vs. the injury group, ^## p < 0.01 vs. the injury group