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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. [ABSTRACT FROM AUTHOR]

Abstract Background Heparan sulfate (HS) degradation mediates pulmonary endothelial hyper-permeability and acute pulmonary edema during acute respiratory distress syndrome (ARDS). The aim of this study was to examine whether histone H4 induced HS degradation by activating heparanase (HPSE) in chlorine gas (Cl2)-induced ARDS. Methods Acute lung injury was induced by Cl2 exposure or histone H4 injection in C57BL/6 mice. Histone H4 in bronchoalveolar lavage fluid (BALF) and plasma was measured by ELISA. HS degradation was measured by immunostaining, ELISA, and flow cytometry. HPSE mRNA and protein were measured by real-time qPCR and western blot analysis, respectively, at preset timepoints. The HPSE inhibitor OGT2115 and specific siRNAs were used to study the role of HPSE during HS degradation caused by Cl2 exposure or histone H4 challenge. Blocking antibodies against TLR1, TLR2, TLR4, or TLR6 were used in vitro to investigate which signaling pathway was involved. The transcriptional regulation of HPSE was studied vis-à-vis NF-κB, which was assessed by nuclear translocation of NF-κB p65 and phosphorylation of I-κBα protein. Results Histone H4 in BALF and plasma increased evidently after Cl2 inhalation. Cl2 exposure or histone H4 challenge caused obvious acute lung injury in mice, and the pulmonary glycocalyx was degraded evidently as observed from endothelial HS staining and measurement of plasma HS fragments. Pretreatment with OGT2115, an HPSE inhibitor, relieved the acute lung injury and HS degradation caused by Cl2 exposure or histone H4 challenge. Targeted knockdown of HPSE by RNA interference (RNAi) significantly inhibited histone H4 induced HS degradation in HPMECs, as measured by immunofluorescence and flow cytometry. By inducing phosphorylation of I-κB α and nuclear translocation of NF-κB p65, histone H4 directly promoted mRNA transcription and protein expression of HPSE in a dose-dependent manner. Additionally, a blocking antibody against TLR4 markedly inhibited both activation of NF-κB and expression of HPSE induced by histone H4. Conclusions Histone H4 is a major pro-inflammatory mediator in Cl2-induced ARDS in mice, and induces HS degradation by activating HPSE via TLRs- and NF-κB-signaling pathways.