Human pulmonary endothelial cell permeability after exposure to LPS-stimulated leukocyte supernatants derived from patients with early sepsis.

Abstract

Systemic immune activation is the hallmark of sepsis, which can result in endothelial injury and the acute respiratory distress syndrome (ARDS). The aim of this study was to investigate heterogeneity in sepsis-mediated endothelial permeability using primary human pulmonary microvascular endothelial cells (HPMECs) and the electric cell-substrate impedance sensing (ECIS) platform. After plasma removal, cellular component of whole blood from 35 intensive care unit (ICU) patients with early sepsis was diluted with media and stimulated with either lipopolysaccharide (LPS) or control media. Resulting supernatants were cocultured with HPMECs seeded on ECIS plates, and resistance was continually measured. A decrease in resistance signified increased permeability. After incubation, HPMECs were detached and cell adhesion proteins were quantified using flow cytometry and immunohistochemistry, and gene expression was analyzed with quantitative PCR. Significant heterogeneity in endothelial permeability after exposure to supernatants of LPS-stimulated leukocytes was identified. ICU patients with sepsis stratified into one of the following three groups: minimal (9/35, 26%), intermediate (18/35, 51%), and maximal (8/35, 23%) permeability. Maximal permeability was associated with increased intercellular adhesion molecule-1 protein and mRNA expression and decreased vascular endothelial-cadherin mRNA expression. These findings indicate that substantial heterogeneity in pulmonary endothelial permeability is induced by supernatants of LPS-stimulated leukocytes derived from patients with early sepsis and provide insights into some of the mechanisms that induce lung vascular injury. In addition, this in vitro model of lung endothelial permeability from LPS-stimulated leukocytes may be a useful method for testing therapeutic agents that could mitigate endothelial injury in early sepsis.