Neutrophils from critically ill septic patients mediate profound loss of endothelial barrier integrity

Introduction: Sepsis is characterized by systemic immune activation and neutrophil-mediated endothelial barrier integrity compromise, contributing to end-organ dysfunction. Studies evaluating endothelial barrier dysfunction induced by neutrophils from septic patients are lacking, despite its clinical significance. We hypothesized that septic neutrophils would cause characteristic patterns of endothelial barrier dysfunction, distinct from experimental stimulation of normal neutrophils, and that treatment with the immunomodulatory drug β-glucan would attenuate this effect.Methods: Blood was obtained from critically ill septic patients. Patients were either general surgery patients (Primary Sepsis (PS)) or those with sepsis following trauma (Secondary Sepsis (SS)). Those with acute respiratory distress syndrome (ARDS) were identified. Healthy volunteers served as controls. Neutrophils were purified and aliquots were untreated, or treated with fMLP or β-glucan. Endothelial cells were grown to confluence and activated with tissue necrosis factor (TNF)-α Electric Cell-substrate Impedance Sensing (ECIS) was used to determine monolayer resistance after neutrophils were added. Groups were analyzed by two-way analysis of variance (ANOVA).Results: Neutrophils from all septic patients, as well as fMLP-normal neutrophils, reduced endothelial barrier integrity to a greater extent than untreated normal neutrophils (normalized resistance of cells from septic patients at 30 mins = 0.90 ± 0.04; at 60 mins = 0.73 ± 0.6 and at 180 mins = 0.56 ± 0.05; p < 0.05 vs normal). Compared to untreated PS neutrophils, fMLP-treated PS neutrophils caused further loss of barrier function at all time points; no additive effect was noted in stimulation of SS neutrophils beyond 30 min. Neutrophils from ARDS patients caused greater loss of barrier integrity than those from non-ARDS patients, despite similarities in age, sex, septic source, and neutrophil count. Neutrophils obtained after resolution of sepsis caused less barrier dysfunction at all time points. β-glucan treatment of septic patients' neutrophils attenuated barrier compromise, rendering the effect similar to that induced by neutrophils obtained once sepsis had resolved.Conclusions: Neutrophils from septic patients exert dramatic compromise of endothelial barrier integrity. This pattern is mimicked by experimental activation of healthy neutrophils. The effect of septic neutrophils on the endothelium depends upon the initial inflammatory event, correlates with organ dysfunction and resolution of sepsis, and is ameliorated by β-glucan.