Evaluation of Sepsis-Related Cytokines on Endothelial Cell Permeability

Abstract

Sepsis elicits a complex immune response in which endothelial cell function is affected by various critical inflammatory mediators and coagulation factors. The endothelial cell lining plays a central role in the pathogenesis of sepsis leading to Severe Inflammatory Response Syndrome (SIRS) and Multiple Organ Failure Syndrome (MOFS). Although diagnostic methods to assess endothelial cell function would be valuable for evaluating the effectiveness of therapeutic interventions designed to repair damaged endothelium in septic patients, no such tools exist at this time. Thus, the overall goal of this research was to characterize vascular endothelial cell monolayer integrity and barrier function changes that may be induced by inflammatory cytokines in vitro. To meet this objective, we recorded dynamic responses of endothelial cell monolayers treated with a selection of sepsis-related cytokines including TNF-, IL-1, IL-6, IL- 8, IL-10, IL-12, IFN-, and TGF- in-vitro using a novel method known as Electric Cell-substrate Impedance Sensing (ECIS). Our results demonstrate that Human Aortic Endothelial Cell (HAoEC) monolayer permeability is significantly increased when treated with TNF-α, IL-1β, IL-6, IL-10, or IFN-γ compared to untreated controls reflected by impedance changes of cell-covered electrodes. The data revealed a significant decrease in normalized impedance (ohm@16kHz) changes at early time-points (<2 hrs.) between untreated control cells compared to: IL1- (p<0.001), IL-6 (p=0.0019), IL-10 (p<0.0001), TNF- (p<0.001), or IFN- (p=0.0288) treatment groups. On the other hand, the response of cells treated with IL-8 (p<0.0001) resulted in a significant increase in the normalized impedance change compared to untreated controls. No significant difference in the normalized impedance at early time-points was observed for the TGF- or IL-12 treated cells. Using the ECIS modeling parameters, significant differences in permeability of cell monolayers treated with sepsis-related cytokines were revealed. A significant reduction in Rb resulting in increased permeability was observed for TNF- (p=0.0112), IL1- (p=0.0196), and IL-10 (p=0.0010) treatment groups. Conversely, the Rb values were significantly increased in the case of IL-6 (p=0.0007), IL-8 (p=0.0016), and TGF- (p<0.0001). No significant difference in Rb was observed for cells treated with IL-12. Significant reductions in Rb values were observed in monolayers treated with cytokines combined. IL-1β combined with TNF-α at ratios of 1:1, 5:1, and 10:1 resulted in significant reduction in Rb compared to mock-treated controls. In the case of IL-12 and IFN- combined, Rb is significantly increased when treated with IL-12:IFN- (10:1) and IL-12:IFN- (5:1) ratios but is significantly decreased when treated with IL-12:IFN- (1:1). Interestingly, Rb of HAoECs is significantly increased when treated with TGF-:TNF- (1:1) (p<0.0001), IL-10:TNF- (1:1) (p<0.0001), IL-10:TNF- (10:1) (p<0.0001), or IL-12:TNF- (5:1) (p<0.0001) compared to TNF- alone. Likewise, preincubation of the anti-TNF-α monoclonal antibody with TNF-α led to loss of cytokine effect and no significant reduction in barrier resistance (Rb) was observed compared to mock treated controls. Results from these in vitro studies provide further evidence to support the translation of new technological approaches from bench-top research tools to clinical applications. More research using the ECIS technology in a clinical setting, using plasma from patients with sepsis, would be of great value to extend these findings and identify early evidence of damage of the endothelial cell integrity of the vasculature and its barrier function. This could potentially lead to more timely treatments and improvement of outcomes in people with sepsis.