Sepsis‐Associated Elastase and Proteinase 3 Induce Endothelial Permeability

Sepsis is a systemic inflammatory response to known or suspected infection, and associated with microvascular dysfunction and capillary leak. The cause of capillary leak may be due to substances released by activated polymorphonuclear neutrophils (PMNs) that adhere to microvascular endothelial cells. Following binding, PMN often degranulate releasing azurophilic enzymes ( e.g. elastase (HLE) and proteinase 3 (PR3)) at the endothelial surface and into plasma. We hypothesized that the actions of these enzymes, particularly HLE and PR3 contribute to the loss of endothelial integrity. Patients with severe sepsis (sepsis with evidence of at least one organ dysfunctioning), were recruited on admission to the intensive care unit. Patient samples were paired with age and sex‐matched controls. The mean age was 54 ± 12 and 52 ± 11 years for sepsis and controls respectively while both groups had 6/15 males. Septic patient pathogens were determined to be 4 gram positive, 7 gram negative, 1 fungal and 3 culture negative. We employed ELISAs to determine plasma HLE and PR3 concentrations and in vitro assays using human umbilical vein endothelial cells (HUVEC) treated with HLE or PR3. HUVEC monolayer resistance and Texas Red‐Dextran flow‐through were used to determine the enzyme's effect on monolayer integrity. The sepsis plasma concentrations were significantly higher (p < 0.0001) compared to healthy volunteers in both HLE (224 ± 66.0 vs 13.9 ± 0.899 ng/mL) and PR3 (52.2 ± 7.63 vs 8.02 ± 2.18 ng/mL). For in vitro assays, we employed concentrations of HLE and PR3 higher than the plasma measurements in order to more closely mimic conditions at the site of degranulation. Treatment with 1, 2 or 5 μg/mL of either enzyme significantly decreased HUVEC monolayer resistance over the course of 90 minutes compared to control. Further, 1 hour of HLE or PR3 treatment significantly increased HUVEC permeability to Texas Red‐Dextran compared to controls. The loss of endothelial integrity secondary to azurophilic enzyme release by adhered PMNs could result in microvascular dysfunction and capillary leak. Endothelial injury could also exacerbate further PMN recruitment. We conclude that neutrophil degranulation, as evidenced by increased plasma concentrations, can lead to increased endothelial permeability through a mechanism which in part involves the actions of HLE and PR3. Support or Funding Information Funded by the Lawson Health Research Institute Internal Research Fund.

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