Integrin VLA3 Mediates Endothelial Barrier Damage by Human Sepsis Patient Neutrophils In Vitro
*Chelsey C. Ciambella1, *Catherine M. Dickinson1, David S. Heffernan1, *Minsoo Kim2, William G. Cioffi1, *Jonathan S. Reichner1
1Brown University School Of Medicine, Division Of Surgical Research, Rhode Island Hospital, Providence, RI, Providence, RI;2University Of Rochester, Department Of Microbiology And Immunology, Center For Vaccine Biology And Immunology, Rochester, NY
Objective: Integrin VLA3 is significantly up-regulated in human neutrophils during sepsis. Our aim is to determine the role of VLA3 in an in vitro model of neutrophil-induced damage of endothelial barrier function.
Design: Prospective study.
Setting: Surgical and Trauma intensive care units at Level 1 Trauma Center.
Patients: Septic patients were identified as those with two or more SIRS criteria with a source of infection confirmed by clinical evidence or microbiological data. Trauma patients were those with injuries severe enough to warrant ICU admission. Patients were enrolled within 24 hours of their diagnosis or admission. Blood was collected from patients and healthy volunteers the same day.
Interventions: Neutrophils were isolated by dextran sedimentation, pretreated with anti-VLA3 antibody or isotype control, and allowed to adhere to TNFα-activated human umbilical vein endothelial cell monolayers.
Main Outcome Measure: Electrical cell-substrate impedance sensing was used to quantify real-time barrier disruption after neutrophil adhesion.
Results: Neutrophils from healthy donors and TICU patients did not induce significant differences in barrier function, measured as a decrease in normalized resistance (nR). Neutrophils from sepsis patients, however, induced a significantly loss of barrier function compared to neutrophils from healthy donors (nR= 0.67+/-0.08 vs 0.87+/-0.05 at 120min; p<0.05) or TICU patients (nR= 0.67+/-0.08 vs. 0.83+/-0.06 at 120 min;p<0.05). Neutrophils from sepsis patients that were pre-treated with the VLA3 blocking antibody showed significantly less damage than isotype control (nR= 0.88+/-0.05 vs. 0.68+/-0.07 at 120min; p<0.05).
Conclusions: Functional blocking of integrin VLA3 attenuated the loss of barrier function by septic patient neutrophils supporting the hypothesis that VLA3 mediates barrier dysfunction. Therefore, VLA3 may serve as a therapeutic target in the treatment of endothelial dysfunction in sepsis.
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