C. M. Dickinson1, X. O’Brien1, D. S. Heffernan1, M. Kim2, W. G. Cioffi1, J. Reichner1 1Brown University School Of Medicine,Division Of Surgical Research, Rhode Island Hospital, Affiliated With Warren Alpert Medical School,Providence, RI, USA 2University Of Rochester,Department Of Microbiology And Immunology, Center For Vaccine Biology And Immunology,Rochester, NY, USA
Introduction: Critical illness induces endothelial dysfunction mediated in part by neutrophil-to-endothelial interactions. Integrins play a major role in the neutrophil-endothelial interface. It was recently reported that the integrin VLA3 (α 3β 1, CD49c/CD29) is significantly up-regulated on the surface of human neutrophils during an infectious cause of inflammation (sepsis), but not in sterile inflammation (SIRS only). Here we sought to determine the role of VLA3 in an in vitro model of neutrophil-induced damage of endothelial barrier function.
Methods: We used TNFα-activated human umbilical vein endothelial cell (HUVEC) monolayers and Electrical Cell-substrate Impedance Sensing (ECIS) to quantify in real-time barrier disruption after neutrophil adhesion. Blood was obtained from healthy volunteers and patients with either sepsis (Surgical ICU) or sterile trauma inflammation (TICU). APACHE II scores were calculated on patients at the time of blood draw. We used HUVEC monolayers grown on fibronectin (a VLA3 ligand) to determine the effect of a function blocking anti-VLA3 antibody (MAb 1992, clone MKID-2) on endothelial barrier dysfunction. Neutrophils from all subjects were isolated by dextran sedimentation, pretreated with anti-VLA3 antibody or isotype control, and allowed adhere to monolayers with and without fMLP stimulation at 30 mins.
Results:Blood was obtained from critically ill TICU patients (n=6) and SICU septic patients (n=5). Healthy donors (n=8) served as controls. Comparing TICU to Septic patients, there was no difference in age (73+/-5.6 vs 56+/-6;p=0.07) or WBC (16.3+/-2.5 vs 16.5+/-1.4;p=0.9). Septic patients had higher APACHEII scores (17.4+/-2.1 vs 5.8+/-0.6;p<0.001). 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 exaggerated loss of barrier function across conditions when 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), with barrier function comparable to healthy donors (nR= 0.88+/-0.05 vs. 0.88+/-0.05 at 120min).
Conclusion: The integrin VLA3 is specifically up-regulated on neutrophils in septic patients.Pretreatment with a function blocking anti-VLA3 antibody was able to attenuate the loss of barrier function to levels indistinguishable from that induced by healthy donors, consistent with the hypothesis that VLA3 mediates the rapid and more pronounced loss of barrier function caused by neutrophils from septic patients. Therefore, VLA3 is a therapeutic target in the treatment of endothelial dysfunction in sepsis warranting further investigation.