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A2891 - Red Blood Cells Attenuate while Its Supernatant Aggravates Inflammatory Response of Pulmonary Artery Endothelial Cells
Author Block: J. Kim1, T. Nguyen1, M. A. Mazzeffi2, K. A. Tanaka2, A. Birukova1, K. G. Birukov2; 1Department of Medicine, University of Maryland, Baltimore, MD, United States, 2Department of Anesthesiology, University of Maryland, Baltimore, MD, United States.
RATIONALE Transfusion of red blood cells (RBC) is a common procedure. However, in certain cases it may lead to development of transfusion related acute lung injury (TRALI) characterized by increased pulmonary vascular leak, lung edema, and inflammation. Numerous studies suggest that old-stored RBC possesses a higher TRALI risk compared with fresh RBC. However, the mechanisms of TRALI and factors defining deleterious effects of old stored RBC lesions remain unclear. We evaluated an impact of washed RBC and supernatant of fresh (less than 2 weeks) and old stored (4~8 weeks) RBC preparations on the human pulmonary endothelial cell (EC) barrier regulation and inflammatory activation. METHODS Pulmonary EC were challenged with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or heat-killed Staphylococcus aureus (HKSA) followed by challenge with washed RBC or RBC supernatants. EC permeability was evaluated by changes in trans-endothelial electrical resistance (TER) monitored by electric cell-substrate impedance sensing (ECIS) system or express permeability assay (XPerT) for macromolecules. EC conditioned media was assessed for IL-8 and sICAM-1 using ELISA, and EC actin remodeling and marker protein expression was monitored by Western blot and immunofluorescence staining. RESULTS Washed RBC decreased, while RBC supernatant increased permeability of pulmonary EC monolayers monitored by TER and XPerT assay. RBC also attenuated EC barrier dysfunction caused by LPS, TNFα or HKSA. In XPerT assay, RBC attenuated permeability increase caused by TNFα and HKSA, while supernatant aggravated effects of inflammatory agonists. RBC supernatant increased actin stress fiber formation, disruption of cell-cell junctions and augmented EC-induced production of IL-8 and sICAM-1 expression of adhesion molecules ICAM-1 and VCAM-1 by stimulated EC. Interestingly, protective effects by washed old-stored RBC were significantly attenuated in comparison to washed fresh RBC. Barrier disruptive and pro-inflammatory effects of RBC supernatants increased with duration of RBC storage. CONCLUSIONS RBC exhibits protective effect on pulmonary EC, while RBC supernatants possess barrier-disruptive and pro-inflammatory properties. RBC storage reduces protective effects of RBC and augments deleterious effects of RBC supernatants. Further studies are warranted to dissect specific mediators and cellular mechanisms defining storage-dependent effects of stored RBC and their supernatants on pulmonary EC barrier.