Paradoxical Effects Of Tumor Necrosis Factor-Alpha On Endothelial Adhesion Of Stored Human Red Blood Cells
Authors: Premkumar Vikram J. , Riccio Daniel A. , Delahunty Martha , Telen Marilyn J. , McMahon Tim J. 
Published: October 2019

Abstract

Introduction
The duration of red blood cell (RBC) storage is currently being examined as a possible determinant of clinical outcomes in transfused patients in large randomized trials in several patient populations, as well as in preclinical models. Biochemical and functional changes coincident with blood storage, referred to as the “storage lesion,” are numerous and complex. Longer storage periods have, for example, been linked to increased tendency of RBCs to adhere to endothelium. How the proadhesive effect of storage influences the transfusion recipient might depend in part on preexisting comorbidities, consistent with a “two-hit” model of transfusion morbidity. We therefore developed a two-hit in vitro model using older stored RBCs and TNF-α pretreatment (activation) of HUVECs in order to better understand mechanisms of post-transfusion complications related to RBC adhesion.

Methods
Red blood cell (RBC) preparation and labeling: Using an IRB-approved protocol, stored packed RBC segments stored in additive solution (AS)-3 were obtained at 35-42 days storage from the Transfusion Service at Duke Hospital. The stored RBCs were removed aseptically and washed 3 times with PBS, labeled with fluorescent dye PKH26, and allowed to incubate for 3 minutes. BSA (1%) in DPBS was added, and after one minute, the labeled cells were washed three times in PBS with Ca++ and Mg++ while avoiding exposure to light. Finally 5 µL of labeled RBCs was suspended in 3 mL of PBS for adhesion assays.
Adhesion assays
Human umbilical vein endothelial cells (HUVECs), grown to confluence on glass slides precoated with a 2% gelatin solution in a 5% CO2 incubator at 37.1°C, were placed in a graduated height-flow chamber. The height was measured at 7 different points along the chamber. After 15 minutes of PBS flow (2 mL min-1), the RBC sample was introduced to the chamber slowly at a rate of 1.5 mL min-1. The RBCs were then allowed to incubate without flow for 5 minutes, and the number of adhered cells at each location (height) was recorded. After 5 minutes of fluidic flow with PBS, the number of adherent cells at each location, corresponding to varying shear stresses, was counted. Percent adhesion and shear stress were calculated at each height.
TNF-α treatment of HUVECs: Where indicated, TNF-α (20 µL) was added to 20 mL of cell media to yield a final concentration of 10 ng mL-1, and allowed to incubate overnight (16 hours).

Results
Adhesion of 35-42-days stored RBCs to non-activated HUVECs varied widely, as reported, and at shear stresses of 1, 2, and 5 dynes/cm2 was 42.92%, 10.77%, and 2.157% respectively, with a typical inverse relation to shear stress (p<0.005). Surprisingly, adhesion of stored RBCs to TNF-α-pretreated HUVECs was consistently lower than that to non-pretreated HUVECs. Mean adhesion of stored RBCs to TNF-α-pretreated HUVECs at 1, 2, and 5 dynes/cm2 was 15.43%, 6.120%, and 1.819%.

Summary
In summary, we find that endothelial adhesion of stored RBCs nearing expiration tends to be inhibited rather than increased by prior treatment of HUVECs with the cytokine TNF-α. Since these stored RBCs had been pre-storage leukoreduced, it is unlikely that there are leukocyte-related confounders. It is possible, therefore, that TNF-α confers a protective effect against RBC adhesion in the microcirculation in the setting of transfusion of older stored RBCs. The mechanism of the paradoxical TNF-α effect is currently under investigation and may involve nitric oxide, which is deficient in stored RBCs. Adverse events related to the RBC storage lesion may depend in a critical and complex manner on the underlying pathophysiology.

Disclosures:
No relevant conflicts of interest to declare.