Extracellular Ubiquitin Increases in Packed Red Blood Cell Units During Storage

Abstract

Ubiquitin (Ub) is involved in intracellular protein metabolism, but may also have extracellular roles in host defense and immunomodulation. Erythrocytes contain high amounts of Ub and hemolysis is one potential source of extracellular Ub in vivo. Since hemolysis also occurs with storage of packed RBC units (pRBCs) in vitro, we hypothesized that Ub is released during storage and that it correlates with immunological properties of pRBCs. Daily aliquots were drawn from pRBCs (n = 3) for 42 days and plasma was isolated. Ub was measured by ELISA. Immunomodulatory properties of plasma were assessed by measuring endotoxin-stimulated cytokine (TNF-alpha, IL-6, IL-8) production of normal whole blood, and cell proliferation in phytohemagglutinin-stimulated peripheral blood mononuclear cells. Plasma Ub linearly increased (49 +/- 2 ng/mL/day; r(2) = 0.82, P < 0.001) 20-fold to 2170 +/- 268 ng/mL on day 42. Plasma inhibited TNF-alpha production but stimulated IL-8 production of normal whole blood, which correlated with time-dependent Ub release (TNFalpha: r(spearman) = -0.626, P < 0.001; IL-8: r(spearman) = 0.427, P = 0.004). Addition of exogenous Ub (equaling day 42 concentration) to day 0-4 plasma inhibited TNF-alpha production by one-third of the effect detected for day 42 plasma, but also inhibited IL-8 production by 40%. IL-6 production and cell proliferation was unchanged between day 0-4 plasma with or without Ub supplementation and day 42 plasma. Extracellular Ub release in pRBCs correlates with in vitro immunomodulatory effects and may partially contribute to transfusion-related immune modulation. Additionally, the linear kinetics of the ubiquitin release during pRBC storage suggest Ub is a suitable in vitro quality control parameter.