Red blood cells are dynamic reservoirs of cytokines

Abstract

Red blood cells (RBCs) have been shown to affect immune function and can induce inflammatory responses after transfusion. The transfusion of washed RBCs can significantly reduce adverse effects, however, the soluble factors that may mediate these effects have not been identified. Previous studies have identified, but not quantified, a small number of chemokines associated with RBCs. We isolated RBCs from healthy volunteers and quantified of a panel of 48 cytokines, chemokines, and growth factors in the lysate, cytosol, and conditioned media of these cells using Luminex® technology. This analysis revealed that, after correcting for white blood cell and platelet contamination, 46 cytokines were detected in RBC lysates, and the median concentration in RBCs was 12-fold higher than in the plasma. In addition, extensive washing of RBCs, such as that performed in proteomics analyses or prior to some RBC transfusions, significantly attenuated the release of six cytokines following incubation at 37 °C. This supports the hypothesis that, alongside its gas exchange function, RBCs play a role in cytokine signalling. This discovery may help supplement disease biomarker research and may shed light on adverse inflammatory processes that can follow RBC transfusion.

Conflict of interest statement

E.K. was a PhD candidate and scholarship holder with the University of Sydney and is now a shareholder and employee of Sangui Bio Pty Ltd. B.H. is a shareholder and director of Sangui Bio Pty Ltd. E.B. was a paid consultant to Sangui Bio Pty Ltd. The data in this manuscript has also been included in two patent applications (WO/2017/059477 and WO/2017/106899).

Figures

Figure 1

Cytokines in RBC lysate and cytosol. Level of cytokines in the total lysate or cytosolic fraction of RBCs as measured by Bio-Plex and reported as fluorescence. Data are presented as mean with minimum and maximum values (n = 10). Data are significant if p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Figure 2

Incubation of recombinant cytokines with RBCs. Boxplot summaries of cytokine log2 transformed raw fluorescence response data for RBC lysates collected using (a) the Bio-Plex human 27-plex panel and (b) the Bio-Plex human 21-plex panel (n = 5). Cells were incubated for 24 hours (37 °C) with or without recombinant cytokines (BP, and control, respectively). Data are statistically significantly different if p < 0.05 (*), or p < 0.001 (***).

Figure 3

Cytokines in RBC conditioned media. Concentration of cytokines in the conditioned media of RBCs (400 × 108 cells per mL) incubated in PBS for 24 hours at 37 °C as measured by Bio-Plex. Data are presented as mean with minimum and maximum values (n = 10).

Figure 4

Cytokines in cell lysates and conditioned media following cell washing. Concentration of cytokines in the conditioned media of RBCs washed once (control RBCs) or ten times (washed RBCs) and subsequently incubated in PBS (400 × 106 cells per mL) for 24 hours at 37 °C as measured by Bio-Plex. Data are presented as mean with minimum and maximum values (n = 10). Data are significant if p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).