Immunosuppressive effects of red blood cells on monocytes are related to both storage time and storage solution

Abstract

Background: Reduced monocyte function is associated with adverse outcomes from critical illness. Red blood cells (RBCs) are thought to impair monocyte function but relationships between RBC storage solution and monocyte suppression are unknown. This study was designed to test the hypothesis that immunosuppressive effects of RBCs on monocytes are related to both storage time and preservative solution.

Study design and methods: Monocytes from healthy adult donors were co-cultured with RBCs that had been stored in AS-1, AS-3, or CPD only for 7, 14, or 21 days. Cells were then stimulated with lipopolysaccharide (LPS) and their supernatants assayed for tumor necrosis factor (TNF)-α and interleukin (IL)-10. Transwell experiments were performed to evaluate the role of cell-to-cell contact. Monocyte mRNA expression was quantified by real-time-polymerase chain reaction.

Results: LPS-induced TNF-α production capacity was reduced compared to controls for all groups, but CPD-only RBCs suppressed monocyte function more than RBCs stored in AS-1 (p = 0.007) and AS-3 (p = 0.006). IL-10 production was preserved or augmented in all groups. A longer storage time was associated with reduced TNF-α production capacity for AS-1 and AS-3 groups but not CPD. Preventing cell-to-cell contact did not eliminate the inhibitory effect of RBCs on monocyte responsiveness. RBC exposure was associated with decreased LPS-induced TNFA mRNA expression (p < 0.05 for all groups).

Conclusions: CPD-only RBCs suppressed monocyte function more than RBCs stored with additive solutions. TNF-α production was reduced even in the absence of cell-to-cell contact and was impaired at the mRNA level. Further work is needed to understand the role of preservative solutions in this process.

Figure 1. Ex vivo LPS-stimulated monocyte cytokine production following exposure to stored PRBC (A,B) or preservative solution alone (C,D)

(A) Monocytes co-cultured with banked PRBC of varying storage times and preservative solutions produced less TNFα upon ex vivo LPS stimulation compared to control (media only) monocytes (p < 0.0001 CPD vs. control, p = 0.02 AS-1 vs. control, p < 0.003 AS-3 vs. control). Impairment of TNFα production was greatest for CPD-preserved compared to AS-1 or AS-3-preserved groups. An effect of storage time was seen for AS-1 and AS-3 groups but not for CPD. (B) Ex vivo LPS-stimulated IL-10 production was not different between PRBC-exposed monocytes and controls. IL-10 production was lower after co-culture with CPD-preserved PRBC compared to AS-1 or AS-3-preserved PRBC. Cytokine production levels by control monocytes (LPS stimulation without PRBC exposure) were as follows: TNFα: 48,525 (±7,832) pg/ml and IL-10: 207.5 (±27) pg/ml. TNFα and IL-10 levels were low (< 50 pg/ml and < 5 pg/ml, respectively) in supernatants prior to LPS stimulation in all groups. (C, D) No differences in ex vivo LPS-induced TNFα (C) or IL-10 (D) production were seen following exposure to preservative solutions alone. Data are expressed as mean (SEM) of percentage of media only controls. * p < 0.05 vs. controls; ** p < 0.01 vs. controls; #p < 0.05 vs CPD.

Figure 2. Ex vivo LPS-stimulated monocyte cytokine production following co-culture with stored PRBC in 0.4 micrometer transwells

Monocytes were co-cultured with PRBC in transwells to prohibit cell-to-cell contact. (A) Exposure to PRBC solution stored in CPD or AS-3 resulted in less monocyte ex vivo LPS-stimulated TNFα production compared to control monocytes (p = 0.003 CPD vs. control, p = 0.02 AS-3 vs. control). TNFα production was lower for CPD-preserved PRBC compared to AS-1-preserved PRBC. (B) Ex vivo LPS-stimulated IL-10 production was higher after exposure to AS-1-preserved PRBC compared to controls and CPD-preserved PRBC. IL-10 production was not different among CPD, AS-3 and controls. Data are expressed as mean (SEM) of percentage of media only controls. * p < 0.05 vs. controls; ** p < 0.01 vs. controls.

Figure 3. Monocyte mRNA expression following co-culture with stored PRBC and stimulation with LPS

(A) LPS-induced expression of mRNA coding for TNFα was decreased in monocytes exposed to PRBC compared to control (media only) monocytes (p = 0.02 CPD vs. control, p = 0.04 AS-1 vs. control, p = 0.03 AS-3 vs. control). (B) LPS-induced expression of IL10 was not different among CPD-preserved groups, AS-1 preserved-groups and controls. IL10 expression tended to be lower after exposure to AS-3-preserved PRBC (p = 0.09 vs. control). Data are expressed as mean (SEM) of relative copy number (RCN). * p < 0.05 vs. controls.