Sickle RBCs Stimulate Human Monocyte Cytokine Expression.
Authors: Xu Ke , Telen Marilyn J. 
Published: April 2020

Abstract
Sickle red cells (SS RBCs) express numerous adhesion molecules, including LW, CD44, CD47, and Lutheran (LU) proteins, as well as phosphatidylserine. Proadhesive SS RBCs bind to endothelial cells, leading to their activation. Sickle monocytes have also been shown to express increased surface CD11b, as well as the cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), indicating their activated state. Chronic inflammation and high plasma levels of IL-1β can stimulate leukocyte proliferation and leukocytosis. An association between leukocytosis and increased morbidity and mortality of sickle cell disease (SCD) has been observed. However, the mechanism of monocyte activation in SCD is still unknown. We therefore sought to determine if SS RBCs played a role in monocyte activation. Blood samples were obtained from six normal adult volunteers, and SS RBCs were obtained from 12 adult patients homozygous for hemoglobin S. Plasma and buffy coat were removed, and 6 μl of SS RBCs were washed with sterile phosphate-buffered saline. After incubation with one million cells of the human promonocytic cell line U937 for 2 hours at 37°C, RBCs were lysed and total U937 RNA was isolated. Following reverse transcription, real-time PCR was performed using 200 nM primers for IL-1β, IL-6 or TNF-α, 50 ng cDNA and 12.5 μl of SYBR green 2× Super Mix in a 25 μl reaction volume. The amplification reaction was carried out over 40 cycles (10 min at 95°C, followed by a two-step cycling program of 15s at 95°C and 30s at 60°C). GAPDH was analyzed in parallel as an internal control. IL-1β, IL-6 and TNF-α U937 mRNA was significantly up-regulated after incubation with SS RBCs vs incubation with normal RBCs (relative to control without RBCs, relative mRNA levels: IL-1β: 2.77 ± 1.79 vs 0.82 ± 0.19, p=0.012; IL-6: 2.17 ± 0.94 vs 0.74 ± 0.34, p=0.003; and TNF-α: 3.01 ± 1.30 vs 1.09 ± 0.17, p=0.003). To identify the adhesion molecules involved in the interaction between SS RBCs and monocytes, 10 μg/ml soluble recombinant Lutheran (srLU) was incubated with U937 cells at 37°C for 2h. After incubation, total RNA was isolated from untreated and treated U937 cells. Real time PCR results showed that srLU treatment activated U937 cell TNF-α expression, (relative mRNA level 1.63 ± 0.02 vs 1.00 ± 0.01, p&lt;0.0001, n=3) but not IL-1β or IL-6 expression (relative mRNA levels sLU treated vs untreated control, n=3-IL-1β: 1.24 ± 0.09 vs 1.00 ± 0.02, p=0.07; IL-6: 1.35 ± 0.30 vs 1.00 ± 0.01, p = 0.31). Neither soluble LW nor soluble CD44 had a similar effect. To verify that LU was involved in the interaction between SS RBCs and monocytes, inhibition assays were also performed. SS RBCs were incubated with anti-LU Ab for 1 h at 4°C to block RBC surface LU before incubation with U937 cells. Data from five paired treated and untreated samples showed decreased TNF-α mRNA expression after anti-LU antibody treatment (1.16 ± 0.14 vs 1.40 ± 0.12, p=0.005). Overall, these data suggest that monocytes can be activated by cell-cell interactions with SS RBCs to express the cytokines IL-1β, IL-6 and TNF-α. This activation is at least partially mediated through SS RBC Lutheran protein, which is over-expressed by these cells and is known to bind to leukocyte α4β1 integrin. These studies thus reveal that intact SS RBCs likely play a direct role in monocyte activation in SCD patients.