Acid sphingomyelinase is activated in sickle cell erythrocytes and contributes to inflammatory microparticle generation in SCD

Abstract

Sphingolipids are a class of lipids containing a backbone of sphingoid bases that can be produced de novo through the reaction of palmitate and serine and further metabolized through the activity of various enzymes to produce intermediates with diverse roles in cellular processes and signal transduction. One of these intermediates, sphingosine 1-phosphate (S1P), is stored at high concentrations (1 μM) in red blood cells (RBCs) and directs a wide array of cellular processes mediated by 5 known G-protein coupled receptors (S1P1-S1P5). In this study, we show that RBC membrane alterations in sickle cell disease enhance the activation acid sphingomyelinase by 13%, resulting in increased production and storage of sphingosine (2.6-fold) and S1P (3.5-fold). We also show that acid sphingomyelinase enhances RBC-derived microparticle (MP) generation. These MPs are internalized by myeloid cells and promote proinflammatory cytokine secretion and endothelial cell adhesion, suggesting that potential crosstalk between circulating inflammatory cells and MPs may contribute to the inflammation-rooted pathogenesis of the disease. Treatment with amitriptyline reduces MP generation in vitro and in vivo and might be used to mitigate inflammatory processes in sickle cell disease.

Figure 1

Sphingolipid metabolism. De novo sphingolipid production starts with the reaction of serine and palmitoyl CoA, mediated by serine palmitoyl transferase. Sphingolipids are metabolized in many reversible and irreversible reactions. Ceramide is at the center of the sphingolipid metabolism network and can result from the hydrolysis of SM, which in turn results in the production of sphingosine and S1P downstream.

Figure 2

SMase activity in SCD. (A) Neutral SMase activity in plasma and (B) RBCs. (C) Acid SMase activity in plasma and (D) RBCs. (E) Acid SMase expression in plasma and RBCs. (F) Acid SMase activity in high- and low-density RBCs (labeled “high ρ” and “low ρ, ” respectively), and (G) representative SEM images of RBCs. (H) Acid SMase activity in whole blood of AS/SS mice immediately after 8 hours normoxic/hypoxic conditioning. *P < .05 measured with an analysis of variance (ANOVA) relative to AA or AS normoxia.

Figure 3

Alkaline CDase, SK, Sph, and S1P in SCD. (A) ACER1 expression in RBCs and (B) plasma. (C-D) SK1 and SK2 expression from equal amounts of RBC protein. (E-F) SK2 levels from equal amounts of plasma protein. (G-I) Sphingosine and S1P levels in whole blood (G), plasma (H), and RBCs (I). *P < .05 measured with a Student t test relative to AA.

Figure 4

RBC-derived MPs and monocyte crosstalk. (A) Transmission electron microscopy images of P2 (10 000g), P3 (37 000g), and P4 (200 000g) MPs. Scale bar = 200 nm. (B) Internalization or CFSE-labeled P3 MPs by PMA-treated THP-1s. Scale bar = 10 μm. (C) Ceramide (Cer) and SM quantification with HPLC-MS. (D) Sphingosine and S1P quantification in RBCs and MPs. (E) Fold change over vehicle in cytokine production, and (F) secretion of PMA-treated THP-1s after non-SCD microparticle (AAMP) and SCD microparticle (SSMP) incubation relative to untreated cells (dotted line). (G) Fold change over vehicle in cytokine production, and (H) secretion of primary AA monocytes after SSMP incubation relative to untreated cells. *P < .05 measured in ANOVA relative to vehicle or AA.

Figure 5

Elevated monocyte adhesion in SCD. (A-B) Monocyte-adhesion after 1 hour with 1 μM S1P, AA plasma, or SS plasma treatment of AA PBMCs. Representative fluorescent images showing adherent DRAQ5 labeled THP-1 cells after 4 hours of adhesion to HUVECs (B). (C-D) RBCs co-incubated with THP-1 monocytes (10:1) for 18 hours. Representative confocal images (C) and monocyte:HUVEC ratio after 4-hour adhesion (D). *P < .05 measured with 1-way ANOVA or Student t test relative to vehicle or AA.

Figure 6

SS RBC-derived MPs enhance endothelial adhesion, express LW, and express more p-ERK1/2 than AA RBC-derived MPs. (A) RBC-derived MPs were incubated with primary AA PBMCs at 3 different concentrations (low, medium, and high) for 18 hours before HUVEC adhesion. Representative fluorescent images show adherent DRAQ5-labeled THP-1 cells after 4 hours of adhesion to HUVECs. (B) AAMPs and SSMPs express surface ICAM-4 (LW). (C-D) p-ERK1/2 is significantly elevated in SSMPs relative to AAMPs (n = 6). P < .05 measured in ANOVA relative to vehicle or AAMPs.

Figure 7

Amitriptyline reduces MP generation. (A-B) Human SS RBCs were incubated in PBS with or without amitriptyline for 1 or 24 hours. Flow cytometry plots of MPs and RBCs 1 hour (A) and 24 hours (B) after amitriptyline treatment. (C) Quantification of MP percentage after treatment. (D) Acid SMase activity in mice after amitriptyline injection. (E) Gating strategy for identifying MPs and EMPs in vivo in WT mice. (F) Quantification of MP production in WT mice after normoxic/hypoxic conditioning with or without amitriptyline. *P < .05 compared with vehicle, and P < .05 compared with 1 μM.