Regular Article

From www.bloodjournal.org by guest on March 12, 2017. For personal use only.

Regular Article RED CELLS, IRON, AND ERYTHROPOIESIS

Acid sphingomyelinase is activated in sickle cell erythrocytes and contributes to inflammatory microparticle generation in SCD Anthony O. Awojoodu,1 Philip M. Keegan,1 Alicia R. Lane,2 Yuying Zhang,1 Kevin R. Lynch,3 Manu O. Platt,1 and Edward A. Botchwey1 1

Department of Biomedical Engineering and 2Department of Biology, Georgia Institute of Technology, Atlanta, GA; and 3Department of Pharmacology, The University of Virginia, Charlottesville, VA

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 • Alterations in RBC in cellular processes and signal transduction. One of these intermediates, sphingosine membranes contribute to 1-phosphate (S1P), is stored at high concentrations (1 mM) in red blood cells (RBCs) dysregulated sphingolipid and directs a wide array of cellular processes mediated by 5 known G-protein coupled metabolism in sickle cell receptors (S1P1-S1P5). In this study, we show that RBC membrane alterations in sickle cell disease (SCD). disease enhance the activation acid sphingomyelinase by 13%, resulting in increased • Increased RBC-derived production and storage of sphingosine (2.6-fold) and S1P (3.5-fold). We also show that MP production enhances acid sphingomyelinase enhances RBC-derived microparticle (MP) generation. These monocyte adhesion and MPs are internalized by myeloid cells and promote proinflammatory cytokine secretion activation in SCD. 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. (Blood. 2014;124(12):1941-1950)

Key Points

Introduction Sickle cell disease (SCD) is a hereditary blood disorder characterized by mutated hemoglobin molecules, which polymerize during deoxygenation to form fibers that deform the erythrocyte membrane. These red blood cells (RBCs) contribute to significant pathology including stroke, avascular necrosis, and vasculopathy.1 Because the sickling of the RBC membrane is pathognomonic in SCD, it is important to understand how modifications in membrane composition play a role in the disease. Sphingolipids are a class of lipids containing a backbone of sphingoid bases. They play diverse roles in cellular processes and are significant components of cell membranes.2 Sphingomyelin (SM), which comprises 10% of the mammalian plasma membrane, is degraded by the hydrolyzing enzyme sphingomyelinase (SMase). In recent studies, membrane curvature and associated increases in mechanical bending stresses in RBCs, activated SMase, thereby reducing SM and increasing ceramide.3 Production of ceramide has a direct effect on a wide range of cellular processes and alters production of other immediately descendent metabolites such as sphingosine and sphingosine 1-phosphate (S1P), which are key regulators of inflammation.4-8 Furthermore, SMase has been implicated in lipid microdomain formation, membrane fragility, vesiculation, and MP formation. 9,10 Although the orientation of membrane lipids in SCD and other hemolytic anemias has been

studied, the consequences of altered distribution and metabolism of sphingolipids have been largely unexplored. Sickle RBCs are a particularly interesting model system for the relationship between membrane stresses, inflammation, and sphingolipid metabolism. Acid SMase is secreted from endothelial and myeloid cells,11 and erythrocyte clearance and chronic inflammation, both of which occur in SCD and enhance the secretion of acid SMase.9,12-14 The role of acid SMase, and sphingolipid dysregulation more generally, has not been studied in the context of SCD. In this study, we hypothesized that dynamic shape distortion of RBCs and the accompanying increases in RBC membrane stresses enhance the activity of acid SMase in the blood, plasma, and RBCs of people living with SCD. This results in a significant increase in sphingosine and S1P in RBCs, as well as the generation of RBCderived MPs (Figure 1 and see supplemental Figure 1 on the Blood Web site). These factors enhance monocyte adhesion to endothelial cells and inflammatory cytokine production. Pharmacologic inhibition of acid SMase with a Food and Drug Administration-approved drug, amitriptyline, reduces MP generation and cytokine production. These results elucidate a novel mechanism for MP-mediated inflammatory cell activation in SCD.15 Additionally, these findings may be relevant to other hemolytic anemias and disorders rooted in aberrant RBC morphology.

Submitted January 10, 2014; accepted July 6, 2014. Prepublished online as Blood First Edition paper, July 29, 2014; DOI 10.1182/blood-2014-01-543652.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. © 2014 by The American Society of Hematology

BLOOD, 18 SEPTEMBER 2014 x VOLUME 124, NUMBER 12

1941

From www.bloodjournal.org by guest on March 12, 2017. For personal use only. 1942


AWOJOODU et al

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.

Methods RBC and MP isolation and fractionation Whole blood samples from donors homozygous for sickle (SS) or normal (AA) hemoglobin were centrifuged against a Ficoll-Paque density gradient (density: 1.077 g/mL; GE Healthcare) for 30 minutes at 400 relative centrifugal force (RCF) at 4°C to separate plasma, peripheral blood mononuclear cells (PBMCs), and packed RBCs (supplemental Figure 2). RBCs were separated into high- and low-density fractions using stacked Percoll solutions centrifuged at 41 000 rpm for 30 minutes using a Beckman SW 41 Ti Rotor.16 RBC-derived MPs were obtained by ultracentrifugation of packed RBCs at 10 000 (P2), 37 000 (P3), or 200 000 (P4) RCF for 1 hour at 4°C. Flow cytometry for MP quantification Whole blood, cultured RBCs, or isolated MPs were incubated with antibodies against glycophorin A (catalog #Ab91163, Abcam) and Annexin V (catalog #640906, BioLegend). Events were counted with AccuCheck counting beads (catalog #PCB100, Life Technologies) and analyzed on a BD FACSAria flow cytometer. Protein and enzyme quantification Enzyme expression and activation were quantified using the following kits: SMase activity (catalog #10006964, Cayman Chemical Company); Acid SMase expression (catalog #SEB360Hu, USCN Life Science); alkaline ceramidase (ACER1) expression (catalog #CSB-EL001151HU and CUSABIO); sphingosine kinase 1 and 2 (SK1/2) western blot (catalog #1000-6822, Cayman Chemical Company and catalog #AB37977, Abcam); intercellular adhesion molecule (ICAM)-4 antibody (catalog #ABIN901654, Antibodies Online); and total and phospho-extracellular signal-regulated kinase (p-ERK1/2) western blot (catalog #9102 and #9101, Cell Signaling). Western blot measurements were standardized by loading an equal volume of protein. An equal volume of blood was used for activity assays. Lipid extraction and quantification Lipids were extracted following a protocol from Shaner et al,17 and analyzed using a Shimadzu LC-10 AD VP binary pump system coupled to a PerkinElmer Series 200 autoinjector coupled to a 4000 quadrupole linear-ion trap (QTRAP) LC-MS/MS system. Monocyte differentiation and MP internalization THP-1 monocytes were seeded at 2 3 105 cells/well on poly-d-lysine–coated coverslips and treated with 100 nM phorbol myristate acetate (PMA) for

72 hours. Primary AA PBMCs were seeded at 1 3 106 cells/mL in 12-well plates in media supplemented with 20 ng/mL macrophage colony stimulating factor (Peprotech) for 24 hours. MPs were isolated from packed RBCs and incubated 1:1 with 1 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Abcam) for 20 minutes at room temperature, followed by ultracentrifugation at 37 000 RCF for 20 minutes at 4°C. The MP pellet was resuspended in phosphate-buffered saline (PBS) and seeded at 1 3 106 particles/well with THP-1 or primary isolated monocytes. After 30 minutes, 2 hours, or 24 hours, conditioned media was collected and the cells were washed three times with PBS before imaging or cell lysis with radioimmunoprecipitation assay buffer. Proteins were assessed for cytokines using a Milliplex MAP Human Cytokine/ Chemokine Premixed 42 Plex Assay (catalog #MPXHCYTO60KPMX42, Millipore) on the Luminex platform. Monocyte adhesion studies GFP-HUVECs (angioproteomie) were grown to confluence in an 8-chamber slide or a 96-well plate. DRAQ5 (eBioscience) or DiI (Life Technologies) labeled THP-1 monocytes or primary isolated PBMCs were pretreated with either vehicle basal RPMI media, 1 mM S1P, or 10% AA/SS plasma for 1 hour, co-incubated with AA/SS RBCs (1:10 Monocyte:RBC) for 18 hours, or co-incubated with AA/SS MPs at high (50%), medium (20%), or low (10%) concentrations for 18 hours. Cells were washed and allowed to adhere to confluent HUVECs for 4 hours. Nonadherent cells were washed away with PBS three times and cells were stained with DAPI (4,6-diamindino-2phenylindole), and imaged with fluorescence or near infrared microscopy. Ex vivo RBC studies RBCs were isolated and resuspended 1:100 in PBS 1 20 mmol/L glucose with 0, 1, 10, or 100 mM amitriptyline and incubated at 37°C for 1, 2, or 24 hours. Cells were resuspended in diH20 for acid SMase activity measurement or PBS for flow cytometric measurement of MPs. Animals Wild-type (WT) C57BL/6 mice were obtained from The Jackson Laboratories; heterozygous (AS) and homozygous (SS) mice were initially obtained from the sickle transgenic breeding colony at Georgia Institute of Technology. Townes’ model sickle transgenic mice were AS or SS for the sickle mutation. Saline and 10 mpk amitriptyline (dissolved in sterile saline) injections were performed via intraperitoneal injection and peripheral blood was drawn via retro-orbital bleed. For hypoxia studies, mice were housed in polypropylene hypoxia chamber (Coy Laboratory Products) and kept at 8% oxygen (using pure O2 and N2 as background gas) for 24 hours. All surgical procedures and animal care protocols were approved by the Georgia Institute of Technology’s Animal Care and Use Committee.

From www.bloodjournal.org by guest on March 12, 2017. For personal use only. BLOOD, 18 SEPTEMBER 2014 x VOLUME 124, NUMBER 12

SPHINGOLIPID METABOLISM IN SICKLE CELL DISEASE

1943

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 r” and “low r, ” 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.

Results Sphingolipid metabolism is dysregulated in SCD

Neutral SMase is known to be activated upon membrane bending.3 Although neutral SMase activity was largely undetectable in plasma and AA RBCs, there was a small but significant (10 pmol/min per mL) increase in neutral SMase activity in SS RBCs but not in plasma (Figure 2A-B). Acid SMase activity in AA plasma was 0.56 nmol/min per mL but was significantly elevated (0.64 nmol/min per mL) in SS plasma (Figure 2C). Acid SMase activity in SS RBCs (98.06 nmol/min per mL) was also significantly elevated in comparison with AA RBCs (87 nmol/min per mL) (Figure 2D). There was a positive correlation (r2 5 0.84) between acid SMase activity in RBCs and plasma suggesting that RBCs may adopt secreted acid SMase from plasma (data not shown). Because both enzyme concentration and activation state can affect overall activity, we quantified acid SMase expression. It was not altered in plasma and RBCs, which suggests that its increased activity in SCD was due to activation (Figure 2E). We sought to determine if sickle RBCs had higher levels of acid SMase activity, so we used Percoll density centrifugation to separate high- (irreversible sickled) and lowdensity fractions of RBCs. 18 Low-density SS RBCs displayed increased acid SMase activity relative to AA RBCs. Irreversible

sickled SS RBCs, however, had the highest acid SMase activity (Figure 2F-G). To confirm the role of RBC sickling in acid SMase activation, we induced sickling in SS mice with hypoxic conditioning for 24 hours. Consistent with humans, SS mice displayed a significant increase in acid SMase activity in whole blood at normoxic baseline. Deoxygenation by hypoxic conditioning further enhanced acid SMase activity in SS mice (Figure 2H). Multiple lipids can be metabolized downstream of SM (Figure 1). ACER1, which converts ceramide to sphingosine19 was measured in RBCs and plasma. ACER1 levels were not altered between AA (73.02 pg/mL) and SS (79.68 pg/mL) RBCs (Figure 3A). ACER1 was present at a concentration of 30.39 pg/mL in SS plasma, but was not detected in AA plasma (Figure 3B). Sphingosine is phosphorylated to form the bioactive signaling lipid, S1P, by SK1/2. Because S1P is stored at high concentrations in RBCs,20 we hypothesized that they would express SK1 and SK2. SK1 was expressed at low levels in both AA and SS RBCs. SK2 was also expressed by both cell types but was significantly elevated in SS RBCs (Figure 3C-D). SK2 levels in plasma were assessed via western blot and were significantly increased in SS relative to AA plasma (Figure 3E-F), suggesting that SK2 may be secreted into circulation in SCD. As these enzymes are upstream of sphingosine and S1P, we hypothesized that these sphingolipids would be elevated in SCD. High performance liquid chromatography mass spectrometry (HPLC-MS) was used to quantify lipids that were


AWOJOODU et al


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.

extracted from whole blood, plasma, or RBCs. Both sphingosine (202.84 nM vs 77.42 nM) and S1P (2080 nM vs 593.5 nM) were significantly elevated in SS whole blood relative to AA blood (Figure 3G). Sphingosine and S1P levels were much lower in the plasma, but there were still significant increases in sphingosine (12.82 nM vs 5.97 nM) and S1P (77.03 nM vs 35.01 nM) levels in SS plasma relative to AA plasma (Figure 3H). SS RBCs also had significantly more sphingosine (369.71 nM vs 73.41 nM) and S1P (1000.22 nM vs 249.3 nM) compared with AA RBCs (Figure 3I).

activity was largely undetectable in MPs (supplemental Figure 4B), suggesting that the enzyme is not internalized within particles upon generation. The particles themselves, however, are internalized by macrophages (Figure 4B). SM and ceramide were measured in RBCs and MPs. As expected, both RBCs and MPs had an increase in the ceramide:SM ratio, indicating a shift toward ceramide production consistent with SMase activity (Figure 4C). Both AA- and SS-derived MPs expressed sphingosine and S1P, and similar to SS RBCs, SS RBCderived MPs contained significantly more sphingosine and S1P (Figure 4D).

RBC-derived MPs express sphingolipids

We used the Townes’ mouse model of SCD to determine whether SS RBCs produced more MPs. SS mice have both mouse a and b hemoglobin knocked out and human a sickle b globin knocked in. Blood was collected from these mice and stained for CD411 platelet MPs and glycophorin A1 RBC MPs.21 Although platelet MPs comprised the vast majority of blood-borne MPs, there was a significant increase in RBC-derived MPs in SS mice relative to AS mice (supplemental Figure 3), corroborating the finding that RBC-derived MPs are significantly elevated in SCD.22 Preliminary image analysis of human RBC-derived MPs revealed a heterogeneous size distribution, so we employed ultracentrifugation at 10 000, 37 000, and 200 000 RCF to isolate P2, P3, and P4 MPs, respectively (Figure 4A). Their size distribution was between 150 and 400 nm (supplemental Figure 4A). Acid SMase

MPs are internalized and modulate cytokine production by monocytes

To determine whether monocytes internalized RBC-derived MPs, PMA-treated THP-1s were incubated with 1 3 106 CFSE-stained MPs for 30 or 120 minutes. Confocal microscopy revealed that MPs were internalized as early as 30 minutes (Figure 4B). Within 30 minutes, 75% of AA MPs and 84% of SS MPs were internalized (supplemental Figure 4C). Surprisingly, after 120 minutes, a smaller proportion of MPs (37% for AA and 40% for SS) was internalized by PMA-treated THP-1s, suggesting that MPs can be internalized and secreted as intact MPs (supplemental Figure 4C). MP internalization increased the production and secretion of many inflammatory cytokines after 24 hours. Interestingly, SS MPs, and not AA MPs, significantly enhanced the production and secretion of IL-6, TNF-a,



1945

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 5 200 nm. (B) Internalization or CFSE-labeled P3 MPs by PMA-treated THP-1s. Scale bar 5 10 mm. (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.

and IL-1b from macrophages, markers of systemic inflammation during SCD,23 relative to vehicle-treated cells (Figure 4E-F). To interrogate the effects of SS MPs on primary AA PBMCs, we incubated PBMCs with SS MPs. SS MPs significantly enhanced the production and secretion of these cytokines in primary AA monocytes by as much as 120-fold (Figure 4G-H). S1P, SS plasma, and SS RBC co-incubation enhances monocyte adhesion to endothelial cells

S1P, an immunomodulatory lipid, signals through the activation of 5 known G-protein coupled receptors (S1P1-5) with diverse biological functions.24 Because SS plasma and MPs express significantly more S1P than AA plasma and MPs, we wished to explore their effects on PBMC adhesion; 1 mM S1P or AA/SS plasma was used to pre-treat primary AA PBMCs for 1 hour before co-incubation with confluent HUVECs for 4 hours. S1P (43%) and SS plasma (28%) but not AA plasma (21.7%) resulted in an elevation in endothelial adhesion relative to vehicle (Figure 5A-B and supplemental Figure 5). Because RBCs are known to interact with monocytes in circulation,25,26 we sought to interrogate the potential of SS RBCs to directly alter monocyte-endothelial adhesion. AA or SS RBCs were incubated with THP-1s at a 10:1 ratio for 18 hours. Thereafter, THP-1s were co-incubated with HUVECs and allowed to adhere for 4 hours (Figure 5C). Coculture with SS RBCs significantly enhanced THP-1 adhesion to endothelial cells, as measured by the

THP-1:HUVEC ratio (0.78) relative to coculture with AA RBCs (0.19) (Figure 5C-D). SS RBC-derived MPs enhance PBMC adhesion and express ICAM-4 and elevated p-ERK1/2

To confirm whether MPs alone could enhance monocyte-endothelial cell adhesion, we incubated primary AA PBMCs with AA- or SS RBC-derived MPs for 24 hours and allowed them to adhere to endothelial cells for 4 hours. Interestingly, AA RBC-derived MP incubation did not enhance adhesion, whereas SS RBC-derived MP incubation resulted in a significant increase in PBMC adhesion at all concentrations (Figure 6A). Zennadi et al established an ERK1/2-mediated mechanism for ICAM-4 activation on SS RBCs, which mediates RBC adhesion to both monocytes and endothelial cells.26,27 Both AA and SS RBC-derived MPs expressed ICAM-4 (Figure 6B). SS RBC-derived MPs, however, expressed significantly activated ERK1/2 as indicated by significantly higher p-ERK1/2 to total ERK1/2 than AA RBC-derived MPs (Figure 6C-D). These results suggest that, similar to SS RBCs, SS RBC-derived MPs may enhance monocyte adhesion in an ERK1/2–ICAM-4 mediated fashion. Acid SMase inhibition reduces MP generation and proinflammatory cytokine production

As spleen dysfunction and reduced MP clearance are characteristic of SCD, we sought to establish a unique role for SMase in MP


AWOJOODU et al


Figure 5. Elevated monocyte adhesion in SCD. (A-B) Monocyte-adhesion after 1 hour with 1 mM 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.

generation. SS RBCs were cultured in media with amitriptyline, a tricyclic antidepressant and acid SMase inhibitor for 1 or 24 hours, and MPs were quantified by flow cytometry. Although 1-hour amitriptyline treatment did not alter the production of MPs at any dose, by 24 hours, amitriptyline had significantly reduced the generation of RBC-derived MPs in vitro in a dose-dependent fashion (Figure 7A-C). We next injected amitriptyline at 4 concentrations into WT C57Bl/6 mice and found that acid SMase activity was significantly reduced after 24 hours of the highest dose (Figure 7D). Hypoxia has

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 5 6). P , .05 measured in ANOVA relative to vehicle or AAMPs.

been shown to enhance MP generation in vivo.28 After 24 hours of hypoxic conditioning, MPs in blood and erythrocyte-derived MPs (EMPs) in blood were significantly elevated relative to prehypoxic conditioning. Amitriptyline reduced hypoxia-induced MP and EMP generation is shown in Figure 7E-F. Additionally, 2 of the 3 cytokines we showed to be modulated by MPs in monocytes in vitro (IL-6 and IL-1b) were reduced in amitriptyline-treated mice relative to saline-treated mice during normoxia (supplemental Figure 6). These results elucidate a role for acid SMase in RBC-derived MP generation and systemic inflammation in vivo, and establishes



1947

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 mM.

pharmacologic modulation of acid SMase as a strategy to modulate these phenomena in SCD.

Discussion SCD is a complex disease with severe pathophysiological effects. In recent years, it has been discovered that MPs, which are elevated in SCD, contribute to SCD pathogenesis.22,29-31 In this study, we examined the dysregulation of sphingolipid metabolism, MP production, and inflammatory cell activation in SCD. López et al found that stresses in RBC membranes transiently enhanced the activation of neutral SMase.3 Similarly, Urbina et al found that SMase activity in unilamellar vesicles in Clostridium perfringens a-toxin increased with membrane curvature.32 Our study shows that SS RBCs, which may undergo membrane stresses due to sickling events, oxidative damage, ion fluxes, and enhanced adhesion, have a significant increase in acid SMase activity relative to AA RBCs (Figure 2D). Irreversibly sickled RBCs had the highest acid SMase activity, corroborating these findings (Figure 2F-G). Furthermore, when RBC sickling was induced in SS mice by hypoxic deoxygenation, there was an increase in acid SMase activity confirming that sickling is upstream of acid SMase (Figure 2H). More studies need to be performed to elucidate the mechanism underlying the sensing of local forces and the subsequent activation of SMase in cell membranes.

SMase activity has been implicated in vascular inflammation.33,34 Becker et al discovered that ceramide production in the lungs contributed to inflammatory cytokine production during cystic fibrosis,35 and amitriptyline reduced ceramide production, inflammatory cytokine production, and inflammatory cell recruitment in a mouse model of cystic fibrosis. 35,36 Other studies have shown that SMase-mediated ceramide synthesis in plasma membranes promotes microvessel formation.9,37 We found that the ceramide:SM ratio in SS RBCs and MPs was increased, which corroborates the finding that acid SMase is activated in SS RBCs (Figure 4C). MPs have been shown to enhance atherosclerosis,31 coagulation, 30 vaso-occlusion in the kidneys, 38 inflammation,39,40 and monocyte adhesion.41 RBC-derived MPs from AA and SS donors were both internalized by THP-1s (Figure 4B). The internalization, processing, and secretion of RBC-derived particles deserve significant attention, and future studies are needed to completely understand these mechanisms. Interestingly, SS MPs enhanced the production and secretion of 3 cytokines consistently associated with inflammation in SCD (ie, TNF-a, IL-1b, and IL-623) to a much larger extent than AA MPs (Figure 4E-H). Additionally, we show that SS MPs, and not AA MPs, enhance monocyte adhesion to endothelial cells (Figure 6A), and similarly, co-incubation of monocytes with SS RBCs enhances monocyte-endothelial adhesion relative to AA RBCs (Figure 5C). Zennadi et al established an ERK1/2-mediated mechanism for ICAM-4 activation on SS RBCs, which mediates monocyte adhesion and activation26,27; therefore,


AWOJOODU et al

we wished to determine whether RBC-derived MPs expressed both ICAM-4 and p-ERK1/2. Interestingly, while both AA and SS RBC-derived MPs expressed roughly the same level of ICAM-4, SS RBC-derived MPs expressed significantly more activated ERK1/2. Taken together, these results suggest that, similar to SS RBCs, SS MPs may enhance monocyte adhesion in an ERK1/2–ICAM-4 mediated fashion. Additional studies are needed to elucidate this mechanism, and lipidomic and proteomic profiling of RBC-derived MPs might provide critical insights into the molecules contributing to these phenotype-specific alterations in inflammatory cell adhesion and cytokine secretion. Other studies have described inflammatory cell activation in SCD,15 and recently, a group discovered that S1P was elevated in SCD and promoted sickling.42 Because both SS plasma, RBCs, and MPs contain significantly more S1P than AA plasma, RBCs, and MPs (Figure 3H-I and Figure 4D), we wished to explore the direct effects of S1P on monocyte adhesion. S1P can be proinflammatory by activating S1P receptors on monocytes,43 inducing E-selectin expression and the adhesion of monocytes to endothelial cells,44 and cathepsins-mediated endothelial cell activation.45 We recently showed that S1P receptor 3 activation enhances the recruitment of anti-inflammatory monocytes to inflamed tissues.7 In this study, we found that both SS plasma and S1P directly enhanced monocyte-endothelial cell adhesion (Figure 5A-B). Although S1P has been shown to alter monocyte morphology and motility through the activation of r-kinase46 and receptor tyrosine kinases,47 more comprehensive studies need to be performed to elucidate the receptor-specific signaling mechanism behind S1P-mediated monocyte adhesion. It is important to note that SCD is characterized by significant hyposplenism. As the spleen is responsible for the removal of cellular debris, the increase in blood MPs observed in SCD is also a result of impaired spleen function.48 We wished to establish a nonredundant role for acid SMase in RBC-derived MP generation, therefore, we interrogated MP generation from SS RBCs in vitro, in the absence of spleen clearance. Amitriptyline and other tricyclic antidepressants have been shown to reduce acid SMase activity and exosome formation.35,36,49 Amitriptyline significantly reduced MP generation from SS RBCs in vitro (Figure 7A-C). Hypoxia has been shown to enhance MP generation28 and we confirmed that in nonsickle mice with normal functioning spleens, MP production was significantly elevated after 24 hours hypoxia. Amitriptyline significantly reduced MP generation in normoxic and hypoxic conditioned WT mice, suggesting a role for acid SMase in MP generation independent of dysfunctional spleen clearance (Figure 7F). Because amitriptyline is not specific to acid SMase and has other targets (ie, NO and PGE2), these studies should be repeated with a putative acid SMase inhibitor or with genetic knockdown. Additionally, these changes occurred in concert with proinflammatory cytokine reduction in vivo (supplemental Figure 6). However, additional studies will be required to demonstrate whether more selective pharmacologic inhibition of SMase is effective in reducing the generation of RBC MPs in SCD. Since amitriptyline acts indirectly on acid SMase by disrupting the electrostatic attraction that attaches SMase to the inner lysosomal membrane, the inhibitory effects of the drug are not specific to acid SMase. Indeed, the drug may act on acid lipase, phospholipase A and C, and other sphingolipid hydrolases by a similar mechanism. Furthermore, amitriptyline, as a tricyclic antidepressant, inhibits the uptake of adrenaline, dopamine, and serotonin, and affects the cholinergic and histaminergic systems.36 The role of systemic hypoxia and local hypoxia in sickle cells due to transient vaso-occlusions in acid SMase


activation, cannot be ruled out by these studies and needs to be explored further. There are multiple lipids metabolically downstream of SM that have diverse biological effects. Erythrocytes convert ceramide into sphingosine with ACER1.19 To date, this enzyme has not been detected in plasma; however, we detected ACER1 in SS plasma (Figure 3B), suggesting that it can be released into circulation through secretion or hemolysis. Ceramide synthesis in erythrocyte membranes is associated with phosphatidylserine exposure and macrophage clearance.50 Additionally, the chronic stress induced by sickled RBCs in circulation during SCD results in an increase in the apoptosis of white blood cells.51 Weigert et al found that cellular apoptosis leads to the activation and secretion of SK2.52 Consistent with these findings, we found that SK2 is increased in both the plasma and RBCs of SCD donors (Figure 3C-F). Although more studies are needed to fully understand the source and mechanism of ACER1 and SK2 secretion into plasma, it is apparent that complex mechanical and biological cues in SCD result in dysregulated sphingolipid enzyme expression and activation, causing a significant increase in sphingosine and S1P (Figure 3G-I). Our findings reveal, for the first time, that sphingolipid metabolism is dysregulated in SCD. Because this dysregulation is a result of altered enzyme activation and expression in damaged cells, these results may not be specific only to SCD, where the damage is due to membrane deformation, but may also be applicable to other RBC disorders. Additionally, they elucidate a novel mechanism for MP generation in SS RBCs. Membrane stresses imposed on RBCs in SCD activate acid SMase, which, in concert with other sphingolipid enzymes, results in elevated S1P and MP production (supplemental Figure 1). SS RBC-derived MPs, containing S1P, activate monocytes by enhancing endothelial cell adhesion and proinflammatory cytokine production. These findings elucidate potential new strategies to regulate inflammatory processes in SCD through modulating sphingolipid metabolism. Modulating sphingolipid metabolism may be a novel way to pharmacologically control systemic inflammation present in many human diseases.

Acknowledgments The authors thank Jingjing Duan, Dr Al Merrill, and Yugesh Kharel for assistance with lipid extraction and HPLC-MS. The authors also thank the Sickle Cell Foundation of Georgia for collecting and recruiting blood donors for these studies. This study was supported in part by grants from the National Institutes of Health National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R01DE019935-01), National Institute of Dental and Craniofacial Research (1R01AR056445-01A2), National Institute of General Medical Sciences (R01GM067958), and Office of the Director (1DP2OD007433-01), and the National Science Foundation (0933643).

Authorship Contribution: A.O.A. and E.A.B. conceived of and designed the study, and wrote the manuscript; P.M.K. and M.O.P. helped with experimental design and data interpretation; A.O.A. and P.M.K. isolated RBCs and plasma from donors; A.O.A., A.R.L., and Y.Z.


performed in vitro adhesion and enzyme assays; and A.O.A. and K.R.L. performed lipidomic analysis. Conflict-of-interest disclosure: The authors declare no competing financial interests.


1949

Correspondence: Edward A. Botchwey, The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 315 Ferst Dr. NW, Atlanta, GA 30332; e-mail: [email protected].

References 1. Gladwin MT, Sachdev V. Cardiovascular abnormalities in sickle cell disease. J Am Coll Cardiol. 2012;59(13):1123-1133. 2. Ohanian J, Ohanian V. Sphingolipids in mammalian cell signalling. Cell Mol Life Sci. 2001;58(14):2053-2068. 3. Lopez ´ DJ, Egido-Gabas M, Lopez-Montero ´ I, et al. Accumulated bending energy elicits neutral sphingomyelinase activity in human red blood cells. Biophys J. 2012;102(9):2077-2085. 4. Olivera A, Allende ML, Proia RL. Shaping the landscape: metabolic regulation of S1P gradients. Biochim Biophys Acta. 2013;1831(1):193-202. 5. Golan K, Vagima Y, Ludin A, et al. S1P promotes murine progenitor cell egress and mobilization via S1P1-mediated ROS signaling and SDF-1 release. Blood. 2012;119(11):2478-2488. 6. Walter DH, Rochwalsky U, Reinhold J, et al. Sphingosine-1-phosphate stimulates the functional capacity of progenitor cells by activation of the CXCR4-dependent signaling pathway via the S1P3 receptor. Arterioscler Thromb Vasc Biol. 2007;27(2):275-282. 7. Awojoodu AO, Ogle ME, Sefcik LS, et al. Sphingosine 1-phosphate receptor 3 regulates recruitment of anti-inflammatory monocytes to microvessels during implant arteriogenesis. Proc Natl Acad Sci USA. 2013;110(34):13785-13790. 8. Ishii M, Kikuta J, Shimazu Y, Meier-Schellersheim M, Germain RN. Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo. J Exp Med. 2010; 207(13):2793-2798. 9. Dinkla S, Wessels K, Verdurmen WP, et al. Functional consequences of sphingomyelinaseinduced changes in erythrocyte membrane structure. Cell Death Dis. 2012;3:e410. 10. Cuschieri J, Bulger E, Billgrin J, Garcia I, Maier RV. Acid sphingomyelinase is required for lipid Raft TLR4 complex formation. Surg Infect (Larchmt). 2007;8(1):91-106. 11. Jenkins RW, Canals D, Hannun YA. Roles and regulation of secretory and lysosomal acid sphingomyelinase. Cell Signal. 2009;21(6): 836-846. 12. Wong ML, Xie B, Beatini N, et al. Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: a possible link between inflammatory cytokines and atherogenesis. Proc Natl Acad Sci USA. 2000;97(15):8681-8686. 13. Marathe S, Schissel SL, Yellin MJ, et al. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling. J Biol Chem. 1998;273(7):4081-4088. 14. Sakata A, Ochiai T, Shimeno H, et al. Acid sphingomyelinase inhibition suppresses lipopolysaccharide-mediated release of inflammatory cytokines from macrophages and protects against disease pathology in dextran sulphate sodium-induced colitis in mice. Immunology. 2007;122(1):54-64.

17. Shaner RL, Allegood JC, Park H, et al. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers. J Lipid Res. 2009;50(8):1692-1707. 18. Kaul DK, Fabry ME, Windisch P, Baez S, Nagel RL. Erythrocytes in sickle cell anemia are heterogeneous in their rheological and hemodynamic characteristics. J Clin Invest. 1983;72(1):22-31. 19. Xu R, Sun W, Jin J, Obeid LM, Mao C. Role of alkaline ceramidases in the generation of sphingosine and its phosphate in erythrocytes. FASEB J. 2010;24(7):2507-2515. 20. Hanel ¨ P, Andreani ´ P, Graler ¨ MH. Erythrocytes store and release sphingosine 1-phosphate in blood. FASEB J. 2007;21(4):1202-1209. 21. Shet AS. Characterizing blood microparticles: technical aspects and challenges. Vasc Health Risk Manag. 2008;4(4):769-774. 22. Allan D, Limbrick AR, Thomas P, Westerman MP. Release of spectrin-free spicules on reoxygenation of sickled erythrocytes. Nature. 1982;295(5850):612-613. 23. Pathare A, Al Kindi S, Alnaqdy AA, Daar S, Knox-Macaulay H, Dennison D. Cytokine profile of sickle cell disease in Oman. Am J Hematol. 2004;77(4):323-328. 24. Rosen H, Gonzalez-Cabrera PJ, Sanna MG, Brown S. Sphingosine 1-phosphate receptor signaling. Annu Rev Biochem. 2009;78:743-768. 25. Hebbel RP, Schwartz RS, Mohandas N. The adhesive sickle erythrocyte: cause and consequence of abnormal interactions with endothelium, monocytes/macrophages and model membranes. Clin Haematol. 1985;14(1): 141-161. 26. Zennadi R, Chien A, Xu K, Batchvarova M, Telen MJ. Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium. Blood. 2008;112(8):3474-3483. 27. Zennadi R, Whalen EJ, Soderblom EJ, et al. Erythrocyte plasma membrane-bound ERK1/2 activation promotes ICAM-4-mediated sickle red cell adhesion to endothelium. Blood. 2012;119(5): 1217-1227. 28. Vince RV, Chrismas B, Midgley AW, McNaughton LR, Madden LA. Hypoxia mediated release of endothelial microparticles and increased association of S100A12 with circulating neutrophils. Oxid Med Cell Longev. 2009;2(1):2-6. 29. Tantawy AA, Adly AA, Ismail EA, Habeeb NM, Farouk A. Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: relation to cardiovascular complications. Platelets. 2013;24(8):605-614. 30. Shet AS, Aras O, Gupta K, et al. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood. 2003;102(7):2678-2683.

15. Manwani D, Frenette PS. Vaso-occlusion in sickle cell disease: pathophysiology and novel targeted therapies. Blood. 2013;122(24):3892-3898.

31. Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res. 2010;107(9): 1047-1057.

16. Zolla L, D’Alessandro A. An efficient apparatus for rapid deoxygenation of erythrocyte concentrates for alternative banking strategies. J Blood Transfus. 2013;2013:896537.

32. Urbina P, Flores-D´ıaz M, Alape-Giron ´ A, Alonso A, Gon˜i FM. Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium

perfringens a-toxin. Biochim Biophys Acta. 2011; 1808(1):279-286. 33. Opreanu M, Tikhonenko M, Bozack S, et al. The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models. Diabetes. 2011;60(9): 2370-2378. 34. Bhatia R, Matsushita K, Yamakuchi M, Morrell CN, Cao W, Lowenstein CJ. Ceramide triggers Weibel-Palade body exocytosis. Circ Res. 2004; 95(3):319-324. 35. Barrett O Jr, Saunders DE Jr, McFarland DE, Humphries JO. Myocardial infarction in sickle cell anemia. Am J Hematol. 1984;16(2):139-147. 36. Becker KA, Riethmuller ¨ J, Luth ¨ A, Doring ¨ G, Kleuser B, Gulbins E. Acid sphingomyelinase inhibitors normalize pulmonary ceramide and inflammation in cystic fibrosis. Am J Respir Cell Mol Biol. 2010;42(6):716-724. 37. Trajkovic K, Hsu C, Chiantia S, et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science. 2008; 319(5867):1244-1247. 38. Camus SM, Gausseres ` B, Bonnin P, et al. Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease. Blood. 2012;120(25):5050-5058. 39. Bardelli C, Amoruso A, Federici Canova D, et al. Autocrine activation of human monocyte/ macrophages by monocyte-derived microparticles and modulation by PPARg ligands. Br J Pharmacol. 2012;165(3):716-728. 40. Vasina EM, Cauwenberghs S, Staudt M, et al. Aging- and activation-induced platelet microparticles suppress apoptosis in monocytic cells and differentially signal to proinflammatory mediator release. Am J Blood Res. 2013;3(2): 107-123. 41. Barry OP, Pratico` D, Savani RC, FitzGerald GA. Modulation of monocyte-endothelial cell interactions by platelet microparticles. J Clin Invest. 1998;102(1):136-144. 42. Zhang Y, Berka V, Song A, et al. Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression. J Clin Invest. 2014;124(6):2750-2761. 43. Fueller M, Wang DA, Tigyi G, Siess W. Activation of human monocytic cells by lysophosphatidic acid and sphingosine-1-phosphate. Cell Signal. 2003;15(4):367-375. 44. Safaya S, Steinberg MH, Klings ES. Monocytes from sickle cell disease patients induce differential pulmonary endothelial gene expression via activation of NF-kB signaling pathway. Mol Immunol. 2012;50(1-2):117-123. 45. Keegan PM, Surapaneni S, Platt MO. Sickle cell disease activates peripheral blood mononuclear cells to induce cathepsins k and v activity in endothelial cells. Anemia. 2012;2012:201781. 46. Harvey KA, Welch Z, Sliva D, Siddiqui RA. Role of Rho kinase in sphingosine 1-phosphatemediated endothelial and smooth muscle cell migration and differentiation. Mol Cell Biochem. 2010;342(1-2):7-19. 47. Yogi A, Callera GE, Aranha AB, et al. Sphingosine-1-phosphate-induced inflammation involves receptor tyrosine kinase transactivation in vascular cells: upregulation in hypertension. Hypertension. 2011;57(4):809-818.


AWOJOODU et al

48. Fontana V, Jy W, Ahn ER, et al. Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients with ITP. Thromb Res. 2008;122(5):599-603. 49. Truman JP, Al Gadban MM, Smith KJ, et al. Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized low-density lipoprotein (LDL) and oxidized LDL immune complexes: role in phagocytosis and


cytokine release. Immunology. 2012;136(1): 30-45. 50. Jilani K, Enkel S, Bissinger R, Almilaji A, Abed M, Lang F. Fluoxetine induced suicidal erythrocyte death. Toxins (Basel). 2013;5(7):1230-1243. 51. Ozdogu H, Boga C, Sozer O, et al. The apoptosis of blood polymorphonuclear leukocytes in sickle cell disease. Cytometry B Clin Cytom. 2007;72(4): 276-280.

52. Weigert A, Cremer S, Schmidt MV, et al. Cleavage of sphingosine kinase 2 by caspase-1 provokes its release from apoptotic cells. Blood. 2010;115(17):3531-3540. 53. Balreira A, Lacerda L, Miranda CS, Arosa FA. Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model. Br J Haematol. 2005;129(5):667-676.

From www.bloodjournal.org by guest on March 12, 2017. For personal use only.

2014 124: 1941-1950 doi:10.1182/blood-2014-01-543652 originally published online July 29, 2014

Acid sphingomyelinase is activated in sickle cell erythrocytes and contributes to inflammatory microparticle generation in SCD Anthony O. Awojoodu, Philip M. Keegan, Alicia R. Lane, Yuying Zhang, Kevin R. Lynch, Manu O. Platt and Edward A. Botchwey

Updated information and services can be found at: http://www.bloodjournal.org/content/124/12/1941.full.html Articles on similar topics can be found in the following Blood collections Red Cells, Iron, and Erythropoiesis (774 articles) Sickle Cell Disease (128 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.