Red Blood Cells Store and Release Interleukin-33

Abstract

Interleukin-33 (IL-33) is a member of the IL-1 cytokine superfamily that potently drives production of a variety of cytokines and contributes to the pathogenesis of inflammatory diseases. The IL-33 is a nuclear protein and is released from apoptotic or necrotic cells. Serum IL-33 levels are increased in various diseases, such as atopic dermatitis, chronic hepatitis C infection, and asthma. Here, we show that red blood cells (RBCs) are one of the major sources of plasma IL-33. The IL-33 levels are significantly increased in supernatants from lysed RBCs. Plasma IL-33 levels are increased in patients during hemolysis, and plasma IL-33 levels show a positive correlation with degree of hemolysis. The IL-33 protein and messenger RNA levels were detected in the late stages of differentiation in ex vivo primary human erythroid progenitor cell cultures, suggesting that IL-33 is expressed during maturation of RBCs. Furthermore, hemoglobin depleted red cell lysates induced IL-8 expression in human epithelial cells. This effect was attenuated in IL-33 decoy receptor expressing cells and was enhanced in IL-33 receptor expressing cells. These results suggest that erythroid progenitor cells produce IL-33 and circulating RBCs represent a major source of IL-33 that is released upon hemolysis.

Keywords

RBC cytokine hemolysis

Interleukin-33 (IL-33), a relatively new member of the IL-1 cytokine superfamily has been reported to play a pathogenic role in inflammatory diseases including acute lung injury,^1,2 asthma,^3,4 pulmonary fibrosis,^5,6 and rheumatoid arthritis.^7–9 The IL-33 binds to ST2, a member of the IL-1 receptor/Toll-like receptor superfamily. ST2 protein consists of 2 types: a soluble (sST2, a decoy receptor) and a membrane-bound (ST2L) isoform.^10–12 The IL-33 activates the mitogen-activated protein kinase signal transduction cascade and increases chemokine release through ligating to ST2L.^2,7,10,13 We and others have shown that downregulation of ST2L attenuated IL-33–induced IL-8 release in human lung epithelial cells.^2,13 ST2 protein expression is regulated at the transcriptional^14,15 and posttranslational levels.² Posttranslational ST2L regulatory mechanisms include phosphorylation and ubiquitination.² The mechanisms that regulate IL-33 expression are incompletely understood. The IL-33 is localized to the nuclei of fibroblasts,¹⁶ endothelial cells,^11,17,18 and epithelial cells.^17,19,20 It is released from apoptotic and necrotic cells and is considered to be a “danger signaling” molecule.^21–23 Increased serum IL-33 levels have been detected in patients with atopic dermatitis,²⁴ rheumatoid arthritis,^25,26 asthma,²⁷ and scleroderma²⁸; however, the source of increased IL-33 in these conditions has not been well studied.

Hemolysis is a general term for excessive breakdown of red blood cells (RBCs). Hemolysis can occur within the circulatory system (intravascular hemolysis) or in the reticuloendothelial system (extravascular hemolysis).²⁹ Hemolysis also occurs during storage of RBCs, and the amount of storage hemolysis increases with the length of time in storage.³⁰ In mammals, circulating mature RBCs maintain a very specialized flexible biconcave discoid shape. Mature RBCs are enucleate and lack organelles providing maximum space for their primary cargo hemoglobin. Circulating RBCs have a limited lifespan (120 days in humans) in the circulation, and old RBCs are removed from the circulation by macrophages in the spleen; thus extravascular hemolysis is part of the natural life cycle of a circulating RBC. Intravascular hemolysis can be caused by bacterial infections, toxins, drugs, medications, autoimmune responses, and alloimmune responses.^31–33 Intravascular hemolysis results in the release of RBC contents into the circulation, which when excessive can cause more hemolysis and vascular dysfunction. Storage hemolysis also causes the release of potentially harmful, vasoactive, RBC-derived components into the RBC unit before transfusion into patients.³⁰ These storage-related changes may harm patients when older RBC units are transfused into patients and contribute to the RBC “storage lesion.”³⁰ Accumulating evidence indicates that stored RBCs have increased cytokine content. Levels of IL-1 and IL-8 are significantly higher in RBC units that have been stored for 40 days compared to the levels of these cytokines observed in freshly collected RBC units.³⁴ Darbonne et al.³⁵ demonstrated that ¹²⁵I-labeled IL-8 rapidly and efficiently bound to RBCs. In addition to IL-8, RBCs also bind monocyte chemotactic peptide-1 (MCP-1).³⁵ A recent study from Lee et al.³⁶ showed that longtime storage of RBCs increases the production of IL-8–bound RBC-derived microparticles. The RBCs also bind insulin and insulin-like growth factors.^37,38 These results support the role of circulating RBCs as carriers of bioactive peptides including cytokines. Diffuse alveolar hemorrhage plays a critical role in the pathogenesis of acute lung injury.^39,40 Recent studies have demonstrated that hemolysis induces inflammatory responses^{34,35,37,38,41,42}; however, the mechanisms have not been well characterized.

Here, we report that RBCs contain IL-33. This is the first study to demonstrate IL-33 expression in differentiating erythroid progenitor cells, and IL-33 is released during hemolysis. IL-33 release during hemolysis may contribute to hemolysis-induced inflammatory responses.

Materials and Methods

Cell Culture and Reagents

Purified human CD34+ progenitor cells were derived from GCSF–treated peripheral blood cells of healthy donors. These cells were grown at 37°C with 5% CO₂ in serum-free medium consisting of Iscove modified Dulbecco medium with 1-thioglycerol, BIT9500 supplement (BITS) (Stem Cell Technologies), bovine serum albunins (Sigma-Aldrich), and the indicated cytokines (PeproTech). The cells initially underwent 72 hours of expansion with 100 ng/mL stem cell factor (PeproTech), 100 ng/mL Feline McDonough Sarcoma-like tyrosine kinase 3 ligand (FLT3 ligand) (PeproTech), 100 ng/mL thrombopoietin (TPO) (PeproTech), and 50 ng/ml IL-3 (PeproTech). After expansion, cells were then seeded in erythroid differentiation medium, which contains recombinant human erythropoietin at 4.5 U/mL (Procrit; Amgen), 10 ng/mL SCF and BIT9500 supplement. Human bronchial epithelial cells (Beas2B) were grown at 37°C with 5% CO₂ in DMEM medium with 10% FBS. Horseradish peroxidase-conjugated goat antirabbit and antimouse antibodies, reverse transcriptase, and real-time polymerase chain reaction (PCR) SosoFast reagents were purchased from Bio-Rad Laboratories (Hercules, CA). Amicon Ultra centrifuge filters were purchased from EMD Millipore (Billerica, MA). Anti-IL-33 antibody was from R&D Systems (Minneapolis, MN). Anti-LarminA/C antibody was from Santa Cruz (Dallas, TX). Anti-GAPDH and β-actin antibodies and RBC lysis buffer were from Sigma (St. Louis, MO).

Rbc Lysates Preparation

Human or mouse whole blood was collected in ethylenediaminetetraacetic acid-treated blood collection tubes, followed by centrifugation at 500 g for 10 min. The RBC fraction was transferred to new tubes and split into 2 groups: the nonlysed control group was incubated in isoosmotic 0.9% NaCl, the hemolyzed group was incubated with RBC lysis buffer. Both groups were incubated at room temperature for 10 minutes. After a brief centrifugation at 500g for 10 minutes, the supernatants of the 2 groups were subjected to IL-33 immunoblotting.

Hemoglobin-Free RBC Lysate Preparation

To remove hemoglobin (64 kDa) from RBC lysates, Amicon ultra (50 kDa) cutoff filter was used. Briefly, RBC lysates were subjected to Amicon ultra (50 kDa) cutoff filter, followed by a centrifugation at 5000g for 20 minutes.

Immunoblotting

Cells were washed with cold phosphate-buffered saline and collected in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM ethylaene glycol tetraacetic acid, 5 mM β-glycerophosphate, 1 mM MgCl₂, 1% Triton X-100, 1 mM sodium orthovanadate, 10 μg/mL protease inhibitors, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin. Equal amounts of cell lysates (20 μg) or RBC supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose membranes, and immunoblotted with indicated antibodies.

Rna Extraction and Real-Time Reverse Transcription Pcr

Total RNA were extracted from cells by TRIzol (Sigma) according the manufacturer's instructions. Reverse transcription was performed using a complementary DNA synthesis kit (Bio-Rad) and real-time PCR was performed to assess cytokine expression using primers specific to human IL-33 and IL-8 messenger (m)RNA transcripts. Amplicon expression in each sample was normalized to its 18S mRNA transcript level. The relative abundance of target mRNA in each sample was calculated using the relative crossing time ratio. As 2 raised to the negative of its threshold cycle value times 10⁶ after being normalized to the abundance of its corresponding 18S (eg, 2^{-(IL-33R2 Threshold Cycle)}/ 2^{-(18S Threshold Cycle)} × 10⁶).

Statistics Analysis

All results were subjected to statistical analysis using 2-way analysis of variance, and, wherever appropriate, analyzed by Student-Newman-Keuls test. Data are expressed as mean ± SD. P less than 0.05 were considered statistically significant.

Results and Discussion

IL-33 Exists in RBC Lysates

Circulating IL-33 levels are increased in patients with allergic, inflammatory, rheumatologic, and infectious diseases.^{5,23,24,26–28} However, the source of IL-33 has not been well characterized. RBCs have been shown to bind plasma cytokines, growth factors, and chemokines.^35,37,38 To investigate whether RBCs are one of the sources of plasma IL-33, we collected mouse whole blood, and compared IL-33 levels in supernatants from lysed RBCs and nonlysed RBC controls by immunoblotting as described in Materials and Methods. Figure 1 shows that the supernatants from hemolyzed samples contain much higher levels of IL-33 compared to nonlysed controls. These results suggest that IL-33 exists in RBC lysates. This is a novel finding and has important implications in hemolytic diseases and RBC storage in blood banking. RBCs are known to bind IL-8, MCP-1, insulin, and insulin-like growth factors,^35,37,38 but have not been shown to bind IL-33 until now. The IL-8 and MCP-1 bind to the Duffy antigen on circulating RBCs.⁴³ Insulin binds to the insulin receptor on the surface of RBCs.⁴⁴ Lee et al.³⁶ have also shown that microparticles from RBCs contain IL-8, suggesting that IL-8 is one of internal components in RBC-derived microparticles. The localization of IL-33 in the RBCs remains unclear.

IL-33 Levels Are Increased in Sickle Cell Patients with Higher Levels of Hemolysis

To further investigate the role of the RBC as a storage pool for circulating IL-33, we compared the IL-33 levels in plasma from sickle cell patients known to have undergone low levels or high levels of hemolysis (Table 1). Sickle cell disease patients from our previous Walk-PHaSST study were randomly selected within the highest and lowest quartiles of hemolytic component. The hemolytic component is a composed index for hemolysis based on a principle component analysis of factors associated with hemolysis, including lactate dehydrogenase, aspartate, aminotransferase, reticulocyte percentage, and total bilirubin in 415 hemoglobin SS patients had a mean of 0 (SD = 1.50).⁴⁵ The IL-33 levels are significantly higher in plasma samples from high hemolyzers compared to the plasma samples from low hemolyzers (Fig. 2). These results support the idea that RBCs are a major source of IL-33 in the circulation. These results also suggest that plasma IL-33 levels may be used as a biomarker for hemolytic disorders, such as sickle cell disease. This finding connects RBC damage during hemolysis directly to a cytokine release.

Table 1

Plasma Samples From Subjects With Low and High Hemolytic Component

Hematopoitic Progenitor Cells Express IL-33

The IL-33 is a nuclear protein. It is unclear how IL-33 gets inside of anucleate RBCs. A speculative mechanism is that IL-33 is left behind in the reticulocyte after enucleation of erythroid progenitor cells during the late stages of maturation. To investigate IL-33 synthesis during human erythropoiesis, IL-33 protein expression was examined during different stages of erythroid progenitor cell differentiation using an ex vivo human CD34+ hematopoietic progenitor cell culture system as described in Materials and Methods. We collected erythroid progenitor cells on days 1, 5, and 9 of erythroid differentiation. As shown in Figure 3, human erythroid progenitor cells show a progressive decrease in expression of the nuclear protein, LaminA/C, whereas IL-33 expression progressively increases, peaking at day 9. Cytoplasm proteins (IQGAP1 and β-actin) remain no changes. Reduction of LaminA/C indicates the erythroid progenitor cells were differentiated to mature RBC-like cells. The IL-33 mRNA, but not IL-8 mRNA, levels progressively increase as erythropoiesis proceeds in the human erythroid progenitor cell cultures (Fig. 4). These results suggest that IL-33 is synthesized during RBC differentiation and maturation and that IL-33 is not stuck on the membrane of RBCs, but is soluble and inside the RBCs. CD34+ progenitors from chronic myeloid leukemia patients have been shown to express ST2L, proliferate, and produce cytokine in response to IL-33,⁴⁶ whereas this study is the first to demonstrate CD34+ progenitors also express its ligand, IL-33. Watari et al.⁴⁷ reported that human hematopoietic progenitor cells produce IL-1β. These results also support the idea that IL-33 is inside circulating erythrocytes.

Hemoglobin Depleted RBCs Lysates Increase IL-8 mRNA Expression in Human Lung Epithelial Cells

We and others have shown that IL-33 treatment increases IL-8 expression in human lung epithelial and endothelial cells.^2,13,21 To investigate if the IL-33 contained in RBC lysates can increase IL-8 expression, independent of hemoglobin, we removed hemoglobin from lysed RBCs using size exclusion filters, because hemoglobin can affect cytokine release. Beas2B cells were treated with hemoglobin depleted RBC lysates for 3 hours, and IL-8 mRNA expression was determined by real-time PCR. As shown in Figure 5, hemoglobin-depleted RBC lysate-induced IL-8 mRNA expression is approximately 23-fold, this effect was attenuated in IL-33 decoy receptor, sST2-overexpressing cells. Further, hemoglobin-depleted RBC lysate-induced IL-8 mRNA expression was enhanced in IL-33 receptor, ST2L overexpressing cells. These results suggest that hemolysis via the release of intra-crythrocyte IL-33 may contribute to IL-8 expression (Fig. 6). Hemolysis can be caused by infection, inflammation, oxidative damage, autoantibodies, alloantibodies, toxins, drugs, medications, and inherited genetic lesions.^29–32 Hemolysis also contributes to inflammatory responses, endothelial barrier dysfunction, and platelet activation, the effects are similar to IL-33 treatment. This study provides new evidence that IL-33 is synthesized in erythroid progenitor cells and can be released from RBCs undergoing hemolysis. The IL-33 may be involved in hemolysis-induced inflammatory responses.

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