Lysis of RBCs during numerous clinical settings such as severe hemolytic anemia, infection, tissue injury, or blood transfusion releases the endogenous damage-associated molecular pattern, hemoglobin (Hb), into the plasma. The redox-reactive Hb generates cytotoxic reactive oxygen species, disrupting the redox balance and impairing the immune-responsive blood cells. Therefore, it is crucial to understand how the immune system defends against the cytotoxic Hb. We identified a shortcut “capture and quench” mechanism of detoxification of Hb by the monocyte scavenger receptor CD163, independent of the well-known dominant antioxidant, haptoglobin. Our findings support a highly efficient two-pass mechanism of detoxification and clearance of Hb: 1) a direct suppression of Hb-pseudoperoxidase activity by CD163, involving an autocrine loop of CD163 shedding, sequestration of Hb, recycling, and homeostasis of CD163 in human monocytes and 2) paracrine transactivation of endothelial cells by the shedded soluble CD163 (sCD163), which further detoxifies and clears residual Hb. We showed that sCD163 and IgG interact with free Hb in the plasma and subsequently the sCD163-Hb-IgG complex is endocytosed into monocytes via FcγR. The endocytosed sCD163 is recycled to restore the homeostasis of CD163 on the monocyte membrane in an autocrine cycle, whereas the internalized Hb is catabolized. Using ex vivo coculture experiments, we demonstrated that the monocyte-derived sCD163 and IgG shuttle residual plasma Hb into the proximal endothelial cells. These findings suggest that CD163 and IgG collaborate to engage monocytes and endothelial cells in a two-pass detoxification mechanism to mount a systemic defense against Hb-induced oxidative stress.

Hemolysis due to tissue injury, trauma (1), or infection by hemolytic microbes (2) ruptures RBCs and releases hemoglobin (Hb) into the plasma. The intrinsic cytotoxicity of the cell-free Hb is well established (3). This is due to the pseudoperoxidase (POX) activity of Hb, which catalyzes the production of free radicals such superoxide anion (O2.−), ferryl Hb, and other reactive derivatives such as hydroxyl radical and hypohalous acid (4).

In an infection, microbial proteases specifically trigger the Hb POX activity, leading to a localized oxidative shock at the site of infection (5, 6). The Hb-induced microbicidal reactive oxygen species (ROS) also damages the host itself when it is not rapidly detoxified and removed from circulation. The interrelationship between ROS and the innate immune system in acute lung injury (7), chronic granulomatous disease (8), hemorrhagic shock, and ischemia (9) has been reported, prompting our systematic analysis of the host defense mechanism(s) against the danger molecule, Hb. Although plasma haptoglobin (Hp) has been reported to bind (10, 11) and mediate the internalization of Hb by monocytes/macrophages via the scavenger receptor membrane-associated CD163 (mCD163) (12, 13), Hp does not alter the reactive properties of the Hb heme group (11, 14). Additionally, neither Hp knockout mice (15, 16) nor humans with anhaptoglobinemia (17) display complete morbidity to hemolysis, suggesting that there are alternative mechanisms of detoxification of Hb. Contrary to the widely accepted mode of clearance of Hb via Hp, a recent study has proposed a possible direct interaction between CD163 and Hb even in the absence of Hp (18). Such Hp-independent clearance mechanism of Hb could be especially crucial during severe hemolysis, when Hp is rapidly bound and exhausted (19). The residual cell-free redox-reactive Hb would have been life-threatening, and yet we survive. Therefore it is conceivable that there are alternative mechanisms of detoxification of Hb even when Hp, the dominant antioxidant of Hb, is depleted. Intriguing questions remain unanswered; for example, although Hb has been shown to be directly recruited by CD163 independent of Hp (18), the functional significance of the CD163-Hb interaction to the redox reactivity of Hb is unknown. Furthermore, during inflammation, mCD163 is proteolytically shed from the monocyte membrane into the plasma, and the soluble CD163 (sCD163) (2022) reportedly binds the Hb-Hp complex in vitro (23). However, the fate of sCD163 under severe hemolytic conditions (when Hp is depleted) remains unclear. All of these findings prompted us to systematically investigate the innate immune mechanisms regulating cell-free Hb, an important danger-associated molecule. Towards this goal, we sought to 1) decipher the direct effect of CD163 on the Hb POX activity and the consequential cell survival when Hp is depleted; 2) elucidate the fate of sCD163; and 3) explore the potential crosstalk between monocytes and endothelial cells under severe hemolytic conditions, which is based on reports that monocytes and endothelial cells are activated during hemolysis, rendering the endothelium adhesive to blood cells (24, 25).

Contrary to the current understanding that Hp is the primary antioxidant of Hb, we show that CD163 confers a two-pass Hb detoxification effect. First, mCD163 directly suppresses the POX activity of Hb in situ on the monocyte membrane, independent of Hp. Consequently, CD163 also rescues monocytes from Hb-induced apoptosis. The shedded sCD163 further complexes with residual plasma Hb. The sCD163-Hb complex then interacts with IgG in the plasma. The IgG bridges the sCD163-Hb complex to the FcγR, enabling the endocytosis of the sCD163-Hb-IgG complex. Subsequently, the endocytosed sCD163 is recycled via endosomes to the membrane to restore homeostasis of mCD163 in an autocrine manner, whereas the internalized Hb undergoes detoxification. Second, the sCD163 elicits a paracrine cycle, transactivating the proximal endothelial cells to scavenge and detoxify the cell-free Hb.

All experiments were performed according to the guidelines on ethics and biosafety (Institutional Review Board, reference code NUS-IRB 08-296).

Purified human Hb, subtilisin A, rabbit polyclonal anti-human Hb, rabbit anti-human IgG, and the protein synthesis inhibitor cycloheximide (CHX) were obtained from Sigma-Aldrich. Mouse monoclonal anti-human FcγRI (CD64) and goat polyclonal anti-human CD163 were purchased from R&D Systems. Purified mouse anti-human FcγRIII (CD16) and mouse anti-human FcγRII (CD32) were from BD Pharmingen. Rabbit anti-human heme oxygenase-1 (HO-1) was from Cell Signaling Technology. Mouse monoclonal anti–plasma membrane calcium (PMCA) ATPase was from Thermo Scientific. The endocytosis and recycling inhibitors, chlorpromazine and monensin, respectively, were from Calbiochem.

Histiocytic lymphoma cell line SU-DHL-1 (DSMZ), also described as monocytic M5-type cells, the only human cell line that expresses high levels of CD163 (13), and Jurkat cells, a human T cell lymphoblast cell line, were cultured in 5% CO2 at 37°C in HEPES-buffered RPMI 1640 (Invitrogen) containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS at a density of 2 × 106 cells/ml. HEK293T and HepG2 cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Human dermal microvascular endothelial cells (HMVEC), which expresses FcγRII (CD32) (26), were cultured in EndoGRO-LS complete medium (Millipore) on gelatin-coated flasks. Primary human monocytes were purified from buffy coat by Ficoll-Paque (GE Healthcare) density gradient centrifugation (27) followed by immunomagnetic cell sorting using a human monocyte enrichment kit (StemCell Technologies) according to the manufacturers’ instructions.

Confluent HMVEC were washed twice with PBS and incubated with freshly isolated primary monocytes or THP-1 cells at a ratio of 1:1 in PBS for 45 min with or without Hb and prepared for immunostaining. For cytokine assays, the cells were cocultured for 24 h in serum-free RPMI 1640 in the presence or absence of Hb, and the supernatants were collected for ELISA.

The generation of free radicals (O2.−) by Hb was monitored by the chemiluminescence of Cypridina luciferin analog (28, 29) using the GloMax 20/20 luminometer (Promega). The relative luminescence units per second specifically measures the dynamics of the generation of O2·.

The native membrane and cytosolic proteins from 2 × 106 SU-DHL-1 cells or primary monocytes were extracted using a native membrane protein extraction kit (ProteoExtract; Calbiochem) according to the manufacturer’s instructions. Briefly, cells were washed twice with ice-cold PBS and incubated for 10 min on ice under gentle agitation with 2 ml ice-cold extraction buffer I supplemented with protease inhibitor mixture. The insoluble material was pelleted by centrifugation at 16,000 × g for 15 min at 4°C and the supernatant enriched in soluble proteins was frozen at −80°C. The cell pellet was then incubated with 1 ml ice-cold extraction buffer I supplemented with protease inhibitor mixture for 30 min on ice, with gentle agitation. The insoluble material was pelleted by centrifugation at 16,000 × g for 15 min at 4°C and the supernatant enriched in membrane proteins was collected and used immediately or frozen at −80°C.

The full-length human CD163 was cloned into pcDNA3.1A (Invitrogen) and expressed in HEK293T cells. HEK293T cells were seeded and grown overnight on 12-well plates (Nunc) at a density of 4 × 105 cells/well in DMEM before transfection. The cells were transfected using TurboFect (Fermentas) according to the manufacturer’s instructions.

The ROS generated within the monocytes was measured using the cell permeant oxidation-dependent fluorogenic dye CM-H2DCFDA (Invitrogen). SU-DHL-1 cells were plated at 2 × 105 cells/well onto 24-well plates in phenol red–free RPMI 1640. The cells were washed and resuspended in PBS containing 10 μM CM-H2DCFDA for 30 min in the dark and stimulated with 15 μM Hb with or without pretreatment with 0.1 μg/ml anti-CD163. The fluorescence of the dye at 495 nm was measured using a microplate reader (BioTek).

Cell viability was measured using a CellTiter-Blue viability assay kit (Promega) following the manufacturer’s instructions. Briefly, HEK293T and HepG2 cells seeded overnight on 96-well plates were stimulated with Hb. CellTiter-Blue was added to each well, and fluorescence was measured (excitation 530 nm, emission 590 nm) after 4 h incubation. The mean fluorescence of triplicate wells was calculated and plotted. Staining of early apoptotic cells was performed using the an annexin V-FITC apoptosis detection kit (eBioscience) and propidium iodide viability staining solutions (eBioscience) according to the manufacturers’ instructions. Briefly, primary monocytes were stimulated with 15 μM native Hb or activated Hb with or without pretreatment with 0.1 μg/ml anti-CD163. The cells were then washed successively with PBS and 1× binding buffer and resuspended in binding buffer at a density of 1 × 106 cells/ml. The cells were incubated with FITC-conjugated annexin V (20:1, v/v) for 15 min at room temperature and washed. Propidium iodide was added at a dilution of 1:20 to the cell suspension and immediately analyzed on a CyAn ADP flow cytometer (Dako).

SU-DHL-1 cells (2 × 106) were washed twice with PBS and fixed in 4% (w/v) paraformaldehyde for 15 min. The cells were then blocked with 2% BSA for 30 min and washed once with PBS (pH 7.4). Subsequently, the cells were sequentially stained with primary goat anti-CD163 (1:100) and NL-557–conjugated secondary Ab (1:200) (donkey anti-goat; R&D Systems). Then the cells were washed three times with PBS, and 104 cells were acquired and analyzed on the CyAn ADP flow cytometer (Dako).

Cultured cells were harvested, pelleted, and protein extraction was performed in ice-cold RIPA lysis buffer (Cell Signaling Technology) containing 1 mM PMSF and 1× protease inhibitor mixture (Sigma-Aldrich). Fifty micrograms total proteins was resolved by 10% SDS-PAGE under nonreducing conditions and then electrotransferred to polyvinylidene difluoride membrane in Tris-glycine buffer with 20% methanol. Membranes were probed with a goat polyclonal Ab against CD163 (R&D Systems) followed by rabbit anti-goat HRP-conjugated secondary Ab (Dako). For loading control, blots were probed with a mouse mAb against a plasma membrane housekeeping protein, PMCA ATPase (Thermo Scientific), followed by goat anti-mouse HRP-conjugated secondary Ab (Dako). Bands were visualized with SuperSignal chemiluminescence substrate (Pierce).

SU-DHL-1 cells, plated at a density of 2 × 106 cells/well in 24-well plates, were stimulated with 15 μM Hb (Sigma-Aldrich) over a time course. This concentration was chosen in view of its pathophysiological relevance (30). The cells were pelleted at 300 × g for 15 min at 22°C, and the concentration of sCD163 in the cell culture supernatants was measured using a human CD163 ELISA kit (Quantikine; R&D Systems).

SU-DHL-1 cells or primary monocytes were seeded at a density of 2 × 105 cells/well onto poly-lysine (Sigma-Aldrich)–coated coverslips and cultured overnight. The cells were then incubated with sCD163-Hb or sCD163-Hb-IgG complex for the indicated time periods. Subsequently, the cells were fixed using 4% (w/v) paraformaldehyde for 10 min, blocked with 1% BSA in PBS, and incubated with a mixture of primary Abs containing goat polyclonal anti-CD163 (1:200) (R&D Systems) and rabbit anti-Hb (1:500) (Sigma-Aldrich) for 60 min at room temperature. Following three washes with PBS (pH 7.4), the cells were incubated with secondary Ab mixture containing NL-557–conjugated donkey anti-goat (1:200) and Alexa 488–conjugated chicken anti-rabbit (1:400). The cells were then washed three times with PBS and mounted on a slide along with the Prolong Gold antifade mounting reagent containing DAPI (Invitrogen). Confocal imaging of the cells was performed on an LSM 510 META microscope (Zeiss) under a ×100 oil immersion objective using the LSM 510 software.

Freshly isolated human primary monocytes at 1 × 108 were washed twice with PBS (pH 7.4) and stimulated with 10−8 M PMA for 2 h at 37°C. sCD163 was isolated from the culture supernatants. The concentration of the affinity-purified sCD163 was determined using a CD163-specific ELISA. sCD163 (0.1 μg/ml) was immobilized onto microplates (Nunc). Increasing concentrations of Hb in PBS were added and the reaction was incubated for 2 h at 37°C. Bound Hb was detected using 1:1000 mouse anti-Hb (Santa Cruz Biotechnology) and 1:2000 goat anti-mouse HRP (Dako Cytomation). The OD at 405 nm was read.

FITC (Thermo Scientific) was conjugated to Hb that had been preactivated by partial proteolysis with a typical bacterial serine protease, subtilisin A (1.5 U). For pulldown of the sCD163-Hb complex, 10 μg anti-CD163 (R&D Systems) in TBS (pH 7.5) was conjugated to protein A-Sepharose (GE Healthcare Life Sciences) overnight at 4°C. Unbound Ab was removed by washing twice with TBS and the bound anti-CD163 was cross-linked to Sepharose by incubating for 60 min in cross-linking buffer containing 50 mM dimethyl pimelimidate (Sigma-Aldrich) and 200 mM triethanolamine (pH 8.9). The Sepharose beads were blocked using 100 mM ethanolamine and then incubated with sCD163 and activated Hb-FITC for 60 min at room temperature with two washes between each binding step. Subsequently, after three washes, the bound proteins were eluted with 2.5% acetic acid into tubes containing neutralization buffer of 1 M Tris-HCl (pH 12.0).

Upon identification of the interaction between IgG and Hb-sCD163, we pulled down the sCD163-Hb-IgG complex by incubating protein A-Sepharose with 5 μg IgG (affinity-purified from human serum) at room temperature for 60 min. The unbound Ab was removed by washing twice in TBS. The beads were incubated with 5 μg Hb-FITC and 5 μg sCD163 for 60 min at room temperature with two washes between each binding step. Subsequently, after three washes, the complex of sCD163-Hb-IgG was eluted using 2.5% acetic acid into tubes containing neutralization buffer.

For purification of sCD163 from cell culture supernatant, 50 μg anti-CD163 in binding buffer (TBS, pH 7.5) was conjugated to protein A-Sepharose by overnight incubation with rotation at 4°C. Unbound Ab was washed twice with binding buffer and the bound anti-CD163 was cross-linked to Sepharose by incubating for 60 min in a cross-linking buffer (50 mM dimethyl pimelimidate in 200 mM triethanolamine [pH 8.9]). The beads were washed twice and incubated with the culture supernatant for 60 min at room temperature. After three washes, bound sCD163 was eluted using 2.5% acetic acid into tubes containing neutralization buffer (1 M Tris-HCl [pH 12.0]).

For purification of IgG from healthy human serum, protein G-Sepharose (GE Healthcare Life Sciences) was incubated with 5 μl serum (contains ∼10 mg/ml IgG) (31) diluted to 400 μl in binding buffer (20 mM sodium phosphate [pH 7.0]) overnight with rotation at 4°C. The beads were washed twice with binding buffer, and the bound IgG was eluted using 0.1 M glycine-HCl [pH 2.7] into tubes containing neutralization buffer (1 M Tris-HCl [pH 12.0]). All experiments were validated using IgG purified from at least three different healthy donors.

The real-time biointeraction between IgG, Hb, and sCD163 was analyzed by surface plasmon resonance using a Biacore 2000 instrument (Biacore International, Uppsala, Sweden). IgG was immobilized on a CM5 chip by amine coupling according to the manufacturer’s instructions. Increasing doses of Hb at 0.2–0.8 μM was injected over the IgG-immobilized chip in running buffer of 50 mM Tris, 145 mM NaCl with 2 mM calcium [pH 7.4] at a flow rate of 30 μl/min. Anti-Hb at 5–20 nM was injected to verify the specificity of interaction between Hb and IgG. sCD163 was buffer-exchanged to the same running buffer using Vivaspin columns (Sartorius Stedim Biotech) and 50 μl sCD163 (2.5–10 ng/ml) was injected over the bound Hb. The dissociation was for 180 s at the same flow rate. Regeneration of the chip surface was performed by injection of 0.1 M NaOH until baseline was restored. The binding affinities were calculated using BIAevaluation software, version 4.1 applying the drifting baseline model assuming 1:1 interaction model. Response units were subtracted from BSA/N-acetylglucosamine–immobilized reference flow cells (negative control).

To validate the role of FcγR in the uptake of IgG-Hb-CD163, we silenced all three types of FcγR, that is, FcγR1 (CD64), FcγRII (CD32), and FcγRIII (CD16). The CD64 targeting small interfering RNA (siRNA) pool was obtained from Dharmacon (Thermo Scientific), and CD32 and CD16 siRNA duplexes were from OriGene Technologies. Primary monocytes (2.5 × 106) were nucleofected with 2 μg siRNA pool using the Amaxa Nucleofector (human monocyte Nucleofector kit, Nucleofector program Y-001). The oligonucleotide sequence of the siRNA pool used to knockdown the FcγR types in primary monocytes are shown in Table I. Scrambled siRNA pool was used as the negative control. Cells were harvested 48 h after nucleofection. The efficiency of knockdown was analyzed by flow cytometry.

Table I.
siRNA pool used to knock-down human FCGRI, FCGRII, and FCGRIII
GenesiRNA Sequence (5′–3′)
FCGRI (CD64) AAACAAAGUUGCUCUUGCA 
GGAAAUGUCCUUAAGCGCA 
GGAACACAUCCUCUGAAUA 
GAGAAGACUCUGGGUUAUA 
FCGRII (CD32) rArGrArArCrArArArGrArGrCrCrCrArArUrUrArCrCrArGAA 
rGrArUrGrUrArGrCrArArCrArUrGrArGrArArArCrGrCrUTA 
rGrArArUrUrArGrArGrArGrGrUrGrArGrGrArUrCrUrGrGTA 
FCGRIII (CD16) rGrCrUrUrCrGrCrUrGrArGrUrUrArArGrUrUrArUrGrArAAC 
rCrGrArUrGrArGrUrCrCrUrCrUrUrArArUrGrCrUrArGrGAG 
rArGrArArArUrArGrCrArGrGrUrArGrUrCrCrArGrGrArUAG 
GenesiRNA Sequence (5′–3′)
FCGRI (CD64) AAACAAAGUUGCUCUUGCA 
GGAAAUGUCCUUAAGCGCA 
GGAACACAUCCUCUGAAUA 
GAGAAGACUCUGGGUUAUA 
FCGRII (CD32) rArGrArArCrArArArGrArGrCrCrCrArArUrUrArCrCrArGAA 
rGrArUrGrUrArGrCrArArCrArUrGrArGrArArArCrGrCrUTA 
rGrArArUrUrArGrArGrArGrGrUrGrArGrGrArUrCrUrGrGTA 
FCGRIII (CD16) rGrCrUrUrCrGrCrUrGrArGrUrUrArArGrUrUrArUrGrArAAC 
rCrGrArUrGrArGrUrCrCrUrCrUrUrArArUrGrCrUrArGrGAG 
rArGrArArArUrArGrCrArGrGrUrArGrUrCrCrArGrGrArUAG 

Sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3′-dithiopropionate (sulfo-NHS-S-S-biotin; Pierce) was used for biotinylation of sCD163. Briefly, 2 μg/ml sCD163 was incubated with 20-fold molar excess of sulfo-NHS-SS-biotin at room temperature for 60 min. Excess biotin reagent was removed using ultracentrifugal spin columns (10K Amicon Ultra-0.5), and the biotin-conjugated sCD163 was buffer exchanged to PBS (pH 7.4). The level of biotin incorporated into sCD163 was quantified to be 18 biotin molecules per sCD163 molecule. Subsequently, primary monocytes were incubated with either bitoin-sCD163 alone or as a complex with Hb and IgG for up to 90 min at room temperature. The membrane and cytosolic fractions isolated from cells were captured on anti-CD163–coated 96-well microplates for 2 h at room temperature. The biotin-labeled protein bound on the plate was detected by HRP-streptavidin conjugate (ZyMax Grade; Invitrogen). ABTS substrate enabled the detection of the HRP conjugate and OD at 450 nm was read. Three washes with PBST were carried out between incubations.

HO-1 activity assay was performed as described earlier (32). Briefly, 50 μl microsomes from cells stimulated with cell-free Hb was added to 250 μl of a reaction mixture containing 0.1 mM NADPH, 1 mM NADP, 1 mM glucose-6-phosphate, 5 mU glucose-6-phosphate dehydrogenase, 2 mg rat liver cytosol (as a source of bilirubin reductase; prepared according to methods in Ref. 33), 100 mM potassium phosphate buffer (pH 7.4), and 1 mg/ml hemin. The reaction was performed at 37°C in the dark for 1 h. The samples were left in an ice bath to terminate the reaction, and 1 ml chloroform was added. The extracted bilirubin was calculated by the difference in absorbance between 464 and 530 nm (ε = 40 mM−1 cm−1). The HO-1 activity was expressed as micromoles of bilirubin per milligram of protein per hour.

The levels of TNF-α, IL-8, and IL-10 in the culture supernatants were measured using commercially available kits (OptEIA human TNF-α, IL-8, and IL-10 ELISA kits; BD Biosciences) following the manufacturer’s instructions.

Data represent means ± SEM of three independent experiments conducted in triplicate each. A p value <0.05 was considered significant by a paired two-tailed Student t test.

Hb was proteolytically activated with a typical bacterial serine protease, subtilisin A, to mimic an infection-mediated proteolysis (5), which released POX-active fragments of <10 kDa (Supplemental Fig. 1A, boxed), in a dose-responsive manner to subtilisin A. Prolonged reaction time led to excessive proteolysis and loss of the 10-kDa Hb POX fragments. To determine whether CD163 affects the Hb POX activity, we knocked in CD163 into HEK293T cells and then incubated the CD163+ HEK293T cells or mock-transfected control cells with activated Hb. The POX activity of the activated Hb was measured by a chemiluminescence assay. Fig. 1A (box) shows that within 10 min, the CD163+ HEK293T cells had reduced the POX activity by ∼80%, whereas the control cells were unresponsive, suggesting that the CD163 effectively blocked the Hb from producing O2.−.

FIGURE 1.

CD163 directly detoxifies Hb and rescues cells from Hb-induced apoptosis. (A) Top panel, Western blotting to confirm the knock-in of CD163 into HEK293T cells. Bottom panel, Hb POX activity was measured over time of incubation of subtilisin A–activated Hb with 2 × 105 CD163+ HEK293T cells or empty vector (EV) only transfected controls. Progressive decrease in Hb POX activity is observed with time and dose (box). ++, Higher dose of CD163+ HEK293T cells (106 cells). (B) Top panel, Western blot of the SU-DHL-1 and HEK293T cell membrane extracts probed for CD163. Bottom panel, The POX activity of 10 μg activated Hb after incubation for 15 min with increasing doses of the membrane protein extracts of SU-DHL-1 or HEK293T cells with or without pretreatment with anti-CD163. Haptoglobin, Hp1-1 (Hp), was used as a positive control. Progressive decrease in Hb POX activity is observed with increasing dose of CD163 (box). (C) Intracellular ROS production in SU-DHL-1 cells incubated with 15 μM native Hb or activated Hb with or without pretreatment with 0.1 μg/ml anti-CD163. (D) Dynamics of apoptosis in primary monocytes stimulated with 15 μM native Hb or activated Hb with or without pretreatment with 0.1 μg/ml anti-CD163. The cells were stained with annexin V-FITC and propidium iodide. Data represent the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.005.

FIGURE 1.

CD163 directly detoxifies Hb and rescues cells from Hb-induced apoptosis. (A) Top panel, Western blotting to confirm the knock-in of CD163 into HEK293T cells. Bottom panel, Hb POX activity was measured over time of incubation of subtilisin A–activated Hb with 2 × 105 CD163+ HEK293T cells or empty vector (EV) only transfected controls. Progressive decrease in Hb POX activity is observed with time and dose (box). ++, Higher dose of CD163+ HEK293T cells (106 cells). (B) Top panel, Western blot of the SU-DHL-1 and HEK293T cell membrane extracts probed for CD163. Bottom panel, The POX activity of 10 μg activated Hb after incubation for 15 min with increasing doses of the membrane protein extracts of SU-DHL-1 or HEK293T cells with or without pretreatment with anti-CD163. Haptoglobin, Hp1-1 (Hp), was used as a positive control. Progressive decrease in Hb POX activity is observed with increasing dose of CD163 (box). (C) Intracellular ROS production in SU-DHL-1 cells incubated with 15 μM native Hb or activated Hb with or without pretreatment with 0.1 μg/ml anti-CD163. (D) Dynamics of apoptosis in primary monocytes stimulated with 15 μM native Hb or activated Hb with or without pretreatment with 0.1 μg/ml anti-CD163. The cells were stained with annexin V-FITC and propidium iodide. Data represent the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.005.

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To test whether in situ mCD163 directly inhibits Hb POX, we added increasing doses of the SU-DHL-1 membrane extract (enriched in mCD163) to Hb. We found that the Hb POX activity diminished dose-dependently of the membrane extract, both in the presence and absence of Hp (Fig. 1B, Supplemental Fig. 1B). Incubation with 50 μg SU-DHL-1 membrane extract reduced 80% of the POX activity. Addition of Hp (Hp1-1 isoform) (34) further reduced the POX activity dose-dependently of the membrane extract. Furthermore, when the SU-DHL-1 membrane extract was preincubated with anti-CD163, the inhibition of the POX activity was abrogated dose-dependently of anti-CD163, suggesting that mCD163 directly and specifically downregulates Hb POX activity.

Next, we measured the dynamics of ROS production within the SU-DHL-1 cells when challenged with Hb, with or without preincubation with anti-CD163. Fig. 1C (left panel) shows that activated Hb induced ∼75% higher ROS production than did native Hb. In the presence of functional CD163, the Hb-generated intracellular ROS was halved compared with when CD163 was preblocked using an Ab (Fig. 1C, right panel). Furthermore, control cells (HEK293T and HepG2) devoid of CD163 succumbed to Hb, showing increased intracellular ROS and concomitant cell death (Supplemental Fig. 1C), supporting the protective role of CD163 against cytotoxic Hb. Ex vivo real-time quenching activity of Hb POX by CD163 was also observed in primary monocytes (Supplemental Videos 1–7, Supplemental Fig. 1D).

To demonstrate the biological significance of CD163-mediated scavenging and inhibition of Hb redox reactivity, we examined the status of the cell survival/death when the Hb-generated intracellular ROS were allowed to accumulate. Additionally, we queried the consequence of blocking CD163 when plasma Hb reaches concentrations as high as those of severe hemolysis during which Hp is depleted (35). We measured the dynamics of apoptosis when primary monocytes were challenged with Hb with or without blocking of CD163 using Ab. FACS analyses using annexin V-FITC and propidium iodide consistently showed that Hb induced ∼50% more apoptosis when CD163 was blocked (Fig. 1D, Supplemental Fig. 1E). Notably, Hb-induced apoptosis was suppressed by the activity of fully functional CD163. Taken together, our findings suggest that CD163 could directly shield monocytes from Hb POX–induced cytotoxicity during a severe hemolysis.

Monocytes exposed to inflammatory stimuli are known to shed CD163 (20). To examine the effects of the highly inflammatory Hb POX on mCD163, we stimulated SU-DHL-1 cells with 15 μM native or proteolytically activated Hb and measured the density of mCD163 on the cells by FACS. We found that the level of mCD163 on the monocytes started to decline within 10 min of stimulation with native Hb, down to ∼60% by 1 h but recovered completely within 3–4 h (Fig. 2A, 2B). In contrast, activated Hb induced a more dramatic and steeper drop of mCD163 to ∼30%, and the cells recovered only up to 50% of the mCD163 after 4 h. Reciprocal to mCD163, the level of sCD163 in the culture supernatant increased during 60 min (Fig. 2C). Compared to native Hb, the activated Hb induced twice the amount of shedding by 60 min. The Hb-mediated regulation of the level of mCD163 was specific because the housekeeping protein PMCA ATPase remained unaffected (Fig. 2B). To preclude any possible effect of endotoxin contamination on the Hb-induced shedding, both native and activated Hb were tested and found to contain ≤0.05 EU/ml. Consistent with FACS analysis (Fig. 2A) and immunoblotting (Fig. 2B), immunofluorescence microscopy showed fewer CD163+ cells at 60 min poststimulation (Fig. 2D). Our data suggest that the monocytes shed mCD163 when they encounter Hb, particularly, the redox active Hb POX.

FIGURE 2.

Hb induces shedding of CD163 from monocyte membrane. (A) FACS analysis of mCD163 density on SU-DHL-1 cells treated with 15 μM native Hb or activated Hb during 4 h. Data were normalized against a plasma membrane–localized housekeeping protein, PMCA ATPase. BSA (15 μM)-treated cells served as negative control. *p < 0.05, **p < 0.005 compared with untreated control. (B) Western blot analysis of mCD163 and PMCA ATPase (loading control) in membrane extracts of SU-DHL-1 cells treated with 15 μM Hb during 4 h. (C) sCD163 in the culture supernatant was measured using a human CD163-specific sandwich ELISA. Data were normalized against untreated cells. *p < 0.05. (D) Immunofluorescence analysis of mCD163 on cells treated with 15 μM native Hb or activated Hb for up to 60 min. Scale bars, 10 μm. Images were acquired using Axio Observer Z1 fluorescence microscope (Zeiss) under ×32 air objective and are representative of three independent experiments.

FIGURE 2.

Hb induces shedding of CD163 from monocyte membrane. (A) FACS analysis of mCD163 density on SU-DHL-1 cells treated with 15 μM native Hb or activated Hb during 4 h. Data were normalized against a plasma membrane–localized housekeeping protein, PMCA ATPase. BSA (15 μM)-treated cells served as negative control. *p < 0.05, **p < 0.005 compared with untreated control. (B) Western blot analysis of mCD163 and PMCA ATPase (loading control) in membrane extracts of SU-DHL-1 cells treated with 15 μM Hb during 4 h. (C) sCD163 in the culture supernatant was measured using a human CD163-specific sandwich ELISA. Data were normalized against untreated cells. *p < 0.05. (D) Immunofluorescence analysis of mCD163 on cells treated with 15 μM native Hb or activated Hb for up to 60 min. Scale bars, 10 μm. Images were acquired using Axio Observer Z1 fluorescence microscope (Zeiss) under ×32 air objective and are representative of three independent experiments.

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Because sCD163 has been shown to bind Hb-Hp complex in vitro (23), we queried whether sCD163 could still bind to Hb when Hp is depleted under conditions of severe hemolysis. We showed that Hb bound directly and dose-dependently to sCD163 in the absence of Hp (Fig. 3A), with the activated Hb binding more strongly than native Hb. Coimmunoprecipitation studies confirmed the specific interaction between sCD163 and Hb (Supplemental Fig. 2A). Redox-active extracellular Hb was reported to aggregate and induce cytotoxicity (36), hence necessitating the rapid inhibition of Hb POX even before its uptake into cells. This prompted us to investigate whether binding of sCD163 to Hb could affect the Hb POX activity extracellularly. We found that the Hb POX activity decreased significantly and dose-dependently of sCD163, correlating with reaction time (Fig. 3B, box). Within 60 min, up to 70% of the POX activity was suppressed in the presence of 10 ng/ml sCD163, whereas the control protein, BSA, had no effect on the POX activity, confirming the specificity of sCD163 toward Hb.

FIGURE 3.

sCD163 binds and quenches Hb POX, and the sCD163-Hb complex is internalized by monocytes. (A) ELISA shows dose-dependent interaction between sCD163 and native or activated Hb (0–1 μM) when 0.1 μg/ml sCD163 was immobilized onto microplates. (B) POX activity of 10 μg activated Hb incubated with sCD163 or BSA (0–10 ng/ml) during 60 min. Red box indicates progressive decrease in Hb POX activity induced by 10 ng/ml sCD163. *p < 0.05, **p < 0.005 compared with untreated control. (C) FACS analysis shows dose-dependent effect of activated Hb on mCD163 over time in the presence or absence of 10 ng/ml sCD163. *p < 0.05 compared with 0 ng/ml sCD163 controls. (D) Purified sCD163-FITC–activated Hb complex (1.5 μM) (green) was incubated for 15–45 min with primary monocytes predepleted of mCD163 and tracked by confocal microscopy. (E) mCD163 predepleted monocytes were incubated with 1.5 μM sCD163-FITC–activated Hb complex and 10 μg/ml Alexa 647-transferrin (early and recycling endosomal marker) for up to 90 min with or without 5 μg/ml CHX pretreatment for 60 min. The localization of sCD163 and Hb was tracked by immunostaining. Images were obtained using the LSM 510 META confocal microscope under ×100 oil objective. Scale bars in (D) and (E), 5 μm. Images are representative of three independent experiments using primary monocytes from a single healthy donor.

FIGURE 3.

sCD163 binds and quenches Hb POX, and the sCD163-Hb complex is internalized by monocytes. (A) ELISA shows dose-dependent interaction between sCD163 and native or activated Hb (0–1 μM) when 0.1 μg/ml sCD163 was immobilized onto microplates. (B) POX activity of 10 μg activated Hb incubated with sCD163 or BSA (0–10 ng/ml) during 60 min. Red box indicates progressive decrease in Hb POX activity induced by 10 ng/ml sCD163. *p < 0.05, **p < 0.005 compared with untreated control. (C) FACS analysis shows dose-dependent effect of activated Hb on mCD163 over time in the presence or absence of 10 ng/ml sCD163. *p < 0.05 compared with 0 ng/ml sCD163 controls. (D) Purified sCD163-FITC–activated Hb complex (1.5 μM) (green) was incubated for 15–45 min with primary monocytes predepleted of mCD163 and tracked by confocal microscopy. (E) mCD163 predepleted monocytes were incubated with 1.5 μM sCD163-FITC–activated Hb complex and 10 μg/ml Alexa 647-transferrin (early and recycling endosomal marker) for up to 90 min with or without 5 μg/ml CHX pretreatment for 60 min. The localization of sCD163 and Hb was tracked by immunostaining. Images were obtained using the LSM 510 META confocal microscope under ×100 oil objective. Scale bars in (D) and (E), 5 μm. Images are representative of three independent experiments using primary monocytes from a single healthy donor.

Close modal

To query whether sCD163 would influence the level of mCD163 when the monocytes encounter activated cell-free Hb, we supplemented the cells with 0, 5, and 10 ng/ml purified sCD163 followed by stimulation with 0.1 or 1 μM activated Hb. Flow cytometry results indicated that the presence of sCD163 dose-dependently reduced the Hb-triggered shedding of mCD163 (Fig. 3C, Supplemental Fig. 2B, 2C). This suggests that sCD163 exerts a negative feedback on Hb-induced shedding of mCD163, implicating a protective role of sCD163 on mCD163, possibly to maintain the level of mCD163 while sequestering Hb.

Pathogens have evolved efficient heme scavenging strategies to usurp iron from the host hemoproteins (37). Because sCD163 appears to influence the level of mCD163, we hypothesized that the sCD163-Hb complex might be recruited back to the monocyte while simultaneously sequestering the heme iron from the microbial invaders. To test our hypothesis, we studied the fate of sCD163 by incubating the complex of sCD163-FITC–conjugated Hb (activated form) with primary monocytes, which had been depleted of mCD163 (Supplemental Fig. 3A, top panel). The fate of sCD163-FITC-Hb complex on and/or in the mCD163-deficient monocytes was tracked by confocal microscopy. We found that the complex was recruited to the cell membrane within 15 min (Fig. 3D) and internalized by 45 min. However, sCD163 by itself or sCD163-FITC-Hb complex, in the absence of serum, did not bind to cells (Supplemental Fig. 3A, bottom panel), suggesting the potential involvement of serum proteins in trafficking the sCD163-Hb complex into the monocytes. The internalized sCD163 was colocalized intracellularly with transferrin, an early recycling endosome marker (38, 39) (Fig. 3E, left panel). By 90 min, the sCD163 reappeared on the cell membrane, which is consistent with the time at which the Hb-treated monocytes started to recover mCD163 (Fig. 2). To examine whether CD163 from the recruited complex of sCD163-Hb reappeared as mCD163 or whether the restored level of mCD163 arose from new protein synthesis, we applied the protein synthesis inhibitor CHX (40) to the cells prior to treatment with the sCD163-Hb complex. Fig. 3E (right panel) shows that CHX treatment did not compromise the recovery of mCD163, indicating that the mCD163 level was not attributable to de novo protein synthesis, but rather, it likely originated from the internalized sCD163.

To identify the potential receptor involved in the recruitment of the sCD163-Hb complex into monocytes, we tested the possible role of FcγR because it mediates uptake of oxidized protein complexes from the plasma (41). This prompted us to examine the potential role of IgG, the known ligand of FcγR, which might participate in the sCD163-Hb interactome. We found that indeed Hb in the sCD163-Hb complex coimmunoprecipitated with IgG from the serum of healthy individuals (Supplemental Fig. 3B). The ELISA results corroborated and established a dose-dependent interaction between the sCD163-FITC-Hb complex and the immobilized IgG (Fig. 4A). No binding occurred with FITC-BSA control, suggesting that Hb but not sCD163 in the Hb-sCD163 complex binds to IgG. Furthermore, in the absence of sCD163, Hb displayed reduced affinity for IgG. Because sCD163 binds Hb (Fig. 3), we sought to test whether purified IgG, Hb, and sCD163 would form a complex in vitro. Real-time biointeraction using surface plasmon resonance analysis showed strong binding between IgG and Hb (KD = 1.15 × 10−7 M) and between IgG, Hb, and sCD163 (KD = 2.25 × 10−9 M), producing shift and supershift, respectively, in a dose-dependent manner when the proteins were injected successively onto the IgG-immobilized chip (Fig. 4B, 4C). The specificity of the interaction between Hb and IgG was affirmed by the supershift produced by anti-Hb (Supplemental Fig. 3C).

FIGURE 4.

FcγR facilitates the endocytosis of the sCD163-Hb-IgG complex into monocytes. (A) ELISA to show the dose-dependent binding of sCD163-FITC-Hb complex to IgG immobilized on MaxiSorp plates. FITC-BSA was used as negative control in place of FITC-Hb. All the readings were subtracted from the values obtained with addition of Hb-FITC alone. *p < 0.05. (B and C) Representative sensograms of three independent surface plasmon resonance experiments showing the dose-dependent binding profiles between immobilized IgG to (B) Hb (0.2–0.8 μM) and (C) Hb (0.2 μM) plus sCD163 (2.5–10 ng/ml). Response units (RU) for (B) were dual referenced against BSA-N-acetylglucosamine–immobilized reference flow cell and BSA (0.2–0.8 μM) whereas (C) was referenced against sCD163 only (without Hb) controls. Dashed lines represent the curve fitting. (D) Wild-type, CD64-, CD32-, and CD16-silenced primary monocytes were incubated with the sCD163-Hb-IgG complex (0.5–2 μM) for 30 min and endocytosis was quantitated using the CyAn ADP flow cytometer on the FITC channel. Data are representative of three independent experiments using primary monocytes from a single healthy donor.

FIGURE 4.

FcγR facilitates the endocytosis of the sCD163-Hb-IgG complex into monocytes. (A) ELISA to show the dose-dependent binding of sCD163-FITC-Hb complex to IgG immobilized on MaxiSorp plates. FITC-BSA was used as negative control in place of FITC-Hb. All the readings were subtracted from the values obtained with addition of Hb-FITC alone. *p < 0.05. (B and C) Representative sensograms of three independent surface plasmon resonance experiments showing the dose-dependent binding profiles between immobilized IgG to (B) Hb (0.2–0.8 μM) and (C) Hb (0.2 μM) plus sCD163 (2.5–10 ng/ml). Response units (RU) for (B) were dual referenced against BSA-N-acetylglucosamine–immobilized reference flow cell and BSA (0.2–0.8 μM) whereas (C) was referenced against sCD163 only (without Hb) controls. Dashed lines represent the curve fitting. (D) Wild-type, CD64-, CD32-, and CD16-silenced primary monocytes were incubated with the sCD163-Hb-IgG complex (0.5–2 μM) for 30 min and endocytosis was quantitated using the CyAn ADP flow cytometer on the FITC channel. Data are representative of three independent experiments using primary monocytes from a single healthy donor.

Close modal

To investigate whether the sCD163-Hb-IgG complex was endocytosed via interaction with FcγR on the primary monocytes, we performed flow cytometry after incubation with increasing doses of purified complex of sCD163, FITC-Hb, and IgG with wild-type cells and FcγR knockdown cells. The efficiency of knockdown of all three types of FcγRs, that is, FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) by the respective siRNA pool (Table I), was verified by the loss of protein at 48 h after nucleofection (Supplemental Fig. 3D). The sCD163-Hb-IgG complex was readily endocytosed by wild-type primary monocytes in a dose-dependent manner (Fig. 4D, top panel). However, the cells knocked down of CD64, CD32, or CD16 showed substantially reduced endocytosis of the sCD163-Hb-IgG complex (Fig. 4D, bottom panel). CD64 knockdown, in particular, compromised the binding of the sCD163-Hb-IgG complex to the greatest extent when compared with CD32 or CD16 knockdown. This could probably be due to the higher affinity of CD64 toward IgG compared with CD32 or CD16 (42). Triple knockdown of all the FcγR types almost completely abrogated the binding of the sCD163-Hb-IgG complex to the cells. The negative controls, BSA, sCD163, sCD163-IgG, and Hb-IgG did not bind to the cells, indicating that the sCD163-Hb complex was specifically endocytosed via interaction with IgG, the ligand that bridges the sCD163-Hb complex to FcγR on the monocyte.

Next, we quantified and tracked the subcellullar localization of CD163 after endocytosis of the sCD163-Hb-IgG complex into primary monocytes. The monocytes were predepleted of mCD163 followed by treatment with CHX to block subsequent de novo synthesis of CD163. Results showed that within 15 min, CD163 was detected in the membrane fraction, indicative of binding of the sCD163-Hb-IgG complex to the membrane (Fig. 5A). Within 30–45 min, CD163 was localized in the cytoplasm, corroborating the endocytosis of the sCD163-Hb-IgG complex, and this was effectively blocked by pretreatment with chlorpromazine, an inhibitor of endocytosis (43). By 90 min, the internalized CD163 reappeared on the membrane and this was abolished when the cells were pretreated with monensin, a known inhibitor of recycling endosomes (44). When the cells were simultaneously pretreated with both chlorpromazine and monensin, CD163 was only observed on the cell membrane throughout the 90 min duration, indicating that both the endocytosis of the sCD163-Hb-IgG complex and subsequent recycling of the endocytosed CD163 were compromised.

FIGURE 5.

The endocytosed sCD163 is recycled into mCD163 whereas the internalized Hb is catabolized. (A) Primary monocytes predepleted of mCD163 and treated with 5 μg/ml CHX were incubated with 1.5 μM sCD163-Hb-IgG complex for up to 90 min with or without pretreatment with 70 μM chlorpromazine (inhibitor of endocytosis) and 20 μM monensin (inhibitor of early endosome recycling) for 60 min. CD163 was quantified in the membrane and cytosol fractions using sandwich ELISA. (B) Top panel, Purity of membrane/cytosol fractions was tested using membrane (CD64) or cytosolic (tubulin) markers. Bottom panel, mCD163-depleted primary monocytes were incubated with either biotinylated sCD163 alone or biotin sCD163-Hb-IgG complex for up to 90 min. Biotinylated sCD163 was quantified in membrane or cytosolic fractions using streptavidin-HRP by ELISA. (C) Primary monocytes were stimulated with increasing doses of Hb (0.1, 1 μM) for up to 180 min, and HO-1 activity (μmoles bilirubin/mg protein/h) was measured by spectrometric quantitation of bilirubin in the presence of excess substrate. Data are representative of three independent experiments using primary monocytes from a single healthy donor. *p < 0.05, **p < 0.005.

FIGURE 5.

The endocytosed sCD163 is recycled into mCD163 whereas the internalized Hb is catabolized. (A) Primary monocytes predepleted of mCD163 and treated with 5 μg/ml CHX were incubated with 1.5 μM sCD163-Hb-IgG complex for up to 90 min with or without pretreatment with 70 μM chlorpromazine (inhibitor of endocytosis) and 20 μM monensin (inhibitor of early endosome recycling) for 60 min. CD163 was quantified in the membrane and cytosol fractions using sandwich ELISA. (B) Top panel, Purity of membrane/cytosol fractions was tested using membrane (CD64) or cytosolic (tubulin) markers. Bottom panel, mCD163-depleted primary monocytes were incubated with either biotinylated sCD163 alone or biotin sCD163-Hb-IgG complex for up to 90 min. Biotinylated sCD163 was quantified in membrane or cytosolic fractions using streptavidin-HRP by ELISA. (C) Primary monocytes were stimulated with increasing doses of Hb (0.1, 1 μM) for up to 180 min, and HO-1 activity (μmoles bilirubin/mg protein/h) was measured by spectrometric quantitation of bilirubin in the presence of excess substrate. Data are representative of three independent experiments using primary monocytes from a single healthy donor. *p < 0.05, **p < 0.005.

Close modal

To validate the recycling of sCD163 into mCD163, we incubated primary monocytes with either biotinylated sCD163 alone or as a preformed complex of biotin sCD163-Hb-IgG and tracked the subcellular localization of sCD163 using streptavidin-HRP. The purity of the membrane/cytosol fractions was assessed using membrane (CD64) or cytosolic (tubulin) markers (Fig. 5B, top panel). By 15 min, sCD163 was detected in the membrane fraction and it was endocytosed within 30–45 min (Fig. 5B, bottom panel). By 90 min, sCD163 reappeared on the membrane, consistent with Fig. 5A, validating that sCD163 from the endocytosed sCD163-Hb-IgG complex was recycled to the membrane. Thus far, our results corroborate that mCD163 plays a major role in frontline defense as it binds Hb to reduce the POX activity, whereas the shedded sCD163 further scavenges plasma Hb, re-enters the monocyte, and undergoes recycling into mCD163, thus completing the autocrine cycle of Hb detoxification and CD163 renewal.

Because the endocytosed sCD163 is recycled to the cell surface, we queried the fate of the internalized Hb. HO-1 is an enzyme responsible for the catabolism of heme into biliverdin, carbon monoxide, and iron (45). Results showed that Hb induced a 70% increase in the HO-1 activity relative to negative control (Fig. 5C). Importantly, activated Hb induced 30% higher HO-1 activity compared with native Hb. Conceivably, this timely induction of HO-1 activity detoxifies the internalized Hb and preempts the avalanche of superoxide radicals resulting from the endocytosed redox-active Hb.

Next, we queried the cellular physiological significance of the monocyte-derived sCD163. Because monocytes are in contact with endothelial cells in vivo, it is conceivable that sCD163 acts in a paracrine fashion to communicate /alert the proximal cells of the imminent presence of cytotoxic Hb. To test this, we used primary HMVEC, which are known to endogenously express FcγRII (CD32) (26) but lack CD163 (46). We then measured the induction of HO-1 in HMVEC incubated for 6 h with increasing doses of the sCD163-Hb-IgG complex. Results showed that when compared with just Hb alone or other negative controls, the sCD163-Hb-IgG complex upregulated HO-1 levels by 3-fold (Fig. 6A). The induction of HO-1 was dose-dependent of the sCD163-Hb-IgG complex (Fig. 6A, box). This indicates that sCD163 and IgG mediate the Hb-induced transactivation of the endothelial cells. After internalization into HMVEC, the Hb is catabolized by HO-1.

FIGURE 6.

Hb induces cell–cell communication between monocytes and endothelial cells via sCD163 and IgG. (A) HMVEC were stimulated with increasing doses of Hb alone or preformed sCD163-Hb-IgG complex for 6 h, and HO-1 protein was quantified by FACS. Hemin was used as positive control. (B) Cytokine production when HMVEC were cocultured for 24 h with CD163+ primary monocytes or CD163 THP-1 cells in the presence of 0.5 mg/ml Hb. (C) Immunostaining to track localization of sCD163 and Hb in HMVEC cocultured with either CD163+ primary monocytes or CD163 THP-1 cells in the presence of Hb for 45 min. All images were obtained using the LSM 510 META confocal microscope under ×100 oil objective. Scale bars, 10 μm. Data represent the means ± SEM of three independent experiments with primary monocytes from single donor. **p < 0.005.

FIGURE 6.

Hb induces cell–cell communication between monocytes and endothelial cells via sCD163 and IgG. (A) HMVEC were stimulated with increasing doses of Hb alone or preformed sCD163-Hb-IgG complex for 6 h, and HO-1 protein was quantified by FACS. Hemin was used as positive control. (B) Cytokine production when HMVEC were cocultured for 24 h with CD163+ primary monocytes or CD163 THP-1 cells in the presence of 0.5 mg/ml Hb. (C) Immunostaining to track localization of sCD163 and Hb in HMVEC cocultured with either CD163+ primary monocytes or CD163 THP-1 cells in the presence of Hb for 45 min. All images were obtained using the LSM 510 META confocal microscope under ×100 oil objective. Scale bars, 10 μm. Data represent the means ± SEM of three independent experiments with primary monocytes from single donor. **p < 0.005.

Close modal

To assess the potential Hb-induced crosstalk between the monocytes and endothelial cells, we cocultured the two cell types in the presence of Hb and measured the cytokine production by the cells. To confirm the significance of CD163 in this process, we employed CD163+ primary monocytes or CD163 THP-1 monocytes (control) (47). Fig. 6B (box) shows a synergistic increase in the production of TNF-α, IL-8, and IL-10 when the HMVEC were cocultured with CD163+ primary monocytes compared with THP-1 or when stimulated in isolation. This synergy was lost when the monocytes were preincubated with anti-CD163, suggesting that the monocyte-derived sCD163 is indispensable for the activation of endothelial cells, which lacks endogenous CD163. Furthermore, stimulation of HMVEC with the sCD163-Hb-IgG complex elicited higher amounts of TNF-α and IL-8 compared with individual protein controls (Supplemental Fig. 4A), corroborating our coculture results (Fig. 6B). Thus, the monocyte-derived sCD163 mediates the paracrine activation of the proximal endothelial cells to systemically alert the human body on the imminent toxicity of plasma Hb.

Next, we tracked CD163 and Hb in the HMVEC, which had been cocultured with primary monocytes or THP-1 monocytes for 45 min. Fig. 6C shows that both CD163 and Hb are colocalized in HMVEC only when cocultured with CD163+ primary monocytes but not with CD163 THP-1 cells. Consistently, the colocalization of Hb and CD163 within the HMVEC was observed only when the two proteins were presented as a complex of sCD163-Hb-IgG (Supplemental Fig. 4B). Additionally, in the absence of IgG, no endocytosis of Hb was detected in HMVEC even when cocultured with CD163+ monocytes, suggesting that IgG is required to bridge the sCD163-Hb complex to FcγR on the HMVEC. Also, when HMVEC and CD163+ monocytes were cocultured in the absence of Hb, no CD163 entered the HMVEC. By live cell imaging, we have demonstrated the sCD163-mediated interaction between monocytes and the proximal endothelial cells in the presence of Hb and IgG (Supplemental Videos 8–11).

Altogether, we have shown that sCD163 is recycled to achieve homeostasis of mCD163 on the monocytes and, simultaneously, the sCD163-Hb complex induces the monocytes to collaborate with the proximal endothelial cells via IgG-FcγR. Whereas the dynamic importation of plasma Hb-sCD163 shuttles the cytotoxic cargo of Hb into the monocytes in an autocrine cycle, it also transactivates the endothelial cells in a paracrine manner to secrete cytokines to raise a systemic alert on the imminent danger from the redox-active Hb (Fig. 7). The internalized Hb is catabolized by HO-1 in both monocytes and endothelial cells.

FIGURE 7.

A hypothetical model of Hp-independent intravascular detoxification and clearance of cell-free Hb by CD163. Hemolysis ruptures RBCs and releases cytotoxic Hb into the plasma. Upon recruiting Hb, the mCD163 directly suppresses the POX activity of Hb in situ on the monocyte membrane. Hb induces shedding of mCD163 into the plasma, and the resulting sCD163 further captures and quenches the residual redox-reactive Hb. Subsequently, IgG interacts with the sCD163-Hb complex. The sCD163-Hb-IgG complex then 1) elicits an autocrine loop of endocytosis via FcγR on the monocyte and subsequent recycling of the internalized sCD163 via endosomes to restore mCD163 homeostasis, whereas the internalized Hb is catabolized by HO-1; and 2) induces the paracrine transactivation of the neighboring endothelial cells (represented by HMVEC tested in this study) lining the blood vessel causing them to upregulate HO-1 and secrete cytokines to mount a systemic defense against Hb.

FIGURE 7.

A hypothetical model of Hp-independent intravascular detoxification and clearance of cell-free Hb by CD163. Hemolysis ruptures RBCs and releases cytotoxic Hb into the plasma. Upon recruiting Hb, the mCD163 directly suppresses the POX activity of Hb in situ on the monocyte membrane. Hb induces shedding of mCD163 into the plasma, and the resulting sCD163 further captures and quenches the residual redox-reactive Hb. Subsequently, IgG interacts with the sCD163-Hb complex. The sCD163-Hb-IgG complex then 1) elicits an autocrine loop of endocytosis via FcγR on the monocyte and subsequent recycling of the internalized sCD163 via endosomes to restore mCD163 homeostasis, whereas the internalized Hb is catabolized by HO-1; and 2) induces the paracrine transactivation of the neighboring endothelial cells (represented by HMVEC tested in this study) lining the blood vessel causing them to upregulate HO-1 and secrete cytokines to mount a systemic defense against Hb.

Close modal

Redox-active extracellular Hb results in oxidative stress and cytotoxicity (36). Hence, it is crucial for our blood cells to counteract the pro-oxidative Hb at the immediate outset even before its uptake into the cells. Although, it is known that mCD163 directly interacts with Hb independent of Hp (18), the functional impact of this interaction on Hb redox reactivity remains enigmatic. We have discovered and mapped in detail a novel two-pass detoxification mechanism of Hb by CD163, independent of Hp. First, at the outset of the encounter with plasma Hb, mCD163 directly inhibits the Hb POX activity in situ and rescues monocytes from Hb-triggerred apoptosis (Fig. 1, Supplemental Fig. 1). The mCD163 is also shedded into the plasma (Fig. 2). The resulting sCD163 scavenges residual free Hb and upon endocytosis of the sCD163-Hb complex via IgG-FcγR, the sCD163 is recycled to restore homeostasis of mCD163 in an autocrine cycle, whereas the internalized Hb is catabolized by HO-1. Second, this novel mechanism of clearance of Hb by CD163 transactivates the proximal endothelial cells in a paracrine fashion, causing these cells to upregulate HO-1 and inducing secretion of cytokines, thus mounting a systemic immune defense against Hb.

Besides suppressing Hb POX activity at the monocyte surface, CD163, which is cotranslocated into the cells, also downregulates the generation of intracellular ROS from the endocytosed Hb (Fig. 1C, Supplemental Videos 1–7). In the absence of such a mechanism as illustrated in this study with CD163 cells, the hydrophobic nature of the Hb heme could readily permeate the cells, inducing free radicals, which would lead to lipid peroxidation and cell death (48, 49). Having established the direct inhibition of the redox activity of Hb by mCD163 (independent of Hp), we then queried the pathophysiological significance of sCD163 under severe hemolytic condition. We found that sCD163 binds excess plasma Hb dose-dependently and rapidly downregulates the Hb POX activity (Fig. 3A, 3B). Thus, it is conceivable that during a severe hemolysis, such a “capture and quench” action by sCD163 would constitute an effective host defense strategy to sequester the heme iron and pre-empt its redox activity. Of particular importance is that the resulting sCD163-Hb complex, which is still redox-active, must be rapidly and efficiently removed from circulation so as to subvert the Hb iron–mediated cytotoxicity. To this end, we identified IgG as a novel interaction partner participating with the sCD163-Hb complex to enable endocytosis of the sCD163-Hb-IgG complex via FcγR into the monocytes (Fig. 4). We found that interaction of the sCD163-Hb complex with IgG is a critical prerequisite for subsequent endocytosis of the complex into monocytes via FcγR (Supplemental Fig. 3).

Following endocytosis of the sCD163-Hb-IgG complex, the internalized sCD163 is recycled via early endosomes to the cell membrane to restore mCD163 (Fig. 5). This is also supported by reports documenting that many endocytic receptors are recycled when internalized into the cell (50) and that early endosomes serve as the focal points of the endocytic pathway, enabling them to undergo fast recycling to the plasma membrane (51). During a severe hemolysis, such a dynamic and efficient recycling of sCD163 would presumably potentiate the recovery of mCD163, which acts to fortify the monocytes against the cytotoxic avalanche of free radicals generated by the cell-free Hb POX. Furthermore, using coculture experiments, we established that sCD163, in collaboration with IgG, confers Hb-scavenging ability to the proximal endothelial cells and also transactivates them to respond against the Hb (Fig. 6, Supplemental Fig. 4). Such a crosstalk between monocytes and endothelial cells (mediated by sCD163-Hb-IgG complex via FcγR) mounts a systemic defense against toxic Hb. Overall, CD163 is dynamically deployed in a two-pass detoxification tactic to engage with and suppress the pro-oxidant activity of plasma Hb, whereas its residential level on the monocyte membrane is restored to homeostasis in an efficient autocrine cycle. Simultaneously, it also transactivates adjacent endothelial cells in a paracrine fashion to metabolize the endocytosed Hb and secrete cytokines to systemically alert the imminent presence of a danger molecule, Hb (Fig. 7).

We thank Tong Yan, National University of Singapore Centre for BioImaging Sciences, for technical assistance with the confocal microscopy.

This work was supported by the Ministry of Education, Singapore Grant T208B3109 and by the Biomedical Research Council/Agency for Science, Technology and Research, Singapore Grant 10/1/21/19/658. K.S. is a research scholar supported by these grants.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CHX

cycloheximide

Hb

hemoglobin

Hp

haptoglobin

HMVEC

human dermal microvascular endothelial cell

HO-1

heme oxygenase-1

mCD163

membrane-associated CD163

PMCA

plasma membrane calcium

POX

pseudoperoxidase

ROS

reactive oxygen species

sCD163

soluble CD163

siRNA

small interfering RNA.

1
Sadrzadeh
S. M.
,
Anderson
D. K.
,
Panter
S. S.
,
Hallaway
P. E.
,
Eaton
J. W.
.
1987
.
Hemoglobin potentiates central nervous system damage.
J. Clin. Invest.
79
:
662
664
.
2
Skaar
E. P.
,
Humayun
M.
,
Bae
T.
,
DeBord
K. L.
,
Schneewind
O.
.
2004
.
Iron-source preference of Staphylococcus aureus infections.
Science
305
:
1626
1628
.
3
Alayash
A. I.
2001
.
Oxidative mechanisms of hemoglobin-based blood substitutes.
Artif. Cells Blood Substit. Immobil. Biotechnol.
29
:
415
425
.
4
Puppo
A.
,
Halliwell
B.
.
1988
.
Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent?
Biochem. J.
249
:
185
190
.
5
Jiang
N.
,
Tan
N. S.
,
Ho
B.
,
Ding
J. L.
.
2007
.
Respiratory protein-generated reactive oxygen species as an antimicrobial strategy.
Nat. Immunol.
8
:
1114
1122
.
6
Du
R.
,
Ho
B.
,
Ding
J. L.
.
2010
.
Rapid reprogramming of haemoglobin structure-function exposes multiple dual-antimicrobial potencies.
EMBO J.
29
:
632
642
.
7
Xiang
M.
,
Fan
J.
,
Fan
J.
.
2010
.
Association of Toll-like receptor signaling and reactive oxygen species: a potential therapeutic target for posttrauma acute lung injury.
Mediators Inflamm
.
8
Hartl
D.
,
Lehmann
N.
,
Hoffmann
F.
,
Jansson
A.
,
Hector
A.
,
Notheis
G.
,
Roos
D.
,
Belohradsky
B. H.
,
Wintergerst
U.
.
2008
.
Dysregulation of innate immune receptors on neutrophils in chronic granulomatous disease.
J. Allergy Clin. Immunol.
121
:
375
382.e9
.
9
Gill
R.
,
Tsung
A.
,
Billiar
T.
.
2010
.
Linking oxidative stress to inflammation: Toll-like receptors.
Free Radic. Biol. Med.
48
:
1121
1132
.
10
Andersen
C. B.
,
Torvund-Jensen
M.
,
Nielsen
M. J.
,
de Oliveira
C. L.
,
Hersleth
H. P.
,
Andersen
N. H.
,
Pedersen
J. S.
,
Andersen
G. R.
,
Moestrup
S. K.
.
2012
.
Structure of the haptoglobin-haemoglobin complex.
Nature
489
:
456
459
.
11
Buehler
P. W.
,
Abraham
B.
,
Vallelian
F.
,
Linnemayr
C.
,
Pereira
C. P.
,
Cipollo
J. F.
,
Jia
Y.
,
Mikolajczyk
M.
,
Boretti
F. S.
,
Schoedon
G.
, et al
.
2009
.
Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.
Blood
113
:
2578
2586
.
12
Kristiansen
M.
,
Graversen
J. H.
,
Jacobsen
C.
,
Sonne
O.
,
Hoffman
H. J.
,
Law
S. K.
,
Moestrup
S. K.
.
2001
.
Identification of the haemoglobin scavenger receptor.
Nature
409
:
198
201
.
13
Law
S. K.
,
Micklem
K. J.
,
Shaw
J. M.
,
Zhang
X. P.
,
Dong
Y.
,
Willis
A. C.
,
Mason
D. Y.
.
1993
.
A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily.
Eur. J. Immunol.
23
:
2320
2325
.
14
Azarov
I.
,
He
X.
,
Jeffers
A.
,
Basu
S.
,
Ucer
B.
,
Hantgan
R. R.
,
Levy
A.
,
Kim-Shapiro
D. B.
.
2008
.
Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin.
Nitric Oxide
18
:
296
302
.
15
Lim
S. K.
,
Kim
H.
,
Lim
S. K.
,
bin Ali
A.
,
Lim
Y. K.
,
Wang
Y.
,
Chong
S. M.
,
Costantini
F.
,
Baumman
H.
.
1998
.
Increased susceptibility in Hp knockout mice during acute hemolysis.
Blood
92
:
1870
1877
.
16
Tolosano
E.
,
Fagoonee
S.
,
Hirsch
E.
,
Berger
F. G.
,
Baumann
H.
,
Silengo
L.
,
Altruda
F.
.
2002
.
Enhanced splenomegaly and severe liver inflammation in haptoglobin/hemopexin double-null mice after acute hemolysis.
Blood
100
:
4201
4208
.
17
Langlois
M. R.
,
Delanghe
J. R.
.
1996
.
Biological and clinical significance of haptoglobin polymorphism in humans.
Clin. Chem.
42
:
1589
1600
.
18
Schaer
D. J.
,
Schaer
C. A.
,
Buehler
P. W.
,
Boykins
R. A.
,
Schoedon
G.
,
Alayash
A. I.
,
Schaffner
A.
.
2006
.
CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.
Blood
107
:
373
380
.
19
Körmöczi
G. F.
,
Säemann
M. D.
,
Buchta
C.
,
Peck-Radosavljevic
M.
,
Mayr
W. R.
,
Schwartz
D. W.
,
Dunkler
D.
,
Spitzauer
S.
,
Panzer
S.
.
2006
.
Influence of clinical factors on the haemolysis marker haptoglobin.
Eur. J. Clin. Invest.
36
:
202
209
.
20
Droste
A.
,
Sorg
C.
,
Högger
P.
.
1999
.
Shedding of CD163, a novel regulatory mechanism for a member of the scavenger receptor cysteine-rich family.
Biochem. Biophys. Res. Commun.
256
:
110
113
.
21
Hintz
K. A.
,
Rassias
A. J.
,
Wardwell
K.
,
Moss
M. L.
,
Morganelli
P. M.
,
Pioli
P. A.
,
Givan
A. L.
,
Wallace
P. K.
,
Yeager
M. P.
,
Guyre
P. M.
.
2002
.
Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163.
J. Leukoc. Biol.
72
:
711
717
.
22
Weaver
L. K.
,
Hintz-Goldstein
K. A.
,
Pioli
P. A.
,
Wardwell
K.
,
Qureshi
N.
,
Vogel
S. N.
,
Guyre
P. M.
.
2006
.
Pivotal advance: activation of cell surface Toll-like receptors causes shedding of the hemoglobin scavenger receptor CD163.
J. Leukoc. Biol.
80
:
26
35
.
23
Møller
H. J.
,
Nielsen
M. J.
,
Maniecki
M. B.
,
Madsen
M.
,
Moestrup
S. K.
.
2010
.
Soluble macrophage-derived CD163: a homogenous ectodomain protein with a dissociable haptoglobin-hemoglobin binding.
Immunobiology
215
:
406
412
.
24
Chen
J.
,
Hobbs
W. E.
,
Le
J.
,
Lenting
P. J.
,
de Groot
P. G.
,
López
J. A.
.
2011
.
The rate of hemolysis in sickle cell disease correlates with the quantity of active von Willebrand factor in the plasma.
Blood
117
:
3680
3683
.
25
Belcher
J. D.
,
Marker
P. H.
,
Weber
J. P.
,
Hebbel
R. P.
,
Vercellotti
G. M.
.
2000
.
Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion.
Blood
96
:
2451
2459
.
26
Gröger
M.
,
Sarmay
G.
,
Fiebiger
E.
,
Wolff
K.
,
Petzelbauer
P.
.
1996
.
Dermal microvascular endothelial cells express CD32 receptors in vivo and in vitro.
J. Immunol.
156
:
1549
1556
.
27
Cao
W.
,
Lee
S. H.
,
Lu
J.
.
2005
.
CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells.
Biochem. J.
385
:
85
93
.
28
Nakano
M.
1990
.
Determination of superoxide radical and singlet oxygen based on chemiluminescence of luciferin analogs.
Methods Enzymol.
186
:
585
591
.
29
Hernuss
P.
,
Müller-Tyl
E.
,
Wicke
L.
.
1975
.
[Ozone and gynecologic radiotherapy]
.
Strahlentherapie
150
:
493
499
.
30
Philippidis
P.
,
Mason
J. C.
,
Evans
B. J.
,
Nadra
I.
,
Taylor
K. M.
,
Haskard
D. O.
,
Landis
R. C.
.
2004
.
Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis: antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery.
Circ. Res.
94
:
119
126
.
31
Stoop
J. W.
,
Zegers
B. J.
,
Sander
P. C.
,
Ballieux
R. E.
.
1969
.
Serum immunoglobulin levels in healthy children and adults.
Clin. Exp. Immunol.
4
:
101
112
.
32
Motterlini
R.
,
Foresti
R.
,
Bassi
R.
,
Calabrese
V.
,
Clark
J. E.
,
Green
C. J.
.
2000
.
Endothelial heme oxygenase-1 induction by hypoxia: modulation by inducible nitric-oxide synthase and S-nitrosothiols.
J. Biol. Chem.
275
:
13613
13620
.
33
Tenhunen
R.
,
Ross
M. E.
,
Marver
H. S.
,
Schmid
R.
.
1970
.
Reduced nicotinamide-adenine dinucleotide phosphate dependent biliverdin reductase: partial purification and characterization.
Biochemistry
9
:
298
303
.
34
Sadrzadeh
S. M.
,
Bozorgmehr
J.
.
2004
.
Haptoglobin phenotypes in health and disorders.
Am. J. Clin. Pathol.
121
(
Suppl
):
S97
S104
.
35
Levy
A. P.
,
Purushothaman
K. R.
,
Levy
N. S.
,
Purushothaman
M.
,
Strauss
M.
,
Asleh
R.
,
Marsh
S.
,
Cohen
O.
,
Moestrup
S. K.
,
Moller
H. J.
, et al
.
2007
.
Downregulation of the hemoglobin scavenger receptor in individuals with diabetes and the Hp 2-2 genotype: implications for the response to intraplaque hemorrhage and plaque vulnerability.
Circ. Res.
101
:
106
110
.
36
Kapralov
A.
,
Vlasova
I. I.
,
Feng
W.
,
Maeda
A.
,
Walson
K.
,
Tyurin
V. A.
,
Huang
Z.
,
Aneja
R. K.
,
Carcillo
J.
,
Bayir
H.
,
Kagan
V. E.
.
2009
.
Peroxidase activity of hemoglobin-haptoglobin complexes: covalent aggregation and oxidative stress in plasma and macrophages.
J. Biol. Chem.
284
:
30395
30407
.
37
Skaar
E. P.
2010
.
The battle for iron between bacterial pathogens and their vertebrate hosts.
PLoS Pathog.
6
:
e1000949
.
38
Barysch
S. V.
,
Aggarwal
S.
,
Jahn
R.
,
Rizzoli
S. O.
.
2009
.
Sorting in early endosomes reveals connections to docking- and fusion-associated factors.
Proc. Natl. Acad. Sci. USA
106
:
9697
9702
.
39
Hopkins
C. R.
1983
.
Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells.
Cell
35
:
321
330
.
40
Shankar
K.
,
Liu
X.
,
Singhal
R.
,
Chen
J. R.
,
Nagarajan
S.
,
Badger
T. M.
,
Ronis
M. J.
.
2008
.
Chronic ethanol consumption leads to disruption of vitamin D3 homeostasis associated with induction of renal 1,25 dihydroxyvitamin D3-24-hydroxylase (CYP24A1).
Endocrinology
149
:
1748
1756
.
41
Huang
Y.
,
Jaffa
A.
,
Koskinen
S.
,
Takei
A.
,
Lopes-Virella
M. F.
.
1999
.
Oxidized LDL-containing immune complexes induce Fc gamma receptor I-mediated mitogen-activated protein kinase activation in THP-1 macrophages.
Arterioscler. Thromb. Vasc. Biol.
19
:
1600
1607
.
42
Ravetch
J. V.
,
Bolland
S.
.
2001
.
IgG Fc receptors.
Annu. Rev. Immunol.
19
:
275
290
.
43
Mueller
A.
,
Kelly
E.
,
Strange
P. G.
.
2002
.
Pathways for internalization and recycling of the chemokine receptor CCR5.
Blood
99
:
785
791
.
44
Schaer
C. A.
,
Vallelian
F.
,
Imhof
A.
,
Schoedon
G.
,
Schaer
D. J.
.
2007
.
CD163-expressing monocytes constitute an endotoxin-sensitive Hb clearance compartment within the vascular system.
J. Leukoc. Biol.
82
:
106
110
.
45
Grochot-Przeczek
A.
,
Dulak
J.
,
Jozkowicz
A.
.
2012
.
Haem oxygenase-1: non-canonical roles in physiology and pathology.
Clin. Sci.
122
:
93
103
.
46
Hiraoka
A.
,
Horiike
N.
,
Akbar
S. M.
,
Michitaka
K.
,
Matsuyama
T.
,
Onji
M.
.
2005
.
Expression of CD163 in the liver of patients with viral hepatitis.
Pathol. Res. Pract.
201
:
379
384
.
47
Bächli
E. B.
,
Schaer
D. J.
,
Walter
R. B.
,
Fehr
J.
,
Schoedon
G.
.
2006
.
Functional expression of the CD163 scavenger receptor on acute myeloid leukemia cells of monocytic lineage.
J. Leukoc. Biol.
79
:
312
318
.
48
Jeney
V.
,
Balla
J.
,
Yachie
A.
,
Varga
Z.
,
Vercellotti
G. M.
,
Eaton
J. W.
,
Balla
G.
.
2002
.
Pro-oxidant and cytotoxic effects of circulating heme.
Blood
100
:
879
887
.
49
Umbreit
J.
2007
.
Methemoglobin—it’s not just blue: a concise review.
Am. J. Hematol.
82
:
134
144
.
50
Jovic
M.
,
Sharma
M.
,
Rahajeng
J.
,
Caplan
S.
.
2010
.
The early endosome: a busy sorting station for proteins at the crossroads.
Histol. Histopathol.
25
:
99
112
.
51
Chen
B.
,
Jiang
Y.
,
Zeng
S.
,
Yan
J.
,
Li
X.
,
Zhang
Y.
,
Zou
W.
,
Wang
X.
.
2010
.
Endocytic sorting and recycling require membrane phosphatidylserine asymmetry maintained by TAT-1/CHAT-1.
PLoS Genet.
6
:
e1001235
.

The authors have no financial conflicts of interest.

Supplementary data