Upregulation and Mitochondrial Sequestration of Hemoglobin Occur in Circulating Leukocytes during Critical Illness, Conferring a Cytoprotective Phenotype
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The classical role of hemoglobin in the erythrocytes is to carry oxygen from the lungs to the tissues via the circulation. However, hemoglobin also acts as a redox regulator and as a scavenger of the gaseous mediators nitric oxide (NO) and hydrogen sulfide (H2S). Here we show that upregulation of hemoglobin (α, β and δ variants of globin proteins) occurs in human peripheral blood mononuclear cells (PBMCs) in critical illness (patients with severe third-degree burn injury and patients with sepsis). The increase in intracellular hemoglobin concentration is a result of a combination of enhanced protein expression and uptake from the extracellular space via a CD163-dependent mechanism. Intracellular hemoglobin preferentially localizes to the mitochondria, where it interacts with complex I and, on the one hand, increases mitochondrial respiratory rate and mitochondrial membrane potential, and on the other hand, protects from H2O2-induced cytotoxicity and mitochondrial DNA damage. Both burn injury and sepsis were associated with increased plasma levels of H2S. Incubation of mononuclear cells with H2S induced hemoglobin mRNA upregulation in PBMCs in vitro. Intracellular hemoglobin upregulation conferred a protective effect against cell dysfunction elicited by H2S. Hemoglobin uptake also was associated with a protection from, and induced the upregulation of, HIF-1α and Nrf2 mRNA. In conclusion, PBMCs in critical illness upregulate their intracellular hemoglobin levels by a combination of active synthesis and uptake from the extracellular medium. We propose that this process serves as a defense mechanism protecting the cell against cytotoxic concentrations of H2S and other gaseous transmitters, oxidants and free radicals produced in critically ill patients.
Although hemoglobin is best known for its role as an oxygen carrier in erythrocytes, many biological functions of hemoglobin predate the evolution of the circulatory system and are used to modulate various redox functions and interactions with gaseous transmitters such as nitric oxide (NO) and hydrogen sulfide (H2S) (1, 2, 3, 4, 5). For instance, clams living in H2S-rich waters upregulate intracellular hemoglobin levels as a protective mechanism against H2S toxicity (5).
The current report describes a significant upregulation of hemoglobin in human peripheral blood mononuclear cells (PBMCs) in two distinct forms of critical illness (sepsis and severe burn injury) and highlights the consequences of this phenomenon in the context of oxidant-, free radical- and gasotransmitter-associated cytotoxicity.
Materials and Methods
All chemicals were obtained from Sigma-Aldrich, unless stated otherwise.
Human monocyte histiocytic lymphoma cells (U937) and human embryonic kidney epithelial cells (HEK239T17) were obtained from ATCC and maintained in RPMI1640 with 10% fetal bovine serum (Life Technologies).
Isolation of Human PBMCs
Blood samples from critically ill patients and from healthy volunteers were collected with the permission of the local institutional review boards (IRBs). For the component of the clinical study involving burn patients, adult patients with burns covering ≥30% of the total body surface area (TBSA) were enrolled. Patients received standard burn care (6) and blood samples were obtained at the time of admission (0 wk) and 3 wks later (3 wks). For the component of the clinical study involving septic patients, adult patients with freshly diagnosed sepsis according to the established sepsis criteria (7) were enrolled at the time of diagnosis (0 wk) and 1 wk later (1 wk). Age-matched healthy human subjects served as healthy controls. Leukocytes and plasma were isolated using Histopaque (Sigma-Aldrich) (8).
Protein Identification by Mass Spectrometry
Protein extract of isolated PBMCs were separated on SDS-PAGE, stained with Coomassie Brilliant Blue (Bio-Rad), and ∼15-kDa bands were excised from gel and subjected for identification by ESI-LC/MS/MS (4000 QTRAP with LC Packings capillary LC system, Applied Biosystems) (9).
Western blotting was performed as before (9, 10, 11) using: anti-hemoglobin α (Santa Cruz Biotechnology), antihemoglobin-β (Santa Cruz Biotechnology) and anti-hemoglobin β/γ/δ (Santa Cruz Biotechnology). Loading was normalized with anti-β-actin-HRP (Santa Cruz Biotechnology). Blots were developed with Supersignal West Pico Chemiluminescent (Thermo Scientific).
List of primers used in the present study.
5-CAG AGA GAA CCC ACC ATG GTG-3
5-GGT GGT GGT TCT GGA TGA AGG-3
5-CAG ACA CCA TGG TGC ATC TG-3
5-CTC CAA GAA ACT CAG GAA ACC C-3
5-GGA AGG CTC CTG GTT GTC TAC C-3
5-CGT TCT TCC ACG ACT GAA GGG-3
5-CAA ACA GAC ACC ATG GTG CAT C-3
5-GTC CAA GGG TAG ACC ACC AG-3
5-CTG GAA TCT ATA AAG AGG CTT GTA CAG C-3
5-GGT TTA GCC GTA GCA TTG GC-3
5-CCT CAG CCA GGA CCT CAC-3
5-CGT CTC CAT TAC CCA CAT ATC TC-3
5-CCA ACA GAA CAG AAA TTT CCA GC-3
5-GGT GAC CAG TGC AAT CAG G-3
5-CCT GAG ATG AGA CAT GTA GAC TGC-3
5-GAG AAC TGA TGG GCC AGA TC-3
5-GGT CAA GCA GAT TGC TAC AGG-3
5-GTC TCA CAG CTT GTC CAA GG-3
5-CCA CAG GTG TTT CAT CAG TAC AGA C-3
5-CCT CTC TCC TGG GAC ATC TGA G-3
5-CCA GAC AGG CTC TTC TCA ACC-3
5-GAT CAG TGC ATA ACA GCC TAA TCT C-3
5-GAC GCA TGT GAA TTT ATC CAG C-3
5-GGA CTC CAT TCA CTC CAG AAG C-3
5-CCC ATT GAC CTC TAC TAC CTT ATG G-3
5-GCC AAA TCC AAT TCT GAA GTC C-3
5-GGA TCC ATC GTG ACT TGT GG-3
5-GGA TAT TCC AGC TTG ACA TGA TGC-3
Measurement of Hemoglobin Concentration
Concentration of hemoglobin in plasma and RPMI was determined using the Hemoglobin Assay Kit (Sigma-Aldrich).
Flow Cytometric Analysis
PMBCs were fixed in 3.7% paraformaldehyde for 30 min at 4°C followed by three washes with PBS and permeabilized with 0.1% Triton-X containing PBS for 30 min, followed by three washes with PBS. CD45 (Santa Cruz Biotechnology Biotechnology) and primary antibody against hemoglobin-β was applied at 500× dilution in blocking buffer (1% BSA in PBS) for 1 h, followed by washing with PBS and subsequent incubation with Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 30 min. Samples were analyzed using a FACSArray Bioanalyzer (BD Biosciences).
U937 cells were treated for 24 h with 2, 20 or 200 mg/dL of purified hemoglobin, isolated from the blood of healthy volunteers, then washed two times with RPMI and fixed with 4% paraformaldehyde in slide chambers (Lab-Tek). Cells were triple stained with: DAPI (Life Technologies), Alexa Fluor 594 conjugated (Mix-n-Stain, Sigma-Aldrich) to anti-CD163 antibody and Alexa Fluor 488 conjugated (Mix-n-Stain, Sigma-Aldrich) to anti-hemoglobin α. Images were visualized using Nikon Eclipse 80i fluorescent microscope with CoolSNAP HQ camera and analyzed with NIS Elements BR3.10 software.
Proximity Ligation Assay (PLA)
In situ protein:protein proximity/interaction studies were performed with Duolink in situ (Sigma-Aldrich) as before (10) using the following antibodies: anti-CD163 (Santa Cruz Biotechnology), anti-hemoglobin α (Abcam), anti-cadherin (Santa Cruz Biotechnology), anti-Na+K+ATPase (Santa Cruz Biotechnology), anti-lamin (Cell Signaling Technology), anti-PDI (Cell Signaling Technology), anti-histone (Cell Signaling Technology), anti-LDH (Cell Signaling Technology), anti-NdufS3 (mitochondrial complex I subunit, Abcam), anti-mitochondrial complex II 70 kDa subunit (Life Technologies), anti-mitochondrial complex IV subunit (cytochrome c oxidase subunit II, Life Technologies), anti-mitochondrial complex V 56 kDa subunit (Life Technologies) and anti-TFAM (mitochondrial transcription factor A, Genetex). Images were visualized using Nikon Eclipse 80i fluorescent microscope with CoolSNAP HQ camera and analyzed with NIS Elements BR3.10 software.
Quantification of Cell Death using LDH Release
U937 cells (5 × 105 cells) were incubated with hemoglobin (2-20-200 mg/dL) for 24 h and washed two times with RPMI media and challenged with 5 mmol/L NaSH, 500 µmol/L H2O2, 100 µmol/L SIN or 500 µmol/L SNAP for 24 h. LDH activity in the culture medium, an indicator of cell necrosis, was measured as described (10).
Measurement of Mitochondrial and Total Cellular Oxidant Production
Detection of mitochondrial superoxide was achieved with MitoSOX (Life Technologies) and total ROS levels were quantified with H2DCF (Life Technologies) as described (11).
Citrate Synthase Activity Measurement
Specific activity of citrate synthase was detected in total cell lysate of 5 × 105 U937 cells previously incubated with hemoglobin (2-20-200 mg/dl) for 24 h using Citrate Synthase Assay Kit (Sigma-Aldrich) as described (11).
Measurement of Mitochondrial Membrane Potential
Mitochondrial membrane potential was measured using TMRE (Life Technologies) in 5 × 105 of U937 cells as described (9).
Measurement of Intracellular ATP Content
ATP content was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) as described (11).
Analysis of Bioenergetic Parameters by Extracellular Flux Analysis
U937 cells (2 × 105) were plated in 24-well Seahorse culture plates (BD Biosciences) followed by 24 h incubation with hemoglobin (2-20-200 mg/dl). Bioenergetic parameters were measured with XF24 Extracellular Flux Analyzer (Seahorse Bioscience) as described (12).
Measurement of Plasma H2S Content
Concentration of H2S in human plasma samples or in samples of animals subjected to burn injury according to a model previously described (13) were detected with 7-azido-4-methylcoumarine (AzMC) H2S-specific fluorescent probe as described (14).
Transient Transfection of HEK293T17 Cells with Hemoglobin
HEK293T17 cells were transfected on 24-well plates with full-length human hemoglobin cDNA inserted into pCMV6-XL4 vector purchased from Origene Technologies. β-galactosidase-inserted plasmid was used as a control (Origene). Transfection of HEK293T17 cells was performed using Lipofectamine 2000 (Life Technologies) with 0.5 µg of plasmid DNA per well. After 48 h HEK293T17 cells were challenged with 7.5 mmol/L NaSH for 1 h. LDH release into the media was quantified and hemoglobin expression was confirmed by Western blotting.
Measurement of Mitochondrial DNA Damage
Damage to mitochondrial DNA was determined as described (10).
All data are presented as means ± SEM and were analyzed using GraphPad Prism software. Statistical analysis was performed by ANOVA followed by Bonferroni’s multiple comparisons.
Hemoglobin Protein and mRNA Levels Are Increased in PBMCs during Critical Illness
Potential Mechanisms of the Upregulation of Hemoglobin in Critical Illness
Another possibility that may contribute to the observed increase in the hemoglobin content of PBMCs may be the uptake of hemoglobin from the circulation, since burn and sepsis is known to induce significant hemolysis, which can markedly increase plasma-free hemoglobin concentrations (16). A significant hemolysis was also confirmed in the present samples (Figure 2B). Flow cytometric analysis showed double staining of PBMCs isolated from burn patients for the presence of hemoglobin and CD45+ (leukocyte common antigen) in the PBMCs of burn patients (Figure 2C), thereby localizing the hemoglobin to the leukocytes. In subsequent studies, we have confirmed the ability of PBMCs and U937 cells to take up hemoglobin from the culture medium in vitro (Figure 2D).
CD163-Mediated Uptake and Mitochondrial Sequestration of Hemoglobin
H2S Induces Hemoglobin Upregulation; Intracellular Hemoglobin Protects from H2S Cytotoxicity
Circulating hemoglobin, or cell-free hemoglobin, as a result of excessive intravascular hemolysis (for instance, as a consequence of infusion of stored blood products, ABO incompatibility or various forms of critical illness) (15,16,20) is generally considered a deleterious molecule, which exerts its toxic effects via several distinct mechanisms including pro-oxidant redox cycles catalyzed by heme (4), scavenging physiologically essential NO from the vascular space, leading to endothelial dysfunction and vasoconstriction (15), activation of proinflammatory pathways via TLR4 receptors (21) and other mechanisms. In this context, uptake of hemoglobin into mononuclear cells from the circulation via CD163 is viewed as a protective pathway that serves to reduce circulating free hemoglobin levels (17).
Several lines of independent studies, however, implicate hemoglobin as a cytoprotective protein. For instance, hypoxia upregulates hemoglobin expression in alveolar epithelial cells in vitro (22) and bacterial lipopolysaccharide upregulates the β hemoglobin subunit in murine macrophages (23). H2O2 increases Hb-α1 and Hb-β expression in HepG2 and HEK293 cells, which, in turn, protects the cells from oxidative stress (24). Hemoglobin has been localized in kidney mesangial cells, where it confers oxidative stress resistance (25). The expression of Hb-α1 and Hb-β also has been demonstrated in mesencephalic dopaminergic neurons and glial cells, and has been linked to the modulation of oxygen homeostasis, oxidative phosphorylation, oxidative stress and NO biosynthesis (26). Finally, red blood cell hemolysate and hemoglobin both reduce the effect of oxidative stress on peripheral blood mononuclear cell DNA damage induced by H2O2 (27). These studies, when taken together with the results of the current study, indicate that upregulation of intracellular hemoglobin (either via uptake from the extracellular space, and/or through de novo biosynthesis) can be a reactive response of the cell to adverse conditions such as oxidative stress, hypoxia or high concentrations of gaseous mediators, which, in turn, serves to protect the cell from the damage.
We conclude that in the current study, the upregulation of hemoglobin in the PBMCs of critically ill patients probably results from a combination of uptake from the extracellular space and an active biosynthesis of hemoglobin, at least in part by immature leukocytes mobilized from the bone marrow. The in vitro data (upregulation of Hb mRNA in U937 cells after incubation with H2S) coupled with the findings showing that (a) circulating H2S levels are increased in critical illness and (b) cells with elevated intracellular hemoglobin levels are protected from H2S-mediated cytotoxicity are consistent with the existence of a reactive process whereby upregulation of hemoglobin serves as a protective mechanism in PBMCs against H2S toxicity in critical illness. This model is not in disagreement with prior work showing that extracellular circulating hemoglobin can have multiple deleterious effects (20); in fact, uptake and sequestration of hemoglobin from the plasma into the PBMCs via CD163 may contribute to a reduction of circulating free hemoglobin levels.
We do not propose that the sole purpose of intracellular hemoglobin in PBMCs is the modulation of H2S homeostasis or protection from H2S-mediated injury. It is more likely that hemoglobin exerts pleiotropic effects in these cells; it modulates the cellular responses to various oxidants and free radicals (NO, superoxide, H2O2, peroxynitrite, and so on); hemoglobin, either through acting on membrane receptors or via modulation of intracellular pathways and signal transduction processes, may also regulate the expression of various oxidant-responsive elements (as evidenced, in our study, by the hemoglobin-induced upregulation of HIF-1α and NRF2). Upregulation of both of these factors is known to confer cytoprotection and ischemic/hypoxic preconditioning via a variety of interacting downstream pathways (28,29).
Intracellular hemoglobin shows remarkable compartmentalization; it concentrates to the mitochondria, where it shows a particularly close association with mitochondrial complex I. It is interesting to note that a recent proteomic analysis of control brains and brains from multiple sclerosis patients has identified the mitochondrial localization of the hemoglobin β chain (30). The functional consequence of mitochondrial hemoglobin remains to be further elucidated; based on the current results, we speculate that it may either contribute to a specific mitochondrial protection (for example, against mitochondrial oxidative stress), or it may play a more specific role in the regulation of mitochondrial electron transport; the latter hypothesis is supported by our extracellular flux analysis findings indicating an increased basal and FCCP-uncoupled maximal rate of mitochondrial respiration in cells with increased mitochondrial hemoglobin content. Our results also indicate that mitochondrial hemoglobin may serve to protect the integrity of mitochondrial DNA from oxidative damage.
The conditions and factors determining the deleterious versus protective roles of hemoglobin in various pathophysiological conditions are likely to depend on the localization of hemoglobin (intracellular versus extracellular), the degree of oxidative stress, the rate of the gaseous transmitters produced, as well as many additional factors. Based on several sets of independent investigations, it appears that when oxidative stress is relatively low, hemoglobin (and its degradation products, for example, heme) increases oxidative stress, and scavenges physiologically necessary amounts of NO, but when the degree of oxidative stress is high, hemoglobin tends to protect from cellular damage (22, 23, 24, 25, 26, 27,31). One also has to keep in mind that hemoglobin decomposes into multiple products, including heme (which increases oxidative stress, but also acts as a substrate of the gasotransmitter carbon monoxide, produced by heme oxygenase), while other degradation products (for example, bilirubin) have significant antioxidant effects (31). These factors remain to be carefully evaluated to dissect the multiple roles of hemoglobin in the regulation of various pathophysiological conditions.
There are a number of limitations of the current study, as well as a number of follow-up issues that remain to be elucidated in future experiments. (a) In addition to the upregulation of Hbs, there are a number of additional protein bands that show enrichment in the PBMCs of critically ill patients (Figure 1); changes in the entire PBMC proteome, as well as additional individual proteins that are up- or downregulated remain to be studied in future experiments. (b) What is the relative contribution of Hb mRNA/protein upregulation and uptake of Hbs from the extracellular space to the observed enrichment of PBMC Hbs in burn and in sepsis? Furthermore, with respect to the upregulation of Hb mRNA, what is the contribution of upregulation in mature PBMCs versus in immature/stem forms that are mobilized into the circulation during the critical illness insult? These issues may be more directly addressed by future preclinical studies (for example, rodent models of sepsis or burns). (c) We have only studied the reactive upregulation of Hbs in response to H2S in the current project. It remains to be elucidated if similar reactive responses can also be elicited by other stimuli (for example, hypoxia, oxidative stress, NO, CO and others) in PBMCs. In this context, it is worth mentioning that, in other cell types, Hb upregulation has been demonstrated in response to bacterial lipopolysaccharide stimulation in murine monocytemacrophages (23), in response to hypoxia in epithelial cells (22) or in response to oxidative stress in hepatocytes (24). (d) What is the exact functional role of Hbs in the mitochondria, and how does Hbs translocation occurs into the mitochondria? Since Hbs are large molecules, the process is likely to involve active transport mechanisms. It also remains to be determined whether Hbs incorporate into respiratory complex I and, if so, do they directly participate in mitochondrial electron transport? One limitation of the results is that PLA studies show close vicinity to the two proteins studied (in this case complex I and Hb) but direct binding requires confirmation by additional methods (for example, confocal microscopy, immunoprecipitation and/or pull-down techniques). In this context it is important to keep in mind that mitochondrial complex I consists of a large number of subunits, including the NADH dehydrogenase module (N module), the electron transfer module (Q module) and the proton translocation module (P module), which are made up of 14 core subunits (32). Specific interaction(s) of Hbs with these subunits remain to be studied in further experiments. (e) The exact kinetics and reactions of Hbs with NO, H2O2 and H2S remain to be further studied in the cytoprotective context. With respect to the reaction of NO with Hbs, the literature is substantial and goes back to a century; it has been put into the biological (vascular) context by the original work of Murad, Ignarro and Furchgott (33, 34, 35, 36), who have identified Hbs as scavengers of NO (as well as of endothelium-derived relaxing factor [EDRF], which later has been demonstrated to be identical with NO). Similarly, with respect to the interactions of Hbs with oxyradicals, substantial work has been conducted already, which can be reviewed in the literature (37, 38, 39). With respect to the interactions of Hbs with H2S, several lines of studies have demonstrated the formation of sulfhemoglobins (40,41), and Banerjee and colleagues have recently outlined several novel aspects of the interaction, especially with respect to the interactions of H2S with methemoglobin (42).
Although much additional work remains to be conducted to explore the above issues, in summary, at least under the conditions studied in the current report, we can conclude that cellular uptake of hemoglobin does not adversely affect the viability of leukocytes, and protects them from various gaseous and oxidative insults. Therefore, we hypothesize that the upregulation of hemoglobin in PBMCs during critical illness constitutes a cytoprotective mechanism.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by the National Institutes of Health (P50GM060338 to DN Herndon and R01GM107846 to C Szabo) and by a grant from the Shriners of North America (#85800) to C Szabo.
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