Published online before print September 28, 2006, doi: 10.1161/01.RES.0000247067.34173.1b
© 2006 American Heart Association, Inc.
Molecular Medicine |
Constitutive Endocytosis of CD163 Mediates Hemoglobin-Heme Uptake and Determines the Noninflammatory and Protective Transcriptional Response of Macrophages to Hemoglobin
From the Departments of Medicine (C.A.S., G.S., A.I., D.J.S.) and Pathology (M.O.K.), University Hospital, Zurich, Switzerland.
Correspondence to Dominik J Schaer, Medical Clinic Research Unit Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland. E-mail dominik.schaer{at}usz.ch
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Abstract |
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Heme toxicity contributes to the pathogenesis of chronic inflammatory diseases, atherosclerosis, and hemolysis associated vasculopathy. Macrophage clearance of cell free hemoglobin (Hb) is thus an essential homeostatic function of these cells. We examined the transcriptional response of human PBMC derived macrophages to Hb by gene array analysis. The observed noninflammatory macrophage response was characterized by induction of an antioxidative and antiinflammatory gene expression pattern with most prominent induction of the inducible heme oxygenase (HO-1). The metabolically active Hb-CD163-HO-1 pathway resulted in synthesis of ferritin—1 of the antioxidative and antiinflammatory end products linked to heme breakdown by HO-1. This response was mediated by the Hb scavenger receptor CD163 and heme and was not related to Hb mediated depletion of reduced glutathione. In contrast to other cellular responses induced by CD163, there was no role of protein phosphorylation dependent CD163 signaling in the protective macrophage response to Hb. Instead, CD163 acted as an Hb transporter, which undergoes constitutive and ligand independent internalization and recycling between the cell surface and early endosomes. The expression of CD163 and HO-1 in macrophages of neovascularized atherosclerotic lesions suggests that the pathway described herein is active in vivo. Noninflammatory Hb clearance and intimately linked HO-1 expression may provide the long sought-after explanation for the antiinflammatory activity associated with CD163-positive macrophages.
Key Words: CD163 • haptoglobin • hemoglobin scavenger receptor • macrophage • oxidative stress
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Introduction |
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Effective clearance of extracellular hemoglobin (Hb) is thought to limit systemic oxidative heme toxicity, which is presumed to contribute to the pathogenesis of atherosclerosis and other chronic inflammatory diseases as well as to hemolysis associated vasculopathy.1,2 Cell-free Hb is tightly bound to haptoglobin (Hp) and subsequently cleared by the macrophage hemoglobin scavenger receptor, CD163.3 Hb that is not bound to Hp can also be cleared via CD163. This Hp-independent, low-affinity Hb binding and uptake by CD163 is thought to be the predominant macrophage Hb clearance pathway after depletion of plasma Hp or during massive Hb release after erythrocyte extravasation on tissue injury.4 Although it is clear that macrophages play a central role in Hb clearance, the cellular pathways involved and the macrophage response to Hb have yet to be resolved.
CD163 is expressed by resident tissue macrophages, and particularly high levels of CD163 have been detected on infiltrating monocytes during the resolution phase of inflammatory reactions. The strong enhancement of CD163 expression by antiinflammatory mediators, such as interleukin-10 and glucocorticoids, supports the notion that CD163 expression is linked to antiinflammatory macrophage functions.5–7
Other evidence suggested a role of CD163 in proinflammatory activation of macrophages.8 Cross-linking of cell-surface CD163 was shown to elicit protein-kinase C (PKC)- and casein-kinase (CK II)-dependent macrophage activation, which was followed by the release of proinflammatory cytokines.9,10 Whether activation of these signaling pathways is attributable to Hb-macrophage interactions is unknown. However, based on clinical experience, macrophage-mediated Hb clearance during hemolysis does not induce an overt inflammatory response. Moreover, experience with Hb based blood substitutes confirms that Hb clearance is a noninflammatory process.2,11
Here we report that Hb elicits a noninflammatory transcriptional response in macrophages, which includes a prominent induction of the antiinflammatory and cytoprotective gene, heme oxygenase (HO-1). Using a heterologous gene expression model, induction of HO-1 by Hb was functionally linked to CD163 and was shown to occur via Hb-heme uptake through ligand-independent internalization of CD163.
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Materials and Methods |
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Cell culture
Human PBMC derived macrophages and wild type and transfected HEK293 cells were cultured as described in the supplement. Endotoxin free Hb was obtained from Hemosol (Ontario, Canada). Porphyrins and Hp (mixed phenotype) were from Sigma-Aldrich (Buchs, Switzerland).
Gene Array Experiments
Gene expression profiling was performed by competitive dual-color hybridization of complementary RNA probes on human 22K 60-mer oligonucleotide microarray chips (Agilent Technologies, Palo Alto, Calif) as described in the online supplement.
Quantitative Real Time RT-PCR
LightCycler-PCR is described in the online supplement.
Quantification of Hb–Hp Uptake and Cell Surface CD163
Hp was labeled with the Alexa-488 or Alexa-633 protein labeling kits (Molecular Probes, Eugene, Ore). Uptake assays are described in the supplement. Cell surface CD163 was determined by FACS using a FITC-conjugated anti-CD163 antibody (5C6-FAT, BMA, Augst, Switzerland).
Measurement of intracellular heme, ferritin, and reduced glutathione (GSH) are described in the online supplement.
Immunohistochemistry and Immunofluorescence
Immunohistochemistry and mmunofluorescence were performed as described.12 Details are given in the online supplement.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 4.0. Treatment groups were compared with analysis of variance (ANOVA) and Bonferroni corrected posttests.
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Results |
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Noninflammatory Transcriptional Responses of Human Macrophages to Hb Exposure: Crucial Role of CD163 and Heme
We compared the messenger RNA (mRNA) expression profiles of untreated macrophages cultured for 8 days and macrophages that were incubated with 500 µg/mL Hb for an additional 12 hour. These experiments were performed in culture medium containing 20% pooled human serum which served as a source of Hp. We found 3455 of 18000 genes which displayed significantly different expression in treated and untreated cells (Figure 1A). A functional classification of all regulated genes is given in supplementary Figure I. Among these genes 68 were either significantly upregulated25 or downregulated45 by more than 2-fold (P<0.001). Several of the genes which were regulated more than 2-fold were oxidative stress-associated genes and included genes encoding a member of the aldo-keto reductase family (AKR1C3), the glutamate-cysteine-ligase modifier subunit (GCLM), flavin containing monooxygenase-1 (FMO1), and the inducible heme breakdown enzyme HO-1 (HMOX1). We have confirmed the Hb-mediated induction of HO-1 by real-time PCR and Western-Blot analysis (Figure 1B). Depending on the inductive stimulus, expression of HO-1 protein is not always associated with significant cellular heme breakdown.13 Therefore, to proof that the HO-1 pathway is active metabolically in Hb treated macrophages we have measured the intracellular concentration of ferritin which is 1 of the end products linked to HO-1 activity.14 The observation that the increase of intracellular ferritin after Hb treatment was inhibited by the HO-1 inhibitor zinc-protoporphyrin (ZnPP IX) confirms the active role of HO-1 in Hb induced ferritin accumulation (Figure 1C). Although we used highly purified Hb for these studies, we specifically focused on the expression of inflammatory response genes, which could indicate inadvertent LPS contamination. Other than a 2.1-fold induction of IL-8 we found no evidence of inflammatory gene expression. In particular, the gene expression pattern observed here displayed no similarity with that induced by a commonly used commercial Hb (ferrous HbAo, Sigma) that we found to be contaminated by endotoxin (supplementary Figure I).
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0.0001), green (significantly downregulated, P
To study the role of CD163 in the macrophage response to Hb, we compared gene expression profiles of CD163-expressing HEK cells in the presence and absence of Hb-Hp (Figure 1B) as well as of CD163-negative and CD163-positive HEK cells exposed to Hb-Hp. Analysis of these data allowed us to segregate the quantitative influence of the 2 independent factors tested, Hb-Hp exposure and CD163 expression. In this model, the regulation of 6 genes was dependent on the concurrent presence of CD163 expression and Hb. Included among these major Hb response genes shared by CD163-HEK293 cells and macrophages were HMOX1, GCLM, and ALAS1. Both HMOX1 and GCLM were induced by Hb-Hp; whereas, [
]-aminolevulinate synthase (ALAS1), the rate limiting enzyme of the heme synthesis pathway, was suppressed (Figure 2A). Thus, these genes represent a distinct set of Hb-CD163 pathway signature genes. Hb-Hp and CD163-dependent induction of HO-1 was confirmed by real-time RT-PCR and Western blot analysis, respectively, and was found to be dose- and time-dependent (Figure 3A and B). No changes in HO-1 mRNA or protein levels were observed in response to Hb-Hp exposure in CD163-negative cells. That treatment of CD163-HEK293 cells but not of CD163-negative cells with Hb-Hp or free Hb, which is a low affinity ligand of CD163, resulted in a dose dependent accumulation of ferritin proofs that the Hb-CD163-HO-1 pathway is enzymatically active in CD163 transduced cells. To exclude that these results depended on extra-cellular heme release or unspecific oxidative side reactions of ferric heme, we confirmed the latter observation with CN-met-Hb. In CN-met-Hb the heme moiety is irreversibly blocked by a cyanide atom and thus cannot be released, nor can it engage in oxidative reactions (Figure 3C).
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Next, we set out to determine whether the changes in the expression of these Hb-CD163 signature genes were because of activation of receptor-dependent signaling or to the increase of intracellular heme concentration which was found to be more than 10-fold higher in CD163-positive compared with CD163-negative cells after incubation with 300 µg/mL Hb-Hp for 24 hour (increase of cell associated heme concentration in HEK293 cells: 1.45±0.35 µmol/mg protein; in CD163-HEK293 cells: 11.78±0.33 µmol/mg protein; P=0.0015). To this end, we examined the gene expression pattern of macrophages after incubation with heme, which enters the cell by a CD163-independent mechanism. As shown in Figure 2B, heme treatment altered expression of the same genes which were previously defined as the Hb-CD163 pathway signature genes. In fact, all the Hb regulated genes selected for verification of their respective regulation by LightCycler-PCR demonstrated similar expression changes in Hb and in heme treated cells (HO-1, GCLM, ALAS1, AKR1C3 and EDRB; supplemental Figure III) Thus, CD163-mediated internalization of Hb-heme seems to be the critical signal for the changes in gene expression induced by Hb and CD163.
Hb Endocytosis, Not Protein Phosphorylation-Dependent Signaling, Is Responsible for CD163-Dependent HO-1 Induction
Several serine/threonine-phosphorylation sites within the CD163 cytoplasmic tail are targets of CKII and PKC. Receptor cross-linking with the anti-CD163 antibody, EDHU1, is associated with activation of these kinases which leads to cell activation and cytokine secretion.9,10 Therefore, we examined whether HO-1 induction is related to protein phosphorylation-dependent signaling triggered by CD163 engagement. To do this, we examined mutations/deletions within the cytoplasmic tail of CD163. Endocytosis of fluorescent Hb-Hp and subsequent HO-1 induction were almost completely abrogated when CD163 was expressed with a truncated cytoplasmic tail containing only 1 or 3 intracellular amino acids (Figure 4). This finding is consistent with the previously recognized role of the CD163 cytoplasmic tail in ligand uptake.15 We then generated a mutant CD163 variant in which all phosphorylation sites were removed. To assure unimpaired ligand-uptake, the YXX
-type endocytosis recognition motif (1091 to 1094 YREM)16 was fused to the membrane proximal part of CD163 (Figure 4A). The mutated receptor was able to internalize Hb-Hp and free Hb equally as well as full length CD163. In addition, we could not detect any difference in CD163-dependent HO-1 induction (Figure 4B-D). Because all proteins were expressed as GFP-fusion proteins, equal expression levels of the different receptor constructs were confirmed by FACS (Figure 4B).
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Hb-CD163 Mediated HO-1 Expression Is Not Related to Reduced Glutathione Depletion
Recent reports described an interrelation of reduced glutathione (GSH) depletion with HO-1 expression under conditions of oxidative stress.17–19 Hb treatment at concentrations which induce maximum HO-1 expression in CD163-HEK293 cells did not reduce levels of GSH in either CD163 positive or CD163 negative HEK293 cells at 2 or 7 hours. Accordingly, concurrent incubation of CD163-HEK293 cells with Hb and the antioxidant and glutathione precursor N-acetylcystein did not attenuate the Hb induced induction of HO-1 mRNA (supplementary Figure IV).
Constitutive Internalization and Recycling Through Early Endosomes Determines CD163-Mediated Intracellular Hb Accumulation
Macrophages were treated with dexamethasone for 36 hour to induce maximal CD163 expression and were incubated with receptor-saturating concentrations of Hb for up to 3 hour. This treatment did not impair the capacity to endocytose Hb-Hp in subsequent uptake assays (Figure 5A). These data intimate that CD163 is not degraded after internalization. Rather, it continually returns to the cell surface to undergo endocytosis. Accordingly, pretreatment of macrophages with saturating concentrations of Hb in the presence of monensin, an inhibitor of endosomal receptor recycling, rapidly reduced cell surface expression of CD163 and, as expected, the capability to internalize fluorescent Hb-Hp (Figure 5A). Monensin treatment reduced cell surface receptor expression as well as Hb-Hp endocytosis even in the absence of Hb, implying that the receptor is constitutively (ligand-independent) internalized.
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), monensin(50 µmol/L; 
), or Hb+monensin (
) for the indicated times and cell surface expression of CD163 was determined by FACS. The results are given as mean± SD of mean channel fluoerescence from 3 experiments. A, right panel, Monocytes were pretreated with or without monensin± Hb for 120 minutes. Hb-Hp uptake was then determined after incubation with fluorescent Hb-Hp488 (5 µg/mL) for 30 minutes. Data represent mean± SD of 3 experiments (Hb vs Hb/monensin or monensin P<0.001; Hb/monensin vs monensin P>0.05). B, Alexa488 Hb-Hp (10 µg/mL) and the monoclonal anti-CD163 antibody 5C6 (10 µg/mL) colocalize with Alexa594 transferrin within the early endosomest when monocytes were concomitantly incubated with the 2 substances for 4 minutes (yellow indicates colocalization in the merged image file). Original magnification 600x; blue: DAPI stained nuclei.After a 5-minute incubation time, both Hb-Hp and anti-CD163 antibody colocalized with fluorescent transferrin, an established marker of the early/recycling endosomes (Figure 5B). In contrast to CD163, which recycles to the cell surface, Hb-Hp is rapidly transferred to LAMP1 positive lysosomes (Figure 6D). We did not observe any differences in the distribution of either fluorescent Hb or Hp within the complex, indicating that both proteins are targeted for degradation in lysosomes. This explains the fact that the Hb-binding protein Hp is rapidly consumed during hemolysis.
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), monensin(50 µmol/L; 
) for the indicated times (left panel). In other experiments, cultures were pretreated with Hb(1 mg/mL; Evidence of constitutive CD163 endocytosis was also found in the CD163-HEK293 cells. Treatment with monensin ±Hb, but not treatment with the protein synthesis inhibitor cycloheximide, inhibited subsequent Hb-Hp uptake (Figure 6A). To directly visualize intracellular CD163 distribution, we generated a HEK293 cell line that expressed CD163 fused to green fluorescent protein (GFP). Fluorescence microscopy revealed a large intracellular receptor pool which displayed a fine vesicular pattern in untreated cells. On inhibition of receptor recycling with monensin, CD163 disappeared from the cell periphery and accumulated in coarse perinuclear vesicles (Figure 6B). The almost complete colocalization of intracellular CD163-GFP with transferrin confirms that the intracellular receptor pool is derived from endocytosed receptor (Figure 6C). Although the CD163 ligands, Hb and Hb-Hp, are transferred to lysosomes after endocytosis, we did not detect significant colocalization of CD163-GFP with LAMP-1 (not shown).
HO-1 Is Expressed by CD163-Positive Macrophages Within Neovascularized Areas of Human Atherosclerotic Lesions
To investigate the potential role of the Hb-CD163-HO-1 pathway in atherosclerosis, we performed immunohistochemical staining for CD163 and HO-1 in atherosclerotic lesions within 26 coronary, carotid, and large peripheral arteries that had been obtained from human autopsies. Based on the expression of CD68, a reference marker for monocyte/macrophage lineage cells, CD163 was expressed by virtually all macrophages in the atherosclerotic lesions (Figure 7A). Although only a minority of macrophages in the fibrous parts of the atherosclerotic lesions stained positive for HO-1 (Figure 7D), a high level of HO-1 labeling was observed within advanced lesions with extensive neovascularization and microhemorrhage, as evidenced by positive iron staining within these areas (Figure 7C). As shown in Figure 7B, high levels of HO-1 labeling were confined to areas rich in CD163-expressing macrophages. CD163/HO-1 double-labeling confirmed that HO-1 expression is restricted to CD163-positive macrophages (Figure 7E). CD163-associated HO-1 immunoreactivity was not detected in the CD68/CD163-positive resident macrophages within the perivascular adipose tissue (supplemental Figure V). This observation argues against a constitutive link between CD163 and HO-1 expression in macrophages but it suggests that an exogenous HO-1-inducing factor, such as Hb, is active in the atherosclerotic environment.
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Discussion |
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In the present study, we used comparative gene expression analysis to define human macrophage responses to Hb. Through these analyses we were able to identify a set of Hb-regulated genes. Included among this set of genes were induced oxidases/reductases (ie, AKR1C3, GCLM, FMO1) as well as HMOX1. The product of HMOX1, the inducible heme oxygenase (HO-1), is linked to heme catabolism and has attracted significant interest because its final catalytic products—carbon monoxide, bilirubin, and ferritin—exert potent antiinflammatory and cytoprotective effects.20 We could proof that this Hb induced protective pathway is active in human macrophages and results in enhanced ferritin synthesis on Hb treatment. Likewise, GCLM, which is among 6 regulated genes classified to the glutathione synthesis pathway, protects against oxidative stress by catalyzing the rate-limiting step in glutathione biosynthesis.21
An important challenge in these experiments was to dissect Hb-specific responses from inflammatory responses, which could be triggered by small molecular contaminants. Endotoxin contamination appears to have confounded earlier investigations using commercial preparations of Hb.22 For example, data from a recent study suggested that IL-10 secretion is a CD163-dependent macrophage response to Hb-Hp treatment,23 leading the authors to infer that Hb may induce specific macrophage responses through receptor signaling by CD163. In our experience, treatment of macrophages with this Hb preparation provoked an inflammatory gene expression profile reminiscent to the response induced by LPS (supplementary Figure I). Endotoxin was subsequently detected as the proinflammatory compound. In our studies, which revealed a noninflammatory macrophage response to Hb, 2 strategies were used to assure that the results were not affected by endotoxin contamination. First, we used a highly purified and endotoxin-free Hb solution. Second, we verified the principle results in the HEK293 cell line, which does not express the endotoxin signaling pathway.
Use of a heterologous gene expression model to dissect CD163-dependent and CD163-independent effects of Hb, provided unequivocal proof of CD163’s role in macrophage responses to Hb. Accordingly, the major Hb-regulated genes were shared by macrophages and HEK293-cells, and they were mutually regulated by Hb and CD163. Because the Hb-CD163 pathway signature genes were also regulated by free heme, we hypothesized that CD163 acts by increasing the intracellular heme concentration. In fact we could show that Hb treatment resulted in a markedly higher increase of intracellular heme concentrations in CD163-positive compared with CD163-negative cells. CD163 may therefore act as an Hb transporter, rather than a signal-transducing receptor, during the macrophage-Hb interaction. This hypothesis was supported by the fact that a mutant CD163 variant, which lacked all protein phosphorylation sites but retained full endocytic capability, supported Hb-induced HO-1 expression equally as well as wild-type CD163. Thus, while protein phosphorylation and CKII-/PKC-interactions with the cytoplasmic domain of CD163 seem to be involved in the macrophage activation and cytokine secretion observed after cross-linking of cell surface CD163 with "agonistic" antibodies, our results suggest that these pathways do not play a significant role in the Hb-induced HO-1 expression. The fact that Hb does not induce an inflammatory pattern of macrophage gene expression is also consistent with the observation that neither systemic hemolysis nor local release of Hb after erythrocyte extravasation induces an overt inflammatory reaction.2 In the latter situation, removal of free Hb by macrophages is an essential step in wound healing and the noninflammatory nature of this process may help to avoid secondary tissue damage.
Oxidative stress is an important trigger of HO-1 expression19,24 and an interrelation exists between HO-1 induction and transient depletion of GSH, the most important nonenzymatic cellular antioxidant.17–19 Our finding that Hb did not reduce cellular GSH levels within the time required to maximally induce HO-1 mRNA in CD163 positive cells and that the antioxidant and glutathione precursor N-acetycystein did not attenuate Hb induced HO-1 expression implies that the Hb-CD163 pathway does not act via the oxidative stress response signaling pathway. It is therefore prudent to speculate that the Hb-CD163 induction of HO-1 is mediated by alternative pathways such as the heme mediated inactivation of the transcritption repressor Bach1.25
Because we found no evidence that any of the signaling pathways previously linked to CD163 were involved in cellular responses to Hb, we examined whether CD163 may share functional properties with a group of receptors that primarily serve as transmembrane transporters of essential molecules (eg, transferrin receptor26). Indeed, we found that also CD163 undergoes ligand-independent endocytosis and is recycled to the cell surface after internalization. This may not only serve to maintain the high endocytic capacity of these receptors, but it also obviates the need to transduce potentially disturbing intracellular signals on ligand engagement. In case of CD163, ligand independent internalization and receptor recycling allows highly efficient clearance of toxic Hb while the subsequent rise in intracellular free heme concentration evokes protective gene expression.
The ability of CD163 to mediate upregulation of HO-1 expression and subsequent synthesis of protective compounds such as ferritin14 in response to extracellular Hb might point to a heretofore unrecognized role for Hb and CD163 in directing monocytes into an antiinflammatory and wound-healing macrophage phenotype after infiltration of damaged tissues. Both CD163 and HO-1 expression have been independently linked to an antiinflammatory and wound-healing macrophage phenotype. These associations are primarily based on the following observations. First, macrophages with high expression of CD163 constitute the predominant macrophage population during the late or resolution phase of inflammatory reactions.27 Second, CD163 expression is strongly induced by antiinflammatory mediators, such as glucocorticoids and IL-10.6,7 Accordingly, the cellular infiltrate observed during the resolution phase of active inflammation in skin wound healing was shown to display high HO-1 expression. Moreover, in the same experimental model, pharmacological inhibition of HO-1 activity resulted in a greatly augmented infiltration of inflammatory cells,28 indicating that HO-1 or its byproducts are directly involved in the downregulation of inflammation.
HO-1 is the rate-limiting enzyme in the catabolism of heme. Its byproducts include carbon monoxide, bilirubin, as well as free iron, which is rapidly sequestered to ferritin. Carbon monoxide, bilirubin, and ferritin have all been assigned a multitude of antioxidative and antiinflammatory properties.20 Further, HO-1 activity has been linked to the induction of other protective pathways such as the antioxidant enzymes extracellular superoxide-dismutase (EC-SOD) and catalase.29,30 The latter changes resulted in favorable changes in the expression of nitric oxide synthetase isoformes (increased endothelial eNOS and decreased inducible iNOS). Accordingly, accumulating evidence suggests that HO-1 plays a protective role in atherosclerosis: Whereas forced expression of HO-1 in the vascular wall suppresses the development of atherosclerosis31 and injury-induced vascular neointima formation,32 targeted deletion of the HO-1 gene accelerates plaque formation and vascular remodeling in the apoE knockout mouse.33 Accordingly, pharmacological inhibition of endogenous HO-1 activity exacerbates atherosclerotic lesion formation.34,35 Our finding that CD163-positive macrophages constitute the major compartment of HO-1 expression in human atherosclerotic lesions not only lends evidence that CD163-positive macrophages limit Hb toxicity within the atherosclerotic vessel wall, but also that the Hb-CD163-HO-1 pathway is operative in vivo.
In conclusion, constitutive endocytosis and recycling of the Hb scavenger receptor drives noninflammatory removal of extracellular Hb by macrophages. Noninflammatory Hb clearance and intimately linked HO-1 expression may, therefore, provide the long sought-after explanation for the antiinflammatory activity of the CD163-positive macrophage population.
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Acknowledgments |
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Sources of Funding
Foundation for Research at the Medical Faculty, Zurich, the Hartmann-Muller Foundation, and the HOLCIM-Foundation (all to D.J.S.).
Disclosures
None.
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Footnotes |
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Original received May 12, 2006; revision received September 12, 2006; accepted September 14, 2006.
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References |
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