Christian A. Schaer

Constitutive Endocytosis of CD163 Mediates Hemoglobin-Heme Uptake and Determines the Noninflammatory and Protective Transcriptional Response of Macrophages to Hemoglobin

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.

Rapid Detection of Pathogenic Fungi from Clinical Specimens Using LightCycler Real-Time Fluorescence PCR

In the study presented here a LightCycler real-time PCR system was used for the diagnosis of fungal infections from clinical tissue samples. Nine specimens were investigated from six patients with suspected or proven invasive fungal infections. Seven of nine samples were positive in a broad-range fungal PCR assay. In four samples, Aspergillus fumigatus was detected both by a species-specific hybridization assay as well as by sequencing of amplification products. In addition, the broad-range fungal PCR assay and PCR sequencing detected and identified, respectively, the following organisms in the specimens noted: Candida albicans in a culture-negative liver biopsy, Histoplasma capsulatum in a bone marrow sample, and Conidiobolus coronatus in a facial soft tissue specimen. Real-time PCR is a promising tool for the diagnosis of invasive fungal infections in human tissue samples and offers some advantages over culture methods, such as rapid analysis and increased sensitivity.

Hemophagocytic macrophages constitute a major compartment of heme oxygenase expression in sepsis

Abstract:  Objectives: Uncontrolled macrophage activation with hemophagocytosis is a distinctive feature of hemophagocytic syndromes (HPS). We examined whether lympho‐histiocytic infiltration of the bone marrow and liver, as well as hemo‐/erythrophagocytosis also occurs during sepsis and whether this process could account for the increased production of anti‐inflammatory heme‐oxygenase (HO‐1) products observed during sepsis. Methods: Hemophagocytosis and expression of CD163, HO‐1, ferritin as well as CD8 and granzyme‐B were examined in post‐mortem bone marrow samples from 28 patients with sepsis and from eight control patients. Results: Comparison of samples from non‐septic patients with samples from patients with fatal sepsis revealed that the latter group displayed dense lympho‐histiocytic bone marrow infiltration with CD163+/HO‐1+/ferritin+ macrophages as well as with CD8+ and granzyme‐B+ T‐cells. Hemophagocytosis with prominent phagocytosis of erythroid cells was readily apparent in septic patients, implying that this process is a likely stimulus for the up‐regulation of macrophage HO‐1 expression. Conclusions: Lympho‐histiocytic activation with hemophagocytosis is a shared pathophysiologic mechanism in HPS and sepsis. Furthermore, the association of hemophagocytosis with an increase in HO‐1 expression may indicate a novel role for this apparently futile process as a negative regulator of inflammation.

CD163-expressing monocytes constitute an endotoxin-sensitive Hb clearance compartment within the vascular system.

Hemoglobin (Hb) is released into the circulation during intravascular hemolysis and exerts toxic effects through oxidative damage and NO scavenging. According to the traditional concept of Hb clearance, free Hb is bound to the plasma protein haptoglobin (Hp), and the Hb‐Hp complexes are cleared by liver and spleen macrophages via the Hb scavenger receptor CD163. Using a novel whole blood assay, we demonstrate that clearance of Hb‐Hp is also mediated by CD14high/CD64high peripheral blood monocytes, which express CD163. Hb‐Hp uptake by these cells is Ca2+‐dependent and is abrogated by the addition of CD163‐blocking antibodies. Accordingly, LPS treatment reduces monocyte surface CD163 and impairs Hb‐Hp uptake. Monocytes likely mediate Hp‐Hb uptake in vivo, as a high expression of the heme breakdown enzyme heme oxygenase‐1 was observed in CD163+ monocytes but not in other leukocyte populations obtained from healthy blood donors. We propose that CD163‐mediated Hb‐Hp uptake by peripheral blood monocytes constitutes an Hb‐Hp clearance pathway, which acts at the site of intravascular hemolysis to reduce Hb‐Hp circulation time and toxicity. Disruption of monocyte Hb‐Hp clearance may increase Hb‐Hp toxicity and contribute to the pathogenesis of systemic inflammatory diseases associated with reduced monocyte CD163 expression.

Glucocorticoid treatment skews human monocyte differentiation into a hemoglobin-clearance phenotype with enhanced heme-iron recycling and antioxidant capacity

Glucocorticoids are used extensively to treat autoimmune hemolytic anemias. Some beneficial effects of glucocorticoid pulse therapy have also been reported in sickle cell disease and paroxysmal nocturnal hemoglobinuria. Based on established concepts of hemoglobin (Hb) toxicity and physiologic Hb scavenger systems, we evaluated whether glucocorticoids could support an adaptive response to extracellular Hb independently of their immunosuppressive activities. Using global proteome and transcriptome analysis with mass-spectrometry (isobaric tag for relative and absolute quantitation and liquid chromatography-mass spectrometry) and gene-array experiments, we found that glucocorticoid treatment in vitro and in patients on glucocorticoid-pulse therapy polarized monocytes into a M2/alternatively activated phenotype with high Hb-scavenger receptor (CD163) expression and enhanced Hb-clearance and detoxification capability. Monocytes concurrently exposed to the interactive activity of glucocorticoids and extracellular Hb were characterized by high expression of a group of antioxidant enzymes known to be regulated by the conserved oxidative response transcription factor nuclear factor E2-related factor. Further, suppressed transferrin receptor, together with high ferroportin expression, pointed to a shift in iron homeostasis directed toward an increased cellular export of heme-derived iron. Therefore, stimulating Hb-endocytosis by CD163 and enhancing antioxidative homeostasis and iron recycling may be an essential activity of glucocorticoids that helps alleviate the adverse effects of extracellular Hb.

Heme carrier protein (HCP-1) spatially interacts with the CD163 hemoglobin uptake pathway and is a target of inflammatory macrophage activation.

Macrophages constitute the major cellular compartment for hemoglobin (Hb) degradation and subsequent recycling of heme‐iron to erythropoiesis. Dysregulation of macrophage iron and heme metabolism is a major pathophysiologic determinant of anemia of chronic disease. In this study, we show that the heme transporter heme carrier protein 1 (HCP‐1) is expressed in human macrophages. Within early endosomes, HCP‐1 colocalizes with endocytosed Hb‐haptoglobin (Hp) complexes, which are taken up via the CD163 scavenger receptor pathway. Hb‐Hp passes the divalent metal transporter 1B/HCP‐1‐positive endosomal compartment on its route from the cell surface to lysosomes. HCP‐1 mRNA and protein expression are down‐regulated by stimulation of macrophages with various TLR agonists and IFN‐γ. The profound suppression of HCP‐1 expression by inflammatory macrophage activation parallels the regulation of the iron exporter ferroportin. In contrast, dexamethasone enhanced HCP‐1 expression significantly. Given the spatial relationship, we propose that the Hb scavenger receptor CD163 and HCP‐1 constitute a linked pathway for Hb catabolism and heme‐iron recycling in human macrophages.

Haptoglobin Preserves Vascular Nitric Oxide Signaling during Hemolysis

RATIONALE Hemolysis occurs not only in conditions such as sickle cell disease and malaria but also during transfusion of stored blood, extracorporeal circulation, and sepsis. Cell-free Hb depletes nitric oxide (NO) in the vasculature, causing vasoconstriction and eventually cardiovascular complications. We hypothesize that Hb-binding proteins may preserve vascular NO signaling during hemolysis. OBJECTIVES Characterization of an archetypical function by which Hb scavenger proteins could preserve NO signaling during hemolysis. METHODS We investigated NO reaction kinetics, effects on arterial NO signaling, and tissue distribution of cell-free Hb and its scavenger protein complexes. MEASUREMENTS AND MAIN RESULTS Extravascular translocation of cell-free Hb into interstitial spaces, including the vascular smooth muscle cell layer of rat and pig coronary arteries, promotes vascular NO resistance. This critical disease process is blocked by haptoglobin. Haptoglobin does not change NO dioxygenation rates of Hb; rather, the large size of the Hb:haptoglobin complex prevents Hb extravasation, which uncouples NO/Hb interaction and vasoconstriction. Size-selective compartmentalization of Hb functions as a substitute for red blood cells after hemolysis and preserves NO signaling in the vasculature. We found that evolutionarily and structurally unrelated Hb-binding proteins, such as PIT54 found in avian species, functionally converged with haptoglobin to protect NO signaling by sequestering cell-free Hb in large protein complexes. CONCLUSIONS Sequential compartmentalization of Hb by erythrocytes and scavenger protein complexes is an archetypical mechanism, which may have supported coevolution of hemolysis and normal vascular function. Therapeutic supplementation of Hb scavengers may restore vascular NO signaling and attenuate disease complications in patients with hemolysis.

Different target specificities of haptoglobin and hemopexin define a sequential protection system against vascular hemoglobin toxicity.

Free hemoglobin (Hb) triggered vascular damage occurs in many hemolytic diseases, such as sickle cell disease, with an unmet need for specific therapeutic interventions. Based on clinical observations the Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hx) have been characterized as a sequential defense system with Hp as the primary protector and Hx as a backup when all Hp is depleted during more severe intravascular hemolysis. In this study we present a mechanistic rationale for this paradigm based on a combined biochemical and cell biological approach directed at understanding the unique roles of Hp and Hx in Hb detoxification. Using a novel in vitro model of Hb triggered endothelial damage, which recapitulates the well-characterized pathophysiologic sequence of oxyHb(Fe(2+)) transformation to ferric Hb(Fe(3+)), free heme transfer from ferric Hb(Fe(3+)) to lipoprotein and subsequent oxidative reactions in the lipophilic phase. The accumulation of toxic lipid peroxidation products liberated during oxidation reactions ultimately lead to endothelial damage characterized by a specific gene expression pattern with reduced cellular ATP and monolayer disintegration. Quantitative analysis of key chemical and biological parameters allowed us to precisely define the mechanisms and concentrations required for Hp and Hx to prevent this toxicity. In the case of Hp we defined an exponential relationship between Hp availability relative to oxyHb(Fe(2+)) and related protective activity. This exponential relationship demonstrates that large Hp quantities are required to prevent Hb toxicity. In contrast, the linear relationship between Hx concentration and protection defines a highly efficient backup scavenger system during conditions of large excess of free oxyHb(Fe(2+)) that occurs when all Hp is consumed. The diverse protective function of Hp and Hx in this model can be explained by the different target specificities of the two proteins.

Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent

The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb), which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM). We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1) response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

Integrative proteome and transcriptome analysis of extramedullary erythropoiesis and its reversal by transferrin treatment in a mouse model of Beta-thalassemia

Beta-thalassemia results from mutations of the β-hemoglobin (Hbb) gene and reduced functional Hbb synthesis. Excess α-Hb causes globin chain aggregation, oxidation, cytoskeletal damage, and increased red blood cell clearance. These events result in anemia, altered iron homeostasis, and expansion of extramedullary erythropoiesis. Serum transferrin (Tf) is suggested to be an important regulator of erythropoiesis in murine models of thalassemia. The present study was conducted to establish a quantitative proteomic and transcriptomic analysis of transferrin-modulated extramedullary erythropoiesis in the spleen of wild type and thalassemic Hbb(th3/+) mice. Our LC-MS/MS protein analysis and mRNA sequencing data provide quantitative expression estimates of 1590 proteins and 24,581 transcripts of the murine spleen and characterize key processes of erythropoiesis and RBC homeostasis such as the whole heme synthesis pathway as well as critical components of the red blood cell antioxidant systems and the proliferative cell cycling pathway. The data confirm that Tf treatment of nontransfused Hbb(th3/+) mice induces a systematic correction of these processes at a molecular level. Tf treatment of Hbb(th3/+) mice for 60 days leads to a complete molecular restoration of the normal murine spleen phenotype. These findings support further investigation of plasma-derived Tf as a treatment for thalassemia.