Gabriele Schoedon

Deactivation of Macrophages with Interleukin-4 Is the Key to the Isolation of Tropheryma whippelii

Whipples disease is a systemic illness caused by a specific agent. Despite recognition of bacteria in lesions, efforts to isolate the causative agent remained futile. A novel strategy was devised to culture Whipple bacilli in deactivated mononuclear phagocytes. Infected tissue was inoculated into human phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone. Within 8-10 days, diastase-resistant periodic acid-Schiff-positive inclusions appeared, corresponding to intact and degenerating bacteria shown to be Tropheryma whippelii by electron microscopy and molecular analyses. T. whippelii was passaged several times in deactivated monocytes and a monoblastic cell line. Time-kinetics growth studies and comparative polymerase chain reaction analysis documented multiplication of T. whippelii in deactivated macrophages. Complementary studies showed that IL-4 rendered phagocytes permissive for T. whippelii, a strong indication that host factors contribute to the pathogenesis of Whipples disease. The propagation of T. whippelii will permit microbiologic, immunologic, seroepidemiologic, and therapeutic studies of this pathogen.

Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification

Detoxification and clearance of extracellular hemoglobin (Hb) have been attributed to its removal by the CD163 scavenger receptor pathway. However, even low-level hydrogen peroxide (H(2)O(2)) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high-affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing alpha-globin cross-links and oxidations of amino acids in critical regions of the beta-globin chain (eg, Trp15, Cys93, and Cys112). As a result of this structural stabilization, H(2)O(2)-exposed Hb-Hp binds to CD163 with the same affinity as nonoxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163-expressing cells were found to be as efficient as with nonoxidized complex. Hp complex formation did not alter Hbs ability to consume added H(2)O(2) by redox cycling, suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H(2)O(2) preserving CD163-mediated Hb clearance under oxidative stress conditions. In addition, our data provide in vivo evidence that unbound Hb is oxidatively modified within extravascular compartments consistent with our in vitro findings.

Induction of the CD163-dependent haemoglobin uptake by macrophages as a novel anti-inflammatory action of glucocorticoids.

Summary.  Highly efficient systems remove toxic and pro‐inflammatory haemoglobin (Hb) from the circulation and local sites of tissue damage. Macrophages are major Hb‐clearing cells; CD163 was recently recognized as the specific haemoglobin–haptoglobin scavenger receptor (HSR). We show that dexamethasone strongly induced the specific uptake of haemoglobin–haptoglobin complexes, CD163 mRNA transcription (13‐fold) and cell surface expression (10‐fold) by human macrophages. In contrast, the TH2‐cytokine interleukin 4 (IL‐4) completely suppressed functional CD163 expression. The range of functional receptor modulation reached a factor of 100 after 4 h of macrophage–ligand interaction. Based on these results, we propose the augmentation of Hb clearance as a novel anti‐inflammatory action of glucocorticoids.

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.

The reaction of hydrogen peroxide with hemoglobin induces extensive α-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways☆

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.

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.

Hemoglobin Can Attenuate Hydrogen Peroxide–Induced Oxidative Stress by Acting as an Antioxidative Peroxidase

Hemoglobin is considered a potentially toxic molecule when released from erythrocytes during hemolysis, inflammation, or tissue injury. The mechanisms of toxicity involve reduced nitric oxide bioavailability and oxidative processes both occurring at the heme prosthetic groups. When the endogenous oxidant H(2)O(2) reacts with Hb, transient radicals are generated during the peroxidative consumption of H(2)O(2). If not neutralized, these radicals can lead to tissue toxicity. The net biologic effect of extracellular Hb in an H(2)O(2)-rich environment will therefore be determined by the balance of H(2)O(2) decomposition (potential protective effect) and radical generation (potential damaging effect). Here we show that Hb can protect different cell types from H(2)O(2)-mediated cell death and the associated depletion of intracellular glutathione and ATP. Importantly, Hb blunts the transcriptional oxidative-stress response induced by H(2)O(2) in human vascular smooth muscle cells (VSMCs). Based on spectrophotometric and quantitative mass spectrometry analysis, we suggested a novel mechanism in which Hb redox-cycles H(2)O(2) and simultaneously internalizes the radical burden, with irreversible structural globin changes starting with specific amino acid oxidation involving the heme proximate betaCys93 and ultimately ending with protein precipitation. Our results suggest that complex interactions determine whether extracellular Hb, under certain circumstances, acts a protective or a damaging factor during peroxidative stress conditions.

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.