Florence Vallelian

Functional Heterogeneity of Memory CD4 T Cell Responses in Different Conditions of Antigen Exposure and Persistence

Memory CD4 T cell responses are functionally and phenotypically heterogeneous. In the present study, memory CD4 T cell responses were analyzed in different models of Ag-specific immune responses differing on Ag exposure and/or persistence. Ag-specific CD4 T cell responses for tetanus toxoid, HSV, EBV, CMV, and HIV-1 were compared. Three distinct patterns of T cell response were observed. A dominant single IL-2 CD4 T cell response was associated with the model in which the Ag can be cleared. Polyfunctional (single IL-2 plus IL-2/IFN-γ plus single IFN-γ) CD4 T cell responses were associated with Ag persistence and low Ag levels. A dominant single IFN-γ CD4 T cell response was associated with the model of Ag persistence and high Ag levels. The results obtained supported the hypothesis that the different patterns observed were substantially influenced by different conditions of Ag exposure and persistence.

Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy

Massive transfusion of blood can lead to clinical complications, including multiorgan dysfunction and even death. Such severe clinical outcomes have been associated with longer red blood cell (rbc) storage times. Collectively referred to as the rbc storage lesion, rbc storage results in multiple biochemical changes that impact intracellular processes as well as membrane and cytoskeletal properties, resulting in cellular injury in vitro. However, how the rbc storage lesion triggers pathophysiology in vivo remains poorly defined. In this study, we developed a guinea pig transfusion model with blood stored under standard blood banking conditions for 2 (new), 21 (intermediate), or 28 days (old blood). Transfusion with old but not new blood led to intravascular hemolysis, acute hypertension, vascular injury, and kidney dysfunction associated with pathophysiology driven by hemoglobin (Hb). These adverse effects were dramatically attenuated when the high-affinity Hb scavenger haptoglobin (Hp) was administered at the time of transfusion with old blood. Pathologies observed after transfusion with old blood, together with the favorable response to Hp supplementation, allowed us to define the in vivo consequences of the rbc storage lesion as storage-related posttransfusion hemolysis producing Hb-driven pathophysiology. Hb sequestration by Hp might therefore be a therapeutic modality for enhancing transfusion safety in severely ill or massively transfused patients.

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.

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.

Structural Stabilization in Tetrameric or Polymeric Hemoglobin Determines Its Interaction with Endogenous Antioxidant Scavenger Pathways

Hemoglobin (Hb) released into the circulation during hemolysis and chemically modified Hb proposed for use as oxygen therapeutics exert toxic effects that are partially attributable to hemes oxidant activity. Native extracellular Hb is scavenged by haptoglobin (Hp) after alphabeta-subunit dimerization. In the absence of Hp, monocyte/macrophage cell-surface CD163 binds and clears Hb. We evaluated several chemically modified Hbs to establish the role of chemical cross-linking patterns and molecular sizes on binding and clearance by each pathway. We found that Hbs possessing beta-globin cross-linking, irrespective of polymerization, demonstrate increased Hp affinity compared with alpha-globin-stabilized Hbs. These data suggest that Hb alpha-subunit accessibility is critical for Hp binding in the absence of dimerization. beta-Globin chain cross-linked tetramers/polymers displayed strong polyvalent Hp binding with increased viscosity and formation of visible gel matrices. Modified Hb interaction with CD163 and cellular uptake demonstrated an inverse relation with molecular size, irrespective of alpha and beta cross-linking. These findings were confirmed by HO-1 induction and intracellular ferritin accumulation in CD163-expressing HEK293 cells. Based on these results, a rational and systematic approach to HBOC design may be used to optimize interaction with endogenous Hb clearance and detoxification pathways.

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.