CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma

CD163 is a highly expressed macrophage membrane protein belonging to the scavenger receptor cysteine rich (SRCR) domain family. The CD163 expression is induced by interleukin-6, interleukin-10 and glucocorticoids. Its function has remained unknown until recently when CD163 was identified as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular hemolysis. Besides having a detoxificating effect by removing Hb from plasma, the CD163-mediated endocytosis of the Hp-Hb complex may represent a major pathway for uptake of iron in the tissue macrophages.The novel functional linkage of CD163 and Hp, which both are induced during inflammation, also reveal some interesting perspectives relating to the suggested anti-inflammatory properties of the receptor and the Hp phenotypes.     

Regulation of CD163 associated casein kinase II activity is haptoglobin genotype dependent

Free hemoglobin is now recognized as a major mediator of a variety of vascular diseases. The abundant serum protein haptoglobin irreversibly binds to hemoglobin and promotes the uptake of hemoglobin via the macrophage CD163 receptor. The haptoglobin gene is polymorphic in man with two common alleles denoted 1 and 2. The haptoglobin genotype specifies the nature of the response of the macrophage to free hemoglobin. Hp 1-Hb complexes stimulate an anti-inflammatory macrophage phenotype while Hp 2-Hb complexes do not. We have previously demonstrated that Hp 1-Hb induced anti-inflammatory cytokine production is critically dependent on casein kinase II. In this study we set out to determine whether the amount or the activity of casein kinase II associated with CD163 was altered by the binding of Hp 1-1-Hb to CD163. Our results indicate that casein kinase II activity is increased by the binding of Hp 1-1-Hb to CD163.     

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-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 Binding to Haptoglobin-Hemoglobin Complexes Involves a Dual-point Electrostatic Receptor-Ligand Pairing

Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.     

Effect of inhibited endocytosis in sinusoidal liver cells on catabolism of equine haptoglobin and haemoglobin in the hen

Catabolism of equine haptoglobin (Hp), haptoglobin-haemoglobin complex (Hp-Hb) and haemoglobin (Hb) in the hen was studied in the liver reticuloendothelial system (RES) in which endocytosis of protein was inhibited by aggregated denatured albumin (Agr-Alb). Intravenous injection of Agr-Alb together with equine [125I]Hp, [125I]Hp-Hb or [125I]Hb partially inhibited elimination of these proteins from hen circulation. The decrease of clearance was less pronounced when the labelled proteins were introduced 30 min after Agr-Alb injection due to stimulation of phagocytosis in RES by Agr-Alb. Elimination of equine proteins (Hp, Hp-Hb and Hb) from hen circulation by RES is only one of the possible metabolic fates of these proteins.     

Corticosteroid enhances heme oxygenase-1 production by circulating monocytes by up-regulating hemoglobin scavenger receptor and amplifying the receptor-mediated uptake of hemoglobin-haptoglobin complex

This study examined the relationship between steroid treatment and CD163-mediated downstream pathways linked to inflammatory resolution. Twelve patients referred for congenital heart disease surgery were divided into two groups based on the severity of intravascular hemolysis during cardiopulmonary bypass surgery. Patients with severe intravascular hemolysis were administered haptoglobin during the procedure. Flow cytometry indicated a peak in monocyte CD163 expression on post-operative day 1 in both groups. Enhanced and prolonged heme oxygenase-1 (HO-1) mRNA expression levels were observed in patients who received haptoglobin. Binding of hemoglobin-haptoglobin complex (Hb/Hp) to CD163 resulted in significant induction of HO-1 by peripheral blood mononuclear cells after exposure to dexamethasone prior to culture. This effect was significantly inhibited by anti-CD163 antibody. Our results demonstrated up-regulation of CD163 expression on the monocyte surface by steroid treatment. Steroid treatment was suggested to facilitate CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induce acceleration of HO-1 synthesis.     

Extracellular hemoglobin polarizes the macrophage proteome toward Hb-clearance, enhanced antioxidant capacity and suppressed HLA class 2 expression

Peripheral blood monocytes and macrophages are the only cell population with a proven hemoglobin (Hb) clearance capacity through the CD163 scavenger receptor pathway. Hb detoxification and related adaptive cellular responses are assumed to be essential processes to maintaining tissue homeostasis and promoting wound healing in injured tissues. Using a dual platform mass spectrometry analysis with MALDI-TOF/TOF and LTQ-Orbitrap instruments combined with isobaric tag for relative and absolute quantitation (iTRAQ), we analyzed how Hb exposure could modulate the macrophage phenotype on a proteome level. We identified and relatively quantified 3691 macrophage proteins, representing the largest human macrophage proteome published to date. The Hb polarized macrophage phenotype was characterized by an induced Hb:Hp-CD163-HO1-ferritin pathway and enhanced antioxidant enzymes while suppression of HLA class 2 was the most prominent effect. Enhanced Hb clearance and antioxidant capacity together with reduced antigen presentation might therefore be essential qualities of Hb polarized macrophages in wounded tissues and hemorrhage or atherosclerotic plaques.     

Oleacein enhances anti-inflammatory activity of human macrophages by increasing CD163 receptor expression

BackgroundOleacein (dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol; 3,4-DHPEA-EDA) have been proven to possess antioxidant and anti-inflammatory activity.PurposeIn this study, we examined whether oleacein could increase CD163 and IL-10 receptor expression as well as HO-1 intracellular secretion in human macrophages.MethodsEffect of oleacein (10 and 20 μmol/l) or oleacein together with complexes of haemoglobin (Hb) and haptoglobin 1-1 (Hp11) or haptoglobin 2-2 (Hp22) on expression of IL-10 and CD163 receptor was determined by Flow Cytometry. Expression of CD163mRNA was measured by real-time quantitative RT-PCR. Heme oxygenase 1 (HO-1) intracellular secretion in macrophages was investigated by enzyme-linked immunosorbent assay (ELISA).ResultsOleacein (OC) together with complexes HbHp11 or HbHp22 stimulated the expression of CD163 (30-100-fold), IL-10 (170-300-fold) and HO-1 secretion (60-130-fold) after 5 days of coincubation. The 2-fold (24 h), 4-fold (48 h) increase of CD163 mRNA level and its final (72 h) decrease was also observed.ConclusionOur results suggested that oleacein enhances anti-inflammatory activity of complexes haemoglobin with haptoglobin 1-1 and 2-2 and could play a potential role in the prevention of inflammatory disease related to atherosclerosis.     

A previously unrecognized protein-protein interaction between TWEAK and CD163: potential biological implications

TWEAK (TNF-like weak inducer of apoptosis) is a TNF superfamily member implicated in several mechanisms. Although fibroblast growth factor inducible 14 (Fn14)/TweakR has been reported as its receptor, an as yet unrecognized surface molecule(s) might modulate TWEAK function(s). Thus, we set out to identify TWEAK-binding proteins by screening a combinatorial peptide library. Cyclic peptides containing a consensus motif (WXDDG) bound to TWEAK specifically. These peptides were similar to CD163, a scavenger receptor cysteine-rich domain family member, restricted to the monocyte/macrophage lineage and responsible for the uptake of circulating haptoglobin-hemoglobin (Hp-Hb) complexes. Sequence profile analysis suggested that TWEAK mimicked the CD163 natural ligand (Hp-Hb). Consistently, we show dose-dependent TWEAK binding to CD163 and blockade by an anti-CD163 Ab. In a competition assay, both soluble CD163 and Fn14/TweakR were able to compete off TWEAK binding to coated Fn14/TweakR or CD163, respectively. Flow-cytometry and immunofluorescence assays showed that human monocytes (Fn14/TweakR negative and CD163 positive) bind TWEAK, thus blocking the recognition of CD163 and reducing the activation mediated by a specific mAb in these cells. We demonstrate that monocytes can sequester TWEAK from supernatants, thus preventing tumor cell apoptosis; this effect was reverted by preincubation with the peptide mimicking CD163 or with a mAb anti-CD163, indicating specificity. Finally, we show that recombinant human TWEAK binding to CD163-transfected Chinese hamster ovary cells is inhibited by the presence of either unlabeled TWEAK or the Hp-Hb complex. Together, these data are consistent with the hypothesis that CD163 either acts as a TWEAK scavenger in pathological conditions or serves as an alternate receptor for TWEAK in cells lacking Fn14/TweakR.     

Studies on free or haptoglobin-bound hemoglobin and derivatives (semihemoglobins and porphyrinated semihemoglobins). Some aspects of their peroxidatic activity.

The peroxidatic activity of hemoglobin (Hb) is known to be enhanced when this hemoprotein is bound to haptoglobin (Hp). The peroxidatic reaction (H2O2, guaiacol as donor) has been kinetically studied (Steady-state) in the presence of free or rabbit-haptoglobin bound human hemoglobin and some of its derivatives, all in ferricyano-form. With free Hb+ CN, we observed linearity of Lineweaver and Burk plots in a wide range of concentrations, the donor's behaviour was therefore assumed to obey the Michaelis-Menten mechanism. When Hp-Hb+ CN is the enzyme, the donor's behaviour is more complicated, analysis shows the existence of two kinds of donor's binding sites. The possibility whether this behaviour might correspond to the intrinsic properties of Hb chains, as revealed after combination with Hp, was examined. The peroxidatic activity of free and Hp-bound alpha and beta chains of Hb were studied. The alpha chains of Hb combine with Hp whereas the beta chains fail to do so. In order to make useful comparisons, the peroxidatic activity of Hp-bound alpha and beta chains were studied by the use of Hp-semihemoglobin complexes where the semihemoglobins carried heme on only one type of chain (alpha or beta). Results did not show an evident correlation between the activities of the two free or bound types of chains and those of the two classes of binding sites revealed in Hp-Hb+ CN. Moreover, it appeared that the heme-free complementary chain might influence the activity of the heme-carrying alpha or beta chain in semihemoglobins and Hp-semihemoglobin complexes. The binding or protoporphyrin on free and Hp-bound semihemoglobins leads to species which exhibit structures close to that of Hb and Hp-Hb complex respectivley. Results of studies on these derivatives brought up new interesting data : when the porphyrin ring alone is bound to the heme deficient chains (alpha or beta), in Hp-semihemoglobin complexes, the same peculiar behaviour, already observed with Hp-Hb complex, is found again. The structural implications of these results are discussed.     

Accumulation of indigestible substances reduces fusion competence of macrophage lysosomes

It is well known that mouse macrophages loaded with indigestible substances become highly vacuolated. However, why this vacuolization occurs and its effect on lysosome function and intracellular transport during endocytosis remain unknown. Here, macrophage vacuoles were formed by incubation with sucrose or a tripeptide of the D-isomer of alanine and were determined to be lysosomal in origin by staining with the lysosomal glycoproteins and lysosomal hydrolases. However, as indicated by confocal and electron microscopy, subsequent delivery of both fluid phase (lucifer yellow, horse-radish peroxidase) and receptor-bound ligands (IgG complexes) was significantly reduced, suggesting that indigestible material reduced the ability of the loaded lysosomes to fuse with endosomes containing newly internalized tracers. Nevertheless, ligands internalized by the vacuolated cells were degraded at almost the normal rate, indicating that degradation occurs in the absence of delivery to the loaded lysosomes. We have also found that this fusion inhibition occurs in human alveolar macrophages loaded with physiologic debris from smoking and asbestos. These results suggest that indigestible material within lysosomes, such as is present in residual bodies in vivo, may affect their fusion competence.     

Molecular Modeling of the Human Hemoglobin-Haptoglobin Complex Sheds Light on the Protective Mechanisms of Haptoglobin

Hemoglobin (Hb) plays a critical role in human physiological function by transporting O₂. Hb is safe and inert within the confinement of the red blood cell but becomes reactive and toxic upon hemolysis. Haptoglobin (Hp) is an acute-phase serum protein that scavenges Hb and the resulting Hb-Hp complex is subjected to CD163-mediated endocytosis by macrophages. The interaction between Hb and Hp is extraordinarily strong and largely irreversible. As the structural details of the human Hb-Hp complex are not yet available, this study reports for the first time on insights of the binding modalities and molecular details of the human Hb-Hp interaction by means of protein-protein docking. Furthermore, residues that are pertinent for complex formation were identified by computational alanine scanning mutagenesis. Results revealed that the surface of the binding interface of Hb-Hp is not flat and protrudes into each binding partner. It was also observed that the secondary structures at the Hb-Hp interface are oriented as coils and α-helices. When dissecting the interface in more detail, it is obvious that several tyrosine residues of Hb, particularly β145Tyr, α42Tyr and α140Tyr, are buried in the complex and protected from further oxidative reactions. Such finding opens up new avenues for the design of Hp mimics which may be used as alternative clinical Hb scavengers.     

Peroxidase Activity of Hemoglobin��Haptoglobin Complexes: COVALENT AGGREGATION AND OXIDATIVE STRESS IN PLASMA AND MACROPHAGES*

As a hemoprotein, hemoglobin (Hb) can, in the presence of H₂O₂, act as a peroxidase. In red blood cells, this activity is regulated by the reducing environment. For stroma-free Hb this regulation is lost, and the potential for Hb to become a peroxidase is high and further increased by inflammatory cells generating superoxide. The latter can be converted into H₂O₂ and feed Hb peroxidase activity. Haptoglobins (Hp) bind with extracellular Hb and reportedly weaken Hb peroxidase activity. Here we demonstrate that: (i) Hb peroxidase activity is retained upon binding with Hp; (ii) in the presence of H₂O₂, Hb·Hp peroxidase complexes undergo covalent cross-linking; (iii) peroxidase activity of Hb·Hp complexes and aggregates consumes reductants such as ascorbate and nitric oxide; (iv) cross-linked Hb·Hp aggregates are taken up by macrophages at rates exceeding those for noncovalently cross-linked Hb·Hp complexes; (v) the engulfed Hb·Hp aggregates activate superoxide production and induce intracellular oxidative stress (deplete endogenous glutathione and stimulate lipid peroxidation); (vi) Hb·Hp aggregates cause cytotoxicity to macrophages; and (vii) Hb·Hp aggregates are present in septic plasma. Overall, our data suggest that under conditions of severe inflammation and oxidative stress, peroxidase activity of Hb·Hp covalent aggregates may cause macrophage dysfunction and microvascular vasoconstriction, which are commonly seen in severe sepsis and hemolytic diseases.As a hemoprotein, Hb,² in the presence of oxidizing equivalents such as H₂O₂, can act as a peroxidase with very high oxidizing potential (1). In red blood cells, this potentially dangerous activity is strictly regulated by the reducing environment and the lack of oxidizing equivalents. The inadvertently appearing ferric forms of Hb are short-lived, and the hemoprotein is effectively converted into ferro-Hb (deoxy-Hb) by metHb reductase (2). Normally, less than 2% of total Hb exists in the form of MetHb because the rate of Hb reduction is far greater than its oxidation (2). For stroma-free Hb, however, this intracellular regulation is lost, and the likelihood for Hb to act as a peroxidase is high. This possibility is markedly increased in the course of severe inflammation (e.g. in sepsis) by the generation of superoxide radicals by immune cells. The latter can be spontaneously or catalytically (by extracellular superoxide dismutase) converted into H₂O₂, a fuel for Hb peroxidase activity. In line with this, several clinical and experimental investigations have established that lethality in sepsis is increased in the setting of hemolysis (3–5).Circulating haptoglobin (Hp) provides an important endogenous defense against the toxic effects of Hb (6, 7). The major biological function of this abundant plasma protein is binding and recycling of stroma-free Hb via the macrophage CD163 receptor-mediated pathway (8, 9). It has been proposed that Hp possesses antioxidant activity and diminishes oxidative stress induced by stroma-free Hb (10–12). The antioxidant action of Hp toward Hb has been associated, at least in part, with weakening of its peroxidase activity (10) or preventing oxidation and cross-linking of Hb (7).Previous work has demonstrated that peroxidase activity of different hemoproteins, including Hb, can induce protein self-oxidation leading to covalent cross-linking and aggregation (13–16). Whether these hetero-oligomeric covalent aggregates retain the peroxidase activity is unknown. If the aggregates retain peroxidase activity, they may continue to be a source of oxidative stress both in circulation as well as in phagocytizing cells involved in their clearance such as macrophages.In the current work, we determined the extent to which: (i) Hb peroxidase activity is decreased by binding with Hp; (ii) peroxidase activity of Hb·Hp complexes initiates cross-linking into covalent hetero-oligomers; (iii) peroxidase activity of Hb·Hp complexes and aggregates utilizes nitric oxide (NO^•); (iv) Hb·Hp aggregates are taken up by macrophages as compared with noncovalent Hb·Hp complexes; and (v) the engulfed Hb·Hp aggregates induce oxidative stress and cytotoxicity. Here, we report that Hb·Hp complexes and aggregates are potent peroxidases capable of inducing oxidative stress in both plasma and macrophages. We further demonstrate the presence of Hb·Hp aggregates in septic plasma.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

The effects of Con A, WGA, Zymosan A on macrophage cytosolic pH and outflow of lysosomal content through exocytosis were studied with SNAFL-calcein and FITC-Dextran on ACAS570. The results showed all three ligands could induce macrophage cytosolic acidification in about 10 min and kept at the same level hereafter; outflow of lysosomal fluorescent probe through exocytosis appeared in 15-20 min. In resting conditions, macrophage lysosomes mainly distributed in cell center; after stimulated for 15 min by three ligands, the number of lysosomes increased in membrane periphery, in 25-30 min lysosomes moved back toward cell center. We proposed that ligands induced lysosomal pH rises was a basic factor for outflow of lysosomal content through exocytosis, cytosolic acidification inhibited receptor-mediated endocytosis. Cytosolic acidification and outflow of lysosomal content through exocytosis were the results of cellular self-regulation and self-protection during receptor-mediated endocytosis.     

受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

本文利用激光扫描共聚焦显微镜A-CAS570从细胞形态学和功能两方面,研究了刀豆素A(Concanavalin A,Con A)、麦芽凝集素(Wheat Germ Agglutinin,WGA)、酵母多糖(Zymosan A,Z.A)对小鼠腹腔巨噬细胞胞质pH和溶酶体内荧光探针FITC—Dextran排出细胞的影响。结果显示三种配体加入细胞外液10min内,胞质pH很快下降,此后维持在该水平;在15min左右细胞外FITC一Dextran迅速增加,20min后变化趋于停止;在三种配体加入后15min左右,细胞内溶酶体在质膜内侧增多;25—30min溶酶体重新向细胞中央运动。根据上述实验结果,我们认为溶酶体pH升高是触发溶酶体内荧光探针通过胞吐作用排出细胞的必要条件,胞质酸化抑制溶酶体内容物通过胞吐作用排出细胞。配体刺激引起的溶酶体内容物通过胞吐作用排出细胞和胞质酸化是细胞自我调节和保护的一种反映。     

Liposomes surface conjugated with human hemoglobin target delivery to macrophages

Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor-mediated endocytosis. The drug delivery system described in this work consists of Hb decorated liposomes that can encapsulate any therapeutic molecule of interest, in this case the model fluorescent dye calcein was used in this study. The results of this study clearly demonstrate that this delivery system is specific towards macrophages and demonstrates the feasibility of using this approach in targeted drug delivery.     

Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products

We have previously shown that oxidized low-density lipoprotein (LDL) induces damage to the macrophage lysosomal membranes, with ensuing leakage of lysosomal contents and macrophage cell death. Cholesterol oxidation products (ChOx) have been reported to be the major cytotoxic components of oxidized LDL/LDL- and also to stimulate cholesterol accumulation in vascular cells. In the present study, we characterized the initial events during macrophage damage induced by cholesterol oxidation products (ChOx). Within 24 h of exposure, ChOx caused lysosomal destabilization, release to the cytosol of the lysosomal marker-enzyme cathepsin D, apoptosis, and postapoptotic necrosis. Enhanced autophagocytosis and chromatin margination was found 12 h after the exposure to ChOx, whereas apoptosis and postapoptotic necrosis was pronounced 24 and 48 h after the exposure. Some lysosomal vacuoles were then filled with degraded cellular organelles, indicating phagocytosis of apoptotic bodies by surviving cells. Because caspase-3 activation was detected in the ChOx-exposed cells, lysosomal destabilization may associate with the leakage of lysosomal enzymes, and activation of the caspase cascade. MnSOD mRNA levels were markedly increased after 24 h of exposure to ChOx, suggesting associated induction of mitochondrial protection repair or turnover. We conclude that ChOx-induced damage to lysosomes and mitochondria are sequelae to the cascade of oxysterol cytotoxic events. The early disruption of lysosomes induced by ChOx, with resultant autophagocytosis may be a critical event in apoptosis and/or necrosis of macrophages/foam cells during the development of atherosclerotic lesions.     

Can Gas Replace Protein Function? CO Abrogates the Oxidative Toxicity of Myoglobin

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N₂. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.     

Apoptotic macrophage-derived foam cells of human atheromas are rich in iron and ferritin, suggesting iron-catalysed reactions to be involved in apoptosis

We investigated the presence of low-molecular-weight iron and ferritin in human atheromas, and their possible relation to the apoptotic process. Arterial wall segments with fatty streaks were collected from coronary arteries and thoracic aortas of 12 clinical autopsy cases with general atherosclerosis. Normal appearing regions from the same cases together with normal coronary arteries from seven young forensic autopsy cases, without any sign of atherosclerosis, were used for comparison. Anti-CD68 (macrophage marker) and anti-ferritin antibodies were applied to serial sections of the arterial wall segments, fixed in formadehyde and embedded in paraffin wax, using an avidin-biotin complex (ABC) technique. Similarly, apoptotic cells were assayed by the TUNEL technique, while low-molecular-weight iron was cytochemically detected by autometallography. Cell counting and computerised image analysis were performed to compare the distribution of macrophages, ferritin- and iron-rich cells, and apoptotic cells in the intima, media, and adventitia of the arteries.

Pronounced ferritin accumulation, occurrence of lysosomal low-molecular-weight iron, and apoptosis mainly concerned CD68-positive cells (macrophages) in the atherosclerotic lesions. No ferritin- or CD68-positivity was found in normal coronary arteries from the young forensic-autopsy cases, while a moderate number of such cells were observed in the intima of normal looking vessel areas from the control cases. In the intima, cytosolic ferritin and low-molecular-weight iron with a lysosomal type distribution were found in many CD68-positive macrophages which frequently were surrounded by erythrocytes. A substantial number of apoptotic cells within the intima, media, and adventitia were registered in all atherosclerotic lesions examined, although mainly in the vulnerable macrophage-enriched areas of the atheroma shoulder.

We suggest that iron may occur within the cytosol, mainly bound in ferritin, but also in low-molecular weight, redox-active form within the acidic vacuolar apparatus of macrophages and macrophage-derived foam cells following erythrophagocytosis or phagocytosis of apoptotic cells. Low-molecular-weight iron within lysosomes, present due to degradation of iron-containing structures, such as ferritin, may partially become exocytosed and contribute to cell-mediated LDL-oxidation. Moreover, such lysosomal iron may also sensitise lysosomes to oxidative stress and induce apoptosis of macrophage/foam-cells that may result in instability and rupture of atherosclerotic plaques.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.