Absence Of Haptoglobin Research Articles

Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent.

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4- to 12-fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb-bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non-heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb-Hpx toxicity was iron-dependent, and was blocked by deferoxamine and ferrostatin-1. Up-regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron-dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

Mechanism of Trypanosoma brucei gambiense resistance to human serum

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic β-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Haptoglobin deficiency facilitates the development of autoimmune inflammation

Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL-6. HP has been demonstrated to modulate the immune response and to have anti-inflammatory activities. To analyze HP's effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp(-/-)) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). Hp(-/-)mice suffered from a more severe disease that was associated with increased expression of IL-17A, IL-6, and IFN-gamma mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL-17+ CD4+ and IFN-gamma+ CD4+ cells persisted in the CNS of Hp(-/-) mice. Absence of HP affected the priming and differentiation of T cells after MOG(35-55) immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp(-/-) T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.

CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin

CD163 mediates the internalization of hemoglobin-haptoglobin (Hb-Hp) complexes by macrophages. Because Hp binding capacity is exhausted during severe hemolysis, an Hp-independent Hb-clearance pathway is presumed to exist. We demonstrate that Hb interacts efficiently with CD163 in the absence of Hp. Not only is Hb internalized into an endosomal compartment by CD163 as a result of active receptor-dependent endocytosis; it also inhibits the uptake of Hb-Hp complexes, suggesting a common receptor-binding site. Free Hb further induces heme oxygenase mRNA expression in CD163+ HEK293 cells, but not in CD163- cells. Additional evidence for Hp-independent Hb-CD163 interaction is provided by the demonstration that CD163 mediates the uptake of alpha alpha-DBBF crosslinked Hb, a chemically modified Hb that forms minimal Hp complexes. Moreover, certain modifications to Hb, such as polymerization or the attachment of specific functional groups (3 lysyl residues) to the beta-Cys93 can reduce or enhance this pathway of uptake. In human macrophages, Hp-complex formation critically enhances Hb uptake at low (1 microg/mL), but not at high (greater than 100 microg/mL), ligand concentrations, lending support for a concentration-dependent biphasic model of macrophage Hb-clearance. These results identify CD163 as a scavenger receptor for native Hb and small-molecular-weight Hb-based blood substitutes after Hp depletion.

Electron Paramagnetic Resonance Study of Cell-Free Hemoglobin in Sickle Cell Disease: Potential Antioxidant Role of Haptoglobin.

It has previously been shown that red blood cell-free oxyhemoglobin (oxyHb) can affect endothelial function in Sickle Cell Disease (SCD) by virtue of its NO scavenging properties (Reiter et al, Nat Med, 2002). This reaction may have down-stream consequence via formation of metHb which has been demonstrated to have pro-oxidant properties and can oxidize low-density lipoprotein in vitro. In this study we have examined the fate of metHb in SCD vs normal plasma by examining the characteristic single line EPR signature of metHb in the g = 6 region. We show that the EPR signature of metHb differs in SCD compared with normals and exhibits a split EPR line indicative of a more rhombic heme geometry (See Figure). [Display omitted] Addition of purified metHb to SCD plasma results in a conversion of the single line EPR spectrum to the rhombic form. By addition of hemin and hemoglobin to plasma constituents we have determined that the rhombic EPR spectrum is consistent with heme bound to serum albumin. In addition, immunoprecipitation of serum albumin from SCD plasma with EPR analysis of the IP fraction revealed the presence of albumin associated metHb. Interestingly, the transfer of heme from metHb to albumin is accelereated by the presence of low-density lipoprotein and inhibited by the addition of haptoglobin suggesting that plasma components can modulate heme release from metHb and that the lack of haptoglobin in SCD plasma allowed the transfer of heme from metHb to albumin. To confirm this we added metHb to SCD plasma in the presence and absence of haptoglobin and observed that the addition of haptoglobin prevented conversion of the metHb spectrum to the albumin associated split (rhombic) species. From these studies we conclude that plasma haptoglobin not only binds hemoglobin but also limits heme release from metHb. To examine potential oxidative consequences of this effect, we incubated LDL with metHb in the presence and absence of haptoglobin. Haptoglobin inhibited LDL oxidation in a dose-dependent manner. Interestingly, the 1-1 isoform of haptoglobin was much more effective than the 2-2 isoform, in agreement with published studies on the efficacy of hemoglobin binding to the two haptoglobin varieties. Additionally, we have measured levels of 8-isoprostanes in the plasma of SCD patients as an index of lipid oxidation and show that 8-isoprostane levels are significantly elevated in SCD patients (295 209 pg/ml, mean S.D., n= 13, vs. 69 37 pg/ml, mean S.D. n= 5 for normal controls). In conclusion, these studies provide evidence that the saturation of the haptoglobin clearance mechanisms in hemolytic disorders such as SCD can not only affect the NO-axis of endothelial function due to increases in cell-free hemoglobin, but can also lead to altered heme metabolism leading to lipid oxidation and formation of pro-inflammatory lipids which have the potential to alter both endothelial and red cell function.

Binding of acellular, native and cross-linked human hemoglobins to haptoglobin: enhanced distribution and clearance in the rat

It is well established that hemoglobin resulting from red cell lysis binds to haptoglobin in plasma to form a complex. The increased molecular size precludes its filtration by the kidneys, redirecting it toward hepatocellular entry. Chemically cross-linked hemoglobins are designed to be resistant to renal excretion, even in the absence of haptoglobin. The manner in which binding to haptoglobin influences the pharmacokinetics of acellular cross-linked and native hemoglobins was investigated after intravenous injection of radiolabeled native human hemoglobin and trimesyl-(Lys82)beta-(Lys82)beta cross-linked human hemoglobin, at trace doses, into rats. Under these conditions, there is sufficient plasma haptoglobin for binding with hemoglobin. In vitro binding assayed by size-exclusion chromatography for bound and free hemoglobin revealed that, at <8 muM hemoglobin, native human hemoglobin was completely bound to rat haptoglobin, whereas only approximately 30% of trimesyl-(Lys82)beta-(Lys82)beta cross-linked hemoglobin was bound. Plasma disappearance of low doses (0.31 mumol/kg) of native and cross-linked hemoglobins was monoexponential (half-life = 23 and 33 min, respectively). The volume of distribution (40 vs. 19 ml/kg) and plasma clearance (1.22 vs. 0.4 ml.min(-1).kg(-1)) were higher for native than for cross-linked hemoglobin. Native and cross-linked human hemoglobins were found primarily in the liver, and not in the kidney, heart, lung, or spleen, mostly as degradation products. These pharmacokinetic findings suggest that the binding of hemoglobin to haptoglobin enhances its hepatocellular entry, clearance, and distribution.

Haemoglobin H Disease in a Turkish Family

Hb H disease was found in a Turkish woman, her mother and two of her siblings. Although all had mild pallor and two had mild splenomegaly, these persons were asymptomatic. In addition to two anodal bands, interpreted as Hb H and Hb Bart's, a third band was seen on Hb‐starch‐gel electrophoresis at pH 7. The slightly elevated plasma Hb level, the presence of methaemalbumin, and the absence of haptoglobin in the serum of the propositus were attributed to haemolytic anaemia.