Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent

Abstract

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.

Keywords: heme; intracerebral hemorrhage; iron; stroke; subarachnoid hemorrhage.

Fig. 1

Study time-line, indicating time points (hours) after hemoglobin (Hb) ± hemopexin (Hpx) treatment at which indicated testing was conducted.

Fig. 2

Effects of hemopexin on hemoglobin and hemin toxicity. (A-C) Phase contrast images of mixed neuron-glia cultures subjected to: (A) sham medium exchange only; phase bright neuron cell bodies cluster in groups and overlie a confluent glial monolayer; B) hemoglobin (Hb) 3 μM for 24 h; (C) Hb 3 μM plus hemopexin (Hpx) 1 mg/ml; neuronal degeneration and a fine precipitate are apparent, while the glial monolayer remains intact. (D-F) Glial-only cultures treated as in A-C; glial monolayer remains intact; precipitate is apparent in Hb+Hpx condition. (G) Bars represent mean neuronal loss (± S.E.M., n = 7 cultures for Hb 3 μM condition, 15 cultures/condition for Hb 10 μM, and 8 cultures/condition for hemin experiments) in mixed neuron-glia cultures as measured by LDH assay. (H) Percentage glial cell loss (± S.E.M., n = 12 cultures/condition) in glia-only cultures treated with Hb alone or with 1 mg/ml Hpx as in G. The low LDH values in sham cultures were subtracted from each mean value to yield the signal associated with neurotoxicity. ***p < 0.001 versus value in Hb or hemin only groups.

Fig. 3

Hemoglobin+hemopexin toxicity is oxidative and iron-dependent. Percentage neuron loss in mixed neuron-glia cultures treated with Hb 3 μM+Hpx 1 mg/ml alone (n = 14 cultures) or with 100 μM ascorbate (Asc, n = 10 cultures), 100 μM Trolox (Tro, n = 10 cultures), 100 μM deferoxamine (DFO, n = 10 cultures), or 10 μM ferrostatin-1 (Ferro, n = 10 cultures). ***p < 0.001 versus Hb+Hpx condition.

Fig. 4

Hemopexin binds heme moieties of Hb. Fluorescence quenching of 1 μM Hpx solution by Hb (0.25 μM containing 1 μM heme) over two hours, consistent with transfer of heme to Hpx. Each point represents mean of 5 replicates.

Fig. 5

Neurons express LRP1 and bind hemopexin. (A-D) Phase contrast and fluorescence images of fixed cultures immunostained with anti-NeuN and anti-LRP1. (E-H) Fixed cultures stained with anti-NeuN and 80 μg/ml solution of Alexa Fluor 568-labeled Hpx for 4 h; the labeled Hpx apoform was used since hemin binding quenched fluorescence when applied to fixed cells. (I, J) Viable culture incubated with 1 μM Alexa Fluor 568-labeled Hpx-hemin solution for 24 h. Uptake by phase bright neuron somata is apparent.

Fig. 6

Hemoglobin precipitation by hemopexin is prevented by haptoglobin. Viable cultures were treated for 24 hours with FITC-conjugated Hb (3 μM) alone (A,B), with unlabeled Hpx 1 mg/ml (C,D), or with Hb+Hpx plus 100 μM ascorbate (Asc, E,F) or 1 mg/ml haptoglobin (Hp, G,H). Bars represent mean fluorescence intensity in cultures treated as above or with deferoxamine (DFO) 100 μM or ferrostatin-1 (FS) 10 μM. *p < 0.05, ***p < 0.001 versus Hb+Hpx condition, n = 6 cultures/condition for sham, Hb and Hb+Hpx, 4 cultures/condition for +DFO, +Asc and +FS, and 5 cultures/condition for +Hp.

Fig. 7

Hemopexin increases hemoglobin precipitation and cell hemoglobin uptake. (A-F) Cultures were fixed after 8h exposure to Hb 3 μM plus Hpx 1 mg/ml, then stained with antibody to α-Hb. Bars represent mean Western blot band intensities (± S.E.M., 13 cultures/condition) from cultures receiving the same treatment, stained with antibodies specific to human α-Hb and β-Hb chains. Gel lane order corresponds with bar order. *p < 0.05 versus sham, ###p < 0.001 versus Hb alone condition.

Fig. 8

Haptoglobin attenuates Hb+Hpx neurotoxicity and nonheme chelatable iron increase. (A) Percentage neuron loss, measured by LDH release assay, in cultures treated for 24 h with Hb 10 μM alone (n = 15 cultures) or with Hpx 1 mg/ml (n = 15 cultures), Hpx 1 mg/ml+Hp 1 mg/ml (n = 8 cultures), or Hp 1 mg/ml without Hpx (n = 8 cultures); (B) nonheme iron content of cultures (n = 7-8) subjected to same treatment, as detected by ferrozine assay; (C) cultures (10/condition) treated with Hb alone or with Hpx as in B, but with addition of 10 μM ferrostatin-1 (F) to prevent cell lysis and neuron iron loss. *p < 0.05, ***p < 0.001 versus Hb alone or Hb + ferrostatin-1 condition, ###p < 0.001 versus Hb+Hpx condition.

Fig. 9

Hemopexin reduces heme oxygenase-1 (HO-1) and ferritin induction by hemoglobin. HO-1 and ferritin expression in cultures treated with Hb 3 μM alone or with 1 mg/ml Hpx for 4 or 8 hours. ***p < 0.001 versus sham, ###p < 0.001 versus Hb alone condition, n = 6 cultures/condition.