Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis

Abstract

Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.

Figure 1

Protease activity of purified Hxs. Enzymatic activity of different purified Hxs (two human and one rabbit) was tested at the same molar concentration (6.7 μmol/L) using chromogenic substrate I: S2238 and II: S2288 that are sensitive to thrombin and serine proteases respectively. Trypsin (0.4 μmol/L) was used as positive control. The substrates were mixed with different Hxs or controls and incubated at 37°C for different times. The absorbance at 405 nm was measured by spectrophotometry. The results represent mean ± SE and are representative of three independent experiments.

Figure 2

Purified Hxs do not inhibit the chemotaxis of neutrophils induced by fMLF and LTB4. Human neutrophils were preincubated with different purified Hxs (hHx-A, rabbit Hx and hHx-B) or control (RPMI 1640 containing 2% FBS) for 1 h. Then 1–2.5 × 10⁵ cells/30 μl cells were applied to the top of the transwell with the lower microplate filled with fMLF (1 nmol/L and 10 nmol/L) (A) or LTB4 (0.1 nmol/L, 1 nmol/L and 10 nmol/L) (B) and incubated for 1 h. Neutrophils migrated to the bottom microplate wells were stained with 0.4% trypan blue and live cells were counted. The percentage of cells migrated over the total cells uploaded to the top of the transwell was calculated. The results represent the mean ± SE and are representative of three independent experiments.

Figure 3

Recombinant Fc-hHx has the same heme-binding activity as purified Hx. Recombinant Fc-hHx prepared as described in Materials and Methods was applied to SDS-PAGE with Coomassie Fluor Orange (Life Technologies) staining (A) and developed as a Western blot with Cy3-labeled goat anti-human Fc antibody (Jackson ImmunoResearch) (B). Fc-hHx is shown in the reduced gels as a high-purity 75-kDa monomer (see arrow). In nonreduced gels, the Fc-hHx is a 150-kDa dimer (not shown) (C) Heme binding assay was done with 10 μmol/L of recombinant Fc-hHx; purified hHx-B; Fc fragment of IgG1; human albumin (hALB). All protein samples or PBS were mixed with free heme at different concentrations (3 and 10 μm) at room temperature for 30 min. The absorbance at 413 nm was measured by Nanodrop ND1000. The results represent the mean ± SE.

Figure 4

Recombinant Fc-hHx inhibits TNF production synergistically induced by LPS and hemin. Mouse BMDMs were incubated with LPS (2 ng/mL) only or with hemin (1 μmol/L) in serum-free medium (SFM) for 9 h in the absence or presence of 1.67 μmol/L of one of the following proteins: Fc fragment of IgG1; Fc-hHx; hHx-B. TNF levels were measured in the supernatants of the culture. The results represent mean ± SE and are representative of four independent experiments. **P < 0.01, compared between cells treated with and without hemin.

Figure 5

Recombinant Fc-hHx has minimal protease activity and no inhibition on the chemotaxis of human neutrophils induced by fMLF and LTB4. Enzymatic activity of recombinant Fc-human Hx (Fc-hHx) was tested by using chromogenic substrates substrate I: S2238 (A) and II: S2288 (B). Fc-hHx and the tag Fc at the concentration of 6.7 μmol/L, positive control trypsin (0.4 μmol/L), negative control human albumin (hALB) and PBS were mixed with the substrate at desired concentrations and incubated at 37°C for different times. The absorbance at 405 nm was measured by spectrophotometry. The small inserts are the sections with small scale and without trypsin control to show the very low increase of the absorbance by Fc-hHx (C) and (D): Fc or Fc-hHx at 100 μg/mL (1.67 μmol/L) were also used to pretreat human neutrophils with control (RPMI 1640 containing 2% FBS) for 1 h. Then 1–2.5 × 10⁵ cells/30 μL cells were applied to the top of the transwell with the lower microplate filled with LTB4 (0.1 nmol/L, 1 nmol/L and 10 nmol/L) (C) or fMLF (1 nmol/L and 10 nmol/L) (D) and incubated for 1 h. Neutrophils migrated to the bottom microplate wells were stained with 0.4% trypan blue and live cells were counted. The percentage of cells migrated over the total cells uploaded to the top of the transwell was calculated. The results represent the mean ± SE and are representative of three independent experiments.

Figure 6

Hx does not suppress neutrophil migration in mice. Thirty minutes after intravenous injection of human Hx (hHx-B) or albumin (hALB) (2 mg/mouse), neutrophil migration was induced by i.p. injection of 500 μl of 4% thioglycolate. At 2 h or 4 h post injection, peritoneal cells were flushed out by PBS and counted. (A) The peritoneal cells were incubated with PE- conjugated anti-Gr-1 and analyzed via FACS to determine the percentage of neutrophils over the total cells. Total neutrophils were calculated. (B) The MPO activity was measured in the total peritoneal cells. The results are representative of five independent experiments and presented as the mean ± SE.

Figure 7

LPS and hemin synergistically induce the production of LTB4 from human monocytes and Hx blocks this synergy by decreasing LTB4 levels and suppresses chemotaxis of neutrophils. (A) Human monocytes were incubated with LPS (10 μg/mL) in the presence or absence of hemin (1 μmol/L) for 1, 3 and 5 h. LTB4 concentrations in the supernatants were measured and compared between the cultures with LPS only and with LPS and hemin. (B) Human monocytes were incubated with LPS (10 μg/mL) and with or without hemin (1 μmol/L) in the absence or presence of hHx-B (1 mg/mL) for 3 h. LTB4 concentrations were measured and compared between the cultures with and without Hx. (C) The supernatants collected from human monocyte cultures at 3 h were loaded to the bottom microplate well of the transmigration system in chemotaxis assay. The percentage of neutrophils migrated to the bottom wells over the total cells loaded to the top of the transwells were calculated and compared between the cultures with and without hHx-B. The results are representative of three independent experiments and presented as the mean ± SE. **P < 0.01 LPS with hemin versus LPS only.

Figure 8

The role of Hx in neutrophil chemotaxis. The pathogenesis of severe infectious diseases is associated with hemolysis and secondary release of cell-free heme. LPS and heme synergize to induce the production of TNF, IL-6 and LTB4 from monocytes/macrophages. LTB4 induces neutrophil migration. Two hypotheses might explain the effect of Hx to suppress chemotaxis of neutrophils: hypothesis I, Hx binds heme and therefore blocks synergistic production of LTB4 and other chemotaxins, secondarily decreasing the signal for the chemotaxis of neutrophils; hypothesis II, Hx itself has protease activity and direct inhibitory effect on the neutrophil migration. The results from this paper support hypothesis I.