Figure 1
Figure 1. Induction of HO-1 in FVIII-deficient mice protects from the development of FVIII inhibitors. (A) Induction of HO-1 in the liver of hemin-treated mice. FVIII-deficient mice were treated by intravenous administration of 150 μL of PBS or hemin (50, 400, and 750μM). After 24 hours, livers (200 mg) were recovered and snap-frozen. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to polyvinylidene fluoride membrane. HO-1 was detected using an anti–HO-1 rat monoclonal IgG (R&D Systems), a horseradish peroxidase–conjugated rabbit anti–rat IgG, and the enhanced chemiluminescence kit. As a control of protein loading, membranes were stained with Protogold after chemiluminescence detection (not shown). Representative of 4 mice per group. Increased HO-1 expression was also detected 48 hours after hemin treatment (not shown). (B-C) Levels of anti-FVIII IgG in hemin-treated mice. Mice were treated with PBS (□) or hemin (750μM, ■) 3 times at weekly intervals. Twenty-four hours after each treatment, mice received intravenously 1 IU FVIII (200 μL). Blood was collected by retro-orbital puncture 1 week after the third FVIII administration. Anti-FVIII IgG were quantified by ELISA using a mouse monoclonal anti-FVIII IgG (mAb6) as a standard. Results are expressed as micrograms per milliliter of anti-FVIII IgG mAb6-equivalent (B). The anti-FVIII inhibitory titer was measured in serum using the Bethesda assay and is expressed in Bethesda units (BU) per milliliter (C). (D) Dose-response of hemin treatment. FVIII-deficient mice (6 mice/group) were treated 3 times at weekly intervals with PBS (□) or with 50 (●), 400 (◇), and 750 (■) μM hemin, 24 hours before administration of 1 IU FVIII. Anti-FVIII IgG were detected in the serum collected 1 week after the third FVIII administration by ELISA. (E) Induction of anti-FVIII IgG by FVIII on neutralization of HO-1. Mice received FVIII (1 IU) 24 hours after treatment with heme (750μM, ■) or PBS (□), 3 times at weekly intervals. Some mice (▵) were treated intraperitoneally with 10 mg/kg SnMP on each day of FVIII administration and 24 hours later. Levels of anti-FVIII IgG were quantified by ELISA. (F) Protective effect of hemin-degradation products by HO-1. Mice received FVIII (1 IU) 24 hours after intravenous injection of heme (750μM, ■), or after intraperitoneal injection of bilirubin (750μM, ◇), or CORM-3 (10 mg/kg, ▵), 3 times at weekly intervals. Bilirubin and CORM-3 were also injected at the time of FVIII administration. Anti-FVIII IgG titers were determined. Statistical differences depicted in all panels were assessed using the nonparametric Mann-Whitney test.

Induction of HO-1 in FVIII-deficient mice protects from the development of FVIII inhibitors. (A) Induction of HO-1 in the liver of hemin-treated mice. FVIII-deficient mice were treated by intravenous administration of 150 μL of PBS or hemin (50, 400, and 750μM). After 24 hours, livers (200 mg) were recovered and snap-frozen. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to polyvinylidene fluoride membrane. HO-1 was detected using an anti–HO-1 rat monoclonal IgG (R&D Systems), a horseradish peroxidase–conjugated rabbit anti–rat IgG, and the enhanced chemiluminescence kit. As a control of protein loading, membranes were stained with Protogold after chemiluminescence detection (not shown). Representative of 4 mice per group. Increased HO-1 expression was also detected 48 hours after hemin treatment (not shown). (B-C) Levels of anti-FVIII IgG in hemin-treated mice. Mice were treated with PBS (□) or hemin (750μM, ■) 3 times at weekly intervals. Twenty-four hours after each treatment, mice received intravenously 1 IU FVIII (200 μL). Blood was collected by retro-orbital puncture 1 week after the third FVIII administration. Anti-FVIII IgG were quantified by ELISA using a mouse monoclonal anti-FVIII IgG (mAb6) as a standard. Results are expressed as micrograms per milliliter of anti-FVIII IgG mAb6-equivalent (B). The anti-FVIII inhibitory titer was measured in serum using the Bethesda assay and is expressed in Bethesda units (BU) per milliliter (C). (D) Dose-response of hemin treatment. FVIII-deficient mice (6 mice/group) were treated 3 times at weekly intervals with PBS (□) or with 50 (●), 400 (◇), and 750 (■) μM hemin, 24 hours before administration of 1 IU FVIII. Anti-FVIII IgG were detected in the serum collected 1 week after the third FVIII administration by ELISA. (E) Induction of anti-FVIII IgG by FVIII on neutralization of HO-1. Mice received FVIII (1 IU) 24 hours after treatment with heme (750μM, ■) or PBS (□), 3 times at weekly intervals. Some mice (▵) were treated intraperitoneally with 10 mg/kg SnMP on each day of FVIII administration and 24 hours later. Levels of anti-FVIII IgG were quantified by ELISA. (F) Protective effect of hemin-degradation products by HO-1. Mice received FVIII (1 IU) 24 hours after intravenous injection of heme (750μM, ■), or after intraperitoneal injection of bilirubin (750μM, ◇), or CORM-3 (10 mg/kg, ▵), 3 times at weekly intervals. Bilirubin and CORM-3 were also injected at the time of FVIII administration. Anti-FVIII IgG titers were determined. Statistical differences depicted in all panels were assessed using the nonparametric Mann-Whitney test.

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