Heme oxygenase-2 is a critical determinant for execution of an acute inflammatory and reparative response

Abstract

Heme oxygenase (HO) represents an intrinsic anti-inflammatory system based on its ability to regulate leukocyte function and inhibit expression of proinflammatory cytokines. This anti-inflammatory function is linked to the inducible isoform HO-1; the role of the constitutive isoform HO-2 is unknown. The current study was undertaken to investigate the role of HO-2 in the regulation of the acute inflammatory and reparative response by using HO-2-null mice and well-established animal models of epithelial injury and antigen-induced peritonitis. Here we show that in vivo deletion of HO-2 disables execution of the acute inflammatory and reparative response after epithelial injury and leads to an exaggerated inflammatory response in antigen-induced peritonitis. HO-2 deletion was associated with impaired HO-1 induction, indicating that HO-2 is critical for HO-1 expression and that the subsequent failure to up-regulate the HO system may contribute to unresolved inflammation and the development of chronic inflammatory conditions. Indeed, supplementation with the HO bioactive product, biliverdin, rescued the acute inflammatory and reparative response in HO-2-null mice. Thus, HO-2 sets in place a basal tone of anti-inflammatory signals that may be a prerequisite for the ordered execution of an inflammatory and reparative response.

Figure 1

Neovascularization and delayed wound healing in HO-2-null mice. A: Fluorescein-stained corneas before and after injury in HO-2-null (HO-2^−/−) and WT mice. B: Wound closure as percent change from day 0 (n = 16 to 20; *P < 0.05 from day 0; ^‡P < 0.05 from WT). C: Photographs of WT and HO-2-null eyes 14 days after injury. D: Neovascularization expressed as total length of penetrating vessels (n = 12; *P < 0.001 from WT). Original magnifications, ×16 (A).

Figure 2

Exaggerated inflammation in HO-2-null mice. A: Corneal MPO activity in WT and HO-2-null (HO-2^−/−) mice (n = 5 to 8; *P < 0.05 from WT). B: H&E staining of uninjured corneas (a, d), 2 days (b, e), and 7 days (c, f) after injury. C: CD68 immunostaining in corneal epithelium (Ep) and stroma (St). D: KC, MIP-2, and MCP-1 levels in uninjured corneas and corneas after injury. Results are the average value from four corneas (n = 2).

Figure 3

Impaired functional expression of HO-1 in HO-2-null mice. A: Real-time PCR analysis of corneal HO-1 mRNA expression in WT and HO-2-null (HO-2^−/−) mice (n = 3, *P < 0.05 versus uninjured, ^‡P < 0.01 versus HO-2-null). B: HO activity (n = 3, *P < 0.05 versus uninjured, ^‡P < 0.05 versus HO-2-null). C: HO-1 immunostaining in the stroma of WT (a, b) and HO-2^−/− (c, d) mice 4 days after injury. Immunopositive macrophages (arrows), fibroblasts (arrowheads), and granulocytes (circles). D: HO-2 immunostaining in WT 4 days after injury (a); immunopositive macrophages (arrows), fibroblasts (arrowheads), and granulocytes (circles). No positive immunostaining in HO-2-null mice (b). Original magnifications, ×100 (C).

Figure 4

Amplification of inflammatory lipid mediator pathways in HO-2-null mice. A: COX-2 immunostaining in stroma of WT and HO-2-null (HO-2^−/−) mice before (a, d), 2 days (b, e), and 4 days (c, f) after injury. Immunopositive macrophages (arrows) and fibroblasts (arrowheads). B: Real-time PCR analysis of CYP4B1 mRNA expression (n = 3, *P < 0.05 versus uninjured, ^‡P < 0.05 versus WT). C: CYP4B1 activity measured as 12-HETrE production (n = 4, *P < 0.05 versus uninjured, ^‡P < 0.05 versus WT).

Figure 5

Amplified leukocyte function in HO-2-null mice. A: Leukocyte number in peritoneal exudates 4 and 14 hours after zymosan A injection in WT and HO-2-null (HO-2^−/−) mice (n = 6 to 9, *P < 0.05 from WT). B: NADPH oxidase activity in peritoneal leukocytes (n = 7, *P < 0.05 from WT). C: KC levels in peritoneal exudates at 14 hours (n = 3, *P < 0.05 from WT). D: HO-1 protein expression in peritoneal leukocytes; levels are normalized to β-actin (AU, arbitrary units; n = 4; *P < 0.05 from WT.

Figure 6

Biliverdin administration rescues impaired inflammatory and reparative responses in HO-2-null mice. A: Photographs of eyes treated with biliverdin or the vehicle (PBS, pH 7.4) 2 days after injury. B: Wound closure as percent re-epithelialization (n = 4, *P < 0.05 versus WT, ^‡P < 0.05 versus vehicle). C: PMN content measured as MPO activity (n = 3, *P < 0.05 versus WT, ^‡P < 0.05 versus vehicle) 7 days after injury. D: Corneal neovascularization in HO-2-null 7 days after injury (n = 4, *P < 0.05 versus vehicle). Original magnifications, ×16.