Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse

Abstract

Induction of heme oxygenase-1 (HO-1) is protective in tissue injury in models of allograft rejection and vascular inflammation through either prevention of oxidative damage or via immunomodulatory effects. To examine the specific role of HO-1 in modulating the immune response, we examined the differences in immune phenotype between HO-1 knockout (HO-1(-/-)) and wild-type (HO-1(+/+)) mice. Consistent with previous findings, marked splenomegaly and fibrosis were observed in HO-1(-/-) mice. The lymph nodes of HO-1-deficient mice demonstrated a relative paucity of CD3- and B220-positive cells, but no such abnormalities were observed in the thymus. Flow cytometric analysis of isolated splenocytes demonstrated no differences in the proportions of T lymphocytes, B lymphocytes or monocytes/macrophages between the HO-1(-/-) and HO-1(+/+) mice. Significantly higher baseline serum IgM levels were observed in HO-1(-/-) versus HO-1(+/+) mice. Under mitogen stimulation with either lipopolysaccharide or anti-CD3/anti-CD28, HO-1(-/-) splenocytes secreted disproportionately higher levels of pro-inflammatory Th1 cytokines as compared to those from HO-1(+/+) mice. These findings demonstrate significant differences in the immune phenotype between the HO-1(-/-) and the HO-1(+/+) mice. The absence of HO-1 correlates with a Th1-weighted shift in cytokine responses suggesting a general pro-inflammatory tendency associated with HO-1 deficiency.

Figure 1

Histological evaluation of tissues from heme oxygenase-1-deficient (HO-1^−/−) mice. A: Hematoxylin-eosin staining of the spleen from the wild-type (HO-1^+/+, left) and HO-1^−/− (right) mice (age 8 to 12 weeks). Insets in both panels represent staining for HO-1 in the spleen using a polyclonal rabbit anti-rat HO-1 antibody (brown color). B: Iron staining with Prussian blue (arrows) of kidney (K) and liver (L) tissue from HO-1^−/− (left) and HO-1^+/+ (right) mice (age 24 weeks). Bar, 100 μm.

Figure 2

Expression of HO-1 and HO-2 protein in spleens from HO-1^+/+ and HO-1^−/− mice. Western blot analysis of HO-1 and HO-2 protein in spleen extracts from HO-1^−/− and HO-1^+/+ mice using anti-HO-1 and anti-HO-2 antibodies as described in Materials and Methods. The blots was stripped and re-probed with an anti-actin antibody to control for loading and transfer. HO-1, HO-2, and actin are identified as positive bands at ∼32-kd, ∼36-kd, and ∼46 kd-sizes, respectively. Each lane represents protein from an individual animal.

Figure 3

Characterization of lymphoid tissue from HO-1^−/− mice. Immunohistochemical staining (blue color) of lymph nodes (A), thymus (B), and spleen (C) from the HO-1^+/+ (left) and HO-1^−/− (right) mice using monoclonal rat antibodies against human CD3, murine B220 (A, B, and C) and murine CD11b (in C). Bar, 100 μm. D: Representative flow cytometric analysis of isolated splenocytes (black, HO-1^+/+; red, HO-1^−/−). The analysis was performed using antibodies against CD3, CD4, CD8, B220, and CD11b.

Figure 4

Serum immunoglobulin levels in HO-1^−/− mice. Baseline serum immunoglobulin profile of the HO-1^+/+ (solid bars) and HO-1^−/− (open bars) mice. Serum levels of IgA, IgM, IgE, IgG2a, and IgG3 were assayed using Beadlyte Mouse Immunoglobulin Isotyping Kit and Luminex¹⁰⁰ LabMAP System (Values are mean ± SEM, n = 3; *, P < 0.05).

Figure 5

Cytokine levels following splenocyte stimulation in HO-1^−/− mice. Profiles of cytokines secreted at 48-hour by isolated splenocytes from the HO-1^+/+ (solid bars) and HO-1^−/− (open bars) mice following stimulation with LPS (A) and antibodies against CD3 and CD28 (B). Levels of 10 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), TNF-α, IFN-γ, and GM-CSF) were measured using a multiplexed kit (Beadlyte Mouse Multi-Cytokine Detection System) and the Luminex¹⁰⁰ LabMAP System. Values are mean ± SEM, n = 3; *, P < 0.05. The hatched bars on the right of each panel represent HO-1^−/− to HO-1^+/+ ratios for each cytokine.