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Comparative Study
. 2004 Sep;165(3):1045-53.
doi: 10.1016/S0002-9440(10)63365-2.

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

Matthias H Kapturczak  1 , Clive Wasserfall, Todd Brusko, Martha Campbell-Thompson, Tamir M Ellis, Mark A Atkinson, Anupam Agarwal
Affiliations
Comparative Study

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

Matthias H Kapturczak et al. Am J Pathol. 2004 Sep.

Erratum in

  • Am J Pathol. 2006 Feb;168(2):714

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.

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Figures

Figure 1
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
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
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
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
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.
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