Catalase overexpression fails to attenuate allergic airways disease in the mouse

Abstract

Oxidative stress is a hallmark of asthma, and increased levels of oxidants are considered markers of the inflammatory process. Most studies to date addressing the role of oxidants in the etiology of asthma were based on the therapeutic administration of low m.w. antioxidants or antioxidant mimetic compounds. To directly address the function of endogenous hydrogen peroxide in the pathophysiology of allergic airway disease, we comparatively evaluated mice systemically overexpressing catalase, a major antioxidant enzyme that detoxifies hydrogen peroxide, and C57BL/6 strain matched controls in the OVA model of allergic airways disease. Catalase transgenic mice had 8-fold increases in catalase activity in lung tissue, and had lowered DCF oxidation in tracheal epithelial cells, compared with C57BL/6 controls. Despite these differences, both strains showed similar increases in OVA-specific IgE, IgG1, and IgG2a levels, comparable airway and tissue inflammation, and identical increases in procollagen 1 mRNA expression, following sensitization and challenge with OVA. Unexpectedly, mRNA expression of MUC5AC and CLCA3 genes were enhanced in catalase transgenic mice, compared with C57BL/6 mice subjected to Ag. Furthermore, when compared with control mice, catalase overexpression increased airway hyperresponsiveness to methacholine both in naive mice as well as in response to Ag. In contrast to the prevailing notion that hydrogen peroxide is positively associated with the etiology of allergic airways disease, the current findings suggest that endogenous hydrogen peroxide serves a role in suppressing both mucus production and airway hyperresponsiveness.

FIGURE 1

Assessment of catalase activity and oxidative stress in lungs or tracheal epithelial cells derived from catalase transgenic or C57BL/6 mice. A, Pulverized lung tissue was homogenized in nine volumes of Na-phosphate buffer, and reacted with 30 mM H₂O₂. Decomposition of H₂O₂ was recorded spectrophotometrically at 240 nm, and compared with a standard curve generated with catalase of known catalytic activity. Data were normalized to protein, and are expressed as units.*, p < 0.05 between Alu/OVA and OVA/OVA. Cat tg, Catalase transgenic mice. B, Tracheal epithelial cells were isolated from catalase transgenic or C57BL/6 mice, grown to confluence and trypsinized for the evaluation of DCF oxidation via flow cytometry, as described in Materials and Methods. Data on x-axis represent log DCF oxidation whereas the y-axis represents cell counts.

FIGURE 2

Ig production following sensitization and challenge with OVA in catalase transgenic and C57BL/6 mice. Plasma from mock or OVA sensitized mice subjected to OVA challenge (Alu/OVA and OVA/OVA, respectively) was analyzed for OVA-specific IgE, IgG2a, and IgG1 by ELISA. Values are corrected mean optical densities (±SEM) from 11 to 13 mice per group.*, p < 0.05 between Alu/OVA and OVA/OVA. Cat tg, Catalase transgenic mice.

FIGURE 3

Airway and tissue inflammation following sensitization and challenge with OVA in catalase transgenic and C57BL/6 mice. A, Lung histopathology was evaluated by staining representative sections from paraffin-embedded lungs with H&E. BAL fluid was collected and total (B) and differential cell counts were performed (C). Values are means (±SEM) from 10 to 13 mice/group.*, p < 0.05 between Alu/OVA and OVA/OVA. Cat tg, Catalase transgenic mice.

FIGURE 4

Mucus metaplasia and mucin gene expression following sensitization and challenge with OVA in catalase transgenic and C57BL/6 mice. A, Representative sections from paraffin-embedded lungs, stained using PAS reagent to visualize mucus producing airway cells. B, Airways with a length to diameter ratio of <2:1 were evaluated for PAS positivity and the percentage of PAS positive airway epithelial cells was recorded. Data are expressed as mean of four mice per group, using multiple airways per mouse (±SEM). RNA was collected from lungs, reverse-transcribed, and analyzed for CLCA3 (C) and MUC5AC (D) expression relative to HPRT by semiquantitative TaqMan PCR. Data are expressed as mean RQ from six mice per group (± SEM).*, p < 0.05 between Alu/OVA and OVA/OVA. Cat tg, Catalase transgenic mice.

FIGURE 5

Collagen deposition and COL1A1 mRNA expression in catalase transgenic and C57BL/6 mice after OVA sensitization and challenge. A, Representative sections from paraffin-embedded lungs that were stained using Pico Sirius red, which stains collagen red in bright-field microscopy (upper panels) and bright green when visualized by differential interference contrast microscopy (lower panels). B, Scoring by two blinded observers of collagen deposition in airways (■) or parenchyma (□) using a scale from 1 to 3. The cumulative score for each mouse was averaged according to treatment group. C, RNA was collected from lungs, reverse-transcribed, and analyzed for COL1A1 mRNA expression by TaqMan PCR. Data are expressed as mean RQ from six mice per group (± SEM), following normalization to the housekeeping gene, HPRT.*, p < 0.05 between Alu/OVA and OVA/OVA.

FIGURE 6

Assessment of airway hyperresponsiveness following OVA sensitization and challenge in catalase transgenic and C57BL/6 mice. Pulmonary hyperresponsiveness to increasing doses of nebulized methacholine was assessed from forced oscillations and expressed as R_n (central airways resistance, A), G_ti (tissue visco-elastic properties and/or heterogeneity, B), and H_ti (elastance, C). Solid lines: Alu/OVA; dotted lines: OVA/OVA; ● C57BL/6; ▲ Cat tg. Data are derived from the constant phase model and are expressed as percentage change from baseline measurements (± SEM) and comprised of 7–8 mice per group.*, p < 0.05 between C57BL/6 Alu/OVA and C57BL/6 OVA/OVA, †, p < 0.05 between Cat tg Alu/OVA and Cat tg OVA/OVA, ‡, p < 0.05 between C57BL/6 Alu/OVA and Cat tg Alu/OVA, §, p < 0.05 between C57BL/6 OVA/OVA and Cat tg OVA/OVA.