Immunohistochemical expression of nuclear factor erythroid-2-related factor 2 and heme oxygenase 1 in normal bovine lung and bovine lung infected with Mannheimia haemolytica

Abstract

Mannheimia haemolytica is an important cause of pneumonia in feedlot cattle. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes, such as heme oxygenase 1 (HO-1), within the lung. The expression of Nrf2 and HO-1 was immunohistochemically evaluated in 4 calves 24 h after experimental infection with M. haemolytica. Calves receiving normal saline served as controls. In the infected lungs, cytoplasmic Nrf2 expression was high in macrophages and bronchioles and low in alveolar epithelium, whereas nuclear expression was high in endothelial cells, macrophages, and bronchioles and lowest in alveolar epithelium. Normal lung samples displayed only faint Nrf2 cytoplasmic staining within bronchiolar epithelium. Expression of HO-1 was detected within the cytoplasm of macrophages and bronchiolar epithelial cells in all infected lung samples, whereas normal lungs displayed only weak cytoplasmic staining in bronchiolar epithelial cells. These findings suggest that bronchiolar epithelial cells and macrophages up-regulate Nrf2 expression early in the course of infection, which results in increased expression of HO-1 within these cells.

Introduction

Respiratory infections account for approximately 75% of all clinically visible disease in cattle and have high mortality and morbidity rates (1–4). One of the most common and serious respiratory infections in feedlot cattle is shipping fever (5,6). The disease is mainly caused by Mannheimia haemolytica, an opportunistic Gram-negative bacterium that is normally present in the upper respiratory tract (7). Antibiotics remain the only known treatment option for this disease in feedlot cattle. However, there is growing public concern about the use of antibiotics to treat animal diseases, owing to the presence of antibiotic residues in meat and the development of bacterial resistance. Therefore, treatment options other than antibiotics are needed.

Alveolar and pulmonary intravascular macrophages have been shown to play central roles in lung inflammation induced by M. haemolytica (8). Acute lung injury results from increased responsiveness of the innate immune system to antigenic stimuli. For example, binding of the lipopolysaccharide (LPS) endotoxin of M. haemolytica to Toll-like receptor 4 on the plasma membrane of macrophages results in the release of proinflammatory mediators that cause host tissue damage (9–11). Although the causes of increased responsiveness of the innate immune system are unknown, the role of oxidative stress in priming innate immune cells such as neutrophils and macrophages for increased responsiveness is well-recognized (12–15).

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant response within the lung (16,17). Under physiological conditions Nrf2 is localized in the cytoplasm, bound to Kelch-like ECH (erythroid cell-derived protein with cap’n’collar homology)-associated protein 1 (Keap1), with a short half-life, up to 20 min (18,19). When oxidative stress occurs, Nrf2 translocates to the nucleus, where it binds to the antioxidant response element sequences, which leads to the activation of antioxidant enzymes (19–21). Among the enzymes activated by Nrf2 is heme oxygenase 1 (HO-1) (21–24), an inducible antioxidant enzyme whose level increases within cells in response to oxidative stress (25). This enzyme catalyzes the degradation of free cellular heme, which is a magnifier of oxidative stress, to iron, carbon monoxide, and biliverdin (26,27). Many cell types in the lung, such as fibroblasts, epithelial cells, alveolar macrophages, endothelial cells, and vascular smooth muscle cells, can express HO-1 during oxidative stress (28), as a protective response to minimize tissue damage.

In human beings, induction of the cytoprotective antioxidant response through Nrf2 activation is a novel approach to the treatment of inflammatory diseases (29,30). However, the expression of Nrf2 and its antioxidant enzymes has never been investigated in cattle. The purpose of this study was to compare the expression of Nrf2 and HO-1 in normal bovine lung and in lung tissue infected with M. haemolytica.

Materials and methods

Tissues

Formalin-fixed paraffin-embedded inflamed lung tissue was obtained from 4 calves infected intratracheally with M. haemolytica in other experiments in our laboratory (8). Briefly, each calf was anesthetized with xylazine (Bayer, Mississauga, Canada) administered intramuscularly [0.1 mg/kg body weight (BW)]. The ventral surface of the neck was clipped and cleaned with antiseptic solution, and the calf was put in the right recumbent position. A 14-gauge needle was inserted between tracheal rings and directed ventrally, then an EZ Cath IV catheter (Desert Pharmaceuticals, Sandy, Utah, USA) was run through the needle, past the tracheal bifurcation. Ten milliliters of a suspension containing 2 × 10⁹ M. haemolytica A1 per milliliter was deposited through the catheter into the lung. The total dose (20 × 10⁹ M. haemolytica) was higher than that used in previous studies and induced acute lung inflammation (31). Four control calves received normal saline in the same manner. All the calves were monitored every 8 h and did not display any overt signs of respiratory distress before euthanasia 24 h after the bacterial challenge.

Light microscopy

Sections 4- to 5-μm thick were prepared from tissue blocks from each animal and placed on slides coated with poly-L-lysine. The slides were then incubated at 55°C for 30 min to improve adherence of the sections to the slides. The sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin for evaluation of tissue architecture before immunohistochemical testing.

Immunohistochemical testing for Nrf2 and HO-1

Immunohistochemical testing was done with the use of an avidin–biotin complex, diaminobenzidine, and methyl green counterstain as previously described (32). Briefly, consecutive 5-μm tissue sections were cut from each paraffin block and then mounted and dried on glass slides. The tissue was deparaffinized in Citrosolv (Fisher Scientific, Ottawa, Ontario), then dehydrated in graded alcohol solutions. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in absolute methanol for 12 min at room temperature. Antigens were retrieved by digestion with 0.2% pepsin (Sigma P5000; Sigma-Aldrich, St. Louis, Missouri, USA) in 0.01 N HCl for 45 min. Nonspecific protein binding was saturated with the use of 1% bovine serum albumin (Sigma-Aldrich) for 30 min in phosphate-buffered saline (PBS) (Invitrogen, Burlington, Ontario). Primary antibodies consisted of polyclonal rabbit Nrf2 and HO-1 against human antigen (both at 1:200 dilution) (antibodies-online Inc., Atlanta, Georgia, USA). All the antibodies were known to cross-react with bovine proteins according to the manufacturer. The diluted primary antibodies were applied to tissue sections and the slides incubated overnight at 4°C, after which they were incubated for 30 min with appropriate secondary antibody conjugated with horseradish peroxidase (5 μg/mL in PBS with 1% bovine serum albumin) (Dako Canada, Mississauga, Ontario). The reaction was visualized by means of a color development kit for peroxidase (Vector Laboratories, Burlingame, California, USA), and sections were counterstained with methyl green. Serial sections were also stained with omission of the primary antibody and with an isotypematched irrelevant antibody. Images were captured with a Nikon Coolpix camera (Nikon Canada, Mississauga, Ontario).

Scoring of Nrf2 and HO-1 expression

The expression of Nrf2 was assessed within bronchiolar and alveolar epithelium, endothelial cells, neutrophils, and macrophages. Within each cell type, cytoplasmic, nuclear, and total expression was determined. In 3 random high-power (600×) fields 300 cells were counted for each of the cell types except endothelial cells, for which only 50 cells were counted. Cytoplasmic expression was calculated by dividing the number of cells displaying brown cytoplasmic staining by the total number of cells counted. Nuclear expression was similarly calculated after counting the number of cells with positive nuclear staining. Total expression was calculated by adding cytoplasmic and nuclear expression and dividing by the total number of cells counted. The expression of HO-1 was assessed within bronchiolar epithelium and macrophages by counting the number of positive cells among 300 cells in 3 random high-power fields. All results were expressed as percentages.

Statistical analysis

Statistical analysis was done with Prism 5.00 software (GraphPad Software, San Diego, California, USA). The Kruskal–Wallis test was used to compare Nrf2 expression among the different cell types and was followed by the Dunnett test. The Wilcoxon rank sum test was used to compare cytoplasmic and nuclear expression in each cell type. All results were expressed as medians and 1st and 3rd quartiles. The strength of association between Nrf2 and HO-1 expression was assessed by Spearman’s rank correlation. A P-value < 0.05 was considered significant.

Results

Tissue from the infected bovine lungs displayed lesions typical of M. haemolytica with moderate to severe fibrinosuppurative bronchopneumonia. Tissue sections tested with isotype-matched antibody did not show any staining, whereas those tested with von Willebrand factor antibody showed vascular endothelial reaction (data not shown). Tissue from the normal lungs showed scattered and faint cytoplasmic staining of Nrf2 and HO-1within bronchiolar epithelial cells, but all other cell types were devoid of staining.

Expression of Nrf2 was detected in all the samples of infected lung and was localized to bronchiolar and alveolar epithelium, endothelial cells, neutrophils, and macrophages (Figure 1). The cytoplasmic, nuclear, and total expression of Nrf2 among the different cell types is shown in Table I. Total Nrf2 expression was highest in endothelial cells and lowest in alveolar epithelium, the difference being statistically significant. Cytoplasmic Nrf2 expression was highest in macrophages and lowest in alveolar epithelium, the difference being statistically significant. Nuclear expression was highest in endothelial cells and lowest in alveolar epithelium, the difference being statistically significant. Nuclear expression was significantly higher than cytoplasmic expression within endothelial cells, but there were no significant differences between nuclear and cytoplasmic expression in the other cell types (Table II). Expression of HO-1 was detected within the cytoplasm of bronchiolar epithelial cells and macrophages in all the infected lung samples (Table III). No correlation was found between Nrf2 expression in all locations within all cell types and HO-1 expression in bronchiolar epithelial cells and macrophages.

Figure 1.

A — Bovine lung infected with Mannheimia haemolytica and displaying lesions typical of fibrinosuppurative bronchopneumonia; inset shows neutrophilic infiltrate within alveoli. Hematoxylin and eosin. Bar = 200 μm. B — Immunohistochemistry omission control for nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1); note absence of staining in all cell types. Bar = 200 μm. C to E — Results of immunohistochemical testing of infected lung for Nrf2: note (C) faint brown cytoplasmic staining (arrows) within neutrophils (bar = 25 μm), (D) brown cytoplasmic (arrow) and nuclear (arrowhead) staining within macrophages (bar = 50 μm), and (E) cytoplasmic staining of bronchiolar and alveolar epithelium (arrows) and nuclear staining within endothelial cells (arrowhead) (bar = 50 μm). F — Results of immunohistochemical testing of normal bovine lung for Nrf2: note cytoplasmic staining within bronchiolar epithelium (arrow). Bar = 50 μm. G — Results of immunohistochemical testing of bovine lung infected with M. haemolytica for HO-1: note brown cytoplasmic staining within bronchiolar epithelium (arrow) and alveolar macrophages (arrowhead). Bar = 50 μm. H — Results of immunohistochemical testing of normal bovine lung for HO-1: note brown cytoplasmic staining within bronchiolar epithelium (long arrows), whereas alveolar macrophages (short arrow), alveolar septa (double arrows), and endothelial cells (arrowheads) are negative. Bar = 50 μm. The staining method involved an avidin–biotin complex, diaminobenzidine, and methyl green counterstain.

Table I.

Percentage of different types of bovine lung cells expressing nuclear factor erythroid-2-related factor 2 (Nrf2) in the cytoplasm and nucleus after 24 h of infection with Mannheimia haemolytica

Cell type and staining site	% of cells displaying staininga
	Calf 1	Calf 2	Calf 3	Calf 4
Bronchiolar epithelium
Cytoplasm	19.3	67	40	10
Nucleus	11.7	14.3	21.7	13
Total	31	81.3	61.7	23
Alveolar epithelium
Cytoplasm	4.3	4	2.4	0
Nucleus	0	1	0.4	3
Total	4	5	2.8	3
Endothelial cells
Cytoplasm	20	6	10	0
Nucleus	36	58	60	40
Total	56	64	70	40
Neutrophils
Cytoplasm	15.6	25.3	11	21
Nucleus	0	0	0	0
Total	15.6	25.3	11	21
Macrophages
Cytoplasm	44	41	34	22
Nucleus	0	16	26	11
Total	44	57	60	33

^aDetermined by immunohistochemical testing with Nrf2-specific antibodies and counting of 300 cells in 3 random high-power fields for all cell types except endothelial cells, for which 50 cells were counted.

Table II.

Median cytoplasmic, nuclear, and total expression of Nrf-2 in the different cell types

	Median % (and 1st and 3rd quartiles)
Cell type	Cytoplasm	Nucleus	Total
Bronchiolar epithelium	29.65 (12.30, 60.25)	13.65 (12.03, 19.85)	46.35 (25.0, 76.4)
Alveolar epithelium	3.2b (0.6, 4.2)	2.0c (0.25, 3.75)	4.65a (2.85, 4.83)
Endothelial cells	8.0 (1.5, 17.5)d	49.0c (37.0, 59.5)d	60.0a (44.0, 68.5)
Neutrophils	18.3 (12.15, 24.23)	0	18.3 (12.15, 24.23)
Macrophages	37.5b (25.0, 43.25)	13.5 (2.75, 23.5)	50.5 (37.75, 59.25)

^a,b,c,dNumbers with the same superscript are significantly different at P < 0.05 according to either the Kruskal–Wallis test^a,b,c or the Wilcoxon rank sum test.^d

Table III.

Percentage of bovine lung cells expressing heme oxygenase 1 (HO-1) in the cytoplasm after 24 h of infection with M. haemolytica

	% of cells displaying staininga
Cell type	Calf 1	Calf 2	Calf 3	Calf 4
Bronchiolar epithelium	18	8	12	16
Macrophages	32	40	18	32

^aDetermined by immunohistochemical testing with HO-1 specific antibodies and counting of 300 cells in 3 random high-power fields.

Discussion

In this study, normal bovine lungs and bovine lungs infected with M. haemolytica were evaluated immunohistochemically for the expression of Nrf2 and HO-1. In the infected samples Nrf2 expression was localized to bronchiolar and alveolar epithelium, endothelial cells, neutrophils, and macrophages, whereas HO-1 expression was detected within only bronchiolar epithelial cells and macrophages.

During periods of increased oxidative stress Nrf2 is known to drive the antioxidant response by increasing the expression of antioxidant enzymes (16,17). Under basal conditions Nrf2 is localized to the cytoplasm, where it is bound to Keap-1, which facilitates its proteasomal degradation. The expression of Nrf2 is increased in response to oxidative stress, which results in translocation to the nucleus, where Nrf2 binds to antioxidant response elements. In the normal bovine lung, faint cytoplasmic staining of Nrf2 was found only within bronchiolar epithelial cells. Our finding of increased cytoplasmic expression of Nrf2 in different lung cell types after M. haemolytica infection is consistent with the finding of others of increased Nrf2 gene expression in response to oxidative stress (33). The cytoplasmic expression of Nrf2 was highest in macrophages and next highest in bronchiolar epithelial cells and was lowest in alveolar epithelial cells. In this experimental setting bronchiolar epithelial cells were the first cell type to be exposed to M. haemolytica. It is therefore not surprising to find increased Nrf2 cytoplasmic expression early in the course of M. haemolytica infection. Given their location, alveolar macrophages are expected to receive infectious particles later; thus, the finding that the cytoplasmic expression of Nrf2 was highest in macrophages suggests that alveolar macrophages up-regulate Nrf2 expression more quickly than other cell types. The low cytoplasmic expression of Nrf2 within alveoli could be due to many factors, including their location down the respiratory tree or the protective role of macrophages that reside in close proximity. Another possible factor is the relatively short duration of this challenge, in which the calves were euthanized after only 24 h of exposure.

Translocation of Nrf2 to the nucleus is required for antioxidant response. In this study, Nrf2 nuclear expression was evident in all cell types except neutrophils and was, surprisingly, highest in endothelial cells and next highest in bronchiolar epithelial cells and macrophages. This finding suggests that endothelial cells are also quick to respond to oxidative stress. The increase in expression of Nrf2 in human aortic endothelial cells (HAECs) has been shown to markedly increase transcriptional activity driven by antioxidant response and to protect HAECs from cytotoxicity mediated by hydrogen peroxide (34).

Contrary to our finding of Nrf2 expression only within bronchiolar epithelial cells in normal bovine lung, Boutten et al (35) reported Nrf2 expression within alveolar macrophages, alveolar epithelial cells, and endothelial cells in normal human lung. This may indicate species variation in the expression of Nrf2 within the lung but could also be related to antibody specificity. Thus, we cannot rule out minimal expression of Nrf2 in other cells within the normal bovine lung.

Excess free heme has proinflammatory properties and is known to generate reactive oxygen species, resulting in cellular damage (26,36). Thus, by degrading excess heme, HO-1 plays an integral role in the antioxidant cytoprotective response. However, a few studies have shown that HO-1 promotes cell injury (37). It is therefore not surprising that the expression of HO-1 is not only inducible but also highly regulated (25). In contrast, other antioxidant enzymes, such as glutathione peroxidase, have constitutive expression (25). In the normal rat lung HO-1 is expressed mainly in macrophages. After intratracheal instillation of LPS, HO-1 expression becomes more intense within macrophages and is also present in bronchiolar and alveolar epithelium (38,39). A similar finding of increased HO-1 expression was reported in smokers compared with nonsmokers (38,39). A few studies have reported scant basal expression within bronchiolar and alveolar epithelium in the normal lung (28,40). In this study, HO-1 was detected within the cytoplasm of macrophages and bronchiolar epithelial cells in all samples of infected lung. The lack of correlation between Nrf2 expression and HO-1 expression could be related to the small sample size. The absence of HO-1 expression in lung endothelium is surprising given the high level of Nrf2 nuclear translocation. The absence of staining in macrophages of the normal bovine lung could be related to species variation in the expression of HO-1.

In summary, this study found widespread cytoplasmic expression of Nrf2 within macrophages and bronchiolar epithelial cells in bovine lungs infected with M. haemolytica, which suggests that these 2 cell types respond early to oxidative stress and induce the expression of antioxidant enzymes such as HO-1.