Induction of ferritin and heme oxygenase-1 by endotoxin in the lung

M. S. Carraway1, A. J. Ghio2, J. L. Taylor1, and C. A. Piantadosi1

1 Department of Medicine, Duke University Medical Center, Durham 27710; and 2 United States Environmental Protection Agency, Chapel Hill, North Carolina 27599

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Heme oxygenase (HO)-1 expression is increased by forms of oxidative stress that also induce ferritin. Even though this could result from release of iron by heme degradation, we hypothesized that ferritin expression in the lung after endotoxin [lipopolysaccharide (LPS)] would occur independently of HO-1 because iron sequestration is an important response to infection. We tested this hypothesis by instilling saline or LPS (1 mg) into lungs of rats and measuring ferritin expression, HO-1 expression and activity, and HO-1 and ferritin mRNAs at different times. Lungs were also inflation fixed for immunohistochemistry for HO-1 and ferritin. Studies were performed with and without the HO inhibitor tin protoporphyrin. Ferritin and HO-1 labeling were minimal (macrophages only) in control lungs. By 4 h after LPS instillation, ferritin staining was present in bronchial epithelium and macrophages, became diffuse at 16 h, and was nearly gone by 48-72 h. HO-1 was detectable in macrophages 4 and 16 h after LPS instillation, increased in macrophages and bronchial epithelium at 24 h, and diffusely increased in bronchial epithelium and the alveolar region at 48-72 h. Lung ferritin content increased significantly by 4 h and peaked at 16 h before declining. HO-1 protein was present by Western blot in control lung, stable at 4 h, and increased by 24 h after LPS instillation, whereas HO enzyme activity had increased by 4 h after LPS instillation. After complete inhibition of HO enzyme activity with tin protoporphyrin, ferritin increased threefold at 4 h and sixfold at 24 h after LPS instillation. HO-1 mRNA increased by 4 h and was sustained at 24 h, whereas ferritin mRNA did not change after LPS instillation. These results indicate that intratracheal LPS rapidly induces ferritin protein in the lung independently of its mRNA synthesis or HO enzyme activity. LPS induces HO-1 mRNA, which is followed by increased expression of protein.

lipopolysaccharide; oxidants; acute lung injury

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

HEME OXYGENASE (HO) catalyzes the degradation of heme into biliverdin, an intermediate in the production of bilirubin (22, 23). Iron and carbon monoxide are also products of this reaction. Three genetic isoforms of HO, designated HO-1, HO-2, and, more recently, HO-3, have been characterized (16, 17). HO-1 is present constitutively in small amounts in many organs including the lung, liver, heart, and spleen, and it is inducible by many stimuli including heme, hyperthermia, heavy metals, lipopolysaccharide (LPS), hyperoxia, and hypoxia (7). Oxidant induction of HO-1 is often accompanied by increased expression of other stress proteins including ferritin (4, 21, 25). Ferritin is regulated posttranscriptionally by an iron-responsive element (IRE) and is rapidly induced in response to iron and heme (1).

Because many oxidants increase HO-1 expression, its induction has been proposed to be a part of the integrated response to oxidative stress (7). Induction of HO-1 before injury protects against oxidant-mediated cytotoxicity in cell culture (14) and prolongs survival after LPS instillation in rats (20). In both cases, this protection is reversed by treatment with competitive inhibitors of the enzyme (19, 20). Because LPS and the early-response cytokines, e.g., interleukin-1, tumor necrosis factor-alpha , and interferon-gamma , influence the intracellular uptake of iron (8, 10, 12), ferritin expression after LPS instillation also may be an early antioxidant response. Induction of ferritin in endothelial cells protects against oxidant-mediated cytotoxicity (2). Several studies (4, 21, 25) have found simultaneous induction of ferritin and HO-1 during in vivo injuries such as glycerol-induced renal failure and hyperoxia- and hemoglobin-induced lung injury in rats.

The antioxidant properties of ferritin are likely mediated by sequestration and storage of iron, thus preventing ferryl or hydroxyl radical generation (13). The specific mechanisms for the cytoprotection by HO-1 have remained uncertain. Biliverdin and bilirubin have been shown to have antioxidant properties, and their production has been suggested as a mediator of the protective effects of HO-1 (7). On the basis of in vivo studies in cultured fibroblasts, another mechanism of cytoprotection postulated for HO-1 is induction of ferritin by iron released from heme degradation (9, 24). In rat lung endothelium, however, methemoglobin induces ferritin even after inhibition of HO-1 activity (3). The interactions between ferritin and HO-1 during inflammation and their relative contributions to protection against tissue injury have not yet been defined in vivo.

Intravenous LPS stimulates HO-1 mRNA, protein, and enzyme activity in the heart, liver, and lungs of rats (20). In the lung, HO-1 is upregulated diffusely in alveolar and bronchial epithelium and inflammatory cells after intravenous LPS (5). Intravenous LPS also increases stainable iron and induces ferritin expression in the spleen (12) and liver (8). In rats, instillation of LPS into the trachea produces a gradually progressive lung injury characterized by increased permeability and influx of inflammatory cells (11, 18). We hypothesized that lung injury in rats caused by tracheal instillation of LPS would rapidly induce ferritin independently of HO-1 expression and enzyme activity. If true, ferritin expression would remain intact after inhibition of HO-1 activity. We tested this hypothesis by measuring ferritin and HO-1 protein and mRNA as a function of time after instilling LPS into the lungs of rats. These experiments were performed with and without the competitive inhibitor of HO, tin protoporphyrin (SnPP). We specifically sought to determine whether HO-1 activity was required for ferritin expression to increase in response to LPS.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Unless otherwise specified, all chemicals were obtained from Sigma (St. Louis, MO).

Experimental and animal protocols. Male Sprague-Dawley (Charles River Laboratories, Research Triangle Park, NC) rats weighing ~250 g were used for the experiments. All animals were handled under protocols approved by the Duke University (Durham, NC) Animal Care and Use Committee in accordance with National Institutes of Health guidelines for the use of laboratory animals. The rats were anesthetized with halothane (Ayerst, Philadelphia, PA), and the trachea was cannulated transorally with an 18-gauge catheter. LPS (Escherichia coli 0111:B4, 1 mg in 0.5 ml of 0.9% NaCl) or NaCl solution alone was rapidly instilled into the trachea. For control rats, NaCl alone was administered intratracheally. To inhibit HO-1, SnPP [100 µM/kg (75 mg/kg); Porphyrin Products, Logan, UT] was given subcutaneously 4 h before LPS instillation. We studied two experimental groups, LPS and LPS+SnPP, and two control groups, saline and saline+SnPP. Lungs were taken from animals killed at 0, 4, 16, 24, 48, and 72 h after intratracheal LPS instillation for immunohistochemistry. At 0, 4, 16, 24, 48, and 96 h after LPS instillation, lungs from four additional rats in each group were obtained for quantitation of ferritin protein. At the early time points (0, 4, and 24 h), lungs from four rats of each group were used to quantitate lung HO-1 enzyme activity and protein and to measure HO-1 and ferritin mRNAs. From four control animals, the spleens were also removed as a positive control for HO-1 and the livers were used to prepare biliverdin reductase for the HO activity assay.

To obtain lungs for immunohistochemistry, the rats were deeply anesthetized with intraperitoneal ketamine (5.0 mg/kg) and valium (0.5 mg/kg). The trachea was isolated and cannulated with an 18-gauge catheter. The lungs were flushed with 50 ml of 0.9% NaCl through the right ventricle while the aorta was transected. The lungs were inflation fixed en bloc with 4% paraformaldehyde at 20 cmH2O fixative pressure. To obtain lungs for the remainder of the analyses, the rats were anesthetized with ketamine and valium (see above). The abdominal and chest cavities were opened, the lungs were flushed with 0.9% NaCl through the right ventricle, and the aorta was transected. The lungs were removed, frozen immediately in liquid nitrogen, and stored at -80°C until used a few days later.

Immunohistochemistry. Inflation-fixed lungs were paraffin embedded and cut into 10-µm sections. Before the tissue sections were labeled, they were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The sections were blocked in a solution of 5% nonfat dry milk, 1% BSA, 5% goat serum in 0.01 M PBS, and 0.1% Triton X-100 before incubation overnight at 4°C with antibodies to ferritin (Dako, Carpenteria, CA) or HO-1 (Stress-Gen, Vancouver, BC) in 1% milk and 1% BSA in 0.01 M PBS and 0.1% Triton X-100 at dilutions of 1:100 and 1:500, respectively. The sections were washed three times with PBS with 0.1% Triton X-100 (5 min each) and incubated with the secondary antibody, biotinylated goat anti-rabbit IgG (Jackson Laboratories), at a dilution of 1:1,000 in 1% milk in 0.01 M PBS and 0.1% Triton X-100 at room temperature for 1 h. The signal was detected with peroxidase-conjugated avidin and diaminobenzidine. The slides were counterstained with hematoxylin. For negative controls, sections were processed as above except that the primary incubation was performed with nonimmune rabbit serum (Jackson Laboratories) instead of the primary antibodies.

Western blot. Samples of lungs and spleen were homogenized on ice in cold lysis buffer [150 mM NaCl, 50 mM Tris, pH 7.6, 1% SDS (Bio-Rad), 3% Nonidet P-40, 5 mM EDTA, 1 mM MgCl2, 2 mM 1,3-dichloroisocoumarin, 2 mM 1,10-phenanthroline, and 0.5 mM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64)]. The homogenate was centrifuged at 10,000 g for 10 min. The supernatant was decanted, and an aliquot was stored at -20°C for measurement of protein concentration. The remaining supernatant was mixed with an equal volume of double-strength Laemmli sample buffer [250 mM Tris · HCl (Bio-Rad), pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% beta -mercaptoethanol], divided into aliquots, and stored at -80°C.

Electrophoresis was performed on 12% polyacrylamide gels under reducing conditions with a minigel system (Hoefer Scientific Instruments, San Francisco, CA). All lanes were loaded with 15 µg of protein, and electrophoresis was performed over 1.5 h under a constant current of 30 mA. The proteins were electrotransferred on a TE series Transphor unit at 100 V (Hoefer Scientific) to a polyvinylidene fluoride membrane (Millipore, Amersham Life Sciences, Cleveland, OH) and blocked overnight at 4°C in Tris-buffered saline (TBS) with 1% polyoxyethylenesorbitan monolaureate (Tween 20; TBS-T) containing 5% nonfat dry milk. The following day, the membranes were washed six times over 30 min in TBS-T at room temperature. Western blots were performed with rabbit polyclonal antibodies against rat HO-1 (Stress-Gen). Incubation with the primary antibody was performed for 1 h at room temperature in TBS-T with 5% milk at a dilution of 1:1,000. After multiple washes in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Jackson Laboratories) at a 1:15,000 dilution in TBS-T with 5% milk. The membranes were then washed six times in TBS-T, and the signal was detected on Biomax film (Eastman Kodak, Rochester, NY) with the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL).

HO-1 activity assay. Lungs were homogenized on ice in one volume of 100 mM phosphate buffer with 2 mM MgCl2 (HO activity buffer). The homogenates were sonicated and centrifuged for 15 min at 18,800 g. The supernatant was used to quantitate HO-1 activity. The reaction mixture consisted of 200 µl of sample homogenate, 100 µl of liver cytosol (source of biliverdin reductase), 20 mM hemin, 0.8 mM NADPH, 2 mM glucose 6-phosphate, and 0.0016 U/µl of glucose-6-phosphate dehydrogenase, and the reaction was performed in duplicate in the dark. The samples were incubated in a 37°C water bath for 1 h. To extract bilirubin, an equal volume of chloroform was added to each tube, and the tubes were mixed thoroughly and centrifuged for 5 min at 15,000 g. The samples were scanned on a spectrophotometer from 464 to 530 nm. Bilirubin concentration was calculated based on the change in optical density at 464 nm from that at 530 nm, with an extinction coefficient of 40 mM/cm. The values are expressed as picomoles of bilirubin formed per hour per milligram of protein. The spleen from a control animal was processed identically to the lung samples to provide a positive control; an NADPH-free reaction mixture provided a baseline against which the measured concentrations were determined.

Slot blots for ferritin. Lung tissue was homogenized (1.0 g/5 ml) in lysis buffer. Fifteen micrograms of protein in the homogenate were vacuum slot blotted onto 0.45-mm nitrocellulose (Schleicher and Schuell, Keene, NH) in saline buffer containing 100 mM Tris, pH 8.0. The blot was air-dried, blocked with 5% milk for 30 min, and incubated with a 1:2,000 dilution of rabbit anti-human ferritin antibody (Dako) in 5% dry milk for 2 h. The blot was washed in PBS-0.05% Tween and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG in 5% dry milk for 1 h. Signals were obtained in a linear portion of the detection curve on film with enhanced chemiluminescence reagents. Band optical densities were quantified with a Millipore digital bioimaging system (Bedford, MA).

RT-PCR. Lung tissue was homogenized (1 g/5 ml) with 4 M guanidine thiocyanate (Boehringer Mannheim, Indianapolis, IN), 50 mM sodium citrate, 0.5% sarkosyl, and 0.01 M dithiothreitol. RNA was pelleted by ultracentrifugation through cesium chloride (Boehringer Mannheim) and 0.1 M EDTA. One hundred nanograms of total RNA were reverse transcribed (M-MLV Reverse Transcriptase, Life Technologies), and the resultant cDNA was amplified for 24, 25, and 24 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ferritin, and HO, respectively, in separate reactions with gene-specific primers. Oligonucleotide sequences were synthesized with an Applied Biosystems 391 DNA synthesizer (Foster City, CA) based on sequences published in the GenBank DNA database. The following sense and antisense sequences, respectively, were employed: GAPDH, 5'-CCATGGAGAAGGCTGGGG-3' and 5'-CAAATTGTCATGGATGACC-3'; ferritin,: 5'-TCGCAGGTGCGCCAGAACTA-3' and 5'AAGGAAGATTCGGCCACCTC-3'; and HO, 5-'ATTGGAGGCTGGAGCTATTCTG-3' and 5-'CCTTCGGTGCAGCTCCTCAG-5'. Amplification products were separated on 2% denaturing agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. The resulting negative (type 55 film, Polaroid, Cambridge, MA) was quantitated with a BioImage densitometer (BioImage, Ann Arbor, MI). For each experimental condition, the integrated optical densities of the ferritin and HO DNA bands were divided by that of the GAPDH DNA band (as a housekeeping gene) to correct for variation in the amount of amplifiable cDNA in each sample.

Biochemical measurements. Protein concentrations were measured in all samples with the bicinchoninic acid assay with BSA as a standard (15). Iron concentrations in LPS and SnPP were measured to exclude iron contamination as a source of error in the experiments. LPS and SnPP were solubilized in 1 N HCl (1.0 mg/1.0 ml). After centrifugation, iron concentrations were determined by inductively coupled plasma emission spectroscopy (model P40, Perkin-Elmer, Norwalk, CT).

Statistical analysis. Group data are expressed as means ± SD. Statistical comparisons were made by two-way analysis of variance, and post hoc comparisons were made with Fisher's exact test with commercially available software (Statview 4.01, Calabasas, CA) on a Macintosh computer (7600/120).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Pathology and immunohistochemistry. At the dose we administered, intratracheal LPS resulted in the development of an inflammatory lung injury over 72 h. This injury was characterized early by increased cellularity in the alveolar capillary region (16-24 h), followed by an accumulation in the alveolar spaces of erythrocytes and inflammatory cells including polymorphonuclear and mononuclear leukocytes. These changes are consistent with the injury reported in earlier studies (11, 18) of intratracheal LPS in rats.

Figure 1 compares representative immunohistochemistry for ferritin (A-D) with HO-1 (E-H) in the bronchial regions of the rat lung 0, 4, 16, and 24 h after LPS exposure. In the control lung, no airway staining of ferritin was detected, and scattered staining for ferritin was found in macrophages (Fig. 1A). Four hours after LPS instillation (Fig. 1B), ferritin staining increased in the bronchial epithelium and macrophages. At 16 h, similar staining for ferritin was present in the bronchial epithelium, macrophages, and alveolar region (Fig. 1C). By 24 h, ferritin staining was no longer present in the bronchial epithelium but remained detectable in the macrophages and alveolar region (Fig. 1D). Negative controls in the lung 16 h after LPS instillation (rabbit serum in place of primary antibody) showed minimal nonspecific staining (data not shown). The contrasting time course of HO-1 induction after intratracheal LPS is shown in Fig. 1, E-H. The control lung [time (t) = 0] showed minimal light staining of macrophages for HO-1, and HO-1 was not present in the airway (Fig. 1E). Four and sixteen hours after LPS instillation, the lung was progressively more cellular, and staining for HO-1 remained limited primarily to the inflammatory cells (Fig. 1, F and G). At 16 h, some staining was also present in bronchial epithelium. By 24 h, there was intense staining of the bronchial epithelium, macrophages, and alveolar region for HO-1 (Fig. 1H). The negative control lung at 72 h after LPS instillation showed minimal nonspecific staining (data not shown).


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Fig. 1.   Immunohistochemistry of bronchial region for ferritin (A-D) and heme oxygenase (HO)-1 (E-H) after intratracheal lipopolysaccharide (LPS) instillation. A: ferritin was not found in bronchial epithelium in control lungs [time (t) = 0]. It was present only in macrophages. B: 4 h after LPS instillation, ferritin staining was present in bronchial epithelium. C: 16 h after LPS instillation, ferritin was still present in bronchial epithelium. D: 24 h after LPS instillation, ferritin staining was almost absent in bronchial epithelium. Influx of mononuclear inflammatory cells, however, stained positively for ferritin. E: control lung (t = 0). F: 4 h after LPS instillation. HO-1 staining was not present in airway epithelium. G: at 16 h, airway staining for HO-1 was barely detectable, whereas macrophages stained intensely. H: at 24 h, definite staining for HO-1 was present in airway. Macrophages stained intensely for HO-1. Original magnification, ×220.

Figure 2, A-C, shows more detailed immunohistochemistry of the alveolar region of the lung for ferritin 0, 4, and 24 h, respectively, after LPS exposure. In the control lung (t = 0), scattered staining for ferritin was found in macrophages (Fig. 2A). Four hours after LPS instillation (Fig. 2B), there were increased inflammatory cells that stained for ferritin, and there was a modest increase in ferritin labeling in the alveolar region. At 16 h, similar staining was present in the macrophages and alveolar region (data not shown). By 24 h, ferritin staining was strong in both the cells and the alveolar region (Fig. 2C).


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Fig. 2.   Immunohistochemistry of alveolar region for ferritin after intratracheal LPS instillation. A: ferritin was present mostly in macrophages (arrowhead) in control lungs (t = 0). B: 4 h after LPS instillation, ferritin stained prominently in macrophages and was present in alveolar region (arrowhead). C: 24 h after LPS instillation, there were increased mononuclear inflammatory cells that stained intensely for ferritin, and staining persisted in alveolar region. Original magnification, ×440.

Figure 3, A-C, shows representative immunohistochemistry for HO-1 0, 4, and 24 h, respectively, after LPS exposure. The control lung (t = 0) showed minimal light staining of macrophages for HO-1 (Fig. 3A). Four hours after LPS instillation, macrophages progressively increased, and these cells stained for HO-1 (Fig. 3B). At 24 h, there was increased staining of the alveolar capillary region for HO-1 and more inflammatory cells, including macrophages, that labeled for HO-1 (Fig. 3C).


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Fig. 3.   Immunohistochemistry of alveolar region for HO-1 after intratracheal LPS instillation. A: HO-1 was barely detectable in macrophages in control lung (t = 0). B: 4 h after LPS instillation, macrophages stained lightly for HO-1 (arrowhead). C: at 24 h, inflammatory cells were increased and macrophages stained intensely for HO-1. HO-1 staining was prominent in alveolar region (arrowhead). Original magnification, ×440.

By 48 h, HO-1 was strongly present in bronchial epithelium, alveolar regions, and inflammatory cells. Ferritin was not present in alveolar regions or bronchial epithelium and was only visible in macrophages, similar to control lungs (data not shown). Figure 4 shows representative immunohistochemistry sections for HO-1 and ferritin 72 h after LPS instillation. Similar to 48 h, the lung showed diffuse infiltration of inflammatory cells and thickened interstitium. Ferritin staining was present only in macrophages, similar to control lungs (Fig. 4A). In contrast, at 72 h, heavy HO-1 staining was present diffusely in the bronchial epithelium, alveolar wall regions, and inflammatory cells (Fig. 4B).


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Fig. 4.   Immunohistochemistry for ferritin (A) and HO-1 (B) 72 h after intratracheal LPS. By 72 h after instillation of LPS, lung showed diffuse infiltration of inflammatory cells. Ferritin staining was limited to macrophages, similar to control rat lungs (t = 0; see Fig. 1A). At 72 h after intratracheal LPS instillation, HO-1 was diffusely increased in alveolar wall region and inflammatory cells. Original magnification, ×220.

Lung HO-1 and ferritin content. The immunohistochemistry findings indicated that, after LPS instillation, ferritin expression increased before that of HO-1. To quantitate these differences, HO-1 and ferritin were measured in the lung at different times after LPS instillation. Figure 5 shows the results of quantitation of ferritin protein from immunoblots of lung samples taken 4, 16, 24, 48, and 96 h after intratracheal LPS instillation compared with the control (t = 0) values. At 4 h, ferritin content in the lung was increased 2.5-fold above the control value. By 16 h, ferritin had peaked eightfold above control value. At 24 and 48 h, ferritin had fallen to four times the control value and by 96 h had returned to baseline.


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Fig. 5.   Ferritin expression in lung after LPS instillation. Values were obtained by densitometry of slot blots, with normalization of post-LPS values to control values (t = 0). In rat lung, ferritin content was increased by 4 h after intratracheal LPS. Ferritin content peaked at 16 h and remained increased over control value at 24 and 48 h. By 96 h, ferritin values were similar to control value.

Because ferritin induction was highest in the first 24 h after LPS instillation, we focused on these first 24 h for quantitative HO-1 protein and activity measurements and experiments with inhibition of HO-1 enzyme activity. Figure 6 is a Western blot of lung samples with antibody to HO-1. The HO-1 protein is identified by Western blot as a strong band at 32 kDa in the spleen, which is rich in HO-1. HO-1 was present in the control lungs and was not increased 4 h after intratracheal LPS. By 24 h after intratracheal LPS instillation, HO-1 protein increased substantially.


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Fig. 6.   HO-1 expression in lung after LPS instillation. Western blot showed a small amount of HO-1 present in rat lungs at baseline (control; t = 0). Four hours after intratracheal LPS instillation, there was no change from baseline. After 24 h, HO-1 protein increased in lung. After pretreatment with tin protoporphyrin (SnPP), HO-1 increased 4 h after LPS exposure and increased further at 24 h. HO-1 also increased at 24 h after SnPP alone. Rat spleen was a positive control for HO-1. All lanes were loaded with 15 µg of protein.

Figure 7 shows HO-1 activity in lung homogenates as measured by bilirubin production and expressed as picomoles per milligram of protein per hour. The control lung generated ~5 pmol bilirubin · mg protein-1 · h-1. Four hours after intratracheal LPS, HO activity was 25 pmol · mg protein-1 · h-1, which was significantly different from the control value (P <=  0.05). After 24 h, there was a further increase in HO-1 activity, to 35 pmol · mg protein-1 · h-1, which was significantly different from the control samples (P <=  0.05) but not different from the samples from the 4-h group.


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Fig. 7.   HO enzyme activity in rat lung. HO enzyme activity was present at baseline in lung samples and increased 5-fold at 4 h after intratracheal LPS instillation (P <=  0.05). Further increases in enzyme activity were detected after 24 h (P <=  0.08 for LPS at 4 vs. 24 h). After pretreatment with SnPP, HO activity did not increase after intratracheal LPS at 4 or 24 h.

Effect of SnPP on ferritin and HO-1. Figure 7 also shows the effect on HO-1 activity of subcutaneous administration of SnPP 4 h before intratracheal LPS. HO-1 activity in the LPS-treated rats at 4 and 24 h was totally inhibited, indicating a long-lasting effect on enzyme activity by the competitive inhibitor. Pretreatment with SnPP, however, did not prevent induction of HO-1 protein by LPS as shown by the Western blot in Fig. 6. There was more total HO-1 per microgram of total protein present by Western blot after 4 and 24 h in the SnPP+LPS-treated rats than in the LPS-alone group at both 4 and 24 h.

Figure 8 summarizes the effect of LPS and LPS+SnPP on the ferritin content of the lung at 4 and 24 h compared with the ferritin content of control lungs. Even after total blockade of HO-1 activity by SnPP pretreatment (see Fig. 7), the ferritin content in SnPP+LPS lungs was increased threefold over the control value 4 h after intratracheal LPS. The SnPP-treated animals also showed a sixfold increase in ferritin over the control value 24 h after intratracheal LPS. At both time points, the increase in ferritin was slightly but not significantly higher than in the LPS-treated lungs. After SnPP+LPS, ferritin was present at 4 and 24 h in amounts similar to those found after LPS alone. After SnPP+saline, ferritin staining was also present but to a lesser degree.


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Fig. 8.   Ferritin content of rat lungs after LPS and LPS+SnPP instillation. By slot blot, ferritin content increased 2.5-fold over control value 4 h after LPS instillation. Four hours after LPS+SnPP instillation, ferritin content increased 3-fold over control value. At 24 h, ferritin increased 4-fold after LPS instillation and 6-fold after LPS+SnPP instillation.

To rule out iron contamination of LPS and saline vehicle as the mechanism by which ferritin and HO-1 protein expression increased, the iron content of the LPS and SnPP solutions was measured. The iron concentration in LPS was 0.328 ± 0.035 part/million and that in SnPP was 0.165 ± 0.031 part/million.

RT-PCR for ferritin and HO-1 mRNAs. Ferritin and HO-1 mRNAs were measured and normalized to GAPDH mRNA (Figs. 9 and 10). Figure 9 shows that ferritin mRNA did not change at either 4 or 24 h after LPS instillation compared with the control value, which is consistent with posttranscriptional regulation of the expression of this iron storage protein. After SnPP, there was also no change in the amount of ferritin mRNA present in the lung.


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Fig. 9.   Ferritin mRNA expression after LPS instillation. Ferritin mRNA, measured by RT-PCR, did not change significantly at either 4 or 24 h after LPS instillation compared with control media. After LPS+SnPP instillation, ferritin mRNA also did not change significantly from the control value at 4 or 24 h. Ferritin mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).


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Fig. 10.   HO-1 mRNA expression after LPS instillation. By RT-PCR, HO-1 mRNA increased by 4 h after LPS instillation compared with control media. HO-1 mRNA content was normalized to GAPDH. At 24 h after LPS exposure, HO-1 mRNA was elevated above control value but was less than 4-h value. In LPS+SnPP-treated lungs, HO-1 mRNA was further increased above control value at 4 and 24 h. * P <=  0.05 compared with control value. ** P <=  0.05 compared with control value.

The control lungs had a ratio of HO-1 to GAPDH mRNAs of 0.75 (Fig. 10). After LPS instillation, there was a significant increase in HO-1 mRNA at 4 h. By 24 h, HO-1 mRNA had decreased from 4 h but still remained significantly increased over the control value. The combination of LPS and SnPP significantly increased the amount of HO-1 mRNA at both 4 and 24 h after LPS instillation.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The results of our experiments show that intratracheal administration of a large dose of LPS significantly increases two antioxidant proteins, ferritin and HO-1, in the lung. Ferritin expression increased by 4 h, continued to increase at 16 h, and returned toward baseline at 48-96 h. This time course was in contrast to the increase in HO-1 protein and enzyme activity, which began later and was sustained for 72-96 h. We also observed that inhibition of HO-1 enzyme activity by SnPP did not prevent the increase in lung ferritin after LPS instillation. The ferritin mRNA content of the lung was also unaffected by LPS, consistent with the known posttranscriptional control of its protein translation by an IRE (1). These new results establish that, in this model of lung injury, early expression of ferritin is independent of HO-1 activity. Also, as the injury progresses, ferritin expression declines to baseline despite intense HO-1 protein expression and increased enzyme activity.

Previous study of these two proteins demonstrated that HO-1 and ferritin can be induced in pulmonary endothelium in rats by intravenous administration of hemoglobin. This response may be adaptive in acute lung injury, e.g., acute respiratory distress syndrome, where endothelial injury can occur by contact with free heme (2, 3). Intravenous LPS also stimulates HO-1 in the rat lung (19, 20) and increases ferritin in the liver and spleen (8, 12). Administration of LPS via the intratracheal route generates an inflammatory response in the lung, originating on the epithelial surface. We used this stimulus to examine the HO-1 and ferritin responses of the lung to epithelial injury because the alveolar surface is frequently exposed to different forms of oxidative stress. Protection of the lung epithelium from oxidative stress has been proposed as a function of ferritin and HO-1, analogous to the effects of the superoxide dismutases and glutathione (7).

The temporal pattern of response of the lung to intratracheal LPS instillation provides important information about the sequence of events leading to ferritin and HO-1 expression. Initially, upregulation of ferritin occurs in the bronchial epithelium, and by 4 and 16 h, labeling of inflammatory cells for ferritin, especially macrophages, is intense. At 16 and 24 h, ferritin staining is no longer apparent in bronchial epithelium but is prominent in the alveolar regions of the lung. The early intense staining for ferritin in the bronchial epithelium is likely due to the high initial exposure of this region to intratracheal endotoxin. The localization of ferritin apparently shifts over time after the injury from the airway and macrophages to the alveolar wall region. This shift could reflect cycling of the ferritin from the epithelial to the endothelial compartment for transport of the storage protein from the lung to the liver. The alternative possibility that the progressive increase in HO-1 stimulates ferritin synthesis in the alveolar region by releasing iron is ruled out by the SnPP experiments.

The precise mechanism by which LPS causes the initial increase in ferritin content in the lung is not completely defined; however, our finding of a rapid ferritin increase after LPS instillation with no change in mRNA suggests activation of the IRE and posttranscriptional protein synthesis. LPS could effect a disequilibrium in metal handling, producing an increase in available iron that would then activate the IRE. Indeed, LPS and other cytokines such as interleukin-1beta , tumor necrosis factor-alpha , and interferon-gamma have been shown to alter iron handling (10) and to increase the ferritin content of other tissues, including the liver and spleen (8, 12). Although LPS can bind measurable amounts of iron (6), the amount of iron we measured was too small to cause a dramatic increase in ferritin content. It is more likely that ferritin induction in the lung after LPS instillation is a direct response of the IRE to the release of cytokines or other inflammatory mediators.

The induction of HO-1 by LPS in the lung follows that of the ferritin. We show that HO-1 protein induction follows an increase in HO-1 steady-state mRNA, as has been shown for hemoglobin by other investigators (4, 20). By Western analysis and immunohistochemistry, HO-1 protein expression increases at 24 h, and the signals continue to intensify at 48 and 72 h. The stimulus for this continued HO-1 expression is not known, but it could be the result of an ongoing requirement to catabolize heme derived from erythrocytes that appear in the lung after injury by LPS. Another possibility is that activated polymorphonuclear leukocytes, which are capable of oxidizing hemoglobin to methemoglobin, provide a potent stimulus for HO-1 production and activity (3). Either of these mechanisms may be operative in the acute lung injury after LPS exposure, as significant inflammatory cell infiltrate and hemorrhage occur after LPS instillation. Another notable finding is the increase in HO-1 activity 4 h after LPS instillation, before the increase in protein. The HO-1 antibody is sensitive to <10 ng of protein, which argues against a lack of sensitivity of our immunodetection methods. This suggests a direct effect of LPS on the enzyme activity that has not previously been considered.

It has been demonstrated in vitro that heme degradation by HO-1 releases iron that, in turn, induces ferritin (9, 24). This sequence of events has been proposed as one of the cytoprotective functions of HO-1 (7, 9, 24). This mechanism, however, does not seem to be a major factor in the lung after LPS exposure because of the different time course of induction and localization of these two proteins. At 72 h after LPS instillation, when HO-1 expression was greatest, ferritin content had returned almost to baseline. Our experiments also clearly show that the early increase in ferritin in the lung is not dependent on HO-1 enzyme activity because it occurred even when HO-1 was totally inhibited for up to 24 h by SnPP. Quantitative measurements of ferritin expression after LPS+SnPP instillation also demonstrate that ferritin content in the lung is higher than after LPS alone. We also ruled out iron contamination of SnPP as the cause of ferritin induction. Although the mechanisms for this difference remain to be explored, they include positive feedback on ferritin by inhibition of HO, e.g., by substrate (e.g., heme), a direct effect of SnPP on ferritin (e.g., through the porphyrin center), or release of ferritin expression from product inhibition (e.g., carbon monoxide).

Despite inactivating HO, SnPP caused significant increases in lung HO-1 protein and mRNA above control values. To explain this observation, it is tempting to invoke a negative feedback mechanism where enzyme inhibition (with loss of product) increased enzyme expression. The time course of induction of HO-1 mRNA by SnPP, however, was so similar to that of LPS that it suggests that a direct stimulus by the protoporphyrin is superimposed on that of LPS. SnPP contains a heme moiety that could stimulate production of the enzyme.

In conclusion, we have demonstrated a distinct difference in the time course and localization of ferritin in relation to HO-1 in the lung during acute alveolar inflammation after intratracheal LPS instillation. We have shown that under these conditions, changes in the expression of ferritin protein does not depend on HO-1. Further studies will be required to understand the cellular mechanisms leading to induction of these proteins after LPS instillation and the protective roles of ferritin and HO-1 in inflammatory lung injury.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. S. Carraway, Division of Pulmonary and Critical Care Medicine, Duke Univ. Medical Center, Box 3315, Durham, NC 27710.

Received 9 January 1998; accepted in final form 14 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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