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(Chest. 2000;118:814-823.)
© 2000 American College of Chest Physicians

Iron Disequilibrium in the Rat Lung After Instilled Blood*

Andrew J. Ghio, MD; Judy H. Richards, BS; Kay M. Crissman, BS and Jacqueline D. Carter, BS

* From the National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, NC.

Correspondence to: Andrew J. Ghio, MD, National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, NC 27711


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: The extravasation of erythrocytes into the human lung occurs in a myriad of pulmonary disorders. Metal that is initially included in hemoglobin has been postulated to precipitate a disequilibrium in iron metabolism, to present an oxidative stress, and to contribute to tissue injury in several lung diseases. The objective of this study is to test the hypothesis that the tracheal instillation of blood in an animal model would have significant effects on iron equilibrium and would be associated with an injury to the lower respiratory tract.

Design: Rats were intratracheally instilled with either 1.0 mL saline solution (n = 36) or 1.0 mL blood (n = 36). Biochemical end points and histochemistry were obtained at times between 20 min and 14 days after the exposure to saline solution or blood.

Results: Total and nonheme iron concentrations in tracheal lavage fluid increased after the instillation of the blood. The percentage of neutrophils in the lavage fluid was elevated 1 day after the instillation of blood and remained at that level for at least 4 days following exposure, while protein concentrations were significantly increased at 1 day and 2 days only. Erythrocytes in the lung tissue were stained for hemoglobin immediately after exposure, but by 4 days after exposure, there was none. Ferritin was elevated between 1 day and 4 days after exposure, but by 7 days after exposure, the expression of this storage protein had returned to baseline values.

Conclusions: We conclude that intratracheal instillation of whole blood in the rat can induce a neutrophilic lung injury that is associated with a disruption of normal iron metabolism. This disruption of the iron equilibrium is made evident by quantifying iron and staining for hemoglobin and ferritin. All indexes of biological effect had corrected by 7 days after exposure.

Key Words: erythrocyte • hemoptysis • hemorrhage • lung diseases • oxidants.


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Free radicals catalyzed by metal can present an oxidative stress to lung tissue. In numerous diseases, indexes of iron metabolism indicate a disequilibrium and suggest an increase in biologically available concentrations. These diseases can include ARDS,1 COPD,2 injury after lung transplantation,3 microlithiasis,4 and disease associated with particle,5 fiber,6 and bleomycin exposures.7 As a result of this potential relationship between catalytically active metal and pulmonary injury, indexes of metal concentrations in both the BAL fluid and lung tissue are being measured with increasing frequency.

The extravasation of erythrocytes into the lower respiratory tract occurs in a myriad of pulmonary disorders. Specific compounds in these cells can function as antioxidants, including superoxide dismutase, catalase, and glutathione. Subsequently, RBCs scavenge extracellular oxidants8 9 and have a protective role in several injuries that are mediated by free radical damage. These cells can protect against damage to both the endothelium and the isolated perfused lung after exposure to hydrogen peroxide.8 10 The tracheal instillation of either human or rat erythrocytes, but not RBC ghosts or lysates, diminishes lung injury in rats after hyperoxia.10 However, extravasated erythrocytes potentially can contribute to concentrations of catalytically active iron in the lower respiratory tract, and metal that is initially included in hemoglobin contained within the erythrocyte also has been postulated to participate in oxidant generation and to contribute to injury.11 We tested the hypothesis that the tracheal instillation of blood in an animal model would have significant effects on iron equilibrium and would be associated with an injury to the lower respiratory tract.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tracheal Instillation of Blood
All animals were kept in pathogen-free facilities and were routinely monitored for pathogens and viruses. Six 60-day old, male Sprague-Dawley rats (Charles River Breeding Laboratories; Raleigh, NC) were anesthetized with halothane (Aldrich; Milwaukee, WI), and blood was collected (approximately 10 mL from each animal) by intracardiac puncture into heparinized syringes and was pooled. Animals then were killed by further exsanguination through the abdominal aorta. Small samples (n = 4) of the pooled blood were placed into microhematocrit tubes (Fisher; Pittsburgh, PA), were centrifuged, and the hematocrit was read (International Equipment; Boston, MA). The mean (± SE) hematocrit of the pooled blood was 43.1 ± 3.3%.

Sixty-day-old, male Sprague-Dawley rats were anesthetized with halothane and intratracheally instilled with either normal saline solution (0.9%) (n = 36) or blood (n = 36). A previous investigation using this method of exposure has demonstrated a uniform distribution of instilled material in the lung.12 No preliminary investigation was done to determine the dose-response of any end points after blood instillation, but, rather, 1.0 mL blood (or 1.0 mL saline solution) was instilled. This volume of blood approximated 10% of the total lung capacity of the rat and, therefore, reflects a large exposure. After the instillation, all rats were allowed to recover from the anesthesia and were returned to animal-care facilities.

Tracheal Lavage
At durations between 20 min and 14 days, rats (n = 4 per time point) were anesthetized with halothane, were killed by exsanguination, and were tracheally lavaged. The volume of saline solution injected was determined from published allometric equations and equaled ~ 90% of the total lung capacity (35 mL/kg body weight). Saline solution was withdrawn after a 3-s pause, was reinjected an additional two times with similar delays, and then was stored on ice. The volume of recovery of the lavage fluid was 78.4 ± 6.9%. There were no significant differences between those animals instilled with blood and those instilled with saline solution.

Employing a modified Wright’s stain (Diff-Quick stain; American Scientific Products; McGaw Park, IL), neutrophils were enumerated and values were expressed as the percentage of total cells recovered. After separation of cells by centrifugation at 600g for 10 min, the level of lavage protein was determined using a protein assay reagent (Coomassie Plus; Pierce; Rockford, IL) modified for automated measurement. Bovine serum albumin served as the standard.

Concentrations of total iron and nonheme iron in the lavage supernatant were quantified using inductively coupled plasma emission spectroscopy (ICPES, model P40; Perkin-Elmer; Norwalk, CT) operating at a wavelength of 238.204. The level of nonheme iron was determined by the addition of a 1.0-mL 6N HCl acid/20% trichloroacetic acid solution to 1.0 mL lavage supernatant, heating to 70°C for 18 h, and centrifugation at 1,200g for 10 min. Standards included ferric chloride in 1% HCl acid.

Lavage Ascorbate, Urate, and Glutathione Concentrations
The lavage fluid supernatant was acidified (35 µL 60% perchloric acid per 1.0 mL supernatant) and was centrifuged at 20,000g for 30 min at 4°C. The supernatant was stored at -80°C until assayed for ascorbate and urate using high-performance liquid chromatography (Waters RCM µ BondaPak C18 column; Millipore; Marlborough, MA) with electrochemical detection (BAS, model LC-4B; Bioanalytical Systems; West Lafayette, IN).13 Levels of nonprotein sulfhydryls, reflecting the amount of total glutathione, also were measured in the supernatant.14

Concentrations of Inflammatory Cytokines in Lavage Fluid
Various mediators are likely to participate in coordinating an acute inflammatory injury. Those cytokines that were assayed were selected as a result of the previous experience of this laboratory. Concentrations of macrophage inflammatory protein (MIP-2) and tumor necrosis factor (TNF) in the supernatants of lavage fluid were measured by enzyme-linked immunosorbent assay using commercially available kits (Quantikine; R&D Systems; Minneapolis, MN).

Stains of Lung Tissue
Lungs (two animals per exposure per time point) were fixed at inflation with 10% formalin (35 mL/kg body weight) (Fisher). Stains included hematoxylin-eosin, hemoglobin by 3,3'-diaminobenzidine with hematoxylin counterstain, Perls’ Prussian blue method for iron, and Turnbull’s blue reaction. The stains employed reflect the current understanding of hemoglobin metabolism, with iron accumulating during significant bleeding and ferritin functioning as the primary storage site for this metal after its release.

Ferritin was stained immunohistologically, employing 4-µm sections heat-fixed to slides, which were deparaffinized, hydrated, and rinsed. Sections were treated with hydrogen peroxide in methanol (1:16) to block endogenous peroxidase. Nonspecific staining of highly charged protein was blocked with incubation in normal goat serum that was diluted 1:20 in phosphate-buffered saline solution (PBS) with 1% bovine serum albumin (BSA) for 10 min at 37°C. The serum was tapped off, and the primary antibody (rabbit {alpha}-ferritin antibody; Dako; Carpinteria, CA) was applied at a dilution of 1:100 in PBS with 1% BSA. After incubation at room temperature (37°C) for 45 min, slides were washed with PBS three times, and goat antirabbit biotinylated IgG diluted 1:200 (Vector Labs; Burlingame, CA) in PBS with 1% BSA was applied for 30 min at room temperature. The tissue then was incubated with a 1:800 dilution of peroxidase-conjugated streptavidin (The Jackson Laboratory; Bar Harbor, ME) in 0.05 mol/L Tris for 30 min at room temperature and was rinsed in PBS. Amino ethyl carbazole (Biomeda; Foster City, CA) was applied for 8 min at room temperature and was rinsed with distilled water. The counterstain employed was hematoxylin (Fisher Scientific; Raleigh, NC). Controls included stains of normal human spleen (positive control) and lung tissue without the polyclonal antibody added (negative control).

Statistics
Data are expressed as mean ± SE. Differences between multiple groups were analyzed employing analysis of variance.15 The post hoc test employed was the Scheffé test. Two-tailed tests of significance were employed. Significance was assumed at p < 0.05.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prior to exposure to blood, the concentration of total iron in the lavage fluid supernatants was measurable (Fig 1 , top) and was equivalent to the nonheme iron [Fe3+] (Fig 1 , bottom). The tracheal instillation of blood corresponded with an increase in the concentrations of both total and nonheme iron measured at 20 min and 1 day after exposure to blood. However, these values returned to baseline values by 2 days after instillation.



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  		Figure 1. Total (top) and nonheme (bottom) iron concentrations in BAL fluid from rats instilled with either saline solution or 1.0 mL blood. Total iron concentrations (top) were measurable before exposure, and values increased significantly after the instillation of the blood. Nonheme iron concentrations (bottom) accounted for almost all of the metal in the BAL fluid. The changes in nonheme iron closely paralleled those of the total metal concentrations. * = significant increase relative to animals instilled with saline solution.

 
Lavage concentrations of low-molecular-weight antioxidants were measured to assess the oxidative stress in the lower respiratory tract after the instillation of the blood. Immediately after exposure to the blood, the lavage concentration of ascorbate was significantly diminished (Fig 2 ). This decrease in lavage ascorbate was not present at >= 2 days after instillation. There were no differences in either glutathione or urate concentrations between the animals exposed to saline solution and to blood.



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  		Figure 2. Concentrations of ascorbate in the BAL fluid from rats instilled with either saline solution or 1.0 mL blood. Concentrations of ascorbate were significantly diminished after exposure to blood but only at 20 min and 1 day after instillation. See Figure 1 for explanation of symbol.

 
The number of cytokines associated with an inflammatory response were increased after instillation of the blood. MIP-2 (Fig 3 , top) was significantly elevated at 1, 2, and 4 days after exposure, while TNF (Fig 3 , bottom) was increased at 1 day and 2 days following instillation.



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  		Figure 3. Concentrations of MIP-2 (top) and TNF (bottom) in the BAL fluid from rats instilled with either saline solution or 1.0 mL blood. After 1 day, levels of both MIP-2 and TNF were significantly elevated. While the level of MIP-2 remained increased for 4 days, that of TNF returned to baseline values after day 2. See Figure 1 for explanation of symbol.

 
Immediately after instillation, RBCs were evident on hematoxylin-eosin staining (Fig 4 , top). There was an influx of inflammatory cells and perivascular edema was prominent 1 day after exposure to the blood (Fig 4 , middle). At 1 day following exposure and at all times after, the hemoglobin, or its degradative products, appeared to form yellow, crystal-like structures in the lower respiratory tract. This was not an artifact, as those erythrocytes fixed within the blood vessels retained a normal biconcave shape. This hematoidin was also apparent at both 2 and 4 days after instillation. Both the cellular incursion and edema persisted to 4 days after exposure but had resolved by day 7 (Fig 4 , bottom). The percentage of neutrophils in the lavage fluid of the animals remained elevated for 4 days (Fig 5 ). Corresponding to the incursion of inflammatory cells, the level of lavage protein was augmented and remained significantly greater than that in animals instilled with saline solution for 2 days (Fig 6 ). This considerable influx of inflammatory cells and injury was corrected by 7 days after the instillation of the blood (Fig 5 , 6) .



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  		Figure 4. Hematoxylin-eosin stain of lung tissue from rats instilled with 1.0 mL blood. Erythrocytes are diffusely distributed in the distal lung 20 min following exposure (top). There is an absence of any inflammatory response to the blood at this time. By 1 day after instillation (middle), there is an obvious influx of macrophages and neutrophils, and injury is evident as perivascular edema (arrow). However, by 7 days after exposure all erythrocytes have been cleared from the lower respiratory tract, and the inflammatory response also has resolved by this time (bottom) (original x200).

 


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  		Figure 5. Percentage of neutrophils in the BAL fluid from rats instilled with either saline solution or 1.0 mL blood. Neutrophils were enumerated, and values were expressed as the percentage of total cells recovered. The percentage of neutrophils was significantly elevated 1 day after the instillation of blood and remained increased for at least 4 days following exposure. See Figure 1 for explanation of symbol.

 


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  		Figure 6. Protein concentrations in BAL fluid from rats instilled with either saline solution or 1.0 mL blood. After the separation of cells by centrifugation at 600g for 10 min, lavage protein was determined using a protein assay reagent modified for automated measurement. BSA served as the standard. BAL protein concentrations were significantly increased at both 1 day and 2 days following exposure of the animal to blood. See Figure 1 for explanation of symbol.

 
To better delineate the changes in both the metal-binding proteins and the metal in the lower respiratory tract after exposure to blood, lung tissue was stained for hemoglobin, iron, and ferritin. The stain for hemoglobin demonstrated an enormous exposure immediately after instillation (Fig 7 ). This was greatly diminished with the uptake of the erythrocytes by alveolar macrophages at 1 day after exposure, and there was no positive staining for hemoglobin by any cells by the fourth day after instillation.



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  		Figure 7. Stain for hemoglobin in lung tissue from rats that were instilled with 1.0 mL blood. Erythrocytes stained brownish-red 20 min after exposure. There was significantly less staining for hemoglobin 1 day after instillation, and by 4 days after exposure there was no staining for hemoglobin in the lung tissue (original x200).

 
Immunohistochemistry for ferritin demonstrated some uptake of the antibody by alveolar macrophages prior to instillation (Fig 8 , top). This did not change immediately after exposure but was greatly increased on days 1 through 4 (Fig 8 , bottom). Comparable to the hemoglobin stain, the uptake for ferritin had decreased to the level of the unexposed rats by day 7.



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  		Figure 8. Stain for ferritin in lung tissue from rats that were instilled with either saline solution or 1.0 mL blood. Ferritin was stained immunohistologically employing a polyclonal antibody (rabbit {alpha}-ferritin antibody). Top: there was some minimal uptake of the antibody by macrophages (arrow) with the instillation of saline solution only. Bottom: ferritin (arrows) was elevated at 1 day after exposure. At 7 days, the expression of this storage protein had returned to baseline values (original x200).

 
There was never any positive staining using Turnbull’s blue reaction. However, a few alveolar macrophages did demonstrate positive staining using Perl’s Prussian blue method for iron. This occurred only at 4 and 7 days after instillation. No index of iron equilibrium, either biochemical or by stain, indicated an imbalance of metal metabolism at 14 days after instillation.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The normal destruction of senescent erythrocytes in the body is presumed to take place in the macrophages of the mononuclear phagocyte system in the spleen, liver, and marrow. In the lung, RBCs can frequently be present in numerous injuries including chronic bronchitis, bronchiectasis, cystic fibrosis, lung cancer, pulmonary embolism, pneumonia, tuberculosis, traumatic injury, and diffuse alveolar hemorrhage. Their elimination from the lower respiratory tract could include several different pathways. The RBCs can be phagocytosed by alveolar macrophages and transported with this phagocyte out of the lung either to regional lymph nodes or to the systemic circulation. The pulmonary alveolar macrophage has been considered to be less effective in the phagocytosis of erythrocytes relative to monocytes and other macrophages.16 However, many of the RBCs instilled into the rats in this investigation were rapidly phagocytosed by alveolar macrophages. The fate of these phagocytes was not determined in this study.

All of the RBCs were not removed to regional nodes by these phagocytes and destruction of a portion of the erythrocytes occurred in the lower respiratory tract as evidenced by the presence of increases in nonheme iron, decrements in ascorbate, a deposition of hematoidin, and an elevated expression of ferritin. These cells could be destroyed by proteases and endogenous oxidants of the inflammatory cells recruited into the lung. A quantity of the hemoglobin originally contained within the erythrocyte was released after the destruction of the cell. Such release could be followed by the binding of this molecule to haptoglobin that is resident in the distal tract.17 This complex then would be cleared and delivered to the parenchymal cells of the liver where the hemoglobin is broken down to bilirubin. Alternatively, hemoglobin originally contained in the erythrocytes could be oxidized to hemin, which is bound to hemopexin in a 1:1 mol/L ratio. The hemin-hemopexin complex is removed from the circulation by hepatic parenchymal cells. A third pathway to handle hemoglobin liberated from RBCs in the distal lung is its catabolism by heme oxygenase in the alveolar macrophages.18 This enzyme utilizes molecular oxygen and nicotinamide adenine dinucleotide phosphate to catabolize the heme with cleavage of the ring by oxidation of the {alpha}-methene bridge. This produces one molecule of biliverdin and one molecule of carbon monoxide. While the carbon monoxide either is carried in the blood in the form of carboxyhemoglobin or is excreted by the lungs, the biliverdin is converted to bilirubin by biliverdin reductase. The bilirubin, or closely related products, precipitate out of solution to produce hematoidin. Hematoidin can be chemically identical to bilirubin and is formed from hemoglobin that is released locally in the tissues, particularly under conditions of reduced oxygen tension. This breakdown product is encountered most commonly within scar tissue, in necrotic debris, and in hemorrhagic infarcts where the appropriate conditions of both released hemoglobin and anaerobiosis are present. In these sites, the pigment occurs as cockle-burr shaped, golden brown granules or occasionally as slender crystals that lie in parallel bundles or sheaths.

These pathways for clearance of either the erythrocyte or the hemoglobin were extremely capable, and exceedingly little iron remained in the lung at 7 days after exposure, with rare positive staining noted on Perl’s Prussian blue reaction. This is consistent with the results of a previous investigation that demonstrated that the removal of these hemosiderin-laden macrophages from the lung was complete by 2 weeks after exposure.19 The hemosiderin-laden macrophages that did persist past 2 to 4 days after exposure were retained in sideromacrophages, likely as an oxidized product of ferritin such as hemosiderin since ferritin does not stain with Perl’s Prussian blue reaction.20 The time required for the formation of hemosiderin in this investigation approximates that (50 h) for the in vivo formation of this protein in the human lung after pulmonary hemorrhage.19 There was minimal staining for ferritin in the lung at 7 days after exposure. The iron that originally was located in hemoglobin contained in the erythrocyte either was transported out of the lung by alveolar macrophages, haptoglobin, or hemopexin or was isolated in the ferritin of these same cells. This storage protein, with its sequestered iron, can be moved out of the lung with the cell. Alternatively, macrophages can release iron both as transferrin-Fe3+ and as metal contained within ferritin.21 22 The reactivity of this iron, both complexed to transferrin and sequestered in ferritin, is likely to be greatly diminished relative to other chelates. However, the iron in both of these proteins is sensitive to chemical reduction by compounds that are present in the alveolar lining fluid (eg, ascorbate). Such reduction could result in the mobilization of the metal, making it available to catalyze oxygen-based radicals and, therefore, presenting an oxidative stress to the lower respiratory tract. This sequence of events would clarify the elevations in nonheme iron concentrations, the decrements in ascorbate, the release of oxidant-sensitive mediators, the neutrophil influx, the injury, the increased expression of ferritin, and the staining for hemosiderin observed after blood exposure in this animal model.

We conclude that intratracheal instillation of whole blood in the rat can induce a neutrophilic lung injury that is associated with a disruption of the normal iron metabolism. This disruption of the iron equilibrium is made evident by quantifying iron and staining for hemoglobin and ferritin. All indexes of biological effect had corrected by 7 days after exposure.


   Footnotes
 
Abbreviations: BSA = bovine serum albumin; MIP = macrophage inflammatory protein; PBS = phosphate-buffered saline solution; TNF = tumor necrosis factor

This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Received for publication May 24, 1999. Accepted for publication February 9, 2000.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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