Environmental toxins, infection, and allergens lead to a transient mucous cell hyperplasia (MCH) in airway epithelia; however, the mechanisms for reducing mucous cell numbers during recovery are largely unknown. This study investigated Bcl-2 expression in mucous cells induced by a neutrophilic or eosinophilic inflammatory response. Brown Norway rats intratracheally instilled with lipopolysaccharide (LPS) showed an inflammatory response characterized primarily by neutrophils. Secreted mucin was increased fourfold at 1 day, and the number of mucous cells was increased fivefold 2, 3, and 4 days post-LPS instillation compared with those in noninstilled rats. None of the mucous cells in non- or saline-instilled control animals expressed Bcl-2, whereas 20–30% of mucous cells were Bcl-2 positive 1 and 2 days post-LPS instillation. Brown Norway rats immunized and challenged with ovalbumin (OVA) for 2, 4, and 6 days showed an inflammatory response characterized primarily by eosinophils. Secreted mucin increased fivefold, and mucous cell number increased fivefold after 4 and 6 days of OVA exposure compared with water-immunized control rats challenged with OVA aerosols. Approximately 10–25% of mucous cells were Bcl-2 positive in OVA-immunized and -challenged rats. These data demonstrate Bcl-2 expression in hyperplastic mucous cells of Brown Norway rats regardless of the type of inflammatory response and indicate that apoptotic mechanisms may be involved in the resolution of MCHs.
the epithelial lining of the conducting airways is exposed to the outer world through the air we breathe; therefore, these cells are constantly exposed to potentially injurious materials from the inhaled air. The mucous layer that overlies the airway epithelia contributes to protection from inhalation of airborne irritants, particles, and microorganisms by trapping foreign material in its viscous matrix and acting as the medium by which cilia clear agents from the respiratory tract. Mucous cells are therefore vitally important for protection of airway epithelia, but chronic inflammation can lead to increased numbers of mucous cells and continuous hypersecretion that overwhelms the normal mucociliary clearance (18, 23). Accumulated mucus can obstruct the airway and impair pulmonary function in pathological conditions such as asthma and chronic bronchitis (10, 17).
Exposure of rats to endotoxin, a lipopolysaccharide (LPS) from gram-negative bacteria, is characterized by infiltration of the alveolar and bronchiolar air spaces by neutrophils (15, 20, 27,29) and induction of mucous cell hyperplasia (MCH) (11). These alterations closely resemble the pathology observed in patients with chronic bronchitis. It is well known that exposure to allergen causes primarily eosinophilic infiltration of the lung (4, 24). The inflammatory response after immunization and challenge of rats to ovalbumin (OVA) is characterized by the predominance of eosinophils, and this inflammatory response is also associated with the development of MCH (4, 24). Changes of the respiratory epithelium under these conditions resemble those observed in asthma patients (2, 9, 25).
Apoptosis is a genetically regulated cellular suicide mechanism that plays a role in the development and maintenance of homeostasis (1). Bcl-2 and related proteins can register diverse forms of intracellular damage, gauge whether other cells have provided a positive or negative death stimulus, and determine the progression or inhibition of the suicide program. This family of proteins is characterized by at least one of the four conserved motifs known as Bcl-2 homology domains, BH1 to BH4. Bcl-2 and Bax are anti- and proapoptotic regulators, respectively, that can heterodimerize, and their relative concentration determines whether a cell lives or dies (22). These proteins function by regulating the release of cytochrome c from mitochondria, which leads to the aggregation of apoptotic protease activating factor-1, proteolytic activation of a cascade of caspases, and the appearance of the apoptotic morphology (8, 32).
Our previous studies demonstrated that exposure of rats to ozone induces MCH in the maxillo and nasal turbinates and that the MCH resolves when exposure to ozone is stopped. The appearance of mucous cells was associated with the expression of Bcl-2, a regulator of apoptosis (28). These observations suggest that apoptotic mechanisms are involved in the resolution of MCH. The objective of the present study was to use a defined animal model to investigate the association of inflammatory cellular responses and the dynamics of mucus secretions and MCH with the expression of Bcl-2 and Bax after exposure to LPS or allergen. Therefore, we examined the time-dependent correlation of mucus secretions and MCH in the airway epithelia of Brown Norway rats intratracheally instilled with endotoxin or immunized and challenged with OVA and demonstrated that Bcl-2 but not Bax is expressed in metaplastic mucous cells regardless of the preceding type of inflammatory response.
MATERIALS AND METHODS
Male Brown Norway rats, 6–8 wk of age, were purchased from Charles River Laboratories. The rats were housed two per polycarbonate cage; each cage was supplied with sterilized hardwood chip bedding and a filter top and placed in animal rooms maintained at 20–22°C, with a relative humidity of 20–50% and a 12:12-h light-dark cycle starting at 6:00 AM. Water supplied by a centralized distribution system with sipper tubes and food (Lab Blox, Allied Mills, Chicago, IL) were provided ad libitum.
Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
Rats were lightly anesthetized with 5% halothane in oxygen before intratracheal instillation with 1.0 mg of endotoxin (LPS fromEscherichia coli 0111:B4, Sigma, St. Louis, MO) in 0.5 ml of pyrogen-free 0.9% NaCl solution or with 0.5 ml of pyrogen-free 0.9% NaCl solution alone. At least four rats were killed 1, 2, 3, 4, 7, and 14 days postinstillation.
Sensitization and allergen exposure.
Rats were sensitized with an intraperitoneal injection with 1 μg OVA/100 μg Al(OH)3 in 0.5 ml of water or, for control animals, with 0.5 ml water/100 μg Al(OH)3 alone ondays 0 and 7. On day 14, rats were exposed for 5 h/day to OVA aerosols at a concentration of 2.3 mg/m3. After 2, 4, or 6 consecutive days of OVA aerosol exposure, the rats were killed, and the lungs were prepared for histopathology.
Necropsy and tissue preparation for histopathology.
Rats were killed with an intraperitoneal injection of 25 mg of pentobarbital sodium (Abbott Laboratories, Chicago, IL) and exsanguination via the renal artery. The thoracic contents were exposed, and the lungs were perfused by cardiac puncture with 0.9% (wt/vol) saline (McGaw, Irvine, CA). The trachea was cannulated with an 18-gauge blunt needle tipped with surgical tubing, and the lungs were lavaged three times with 5 ml of ice-cold PBS containing 1% fetal bovine serum. The bronchoalveolar lavage (BAL) fluid was collected. The lungs were then inflated with 10% zinc Formalin (Stephens Scientific, Riverdale, NJ) at a pressure of 25 cmH2O for 6 h and immersed in a large volume of the same fixative overnight as described previously (29).
A stratified random-sampling scheme was used to cut the fixed lung lobes into cross-sectional slices, each ∼0.4 cm thick. Depending on the size of the lung, five or six slices were prepared and numbered from the proximal (slice 1) to the distal (slice 5 or 6) end. The slices were then embedded in paraffin and sectioned (5 μm). The sections were used for Alcian blue (AB) and immunohistochemical staining.
Quantification of neutrophils and macrophages.
The cells recovered by lavage were enumerated with a hemacytometer. Cytological preparations were made and stained with Wright-Giemsa (American Scientific Products, McGraw Park, IL) to determine the different types of cells present in the BAL fluid. At least 400 cells were counted from each slide, and the distribution of macrophages, neutrophils, eosinophils, and lymphocytes was determined.
Analysis of mucin content in BAL fluid.
The recovered BAL fluid was reproducibly ∼11 ± 1 ml; therefore, the same sample volume was used for mucin assays from all samples. Anti-mucin antibody 10G5 was a generous gift from Dr. Carol Basbaum (University of California, San Francisco, CA). The specificity of the antibody was tested in other experiments. However, before the final assay was performed with the BAL fluid samples, preliminary experiments were carried out with several dilutions to find the linear range for the assay. Aliquots (50 μl, in duplicate, diluted 1:1 with 50 mM NaHCO3) of the BAL fluid were transferred to the wells of polyvinyl chloride, high protein-binding, 96-well plates previously rinsed with 50 mM NaHCO3 buffer, pH 8.0. The samples were allowed to dry at 37°C overnight. The wells were blocked with 100 μl of PBS with 2.5% nonfat dry milk (Carnation) in 0.3% Triton X-100 and 1% normal goat serum (PMTG). The mucin antibody was applied at a dilution of the hybridoma culture supernatant of 1:10 in PMTG. The plate was incubated for 90 min on a rotating platform, and the wells were washed four times each with 50 μl of PMTG. A Vector Laboratories (Burlingame, CA) ABC kit was used to detect the bound anti-mucin antibody with the secondary antibody (biotinylated rat-adsorbed goat anti-mouse) at a dilution of 1:100 in PMTG as described by the manufacturer. Reaction of the p-nitrophenyl phosphate substrate in diethanolamine buffer (Bio-Rad, Hercules, CA) was detected at 410 nm (Dynex MR600) after 1 h of incubation at room temperature. Readings from the duplicate wells were averaged.
Western blot analysis.
Several cell types of the lung, e.g., type II cells and macrophages, express Bcl-2 (31, 35). The following procedure was carried out to enrich for LPS-induced metaplastic mucous cells and to demonstrate that the antibody used for immunohistochemistry reacts with the 28-kDa Bcl-2 in protein homogenates. Brown Norway rats were intratracheally instilled with 1 mg of LPS in 0.5 ml of saline or with 0.5 ml of saline alone and killed 2 days later. The lungs were removed, and the axial airways were immediately microdissected under a dissection scope (Wild Heerburg) and stored at −80°C until further use. Protein was extracted from these microdissected tissues by homogenization in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 5 mM EDTA) supplemented with the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), pepstatin (10 μg/ml), leupeptin (2 μg/ml), and bestatin (2 μg/ml). The protein concentration was determined with the BCA assay kit (Pierce, Rockford, IL); 120 μg of protein from saline- and LPS-instilled rats were loaded on each lane. Western blotting was carried out as described earlier (30), and filters were stained with Ponceau S to confirm that equivalent amounts of protein had been loaded on each lane. The polyclonal rabbit anti-mouse Bcl-2 (PharMingen, San Diego, CA) was used at a 1:1,000 dilution. The signal on the Western blot was quantified via densitometry with the Fluor-S MAX Imager and Quantity One software (Bio-Rad).
Endogenous peroxidase activity was blocked by incubating the sections in 2% hydrogen peroxide-methanol for 1 min. Slides were washed in deionized water; all subsequent washes were in 0.05% Brij 35-Dulbecco's PBS (pH 7.4). Bcl-2 or Bax was unmasked by immersing the slides for 20 min in antigen-retrieval solution (BioGenex) that was heated to boiling temperature. After preincubation in 1% normal goat serum in 2% BSA-0.1% Triton X-100 in buffer consisting of Tris, pH 7.7, 550 mM NaCl, and 10 mM KCl, the primary antibody or the normal rabbit serum was applied at the respective dilutions. The dilutions for the Bcl-2 (PharMingen) and Bax (a generous gift from Dr. John Reed, Burnham Institute, La Jolla, CA) antibodies were 1:1,000 and 1:200, respectively. After an overnight incubation at room temperature, the immunoreaction was visualized with the Vectastain ABC kit and the peroxidase substrate diaminobenzidine (Vector Laboratories) as described by the manufacturer. Epithelial cells with mucosubstances were detected by staining tissue sections with AB (15).
Results are presented as means ± SD. Multiple comparisons between groups were made with Bonferroni (Dunn) t-tests. A value ofP < 0.05 was considered significant.
Inflammatory cells in airways of rats instilled with saline or LPS.
Rats instilled with 0.5 ml of saline showed no significant changes in the number of inflammatory cells in the BAL fluid except for a tendency toward fewer macrophages on days 1 and 2postinstillation compared with the age-matched, noninstilled control rats (Fig. 1). Endotoxin instillation induced an increase in the number of neutrophils in the BAL fluid over a period of 2 days, and this inflammatory response decreased to background levels over 4 days (Fig. 1). The number of macrophages showed a significant peak 4 days post-LPS instillation compared with that in non- or saline-instilled rats and decreased to background levels on days 7 and 14. No significant increase in eosinophils was observed at any time point; however, 3 days post-LPS instillation, the number of lymphocytes was significantly elevated compared with that in non- or saline-instilled rats.
Secreted and stored mucosubstances in rats instilled with saline or LPS.
The relative amounts of mucosubstances in the BAL fluid of air control and saline-instilled rats were not significantly different at any time point (Fig. 2). However, a twofold increase relative to saline instillation was detected in rats onday 1 post-LPS instillation. The levels decreased to the levels observed in saline- or noninstilled control rats by 2 days post-LPS instillation and remained at similar levels over the 14 days tested (Fig. 2).
Twenty-four hours post-LPS instillation, the epithelia lining the axial airways and some of the preterminal bronchioles were markedly thickened, and columnar AB-periodic acid-Schiff (PAS)-positive cells were observed, primarily in the intrapulmonary axial airways (generation 3). Therefore, the number of AB-PAS-positive cells was quantified in tissue sections prepared from the axial airway of the left lung lobe. No significant differences were observed between noninstilled control and saline-instilled rats 1, 2, and 4 days postinstillation. The number of AB-PAS-positive cells was significantly increased in LPS-exposed rats 2, 3, and 4 days after instillation compared with that in noninstilled rats. The number of mucous cells decreased to levels observed in air control animals 7 and 14 days post-LPS instillation (Fig. 3). Morphometric analyses of stored mucosubstances in airway epithelia from the same tissue sections showed the same results (data not shown).
Bcl-2-immunopositive mucous cells in rats instilled with saline or LPS.
Bcl-2 expression was increased in ozone-induced metaplastic mucous cells in the nasal epithelia of Fischer 344/N rats (28). To investigate whether Bcl-2 levels were increased in LPS-induced MCH in airways of Brown Norway rats, Bcl-2 expression was assessed by immunohistochemistry and Western blot analyses. Bcl-2-positive mucous cells were increased significantly after LPS instillation compared with those in non- and saline-instilled rats (Fig.4 A). Western blot analysis of protein extracts prepared from microdissected axial airways showed that the antibody was specific for the 28-kDa Bcl-2 protein and that this protein was increased in LPS-instilled rats (Fig. 4 B). As determined by densitometric analysis, the Bcl-2 protein was increased twofold in tissues from LPS- compared with saline-instilled rats. The microdissected tissues contained many cell types including ciliated, basal, Clara, some smooth muscle, and parenchymal cells and contributed to the dilution of the impressive increases observed in mucous cells. Only 0–1% of mucous cells were immunopositive for Bcl-2 in saline- or noninstilled control rats, whereas a significant 20–30% of mucous cells were Bcl-2 positive on days 1and 2 after LPS instillation, and ∼10% remained positive until day 4 postinstillation (Fig. 4 C). The numbers of Bcl-2-positive cells decreased to ∼3% on days 7 and 14 (Fig. 4 C). Less than 1% of mucous cells immunostained for Bax in control or LPS-instilled rats (data not shown).
Inflammatory cells in rat airways exposed to allergen.
The number of inflammatory cells in rats immunized with water and exposed to OVA aerosols did not change significantly from those observed in air control rats. However, in rats immunized and challenged with OVA, the number of macrophages increased threefold on day 2 of OVA challenge and decreased to background level on days 4 and 6. The number of recovered eosinophils in the BAL fluid increased ∼25-fold in rats sensitized and challenged with OVA compared with control rats, and this increase was sustained over the 6-day period of exposure (Fig.5). The number of neutrophils also increased significantly after 2 days of challenge with OVA aerosols, and there was a tendency toward an increase in lymphocytes compared with air control or water-immunized and OVA-exposed rats.
Secreted and stored mucosubstances in rats exposed to allergen.
Concurrent with these inflammatory changes, secreted mucosubstances in OVA-sensitized and -challenged rats increased fivefold compared with air control and water-sensitized and OVA-challenged rats (Fig.6). After 2 days of OVA exposure, the number of mucous cells in the surface epithelia lining the intrapulmonary axial airways was similar in water- and OVA-sensitized rats exposed to OVA aerosols (Fig. 7). However, on days 4 and 6 of exposure to OVA aerosols, the OVA-sensitized rats exhibited approximately fivefold more mucous cells compared with the water-sensitized rats (Fig. 7). Similar results were observed when stored mucosubstances were quantified morphometrically (not shown).
Bcl-2-positive mucous cells in rats exposed to allergen.
Only 1–2% of the mucous cells of air control and water-sensitized and OVA-challenged rats were immunopositive for Bax and Bcl-2, similar to the control rats in the LPS instillation experiment (data not shown). Similarly, only 1–2% of OVA-sensitized and -challenged rats were Bax positive (data not shown). However, in OVA-sensitized rats, 3% of the mucous cells expressed Bcl-2 on day 2 of OVA challenge, and 10 and 25% of mucous cells were Bcl-2 positive ondays 4 and 6 of OVA challenge, respectively (Fig.8).
The main conclusion of this study is that Bcl-2, but not Bax, is expressed in metaplastic mucous cells in Brown Norway rats regardless of whether LPS instillation or exposure to allergen induced the inflammatory response and the resulting MCH. The inflammatory responses caused by these exposures are very different and are characterized by infiltration of the airways by, primarily, neutrophils for LPS instillation or eosinophils for allergen exposure.
A twofold increase of secreted mucosubstances was observed in the BAL fluid of LPS-instilled rats compared with the saline-instilled rats 1 day postinstillation, indicating that LPS induces mucin secretion. Secretion may be caused directly by stimulation of certain receptors, may be related to the process of neutrophilic influx through the epithelial layer, or may also be a result of proteases released by neutrophils. Secretion of mucosubstances would be expected to correlate with a transient depletion of stored mucosubstances in airway epithelia. Studies that analyzed earlier time points after LPS instillation have shown that a transient depletion of epithelium-stored mucosubstances occurs in nasal epithelia (14) and in airway epithelia (26) of Fischer 344/N rats. The fact that the number of mucous cells in airway epithelia of LPS-instilled rats remained the same as in noninstilled control animals after 1 day also agrees with these studies. However, at this time point, the percentage of Bcl-2-positive mucous cells increased significantly from 0 to 20%. Previous studies (12, 13, 29) show that epithelial cells proliferate after endotoxin instillation but not after saline instillation, and an increase in epithelial cell number occurs 2 days post-LPS instillation. Therefore, as early as 1 day post-LPS instillation, Bcl-2-expressing mucous cells may be, at least in part, not newly formed but existing cells that start to synthesize and store mucus in larger quantities than secreted amounts.
Two days post-LPS instillation, the number of mucous cells increased significantly, and ∼30% of these mucous cells were Bcl-2 positive. At this time point, Fischer 344/N rats instilled with endotoxin had ∼30% more epithelial cells per millimeter of basal lamina than saline-instilled control rats (11). This increase was shown to be predominantly a result of an increase in the number of mucus secretory cells and not in the number of ciliated or basal cells, indicating that the cellular response to endotoxin exposure is not a substitution of one epithelial cell type for another (11). Together, these data suggest that on day 2 post-LPS instillation, the Bcl-2-positive mucous cells may, at least in part, represent newly formed cells.
The number of Bcl-2-expressing mucous cells decreased to background levels at 3 days, whereas the number of mucous cells was still elevated 4 days post-LPS instillation. Therefore, mucous cell numbers were decreased to background levels at least 2 days after the percentage of Bcl-2 positivity decreased to background levels. These data support the hypothesis that Bcl-2 as an inhibitor of apoptosis must be downregulated before mucous cell numbers can be reduced as a result of programmed cell death. It is possible, however, that Bcl-2 expression is not directly related to the resolution of MCHs and may represent an epiphenomenon without any direct role in the pathophysiological response.
The acute induction of MCH after LPS instillation is preceded by massive neutrophilic inflammation. Several reports (5, 16,21) have described that a single intratracheal instillation of purified neutrophil elastase causes the development of MCH and that a selective neutrophil elastase inhibitor reduces MCH by 37% (27), suggesting that neutrophil elastase contributes significantly to the observed MCH. Pseudomonas aeruginosaexoproducts directly induce MUC2 transcription (3). Exposure to LPS and recruitment of neutrophils into the air spaces of the lung are primarily associated with tumor necrosis factor-α, interleukin-1, and interleukin-6 (19, 36). Whether neutrophil elastase or the inflammatory mediators are directly associated with mucin synthesis and Bcl-2 expression is currently being studied in our laboratory.
The number of eosinophils in the BAL fluid of rats immunized and challenged with OVA had already reached maximum levels on day 2 of allergen exposure. However, the mucous cell numbers remained constant after reaching a maximum at 4 days, and the percentage of Bcl-2-positive mucous cells increased from 3 to 25% over the 2, 4, and 6 days of the exposure period. Another study (6) has shown that eosinophils are not directly responsible for the MCH. Similarly, there is no evidence that macrophages directly induce MCH, although a threefold increase in alveolar macrophages was observed 2 days after exposure to OVA. Schneider et al. (24) reported the same observation for Brown Norway rats exposed to allergen. However, the modest increase in neutrophils and lymphocytes in the BAL fluid observed in our study in OVA-immunized rats 2 days after challenge with OVA aerosols may play a role in MCH on days 4 and6. Passive transfer of antigen-primed CD4+ T lymphocytes into naive Brown Norway rats induces an asthma phenotype (33, 34), and activated helper T cell type 2 lymphocytes are important in inducing MCH in mouse airways (7). However, whether these lymphocytes are associated with induction of Bcl-2 expression in mucous cells is not known.
Secreted mucosubstances increased twofold after LPS instillation, whereas they increased fivefold in rats exposed to allergen for 2, 4, and 6 days compared with that in air control animals. Interestingly, both LPS instillation and exposure to OVA caused a five- to sevenfold increase in mucous cell numbers compared with those in air control subjects. Steiger et al. (26) also reported a fivefold increase of epithelium-stored mucosubstances at the same location in Fischer 344/N rats instilled with LPS. Whether continuous exposure to LPS would have increased mucus secretions to the levels observed after continuous exposure to allergen is not known. These results also suggest that continuous exposure to allergen may cause sustained Bcl-2 expression to maintain the newly formed mucous cells, ensuring a steady state of mucin gene expression, maximum storage of mucosubstances, and secretion of excess mucus to protect the airway epithelia until the insult subsides.
Inflammatory responses induced by both LPS instillation and exposure to allergen caused Bcl-2 expression in ∼15–30% of mucous cells. These numbers represent Bcl-2-positive cells at any given time point, so it is not clear whether the nonpositive cells remain Bcl-2 negative or may express Bcl-2 at earlier or later time points. This study demonstrates that Bcl-2 expression in mucous cells of rat airway epithelia is associated not only with neutrophilic but also with eosinophilic inflammation. Expression of Bcl-2, regardless of the preceding type of inflammatory response, suggests that Bcl-2 expression is induced by inflammatory factors found in both types of inflammatory responses, although they are characterized by different cytokines and leukocytes. A previous study by Tesfaigzi et al. (28) shows that Bcl-2 induced by ozone exposure in rat nasal epithelia is expressed in metaplastic mucous cells. Together, these observations suggest that expression of Bcl-2 in mucous cells occurs independent of airway location in both airway and nasal epithelia. It is possible, therefore, that Bcl-2 is inherently an important regulator of cell numbers in rat airway epithelia, may represent an intricate response to MCH, and, therefore, is expressed in hyperplastic mucous cells that are preceded by epithelial cell proliferation. Because the epithelial cells proliferate after exposure to environmental toxin, certain cells must be deleted to reduce the numbers of epithelial cells during the “healing process” (13). Our findings suggest that apoptotic mechanisms are important in ensuring a correct transition in retaining the cells for the recovered epithelium and in discarding the cells that are no longer needed. The exact role of Bcl-2 in hyperplastic mucous cells is currently under investigation. Understanding these processes will be important for the development of novel approaches for reducing mucus hypersecretion in asthmatic and chronic bronchitis patients.
We thank Yoneko Knighton for the preparation of tissue samples and Justin Kubatko for solving the statistical problems.
These studies were sponsored by the Office of Biological and Environmental Research, US Department of Energy, under Cooperative Agreement No. DE-FC04-96AL6406; a grant from the American Lung Association; and National Institute of Environmental Health Sciences Grant ES-09237 (to Y. Tesfaigzi).
Address for reprint requests and other correspondence: Y. Tesfaigzi, Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185 (E-mail:).
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. Section 1734 solely to indicate this fact.
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