1 Research, Anesthesiology, and Medicine Services, Veterans Affairs San Diego Healthcare System, San Diego 92161; 2 Departments of Anesthesiology and Medicine, University of California San Diego, La Jolla, California 92093; 3 Department of Pediatrics (Neonatology), State University of New York at Buffalo, Buffalo 14214; and 4 Department of Pediatrics (Neonatology), Strong Children's Research Center, University of Rochester, Rochester, New York 14642
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ABSTRACT |
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Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 ± 710 pg/ml (mean ± SE) at 48 h of hyperoxia. Levels fell to 299 ± 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h (P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 ± 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 ± 153 pg/ml at 48 h of recovery (P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD50, concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.
adult respiratory distress syndrome; calcitropic hormones; growth factor; receptor; type II pneumocyte
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INTRODUCTION |
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PARATHYROID HORMONE-RELATED PROTEIN (PTHrP) was originally discovered in a lung squamous cell carcinoma as the mediator of humoral hypercalcemia of malignancy. The protein is named for its similarity to parathyroid hormone. The amino-terminal portions of the two molecules have significant homology in primary and secondary structure (18). Thus PTHrP 1-34 binds to the classic type 1 PTH receptor and mimics all of the effects of PTH, including hypercalcemia, in tissues that bear the receptor (23). In contrast to PTH, PTHrP is not found in detectable levels in the circulation under normal conditions and does not act as a circulating hormone. PTHrP expression is distributed in a wide variety of normal tissues, including the lung, and many of its physiological functions are manifest as paracrine or autocrine effects on growth (27). PTHrP acts as a growth inhibitor in many tissues and cell types, including keratinocytes, vascular smooth muscle cells, and lung cancer cells (17, 19, 22).
Our studies have demonstrated that alveolar type II epithelial cells produce and secrete PTHrP, that they express the PTHrP receptor, and that they respond to changes in ambient PTHrP concentration with alterations in function and growth (10, 13, 14). The amino-terminal portion of the molecule, PTHrP 1-34, stimulates characteristics that are typical of the differentiated type II cell phenotype, such as production of disaturated phosphatidylcholine and expression of alkaline phosphatase (13). Treating cultured type II cells with neutralizing PTHrP antibodies induces type II cell proliferation, whereas instillation of the same antibodies into normal lungs causes type II cell proliferation (14). Thus endogenous PTHrP 1-34 is an autocrine inhibitor of type II cell growth. PTHrP also appears to be involved in the epithelial response to injury. Lung PTHrP levels are decreased between 4 and 8 days of exposure to 85% oxygen in rats, concurrent with increased expression of proliferating cell nuclear antigen (PCNA) and increased incorporation of 5-bromo-2-deoxyuridine (BrdU) into DNA in alveolar type II cells (9). Instillation of PTHrP 1-34 into rat lungs on the fourth day of hyperoxia reduces BrdU incorporation. These data suggest that changes in lung PTHrP levels are involved in regulating type II cell growth in this model.
Type II cells divide, spread across a damaged alveolar surface, and differentiate into type I cells (1). However, changes in lung PTHrP levels have not been measured in other types of lung injury, in species other than rats, nor in neonatal animals. Alveolar type II cell proliferation is a common response to lung injury of many etiologies. Lung injury models may vary in severity, time course, and proliferative response. For example, 100% oxygen causes a more severe injury than 85% oxygen. Adult rats die within 72 h of exposure to 100% oxygen but can survive indefinitely at the lower concentration. Type II cell proliferation occurs throughout exposure to 85% oxygen, but becomes prominent with injury due to 100% oxygen when the animals are transferred from hyperoxia to room air (7, 25). Newborn animals are more tolerant of 100% oxygen than adults. They demonstrate inhibited type II cell proliferation during the acute injury phase and robust proliferation during the recovery phase (6, 8). Finally, the pattern of cell proliferation after lung injury may vary among different species in time course and magnitude (26). The goal of this study was to investigate the time course for changes in lung PTHrP levels relative to type II cell proliferation in adult rats exposed to and recovered from >95% oxygen. We compared the results in rats with the changes in PTHrP expression in hyperoxic adult and newborn rabbits.
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METHODS |
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Animals
Male pathogen-free Sprague-Dawley rats (250-300 g) were ordered from Harlan Laboratories (San Diego, CA) for experiments at the University of California San Diego. Experiments were also performed on adult New Zealand White rabbits weighing 1.9-2.2 kg (Buffalo, NY) and newborn (NB) rabbits delivered vaginally at term (Rochester, NY). All animal protocols were reviewed and approved by the institutional animal care and use committee at the institution where the experiments were performed.Hyperoxic Lung Injury in Adult Rats
Rats were housed two to a cage in a 50-l Plexiglas chamber ventilated with 6 l/min of >95% oxygen in nitrogen for 24, 48, or 60 h. Additional rats were exposed to oxygen for 48 h and then allowed to recover in the chamber ventilated with ambient air for 24, 48, or 72 h. These experimental groups will be labeled 24/0, 48/0, 60/0, 48/24, 48/48, and 48/72, with the first number referring to the hours of hyperoxia and the second number listing the recovery hours in air. The oxygen concentration and carbon dioxide tension of outflow gas were measured in line daily with an Ohmeda 5250 RGM gas analyzer (Englewood, CO). Oxygen concentrations were always >95% during the hyperoxic period and carbon dioxide tensions never exceeded 3 mmHg. Control rats were housed in the chamber ventilated with ambient air at the same flow rate for 48 and 96 h. Additional controls consisted of age-matched rats housed in the vivarium until death. Rats had free access to food and water. Cages were exchanged for clean cages every 2 days. The exchange took <1 min. At the end of the experimental period, rats were anesthetized with 80 mg/kg intraperitoneal pentobarbital sodium and exsanguinated by cutting the abdominal aorta. The thoracic cavity was opened, and pleural fluid was measured and saved for PTHrP assay. The right lung was removed, weighed, and homogenized distal to the primary bronchus for PTHrP assay. In five animals per time point in control, 48/0, and 60/0 groups, the lung was lavaged with four 5-ml aliquots of PBS plus protease inhibitors before homogenization (9). Bronchoalveolar lavage (BAL) was centrifuged at 1,200 g to separate cells and supernatant for subsequent PTHrP assay. The left lung was fixed by tracheal instillation of 6 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at 37°C, pH 7.4, and processed for histochemical staining or terminal deoxynucleotidyl transferase (TDT) dUTP nick-end labeling (TUNEL).Hyperoxic Injury in Adult Rabbits
Rabbits were allowed access to food and water ad libitum. Animals were exposed to 100% oxygen in individual Lucite chambers for 48 or 64 h (48/0 and 64/0 groups). After the 64-h exposure, some animals were exposed to room air for an additional 24-200 h (64/24, 64/48, 64/72, 64/96, 64/160, and 64/200 groups).Lung preparation. At the end of an experiment, the rabbit was heparinized and administered an overdose of intravenous pentobarbital sodium (80 mg/kg). The chest was opened, and the lungs were perfused through a catheter placed in the pulmonary artery with ice-cold buffered saline until free of blood. Blocks of lung (0.3-1.5 g) were frozen in liquid nitrogen for later PTHrP assay. Lung PTHrP levels were measured in all groups from 48/0 through 64/72.
Type II cell preparation. In separate animals, the lungs were perfused as described above and then excised and lavaged with 400 ml of Ca2+-free balanced salt solution (BSS). The lungs were inflated with 50 ml of BSS containing 5 mg of BaSO4 particles and incubated for 10 min at 37°C. The lungs were then processed for type II cell isolation as described previously (16). Briefly, the lungs were digested with low concentrations of elastase, minced, filtered through Nitex nylon gauze filters, and washed free of protease. Type II cells were isolated from the crude cell preparation by centrifugation on a Ficoll discontinuous density gradient. Cell counts were determined with a hemocytometer, and cell viability was assessed by trypan blue exclusion. The purified type II cell population generally contained 85-90% type II cells with >95% viability. Purity was initially determined by the use of Papanicolaou staining of air-dried smears and later by a laser-flow cytometric determination of phosphine 3R fluorescence staining of lamellar bodies in individual type II cells. The yield, purity, and viability of type II cells from lungs of rabbits exposed to 100% oxygen for 64 h did not change compared with controls.
Cell cycle determinations. Cell cycle status based on DNA analysis was determined on isolated type II cells by flow cytometry. Pellets containing 5 × 106 cells were resuspended in a solution of 3.7 mM sodium citrate, 0.1% Triton X-100, and 1.74 mM propidium iodide and analyzed within 15 min on an EPICS-V flow cytometer (Coulter Electronics).
Hyperoxic Lung Injury in NB Rabbits.
Samples were generated from a previously described NB rabbit hyperoxia model (6, 8). Briefly, New Zealand White term NB rabbit litters at <24 h of age (0 days of age) were placed in a large Plexiglas chamber (33 × 40 × 70 cm) and exposed to humidified oxygen or room air. Animals were exposed to 100% until 9 days of age and then recovered in 60% oxygen up to 14 days of age. Animals from these litters and room-air control age-matched animals were killed at 0, 2, 4, 8, 10, 12, and 14 days. Additional animals were studied after exposure to >95% oxygen to a point at which 50% of the pups died because of severe respiratory distress (LD50). The LD50 was 8 days in one litter and 11 days in another. Data were collected from surviving animals. At least two litters (of six animals each) were exposed to each condition, and no litter contributed more than two animals per condition per time point (except for the LD50). Animals were killed by an intraperitoneal injection of 200 mg/kg of pentobarbital sodium. A total of 42 hyperoxia-exposed and 41 room air-exposed NB rabbits were examined.Lung preparation. Paraffin sections were obtained from three to six animals per time point. Lungs were processed as previously described (6, 8). Briefly, a thoracotomy was performed immediately after the animal was killed, the right main stem bronchus was clamped, and the right lung was removed and flash-frozen in liquid nitrogen for other studies. The left lung was instilled in situ with phosphate-buffered 10% formalin under 25 cmH2O pressure for 30 min. It was then removed for 16-24 h of additional formalin fixation and preserved in 70% ethanol until embedded in paraffin for sectioning.
BAL.
Separate animals were used for lavage, two to three animals per time
point. Lungs were exposed by thoracotomy, perfused in situ with Hanks'
balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD), and
removed. Isolated lungs were lavaged with five aliquots of ice-cold
HBSS with each aliquot fully distending the lung (and ranging from 5 to
40 ml, depending on the size of the animal, and equal for age-matched
animals). Lavages were pooled, and cells were sedimented at 300 g for 6 min. Lavage supernatants were treated with 1 µM
Pefabloc protease inhibitor (Roche, Indianapolis, IN) and stored at
80°C before PTHrP assay.
Radioimmunoassay of PTHrP
Lungs were homogenized in a standard 3:1 vol/wt of tissue lysis buffer with protease inhibitors. Homogenates were centrifuged at 16,000 g for 30 min (9). PTHrP levels were determined in the lung supernatants or BAL with a previously described radioimmunoassay (14, 24). Assays were performed in triplicate and using multiple dilutions that paralleled the corresponding standard curve. PTHrP values were reported from measurements that fell on the linear portion of the standard curve (fractional binding values between 0.25 and 0.75). In some cases, PTHrP values were normalized to DNA measured fluorometrically after reaction with Hoechst 33258 dye.Control assay experiments were performed to test whether substances that might interfere with the PTHrP assay were present in homogenates of normal or injured lung. Homogenates from control lung, 48/0 lung, and 60/0 lung were added to the samples containing known quantities of PTHrP standards. The vol/vol ratio of homogenate to total assay volume was 1:5, matching the greatest proportion of homogenate in assays of any of the unknown samples. When measured PTHrP concentration was plotted vs. expected concentration, the points fell close to the line of identity and were indistinguishable for the control lung, 48/0, lung and 60/0 lung homogenates; these results indicate complete recovery of PTHrP and demonstrate that the assay measured changes in PTHrP levels in normal and injured lung homogenates with similar accuracy.
Immunohistology
Lung blocks were dehydrated through an ethanol/butanol series and embedded in paraffin. Sections (5 µm) were deparaffinized in xylene and hydrated through a graded ethanol series. Sections were microwaved in a 1% zinc sulfate solution for antigen enhancement and retrieval. Nonspecific protein binding was blocked with 10% goat serum. For PCNA immunostaining, sections were incubated with primary antibody (PC10; DAKO, Carpinteria, CA) at a 1:20 dilution with 0.5% bovine serum albumin in PBS, overnight at 4°C. Primary antibody was omitted on sections serving as negative controls. PTHrP was stained with the rabbit polyclonal antibody R759, directed against PTHrP 67-86, or the monoclonal antibody 8B12, directed against PTHrP 1-34, both at concentrations of 10 µg IgG/ml. These antibodies were developed in our laboratory (13). For negative controls, the primary antibody was preadsorbed with a 100-fold molar excess of PTHrP 1-34 or PTHrP 67-86. Secondary antibody (biotinylated goat anti-mouse or anti-rabbit; Calbiochem, La Jolla, CA), diluted at 1:33 with 0.01% goat serum in PBS, was applied for 25 min. Staining was completed by incubation with alkaline phosphatase-streptavidin conjugate and vector red or fast blue alkaline phosphatase substrate (Vector Laboratories, Burlingame, CA) with 0.01% Tween and 0.01% levamisole. Staining experiments were performed on lung sections from at least three animals per time point. Staining was repeated at least once for each block of animals. PTHrP immunoreactivity and PCNA immunoreactivity were quantified as a function of experimental manipulation by evaluating the percentage of stained corner cells. Cells were counted in six high-power fields per slide from at least three animals per time point. The percentage of immunoreactive corner cells was scored on an ordinal scale, with <2, 2-25, 26-50, 51-75, and >75% corresponding to scores of 0-4, respectively.TUNEL
TUNEL staining was performed with a kit from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Cell nuclei in tissue sections were stripped of proteins by incubation with 20 µg/ml of proteinase K for 10 min before tissue was fixed in 4% paraformaldehyde at room temperature for 5 min. Slides were washed with 0.5% Triton X-100 in PBS for 5 min and then incubated in 1% glycine in PBS and rinsed in deionized water. Sections were covered in TDT (0.3 U/µl) and biotin-16-dUTP in TDT buffer (30 mM Tris at pH 7.2, 140 mM sodium cacodylate, and 1 mM cobalt chloride) and incubated in a humidified chamber at 37°C for 60 min. Slides were washed in PBS, incubated with streptavidin-horseradish peroxidase conjugate for 30 min at 37°C, and washed 1 min 3× in PBS. Then they were immersed in buffer containing 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide for 30 min in the dark. Sections were lightly counterstained with methyl green. TUNEL staining was quantified as described in Immunohistology.In Situ Hybridization
Expression of PTHrP mRNA was investigated by nonradioactive in situ hybridization, as previously described (13). The probe was a digoxigenin-dUTP-labeled single-stranded cDNA fragment complementary to amino acids 15-120 of PTHrP. Sense strands were used as controls. Probes were generated by asymmetric polymerase chain reaction. Presence of the probe was visualized in lung sections after hybridization with an immunohistochemical technique using an alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim, Indianapolis, IN).Statistics
Lung homogenate PTHrP levels, PTHrP immunoreactivity, PCNA immunoreactivity, and TUNEL staining were compared among groups by nonparametric ANOVA. Lung weights and BAL total protein contents were compared among groups by ANOVA. Hyperoxic treatment and time effects on NB rabbit BAL PTHrP levels were evaluated with two-way ANOVA. The Tukey and Dunn tests were used for post hoc pair-wise parametric and nonparametric comparisons, respectively (28). Data are reported as means ± SE. Significance was accepted if the probability of a type I error was <0.05. ![]() |
RESULTS |
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Hyperoxic Lung Injury in Adult Rats
Extent of injury.
Rats in the 48/0 and 48/48 hyperoxic groups showed an influx of
inflammatory cells but near-normal lung architecture compared with
normoxic control animals (Fig. 1, A, B, and
D). Lungs from the 60/0 group contained thickened alveolar
septa, alveolar exudate, and hemorrhage (Fig.
1C). Lung weight and BAL total
protein levels varied significantly with exposure to hyperoxia (Table
1, P < 0.001). Rats in
the 60/0 hyperoxic group had greater signs of injury than animals from
the other groups. Average lung weight was 80% greater than control
lungs, and BAL total protein was six times the control level. Lung
weight normalized to body weight was 0.44 ± 0.03 mg/g in 60/0
rats compared with 0.22 to 0.29 in all other groups (P < 0.05). All of the 60/0 animals had pleural effusions, ranging from 2 to 7 ml in volume, whereas none of the other animals had effusions.
Lung weight and BAL protein were significantly greater in 48/0 animals
than control animals (P < 0.05).
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Lung PTHrP content.
Exposure to >95% oxygen caused significant changes in rat lung PTHrP
levels with time (Fig. 2). PTHrP levels
were 614 ± 18 pg/ml in the 24/0 group, unchanged from control
lung, but then increased 10-fold to 6,356 ± 710 pg/ml in the 48/0
group (P < 0.05). Lung PTHrP content fell abruptly
below control levels to 299 ± 78 pg/ml after an additional
12 h of hyperoxic exposure in the 60/0 group (P < 0.05). Lung PTHrP concentrations returned to control levels in 21 of 31 rats allowed to recover in room air after 48 h of >95% oxygen,
but remained elevated in 10 animals. To investigate whether the changes
in PTHrP were due to the hyperoxia or to exposure to the chamber, we
also studied control animals that were housed in the vivarium or in the
environmental chamber ventilated with ambient air for 48 and 96 h.
Lung PTHrP levels did not vary among the three control groups. The mean
lung PTHrP levels were 691 ± 41, 757 ± 104, and 790 ± 81 pg/ml for vivarium (n = 25), 48-h chamber
(n = 5), and 96-h chamber (n = 6)
animals, respectively. Because of concerns that PTHrP levels should be normalized to a measure of cell number, we repeated the analysis, normalizing supernatant PTHrP levels to supernatant DNA content. The
results of the analysis were unchanged (data not shown).
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PTHrP immunoreactivity.
PTHrP immunoreactivity was found in normoxic and hyperoxic lung in
cells that were the size, shape, and location expected for type II
epithelial cells (Fig. 3). Exposure to
hyperoxia caused significant time-dependent changes in PTHrP expression
(Table 2). Up to 50% of corner cells
were immunoreactive in control lungs and lungs from the 24/0 and 48/0
groups. The fraction of type II cells containing PTHrP reaction product
increased to 51-75% in the 60/0 groups (P < 0.05 vs. all other groups), even though lung PTHrP content measured by
immunoassay fell below control levels. All animals at this time point
demonstrated a qualitative increase in the intensity of PTHrP staining
(Fig. 3C). In the room-air recovery groups, the fraction of
corner cells containing PTHrP reaction product diminished to <25% of
cells in all animals (Fig. 3, D and E;
P < 0.05 for 48/72 group vs. normoxic control group).
The fraction of type II cells that was immunoreactive for PTHrP did not
change with time in control animals exposed to room air in the
environmental chamber (data not shown). Preadsorption of the antibody
with the antigenic peptide abolished staining (not shown).
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PTHrP in situ hybridization.
Reaction product for the digoxigenin-labeled PTHrP mRNA antisense probe
was found in corner cells of normoxic and 48/0 hyperoxic lung (Fig.
4). Expression of PTHrP mRNA appeared to
be greatly diminished in 60/0 lung. Reaction product was greatly
reduced when sections were hybridized with the sense probe compared
with the antisense probe. Results were verified in lung sections from three animals per group.
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BAL PTHrP levels. Because of the discordance between the changes in lung PTHrP content and lung cell PTHrP immunoreactivity at 60 h, we examined the distribution of PTHrP between lung and BAL in several rats. The percentage of total lung PTHrP found in BAL was 71 ± 4%, 71 ± 4%, and 50 ± 7% in age-matched control animals housed in the vivarium, room-air control animals housed in the chambers, and 48/0 hyperoxic animals, respectively (no significant difference). However, in 60/0 hyperoxic animals, BAL PTHrP accounted for only 9 ± 9% of the total (P < 0.05 vs. all other groups). Less than 0.1% of the total lung PTHrP was present in the BAL cell pellet at any time point. PTHrP was undetectable in the pleural fluid samples, all from animals in the 60/0 group.
Alveolar cell proliferation.
PCNA immunoreactivity was present in corner cells in rats from the
48/0, 60/0, and recovery groups but not at earlier time points (Fig.
5). The fraction of PCNA-positive corner
cells was significantly greater in the 60/0 group than in the other
groups (Table 3). The staining intensity
was qualitatively greatest in the 60/0 group as well (Fig.
5C). The data in Table 3 have been incorporated into Fig. 2
to demonstrate the temporal relationship between changes in lung
PTHrP expression and type II cell proliferation. The solid line beneath
the graph in Fig. 2 marks the period of type II cell proliferation. The
changes in PTHrP expression coincided with type II cell proliferation.
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Alveolar apoptosis.
TUNEL-positive corner cells were found in animals from the 60/0, 48/0,
48/24, 48/48, and 48/72 groups but were absent from the lungs of
control rats and rats in the 24/0 group (Fig.
6). The 60/0 group had a significantly
greater incidence of TUNEL-positive corner cells than the other groups.
The fraction of corner cells that were apoptotic was >25% in
three out of three animals in this group but <25% in all other
animals (P < 0.05).
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Hyperoxic Lung Injury in Adult Rabbits
Lung PTHrP.
PTHrP levels in adult rabbit lung varied significantly with exposure to
>95% oxygen and subsequent recovery in room air (Fig. 7). The highest levels, 3,289 ± 230 pg/g lung, occurred in the 64/24 group, which had 24 h of room-air
recovery. In the 64/48 group 24 h later, levels had dropped
significantly to 1,629 ± 153 pg/mg lung (P < 0.05). PTHrP levels in control lung were 2,289 ± 181 pg/g lung.
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Type II cell proliferation. No more than 2% of type II cells were in S phase in control rabbits and rabbits during hyperoxic exposure (Fig. 7, dashed line). The percentage increased to 36 ± 3% at 72 h of recovery (P < 0.05). The increase in S phase coincided with the onset of type II cell PCNA expression at 48 h of recovery (solid line beneath the graph), based on data from in previous studies (6, 16). The greatest percentage of type II cells in G2/M phase was found in the 64/96 group, 24 h after the peak in S phase cells. G2/M phase represented 9 ± 2, 4 ± 0.3, 7 ± 2, 16 ± 2,* and 4 ± 0.3% of type II cells in control, 64/0, 64/72, 64/96, and 64/200 rabbits (*P < 0.05 vs. other groups).
Hyperoxic Lung Injury in NB Rabbits
PTHrP in NB BAL.
BAL PTHrP concentrations in normoxic NB rabbits did not vary
significantly from 2 to 14 days and averaged 396 ± 29 pg/ml (Fig. 8). Exposure to hyperoxia had a
significant effect on BAL PTHrP concentrations (P < 0.01). PTHrP levels were increased ~150% compared with the
air-breathing control animals in rabbits breathing 100% oxygen until 8 days and animals breathing 60% oxygen until 14 days (P < 0.05). In contrast, NB rabbits maintained in 100% oxygen past 8 days to the LD50 point showed a significant decrease in BAL
PTHrP levels. PTHrP concentrations averaged 313 ± 53 pg/ml in
LD50 animals, ~50% lower than levels in the other
hyperoxic groups (P < 0.05). The lines beneath the
graph in Fig. 8 indicate the periods of type II cell proliferation in
this model as determined in a previous study (6). In
contrast to adult animals, NB rabbits demonstrate a moderate degree of
type II cell proliferation under control conditions. Animals breathing
>95% oxygen for 4 and 8 days show decreased type II cell
proliferation compared with normoxic control animals (dotted line).
Proliferation in LD50 rabbits and rabbits maintained in
60% oxygen from 8 until 14 days is greater than in the air-breathing
control animals (heavy line).
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PTHrP immunoreactivity in NB rabbits.
PTHrP immunoreactivity was present in bronchial cells, corner
alveolar cells (type II cells), and in noncorner alveolar cells (type I
cells) in both normoxic and hyperoxic groups (Fig.
9). The fraction of type II cells that
contained PTHrP reaction product was <25% in air-breathing NB rabbits
at all time points (Table 4). PTHrP
immunoreactivity was present in a significantly greater fraction of
type II cells in hyperoxic rabbits, as high as 75% in some rabbits) at
days 10 and 12 (P < 0.05). PTHrP
immunoreactivity in type I cells and bronchial cells was not
significantly affected by hyperoxia (data not shown).
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DISCUSSION |
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PTHrP Expression During Lung Injury in Adult Rats
The studies described in this paper addressed the hypothesis that lung PTHrP expression changes as a general response to lung injury of various etiologies and that the changes are temporally related to periods of type II cell proliferation. A previous study from our laboratory found that lung PTHrP levels rose above control levels during the first 2 days of exposure to 85% oxygen in adult rats (9). PTHrP levels fell significantly below control values between days 4 and 8, approximately the same time that type II cells were dividing. In the current studies, we found that exposing adult rats to >95% oxygen caused a similar pattern of PTHrP expression relative to type II cell proliferation. Lung PTHrP peaked at 48 h of exposure (48/0 group) and fell dramatically at 60 h. PTHrP mRNA was decreased at 60 h as well. Pneumocyte PCNA expression was greatest in the 60/0 group. Thus lung PTHrP expression changes with the same temporal pattern in both the >95% oxygen and the 85% oxygen injury models in rats. PTHrP levels increase early after injury and then decrease during the period when type II cells are proliferating.In addition to affecting overall levels of expression, lung injury altered the distribution of PTHrP between the air spaces and the lung parenchyma. Approximately 70% of lung PTHrP was present in BAL (representing PTHrP secreted into the air spaces) in control adult rats and in hyperoxic rats after 48 h of exposure or less. However, BAL PTHrP levels fell dramatically at 60 h, the point of greatest injury and highest PCNA expression. In addition, no PTHrP was detected in pleural fluid at this time point. Thus secretion of PTHrP appears to be inhibited by severe hyperoxic lung injury. BAL PTHrP levels also decreased with severe lung injury in NB rabbits. We did not measure air space PTHrP in the adult rabbit experiment. Changes in PTHrP secretion may be significant because PTHrP can exert its effects through multiple pathways. When it is secreted, PTHrP can exert paracrine or autocrine effects at cell surface receptors. If it is not secreted, PTHrP may be transported to the nucleus or nucleolus where it can exert effects on cell function, a process termed "intracrine" signaling (15, 19). The PTHrP molecule contains clusters of multibasic residues in the 87-106 region that are similar in configuration to the bipartite nuclear/nucleolar localization sequences in viral transcription factors and growth factors, such as human immunodeficiency virus-1 transactivator of transcription and fibroblast growth factor (FGF)-2. This sequence mediates localization of PTHrP to the nucleus in several cell types (15). Interestingly, the intracrine and paracrine effects of PTHrP on growth may oppose each other. Massfelder and coworkers (19) showed that treatment with exogenous PTHrP inhibited proliferation in vascular smooth muscle cells. In contrast, introducing PTHrP into the cells by gene transfer augmented cell growth. In our experiments, the decrease in secretion of PTHrP at the height of lung injury suggests that paracrine signaling may be greatly diminished while intracrine stimulation continues. Nuclear localization of PTHrP has been observed in type II cells (10, 13, 14) but has not been formally studied. The intracrine effects of PTHrP in lung cells are unknown.
This study also identified a temporal relationship between changes in PTHrP expression and onset of apoptosis in lung cells in adult rats. Type II cell TUNEL staining was minimal in hyperoxic rat lungs until 60 h of exposure, the point at which PTHrP protein and mRNA levels were lowest. PTHrP has a role in inhibiting apoptosis in chondrocytes and cerebellar neurons (3, 15, 21). PTHrP increases expression of Bcl-2 in growth plate chondrocytes, leading to a delay in their maturation and apoptotic death (3). Overexpression of PTHrP in transgenic mice causes marked delays in skeletal development, whereas mice homozygous for PTHrP gene ablation exhibit skeletal deformities due to accelerated chondrocyte differentiation (2, 3). If PTHrP also inhibits apoptosis in lung epithelial cells, apoptosis after lung injury could be regulated by a fall in lung PTHrP levels. Preliminary reports suggest that PTHrP treatment could reduce type II cell apoptosis after silica lung injury (11, 12), but a definitive study has not been completed.
PTHrP Expression During Lung Injury in Adult and Newborn Rabbits
Hyperoxic adult rabbits showed an increase in lung PTHrP, followed by a decrease, just as in the adult rats. In both species, the changes were temporally related to alveolar epithelial proliferation. In the rabbits, PTHrP decreased at 48 h of recovery and remained below peak levels at 72 h, in close proximity to the onset of type II cell division. Type II cells express PCNA at 48 and 72 h of recovery (6), and the fraction of pneumocytes in S phase increases in adult rabbits at 72 h of recovery (64/72 group). These results are consistent with PTHrP playing a role in regulating type II cell proliferation. However, causality between the changes in PTHrP and type II cell growth has not been demonstrated.Hyperoxic exposure in NB rabbits was associated with an initial increase in BAL PTHrP levels compared with air-breathing rabbits. BAL PTHrP levels were 50% greater in 4- and 8-day hyperoxic animals than in control animals. In previous studies, we observed decreased type II cell proliferation in hyperoxic animals at these time points compared with the normoxic controls (6). PTHrP levels decreased in NB rabbits taken to the LD50 point, just as they decreased in adult rats in the 60/0 group, the group with the most severe lung injury in that model. In both cases, the decrease in PTHrP level was associated with increased type II cell PCNA expression, consistent with our overall hypothesis that PTHrP inhibits type II cell growth. In our previous study with the NB rabbit model, we investigated changes in keratinocyte growth factor (KGF, FGF-7), a positive regulator of type II cell growth. The changes in KGF followed a pattern that was the mirror image found for PTHrP. KGF levels increased after 6-8 days of 100% oxygen, peaking when PTHrP levels were falling. KGF then fell dramatically after the rabbits are placed in 60% oxygen and at the LD50, when PTHrP levels were increased. The changes in expression of both PTHrP and KGF are consistent with a role in regulating pneumocyte proliferation after injury.
The demonstration that PTHrP is expressed in neonatal type I epithelial cells and Clara cells as well as type II cells was a new finding. PTHrP expression has not been observed in type I cells or Clara cells in adult rat lungs. The presence of PTHrP in other cell types in NB lung may be a residual of the distribution of PTHrP in the prenatal period. PTHrP immunoreactivity is present in bronchial epithelium at week 20 in human fetal lung and at day 18 in fetal rat lung (5, 20).
Critique of Methods and Results
Lung supernatant PTHrP levels fell after 60 h of hyperoxia whereas PTHrP immunoreactivity paradoxically increased. As discussed above, this finding might be produced by a decrease in total expression of PTHrP along with a decrease in secretion, resulting in less PTHrP in the lavage, less total PTHrP, but greater PTHrP immunoreactivity. The demonstration by in situ hybridization that PTHrP mRNA expression was downregulated at 60 h supports the assertion that PTHrP protein was also downregulated at this point. However, alternative explanations are possible. The fall in PTHrP levels measured by immunoassay could result from alterations in PTHrP binding rather than changes in total PTHrP levels, or substances present in injured lung might interfere with the immunoassay in vitro, spuriously reducing the apparent lung PTHrP levels. We investigated these possibilities by including lung homogenate in assays of known quantities of PTHrP standard in volumes equivalent to those present in assays of unknown samples. The measured PTHrP levels agreed with the predicted levels. If binding proteins or other interfering substances were present, the measured PTHrP levels would have been less than the true values. More importantly, the relationship between measured and true PTHrP concentrations was the same whether control lung homogenate, 48/0 lung homogenate, or 60/0 lung homogenate was added. Thus an artifact in the assay cannot explain the decreased PTHrP immunoassay levels in the 60/0 lungs compared with control or 48/0 lungs.We observed PCNA expression in rat type II cells after 48 and 60 h in >95% oxygen. Previous studies have found that total lung type II cell numbers do not change during exposure to near 100% oxygen. Pneumocyte numbers increase only after rats are returned to room air (7, 25). However, increases in type II cell numbers may lag behind the onset of proliferation, especially if proliferation is also accompanied by cell death. Tryka and coworkers (26) found that the labeling index for lung cells incorporating thymidine was increased sevenfold in rats after 48 h of hyperoxia compared with control animals. Bui and colleagues (4) have observed that the number of rat type II cells in S phase and G2/M phase increases with 48 h of exposure to 100% oxygen. These findings are consistent with our results and indicate that type II cell proliferation begins during the hyperoxic period. We used PCNA staining and TUNEL as measures of cell proliferation and apoptosis, respectively. However, concurrent high PCNA and TUNEL staining could alternatively be explained by ongoing repair of DNA damage. This study only examined the temporal relationship between changes in PTHrP and type II cell proliferation. Additional studies in which lung PTHrP levels are manipulated will be necessary to test causal relationships.
Summary
Similar patterns were observed for PTHrP expression in adult rat, adult rabbit, and NB rabbit models of hyperoxic lung injury. PTHrP initially increases in a period with little type II cell proliferation. Levels then decrease near the time in which type II pneumocytes are dividing. The pattern was similar whether the injury was induced by 85% or >95% oxygen, although the time frame varied. Newborn rabbits demonstrated a short lag time between fall in PTHrP levels and onset of type II cell proliferation. Differences in temporal pattern might be related to species differences, differences in the experimental design, and differences in patterns of expression of other growth factors. Severe lung injury was accompanied by inhibition of PTHrP secretion, a situation in which paracrine stimulation would be reduced but the intracrine effect of PTHrP might continue. The importance of nuclear localization in mediating the effects of PTHrP and the nature of intracrine effects in the lung are unknown. The results of this study and our previous investigations support the hypothesis that PTHrP regulates alveolar cell proliferation and alveolar repair after injury. Further work is necessary to test whether changes in lung PTHrP expression bear a causal relationship with type II cell proliferation and apoptosis during lung injury caused by >95% oxygen. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Rebeca Sandoval for performing the in situ hybridization.
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FOOTNOTES |
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This work was supported by Veterans Affairs Merit Review grants (R. H. Hastings and L. J. Deftos); National Institutes of Health Grants ES-09227 (R. H. Hastings), DK-60588 (L. J. Deftos), HL-03493 (C. T. D'Angio), and HL-56176 (B. A. Holm); by the American Heart Association (to C. T. D'Angio); and the March of Dimes (R. M. Ryan).
Address for reprint requests and other correspondence: R. H. Hastings, VA Medical Center (125), 3350 La Jolla Village Dr., San Diego, CA 92161-5085 (E-mail address: rhhastings{at}ucsd.edu).
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.
First published January 4, 2002;10.1152/ajplung.00139.2001
Received 20 April 2001; accepted in final form 21 December 2001.
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