1 Division of Neonatal Medicine, Department of Pediatrics, Neonatal-Perinatal Research Institute, and 3 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Duke University Medical Center, Durham 27710; and 2 Comprehensive Center for Inflammatory Disorders, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Inflammation may contribute to lung injury and impaired alveolar development in bronchopulmonary dysplasia. We treated hyperoxia-exposed newborn rats with antibodies to the neutrophil chemokine cytokine-induced neutrophil chemoattractant-1 (CINC-1) during 95% O2 exposure to reduce adverse effects of hyperoxia-induced inflammation on lung development. Rats were exposed at birth to air, 95% O2, or 95% O2 + anti-CINC-1 (injected on days 3 and 4). Bromodeoxyuridine (BrdU) was injected 6 h before death. Anti-CINC-1 treatment improved weight gain but not survival at day 8. Anti-CINC-1 reduced bronchoalveolar lavage neutrophils at day 8 to levels equal to air controls. Total detectable lung CINC-1 was reduced to air control levels. Lung compliance was improved by anti-CINC-1, achieving air control levels in the 10-µg anti-CINC-1 group. Anti-CINC-1 preserved proliferating cell nuclear antigen expression in airway epithelium despite 95% O2 exposure. BrdU incorporation was depressed by hyperoxia but preserved by anti-CINC-1 to levels similar to air control. Alveolar volume and surface density were decreased by hyperoxia but preserved by anti-CINC-1 to levels equal to air control. Blockade of neutrophil influx in newborns may avert early lung injury and avoid alveolar developmental arrest that contributes to bronchopulmonary dysplasia.
chemotactic factors; proliferation; bromodeoxyuridine; proliferating cell nuclear antigen
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INTRODUCTION |
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BRONCHOPULMONARY DYSPLASIA (BPD) is a frequent sequela of extreme prematurity, believed to be a consequence of surfactant deficiency, inadequate antioxidant defenses, and an inflammatory response to consequent lung injury (31).
Delayed or arrested alveolar development has been increasingly recognized as a central feature of modern-day BPD and may be a consequence of oxidant stress and cellular injury (24). Hyperoxic exposure leads to delayed alveolar development (42). The mechanism by which hyperoxia leads to abnormal alveolar development is unknown. Inflammatory responses to hyperoxia-induced lung injury may amplify alveolar epithelial injury. Death of type I pneumocytes and delay or arrest of repair because of hyperoxic effects on type II cell function may contribute to arrest of alveolar development.
Inflammation has long been recognized as an inciting or exacerbating factor in BPD (34). Elevations of proinflammatory cytokines are found in the tracheal aspirates of newborns who later develop BPD (27, 28). Clinical therapies aimed at blocking inflammation have consisted largely of glucocorticoids, which acutely decrease inflammatory markers and improve pulmonary function but do not provide lasting benefit. Instead, they have been implicated in adverse pulmonary development and impaired neurodevelopmental outcome (5, 23, 33).
To determine whether targeted blockade of inflammation would prevent adverse effects of hyperoxia on lung development, we chose specifically to block neutrophil influx with a neutralizing antibody directed against the major rat neutrophil chemokine cytokine-induced neutrophil chemoattractant-1 (CINC-1). Neutrophil accumulation in injured lung can propagate further injury because of elaboration of proteases, oxidant stress (32), and further chemokine release (38). Depleting neutrophils had an additive beneficial effect on airway injury, for example, in hyperoxia-exposed mice overexpressing extracellular superoxide dismutase (18). Strategies aimed at preventing neutrophil accumulation have been beneficial in some acute lung injury models (6, 22) and in selected clinical conditions, such as cardiopulmonary bypass (4, 19).
We previously reported that exposure to hyperoxia alone is sufficient to induce inflammation and accompanying neutrophil chemokine accumulation in neonatal rats (15). Neutrophil influx after 6 days of 95% O2 was ameliorated by treatment with neutralizing antibodies to the rat neutrophil CINC-1 and macrophage inflammatory protein-2. We performed additional studies to determine whether treatment with anti-CINC-1 would lead to improved pulmonary development in a rat model of neonatal BPD. Our findings demonstrate that treatment with anti-CINC-1 prevents airway neutrophil influx and preserves lung compliance, alveolar development, and lung cell proliferation in newborn rats exposed to 95% O2 for 8 days.
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METHODS |
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Hyperoxic Exposure
Newborn rats were exposed to 95% O2-5% air or air alone beginning on the day of birth, within 10 h of delivery, as previously described in detail by Bruce and colleagues (9). Rat pups from four litters were randomly sorted into four recombined litters and were exposed to 95% O2 or air in sealed Plexiglas cages measuring 60 × 45 × 25 cm, which we have previously described in detail (1). O2 was measured in each cage every 15 min with an analyzer (model 572; Servomex, Norwood, MA). CO2 was removed with a soda-lime trap. Humidity was routinely 70%. Nursing rat dams were switched between air and 95% O2 every 24 h. Exposure to ambient O2 was <10 min/day for weighing and cage cleaning. Litter size was adjusted to 10 pups using air-exposed controls throughout the exposure to control for effects of litter size on nutrition and growth. Two litters were exposed in each treatment condition. Recombined litters were assigned to a single treatment per litter per cage.On days 3 and 4, hyperoxia-exposed pups were injected intraperitoneally with purified goat anti-rat CINC-1 (R&D Systems, Minneapolis, MN) at 1, 5, or 10 µg (~0.2, 1, and 2 mg/kg body wt, respectively) or with 5 µg of nonimmune goat IgG (Sigma, St. Louis, MO). Air-exposed animals were not injected. Four animals in the air, 95% O2 + IgG, and 95% O2 + 10-µg anti-CINC-1 groups were injected 6 h before death with bromodeoxyuridine intraperitoneally to evaluate lung cell proliferation.
At 8 days, pups were killed with 150 mg/kg ip pentobarbital sodium, and a tracheal cannula was placed. An abdominal incision was made, and the diaphragm was punctured carefully to collapse the lungs. After midline thoracotomy, the pulmonary artery was cannulated with a 23-gauge needle, and the left atrial appendage was clipped. The lungs were gently perfused with 10 ml of 0.9% NaCl and 1 mM EDTA, pH 7.4, to remove blood. Bronchoalveolar lavage (BAL) was performed via the tracheal cannula using four exchanges of 1 ml of 0.15 M NaCl and 1 mM EDTA.
Measurements of Inflammation
BAL cell counts.
To determine whether anti-CINC-1 treatment on days 3 and
4 would lead to sustained reductions of inflammation, we
measured BAL cell counts. Lavage volumes were recorded, and cell counts were determined with a hemacytometer. Differential counts of 200 cells were obtained using Wright-Giemsa stain. Samples with gross hemorrhage and with <70% BAL recovery were not analyzed.
Neutrophil chemokine expression.
Effects of anti-CINC-1 treatment on days 3 and 4 on chemokine CINC-1 expression in BAL and whole lung homogenates at
day 8 of 95% O2 exposure were measured by
ELISA. CINC-1 measurements were performed on BAL supernatants and from
whole lung homogenates from animals that were perfused and lavaged. We
used a sandwich ELISA previously described (20) with the
following modifications. Plates were coated with affinity-purified
polyclonal goat anti-rat CINC-1 at 10 µg/ml (kind gift of Dr. John
Zagorski), and standard curves were prepared using rat CINC-1
(Chemicon, Temecula, CA). Biotinylated rabbit anti-rat CINC-1 was used
as the detection antibody (0.5 µg/ml; R&D Systems) and was detected
with avidin-horseradish peroxidase conjugate (Vector) and
o-phenylenediamine hydrochloride substrate at 490 nm, with a
background correction at 550 nm, in a microplate reader. Concentrations
were calculated using software supplied with the plate reader according
to the manufacturer's directions (BioTek, Winooski, VT). The usable
range of the assay was 25 pg/ml to 5 ng/ml. Standards were assayed in
triplicate, and samples were assayed in duplicate. Results for the
assay of standards or samples were accepted when the coefficient of
variation was 10%. CINC-1 measurements were normalized to whole
lung. Five animals per treatment group were analyzed.
Lung Mechanics
Five animals per treatment group were killed with pentobarbital sodium and had placement of tracheal cannulas but were not perfused or lavaged. After the diaphragm was punctured and the chest wall was removed, lungs were carefully inflated with a 1- or 3-ml syringe connected to a pressure transducer (Baxter Products, Round Lake, IL) and chart recorder (AstroMed, West Warlock, RI) to a peak pressure of 25 cmH2O three times. Deflation measurements of pressure and volume were made an additional three times. Static compliance was calculated at 20 cmH2O inflation pressure, a point between total lung capacity and middeflation. Data were normalized to lung weight. Means from each treatment group were compared by ANOVA.Histological Morphometric Evaluation
After 8 days, lungs from four nonperfused, nonlavaged animals in each group were inflation fixed via a tracheal cannula at 20 cmH2O for 30 min using 10% phosphate-buffered formalin. After overnight immersion in fixative, the lungs were embedded in paraffin, cut into 5-µm-thick sections, and stained with hematoxylin and eosin for light microscopy. Lungs were examined qualitatively, and a quantitative analysis was performed using light-microscopic morphometry according to methods described by Bolender et al. (8). A stratified random sample was obtained for morphometry by analyzing 3 of 10 sagittal sections obtained through the center of each tissue block. These sections contained tissue from at least three of the four lung lobes of each animal. Sections were viewed at ×400 magnification, and images were captured for counting with a Hitachi charge-coupled device video camera connected to a frame grabber. Stage micrometer images were obtained to calibrate the tissue images and the counting grid. Five fields on each section were chosen at random using a computer software random number generator (Microsoft Excel 5.0, Redwood, WA) to choose coordinates within the vertical and horizontal axes of the microscope stage. Images were overlaid with a 10 × 10 quadratic grid (Metamorph; Universal Imaging, West Chester, PA) for point (P) and intercept (I) counting of the alveolar septa. Large vascular and bronchial structures were omitted. Alveolar tissue volume density was calculated from Palveoli/Pparenchyma and alveolar surface density from 2Ialveoli/LT, where LT was the test line length within the lung parenchyma. Data from each group are expressed as means ± SE.Lung Cell Proliferation
DNA content. Lungs were weighed, flash-frozen under liquid nitrogen, and later homogenized and extracted in a phenol-based mixture according to the manufacturer's directions (TRIzol). DNA was quantitated by ultraviolet absorbance at 260 nm.
Proliferating cell nuclear antigen immunohistochemistry. Sections from each treatment group were studied to evaluate the effect of anti-chemokine treatment on lung cell proliferative state as determined by expression of the cell cyclin proliferating cell nuclear antigen (PCNA). Sections from 8-day air, 8-day 95% O2, 8-day 95% O2 + IgG, and 8-day 95% O2 + 1-, 5-, and 10-µg anti-CINC-1-exposed pups were used to immunolocalize PCNA expression. Sections were permeabilized by microwaving at 900 watts in 0.1 M sodium citrate for 10 min, blocked in 2% horse serum in PBS, incubated in 1:200 monoclonal mouse anti-PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), and detected with secondary biotinylated rabbit-anti-mouse antibody at 1:1,000. The avidin-biotin complex Elite kit was used to develop the chromophore using a peroxidase substrate yielding a purple precipitate and counterstained with methyl green according to the manufacturer's directions (VIP; Vector, Burlingame, CA). The details of the immunohistochemical procedures have been described previously (3). Sections were photographed with a Vanox-S AH-2 microscope (Olympus America, Melville, NY).
Bromodeoxuridine incorporation. Random sections from the bromodeoxyuridine (BrdU)-injected 95% O2 + IgG, 95% O2 + 10-µg anti-CINC-1, and air treatment groups were detected. Sections were incubated with 1:500 biotinylated mouse monoclonal anti-BrdU and detected according to the manufacturer's directions, as previously described (Zymed, South San Francisco, CA; see Ref. 40). Detection was performed as described above. Five random fields from two random sections from each of four animals per treatment group were examined and calculated, with a minimum of 500 cells/animal counted. Mean labeling indexes (labeled cells/total cells) for each treatment group were compared.
Statistical Analysis
Grouped data are expressed as means ± SE except where noted. Between-group analysis was tested using single-factor ANOVA. Post hoc comparisons were made using Newman-Keuls analysis. Results were computed using statistical software (JMP; SAS, Cary, NC). ![]() |
RESULTS |
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Survival and Weight Gain
Survival was 97% in the air-exposed group. Survival was decreased in all O2-exposed groups as follows: 95% O2 + IgG = 64%; 95% O2 anti-CINC-1: 1 µg = 69%, 5 µg = 76%, 10 µg = 62%. Animals exposed to 95% O2 were smaller at 8 days than air-exposed animals, but 95% O2 + 10-µg anti-CINC-treated animals were larger than 95% O2 goat IgG-treated animals at 8 days (Fig. 1).
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Inflammatory Markers: BAL Neutrophils, Chemokine Expression
Anti-CINC-1 treatment on days 3 and 4 markedly reduced BAL neutrophil accumulation in 95% O2-exposed rat pups on day 8 in a dose-response pattern, reducing neutrophils in the highest dose treatment group to levels comparable with air-exposed controls (Fig. 2). Hyperoxia induced CINC-1 accumulation in BAL, but anti-CINC-1 treatment did not affect BAL CINC-1 levels at day 8 (Fig. 3). Two BAL samples were not analyzed because of gross hemorrhage. This occurred in one of the control 95% O2 + IgG animals and in one of the 1-µg anti-CINC-1-treated animals. CINC-1 accumulation in whole lung homogenates from the 95% O2 + goat anti-CINC-1 treatment groups was reduced to air-exposed control levels at all doses (Fig. 3). We found varying amounts of detectable goat IgG by ELISA in lung homogenates from each injected treatment group in day 8 pups (0.1-5 ng/lung, representing a IgG-to-CINC molar ratio
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Static Lung Compliance
To determine whether blocking neutrophil influx would lead to functional lung improvements, we measured static compliance on day 8 in all treatment groups (n = 5/group). Anti-CINC-1 treatment in 95% O2-treated animals significantly improved lung compliance in a dose-dependent manner compared with 95% O2 + IgG controls (Fig. 4), parallel to its effects on BAL neutrophils (Fig. 1). Anti-CINC-1 (10 µg) treatment at days 3 and 4 preserved static lung compliance in 95% O2 8-day exposed rats at levels comparable to air-exposed controls.
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Lung Cell Proliferation
DNA accumulation. Air-exposed animals had a higher DNA content per milligram lung than did O2-exposed animals. Anti-CINC-1 had no statistically significant effect on DNA concentration (mean ± SE, µg DNA/mg wet lung) among the O2-treated groups at any dose as follows: 95% O2 + IgG = 3.0 ± 0.3; O2 + anti-CINC: 1 µg = 3.0 ± 0.4, 5 µg = 2.8 ± 0.2, and 10 µg = 2.5 ± 0.2. There was no effect of anti-CINC-1 on DNA content per whole lung after normalization to lung weight (data not shown).
PCNA expression.
The 95% O2 + vehicle exposure markedly depressed PCNA
expression in all cell types compared with air-exposed controls at 8 days, as shown in representative photomicrographs in Fig.
5. In 95% O2 + 10-µg
anti-CINC-treated animals, PCNA expression was preserved in part in
parenchymal cells and bronchiolar epithelium. PCNA expression was most
prevalent at the periphery of the lung and in conducting airways in
both air and 95% O2 + anti-CINC-1 treatment groups.
Loss of PCNA signal in the 95% O2-treated group was
particularly evident in peripheral parenchymal cells. Anti-CINC-1 treatment appeared to preserve alveolar epithelial PCNA expression, acknowledging the limits of light microscopy at this magnification. Double labeling with alveolar epithelial markers would confirm our
identification of alveolar epithelium.
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BrdU incorporation.
Effects of hyperoxia and hyperoxia + anti-CINC-1 on the BrdU
labeling index were parallel to the effects on PCNA. The 95% O2 exposure depressed BrdU incorporation at 8 days, as
estimated by the labeling index, compared with air-exposed animals
(Fig. 6). In contrast, 95%
O2 + 10-µg anti-CINC-1-treated animals demonstrated a labeling index comparable to air controls and 95%
O2-exposed animals. The spatial pattern of
hyperoxia-suppressed BrdU labeling and its preservation by anti-CINC-1
was similar to the pattern of effects on PCNA expression.
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Effects of Anti-CINC-1 on Alveolar Development: Histopathology
Histology.
Representative photomicrographs demonstrate loss of alveolar
septation and dilated terminal airspaces in the 95%
O2 + IgG control group, with preservation of alveolar
size and complexity in the 95% O2 + anti-CINC groups
(Fig. 7). Leukocyte
infiltration was present on the 95% O2 + IgG control.
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DISCUSSION |
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Our results demonstrate that targeted anti-neutrophil chemokine treatment can partly preserve alveolar development, maintain normal lung compliance, improve weight gain, and suppress airway inflammation in 95% O2-exposed newborn rats in the short term. The effects on alveolar development were mediated in part by preservation of epithelial cell proliferation as measured by BrdU incorporation. There were no effects of anti-CINC-1 treatment on mortality. Weight gain in the surviving high-dose anti-CINC-1-treated animals was significantly improved.
Anti-CINC-1 treatment did not increase overall DNA per lung in the 95% O2 groups. This may have been because of the variability of inflammatory influx and adherence of inflammatory cells, which might affect total lung DNA. Decreased DNA lung content resulting from hyperoxic effects on growth may have been offset by DNA attributable to inflammatory influx, with the reverse process expected with anti-inflammatory treatment. Prolonged studies over 2-3 wk with recovery at lower inspired O2 concentrations might be expected to permit resolution of acute inflammation and recovery of lung cell proliferation sufficient to evaluate the effects of anti-CINC-1 treatment on lung growth. Edema from 95% O2-induced injury would also be expected to affect the DNA/lung concentration, but we did not observe any effects of anti-CINC treatment on DNA accumulation when DNA concentration was normalized to whole lung (data not shown).
We used nonimmune IgG as a control to account for effects of injection and potential nonspecific effects of IgG on inflammation. Although some have reported that IgG can affect lung cell proliferation in vitro, similar hyperoxic exposures in vivo showed that IgG had no effect on proliferation as measured by [3H]thymidine incorporation (10).
Because inflammation exacerbates many types of lung injury
(39), we blocked neutrophil influx with neutralizing
antibodies to the chemokine CINC-1, since neutrophil-derived oxidant
stress and proteolytic damage would be expected to significantly
contribute to the pathophysiology of BPD (25). Neutrophil
chemoattraction is mediated by several pathways, including the
interleukin-8 receptors C-X-C-receptor (CXCR) 1 and 2, the formyl
peptide receptor, the C5a receptor, and the leukotriene B4
receptor (39). Our previous study determined that
blocking neutrophil chemokines alone was sufficient to reduce
inflammation in hyperoxia-exposed newborn rats at day 6 (15). Although we did not test whether other
chemoattractants were elevated in our model, anti-CINC-1 treatment was
sufficient to substantially reduce inflammation as has been
demonstrated in other acute lung injury studies (41). We
therefore concluded that the CINC-1CXCR2 pathway of neutrophil
chemotaxis is predominant in this model of neonatal hyperoxia-induced
lung injury.
The anti-inflammatory effects of the anti-chemokine treatment appear to
be mediated in part by persistent depression of CINC-1 levels in whole
lung on day 8 after anti-chemokine treatment on days 3 and 4. We found persistent goat IgG in
the lungs of nonimmune IgG and goat anti-rat CINC-1-injected animals.
The apparently reduced CINC-1 levels that we observed in whole
lung at 8 days may therefore be a result of detection interference from
residual injected anti-CINC-1 IgG. Reductions in bioavailable/bioactive CINC-1 (whether by decreased production or by neutralization by anti-CINC-1) would presumably have the same effect on chemokinesis. Alternatively, neutrophils and macrophages are sources of chemokines (36), and preventing their influx in lung parenchyma may
interrupt the cycle of injuryinflammation
injury, which may in
turn result in reduced lung chemokine expression.
In contrast, anti-CINC-1 treatment at 3 and 4 days did not reduce CINC-1 in BAL from 8-day 95% O2-treated animals. Decreases in lung parenchymal CINC-1 may precede the eventual depletion of CINC-1 from the airway compartment accessible to BAL, although this was not tested directly.
The most striking finding was the significant preservation of alveolar development as reflected by improved lung compliance (Fig. 4), alveolar volume density, and alveolar surface density (Fig. 8). These effects suggest that the compliance improvements we observed with anti-CINC-1 treatment can be attributed in part to preserved alveoli. Edema resulting from 95% O2-induced injury might be expected to increase alveolar septal volume and hence alveolar volume density, resulting in overestimated alveolar development in the 95% O2 + IgG treatment group. This would make the anti-CINC-1-associated improvements in volume density we observed even more significant. The improvements in BrdU incorporation and PCNA expression reflect preserved alveolar proliferative capacity and preserved alveolar proliferation and suggest that the improvements in lung volume, alveolar volume density, and alveolar surface density are due in part to preserved lung growth. Increased BrdU uptake and PCNA expression may also represent DNA repair, which is likely taking place in response to inflammation and hyperoxia-induced injury (26). Quantitative lung cell counts, e.g., number of type II epithelial cells, would help to distinguish whether the effects on alveolar development were predominantly due to preserving normal proliferation or avoiding cell death.
The mechanisms by which oxidant stress or inflammation impair neonatal alveolar development are not thoroughly understood. Hyperoxia has been demonstrated to arrest alveolar development in a number of neonatal animal models (2, 14, 35, 42). Whether this effect is predominantly a failure of cell division or increased cell death, perhaps because of apoptosis in type II alveolar cells, is not known (11). Both cell culture and in vivo experiments have demonstrated depressed cell proliferation factors in response to hyperoxia, with recovery of expression of cell cyclins A and D (PCNA) after recovery from hyperoxia (12). Treatment with antioxidants reversed the hyperoxia-associated effects on PCNA expression (29). Our anti-neutrophil chemokine treatment was directed at reducing neutrophil accumulation and may have reduced neutrophil-borne oxidant stress, thereby indirectly ameliorating oxidative or nitrosative effects on cell proliferation (26, 37).
We chose a neonatal animal model that incorporated some of the important features of lung injury associated with and implicated in the pathophysiology of clinical and experimental BPD, namely acute oxidant stress-associated inflammatory influx associated with alveolar growth arrest (24). Mechanisms of inflammatory effects on alveolar development have not been established. Studies using neonatal animal models of BPD have described the predominance of neutrophilic alveolitis during the first days of exposure to supplemental O2 and mechanical ventilation (2, 14, 42). Because we found improvement in alveolar development as a consequence of targeted anti-neutrophil chemokine treatment, we speculated that inflammation in addition to hyperoxia may play a role in the arrest of alveolar development in BPD.
The effects we observed on lung development using a specific anti-inflammatory approach contrasts with the effects of postnatal glucocorticoid treatment (7) or combined glucocorticoid treatment and hyperoxic exposure, which have shown impaired alveolar development (13). It is likely that these effects are attributable to the well-known effects of glucocorticoids on cell progression and division (16). Clinical trials of glucocorticoid treatment to prevent BPD have aimed at lower doses and inhaled agents and have been successful at reducing side effects of systemic steroids but have not succeeded in reducing acute inflammation (21).
Specific, targeted immunomodulatory therapy may provide a better strategy than glucocorticoids. Direct effects of inflammatory cells in the neonatal lung may cause alveolar cellular damage, leading to death, and thereby impair alveolar development. A number of studies have implicated hyperoxia (11, 30) and inflammation (17) in promoting apoptosis, for example. We did not study cellular loss or death in this injury model. The improvement we observed in alveolar development may have been a combination of preserved alveolar cellular growth and decreased cell death.
In summary, anti-CINC-1 blockade of neutrophil influx accompanying hyperoxia in a neonatal rat model of BPD preserves normal lung compliance, normal alveolar surface, and volume density by preventing the hyperoxia-induced arrest of lung cell proliferation. If these findings are relevant in human newborns, then we speculate that brief, specific blockade of neutrophil influx in newborns might avert the early lung injury and attenuate alveolar developmental arrest that contribute to chronic lung disease of prematurity. Evaluation of this strategy and other anti-neutrophil approaches in longer-term studies of lung development after recovery from early hyperoxia-induced injury will help to determine whether this approach can lead to permanent benefits.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical support of Carol Torres and the careful review of the manuscript by Drs. Claude Piantadosi and Steve Young.
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FOOTNOTES |
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This work was supported by grants from the North Carolina American Lung Association and the Children's Miracle Network and National Institute of Dental and Craniofacial Research Grant 1-P60-DE-13079.
Address for reprint requests and other correspondence: R. L. Auten, DUMC Box 3179, Durham, NC 27710 (E-mail: auten{at}duke.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.
Received 2 October 2000; accepted in final form 26 February 2001.
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