Published ahead of print on September 3, 2004, doi:10.1164/rccm.200402-215OC
© 2004 American Thoracic Society doi: 10.1164/rccm.200402-215OC
Opposing Effects of 60% Oxygen and Neutrophil Influx on Alveologenesis in the Neonatal RatCanadian Institutes of Health Research (CIHR) Group in Lung Development, Lung Biology Programme, Hospital for Sick Children Research Institute and Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada; Departments of Pediatrics, Medicine, and Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah; Neonatal Perinatal Research Institute, Division of Neonatal Medicine, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina Correspondence and requests for reprints should be addressed to Dr. A. Keith Tanswell, Division of Neonatology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: keith.tanswell{at}sickkids.ca
The lungs of newborn rats exposed to 60% oxygen for 14 days develop an injury that shares morphologic similarities to human bronchopulmonary dysplasia (BPD). Neutrophil influx into the lung, as part of an inflammatory response, may play a pivotal role in the development of BPD. A neutrophil chemokine, cytokine-induced neutrophil chemoattractant-1, which signals through the neutrophil CXC chemokine receptor-2, is increased in the lung tissue of newborn rats exposed to 60% oxygen. The purpose of this study was to explore the role of neutrophils in the rat model of BPD by inhibiting neutrophil influx using SB265610, a selective CXC chemokine receptor-2 antagonist. SB265610, administered to 60% oxygen–exposed newborn rats from birth to 14 days, completely inhibited neutrophil influx. It also attenuated increased production of reactive oxygen species in newborn rat lung tissue after exposure to 60% oxygen for 4 days. Lung morphometric analysis revealed that 60% oxygen for 14 days, when accompanied by treatment with SB265610 to prevent neutrophil accumulation, increased alveolar formation over that seen in newborn rats exposed to air. These data suggest that exposure of the neonatal lung to moderate hyperoxia may enhance postnatal lung growth, provided postnatal pulmonary inflammation is suppressed.
Key Words: bronchopulmonary dysplasia • CXC chemokine receptor-2 • inflammatory response • lung injury • oxygen toxicity Bronchopulmonary dysplasia (BPD) is a major cause of pulmonary morbidity after premature birth (1). The traditional view has been that BPD is of multifactorial origin with significant contributions from oxygen- and ventilation-mediated injuries. Although the pathogenesis of BPD is incompletely understood, prolonged exposure to sublethal hyperoxia in animal models recapitulates some of the processes observed during the development of BPD (2, 3). Recent studies in premature baboons (4) leave little doubt that oxidant injury alone can produce the pathologic features of BPD as originally described (5). It is not clear whether the major oxidant stress is derived from excess reactive oxygen species formed in constitutive lung cells under hyperoxic conditions, or from phagocytes invading the lung as part of the inflammatory process induced by hyperoxia (6).
Neutrophils are believed to play an important role in exacerbating the inflammatory process of chronic lung disease in premature infants (7). A persistent increase in the number of neutrophils is found both in the interstitium and in the airspaces of the lungs of infants with BPD (8). Activated neutrophils undergo a respiratory burst, generating various reactive oxygen species (9). These include superoxide anion, which can spontaneously or enzymatically dismute to form hydrogen peroxide, which can then react with superoxide to form the hydroxyl radical. These reactive oxygen species oxidize active Cytokine-induced neutrophil chemoattractant-1 is a CXC chemokine that is a functional homolog of human CXC chemokines, and shares the ability to signal through the CXC receptor-2 (13–16). Neutrophil infiltration is due, in part, to the interaction of CXC chemokines with their major receptor, CXC receptor-2. Blockade of this receptor using SB265610, a selective antagonist, was effective in preventing pulmonary neutrophil influx in newborn rats exposed to 95% oxygen (16). Unlike newborn rats exposed to 95% O2, in which there is a simple homogeneous arrest of lung growth and DNA synthesis (17), newborn rats exposed to 60% oxygen for 14 days develop a heterogeneous parenchymal lung injury with areas of arrested alveolarization and growth mixed with areas of interstitial thickening with active DNA synthesis (18). Unlike the situation of global arrest of lung growth with 95% oxygen, there is no experimental evidence to support or refute a critical role for neutrophils in the heterogeneous changes observed in lung tissue after exposure to 60% oxygen, which could occur through a completely neutrophil-independent mechanism such as dysregulation of growth factor expression. A significant macrophage influx after 7 days of exposure to 60% oxygen is associated with the development of pulmonary hypertension (19). We hypothesized that an early neutrophil influx, in response to an exposure to 60% oxygen, would contribute to the development of the heterogeneous parenchymal tissue injury. As described below, SB265610 was effective in inhibiting activated neutrophil influx and reduced superoxide formation in lung tissue exposed to 60% oxygen. Quantitative assessment of postnatal alveolar development revealed an hitherto unsuspected permissive role for 60% oxygen, in lungs in which activated neutrophil influx had been inhibited by treatment with SB265610. Some of the results of these studies have been previously reported in the form of an abstract (20).
In Vivo Interventions Animal studies were conducted according to criteria established by the Canadian Council for Animal Care. Rat pups were maintained in paired chambers (air or 60% oxygen) for 4-, 7-, 10-, or 14-day exposure periods as previously described (18, 19). Lung macrophage influx was inhibited with 10 mg/kg gadolinium chloride on Day 1 of life only (19). For inhibition of neutrophil influx, paired litters received either 0.1% dimethyl sulfoxide in saline (vehicle control) or SB-265610 (provided by Dr. Skip Sarau, GlaxoSmithKline, King of Prussia, PA) in 0.1% dimethyl sulfoxide in saline (2 µg/g and 5 µl/g [16]) by daily intraperitoneal injection into the right iliac fossa via a 30-gauge needle.
Lung Homogenate Assays
Immunohistochemistry
Morphometric Analyses
Data Presentation
Whole lung content of the neutrophil chemoattractant CINC-1 significantly increased (p < 0.05) in newborn rats exposed to 60% oxygen for 7 days, compared with air-exposed controls (Figure 1). By 14 days, there was no statistical difference in lung content of CINC-1 between the air-exposed and the 60% oxygen–exposed groups (p > 0.05). Immunohistochemistry for neutrophils demonstrated an increase in lung neutrophil content on exposure to 60% oxygen for 4 days, which was inhibited by treatment with SB265610, the CXCR2 receptor antagonist (Figure 2). Similar findings were evident at Days 7 and 14 (Figures E2 and E3). Myeloperoxidase activity, a marker of neutrophil recruitment and degranulation (30) used to quantitate the neutrophil influx, was significantly increased (p < 0.05) in the lungs of 60% oxygen–exposed pups at Day 4 (Figure 3A), Day 7 (Figure 3B), and Day 14 (Figure 3C), compared with air-exposed control animals, and was attenuated by daily intraperitoneal injections of SB265610.
Cyanide-resistant oxygen consumption, a measure of tissue superoxide production, was calculated from the slope of the polarograph output. The steepest slopes of the polarograph output, before and after the addition of potassium cyanide, were observed in 60% oxygen–exposed and vehicle-treated animals, compared with 60% oxygen–exposed and SB265610-treated animals or air-exposed and SB265610-treated animals, which had similar slopes (Figure E1). As shown in Figure 4A, administration of SB265610 for 4 days completely attenuated the increase in cyanide-resistant oxygen consumption observed in tissue from 60% oxygen–exposed and vehicle-treated animals relative to air-exposed and vehicle-treated animals (p < 0.05). The addition of 100 µM SB265610 (16) to lung homogenates from Day 4 vehicle-treated animals had no independent effect on cyanide-resistant oxygen consumption (data not shown). Treatment of animals with SB265610 alone for 7 days only partially inhibited the increase in cyanide-resistant oxygen consumption mediated by 60% oxygen exposure (Figure 4B), which remained significantly increased (p < 0.05). However, administration of SB265610 for 7 days, together with gadolinium chloride administered on the day of birth to prevent macrophage influx, completely attenuated the increase in cyanide-resistant oxygen consumption observed in 60% oxygen–exposed and vehicle-treated animals (Figure 4C).
Vehicle-treated animals exposed to 60% oxygen for 14 days had significantly decreased (p < 0.05) post-fixation whole lung volumes when compared with the 60% oxygen–exposed and SB265610-treated group or either air-exposed group (Table E1). The normal morphology of the lung in air-exposed and vehicle-treated pups (Figure 5, A14 vehicle) was unaffected by treatment with SB265610 (Figure 5, A14 SB265610). As we have previously described (18), exposure to 60% oxygen for 14 days induced an heterogeneous change in lung morphology with patchy areas of parenchymal thickening and small airspaces interspersed with areas of enlarged airspaces (Figure 5, O14 vehicle). Not only were these heterogeneous changes not present in animals that had been both exposed to 60% oxygen and treated with SB265610, but the size of the distal airspaces appeared reduced (Figure 5, O14 SB265610) compared with those of air-exposed animals. Consistent with these images, the fractional area occupied by tissue was significantly (p < 0.05) increased after exposure to 60% oxygen for 14 days, when compared with air-exposed control pups (Figure 6A). This, and the reduced postinflation lung volumes following exposure to 60% oxygen, are consistent with previously reported abnormal volume–pressure loops in such animals (18). After treatment with SB265610, the increase in tissue fraction in 60% oxygen– relative to air-exposed control pups (Figure 6A) was no longer statistically significant (p > 0.05), and any residual increase might reflect what appeared to be a reduction in alveolar size. Results of a quantitative assessment of alveolar development, using mean linear intercepts, are shown in Figure 6B. There was no significant effect of 60% oxygen alone on the average mean linear intercept, but there was a significant effect (p < 0.05) on the variance of mean linear intercept, reflecting the heterogeneous effect on development. Animals exposed to 60% oxygen and treated with SB265610 for 14 days had a significantly reduced variance of mean linear intercept and overall mean linear intercept (p < 0.05), when compared with animals exposed to 60% oxygen and treated with vehicle, consistent with a reduction in airspace diameter and enhanced alveolar formation. Consistent with the results for mean linear intercept, alveolar surface area per unit lung volume was significantly increased (p < 0.05), when compared with animals exposed to 60% oxygen and treated with vehicle (Figure 6C), consistent with enhanced alveolar formation. The apparent reduction in mean linear intercept and increase in alveolar surface area per unit lung volume, in the lungs of animals exposed to 60% oxygen and treated with SB265610 relative to those exposed to air, could not be attributed to artifact induced by poor inflation, as shown by postinflation lung volumes (Table E1). Immunohistochemistry for lung elastin was used to highlight secondary crest formation, the initial step in the development of alveoli from saccules. Secondary crests were densely stained for elastin at their tips (29). Long thin secondary crests were evident in air-exposed Day 14 rats that had received vehicle or SB265610 (Figures 7a and 7b). In lungs from 60% oxygen–exposed and vehicle-treated pups, the secondary crests in areas with large distal airspaces were shortened and blunted (Figure 7c). Treatment with SB256510 appeared to completely reverse this morphologic change, with restoration of long thin secondary crests (Figure 7d).
To further quantitatively assess alveolar development, we calculated the secondary crest number/field and the secondary crest/tissue ratio in elastin-stained lung sections from rats that had been exposed to air or 60% oxygen for 14 days and had received either vehicle or SB265610. As shown in Figure 8A, there was a significant increase in the secondary crest number/field in the lungs of 60% oxygen–exposed and SB265610-treated animals (p < 0.05), when compared with air-exposed or 60% oxygen–exposed and vehicle-treated animals. Similarly (Figure 8B), there was a significant increase in the secondary crest/tissue ratio in the lungs of 60% oxygen–exposed and SB265610-treated animals (p < 0.05), when compared with air-exposed or 60% oxygen–exposed and vehicle-treated animals. To determine if similar changes could be detected at an earlier time point, when interstitial thickening is not grossly obvious, sections were examined from pups that had been similarly exposed, but for only 7 days. These slides were additionally stained for immunoreactive proliferating cell nuclear antigen, as a marker of cell DNA synthesis. As shown in Figures 9A and 9B, there were significant increases in the proliferating cell nuclear antigen–positive and total secondary crest/tissue ratios in the lungs of 60% oxygen–exposed and SB265610-treated animals (p < 0.05), when compared with air-exposed or 60% oxygen–exposed and vehicle-treated animals.
In the rat, the formation of alveoli from saccules occurs postnatally, by a process in which saccules are subdivided into smaller compartments by the outgrowth and elongation of secondary septa from the primary septa. The most rapid rate of alveolar formation occurs between Days 3 and 8 of life (31) and is largely completed by Day 14 (32). Newborn rats exposed to 60% oxygen for 14 days develop an heterogeneous lung injury with morphologic similarities to human infants with BPD, including an arrest of DNA synthesis in those parts of the lung having enlarged distal airspaces (18), consistent with an arrest of alveologenesis. We have previously demonstrated that one component of the inflammatory response induced by 60% oxygen, a macrophage influx during the latter part of the 2-week exposure period, was responsible for the pulmonary hypertension seen in this model (19). Furthermore, inhibition of the macrophage influx during exposure to 60% oxygen did not prevent the parenchymal injury (19). Neonatal rats exposed to 95% oxygen have a different pattern of lung injury to that seen with 60% oxygen, in that they have an homogeneous arrest of lung cell DNA synthesis (33), with arrested alveologenesis (34). This arrest of alveologenesis could be prevented by treatment of 95% oxygen-exposed animals with an anti–CXC receptor-2 chemokine intervention (34). As described above, we have been able to demonstrate that newborn rats exposed to 60% oxygen, in common with those exposed to 95% oxygen, have an increased expression of the neutrophil chemokine CINC-1 and an early and sustained influx of neutrophils. The influx of neutrophils preceded the observed 60% oxygen-induced increase in CINC-1 concentration. However, we did not test all potential ligands for the CXC receptor-2, such as CINC-2  , CINC-2ß, or MIP-2, an increase of any one of which could have preceded the increase in CINC-1. The source of CINC-1 is unclear, in that the neutrophil influx appears to precede that of alveolar macrophages, a major source of secreted cytokines, in this model (35). Increased CINC-1 expression was induced by 95% oxygen in newborn rat lung epithelium, as well as in alveolar macrophages (21). Furthermore, the observation that freshly isolated rat type II pneumocytes express ELR-positive chemokines and CXC receptor-2 at much higher levels than do freshly isolated alveolar macrophages (36) suggests that the distal lung epithelium may be the source. We adopted the previously described strategy of using a nonpeptide CXC receptor-2 antagonist to block neutrophil influx into the lung, to determine if this intervention could prevent the heterogeneous parenchymal changes observed with a 60% oxygen exposure. The efficacy of this intervention in preventing activated neutrophil influx was confirmed by immunohistochemistry and myeloperoxidase assay for neutrophil content, and by reduced superoxide production, as assessed by tissue cyanide–resistant oxygen consumption. Because our measurements of tissue cyanide–resistant oxygen consumption were performed after homogenates were equilibrated with air, they do not reflect the increase in superoxide production in constitutive lung cells brought about by an increase in tissue oxygen concentration. Our current, and previous (35), observations suggest that the increased superoxide production evident in equilibrated homogenates from 60% oxygen–exposed lung tissue can be accounted for largely by macrophages at Day 14, by neutrophils at Day 4, and by a combination of both neutrophils and macrophages at Day 7. The tissue fraction of lungs from pups exposed to 60% oxygen was increased relative to that of air-exposed pups, consistent with the appearance of the tissue under the microscope. However, our morphometric analyses, using mean linear intercept, alveolar volume, and secondary crest density, did not demonstrate "whole lung" differences between air- and 60% oxygen–exposed lung due to the heterogeneous nature of the lung injury induced by 60% O2. That classic morphometric techniques may not always be effective in quantitating structural changes that are quite obvious to the eye is well recognized (37). Indeed, the lack of any effect of 60% oxygen on secondary crest/tissue ratio, despite an increase in tissue fraction but no change in secondary crests/field, provides a seeming paradox. One explanation might be an increase in secondary crest volume, rather than number, equivalent in degree to the increase in tissue fraction induced by 60% oxygen.
We did observe that blocking the neutrophil influx with the CXCR2 antagonist, SB265610, resulted in enhanced alveolar formation: airspace size variance was reduced, mean linear intercept was decreased and secondary crest formation was enhanced. We interpret these findings to suggest that the heterogeneous parenchymal lung injury observed in 60% oxygen–exposed neonatal rats reflects a balanced mixture of stimulation of alveologenesis by 60% oxygen and an indirect inhibition of alveologenesis by a secondary neutrophil influx. That alveologenesis is inhibited by a 60% oxygen–mediated secondary neutrophil influx is entirely consistent with previous observations in neonatal rats exposed to 95% oxygen (34). That inhibition of neutrophil influx resulted in enhanced alveologenesis in the 60% oxygen–exposed neonatal rat lung, and only preserved alveologenesis in the 95% oxygen–exposed neonatal rat lung, presumably reflects the decreased amount of reactive oxygen species generated at the lower oxygen concentration. How the adverse effect of a neutrophil influx on alveologenesis is mediated is unclear. It may be through an excess production of oxidants (4), over and above that produced by constitutive lung cells under hyperoxic conditions, or through the release of their granule-associated proteins such as myeloperoxidase (38). In addition to myeloperoxidase, neutrophils release numerous other enzymes from azurophilic granules, including hydrolases, lysozyme, and neutral serine proteases. Among the neutral serine proteases, elastase represents quantitatively the major granule component (11, 12) and is the only neutrophil protease capable of degrading elastin (30). The major elastase inhibitor is The most intriguing of our findings was the enhanced alveolar development, as determined by secondary crest formation, evident in 60% oxygen–exposed neonatal rats that had the lung neutrophil influx suppressed. That moderate hyperoxia can stimulate lung parenchymal cell DNA synthesis is well recognized in the adult rat exposed to 85% oxygen (43), but has not, to our knowledge, been recognized to do so in neonatal rats. As we have previously reported, increased lung cell DNA synthesis in the adult rat exposed to 85% oxygen is associated with the up-regulation of a number of growth factors and growth factor receptors (44–46). We have also observed an increase in the expression of the insulin-like growth factor-1 receptor (18) and the platelet-derived growth factor ß-receptor (47) in those areas of the 60% oxygen–exposed neonatal rat lung in which growth was preserved, making these receptors and their ligands likely candidates to mediate, at least in part, the alveologenesis-stimulating effect of 60% oxygen over a 2-week exposure. Alternatively, as reactive oxygen species are important intracellular signaling molecules downstream from binding of many ligands, including growth factors, to their receptors (48, 49) increased intracellular reactive oxygen species concentrations may increase DNA synthesis without changes in expression of growth factors or their receptors. Whatever the mechanism, the observation that moderate hyperoxia can stimulate alveolar formation when inflammation is suppressed has significant clinical and therapeutic implications.
A.K.T. holds the Hospital for Sick Children Women's Auxiliary Chair in Neonatal Medicine. M.P. holds a Tier-1 Canada Research Chair from the Canadian Institutes of Health Research. R.P.J. is supported by Postdoctoral Research Awards from the Canadian Institutes of Health Research and the Canadian Lung Association and a Clinician-Scientist Training Fellowship from the Hospital for Sick Children Research Institute. R.L.A. is supported by the National Institutes of Health HL-067021.
Supported by a Group Grant from the Canadian Institutes of Health Research (A.K.T., M.P.), NIH HL 067,021 (R.L.A.), and HL 62,875 (K.H.A.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Conflict of Interest Statement: M.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.P.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; I.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; N.B.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.H.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.L.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.K.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form February 19, 2004; accepted in final form August 29, 2004
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