Main

Chronic neonatal lung injury, or BPD, remains an important cause of morbidity and mortality in preterm infants requiring respiratory support with mechanical ventilation and prolonged use of supplemental O2(1–3). Despite well-recognized changes in the usual histopathological features of BPD over the last three decades (4), infants with early respiratory failure (5) or established BPD (6) remain at increased risk of developing pulmonary hypertension, which is a recognized predictor of subsequent morbidity and mortality (7–9). Although the pathologic changes in the pulmonary vasculature of infants with severe BPD are well characterized (10, 11), the causes of aberrant pulmonary vascular development seen in BPD are poorly understood. Current evidence implicates ROS, generated in excess of antioxidant defenses, in the pathogenesis of BPD (12). However, it is unclear whether ROS contribute specifically to the development of pulmonary hypertension.

Pulmonary macrophages are known to produce many vasoactive, mitogenic, and proinflammatory cytokines that have been implicated in tissue injury (13–15). The macrophage content in the lung is normally negligible in utero, though it increases slightly toward term with further increases in the early postnatal period (16). Interstitial and alveolar macrophages are seen in exaggerated numbers in preterm infants who are destined to develop BPD (17). They are also the predominant inflammatory cell type in infants with established BPD (18). Macrophages isolated from the airways of infants with BPD demonstrate an increase in ROS production relative to macrophages from infants without BPD (18).

Normal pulmonary arterial development in the postnatal rat lung is very similar to that seen in the human (19, 20), making the newborn rat a useful model for the study of pathologic changes in the pulmonary vasculature. We have described a newborn rat model with BPD-like histopathology (21) and pulmonary hypertension (22) induced by exposure to 60% O2 for 14 d. Pathologic changes in the distal lung include both areas of parenchymal thickening and areas with an emphysema-like appearance. This appearance is consistent with failure of septation from inhibition of peripheral cell growth (21, 23), as described for human infants (4). Manifestations of pulmonary hypertension in this model, which include RVH and pulmonary arterial smooth muscle hyperplasia, were reversed by treatment with a 21-aminosteroid antioxidant, U74389G (24). The U74389G-mediated effect on pulmonary hypertension seemed to be secondary to inhibition of an 8-isoprostane-induced up-regulation of ET-1 expression by 60% O2(24). As described below, we subsequently observed that 60% O2-exposed animals treated with U74389G had an attenuated macrophage influx into the lung. This led us to hypothesize that vasoactive compounds such as ET-1 (14), derived directly or indirectly from alveolar macrophages, may play a causative role in the pulmonary vascular changes observed in this model. To test this hypothesis, we examined the modifying effect of GdCl3 on lung macrophage content after exposure to 60% O2. GdCl3 is a rare earth lanthanide that has been used to abrogate macrophage migration and activation in vivo to delineate their role in disease. In animals, it has been reported to be effective in preventing pulmonary injury induced by ozone inhalation (25) and ovine lentivirus infection (26).

METHODS

Materials.

GdCl3. 6H2O and BSA type V were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). U74389G and CS-4 vehicle (20 mM citric acid monohydrate, 3.2 mM sodium citrate dihydrate, 77 mM NaCl, pH 3.0) were provided by Dr. D. Zimmerman (Pharmacia & Upjohn, Kalamazoo, MI, U.S.A.). SB217242 was kindly provided by Dr. Douglas Hay (SmithKline Beecham Pharmaceuticals, King of Prussia, PA, U.S.A.). Organic solvents were of HPLC grade. Peroxynitrite and a rabbit polyclonal antibody to nitrotyrosine were from Upstate Biotechnology (Lake Placid, NY, U.S.A.). Rabbit polyclonal antibody to human ET-1 was purchased from Chemicon (Temecula, CA, U.S.A.). Goat anti-rabbit IgG-peroxidase antibody was from Boehringer Mannheim (Mannheim, Germany) and goat anti-mouse IgG-peroxidase was from Calbiochem (La Jolla, CA, U.S.A). Goat anti-rabbit and goat anti-mouse IgG-biotin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Mouse MAb to rat TPRM-2 macrophage protein, a cytoplasmic protein specific to rat macrophages, was obtained from BMA (Augst, Switzerland). Mouse MAb to α-smooth muscle actin was from Neomarkers (Fremont, CA, U.S.A.). Avidin-biotin-peroxidase and alkaline phosphatase complex immunohistochemistry kits were purchased from Vector Laboratories (Burlingame, CA, U.S.A.). A total nitric oxide colorimetric assay kit was from R&D Systems (Minneapolis, MN, U.S.A.). ET-1 and 8-isoprostane enzyme immunoassay kits were from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.), a total protein assay kit was from BioRad (Hercules, CA, U.S.A.), and Sep Pak C18 cartridges were from Waters (Mississauga, ON, Canada). [3H]Prostaglandin F2α was purchased from Dupont NEN Products (Boston, MA, U.S.A.). Gels and membranes were from Novex (San Diego, CA, U.S.A.). Cell culture media, antibiotics, and trypsin were from GIBCO (Burlington, ON, Canada). Gelfoam sponges were from Pharmacia & Upjohn (Toronto, ON, Canada). FBS was from Flow Laboratories (McLean, VA, U.S.A.), and collagenase and DNase were from Worthington (Freehold, NJ, U.S.A.).

Institutional review.

All procedures involving animals were conducted according to criteria established by the Canadian Council for Animal Care. Approval for the study was obtained from the Animal Care Review Committee of the Samuel Lunenfeld Research Institute, Mount Sinai Hospital.

Exposure system.

The exposure system has been described in detail previously (21, 23, 24). Briefly, pathogen-free, timed pregnant Sprague Dawley rats (250–275 g) were obtained from Charles River (St. Constant, QC, Canada). Experiments were conducted as paired exposures, with one chamber receiving 60% O2 and the other receiving air. On the anticipated day of delivery, each dam was placed in a 60 × 48 × 25-cm plastic chamber with 12 h/12 h light-dark cycles, with temperature maintained at 25 ± 1°C, minimal humidity, and a CO2 concentration of <0.5%. Equal litter sizes (10–12 pups) were maintained between paired chambers. Food and water were available ad libitum. Dams were exchanged daily between chambers to prevent maternal O2 toxicity. At the termination of each exposure period, animals were killed by ether inhalation.

Interventions.

Pups were maintained in four paired chambers (air and O2) for a 14-d exposure period. Injections (5 μL/g body weight) were given i.p. via a 30-gauge needle into the right iliac fossa, as previously described for delivery of liposomes, drugs, and antibodies to the lungs of neonatal rats (23, 24, 27, 28). For the intervention with GdCl3, each pair received either 0.9% NaCl (vehicle control) or GdCl3. 6H2O in 0.9% NaCl (2 mg/mL and 10 mg/kg) within 6 h of birth and on day 7. For the intervention with U74389G, a 21-aminosteroid antioxidant, each pair received either CS-4 (vehicle control) or U74389G in CS-4 (2 mg/mL and 10 mg/kg) within 6 h of birth and daily thereafter as previously described (24). For the intervention with SB217242, a mixed endothelin receptor antagonist (29), each pair received either 0.9% NaCl (vehicle control) or SB217242 (1 mg/mL and 5 mg/kg) within 6 h of birth and daily thereafter.

Examination of RVH.

RVH is a well-established index of pulmonary hypertension (30), and has been shown to have a direct correlation with vascular smooth muscle hyperplasia in this model (22). At sacrifice, the thoracic contents were removed en bloc. The heart was then separated from the lungs and the right ventricle was dissected free from the left ventricle with the septum. Each component was freeze-dried and weighed separately.

Immunohistochemistry.

Animals were anesthetized with i.p. ketamine (80 mg/kg) and xylazine (20 mg/kg). The pulmonary circulation was flushed with PBS containing 1 U/mL heparin to clear the lungs of blood, and perfusion fixed with 4% (wt/vol) paraformaldehyde while a constant airway pressure of 10 cm H2O was maintained via a tracheal catheter. Before immunohistochemical staining, lung sections were examined to confirm a normal lung structure from air-exposed control pups, and the presence of patchy areas of interstitial thickening and emphysema in 60% O2-exposed pups (21). When lung sections were examined for the presence of macrophages, dilutions of the primary and secondary antisera were 1:100 and 1:250, respectively. Interstitial and alveolar macrophage numbers were quantified by counting of positively stained cells per high-power field (four animals per group and four fields per animal). For α-smooth muscle actin, dilutions of the primary and secondary antisera were 1:1200 and 1:300, respectively. For ET-1, dilutions of the primary and secondary antisera were 1:300 and 1:200, respectively. For the detection of nitrotyrosine residues, dilutions of both primary and secondary antibodies were 1:300. Several methods were used as controls for the specificity of nitrotyrosine immunostaining as described in detail by Viera et al.(31). Briefly, these included blocking the primary antiserum with 10 mM nitrotyrosine and reduction of nitrotyrosine in situ to aminotyrosine. A positive control for nitrotyrosine immunostaining was generated by addition of 100 mM peroxynitrite to tissue sections. For all other antibodies, antibody specificity was verified by omitting the primary antiserum. After completion of immunohistochemical studies, using an avidin-biotin-peroxidase complex method (32), slides were counterstained with Carazzi hematoxylin, dehydrated, cleared in xylene, and mounted.

Total (free and esterified) 8-isoprostane measurement.

To prevent auto-oxidation, lung tissue or cell culture medium was immediately flash frozen with liquid N2 and stored at −80°C until analysis. Upon thawing, 0.005% (wt/vol) butylated hydroxytoluene was added to cell culture medium or tissue homogenized in 1 × PBS. [3H]Prostaglandin F2α (5000 cpm) was added to quantify recovery after purification. Proteins were precipitated by ethanol and removed by centrifugation. The supernatant was incubated with an equal volume of 15% (wt/vol) potassium hydroxide at 40°C for a 1-h alkaline hydrolysis of esterified lipid before solid phase extraction using Sep Pak C18 cartridges as previously described (28). After purification, samples were analyzed in duplicate for 8-isoprostane content using a commercially available enzyme immunoassay kit. Recovery from the purification step was analyzed by liquid scintillation counting of the extract and values expressed as picograms per milligram protein for tissue or picograms per milliliter for cell culture medium.

Lung volume-pressure loops.

Animals were anesthetized with sodium pentobarbitone (5–10 mg/kg) and paralyzed with pancuronium bromide (0.3 mg/kg). After tracheotomy, the lungs were degassed and volume-pressure loops in the open chest recorded as previously described (21).

Protein analysis.

Western blot analysis on lung tissue was performed using protein from lung homogenates. Samples and recombinant protein standards were fractionated by SDS PAGE for 2 h at 120 V. For α-smooth muscle actin (43 kD), 20 μg per lane of protein was fractionated under reducing conditions on 8% to 16% (wt/vol) graded tris-glycine gels and transferred to PVDF membranes. For ET-1 (2.5 kD), 40 μg per lane of protein was fractionated under nonreducing conditions on 16% (wt/vol) tricine gels and transferred to PVDF membranes. Membranes were blocked with 3% nonfat milk for >1 h, washed in TBS (Tris base 20 mM, NaCl 137 mM, pH 7.6) with 0.1% Tween 20, incubated with appropriate primary antibodies for >1 h followed by further washing and secondary antibody for >1 h. For α-smooth muscle actin, dilutions of primary and secondary antibodies were 1:5000 and 1:2800, respectively. For ET-1, dilutions of primary and secondary antibodies were 1:1000 and 1:20,000, respectively. The protein bands were imaged using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, U.S.A) and exposed for 30–120 s on Kodak X-Omat Blue XB-1 film (Eastman Kodak, Rochester, NY, U.S.A.). The films were electronically scanned and the band densities were quantified using Scion Image software (Version 1.6, National Institutes of Health, Bethesda, MD, U.S.A.). Equal protein loading was confirmed by Coomassie blue staining of all gels.

Strain of fetal lung cells in organotypic culture.

Primary mixed lung cell cultures were prepared from d-19 rat fetuses as previously described (33). Briefly, after removal of the heart, major vessels, and airways, the combined lungs were minced with scissors. The tissue was then subjected to sequential enzymatic dissociation, initially using 1 mg/mL trypsin and 0.01 mg/mL DNase. Disaggregated fetal lung cells reaggregate within Gelfoam sponges to form highly organized alveolar-like structures (34). Cells were cultured on sponges as described previously (35). Briefly, cells were inoculated on 2 × 2 × 0.25-cm Gelfoam sponges at a density of 10 × 106 cells per sponge and incubated for 72 h in DMEM with 10% (vol/vol) FBS at a gas phase of 21% O2, 5% CO2, and 74% N2 at 37°C. The organotypic cell cultures were then subjected to mechanical strain for comparison with unstrained control cultures. Sponges were placed in DMEM or DMEM with GdCl3 (0.1, 1, or 10 μM). The mechanical strain device has been described in detail elsewhere (35). Briefly, the unit consisted of a programmable burst timer, a control unit, a regulated DC power supply, and a set of solenoids. A culture dish with a Gelfoam sponge was placed in front of each solenoid. One end of each sponge was fixed to the dish and the other end was attached to a movable metal bar. The movement of the metal bar and sponge was driven by the magnetic force and the recoil property of the sponge. The whole solenoid strain unit was placed in an incubator at a gas phase of 21% O2, 5% CO2, and 74% N2 at 37°C. As previously described for optimal DNA synthesis (35), intermittent strain was at 60 cycles/min with 15-min strain/h and a 5% elongation of the sponge for 24 h.

Total nitric oxide assay.

Culture medium in which Gelfoam sponges had been immersed was analyzed for total nitrite (NO2−) as a quantitative measure of nitric oxide production using a commercially available colorimetric assay. This assay is based on the enzymatic conversion of nitrate [NO3− to NO2− by nitrate reductase followed by measurement of NO2− by the Griess reaction (36)]. The detection limit of the assay is ≥1.35 nmol/mL.

ET-1 measurement.

Unpurified culture medium was analyzed for ET-1 content using a commercially available enzyme immunoassay kit with a detection limit of ≥32 pg/mL.

Data presentation.

Unless otherwise stated, all values are for the mean ± SEM of four litters. Statistical significance (p < 0.05) was determined by ANOVA followed by assessment of differences using Duncan's multiple range test (37). Error bars are not evident in figures where they fall within the plot point.

RESULTS

Immunohistochemical staining of lung sections for macrophage TPRM-2 antigen are shown in Figures 1 and 2. Compared with air controls (Fig. 1A), pulmonary accumulation of macrophages was increased after 60% O2 exposure for 14 d (Fig. 1B). Air-exposed animals treated with the 21-aminosteroid, U74389G (Fig. 1C), had similar macrophage content to vehicle-treated controls. This 60% O2-mediated increase was completely abrogated by treatment with U74389G (Fig. 1D).

Figure 1
figure 1

Lung macrophage content and the effect of treatment with U74389G. Immunohistochemistry for macrophage TPRM-2 antigen (brown stain) in lung tissue after exposure to air or 60% O2 for 14 d. Newborn rats received daily i.p. injections of U74389G (10 mg/kg) in CS-4 vehicle or CS-4 vehicle alone. Bar length = 250 μm. (A) Interstitial macrophages were evident in air-exposed pups that received vehicle. (B) The macrophage content of the lungs of 60% O2-exposed pups that received vehicle was markedly increased. (C) The lungs of air-exposed pups that received U74389G. (D) The 60% O2-exposed pups that received U74389G had a similar macrophage content to air-exposed control animals.

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Figure 2
figure 2

Lung macrophage content and the effect of treatment with GdCl3. Immunohistochemistry for macrophage TPRM-2 cytoplasmic antigen (brown stain) in lung tissue after exposure to air or 60% O2 for 7 d. Newborn rats received i.p. injections on d 0 and d 7 of GdCl3 (10 mg/kg) in 0.9% saline vehicle or 0.9% saline vehicle alone. Bar length = 250 μm. (A) Interstitial macrophages were evident in air-exposed pups that received vehicle. (B) The macrophage content of the lungs of 60% O2-exposed pups that received vehicle was markedly increased. (C) The lungs of air-exposed pups that received GdCl3 had a slight reduction in macrophage content. (D) The 60% O2-exposed pups that received GdCl3 had a similar macrophage content to air-exposed control animals. (E) The lungs of pups on d 0, before GdCl3 injection, had a negligible macrophage content. (F) Control slides, from which the primary antiserum was omitted, showed no staining.

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As shown in Figure 2, compared with air controls at 7 d (Fig. 2A), pulmonary accumulation of macrophages was markedly increased (29.25 ± 0.5 macrophages per high-power field versus 10.25 ± 2 in air-exposed vehicle-treated animals;p < 0.05) after exposure to 60% O2 for 7 d (Fig. 2B). Treatment with GdCl3 (Fig. 2, C and D) markedly reduced the macrophage content of O2-exposed lungs (Fig. 2D; 10.75 ± 1.2 macrophages per high-power field versus 29.25 ± 0.5 in vehicle-treated animals;p < 0.05) to levels comparable to air controls (Fig. 2A), in keeping with the known inhibitory effect of GdCl3 on macrophage chemotaxis and survival. In contrast to air-exposed animals at 7 d (Fig. 2A), pulmonary macrophages were not evident on the day of birth (Fig. 2E). A negative control in which the primary antibody was omitted is shown in Figure 2F.

Vehicle-treated 60% O2-exposed animals had significant RVH (p < 0.05), as assessed by the ratio of the right ventricle to the left ventricle and septum dry weights, compared with both vehicle- and GdCl3-treated air-exposed control animals (Fig. 3). This 60% O2-induced RVH, an index of pulmonary hypertension, was completely attenuated (p < 0.05) in animals treated with GdCl3. Smooth muscle mass was assessed by α-smooth muscle actin immunohistochemistry (Fig. 4) and Western blot analysis (Fig. 5). Compared with air-exposed controls (Fig. 4A), vehicle-treated 60% O2-exposed animals showed a marked increase in immunoreactive arterial smooth muscle (Fig. 4B), and an increase in total lung α-smooth muscle actin content (Fig. 5;p < 0.05). Animals treated with GdCl3 (Fig. 4, C and D) did not have evidence of these O2-induced smooth muscle changes (Fig. 4D and Fig. 5;p < 0.05).

Figure 3
figure 3

RVH. Right ventricular (RV) dry weight compared with that of the combined left ventricle and septum (LV+S), as an index of RVH, after exposure to air or 60% O2 for 14 d. Newborn rats received i.p. injections on d 0 and d 7 of GdCl3 (10 mg/kg) in 0.9% saline vehicle (closed circles) or 0.9% saline vehicle alone (open circles). The injection of GdCl3 attenuated the 60% O2-mediated increase in RVH. Plot points represent mean ± SEM for four litters. *p < 0.05, by one-way ANOVA, for vehicle-treated animals exposed to 60% O2 compared with those in air. #p < 0.05, by one-way ANOVA, for GdCl3-treated animals compared with vehicle-treated animals exposed to 60% O2.

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Figure 4
figure 4

Effect of GdCl3 on 60% O2-mediated increased pulmonary vascular smooth muscle mass. Immunohistochemistry for α-smooth muscle actin (brown stain) in medium-sized pulmonary vessels (v) after exposure to air or 60% O2 for 14 d. Newborn rats received i.p. injections on d 0 and d 7 of GdCl3 (10 mg/kg) in 0.9% saline vehicle or 0.9% saline vehicle alone. Bar length = 250 μm. (A) Smooth muscle was evident around vessels of air-exposed pups that received vehicle. (B) A large increase in smooth muscle was evident around the vessels of 60% O2-exposed pups that received vehicle. Inset: negative control where the primary antiserum was omitted. (C) The smooth muscle around vessels in the lungs of air-exposed pups that received GdCl3 did not appear different from air-exposed control animals. (D) The lungs of 60% O2-exposed pups that received GdCl3 did not show an increase in perivascular smooth muscle.

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Figure 5
figure 5

Effect of GdCl3 on α-smooth muscle actin content of whole lung. (A) Lung α-smooth muscle actin protein increased after exposure to 60% O2 for 14 d relative to animals exposed to air. Newborn rats received i.p. injections on d 0 and d 7 of GdCl3 (10 mg/kg) in 0.9% saline vehicle (closed circles) or 0.9% saline vehicle alone (open circles). GdCl3 prevented the 60% O2-mediated increase in α-smooth muscle actin. Plot points represent mean ± SEM for 3 litters. *p < 0.05, by one-way ANOVA, for animals exposed to 60% O2 compared with those in air in the same treatment group. #p < 0.05, by one-way ANOVA, for GdCl3-treated animals compared with vehicle-treated animals exposed to 60% O2. (B) Examples of Western blots for α-smooth muscle actin in whole lung after exposure to air or 60% O2 for 14 d. Newborn rats received i.p. injections of either GdCl3 (GADOLINIUM) or vehicle alone (VEHICLE). Protein size is in kilodaltons.

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Nitrotyrosine formation in lung tissue, a marker of protein nitration by reactive nitrogen species such as peroxynitrite, was assessed by immunohistochemistry (Fig. 6). When compared with air-exposed controls (Fig. 6A), vehicle-treated animals exposed to 60% O2 for 7 d (Fig. 6B) showed abundant nitrotyrosine formation. Treatment with GdCl3 (Fig. 6, C and D) prevented this O2-mediated increase in nitrotyrosine (Fig. 6D). Positive (addition of 100 mM peroxynitrite;Fig. 6E) and negative (omission of primary antibody;Fig. 6F) controls are shown.

Figure 6
figure 6

Immunohistochemistry for nitrotyrosine (brown stain), a marker of protein nitration, in lung tissue after exposure to air or 60% O2 for 7 d. On the day of birth, rat pups received a single i.p. injection of GdCl3 (10 mg/kg) in 0.9% saline or 0.9% saline alone. Bar length = 250 μm. (A) Air-exposed pups that received vehicle had negligible nitrotyrosine staining. (B) The 60% O2-exposed pups that received vehicle had abundant nitrotyrosine formation. (C) Lung tissue from air-exposed pups that received GdCl3 had a similar appearance to air-exposed control pups. (D) The 60% O2-exposed pups that received GdCl3 had a marked reduction in nitrotyrosine staining. (E) Positive control staining produced by the addition of 100 mM peroxynitrite. (F) Negative control staining in which the primary antiserum was blocked with 10 mM nitrotyrosine.

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As previously described (21), the lungs of rat pups exposed to 60% O2 for 14 d have areas of apparent emphysema interspersed with patchy areas of parenchymal thickening and, consistent with these changes, abnormal lung mechanics. When compared with these previously reported changes (21), we could not detect any obvious effect of treatment with GdCl3 on either macroscopic structural changes (data not shown) or on lung volume-pressure loops (Fig. 7, A and B).

Figure 7
figure 7

Lung mechanics. The effect of air (open circles) or 60% O2 (closed circles) on lung volume-pressure loops at d 14 in newborn rats that received 0.9% NaCl vehicle (A) or GdCl3 (10 mg/kg) in 0.9% NaCl vehicle (B) on d 0 and d 7 by i.p. injection. Plot points represent mean ± SEM for four or five pups. *p < 0.05, by one-way ANOVA, for O2-exposed animals compared with air controls for the same pressure.

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The effect of treatment with GdCl3 on possible mediators of O2-induced pulmonary hypertension was also examined. ET-1 expression was studied by Western blot analysis (Fig. 8), whereas 8-isoprostane (Fig. 9) was quantified by enzyme immunoassay. Exposure of vehicle-treated animals to 60% O2 for 14 d led to an increase in ET-1 in lung tissue (Fig. 8). This 60% O2-mediated increase in ET-1 was prevented (p < 0.05) by treatment with GdCl3 (Fig. 8). There seemed to be a small effect of treatment with GdCl3 on ET-1 expression in the lung tissue of air-exposed control animals, but this was not statistically significant (p > 0.05). Similarly, the 60% O2-mediated increase in lung 8-isoprostane content was completely attenuated (p < 0.05) by GdCl3 (Fig. 9).

Figure 8
figure 8

Effect of GdCl3 on ET-1 content in whole lung. (A) ET-1 expression increased after exposure to 60% O2 for 14 d relative to values for air-exposed animals. Newborn rats received i.p. injections on d 0 and d 7 of GdCl3 (10 mg/kg) in 0.9% saline vehicle (closed circles) or 0.9% saline vehicle alone (open circles). Treatment with GdCl3 attenuated the 60% O2-dependent increase in ET-1 expression. Plot points represent mean ± SEM for three litters. *p < 0.05, by one-way ANOVA, for animals exposed to 60% O2 compared with those in air in the same treatment group. #p < 0.05, by one-way ANOVA, for GdCl3-treated animals compared with vehicle-treated animals exposed to 60% O2. (B) Example of Western blot analysis for ET-1 in whole lung after exposure to air or 60% O2 for 14 d. Newborn rats received i.p. injections of either GdCl3 (GADOLINIUM) or vehicle alone (VEHICLE). Protein size is in kilodaltons.

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Figure 9
figure 9

Effect of GdCl3 on total (free and esterified) 8-isoprostane in whole lung after exposure to air or 60% O2 for 14 d. Newborn rats received i.p. injections on d 0 and d 7 of GdCl3 (10 mg/kg) in 0.9% saline vehicle (closed circles) or 0.9% saline vehicle alone (open circles). Treatment with GdCl3 attenuated the 60% O2-dependent increase in 8-isoprostane. Plot points represent mean ± SEM for four litters. *p < 0.05, by one-way ANOVA, for animals exposed to 60% O2 compared with those in air in the same treatment group. #p < 0.05, by one-way ANOVA, for GdCl3-treated animals compared with vehicle-treated animals exposed to 60% O2.

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Because any effects of GdCl3 could be mediated through inhibition of stretch-activated cation channels, in addition to inhibition of macrophage accumulation and activation, we examined the effect of GdCl3 on organotypic cultures of rat fetal lung cells exposed to mechanical strain. After 24 h of mechanical strain, ET-1 was significantly (p < 0.05) increased in cell culture medium (660 ± 93 pg/mL versus 303 ± 58 pg/mL in unstrained control cultures;n = 4). GdCl3 (0.1–10 μM) had no effect (p > 0.05) on this stretch-mediated increase. Mechanical strain also had no effect (p > 0.05) on 8-isoprostane or nitrite content in cell culture medium (data not shown).

Neonatal pups treated with a mixed ET-1 receptor antagonist, SB217242, did not develop 60% O2-mediated RVH (Fig. 10). Perivascular smooth muscle was assessed by α-smooth muscle actin immunohistochemistry (Fig. 11). Compared with vehicle-treated air-exposed controls (Fig. 11A), 60% O2-exposed vehicle-treated animals had a marked increase in perivascular smooth muscle mass (Fig. 11B). Treatment with SB217242 (Fig. 11, C and D) prevented this O2-mediated increase in smooth muscle mass (Fig. 11D). SB217242 had no impact on 60% O2-mediated lung structural changes (data not shown). These findings confirm that the increase in ET-1 observed with exposure to 60% O2 was causally related to the development of pulmonary hypertension.

Figure 10
figure 10

Effect of the mixed endothelin receptor antagonist, SB217242, on 60% O2-mediated RVH. Right ventricular (RV) dry weight compared with that of the combined left ventricle and septum (LV+S), as an index of RVH, after exposure to air or 60% O2 for 14 d. Newborn rats received daily i.p. injections of SB217242 (5 mg/kg) in 0.9% saline vehicle (closed circles) or 0.9% saline vehicle alone (open circles). The injection of SB217242 attenuated the 60% O2-mediated increase in RVH. Plot points represent mean ± SEM for four litters. *p < 0.05, by one-way ANOVA, for vehicle-treated animals exposed to 60% O2 compared with those in air. #p < 0.05, by one-way ANOVA, for SB217242-treated animals compared with vehicle-treated animals exposed to 60% O2.

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Figure 11
figure 11

Effect of SB217242 on 60% O2-mediated increased pulmonary vascular smooth muscle mass. Immunohistochemistry for α-smooth muscle actin (brown stain) in medium-sized pulmonary vessels (v) after exposure to air or 60% O2 for 14 d. Newborn rats received daily i.p. injections of SB217242 (5 mg/kg) in 0.9% saline vehicle or 0.9% saline vehicle alone. Bar length = 100 μm. (A) Smooth muscle was evident around vessels of air-exposed pups that received vehicle. (B) A large increase in smooth muscle was evident around the vessels of 60% O2-exposed pups that received vehicle. (C) The smooth muscle around vessels in the lungs of air-exposed pups that received SB217242 did not appear different from air-exposed control animals. (D) The lungs of 60% O2-exposed pups that received SB217242 did not show an increase in perivascular smooth muscle.

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DISCUSSION

Macrophages are differentiated mononuclear phagocytes that may reside in tissues for several months. They are essential for tissue remodeling and wound healing, and congregate during subacute or chronic inflammation. In human preterm infants with respiratory distress, pulmonary macrophage numbers increase early in the second week of life (17), remain elevated in infants who later develop clinical and radiologic features of BPD, and decline in those who recover (18). In adult animals, pulmonary macrophages are present in exaggerated numbers in pulmonary hypertension induced by hypobaric hypoxia (38). Pulmonary macrophages are central to the pathogenesis of pulmonary hypertension induced by monocrotaline injection in rats (39–41) and in antiplatelet serum-induced pulmonary hypertension in sheep (42). As described above, we found pulmonary interstitial macrophages to be greatly increased after exposure to 60% O2. Inhibition of this increase in lung macrophage content and secondary up-regulation of ET-1 prevented RVH and the concomitant increase in smooth muscle cell mass but not other morphologic aspects of lung tissue injury or abnormal lung mechanics.

Abnormalities in vascular tone and smooth muscle mass underlie the pathophysiology of pulmonary hypertension in BPD, although the cellular mechanisms are not fully understood. The pathogenesis of BPD is clearly multifactorial, but it is widely believed that oxidant injury plays a critical role (12). ROS can be generated from multiple sources during hyperoxia. Intracellular production is greatly increased (43, 44), and extracellular formation may be induced by activation of polymorphonuclear leukocytes and macrophages (13, 15). These cells produce other mediators implicated in lung injury, such as ET-1 (14), 8-isoprostane (24), and various growth factors (13).

GdCl3 abrogates macrophage accumulation by induction of apoptosis after phagocytosis (45), but has little effect on polymorphonuclear leukocytes. We found that a commonly used dose of GdCl3 abrogated 60% O2-induced macrophage accumulation in newborn rats. GdCl3 also inhibited O2-mediated nitrotyrosine formation in the lung. Another known effect of GdCl3 is the blockade of stretch-mediated (mechanogated) cation channels, which are involved in cell proliferation and altered gene expression (46). We attempted to elucidate the mechanism of GdCl3 action in our in vivo model by subjecting fetal cells in organotypic culture to mechanical strain. We have previously demonstrated that 10 μM GdCl3 inhibits strain-induced DNA synthesis in this in vitro model (47). Our findings in vitro suggest that the observed effects of GdCl3in vivo were the result of inhibition of lung macrophage content rather than a blockade of mechanogated ion channels.

Cytokines, many of which are produced by macrophages, act as modulators of cell proliferation in many diseases that share common pathogenic elements. Pulmonary pathologies that are likely to be mediated by abnormal cytokine expression include pulmonary hypertension and pulmonary fibrosis, both of which are components of BPD. As described above, we found that ET-1 and 8-isoprostane are increased during O2 exposure and are abrogated by inhibition of macrophage accumulation. This indicates that macrophages are directly or indirectly involved in the up-regulation of these factors. ET-1 and 8-isoprostane elicit a number of biologic effects in the lung, including smooth muscle cell contraction and proliferation (48, 49) and fibroblast chemotaxis and proliferation (50). Macrophages can synthesize ET-1 (14), or be primed by ET-1 to produce increased quantities of ROS (51). Moreover, the production of ET-1 may be greatly augmented by 8-isoprostane in the lung (24) and other organs (49, 52). Our previous studies (24) demonstrated a 60% O2-mediated, and 8-isoprostane-induced, up-regulation of ET-1 in association with the development of pulmonary hypertension. As described herein, a causal relationship between increased ET-1 expression and the development of pulmonary hypertension has been confirmed by the use of a mixed endothelin (ETA/ETB) receptor antagoniSt. Whether macrophages are the primary source of the ET-1 and 8-isoprostane generated during O2 exposure, or whether a macrophage product enhances their formation by another cell type, requires further elucidation. Whatever the source of ET-1 and 8-isoprostane, it is clear that their enhanced production, and the resultant pulmonary hypertension, after exposure to 60% O2 are macrophage dependent.

Taken together, the findings reported above suggest that pulmonary macrophages are important in the pathogenesis of O2-mediated pulmonary hypertension. The findings that GdCl3 did not alter either abnormal lung morphology or lung mechanics induced by 60% O2 indicate that reactive nitrogen species, 8-isoprostane, and ET-1 may not be involved in these aspects of O2-induced lung injury. These findings are consistent with our previously reported observations with an antioxidant intervention (24), in that different components of O2-induced lung injury seem to be regulated by different mediators and cell types.