Bombesin inhibits alveolarization and promotes pulmonary fibrosis in newborn mice

Abstract

Rationale: Bombesin-like peptides promote fetal lung development. Normally, levels of mammalian bombesin (gastrin-releasing peptide [GRP]) drop postnatally, but these levels are elevated in newborns that develop bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested alveolarization. In premature baboons with BPD, antibombesin antibodies reduce lung injury and promote alveolarization.

Objectives: The present study tests whether exogenous bombesin or GRP given perinatally alters alveolar development in newborn mice.

Methods: Mice were given peptides intraperitoneally twice daily on Postnatal Days 1-3. On Day 14 lungs were inflation-fixed for histopathologic analyses of alveolarization.

Measurements and main results: Bombesin had multiple effects on Day 14 lung, when alveolarization was about half complete. First, bombesin induced alveolar myofibroblast proliferation and increased alveolar wall thickness compared with saline-treated control animals. Second, bombesin diminished alveolarization in C57BL/6 (but not Swiss-Webster) mice. We used receptor-null mice to explore which receptors might mediate these effects. Compared with wild-type littermates, bombesin-treated GRP receptor (GRPR)-null mice had increased interstitial fibrosis but reduced defects in alveolarization. Neuromedin B (NMB) receptor-null and bombesin receptor subtype 3-null mice had the same responses as their wild-type littermates. GRP had the same effects as bombesin, whereas neither NMB nor a synthetic bombesin receptor type 3 ligand had any effect. All effects of GRP were abrogated in GRPR-null mice.

Conclusions: Bombesin/GRP can induce features of BPD, including interstitial fibrosis and diminished alveolarization. GRPR appears to mediate all effects of GRP, but only part of the bombesin effect on alveolarization, suggesting that novel receptors may mediate some effects of bombesin in newborn lung.

Figure 1.

Bombesin (BN) increases alveolar cell proliferation in newborn Swiss-Webster mice as assessed by proliferating cell nuclear antigen (PCNA) immunostaining. Swiss-Webster mice were treated with BN or phosphate-buffered saline (PBS) prenatally (E16–E18) or postnatally (P1–P3), as detailed in Methods. All of the lungs analyzed in this study were from Postnatal Day 14 animals. Immunostaining for PCNA was performed to evaluate the prevalence of alveolar cell proliferation in lung tissue sections. Details of computerized image analysis are given in Methods. (A) Representative section of lung from mouse given PBS postnatally. Note several PCNA-positive nuclei lining airspaces (red arrows). There are also scattered PCNA-positive cells in the airway epithelium (black arrows). (B) Lung from mouse given BN (200 μg/kg) postnatally. There are numerous PCNA-positive nuclei in the alveolar walls (many are indicated by red arrows). There are also multiple PCNA-positive cells in the airway epithelium (some indicated by black arrows). (C) Results of morphometric analyses determining the percentage of nuclei in the alveolar wall that are PCNA-positive. Mice treated with BN either prenatally or postnatally have over a threefold increase in the percentage of PCNA-positive cells (*p < 0.0001 compared with corresponding PBS-treated control group). (D) Immunohistochemistry using lung slides from mice treated with BN demonstrated PCNA labeling by bright-field microscopy merged with SMA immunofluorescence (arrows indicate cells with green cytoplasm and PCNA-positive nuclei). (E) In contrast, PCNA positivity only infrequently colocalized with surfactant protein C (SPC), a marker of alveolar type II cells. Note red cytoplasm indicating SPC immunoreactivity in a cell with PCNA positivity (arrow). Scale bar in lower left corner of (A) and (B) = 50 μm. L = airway lumen; V = vessel.

Figure 2.

Bombesin-induced alveolar cell proliferation in GRPR-WT and GRPR-KO mice. GRPR KO and WT mice on a C57BL/6 background were treated with BN or PBS postnatally (P1–P3), and immunostaining for PCNA was performed as described in Figure 1. (A) Results of morphometric analyses determining the percentage of nuclei in the alveolar wall that are PCNA-positive. There was no significant difference in PCNA labeling between untreated WT and untreated KO mice (KO is 1.4-fold greater than WT; p = 0.30). KO mice treated with BN, however, had significantly more PCNA-labeling than BN-treated WT littermates (†p < 0.02). Both WT and KO mice had significant BN-induced responses compared with the corresponding untreated control animals (**p < 0.0001 for KO mouse responses, and *p < 0.005 for WT mouse responses). (B) Representative section of lung from GRPR-WT mouse given BN. Note several PCNA-positive nuclei in developing alveoli (red arrows). (C) Representative section showing PCNA immunostaining of lung from BN-treated GRPR-KO mouse. There are numerous PCNA-positive nuclei in the alveolar walls (many are indicated by red arrows). L = airway lumen.

Figure 3.

Bombesin increases SMA-positive cells in alveoli of newborn mice. Swiss-Webster mice were treated with BN or PBS prenatally (E16–E18) or postnatally (P1–P3), as described in Methods. Immunostaining for SMA was performed to determine the prevalence of myofibroblasts in developing alveoli. Black arrows indicate SMA-positive cells along the surface of alveolar spaces, consistent with developing septa. Red arrows indicate SMA-positive cell(s) within the interstitium. (A) Representative section of lung from mouse given PBS postnatally. Inset: note SMA-positive cells predominantly at the tips of developing septa (a few are indicated by black arrows). (B) Representative section of lung from mouse given BN postnatally. Compared with (A), there is an increased volume percent of SMA-positive cells, most of which occurs in the alveolar interstitum (red arrows), as shown at higher magnification in the inset. V = vessel, also indicated by red asterisk. Scale bars in lower left corners of (A) and (B) = 50 μm. (C) Results of morphometry assessing SMA-positive cells in developing alveoli. Mice treated with BN either prenatally or postnatally had ∼ threefold increased volume percent of myofibroblasts (*p < 0.0001 and **p < 0.01 compared with corresponding PBS-treated control group). There was no significant difference between mice given PBS prenatally versus postnatally.

Figure 4.

Bombesin-induced SMA-positivity is enhanced in GRPR-KO mice. GRPR-WT and GRPR-KO mice were treated with BN postnatally (P1–P3), as described in the legend to Figure 2. Immunostaining for SMA was performed as in Figure 3. Black arrows indicate SMA positivity consistent with developing septa. Red arrows indicate SMA-positivity within the interstitium. (A) Results of morphometry assessing the volume percent of SMA-positive cells in developing alveoli. Mice treated with BN have ∼2 to 3-fold increased volume percent of myofibroblasts (*p < 0.0001 compared with the corresponding untreated control group). Bombesin-treated KO mice have significantly more SMA staining than BN-treated WT mice (twofold greater volume percent of SMA; ^†p < 0.001). (B) Representative section of lung from GRPR-WT mouse given BN. (C) Representative section of lung from GRPR-KO mouse given BN. Compared with Figure 2B, there is an increased prevalence of SMA-positive cells, especially in the alveolar interstitum (red arrows). V = vessel, also indicated by red asterisk.

Figure 5.

Bombesin increases alveolar wall thickness in alveoli of newborn mice. Swiss-Webster mice were treated with BN or PBS, as described in the legend to Figure 1. Lung tissue sections were stained with hematoxylin and eosin (H&E) to demonstrate alveolar architecture. Representative red lines are drawn at 90° across alveolar septa. (A) Representative section of lung from a mouse given PBS postnatally. (B) Representative section of lung from a mouse given BN postnatally. Scale bar in lower right hand corners = 30 μm. (C) Results of morphometry assessing alveolar wall thickness. Mice treated with BN either prenatally or postnatally have a ∼ 20–25% increase in alveolar wall thickness (*p < 0.0001compared with the corresponding PBS-treated control group).

Figure 6.

Bombesin and GRP arrest alveolarization. GRPR-WT and GRPR-KO mice were treated with BN or left untreated, as described in Methods and in the legend to Figure 2. Lung tissue sections were stained with H&E to demonstrate alveolar architecture. Representative sections are given in (A)–(D): (A) GRPR-WT, untreated. (B) GRPR-KO, untreated. (C) GRPR-WT treated with BN. (D) GRPR-KO treated with BN. * Pleural surface. Scale bar in lower left hand corner = 50 μm. (E) Results of morphometry for mean linear intercept (MLI) in the above BN-treated groups (n = 8–10 animals per group; 10–12 20× fields per animal) (*p < 0.0001 compared with corresponding untreated group; ^†p < 0.001 compared with BN-treated WT pups). (F) Results of morphometry for MLI in similar groups, but treated with GRP rather than BN (n = 6–10 animals per group) (*p < 0.0001 compared with either the GRP-treated KO group or the corresponding PBS-treated WT group).