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Published ahead of print on June 13, 2003, doi:10.1164/rccm.200212-1434OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 601-611, (2003)
© 2003 American Thoracic Society

Original Article

Bombesin-like Peptides and Mast Cell Responses

Relevance to Bronchopulmonary Dysplasia?

Meera Subramaniam, Kumiya Sugiyama, David H. Coy, Yanping Kong, York E. Miller, Peter F. Weller, Keiji Wada, Etsuko Wada and Mary E. Sunday

Department of Pathology and Pulmonology, Children's Hospital Boston; Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School; and Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts; Department of Medicine, Peptide Research, Tulane University Health Sciences, New Orleans, Louisiana; Department of Medicine, Veterans Affairs Medical Center, University of Colorado, Denver, Colorado; and Institute of Neuroscience, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Mary E. Sunday, M.D., Ph.D., Brigham and Women's Hospital, Department of Pathology, 75 Francis Street, Boston, MA 02115. E-mail: sunday{at}tch.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bombesin-like peptides (BLPs) are elevated in newborns who later develop bronchopulmonary dysplasia (BPD). In baboon models, anti-BLP blocking antibodies abrogate BPD. We now demonstrate hyperplasia of both neuroendocrine cells and mast cells in lungs of baboons with BPD, compared with non-BPD controls or BLP antibody-treated BPD baboons. To determine whether BLPs are proinflammatory, bombesin was administered intratracheally to mice. Forty-eight hours later, we observed increased numbers of lung mast cells. We analyzed murine mast cells for BLP receptor gene expression, and identified mRNAs encoding bombesin receptor subtype 3 and neuromedin-B receptor (NMB-R), but not gastrin-releasing peptide receptor. Only NMB-R-null mice accumulated fewer lung mast cells after bombesin treatment. Bombesin, gastrin-releasing peptide, NMB, and a bombesin receptor subtype 3-specific ligand induced mast cell proliferation and chemotaxis in vitro. These observations support a role for multiple BLPs in promoting mast cell responses, suggesting a mechanistic link between BLPs and chronic inflammatory lung diseases.

Key Words: bronchopulmonary dysplasia • chemotaxis • infant, premature • neuromedin B • pulmonary fibrosis

Bombesin is a 14-amino acid bioactive peptide originally isolated from frog skin (1). Bombesin-like peptides (BLPs) are mammalian homologs of bombesin that are elevated in newborns who later develop bronchopulmonary dysplasia (BPD) (2). BLPs comprise a family of related peptides, of which several could account for BLP immunoreactivity localized to the pulmonary neuroendocrine (NE) cells, with highest BLP levels occurring in fetal lung (3). One major pulmonary BLP was later identified as gastrin-releasing peptide (GRP) (1). GRP and bombesin are highly similar both structurally and functionally, and thus are collectively referred to as "BLPs"; neuromedin B (NMB) is also closely related to bombesin (1, 4). BLPs are growth factors for 3T3 fibroblasts, normal airway epithelial cells, and many cancer cell lines (1, 5); have neuroregulatory functions in the brain and gut; and act as potent bronchoconstrictors (6). Multiple receptor subtypes mediate effects of bombesin-related peptides, and three mammalian genes for these G protein-coupled receptors have been cloned: the GRP/bombesin (BLP)-preferring receptor (GRP-R), the neuromedin B receptor (NMB-R), and the orphan bombesin receptor subtype-3 (BRS-3), for which no natural ligand is known (7). A related receptor has been cloned in the frog and is known as bombesin receptor subtype-4 (8). In fetal lung, GRP-R mRNA is localized predominantly in the undifferentiated mesenchyme around developing airways and blood vessels, with lower levels in airway epithelium (9). GRP-R, NMB-R, and BRS-3 have also been identified in many human cancers and cancer cell lines and some corresponding normal cell types (1, 10–15).

In previous studies, we demonstrated that BLPs promote fetal lung development, including lung branching morphogenesis, epithelial and mesenchymal cell proliferation, and Type II cell differentiation (1). GRP and GRP-R mRNAs fall to undetectable levels after birth but may be upregulated postnatally in premature baboons with bronchopulmonary dysplasia (BPD, also known as chronic lung disease of prematurity or CLD) (16). Increased numbers of BLP-positive NE cells occur in lungs of infants dying of BPD (17, 18). Elevated levels of BLPs and/or increased numbers of BLP-positive NE cells have been associated with other chronic inflammatory lung diseases (1), including cystic fibrosis, lung cancer, eosinophilic granuloma, tobacco-related lung disorders, and pulmonary hypertension. A few previous reports suggested a possible role for BLPs in promoting cellular responses that may contribute to inflammatory processes in vitro, including macrophage activation and phagocytosis (1), chemotaxis of macrophages and fibroblasts (1, 19, 20), smooth muscle constriction (21), and proliferation of fibroblasts, lymphocytes, and endothelial cells (20, 22, 23). In spite of all these observations, no mechanistic link has been established between BLPs and pulmonary inflammation in vivo.

BPD is the most common chronic lung disease in infants in the United States (24, 25). Recognized contributing factors include mechanical ventilation, oxygen toxicity, infection, and pulmonary immaturity. Nonetheless, the pathophysiology of BPD remains enigmatic. It is unclear why only a subset of premature infants develops BPD (24). Two different premature baboon models of BPD have been valuable tools for investigating the pathophysiology of this disorder (26, 27): "hyperoxic" animals gestated for 140 days (140d/100%), and "interrupted gestation" (125-day) animals given maintenance levels of oxygen (125d/PRN [pro re nata, as needed]). Previously (18), we observed elevated urine BLP levels in both of these baboon models of BPD. Administration of anti-BLP blocking antibody 2A11 to baboons in either model abrogates the lung injury characteristic of BPD (18). We demonstrated elevated BLPs shortly after birth in premature human infants who later develop BPD (28). Cumulatively, these observations prompted us to test the hypothesis that BLPs function postnatally as proinflammatory cytokines. In the present study, we focus our analysis on mast cells, which are present in diverse inflammatory lung diseases.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animals
Mice housed at Children's Hospital Boston (Boston, MA) included 5- to 6-week-old Swiss–Webster females (Taconic Farms, Germantown, NY) and GRP-R–null, NMB-R–null, and BRS-3–null mice (29–31). Protocols were approved by the Institutional Animal Care and Use Committee of Children's Hospital Boston.

Baboons were studied at the Southwest Foundation for Biomedical Research (San Antonio, TX) as described (16, 18).

Premature baboons from the hyperoxic model of BPD (140d/100% x 10d) (26) were given anti-BLP antibody (2A11) or the control IgG1, MOPC21 (18). On Day 10, right middle lobes inflated with 4% paraformaldehyde were fixed overnight. Sections chosen included complete lung cross-sections from proximal (cartilaginous) conducting airways down to the distal parenchyma (alveoli and pleura).

Intratracheal Bombesin
Seventy-five µl of sterile phosphate-buffered saline (PBS)–1% bovine serum albumin (BSA)–0.5% Evans blue was instilled into anesthetized mice via a transoral/intratracheal catheter with or without bombesin (5 µg). There was no mortality of the mice in any experimental groups. After CO2 euthanasia, lungs were inflated in situ with 4% paraformaldehyde.

Histochemistry
Five-micrometer baboon lung paraffin sections were immunostained for tryptase with monoclonal antibody clone AA1 and the Animal Research Kit peroxidase system (Dako Laboratories, Carpinteria, CA). BLP immunostaining is described elsewhere (18). Alcian blue–safranin histochemistry was performed to detect murine mast cells (32).

Morphometry
Positive cells (NE cells or mast cells) were counted across entire slides. Tissue section areas were determined by computer-assisted image analysis (Scion Image, version 1.62b; Scion, Frederick, MD) and results were expressed as number of cells per unit area (square millimeters for baboons, square centimeters for mice) (33).

Cultured Mast Cell Line
MC/9 cells were cultured as described (American Type Culture Collection, Manassas, VA) (34, 35). This cloned normal murine mast cell line bears IgE receptors and produces histamine and leukotrienes.

Bone Marrow-derived Mast Cells
Bone marrow cells from Swiss–Webster mice were cultured as described (36). Purity was assessed by flow cytometry for surface IgE (36).

Reverse Transcription-Polymerase Chain Reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was performed as described (37): 25 cycles for glyceraldehyde-3-phosphate dehydrogenase and 35 cycles for GRP-R, NMB-R, and BRS-3 (4, 38, 39). A second round of PCR (10 cycles) used nested primers for GRP-R, BRS-3, and NMB-R. Glyceraldehyde-3-phosphate dehydrogenase primers were from BD Biosciences Clontech (Palo Alto, CA). PCR primers for murine GRP-R, NMB-R, and BRS3 are described in the online supplement.

Mast Cell Proliferation
MC/9 cells were cultured at 37°C in 5% CO2 and serum-free Dulbecco's modified Eagle's medium (DMEM)–0.1% BSA (DMEM–BSA) for 18 hours before assay. Cells (15 x 104/ml) were plated in 6-well plates with DMEM–BSA with or without 0.1–100 nM bombesin (NMB; Peninsula Laboratories, San Carlos, CA), or with BRS-3–specific synthetic peptide 3209 [D-Tyr6,ß-Ala11,Phe13,Nle14]bombesin(6–14) (BRS-3L) (40). After 24 hours, cells were harvested for Ki-67 flow cytometry (41, 42). GRP-R–specific antagonist D-Tyr6-bombesin(6–13) methyl ester (GRA) was also used (43). No ligand or antagonist had any effect on cell viability, as determined by trypan blue exclusion.

Chemotaxis
MC/9 cells (106 in 100 µl of PBS–BSA) were plated in the upper chambers of Transwell inserts (pore size, 5 µm; 6.5 mm in diameter; polycarbonate) in 24-well plates and incubated for 3 hours at 37°C. The lower chambers had 600 µl of PBS–BSA with or without 0.1–10 nM bombesin, GRP(1–27), GRP(1–14), GRP(16–27), NMB (Peninsula Laboratories), or BRS-3L (40). Upper chamber cells were sometimes treated with GRA (43). Lower chamber cells were counted with a hemacytometer. Numbers were expressed as a percentage of total number of cells added to the upper wells.

Statistics
For statistical analyses we used an unpaired Student t test or analysis of variance.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pulmonary NE Cells in BPD and Non-BPD Baboons
Increased urine BLP levels are known to occur in the hyperoxic preterm baboon model of BPD (140d/100% baboons) (18). One of the main sources of this elevated BLP immunoreactivity is believed to be increased BLP production by pulmonary NE cells. To test this hypothesis, we performed morphometric analyses of BLP-positive cells in lung sections from these baboons. Similar to our earlier studies of hyperoxic hamsters, there was an increase in both the total number of clusters (neuroepithelial body-like foci) and in the number of nucleated cells per cluster. Note that by definition, neuroepithelial bodies must be innervated and this could not be evaluated on the given slides because pulmonary nerve fibers are negative for BLP immunostaining. NE cells staining for the NE/neural cytoplasmic marker PGP9.5 were increased, similarly to BLP-positive cells, but abundant PGP9.5-positive mesenchymal cells obscured evaluation of nerve fibers (M. Sunday, data not shown). We quantified the total number of BLP-positive pulmonary neuroendocrine cells (PNECs) per unit area of lung to take into account both the increased number of clusters and the increased number of cells per cluster. Representative photomicrographs are given in Figures 1a–1c . The pooled results from morphometric analyses are summarized in Figure 1d. Compared with non-BPD baboons treated for 10 days with O2 PRN (pro re nata, sufficient to maintain hemoglobin saturation at about 90%) (Figures 1a and 1d), there was an overall ninefold increase in BLP-positive NE cells in 140d/100% baboons with BPD (Figures 1b and 1d) (p < 0.0004). Most of the increased NE cells were localized to small bronchioles and alveolar ducts. A similar increase in PGP9.5-positive NE cells was observed (18). To determine whether these changes are BLP dependent, one experimental group of 140d/100% animals was treated with the blocking anti-BLP antibody 2A11, which is free of any BLP agonist activity (18). The 2A11 antibody was previously shown to attenuate the histopathologic and clinical features of BPD in this model (18). As shown in Figure 1c, 2 A11 treatment completely abrogated the increase in NE cells (compare Figures 1c and 1b) (p < 0.0004). There was no effect of an irrelevant murine monoclonal IgG1 antibody, MOPC21, given at the same dose and according to the same schedule as in the 2A11-treated group (data not shown). Thus, the urine BLP levels occurring in these groups are directly correlated with relative numbers of BLP-positive NE cells in the lung, supporting the concept that pulmonary production of BLPs is increased in infants with BPD.



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  		Figure 1. BLP-positive pulmonary NE cells in the hyperoxic baboon model of BPD: representative photomicrographs demonstrate BLP-positive NE cells in clusters (indicated by arrows; magnification bars, 100 µm) in the lungs of baboons treated with (a) O2 PRN (non-BPD controls, n = 6), (b) 100% O2 (hyperoxic model of BPD, n = 7), or (c) 100% O2 plus anti-BLP antibody 2A11 (n = 5). (d) Morphometric analyses demonstrate a ninefold increase in numbers of BLP-positive NE cells in the lungs of baboons with BPD (100% O2) compared with controls (PRN O2) (p < 0.0004). Postnatal treatment of BPD baboons with anti-BLP antibody 2A11 (100% O2 + 2A11) is associated with significantly fewer NE cells (p < 0.0004), which are at levels similar to control animals (values are expressed as means ± SE).

 


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  		Figure 2. Lung mast cells in baboons with BPD. Mast cells were immunostained for murine tryptase (indicated by red arrows in a–c; magnification bar, 200 µm) or stained red, using chloroacetate esterase histochemistry (d; magnification bar, 50 µm). Mast cells were observed in the lung tissue of (a) non-BPD control animals (PRN O2: gestation, 140 days; oxygen as needed), (b) BPD baboons (100% O2: gestation, 140 days; 100% O2), or (c and d) BPD baboons given 2A11 (c—100% O2 + 2A11: gestation, 140 days; animals given 100% O2 and 2A11; d—CAE: gestation, 140 days; animals given 100% O2 and 2A11; cells stained with chloroacetate esterase). In the control animals (PRN O2) and the animals treated with anti-BLP antibody (100% O2 + 2A11), mast cells were distributed mainly near the blood vessels and major bronchi, similar to gestational controls. There was a fourfold increase in total tryptase-positive mast cells per square millimeter in the lungs of hyperoxic BPD baboons (given 100% O2) compared with non-BPD controls (given O2 as needed) (p < 0.005). Treatment of BPD baboons with anti-BLP antibody 2A11 (100% O2 + 2A11) significantly reduced the accumulation of mast cells (*p < 0.002 for 140d/100% animals versus 100% O2 + 2A11 animals). (The numbers of animals per group are given in the legend to Fig. 1; values are expressed as means ± SE). L = lumen.

 
Mast Cells in Lungs of Baboons with BPD
Considering one report of mast cell hyperplasia in lungs of human infants with BPD (44), we quantitated the numbers of lung mast cells occurring in hyperoxic baboons with BPD (140d/100%, BPD group). These results were compared with those of the 140d/PRN, non-BPD group and with 140d/100% baboons treated with anti-BLP antibody 2A11. We determined that immunostaining for human tryptase is the most sensitive and specific method for detecting mast cells in baboon lung (Figures 2a–2c). Similar results were observed when mast cells were stained red, using chloroacetate esterase histochemistry (Figure 2d). Tryptase-positive mast cells were quantified in lung sections by two types of computerized image analysis: (1) as total mast cells per square millimeter of lung tissue (Figure 2e), and (2) as mast cells present in the distal lung parenchyma (i.e., interstitial mast cells, excluding those associated with blood vessels and conducting airways). The total number of mast cells was increased fourfold in the 140d/100% group (14.7/mm2 lung tissue) compared with 140d/PRN baboons (3.6/mm2) (p < 0.005). In contrast, 2A11-treated 140d/100% animals had 2.8 total mast cells per square millimeter, which is fivefold less than in the 140d/100% animals not receiving 2A11 (p < 0.002). 140d/100% animals treated with the isotype-matched negative control antibody MOPC21 were not significantly different from the 140d/100% animals that received no antibody treatment (data not shown). Furthermore, parenchymal mast cells were increased ninefold in 140d/100% animals compared with the non-BPD group. 140d/100% animals treated with blocking anti-BLP antibody 2A11 demonstrated significantly fewer mast cells in the lung parenchyma, with mean values similar to those of the non-BPD group (Figure 2c and data not shown).

Lung Mast Cells in Mice Given BLP Intratracheally
To directly test the hypothesis that BLPs can function as proinflammatory cytokines in the lung, we administered bombesin (5 µg in 75 µl of PBS–BSA) or vehicle alone (PBS–BSA) intratracheally to anesthetized mice. Lungs were harvested after 4 hours, 48 hours, 7 days, or 14 days and paraffin-embedded sections were stained with Alcian blue–safranin (32) to demonstrate mast cells, which we determined to be the most sensitive method for detection of these cells in mouse lung (Figures 3a and 3b) . The relative density of mast cells was lower in lung sections from mice given a single BLP treatment as compared with sections from BPD baboons treated with hyperoxia for more than 1 week. We observed a twofold increase in numbers of mast cells per square centimeter of lung tissue 48 hours and 7 days after bombesin administration (Figures 3a and 3c) as compared with mice given saline vehicle alone (Figure 3b) (p < 0.01). There was no significant effect only 4 hours after bombesin administration (Figure 3c). In a second series of three experiments, we also tested the effects of intratracheal administration of GRP, which has effects similar to those elicited by bombesin. We compared the responses to GRP with those elicited by the active half of GRP [GRP(16–27)] versus the inactive half of GRP [GRP(1–14)], which does not bind to GRP-R or NMB-R (Figure 3d). We used a fourfold larger dose of GRP compared with bombesin because GRP has a twofold greater molecular weight than bombesin or NMB and a three- to fivefold lower affinity for the GRP-R compared with bombesin and a twofold lower affinity for the NMB-R (45). Lung mast cell numbers increased threefold 48 hours after GRP treatment compared with saline treatment (p < 0.001); no significant difference was seen between the control and the GRP(1–14) group. There was a twofold difference in the GRP(16–27)-induced increase in mast cells compared with saline-treated control mice (p < 0.02).





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  		Figure 3. Bombesin promotes accumulation of mast cells in murine lung. (a) Intratracheal administration of bombesin (5 µg) to mice promotes increased numbers of Alcian blue–safranin-positive mast cells (indicated by arrows) in the interstitial and subpleural (PL) regions compared with control mice (magnification bar, 30 µm) (n = 7, 16, and 7, respectively, at 4 hours, 48 hours, and 7 days, representing 2, 3, and 2 experiments, respectively). (b) Control mice have scant mast cells (indicated by arrow), usually one or two per lung cross-section, located around large blood vessels (v) (magnification bar, 30 µm) (n = 7, 12, and 6, respectively, at 4 hours, 48 hours, and 7 days). (c) Morphometric analyses demonstrate a twofold increased number of Alcian blue–safranin-positive mast cells per square centimeter of lung 48 hours and 7 days after bombesin administration (*p < 0.05) (the number of animals per group is as in a and b; experimental values are expressed as means ± SE). (d) GRP(1–27) (1.0 nM, n = 5) induced a threefold increase in mast cell accumulation (*p < 0.001). The inactive N-terminal segment of GRP, GRP(1–14) (n = 5), had no effect on relative numbers of mast cells, whereas the active C-terminal GRP analog GRP(16–27) (n = 5) induced a twofold increase (**p < 0.02) (n = 5 for untreated control group). One representative experiment (of three similar experiments) is shown. (e) NMB-R–null mice (n = 3) have diminished mast cells in response to bombesin compared with their normal littermates (n = 3). Intratracheal administration of bombesin (5 µg) to NMB-R, GRP-R, and BRS-3–null mice (n = 3, 4, and 5, respectively) demonstrates significantly reduced numbers of Alcian blue–safranin-positive mast cells in the lungs of NMB-R–knockout mice compared with their wild-type littermates 48 hours after bombesin administration (p < 0.03). No significant difference was observed between wild-type littermates (five per genotype) and the GRP-R and BRS-3–null mice either at baseline or after bombesin treatment (data not shown). (Experimental values are expressed as means ± SE).

 
To determine which of the three cloned mammalian BLP receptors might mediate these mast cell responses, we administered bombesin intratracheally (5 µg/mouse) to mice genetically deficient for GRP-R, NMB-R, or BRS-3, and their normal littermates, keeping in mind the caveat that such mice can have altered expression of other cytokine/mediator-signaling pathways. Knockout mice were used rather than BLP receptor-specific antagonists because most antagonists are not entirely specific for only one BLP receptor subtype and partial agonist effects are common. The only exception is the GRP-R–specific antagonist (D-Tyr6-bombesin(6–13) methyl ester, or GRA), which we were able to obtain in only small quantities that precluded its use for in vivo blocking studies. Furthermore, the use of the BLP blocking antibody 2A11 would not give us any pharmacologic information about the receptors involved, but would simply confirm that 2A11 binds BLPs sharing the same C-terminal 7-amino acid sequence as bombesin and GRP.

Only experiments with NMB-R-null mice resulted in 50% fewer mast cells accumulating in lung tissues, compared with wild-type littermates at 48 hours (Figure 3e). At baseline, there was no significant difference in mast cell numbers between NMB-R–null and their wild-type littermates. There was no difference in the numbers of BLP-induced mast cells between wild-type littermates and GRP-R–null or BRS-3–null mice. We did not see a significant difference in the number of mast cells at baseline in any of the untreated mice (data not shown).

GRP-R, NMB-R, and BRS-3 Gene Expression by Mast Cells
To determine whether bombesin might directly trigger murine mast cell responses, we analyzed gene expression for BLP receptors in MC/9, a cloned mouse mast cell line, and in murine bone marrow–derived mast cells (more than 99% pure by flow cytometry and more than 99% viable by trypan blue exclusion). Semiquantitative RT-PCR was performed to assess relative levels of the mRNAs encoding the three BLP receptors: GRP-R, NMB-R, and BRS-3. NMB-R and BRS-3 mRNAs were detected in both types of mast cells (Figures 4A and 4B) . GRP-R mRNA was not detected by RT-PCR even when nested primers were used to reamplify the first RT-PCR products in a second round of PCR for up to 10 cycles. The identities of the PCR products were confirmed by sequencing.



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  		Figure 4. BLP receptor transcripts in mast cells. Gene expression for the BLP receptors NMB-R and BRS-3 was demonstrated in the murine mast cell line MC/9 (A) and in bone marrow–derived mast cells (BMMCS; B), using RT-PCR. Lanes 1–3 give results of a single round of PCR for GRP-R, NMB-R, and BRS-3 mRNAs, and lanes 4–6 give the results of the corresponding reamplifications using nested primers. Lanes 1 and 4, positive controls (+) (E17 fetal lung for GRP-R, mouse testis for NMB-R and BRS-3); lanes 2 and 5, negative control (-) with water replacing RNA in the RT reaction; lanes 3 and 6, note absence of GRP-R mRNA even after reamplification with nested primers. Note that MC/9 cells are positive for NMB-R and BRS-3 receptors. Similar results are seen in the bone marrow–derived mast cells shown in lanes 3 and 4. G3PDH = glyceraldehyde-3-phosphate dehydrogenase.

 
BLP-induced Proliferation of Mast Cells
We hypothesized that cell proliferation is one mechanism by which bombesin could increase mast cell numbers in mouse lung. Also, NMB is known to act as a growth factor for lung cancer cell lines (46). The cloned mast cell line MC/9 was chosen for our studies because the numbers of bone marrow–derived mast cells was insufficient for carrying out controlled experiments with samples in duplicate or triplicate to ensure reproducibility. Mast cell proliferation induced by several different BLP ligands was evaluated by flow cytometry for Ki-67, a well characterized marker of proliferating cells. Significantly increased Ki-67 labeling was observed with bombesin at 10 nM (1.7-fold increase over baseline, p < 0.02) (Figures 5A and 5B) . Although it can interact with both NMB-R and GRP-R, bombesin has the highest affinity for GRP-R (46, 47), with peak effects typically in the 0.1–1.0 nM range. These data suggest that GRP-R is not the leading candidate for mediating mast cell proliferation. This was confirmed by demonstrating that a potent and selective GRP-R–specific antagonist did not block bombesin-induced proliferation of MC/9 cells (Figure 5C).




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  		Figure 5. BLPs induce mast cell proliferation. (A) Proliferation of MC/9 cells treated with BLPs for 24 hours under serum-free conditions was evaluated by fluorescent labeling of the cells for Ki-67, a marker of cell proliferation. Increased Ki-67 labeling was demonstrated by flow cytometry of MC/9 cells treated with each of the ligands. The representative flow cytometric analysis given here shows a 1.8-fold increase in MC/9 cell proliferation with 10 nM bombesin (80% increase = [(10.4 – 5.8)/5.8] x 100). (B) Bombesin (BN, 10 nM) induces a 1.7-fold increase (equivalent to 170% of the saline control value, *p < 0.02) in Ki-67 labeling, indicating MC/9 cell proliferation (depicted by solid triangles). Note that we are comparing this response with the saline control response, which is 100%. BRS-3L at 1.0 nM (solid circles) induced a 1.4-fold increase (equivalent to 140% of the saline control value, *p < 0.02) in MC/9 proliferation. NMB (open squares) elicited 1.4-fold increased Ki-67 labeling at both the 1.0 and 10 nM doses (equivalent to 140% of the saline control value, **p < 0.02 and 0.04, respectively). Results represent pooled data from three experiments (five or six duplicate samples per group per experiment), and are expressed as mean values ± SE.

 
To determine whether NMB-R and/or BRS-3 could be implicated in the MC/9 proliferative response, we tested two other ligands. NMB induced a 1.4-fold increase in MC/9 proliferation at both 1.0 and 10 nM (p < 0.02 and p < 0.05, respectively) (Figure 5B). The synthetic BRS-3L induced a 1.4-fold increase in Ki-67 labeling of MC/9 cells at 1.0 nM (p < 0.02) (Figure 5B). These peak responses occurring at 1.0 nM suggest that high-affinity NMB-R and BRS-3 receptors might be implicated in the observed mast cell proliferation. The magnitude of these responses was consistently about 40% above baseline, and the values in each group had minimal variability.

Mast Cell Chemotaxis in Response to BLPs
Increased mast cells in the lungs of BPD baboons were observed primarily in the distal lung parenchyma, whereas control non-BPD baboons had a more proximal distribution of these cells. We tested the ability of BLPs to induce mast cell (MC/9) chemotaxis. MC/9 chemotaxis was significantly increased in the presence of bombesin (2.5- to 3.5-fold increase at 1 and 10 nM bombesin, respectively; p < 0.003) (Figure 6A and data not shown), NMB (fivefold increase at 0.01 nM, p < 0.05) (Figure 6B), or BRS-3L (more then twofold at 1.0 nM, p < 0.03) (Figure 6C). The GRP-R-specific antagonist (GRA) did not significantly reduce bombesin-induced chemotaxis (Figure 6A), consistent with no role for GRP-R in bombesin-induced mast cell chemotaxis. NMB induced significantly increased mast cell chemotaxis only at the 0.01 nM dose (Figure 6B), similar to other systems (19), and the GRP-R–specific antagonist did not abrogate this chemotaxis. Finally, the BRS-3 synthetic peptide ligand induced significant chemotaxis at 1.0 nM and the GRP-R–specific antagonist did not abrogate BRS-3 ligand–induced chemotaxis (Figure 6C).





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  		Figure 6. BLPs induce mast cell chemotaxis. Mast cell chemotaxis was assayed with Transwell filters to separate upper and lower chambers, as detailed in METHODS. MC/9 cells were added to the upper chamber and various BLPs were added to the lower chamber. The negative control well (Neg) included medium alone in both chambers. For some experimental groups, 100 nM GRP-R–specific antagonist (GRA) was added to the upper chamber with the cells. Data shown represent pooled results of three separate experiments, with five replicates per group per experiment. (A) Bombesin (BN, 1.0 nM) induced a 2.5- to 3.5-fold mean increase in chemotaxis (*p < 0.003). GRP-R–specific antagonist (GRA, 100 nM) did not significantly block chemotaxis induced by 1.0 nM bombesin ({dagger}p = 0.12, comparing BN with BN+GRA). (B) NMB (0.01 nM) induced a fivefold increase in chemotaxis (*p < 0.05). GRA at 100 nM did not inhibit chemotaxis induced by 0.01 nM NMB ({dagger}p > 0.99, comparing NMB with NMB+GRA). (C) BRS-3 ligand (BRS-3L, 1.0 nM) induced a more than twofold increase in chemotaxis (*p < 0.03). GRA at 100 nM did not inhibit chemotaxis induced by 1.0 nM BRS-3L ({dagger}p = 0.60, comparing BRS-3L with BRS-3L+GRA).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates two major lines of evidence supporting a previously missing direct link between BLPs and mast cell hyperplasia. First, in lungs of hyperoxic premature baboons, endogenous BLPs mediate hyperplasia of NE cells and mast cells: anti-BLP blocking antibody (2A11) abrogates these increases. Previously, we showed that 2A11 treatment abrogates the development of BPD in these same hyperoxic hyperoxic baboons, both clinically and pathologically (18). Second, in mice, intratracheal bombesin induces lung mast cell accumulation. Cumulatively, these observations suggest that BLPs could play a role in the pathophysiology of chronic inflammatory lung diseases by triggering a cellular cascade including mast cells.

Hyperplastic pulmonary NE cells are likely to be the source of elevated urine BLP levels in newborns with BPD (18, 28). This hyperplasia could be induced by oxidant injury (47), tumor necrosis factor-{alpha} (49), or other cytokines, including BLPs (50, 51). It is known that PNECs in normal lung occur both as isolated cells (mainly in large airways) and as small clusters called neuroepithelial bodies that are concentrated at branch points within the epithelium of branching airways. Hyperplastic PNECs induced by hyperoxia, nitrosamine carcinogens, cigarette smoking, or in idiopathic primary PNEC hyperplasia are often preferentially localized to the small bronchioles and alveolar ducts (2, 18, 52–55). Similarly, there are also more mast cells and other inflammatory infiltrates in the small airways in patients with nonfatal asthma or status asthmaticus (56, 57). Cumulatively, these observations suggest that NE cells can function as proinflammatory cells.

BLPs could promote lung remodeling in BPD by triggering proliferation of both epithelial and mesenchymal cells (58). BLP levels are increased shortly after birth in infants who later develop BPD (18, 28). Mast cells can function in both acute and chronic inflammation, promoting both innate and acquired immune responses including leukocyte recruitment by chemotaxis (59, 60), and leading to lung injury and fibrosis in later stages (61–63). The current study demonstrates increased mast cells in the lung in response to BLPs. This inflammatory response may have a role in the pathogenesis of BPD. Proinflammatory cytokines, adhesion molecules, and inflammatory cells including mast cells have been demonstrated in the lung parenchyma of infants with BPD (44, 64, 65). The concept that BLP-induced mast cell responses might be an early event leading to lung injury in BPD represents a paradigm shift in current understandings of BPD. These observations could be of further relevance because BPD is associated with bronchospasm and inflammation, similar to asthma (61, 62).

BLP is well known as a growth factor and neuroregulatory peptide (1, 22, 23). However, no prior study has documented gene expression for BLP receptors in hematopoietic cells, in particular mast cells. Two mechanisms by which BLPs could increase mast cells in the lung are by directly triggering mast cell proliferation and/or chemotaxis. We demonstrate that two of the three genes encoding mammalian BLP receptors, BRS-3 and NMB-R, are expressed by murine mast cells (MC/9 and bone marrow–derived mast cells). The functional relevance of this receptor gene expression is supported by the ability of BRS-3- and NMB-R–specific ligands to elicit significant mast cell proliferation and chemotaxis at low doses (1.0 nM or less). Proliferation induced by 10 nM bombesin is probably mediated via NMB-R on mast cells (66), because bombesin has lower affinity for NMB-R compared with NMB. Bombesin has low affinity for BRS-3 in pharmacologic studies (67). It is unlikely that the GRP-R is involved in bombesin-induced mast cell responses because GRP-R mRNA is undetectable in mast cells, even using nested RT-PCR. Furthermore, a GRP-R–specific antagonist (GRA) (42, 68) does not significantly inhibit bombesin-induced mast cell proliferation or chemotaxis. The observation that mast cell responses can be elicited by both bombesin and GRP, but not by inactive GRP analogs, suggests the involvement of additional bombesin/GRP–preferring receptors, such as a phyllolitorin receptor (50, 67) or a homolog of the frog BB4 receptor (8). Previously, NMB-R and GRP-R have been implicated in cell proliferation in vitro (69, 70), whereas BRS-3 signaling increases calcium mobilization and metabolism but not mitogenesis in cancer cell lines (14). Our observed stimulation of mast cell proliferation using a BRS-3–specific ligand represents a novel function for this BLP receptor subtype, and suggests a role for BRS-3 in inflammatory responses.

Functional studies in genetically deficient mice indicate that null mutations of the three cloned murine BLP receptors yield distinct phenotypes at baseline (29–31). However, none of the previous studies addressed the effects of BLPs in BLP receptor-null mice. We now observe that NMB-R–null mice treated with bombesin have significantly less mast cell hyperplasia compared with wild-type littermates, despite there being no difference in mast cell numbers or distribution at baseline. The lack of such a difference between wild-type controls and GRP-R or BRS-3–null mice is logical: murine mast cells lack GRP-R, and bombesin has low affinity for BRS-3 receptors.

In hyperoxic baboons with BPD, we observed the greatest number of mast cells in the lung parenchyma (alveolar interstitium) distal to the conducting airways. Normally, lung mast cells occur around blood vessels, large airways, and nerves. Others have noted a similar distribution of tryptase-positive mast cells in lungs of human infants dying of BPD (44). BLP-positive NE cells are also found predominantly in the small bronchioles and alveolar ducts in infants with BPD (2, 18). The localization of mast cells to the distal lung parenchyma in infants with BPD may be due to mast cell migration in response to high local concentrations of BLPs. Similarly, mice given bombesin intratracheally have a higher density of mast cells in the lung parenchyma than in the whole lung. We demonstrate chemotaxis of mast cells in response to BLPs, similar to macrophages, epithelial cells, and fibroblasts (1, 20). Cumulatively, these data suggest that BLPs can promote increased parenchymal mast cells in BPD, which in turn may be involved in promoting the chronic inflammation and interstitial fibrosis characteristic of BPD. At present, we cannot rule out additional mechanisms for BLP-elicited accumulation of mast cells.

Genetic variation could explain why only some premature infants develop BPD. A family history of asthma is associated with a 12-fold increased risk of BPD (72, 73). Similarly, BLPs may be implicated in the pathophysiology of asthma, either as direct bronchoconstrictors (6) and/or by modulating the numbers of mast cells in the lung. Our present observations suggest a possible link between NE cells and the pathophysiology of asthma. There have been several reports of NE cell hyperplasia in animals sensitized for allergic airway reactions, with NE cell degranulation occurring after specific antigen challenge (1). Abnormal NE cells and/or NE cell hyperplasia have been identified in lungs of infants exposed to smoke in utero; such children of mothers who smoke are at a threefold increased risk of developing pediatric asthma (1). Finally, many of the bioactive peptides produced by pulmonary NE cells are potent bronchoconstrictors (1). The present study indicates that NE cell–derived peptides may have multiple roles in promoting airway inflammatory responses.

In conclusion, hyperoxic lung injury in premature baboons can be mediated by excessive production of BLPs (18), apparently derived from hyperplastic BLP-positive pulmonary NE cells. Cellular mechanisms for this injury include a cascade leading from NE cells to mast cells, via BRS-3 and NMB receptors on mast cells. Bronchoconstriction can occur early in the course of BPD (74), possibly secondary to mast cell accumulation elicited by BLPs. The definitive significance of mast cells for the pathogenesis of BPD in hyperoxic baboons remains to be clarified. As part of the innate immune system, lung mast cells can promote both innate and acquired immune responses by production of cytokines leading to inflammation, injury, and pulmonary fibrosis in later stages (61–63). Finally, the role of mast cells in the pathogenesis of "new BPD" that is currently observed in very low birthweight infants (24) is unclear but remains under investigation in the extremely premature baboon model of BPD (27). In conclusion, the current study opens new avenues for investigation of the pathophysiology of chronic inflammatory lung diseases.


   Acknowledgments
 
The authors thank the NIH for support of the Collaborative Program in BPD, headed by Dr. Jacqueline Coalson, including Dr. Brad Yoder, the Department of Pathology staff at the University of Texas Health Sciences Center (San Antonio, TX), the NICU and animal production staffs at the Southwest Foundation for Biomedical Research (San Antonio, TX), and Vicki Winter. The authors also thank Dr. James Crapo for helpful discussions.


   FOOTNOTES
 
Supported by NIH grants HL52638 (M.E.S.), HL52636 (Animal Core), HL56386 (P.F.W), and AI20241 (P.F.W.).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: M.S. has no declared conflict of interest; K.S. has no declared conflict of interest; D.H.C. has no declared conflict of interest; Y.K. has no declared conflict of interest; Y.E.M. has no declared conflict of interest; P.F.W. has no declared conflict of interest; K.W. has no declared conflict of interest; E.W. has no declared conflict of interest; M.E.S. has no declared conflict of interest.

Received in original form December 9, 2002; accepted in final form June 4, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sunday ME. Neuropeptides and lung development. In: McDonald JA, editor. Lung growth and development. New York: Marcel Dekker; 1997. p. 401–494.
  2. Johnson DE, Lock JE, Elde RP, Thompson TR. Pulmonary neuroendocrine cells in hyaline membrane disease and bronchopulmonary dysplasia. Pediatr Res 1982;16:446–454.[Medline]
  3. Wharton J, Polak JM, Bloom SR, Ghatei MA, Solcia E, Brown MR, Pearse AGE. Bombesin-like immunoreactivity in the lung. Nature 1978;273:769–770.[CrossRef][Medline]
  4. Wada E, Way J, Shapira H, Kusano K, Lebacq-Verheyden AM, Coy D, Jensen R, Battey J. cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor. Neuron 1991;6:421–430.[CrossRef][Medline]
  5. Siegfried JM, Guentert PJ, Gaither AL. Effects of bombesin and gastrin-releasing peptide on human bronchial epithelial cells from a series of donors: individual variation and modulation by bombesin analogs. Anat Rec 1993;236:241–247.[CrossRef][Medline]
  6. Impicciatore M, Bertaccini G. The bronchoconstrictor action of the tetradecapeptide bombesin in the guinea-pig. J Pharm Pharmacol 1973;25:872–875.[Medline]
  7. Kroog GS, Jensen RT, Battey JF. Mammalian bombesin receptors. Med Res Rev 1995;15:389–417.[Medline]
  8. Nagalla SR, Barry BJ, Creswick KC, Eden P, Taylor JT, Spindel ER. Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4). Proc Natl Acad Sci USA 1995;92:6205–6209.[Abstract/Free Full Text]
  9. Sunday ME. Bioactive peptides and lung development. In: Gaultier C, Bourbon JR, Post M, editors. Lung development. New York: Oxford University Press; 1999. p. 304–326.
  10. Toi-Scott M, Jones CL, Kane MA. Clinical correlates of bombesin-like peptide receptor subtype expression in human lung cancer cells. Lung Cancer 1996;15:341–354.[CrossRef][Medline]
  11. Kiaris H, Schally AV, Nagy A, Sun B, Armatis P, Szepeshazi K. Targeted cytotoxic analogue of bombesin/gastrin-releasing peptide inhibits the growth of H-69 human small-cell lung carcinoma in nude mice. Br J Cancer 2001;81:966–971.
  12. Sun B, Schally AV, Halmos G. The presence of receptors for bombesin/GRP and mRNA for three receptor subtypes in human ovarian epithelial cancers. Regul Pept 2000;90:77–84.[CrossRef][Medline]
  13. Sun B, Halmos G, Schally AV, Wang X, Martinez M. Presence of receptors for bombesin/gastrin-releasing peptide and mRNA for three receptor subtypes in human prostate cancers. Prostate 2000;42:295–303.[CrossRef][Medline]
  14. Ryan RR, Weber HC, Mantey SA, Hou W, Hilburger ME, Pradhan TK, Coy DH, Jensen RT. Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells. J Pharmacol Exp Ther 1998;287:366–380.[Abstract/Free Full Text]
  15. DeMichele MA, Davis AL, Hunt JD, Landreneau RJ, Siegfried JM. Expression of mRNA for three bombesin receptor subtypes in human bronchial epithelial cells. Am J Respir Cell Mol Biol 1994;11:66–74.[Abstract]
  16. Cullen A, Emanuel RL, Haley KJ, Torday JS, Asokananthan N, Sikorski KA, Sunday ME. Bombesin-like peptide (BLP) and BLP receptors in two different baboon models of bronchopulmonary dysplasia: diversity in gene expression yet similarity in function. Peptides 2000;21:1627–1638.[CrossRef][Medline]
  17. Johnson DE, Anderson WR, Burke BA. Pulmonary neuroendocrine cells in pediatric lung disease: alterations in airway structure in infants with bronchopulmonary dysplasia. Anat Rec 1993;236:115–119.[CrossRef][Medline]
  18. Sunday ME, Yoder BA, Cuttitta F, Haley KJ, Emanuel RL. Bombesin-like peptide mediates lung injury in a baboon model of bronchopulmonary dysplasia. J Clin Invest 1998;102:584–594.[Medline]
  19. Kim JS, McKinnis VS, White SR. Migration of guinea pig airway epithelial cells in response to bombesin analogues. Am J Respir Cell Mol Biol 1997;16:259–266.[Abstract]
  20. Yule KA, White SR. Migration of 3T3 and lung fibroblasts in response to calcitonin gene-related peptide and bombesin. Exp Lung Res 1999;25:261–273.[CrossRef][Medline]
  21. Erspamer GF, Mazzanti G, Farruggia G, Nakajima T, Yanaihara N. Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations. Peptides 1984;5:765–768.[CrossRef][Medline]
  22. Del Rio M, Hernanz A, De la Fuente M. Bombesin, gastrin-releasing peptide, and neuromedin C modulate murine lymphocyte proliferation through adherent accessory cells and activate protein kinase C. Peptides 1994;15:15–22.[CrossRef][Medline]
  23. Marion-Audibert AM, Nejjari M, Pourreyron C, Anderson W, Gouysse G, Jacquier MF, Dumortier J, Scoazec JY. Effects of endocrine peptides on proliferation, migration and differentiation of human endothelial cells [in French]. Gastroenterol Clin Biol 2000;24:644–648.[Medline]
  24. Northway WH. Bronchopulmonary dysplasia: twenty-five years later. Pediatrics 1992;89:969–973.[Abstract/Free Full Text]
  25. Miller VL, Rice JC, DeVoe M, Fos PJ. An analysis of program and family costs of managed care for technology-dependent infants with bronchopulmonary dysplasia. J Pediatr Nurs 1998;13:244–251.[CrossRef][Medline]
  26. Coalson JJ, Kuehl TJ, Escobedo MB, Hilliard JL, Smith F, Meredith K, Null DM, Walsh W, Johnson D, Robotham JL. A baboon model of bronchopulmonary dysplasia. II. Pathologic features. Exp Mol Pathol 1982;37:335–350.[CrossRef][Medline]
  27. Coalson JJ, Winter VT, Siler-Khodr T, Yoder BA. Neonatal chronic lung disease in extremely immature baboons. Am J Respir Crit Care Med 1999;160:1333–1346.[Abstract/Free Full Text]
  28. Cullen A, Van Marter LJ, Moore M, Parad R, Sunday ME. Urine bombesin-like peptide elevation precedes clinical evidence of bronchopulmonary dysplasia. Am J Respir Crit Care Med 2002;165:1093–1097.[Abstract/Free Full Text]
  29. Wada E, Watase K, Yamada K, Ogura H, Yamano M, Inomata Y, Eguchi J, Yamamoto K, Sunday ME, Maeno H, et al. Generation and characterization of mice lacking gastrin-releasing peptide receptor. Biochem Biophys Res Commun 1997;239:28–33.[CrossRef][Medline]
  30. Ohki-Hamazaki H, Watase K, Yamamoto K, Ogura H, Yamano M, Yamada K, Maeno H, Imaki J, Kikujama S, Wada E, et al. Mice lacking bombesin receptor subtype-3 develop metabolic defects and obesity. Nature 1997;390:165–169.[CrossRef][Medline]
  31. Ohki-Hamazaki H, Sakai Y, Kamata K, Ogura H, Okuyama S, Watase K, Yamada K, Wada K. Functional properties of two bombesin-like peptide receptors revealed by the analysis of mice lacking neuromedin B receptor. J Neurosc 1999;19:948–954.[Abstract/Free Full Text]
  32. Culling CFA, Allison RT, Barr WT. Cellular pathology technique. London: Butterworths; 1985.
  33. Whimster WF. Diagnostic morphometry in emphysema and chronic bronchitis. Anal Quant Cytol Histol 1985;7:183–186.[Medline]
  34. Ito F, Toyota N, Sakai H, Takahashi H, Iizuka H. FK506 and cyclosporin A inhibit stem cell factor-dependent cell proliferation/survival, while inducing upregulation of c-kit expression in cells of the line MC/9. Arch Dermatol Res 1999;291:275–283.[CrossRef][Medline]
  35. Bryant RW, She HS, Ng KJ, Siegel MI. Modulation of the 5-lipoxygenase activity of MC-9 mast cells: activation by hydroperoxides. Prostaglandins 1986;32:615–627.[CrossRef][Medline]
  36. Pivniouk VL, Martin TR, Lu-Kuo JM, Katz HR, Oettgen HC, Geha RS. SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells. J Clin Invest 1999;103:1737–1743.[Medline]
  37. Willett CG, Smith DI, Shridhar V, Wang M-H, Emanuel RL, Patidar K, Graham SA, Zhang F, Hatch V, Sugarbaker DJ, et al. Differential screening of a human chromosome 3 library identifies hepatocyte growth factor-like/macrophage-stimulating protein and its receptor in injured lung: possible implications for neuroendocrine cell survival. J Clin Invest 1997;99:2979–2991.[Medline]
  38. Battey JF, Way JM, Corjay MH, Shapira H, Kusano K, Harkins R, Wu JM, Slattery T, Mann E, Feldman RI. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc Natl Acad Sci USA 1991;88:395–399.[Abstract/Free Full Text]
  39. Fathi Z, Corjay MH, Shapira H, Wada E, Benya R, Jensen R, Viallet J, Sausville EA, Battey JF. BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J Biol Chem 1993;268:5979–5984.[Abstract/Free Full Text]
  40. Mantey SA, Coy DH, Pradhan TK, Igarashi H, Rizo IM, Shen L, Hou W, Hocart SJ, Jensen RT. Rational design of a peptide agonist that interacts selectively with the orphan receptor, bombesin receptor subtype 3. J Biol Chem 2001;276:9219–9229.[Abstract/Free Full Text]
  41. Caggiari L, Zanussi S, Crepaldi C, Bortolin MT, Caffau C, D'Andrea M, De Paoli P. Different rates of CD4+ and CD8+ T-cell proliferation in interleukin-2-treated human immunodeficiency virus-positive subjects. Cytometry 2001;46:233–237.[CrossRef][Medline]
  42. Keng PC, Siemann DW. Measurement of proliferation activities in human tumor models: a comparison of flow cytometric methods. Radiat Oncol Investig 1998;6:120–127.[CrossRef][Medline]
  43. Jensen RT, Mrozinski JE, Coy DH. Bombesin receptor antagonists: different classes and cellular basis of action. Recent Results Cancer Res 1993;129:87–113.[Medline]
  44. Lyle RE, Tryka AF, Griffin WS, Taylor BJ. Tryptase immunoreactive mast cell hyperplasia in bronchopulmonary dysplasia. Pediatr Pulmonol 1995;19:336–343.[Medline]
  45. Tokita K, Hocart SJ, Coy DH, Jensen RT. Molecular basis of the selectivity of gastrin-releasing peptide receptor for gastrin-releasing peptide. Mol Pharmacol 2002;61:1435–1443.[Abstract/Free Full Text]
  46. Moody T, Staley J, Zia F, Coy D, Jensen R. Neuromedin B binds with high affinity, elevates cytosolic calcium and stimulates the growth of small-cell lung cancer cell lines. J Pharmacol Exp Ther 1992;263:311–317.[Abstract/Free Full Text]
  47. Battey J, Wada E. Two distinct receptor subtypes for mammalian bombesin-like peptides. Trends Neurosci 1991;14:524–528.[CrossRef][Medline]
  48. Chang L, Subramaniam M, Yoder BA, Day BJ, Coalson JJ, Sunday ME, Crapo JD. A catalytic antioxidant attenuates alveolar structural remodeling in bronchopulmonary dysplasia. Am J Respir Crit Care Med 2003;167:57–64.[Abstract/Free Full Text]
  49. Haley KJ, Patidar K, Zhang F, Emanuel RL, Sunday ME. Tumor necrosis factor induces neuroendocrine differentiation in small cell lung carcinoma cell lines. Am J Physiol 1998;275:L311–L321.
  50. Emanuel RL, Torday JS, Mu Q, Asokananthan N, Sikorski KA, Sunday ME. Bombesin-like peptides and receptors in normal fetal baboon lung: roles in lung growth and maturation. Am J Physiol 1999;277:L1003–L1017.
  51. King KA, Torday JS, Sunday ME. Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a specific receptor-mediated mechanism. Proc Natl Acad Sci USA 1995;92:4357–4361.[Abstract/Free Full Text]
  52. Nakatani Y. Pulmonary endocrine cells in infancy and childhood. Ped Pathol 1991;11:31–48.
  53. Elizegi E, Pino I, Vicent S, Blanco D, Saffiotti U, Montuenga LM. Hyperplasia of alveolar neuroendocrine cells in rat lung carcinogenesis by silica with selective expression of proadrenomedullin-derived peptides and amidating enzymes. Lab Invest 2001;81:1627–1638.[Medline]
  54. Aguayo SM, Miller YE, Waldron JA, Sunday ME, Staton GW, King TE. Brief Report: idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells and airways disease. N Engl J Med 1992;327:1285–1288.[Medline]
  55. Aguayo SM. Pulmonary neuroendocrine cells in tobacco-related lung disorders. Anat Rec 1993;236:122–127.[CrossRef][Medline]
  56. Carroll NG, Mutavdzic S, James AL. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur Respir J 2002;19:879–885.[Abstract/Free Full Text]
  57. Balzar S, Wenzel SE, Chu HW. Transbronchial biopsy as a tool to evaluate small airways in asthma. Eur Respir J 2002;20:254–259.[Abstract/Free Full Text]
  58. Johnson DE, Georgieff MK. Pulmonary neuroendocrine cells, their secretory products and their potential roles in health and chronic lung disease in infancy. Am Rev Respir Dis 1989;140:1807–1812.[Medline]
  59. Weber S, Babina G, Henz BM. Human leukemic (HMC-1) and normal skin mast cells express ß2-integrins: characterization of ß2-integrins and ICAM-1 on HMC-1 cells. Scand J Immunol 1996;45:471–481.
  60. Olsson N, Rak S, Nilsson G. Demonstration of mast cell chemotactic activity in bronchoalveolar lavage fluid collected from asthmatic patients before and during pollen season. Clin Immunol 2000;105:455–461.
  61. Temann UA, Geba GP, Rankin JA, Flavell RA. Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J Exp Med 1998;188:1307–1320.[Abstract/Free Full Text]
  62. Galli SJ, Gordon JR, Wershil BK. Mast cell cytokines in allergy and inflammation. Agents Actions 1993;43:209–220.[CrossRef]
  63. Akers IA, Parsons M, Hill MR, Hollenberg MD, Sanjar S, Laurent GJ, McAnulty RJ. Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2. Am J Physiol 2000;278:L193–L201.
  64. Groneck P, Speer CP. Pulmonary inflammation in the pathogenesis of bronchopulmonary dysplasia. Pediatr Pulmonol Suppl 1997;16:29–30.[Medline]
  65. Raghavender B, Smith JB. Eosinophil cationic protein in tracheal aspirates of preterm infants with bronchopulmonary dysplasia. J Pediatr 1997;130:944–947.[CrossRef][Medline]
  66. Benya R, Kusui T, Pradhan T, Battey J, Jensen R. Expression and characterization of cloned human bombesin receptors. Mol Pharmacol 1995;47:10–20.[Abstract]
  67. Ryan RR, Weber HC, Hou W, Sainz E, Mantey SA, Battey JF, Coy DH, Jensen RT. Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. J Biol Chem 1998;273:13613–13624.[Abstract/Free Full Text]
  68. Coy DH, Mungan Z, Rossowski WJ, Cheng BL, Lin JT, Mrozinski JE Jr, Jensen RT. Development of a potent bombesin receptor antagonist with prolonged in vivo inhibitory activity on bombesin-stimulated amylase and protein release in the rat. Peptides 1992;13:775–781.[CrossRef][Medline]
  69. Aguayo SM, Kane MA, King TE, Schwarz MI, Grauer L, Miller YE. Increased levels of bombesin-like peptides in the lower respiratory tract of asymptomatic cigarette smokers. J Clin Invest 1989;84:1105–1113.
  70. Moody TW, Jensen RT, Garcia L, Leyton J. Nonpeptide neuromedin B receptor antagonists inhibit the proliferation of C6 cells. Eur J Pharmacol 2000;409:133–142.[CrossRef][Medline]
  71. Burghardt B, Wenger C, Barabas K, Racz G, Olah A, Flautner L, Coy DH, Gress TM, Varga G. GRP-receptor-mediated signal transduction, gene expression and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF. Peptides 2001;22:1119–1128.[CrossRef][Medline]
  72. Nickerson BG, Taussig LM. Family history of asthma in infants with bronchopulmonary dysplasia. Pediatrics 1980;65:1140–1144.[Abstract/Free Full Text]
  73. Evans M, Palta M, Sadek M, Weinstein MR, Peters ME. Associations between family history of asthma, bronchopulmonary dysplasia, and childhood asthma in very low birth weight children. Am J Epidemiol 1998;148:460–466.[Abstract/Free Full Text]
  74. Motoyama EK, Fort MD, Klesch KW, Mutich RL, Guthrie RD. Early onset of airway reactivity in premature infants with bronchopulmonary dysplasia. Am Rev Respir Dis 1987;136:50–57.[Medline]



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