Bronchopulmonary dysplasia

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.

Keywords: 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 poslnalally 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

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 hypcroxic model of BPD (140/100% 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 [mu]l 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 [mu]g). There was no mortality of the mice in any experimental groups. After CO^sub 2^ euthanasia, lungs were inflated in situ with 4% paraformaldehyde.

Histochemistry

Five-micrometer baboon lung paraffin sections were immunostained for tryplase 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 histaminc 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[degrees]C in 5% CO^sub 2^ and serum-free Dulbecco's modified Eagle's medium (DMEM)-0.1% BSA (DMEM-BSA) for 18 hours before assay. Cells (15 X 10^sup 4^/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-, Tyr^sup 6^,[beta]-Ala^sup 11^,Phe13,Nle^sup 14^]bombesin^sub (6-14)^ (BRS-3L) (40). After 24 hours, cells were harvested for Ki-67 flow cytometry (41, 42). GRP-R-specific antagonist D-Tyr^sup 6^-bombesin^sub (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 (10^sup 6^ in 100 [mu]l of PBS-BSA) were plated in the upper chambers of Transwell inserts (pore size, 5 [mu]m; 6.5 mm in diameter; polycarbonate) in 24-well plates and incubated for 3 hours at 37[degrees]C. The lower chambers had 600 [mu]l of PBS-BSA with or without 0.1-10 nM bombesin, GRP^sub (1-27)^, GRP^sub (1-14)^, GRP^sub (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 hemacytomeler. 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 / test or analysis of variance.

RESULTS

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 lest 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 O^sub 2^ 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

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 (141d/ 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/mm^sup 2^ lung tissue) compared with 140d/PRN baboons (3.6/mm^sup 2^) (p

Lung Mast Cells in Mice Given BLP Intratracheally

To directly test the hypothesis that BLPs can function as proinfiammatory cytokines in the lung, we administered bombesin (5 [mu]g in 75 [mu]l 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

To determine which of the three cloned mammalian BLP receptors might mediate these mast cell responses, we administered bombesin intratracheally (5 [mu]g/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-Tyr^sup 6^-bombesin^sub (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.

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

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

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

DISCUSSION

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.

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.

Acknowledgment: 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.

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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

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

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

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@tch.harvard.edu

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

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