Acute Lung Injury with Endotoxin or NO2 Does Not Enhance ...

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doi:10.1016/j.ymthe.2005.05.007

Acute Lung Injury with Endotoxin or NO2 Does Not Enhance Development of Airway Epithelium from Bone Marrow Travis Beckett, Roberto Loi, Robert Prenovitz, Matthew Poynter, Kaarin K. Goncz, Benjamin T. Suratt, and Daniel J. Weiss* Pulmonary and Critical Care, Vermont Lung Center, University of Vermont College of Medicine, 226 Health Sciences Research Facility, Burlington, VT 05405, USA *To whom correspondence and reprint requests should be addressed. Fax: +1 802 656 8926. E-mail: [email protected].

Available online 18 July 2005

Adult marrow-derived stem cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. Lung injury increases recruitment of the marrow-derived cells. We speculated that comparing patterns of lung engraftment following different lung injuries would provide insight into potential mechanisms by which marrow-derived cells were recruited to lung. To evaluate this, adult female C57Bl/6 mice irradiated and engrafted with marrow from adult male transgenic GFP mice were exposed to either intranasal inhalation of endotoxin (25 Ag/mouse) or 3 days of 25 ppm NO2 and then compared 1 or 3 months later to transplanted but otherwise uninjured mice. In all cases, the majority of marrow-derived cells recruited to lung were CD45+ leukocytes. In lungs of transplanted but otherwise uninjured mice, small numbers of CD45 donor-derived cells in alveolar septae stained positively for pro-surfactant protein C. Rare donor-derived cells located in the airway epithelium stained positively with cytokeratin. Subsequent exposure of engrafted mice to NO2 or endotoxin did not significantly increase the number or pattern of donor-derived CD45 cells found in recipient lungs. These results suggest that NO2 or endotoxin lung injury does not result in significant engraftment of marrow-derived cells in lung. Key Words: lung, airway epithelium, stem cell, bone marrow transplant, endotoxin, NO2

INTRODUCTION Stem cells derived from adult murine bone marrow can, following systemic administration to adult recipient mice, localize to and acquire immunophenotypic characteristics of different types of lung cells [1–6]. Similar chimerism has also been demonstrated in lungs of clinical hematopoietic stem cell and lung transplant recipients [7,8]. The phenotypes most frequently ascribed to the incorporated cells are those of type 1 and 2 alveolar epithelial cells and of interstitial cells [5–11]. In several instances, functional behavior of the marrow-derived cells has been suggested, particularly in models of acute lung injury or of lung fibrosis [4,5,11–13]. Some reports also suggest that adult marrow-derived cells acquire phenotypic markers of airway epithelial cells [2]. These results suggest that adult bone marrow-derived cells might be utilized therapeutically to replace or repopulate defective tissues in diseased lungs [6,14]. However, the mechanisms by which marrow-derived stem cells are recruited to lung and acquire immuno-

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phenotypic markers and functional properties consistent with lung epithelial or interstitial cells are unknown. Injury to the lung increases recruitment of marrowderived stem cells to lung and phenotypic conversion to lung cells [3–5,9,11–13]. However, lung injury also increases recruitment of mature inflammatory cells, i.e., leukocytes, to lung. In studies presented to date, it is not always clear whether cells of donor marrow origin found in lung, for example as determined by presence of the Y chromosome following male to female transplantation, are donor marrow-derived mature leukocytes or are donor-derived marrow stem cells that have acquired the immunophenotype of lung epithelial or interstitial cells. We are interested in the potential for adult marrowderived cells to repair defective airway epithelium in disease such as cystic fibrosis. However, at present the recruitment of only small numbers of chimeric airway epithelial cells has been demonstrated. Understanding the conditions under which marrow-derived cells are

MOLECULAR THERAPY Vol. 12, No. 4, October 2005 Copyright C The American Society of Gene Therapy 1525-0016/$30.00

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recruited to lung, particularly to airway epithelium, and are induced to undergo phenotypic conversion to airway epithelial cells will allow development of strategies to increase the efficiency of these processes. As an initial inquiry, we postulated that different methods of inducing lung injury or stimulating inflammatory signaling from airway epithelium would result in different patterns of marrow-derived cell recruitment and phenotypic conversion to airway epithelial cells. To investigate this, we compared the recruitment of marrow-derived cells to airway epithelium in chimeric mice after radiationinduced lung injury alone or after radiation-induced lung injury followed by stimulation of airway epithelial inflammatory signaling in vivo by exposure either to intranasally instilled endotoxin or to inhaled NO2.

RESULTS Transplantation and Lung Injury The survival rate of adult female C57Bl/6 mice myeloablated by total body irradiation and transplanted with total marrow cells from male transgenic green fluorescent protein (GFP)-expressing mice was 100%. After 1 month we engrafted all recipient mice with an average of 82 F 17% peripheral leukocytes of donor origin as determined by the percentage of peripheral leukocytes expressing GFP using flow cytometry. To confirm that lung injury had been induced by either endotoxin or NO2 administration, we assessed a parallel group of nonablated mice either 24 h following endotoxin or 48 h following NO2, correlating with the peak injury for each respective model. These mice demonstrated airway and alveolar abnormalities characteristic of each respective injury. Photomicrographs of nonablated endotoxin or NO2injured mouse lungs demonstrate characteristic features of lung damage, including inflammatory cell infiltration, peribronchial collections of inflammatory cells, and alveolar wall thickening (Figs. 1A and 1B) [16,17]. Shown also for comparison are lungs from mice assessed 2 months after irradiation and transplantation in which no obvious inflammatory injury is evident (Fig. 1C). Assessments of lungs of transplanted mice 1 month after NO2 administration (corresponding to 2 months after irradiation and transplantation) demonstrate no obvious evidence of inflammatory injury (Fig. 1D). Mouse lungs assessed 1 month after endotoxin injury, similarly corresponding to 2 months after irradiation and transplantation, demonstrate only small residual foci of inflammatory injury mostly consisting of peribronchial inflammatory cell collections (Fig. 1E). The majority of lung airways and parenchyma appear normal. These results demonstrate that acute lung injury induced by irradiation with or without subsequent endotoxin or NO2 exposure had largely resolved at the times lungs in each experimental group were assessed for the presence

MOLECULAR THERAPY Vol. 12, No. 4, October 2005 Copyright C The American Society of Gene Therapy

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of donor-derived marrow cells (2 and 4 months after transplantation). Localization of Donor Marrow-Derived Cells to Lung Epithelium in Chimeric Mice Photomicrographs of irradiated-only chimeric mouse lungs assessed at 2 or 4 months after transplantation demonstrate numerous donor-derived cells staining positively for the Y chromosome (Fig. 2). Two months after transplantation, the majority of donor-derived cells were located in lung parenchyma and co-stained for CD45, indicating their identity as donor-derived leukocytes (Figs. 2A and 2B). Some CD45 donor-origin cells were also located in the interstitium just below the airway epithelium (Fig. 2A) and in the alveolar septa (Fig. 2B). However, we did not further characterize these cells. We observed similar findings 4 months after transplantation (Figs. 2C and 2D). At both 2 and 4 months, some Y+ CD45 cells in the alveolar septa stained for prosurfactant protein C (pro-SPC), suggesting an immunophenotype consistent with type 2 alveolar epithelial cells (Figs. 2B and 2D). There was no obvious difference in number or localization of CD45 donor-derived cells between lungs harvested at 2 or 4 months after transplantation. We observed no obvious histologic indication of pneumonitis, suggesting that any radiation-related lung injury induced by myeloablation (total body irradiation) at the time of transplantation had resolved by the times of lung assessment. Localization of Donor Marrow-Derived Cells to Lung Epithelium Following NO2 or Endotoxin Lung Injury in Chimeric Mice One month after NO2 administration to irradiated chimeric mice (corresponding to 2 months after transplantation), we similarly observed numerous Y+ donorderived cells in the lung (Fig. 3). The majority of these were located in the parenchyma with a few located beneath the airway epithelium. As in the irradiated-only chimeric mice, most cells also stained positively for CD45. We observed rare cells in the airway and alveolar epithelium, particularly in distal airways, that were Y+ and cytokeratin+ but CD45 (Fig. 3A). Similarly, some cells in the alveolar walls were Y+ CD45 pro-SPC+, suggesting an immunophenotype consistent with type 2 alveolar epithelial cells (Fig. 3B). Three months after NO2 administration (4 months after transplantation), more donor-derived cells were apparent in the airway epithelial wall. While the majority of these were CD45+, some were CD45 and were localized beneath Clara cell secretory protein (CCSP)-positive airway epithelial cells (Fig. 3C). As at 1 month, we observed scattered Y+ CD45 pro-SPC+ cells in alveolar walls (Fig. 3D). We observed a different pattern of donor-derived cells in lungs 1 month after endotoxin administration (2 months after transplantation) to irradiated chimeric mice

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FIG. 1. Photo micrographs of hematoxylin and eosin-stained lung sections from (A) naRve (unirradiated) C57Bl/6 mouse lung 24 h after endotoxin administration, (B) naRve (unirradiated) C57Bl/6 mouse lung 48 h after NO2 administration, (C) female C57Bl/6 mouse 2 months after total body irradiation and transplantation of total marrow cells from male GFP transgenic mouse, (D) irradiated and transplanted mouse 1 month after NO2 administration (2 months after irradiation and transplantation), (E) irradiated and transplanted mouse 1 month after NO2 administration (2 months after irradiation and transplantation), and (F) naRve female mouse lung. Several features of acute lung injury are noted in A and B, including inflammatory cell infiltrates and alveolar wall thickening (arrows). Mouse lungs depicted in C–F display no obvious features of acute lung injury. Original magnification: 100.

FIG. 2. Photomicrographs of lung sections from female C57Bl/6 mice irradiated and transplanted with adult male donor cells and subsequently assessed either 2 (A, B) or 4 (C, D) months later. FISH for the Y chromosome is indicated in red and immunostaining for CD45 is indicated in blue. Green immunostaining indicates either Clara cell secretory protein (A, C) or pro-SPC (B, D). Nuclear staining in B is indicated in gray and by light blue arrows. Examples of specific donor-derived cells are indicated as follows: dark pink arrows, Y+CD45 cells in subairway interstitium (A, C); yellow arrows, Y+CD45+ donororigin leukocytes; white arrows, Y+CD45 cells in the alveolar septa; orange arrow, Y+CD45 pro-SPC+ cell in the alveolar septa (B, D). Asterisks indicate region of magnified insets for each image. Samples are representative of sections from two mice per experimental condition. Original magnifications: A, D, 200; B, C, 400.

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FIG. 3. Photomicrographs of lung sections from irradiated chimeric female C57Bl/6 mice subsequently exposed to NO2 and assessed either 1 (A, B) or 3 (C, D) months later. FISH for the Y chromosome is indicated in red and immunostaining for CD45 is indicated in blue. Green immunostaining indicates cytokeratin (A), Clara cell secretory protein (C), or pro-SPC (B, D). Examples of specific donor-derived cells are indicated as follows: light pink arrows, Y+CK+ cells in airway and alveolar walls (A); yellow arrows, Y+CD45+ donor-origin leukocytes; dark pink arrows, Y+CD45 cells in the subairway interstitium (C); white arrow, Y+CD45 cell in the alveolar septa (B); orange arrow, Y+CD45 pro-SPC+ cell in the alveolar septa (B, D). Asterisks indicate region of magnified insets for each image. Samples are representative of sections from two mice per experimental condition. Original magnifications: A, B, C, 600; D, 400.

FIG. 4. Photomicrographs of lung sections from irradiated chimeric female C57Bl/6 mice subsequently exposed to intranasal endotoxin (25 Ag/mouse) and assessed either 1 (A, B) or 3 (C, D) months later. FISH for the Y chromosome is indicated in red and immunostaining for CD45 is indicated in blue. Green immunostaining indicates cytokeratin (A), pro-SPC (B, D), or CCSP (C). Examples of specific donor-derived cells are indicated as follows: light pink arrows, Y+CK+ cells in airway and alveolar walls (A); yellow arrows, Y+CD45+ donor-origin leukocytes; white arrows, Y+CD45 cell in the alveolar septa (B, D); orange arrows, Y+CD45 pro-SPC+ cell in the alveolar septa (B, D). Asterisks indicate region of magnified insets for each image. Samples are representative of sections from two mice per experimental condition. Original magnifications: A, 400; B, D, 200; C, 600.


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TABLE 1: Summary of FISH immunofluorescence data

Experimental group (n) Irradiation Irradiation Irradiation Irradiation Irradiation Irradiation a

only 2 months (2) only 4 months (2) + NO2 1 month (2) + NO2 3 months (2) + LPS 1 month (2) + LPS 3 months (2)

% donor-derived cells of total cells

Normalized % donor-derived cells of total cellsa

% CD45+ cells of Y+ cells

% pro-SPC+ cells of Y+ cells

% CCSP+ cells of Y+CD45– cells

18.2 15.5 20.8 22.4 23.0 30.2

24.2 20.7 27.7 29.9 30.7 40.3

94.5 93.0 95.7 95.2 96.4 96.6

0.3 0.3 0.2 0.2 0.2 0.2

0.0 0.0 0.0 0.0 0.0 0.0

The detection frequency of donor-derived cells was normalized to the fraction of cells showing Y positivity by FISH in a male control (75%) to compensate for partial nuclear sampling.

(Fig. 4). We observed numerous Y+ CD45+ cells in clusters in the interstitium, frequently just under the airway epithelium (Fig. 4A). These corresponded with collections of lymphoid-appearing cells as assessed by light microscopy of the same slides (Fig. 1E). However, we also observed rare cells in the airway and alveolar epithelial walls that were Y+ but CD45 and stained with either cytokeratin or pro-SPC (Figs. 4A and 4B). Three months after endotoxin administration (4 months after transplantation), more donor-derived cells were apparent in the airway epithelium and parenchyma (Figs. 4C and 4D). Similar to lungs harvested 3 months after NO2 administration, the majority of donor-derived cells were CD45+. However, donor-derived CD45 and pro-SPC+ cells were detectable (approximately 0.5%), suggesting acquisition of an epithelial phenotype. We observed similar percentages by cell counting in the irradiatedonly and NO2-treated mice. Quantitative results from all experiments are summarized in Table 1.

DISCUSSION In summary, these results demonstrate that, following transplantation of adult total bone marrow cells, the majority of donor-derived cells found in lungs were leukocytes. Only a very small number of donor-derived cells appear to have acquired immunophenotypic markers of lung epithelium. Of those demonstrating lung-specific markers, most were consistent with type 2 alveolar epithelial cells. Further epithelial stimulation in chimeric mice with either endotoxin or NO2 did not increase the number of donor-origin cells acquiring type 2 alveolar epithelial cell phenotype or localizing to the airway wall. Under all experimental conditions evaluated, only rare CD45-negative donor-origin cells appeared to localize to airway epithelium or subairway epithelial areas. We have not yet further characterized these cells. A number of recent reports have suggested that more extensive chimerism of lung epithelium and interstitium may occur in mouse models [2,5,7,9,13]. These studies have utilized a variety of lung injury models, and donor cells have included total marrow, selected populations of

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hematopoietic stem cells, mesenchymal stem cells, and fetal liver cells. As such, there is as yet no clear pattern wherein specific populations of donor marrow cells acquire phenotypic markers of specific lung epithelial or interstitial cells. Radiation-induced lung injury is recognized as contributing to engraftment of lung with marrow-derived cells [9,12]. To attempt to minimize radiation effects, we had utilized a lower dose, 1000 cGy, than the more commonly used dose of 1200 cGy utilized in studies that have reported higher levels of radiation-related engraftment [9,11,12]. While the difference between 1000 and 1200 cGy might seem slight, it might be around a threshold of lung injury that could explain the differences in outcomes. However, one other study that utilized a similar radiation dose of 1000 cGy, delivered as two doses of 500 cGy given 3 h apart, reported that up to 27.5% of Col1-expressing fibroblasts were of donor origin in mouse lungs injured with bleomycin [13]. Another possible explanation for the discrepancy between our results and the published results is the improved histologic techniques utilized to perform FISH and cell-specific immunostaining on the same sections [15]. Using this approach demonstrated that the majority of donor-origin cells found in lung are in fact donor leukocytes as indicated by CD45 expression. Nonetheless, we did observe rare donor marrow-derived cells that were CD45 negative and that localized to airway epithelial areas and to alveolar septa. Although the histologic technique we utilized is powerful, it is possible that even these cells may have reflected a juxtaposition of a CD45positive leukocyte with a lung epithelial or interstitial cell such that the CD45 immunophenotyping was not evident. We are expanding these studies to characterize more extensively donor marrow-derived cells using flow cytometry and other techniques to identify better any potential donor marrow-derived cells that have other than a leukocyte phenotype. The different lung injuries investigated in this study did result in different patterns of inflammatory cell, i.e., mature leukocytes of donor hematopoietic stem cell origin, recruitment to lung. However, the different mechanisms of inducing lung injury did not result in


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substantially different patterns of nonleukocyte donor marrow-derived cells that engrafted as lung. Each of the lung injuries evaluated results in release of a different pattern of chemokines and cytokines as well as expression of different cell surface markers and extracellular matrix proteins [16,17]. Based on previous studies suggesting that soluble chemoattractant molecules as well as specific extracellular matrix proteins may function to recruit stem cells to lung [4,18], we had expected to find different patterns of lung epithelial and interstitial engraftment with the different injury models. However, as there were only rare donor marrow-derived cells that did not appear to be leukocytes, potential differences in patterns of chimerism may not have been obviously evident. Nonetheless, we were surprised to find that further epithelial stimulation with either endotoxin or NO2 had no significant effect on lung engraftment in the chimeric mice. The endotoxin results are in contrast to those in which transplantation with fetal liver cells mitigated lung injury following endotoxin administration to irradiated mice [5]. In that study, marrow origin cells appeared to acquire immunophenotypic markers of both type 1 alveolar epithelial cells and endothelial cells. However, in that study, both a different donor cell population and a higher radiation dose was utilized. Further, radiation was delivered as a split dose in contrast to our approach, which utilized a single dose. The effect of single-dose irradiation [2,9] vs split-dose irradiation on marrow stem cell engraftment in lung is unclear. The presence of mature donor-origin leukocytes in the lung at 1 or 3 months after either endotoxin or NO2 exposure in this study was unanticipated. In both of these models, lung injury should have resolved by these times [16,17]. The results suggest that the combination of irradiation with either endotoxin or NO2 results in prolonged inflammation yet does not increase apparent transdifferentiation of marrow-derived cells into lung cells. These observations are in contrast to those recently noted in murine gastric epithelium in which chronic inflammation elicited by Helicobacter infection resulted in robust derivation of epithelium from marrow-derived cells [19]. None of the lung injury models evaluated in our study was specific for airway epithelium. Radiation, endotoxin, and NO2 can all also result in alveolar and interstitial injury. Use of injury models more selective for airway epithelium may result in higher levels of airway epithelial chimerism. This study also did not address the issue of whether donor-origin cells fused with lung epithelial or interstitial cells. Fusion is well documented in liver and muscle but whether this occurs in lung is less clear [20–22]. In summary, chimerism of lung epithelium following transplantation of adult whole bone marrow is a rare event, even in the presence of clinically relevant lung injuries that can affect airway and alveolar epithelium. If


repopulation of airway epithelium with adult marrow origin cells is to be considered as a potential therapeutic approach for airway diseases, further strategies need to be devised to increase airway epithelial chimerism.

METHODS Animals All studies were subject to IACUC review at the University of Vermont (UVM) and conformed to institutional and AAALAC standards for humane treatment of laboratory animals. Mice were housed in pathogen-free barrier facilities (ventilator racks) in the Small Animal Facility at UVM. Adult male (6–12 weeks) transgenic C57Bl/6 mice constitutively expressing GFP under the control of the ubiquitin promoter (courtesy of Phillippa Marrack, Ph.D., National Jewish Medical Center, Denver, CO, USA) were utilized as donors. Adult female C56Bl/6 mice (8–12 weeks; The Jackson Laboratory) were used as recipients. Bone Marrow Harvest Adult male GFP mice (6–12 weeks) were euthanized by intraperitoneal overdose of sodium pentobarbital. The legs and pelvis were removed and overlying skin, muscle, and soft tissue removed by blunt dissection. The femur and tibia from each leg and the pelvis were isolated and residual tissue was removed by rubbing with a sterile-gauze cover sponge. The bones were rinsed in 70% ethanol and stored in culture medium (RPMI 1640; Cellgro) with 2% FBS, 1% l-glutamine, 1% P/S. Under sterile conditions, the ends of each bone were cut off with a scalpel blade and the marrow was flushed out by inserting a 25-gauge syringe and rinsing with medium until clear. Recovered total marrow cells were centrifuged (500g 5 min), washed once with fresh medium, and then resuspended in fresh medium (DulbeccoTs modification of EagleTs medium; Cellgro) with 15% FBS, 1% l-glutamine, 1% P/S [2,3]. The cells were passed through a 100Am filter to remove bone fragments and debris and total number of cells was counted with a hemacytometer. On average, approximately 150 106 total marrow cells were recovered from a single mouse. Myeloablation and Transplantation On the day of donor cell administration, 12 adult female C57Bl/6 (recipient) mice underwent total body irradiation (1000 cGy using a cesium-137 cell irradiator (Nordion International ISO 1000, Model B; Vancouver, BC, Canada)). Approximately 4 h after irradiation, mice received resuspended total bone marrow cells (20–40 106 cells/mouse) by tail vein injection. Mice were subsequently housed under barrier conditions and fed sterilized food and antibiotic-treated water. Engraftment was assessed 30 days after transplantation by determining the percentage of peripheral leukocytes expressing GFP using flow cytometry. In Vivo Stimulation of Airway Epithelial Inflammatory Signaling Some of the engrafted mice were subjected to NO2 or endotoxin administration 1 month after transplantation. NO2. Four chimeric mice were subjected to whole-body exposure to 25 ppm of NO2 for 6 h/day for 3 consecutive days at the Inhalation Toxicology Facility on the University of Vermont campus. This approach induces a neutrophil-dominated airway inflammatory response as well as injury to type 2 alveolar epithelial cells [16]. Indicative of the transient nature of this model, NO2-injured mice had fully recovered and were indistinguishable from air-exposed controls by 1 week following exposure. Endotoxin. Four transplanted chimeric mice were anesthetized by ip injection of Avertin (5–10 mg/kg of 25% stock solution). Twenty-five micrograms of endotoxin (Escherichia coli 0111:B4 endotoxin; Sigma) diluted in 50 Al of sterile saline was then administered by intratracheal instillation [17]. This results in acute lung injury marked by lung edema, increase in proinflammatory cytokines, and recruitment of inflammatory cells to the lung within 3–6 h. Mice recover from injury approximately 2–3 days after administration [17].

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Both the endotoxin and the NO2 injury protocols are well established in our laboratory. Nonetheless, as the acute lung injury resulting from either endotoxin or NO2 would normally have resolved at the time of lung assessment in this study, a separate group of naRve (unirradiated) C57Bl/6 mice were concurrently exposed to either endotoxin or NO2 along with the transplanted mice. The naRve injured mice were euthanized and lungs assessed either 1 or 3 days, respectively, after endotoxin or NO2 exposure. Increases in bronchoalveolar lavage fluid content of inflammatory cells and total protein, as well as characteristic airway and alveolar abnormalities consistent with each type of acute lung injury, were observed. These observations confirm that acute lung injury was induced with the methods utilized. Assessment of Donor Cells in Recipient Lungs Chimeric mice were euthanized by lethal overdose of pentobarbital at either 1 or 3 months after exposure to endotoxin or NO2 (n = 2 for each experimental condition). This corresponded, respectively, to 2 or 4 months after myeloablation and transplantation. Control chimeric mice not exposed to either agent were similarly euthanized 2 or 4 months after transplantation (n = 2 for each experimental time point). The chest was opened in situ and the vasculature perfused by infusing 10–20 cc of normal saline into the right ventricle. The trachea was cannulated with a small-gauge butterfly syringe and the lungs were removed en bloc. Four percent paraformaldehyde was infused into the trachea until the lungs were inflated and the lungs were subsequently gravity fixed at 20 cm pressure for 2 h at 48C. Lungs were also immersed in the paraformaldehyde during this period. Following fixation, lungs were paraffin embedded and 5-Am cut sections mounted on glass slides. Hematoxylin and eosin-stained sections were assessed for inflammation and injury. Detection of donor-derived cells was determined by the presence of Ychromosome-positive cells in lung sections detected using the method we have developed [15]. In brief, murine Y paint probe was digoxigenin labeled (DIG-Nick Translation Mix; Roche, Penzberg, Germany), denatured, and reannealed in the presence of mouse Cot-1 DNA (Invitrogen, San Diego, CA, USA) to reduce nonspecific hybridization. Deparaffinized 5-Am mounted lung sections were immersed in 10 mM sodium citrate for 15 min at 968C. After denaturation in 60% formamide/2 SSC at 708C, slides were incubated overnight with digoxigenin-labeled Y paint probe. After being washed in 50% formamide/2 SSC at 428C for 2 min to remove unbound probe, slides were incubated for 1 h at room temperature with Cy3-conjugated mouse anti-digoxigenin (Jackson ImmunoResearch, West Grove, PA, USA; 1:100 dilution). The presence of probe hybridization signal was assessed by fluorescence microscopy prior to the immunohistochemical detection step. Detection of cell-type-specific phenotypic markers was subsequently done on the same histologic sections using a set of antibodies directed against epithelium and leukocytes. Antibodies that were used include rabbit anti-cow pancytokeratin (DakoCytomation, Carpinteria, CA, USA; 1:500 dilution), goat anti-mouse Clara cell secretory protein (gift from Barry Stripp, Ph.D., University of Pittsburgh, PA, USA; 1:5000 dilution), rabbit anti-human pro-SPC (Chemicon, Temecula, CA, USA; 1:1000 dilution), rat anti-mouse CD45 (Caltag Laboratories, Burlingame, CA, USA; 1:500 dilution). Secondary detection was done using AlexaFluor 488 donkey anti-goat, AlexaFluor 488 goat anti-rabbit, and AlexaFluor 647 goat anti-rat antibodies (Molecular Probes, Eugene, OR, USA) at a 1:300 dilution. All antibodies were diluted in 10 mM PBS containing 0.5% BSA and 0.1% Triton X. For nuclear staining, sections were incubated with Hoechst 33342 dye (Molecular Probes) at room temperature for 10 min. Sections were systematically visualized with an Olympus BX50 confocal microscope equipped with a krypton–argon mixed-gas multiline laser using the following settings: green excitation 488 nm, filter cube emission 522DF32; red excitation 568 nm, filter cube emission 605DF32; far-red excitation 647 nm, filter cube emission 680DF32. Images were acquired by Bio-Rad Lasersharp 2000 software (PSI, Inc., League City, TX, USA). Images with nuclear staining were captured with a Zeiss LSM 510 META confocal microscope. Cell counts of Y-chromosome-positive, CD45-positive cells (representative of mature donor-derived leukocytes) and of Y-chromosome-

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positive, CD45-negative, pro-SPC-positive cells (representative of donorderived type II pneumocytes) were done on 20 randomly selected 60 power fields per lung.

ACKNOWLEDGMENTS The authors acknowledge Justin Robbins and David Hemenway, Ph.D., of the Inhalation Toxicology Facility and Department of Civil and Environmental Engineering at the University of Vermont for assistance with NO2 exposures and Winifred Trotman and Douglas Taatjes, Ph.D., of the University of Vermont Imaging Core and Department of Pathology for assistance with fluorescence and confocal microscopy. The authors also thank Diane Krause, M.D., Yale University, for the Y probe and for helpful advice and discussion. These studies were supported by NHLBI HL03864, NCRR P20 RR15557, and the Cystic Fibrosis Foundation. RECEIVED FOR PUBLICATION APRIL 13, 2005; REVISED MAY 16, 2005; ACCEPTED MAY 24, 2005.

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