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Stem Cells Vol. 24 No. 8 August 2006, pp. 1986 -1992
doi:10.1634/stemcells.2005-0579; www.StemCells.com
© 2006 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Threshold of Lung Injury Required for the Appearance of Marrow-Derived Lung Epithelia

Erica L. Herzoga, John Van Arnamb, BuQu Hub, Diane S. Krauseb

Departments of aInternal Medicine and
bLaboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

Key Words. Stem cell plasticity • Lung injury • In vivo • Mice

Correspondence: Erica Herzog, M.D., Ph.D., Department of Medicine, Pulmonary and Critical Care Division, 333 Cedar St. TAC 441-S, New Haven, Connecticut 06520, USA. Telephone: 203-785-3627; Fax: 203-785-3826; e-mail: erica.herzog{at}yale.edu

Received November 22, 2005; accepted for publication May 1, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Bone marrow-derived cells (BMDCs) can adopt an epithelial phenotype in the lung following bone marrow transplantation (BMT). This phenomenon has been assumed to result from the lung injury that occurs with myeloablative radiation. To date, no study has related the degree of epithelial chimerism following bone marrow transplantation to the lung damage induced by preconditioning for BMT. Such a goal is crucial to understanding the local host factors that promote the engraftment of BMDCs as lung epithelia. We undertook this aim by performing sex-mismatched bone marrow transplantation using a variety of preconditioning regimens and comparing measurements of lung injury (bronchoalveolar lavage [BAL] cell count, alveolar-capillary leak assayed by BAL protein levels, and terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis on epithelial cells) with rigorous methods to quantify bone marrow-derived lung epithelia (costaining for epithelial and donor markers on tissue sections and isolated lung epithelia in recipient mice). We found that only at doses that induced lung injury could marrow derived lung epithelium be identified following BMT. With irradiation doses less than 1,000 centigray (cGy), there was little to no apparent injury to the lung, and there were no marrow-derived pneumocytes despite high levels of hematopoietic chimerism. In contrast, 4 days after either split or single-dose 1,000 cGy irradiation, nearly 15% of lung epithelia were apoptotic, and with this dose, marrow-derived type II pneumocytes (0.2%) were present at 28 days. These data indicate a critical relationship between lung injury and the phenotypic change from BMDCs to lung epithelial cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
An increasing body of literature documents the surprising ability of bone marrow-derived cells (BMDCs) from adults to adopt epithelial phenotypes in the lung [1–8]. Although previous paradigms of lung injury have included re-epithelialization deriving from pre-existing alveolar or bronchiolar cells [9], the ability of circulating BMDCs to contribute to this process has sparked a new area of interest in the field of lung repair. Initially termed "plasticity," this process occurs in murine models of both fibrotic and emphysematous lung disease, raising questions about the lung conditions necessary for BMDCs to adopt an epithelial phenotype. It has been suggested that the lung injury associated with radiation creates a milieu that induces marrow progenitor cells to adopt the gene expression pattern of mature pneumocytes and bronchial epithelium, either through differentiation [10] or by fusion with mature epithelia [11]. This hypothesis has never been systematically tested. Thus, in this study, to test the hypothesis that bone marrow preconditioning using doses of irradiation below the known lung injury threshold of 750 centigray (cGy) [12] would not result in the engraftment of marrow-derived lung epithelia, we performed bone marrow transplants (BMT) in mice using preconditioning with 400, 600, or single- or split-dose 1,000 cGy irradiation. The resulting lung injury and the extent of hematopoietic and lung epithelial engraftment by the injected bone marrow cells were then quantified. We found that only at total body irradiation (TBI) doses high enough to induce lung injury does BMT lead to marrow-derived lung epithelia (MDLE), suggesting an association between lung injury and the development of MDLE.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Bone Marrow Transplantation
Four- to six-week-old wild-type BALB/c mice underwent BMT as previously described [13]. Briefly, female mice were exposed to 400, 600, or 1,000 cGy TBI. Mice receiving 1,000 cGy were exposed either to two doses of 500 cGy 12 hours apart or a single dose of 1,000 cGy. Six hours following dose completion, the mice received 2 million male whole bone marrow (BM) cells via tail vein injection. Following BMT, mice were maintained on sulfatrim and received autoclaved food and water. All animals were treated in accordance with Institutional Animal Care and Use Committee guidelines. Five mice were included in each group, and the experiment was performed twice for a total of 10 mice per group at each time point.

Bronchoalveolar Lavage and Tissue Harvest
To quantify lung damage, 4 days following transplant, mice anesthetized with ketamine/xylazine underwent bronchoalveolar lavage (BAL) as previously described followed by harvesting of lung tissue [14]. A BAL cell count was performed using a Neubauer hemacytometer, and protein levels were quantified using the ABC Kit (Pierce, Rockford, IL, http://www.piercenet.com). Four weeks following transplant, a separate cohort of recipient mice anesthetized with ketamine/xylazine underwent thoracotomy and right ventricular perfusion as described previously [14]. To prepare single-cell suspensions and cytospins, the left lung was digested using a modification of the procedure of Corti et al. [13], in which the left mainstem bronchus was cannulated using a 23-gauge angiocatheter (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and instilled with 1 ml of dispase 2.4 U/ml (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) followed immediately by 1% low-melt agarose in phosphate-buffered saline (PBS) at 42°C. This lung was placed on ice for 2 minutes, after which it was digested with an additional 1 ml of dispase for 45 minutes at room temperature, minced in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml DNase I (Roche Diagnostics), and passed through a 40 micron filter and a 22 micron nytex mesh (Sefar America, Kansas City, MO, http://www.sefar.com). The cells were centrifuged at 800g for 10 minutes, after which red blood cells were lysed using BD Pharmlyse according to the manufacturer’s instructions (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). The cells were washed with PBS and resuspended in PBS supplemented with 3% bovine serum albumin and 5 mM EDTA at 100,000 cells per milliliter. Cells from digested lungs were spun onto Surgipath (Richmond, IL, http://www.surgipath.com) precleaned slides in a Thermo-Shandon cytospin (Pittsburgh, http://www.thermo.com) at 800 rpms for 5 minutes, after which they were fixed in 1:1 methanol-acetone for 10 minutes at –20°C and air-dried at room temperature for at least 1 hour prior to storage at –80°C. Cytospins of BM were fixed in 1:1 methanol-acetone as described above.

Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Analysis/Cytokeratin Costaining on Sections
The right lung was inflated to 20 cm H2O with PBS, tied off at the right mainstem bronchus, and removed. After fixation in 3.7% formaldehyde for 4 hours, the tissues were transferred to 70% ethanol and paraffin-embedded, and 3-µm sections were cut. Paraffin-embedded sections were heated to 60°C, cleared with Histo-Clear (Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com), serially rehydrated with 100%:95%:70% ethanol, and then incubated with 20 µg/ml Proteinase K (Roche Diagnostics) for 20 minutes at 37°C. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis was performed using the Roche Diagnostics in situ cell death kit (no. 2156792 TMR red) according to the manufacturer’s instructions. Staining for cytokeratin was achieved by incubation with rabbit polyclonal antibody against wide spectrum cytokeratin (DAKO USA, Carpinteria, CA, http://www.dakousa.com) overnight at 4°C and detection using goat-anti-rabbit fluorescein (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) at 1:500 for 30 minutes at 37°C. Slides were washed and mounted using Vectashield with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Positive controls for TUNEL staining were lung sections from transgenic mice that, due to overexpression of transforming growth factor ß-1 under the CC10 promoter, exhibit marked apoptosis of pulmonary epithelia upon exposure to doxycycline (kind gift of Dr. Jack Elias, Yale University, New Haven, CT) [15]. Negative controls were uninjured, wild-type mice.

Simultaneous Assessments for Cytokeratin or Thyroid Transcription Factor-1/CD45/Y Fluorescent In Situ Hybridization on Paraffin Sections
After rehydration, slides were heated in antigen retrieval solution (BD Retrievagen; BD Pharmingen) for 30 minutes, cooled to room temperature, digested in 0.2 N HCl for 12 minutes, heated in 1 M sodium thiocyanate for 20 minutes, ethanol-dehydrated using 70%:95%:100% ethanol, and air-dried. Digoxigenin-labeled whole Y chromosome paint was then applied to the slides, incubated overnight, washed, and detected with anti-digoxigenin rhodamine as previously described [16]. Following a 15-minute PBS wash, slides were incubated overnight at 4°C in 1:500 rat anti-mouse CD45 (Caltag, Carpinteria, CA, http://www.caltag.com), washed for 15 minutes, and detected using 1:500 anti-rat 647 (Molecular Probes) at 37°C for 2 hours. For a more specific assessment of alveolar type II cells, slides were assayed for the presence of a Y chromosome in cells expressing thyroid transcription factor-1 (TTF-1), a transcription factor critical for the production of surfactants, which is a specific marker of type II cells in the alveolus. Mouse anti-TTF-1 (DAKO USA) was applied 1:50 overnight at 4°C. Detection was performed using the Vector MOM kit and a 30-minute incubation with streptavidin-fluorescein isothiocyanate (SA-FITC; Molecular Probes) at 1:500. Cytokeratin was detected after a 10-minute fixation in 0.15% formaldehyde, trypsin digestion at 37°C for 20 seconds and then visualized as described above. Isotype control antibodies were obtained from DAKO USA and were run simultaneously with the above.

Simultaneous Assessments for X/Y Fluorescent In Situ Hybridization and Cytokeratin Immunofluorescence on Cytospins
Lung cytospins that had been fixed and permeabilized using methanol-acetone were brought to room temperature and air-dried. Digoxigenin-labeled Y chromosome [17] and biotin-labeled whole X chromosome paint (Vysis, Downer’s Grove, IL, http://www.vysis.com) were applied to the slide, coverslipped, sealed using rubber cement, and denatured at 73°C for 5 minutes. Slides were incubated at 37°C overnight, decoverslipped, and stringently washed. Cytokeratin was detected using pankeratin antibody diluted 1:200 in a fluorescent in situ hybridization (FISH) block at 4°C overnight. Following PBS washing, X and Y chromosomes were detected using 1:500 SA-647 (Molecular Probes) and 1:10 anti-digoxigenin rhodamine, respectively, for 30 minutes at room temperature. Slides were then washed for 30 minutes in PBS, and cytokeratin was detected using 1:500 anti-rabbit fluorescein for 30 minutes at 37°C. Slides were washed, rinsed in water, and allowed to air-dry. Coverslipping was performed with Vectashield/DAPI as described above. Y FISH on bone marrow was performed as described previously [16].

Data Acquisition and Analysis
Microscopy was performed on an Olympus BX51 microscope equipped with a SensiCamQE camera (Applied Scientific, Eugene, OR, http://www.asiimaging.com). IPLab software (Scanalytics, Fairfax, VA, http://www.scanalytics.com) was used to capture images. Photoshop was used to crop images. Differences between means were calculated by Student’s t test. Statistical significance was achieved when the p value was <.05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Varying Doses of Irradiation Do Not Affect Bone Marrow Engraftment
To assess whether differences in marrow-derived pneumocytes correlate with the degree of irradiation-induced lung damage, mice were transplanted with BMDCs after receiving different doses of irradiation. Based on Y-FISH on marrow preparations from the transplanted mice, increasing doses of TBI from 400 to 600 to 1,000 resulted in mean marrow engraftments of 85%, 93%, and 96%, respectively (Table 1). Although there was a difference between the means for the 400 cGy and 1,000 cGy (p < .02), the marrow engraftment of mice receiving 600 cGy and 1,000 cGy did not differ significantly (p < .08), nor was there any difference between the marrow engraftment of mice receiving single- or split-dose 1,000 cGy (96.2% vs. 96.4%); however, there was a difference in the presence or absence of donor-derived epithelia among these mice (see below). Thus, the degree of hematopoietic engraftment does not correlate with the presence of donor-derived epithelia in the recipient lung.


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  		Table 1. TUNEL, marrow, and lung engraftment related to total body irradiation dose

 
Indices of Lung Damage Are Highest in Mice Receiving 1,000 cGy Irradiation in Single or Split Dose
Lungs were examined 4 days post-transplant for indices of damage. In contrast to the extensive histological evidence of damage (inflammation, alveolar exudates) observed on H&E-stained lung sections from mice that received total doses of 1,000 cGy TBI, there was no histological evidence of lung injury in mice that received 400 and 600 cGy (data not shown). There was a high level of TUNEL+ epithelial cells in lungs from mice that received 1,000 cGy. The percentages of cytokeratin (CK)+ lung cells that had TUNEL+ nuclei were 12.4 ± 3.6% and 13.1 ± 2.1% of CK+ for split-dose and single-dose 1,000 cGy irradiation (XRT), respectively (Fig. 1; Table 1). These means were not significantly different. The 400 and 600 cGy recipients did not have TUNEL signal above baseline (0.2%; Table 1; Fig. 1). The differences between these values and those of the mice receiving 1,000 cGy were statistically significant (p < .02).


Figure 1
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  		Figure 1. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining is increased in mice receiving 1,000 centigray (cGy) irradiation. (A): Cytokeratin (green cytoplasmic stain) and TUNEL (magenta) in the lung of an unirradiated mouse. No TUNEL reactivity is noted, indicating a low level of cell death at baseline. (B): Murine lung 4 days after irradiation with 1,000 cGy. A marked increase in cytokeratin-positive TUNEL+ cells is seen. Slides were counterstained with 4',6-diamidino-2-phenylindole nuclear stain (blue).

 
The functional parameters of lung damage were also increased over baseline in the mice that received 1,000 cGy but not in those that received 400 and 600 cGy. An approximately 10-fold increase above baseline (6.6 x 103 cells per milliliter) in BAL cellularity was noted in mice that received 1,000 cGy as a single or split dose (5.4 x 104 vs. 5.2 x 104 cells per milliliter). Mice receiving 400 cGy contained 3.69 x 103 cells per milliliter, and those receiving 600 cGy had 5.41 x 103 cells per milliliter in the BAL fluid. Both of these means differed significantly from the mice that received 1,000 cGy and did not differ from the baseline levels. Likewise, an approximately fourfold increase in alveolar capillary leak (measured by protein level in BAL fluid) was detected in mice that received 1,000 cGy in either split or single doses (0.482 ± 0.12 vs. 0.461 ± 0.10 mg/ml, respectively; p < .00008 and p < .00003, respectively), whereas there was no change from baseline (0.184 ± 0.02 mg/ml) in those that received 400 (0.125 ± 0.03 mg/ml) or 600 (0.134 ± 0.02 mg/ml) cGy (p < .99 for each). These data, presented in Table 1, complement the histologic data described above.

Bone Marrow-Derived Lung Epithelial Cells Found Only in 1,000 cGy Recipients
Although significant hematopoietic engraftment occurred in the mice that received levels of irradiation below the threshold for lung injury, no marrow-derived cytokeratin-positive lung cells were detected in these animals (Table 1; Fig. 2A). This contrasted sharply with the data from mice that received 1,000 cGy, in which ~1/500 of CK cells was donor-derived (Table 1; Fig. 2). Mice receiving split- and single-dose XRT had the same proportion of MDLE. Cells were considered to be cytokeratin-positive if their nuclei were ringed with cytokeratin signal and there was no staining for CD45 (Fig. 2B, 2C). Twenty randomly chosen high-power (x40) fields on 3-µm sections were examined for each mouse for a total of ~20,000 nuclei per mouse.


Figure 2
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  		Figure 2. Marrow-derived cytokeratin (CK)-positive epithelial cells are detected after 1,000 centigray (cGy) irradiation. (A–C): CK (green), Y (red), and CD45 (yellow) staining on paraffin-embedded sections is shown from the 400-cGy transplant (A), a single-dose 1,000-cGy transplant (B), and the same field with the cell of interest enlarged (C). All nuclei are counterstained blue with 4',6-diamidino-2-phenylindole (DAPI). Despite hematopoietic engraftment in the mice preconditioned with 400 cGy, no CK-expressing cells contained the Y chromosome. Conversely, mice that received 1,000 cGy have donor-derived cytokeratin-expressing cells (arrow). (D): CK (yellow), X (green), and Y (red) staining on the cytospin of lung tissue from a mouse that received a split-dose 1,000 cGy. In this single-cell preparation, overlay is not an issue, and stable fusion is ruled out by the presence of only one X and Y chromosome in donor-derived cells (cell on left). (E–H): Separate channels used to create Figure 2C. (E): DAPI; (F): fluorescein isothiocyanate; (G): rhodamine; and (H): Cy5.

 
To address the possibility that rather than representing mature, resident lung epithelia, circulating CK+ marrow cells that express CD45 were responsible for the appearance of donor-derived CK+/Y+ cells in the lung, we also assayed for donor-derived epithelial cells by performing Y FISH and TTF-1/CD45 immunofluorescent costaining. TTF-1 is a transcription factor that in the lung exclusively stains type II pneumocytes and Clara cells [18]. In mice that received 400 and 600 cGy XRT as transplant preconditioning, no Y+/TTF-1+/CD45– cells were detected. In contrast, in lungs from mice that received 1,000 cGy, roughly the same fraction of TTF-1+ cells contained the Y chromosome as did cytokeratin-positive cells (Table 1; Fig. 3C). This proportion did not vary between the mice receiving single- or split-dose TBI.


Figure 3
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  		Figure 3. Detection of overlay (A, B) and marrow-derived epithelial cells (C) with a combination of thyroid transcription factor-1 (TTF-1) (green), Y fluorescent in situ hybridization (red), and CD45 (yellow) immunostaining on sections from a female mouse that received male marrow following 1,000 centigray total body irradiation. Nuclei are stained blue with 4',6-diamidino-2-phenylindole. In (A), there are two TTF-1+ cells that appear to contain the Y chromosome. However, in (B), with the addition of CD45 staining, it is clear that these cells are CD45+. This image demonstrates that one can be fooled by microscopy artifact, as the CD45 staining shows that even on 3-µm sections, overlay can occur. Panel (C) demonstrates a TTF-1+, Y+ cell that is not concomitantly positive for CD45, which indicates that it is a marrow-derived type II cell (white arrow).

 
Additional analysis on single cells from digested lungs was employed for two reasons: (a) to rule out overlay, and (b) to optimize the visualization of whole nuclei after in situ hybridization for X and Y chromosomes. As shown in Figure 3A and 3B, it is possible on paraffin sections in the absence of costaining for CD45 to mistakenly identify cells as being donor-derived cytokeratin-positive epithelial cells when in fact this appearance results from nuclear overlay. This problem can be further avoided through the use of confocal microscopy or the use of single-cell analysis (which is employed here), both of which allow for sampling of cells on a single plane. An additional reason for the use of cytospin analysis is that on thin sections the nucleus is not completely sampled; thus, one or both sex chromosomes may not be detected on a certain cut [19]. For this reason, thin sections are also not optimal for the assessment of extra copies of the sex chromosomes, which could be present in fused cells. Cytospins create a monolayer that allows for visualization of whole nuclei with the FISH- and cytokeratin-staining protocols. Using this method, six slides containing 10,000 cells each were counted per mouse for a total of 60,000 single cells per mouse. The number of CK+ cells expressing the Y chromosome mirrored that of the data from paraffin sections with a frequency of 1:500 (Table 1; Fig. 3C). All Y+ cells examined in this manner contained only one X chromosome. In no animal did we find an XXXY cell as would be expected to result from stable fusion. No CD45 staining is necessary on these cells, because, as a monolayer, the possibility of nuclear overlay is not an issue. Due to technical limitations, TTF-1/X/Y staining was not performed on cytospins.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
The present study addresses an important concept in the emerging field of bone marrow plasticity, which is that lung injury plays a critical role in the engraftment of marrow-derived pneumocytes. By injecting whole marrow into mice that had received varying doses of TBI and quantifying the degree of lung damage associated with XRT, we demonstrate a dose-response effect showing that doses of XRT that do not cause acute lung injury (400 and 600 cGy) allow for a high degree of hematopoietic chimerism but do not result in any detectable MDLE. Conversely, doses of TBI that induce histologic, functional, and molecular markers of acute lung injury are conducive for low-level engraftment of MDLE 1 month following transplant.

We chose day 4 as our time point to assess injury, because this is within the time of maximal acute lung injury in the murine model of radiation pneumonitis [20]. We augmented the histologic evidence of damage seen on H&E sections with functional markers of lung damage to quantify the severity of injury necessary for the appearance of marrow-derived pneumocytes. The increases in BAL cellularity and capillary leak demonstrate a significant injury response to 1,000 cGy. The TUNEL data show that between 10% and 15% of all CK+ cells in the lung were dead or dying 4 days following irradiation, a further assessment of the tissue damage induced with either single- or split-dose 1,000 cGy, and this was the only irradiation dose at which marrow-derived epithelial cells were detected.

TUNEL, although originally developed as a tool to assess programmed cell death (apoptosis) [21], has been increasingly recognized as a nonspecific marker of cell death [22]. Both necrotic and apoptotic cells can be labeled in this reaction. Thus, it is not possible to ascertain whether apoptosis or necrosis is the culprit in this process. Accumulating evidence reveals this distinction to be artificial, as it seems that apoptosis and necrosis occupy separate ends of the same continuum of tissue damage ([23–26]). This is important, because it is likely that the local microenvironment created in the lung during times of injury creates a favorable environment for the engraftment of BMDCs as CK+, TTF+, and CD45– cells in the lung.

To maximize the specificity of our findings, we assessed marrow-derived epithelial cells using multiple complementary approaches. By using cytokeratin and CD45 costaining in addition to Y chromosome FISH on serial sections, we were able to avoid artifacts of microscopy such as overlay or macrophage ingestion of epithelial cells that could contribute to false-positive results [27]. The use of simultaneous TTF-1 immunofluorescence and Y-FISH allowed for nuclear colocalization to confirm these findings. By performing single-cell analysis on whole lung digests, we were able to rule out overlay and assess for fusion. Since the results gleaned from all of these approaches were comparable, it is likely that these complementary techniques give a true assessment of the frequency of marrow-derived epithelial cells following BMT.

The proportion of MDLE reported herein differs greatly from that first reported by our laboratory [1] but is similar to that reported by investigators using similar techniques [28, 29]. Many factors may explain the lower levels of MDLE reported herein. Initial work from our laboratory found MDLE at a proportion of 15%–20% [1]. This is unlikely to be explained by mouse strain since the strain used herein (BALB/c) demonstrates equal/greater radiosensitivity [20] than the C57Bl6 strain used in our prior work. Possible explanations may be found in the timing of the analysis. Radiation pneumonitis has three phases of damage, an early initial phase followed by a period of latency that is then followed by a second wave of injury many weeks after the initial insult. Like the early phase, the timing and acuity of this late onset damage is strain-dependent [20]. In the current work, we chose to focus on the acute phase of damage, whereas our prior work looked at much later time points. Thus, it is plausible that the second, later stage led to much higher numbers of MDLE. Such a hypothesis needs to be tested. Another possible reason for the apparent discrepancy between the two sets of data is the technical limitations of the earlier study. The analysis in the first study was done without CD45 costaining, which, as noted herein, may result in false-positives when MDLE are detected by combined cytokeratin immunostaining and FISH.

Some studies suggest that there are cytokeratin-positive cells in the bone marrow [30, 31]. A recent study by Summer et al. [32] showed that a small fraction of marrow-derived side-population cells coexpress cytokeratin and the myofibroblast marker {alpha}-smooth muscle actin while being negative for CD45. Likewise, since the initial description of cytokeratins as being pathognomonic of epithelia, sporadic studies have found them to be expressed in other cell types [33]. These findings imply that cytokeratin-expressing cells in the lung could derive from the circulation and be either CD45+ or CD45– and not necessarily resident lung cells. Our use of TTF-1, which is not expressed in cells in the bone marrow [18], is therefore necessary to validate that the BM-derived Y+/CK+/CD45– cells represent a true resident epithelial population. Assiduous use of isotype and negative controls in our staining also heightens the specificity of our findings.

We found that the lungs of animals exposed to doses of irradiation above the known injury threshold of 750 cGy contained marrow-derived epithelial cells. On sections, the morphology and location of these cells was consistent with type II pneumocytes. The section analysis did not reveal any Y chromosome-positive bronchial epithelial cells. Because of the difficulty in definitively identifying type I cell nuclei, it was not possible to use chromosomal analysis on paraffin sections to identify these cells.

To date, no clear consensus exists on the degree of lung chimerism following BMT. Data from a large number of laboratories using widely varied approaches show the derivation of many types of lung epithelia [1–4, 34] and fibroblasts [35–37] from bone marrow. Similarly, other laboratories have found only very rare or absent [38] evidence of these BM-derived nonhematopoietic cells. The differences in these data, including those reported herein, are likely due to differences in donor cell type, injury models, time after bone marrow transplantation, and detection techniques.

A number of recent studies have called the entire field on BM cell plasticity into question. Using transgene-marked marrow in models of murine BMT, some studies have failed to demonstrate transgene expression in recipient lung epithelia. These findings could be explained by lack of lung injury and suboptimal delivery models [39] and/or silenced transgene expression in donor cells [40]. One study compared transgene expression (lacZ) and chromosomal analysis following sex-mismatched BMT of ROSA26 marrow and found absent lacZ expression in lung epithelium but found 0.8% of airway epithelia to contain donor chromosomal material [29]. These differences highlight the inherent difficulties in use of transgenes to identify these very rare cells, as has already been reported for other organs [41]. It is for these reasons that we used the Y chromosome as our donor marker.

The finding that transplanted marrow cells adopt an epithelial phenotype in the lung only in the presence of significant lung damage will require more investigation. For example, the exact marrow subpopulation(s) with the ability to become epithelia needs to be discerned, as well as those factors in the lung microenvironment that facilitate this change. Furthermore, although we did not find evidence of XXXY pneumocytes, which would indicate stable cell-cell fusion in this model, it may be that fusion had occurred and was followed by reductive cell division, or that in other types of injury-induced heterokaryon formation does occur. Despite the remaining questions, these findings represent an important advance in our understanding of the marrow-epithelial continuum and show that induction of lung damage may be required if these findings are to be translated for clinical use.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Dr. Jack Elias for the use of CC10-rtTA-tTS-transforming growth factor-ß1 mice and to Dr. Patty Lee for assistance with the lung protein assays. Thanks also to Stephanie Donaldson for excellent animal care. This work was supported in part by NIH Grants DK061846 and HL073742 (to D.S.K.) and 1K08HL079066 (to E.L.H.). E.L.H. was also a Parker B. Francis Fellow in Pulmonary Research in 2004 and the recipient of the Yale Center for Excellence in Molecular Hematology Pilot Award.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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