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Published ahead of print on July 28, 2004, doi:10.1164/rccm.200307-908OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1158-1163, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200307-908OC

Original Article

Cells Derived from the Circulation Contribute to the Repair of Lung Injury

Shinji Abe, Craig Boyer, Xiangde Liu, Fu Qiang Wen, Tetsu Kobayashi, Qiuhong Fang, Xingqi Wang, Mitsuyoshi Hashimoto, J. Graham Sharp and Stephen I. Rennard

Department of Internal Medicine, Pulmonary and Critical Care Medicine Section; Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska

Correspondence and requests for reprints should be addressed to Stephen I. Rennard, M.D., University of Nebraska Medical Center, Department of Internal Medicine, Pulmonary and Critical Care Medicine Section 985125 Nebraska Medical Center, Omaha, NE 68198–5125. E-mail: srennard{at}unmc.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone marrow (stem/progenitor) cells have been shown to "differentiate" into cells in multiple tissues, including lung. A low number of hematopoietic stem/progenitor cells also circulate in peripheral blood. The physiologic roles of these cells are still uncertain. This study was designed to test, using parabiotic mice that were joined surgically, whether stem/progenitor cells in blood contributed to the regeneration of lung after injury. Parabiotic mice were generated surgically by joining green fluorescent protein transgenic mice and wild-type littermates. These mice developed a common circulation (approximately 50% green cells in blood) by 2 weeks after surgery. The wild-type mouse was either uninjured or lethally irradiated or received intratracheal elastase or the combination of radiation with intratracheal elastase injection. Radiation or the combination of radiation with elastase significantly increased the proportion of bright green cells in the lungs of the wild-type mice. Morphologically, interstitial monocytes/macrophages, subepithelial fibroblast-like interstitial cells, and additionally type I alveolar epithelial cells immunostained for green fluorescent protein in wild-type mice. Approximately 5 to 20% of lung fibroblasts primary cultured from injured wild-type mice were green fluorescent protein expressing cells, indicating their blood derivation. This study demonstrates that stem/progenitor cells in blood contribute to the repair of lung injury in irradiated mice.

Key Words: lung repair • parabiosis • stem/progenitor cell in blood

There is accumulating evidence that the tracheal epithelium contains cells with generally accepted properties of stem/progenitor cells (1–5), and like bone marrow stem cells (6), these cells may reside in niches. It is still less clear that there is a single multipotent stem/progenitor cell in the lung for different lineages, for example, tracheal gland duct cells versus tracheal basal cells versus Clara cells versus pulmonary neuroendocrine cells versus type II alveolar epithelial cells. Traditionally, type II alveolar epithelial cells have been believed to be progenitor cells of type I cells (7, 8).

Recently, bone marrow or bone marrow stem/progenitor cells have been reported to differentiate into cells in multiple tissues, including lung (9–13) in mice. In these reports, bronchial epithelial cells (9), type I alveolar epithelial cells (11), and type II alveolar epithelial cells (9, 10) have been demonstrated to be derived from donor bone marrow cells.

A low number of hematopoietic stem/progenitor cells also circulate in blood. The physiologic roles of these cells are still uncertain. In human studies after hematopoietic stem cell transplantation, "chimerism" in lung has been reported (14, 15). Similarly, significant chimerism of epithelial and endothelial cells in lungs of recipients has been demonstrated (14).

Transplant models have been used to demonstrate population of the lung by circulating stem/progenitor cells. An interesting potential role for such cells is repair of the lung after injury. However, in transplant models, it is unclear when transplantation should be performed relative to the injury. It is also unclear whether systemic (irradiation) or local injuries of the lung are needed for stem cell engraftment.

Consequently, this study employed parabiotic pairs of transgenic enhanced green fluorescent protein (GFP) expressing and wild-type littermate mice in which the wild-type mouse was either uninjured or lethally irradiated (9.5 Gy, total body irradiated) or received intratracheal elastase (0.05 U/g) or both irradiation and intratracheal elastase. Blood cells were examined to determine whether parabionts shared vascular communication. GFP+ cells in the lungs of both partners were detected by fluorescence-activated cell sorter analysis and immunocytochemically. Primary lung fibroblasts were cultured from wild-type mice of parabionts to detect whether any cells were donor derived.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Transgenic GFP C57Bl/6-TgN (ACTbEGFP, 4–5 weeks) and wild-type littermate C57Bl/6 (4–5 weeks) mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Parabiosis
For parabiosis of GFP and wild-type mice, mice were anesthetized to full muscle relaxation with xylazine (0.04 mg per mouse) and ketamine HCl (1 mg per mouse) by intraperitoneal injection and were joined by the technique previously described with minor modifications (16, 17). Mortality from the procedure was nil, and the health of animals after parabiosis was excellent, as is commonly observed (16, 17).

Lung Injury
After confirming complete vascular communications of parabionts, wild-type mice were given porcine pancreatic elastase (0.05 U/g) or the same volume of saline intratracheally. In some parabionts, wild-type mice only were total body irradiated (9.5 Gy) and after 2 weeks were given elastase (0.05 U/g) or saline intratracheally. All mice were necropsied 4 weeks after elastase injection into wild-type mice.

Flow Cytometry Analysis of the Lung and Bone Marrow
For fluorescence-activated cell sorter analysis, phosphate-buffered saline (PBS)-perfused lungs were minced and digested in Dulbecco's modified Eagle medium containing 10-U/g porcine pancreatic elastase and 125-U/g collagenase solution, as previously reported, with minor modifications (18) in bath for 25 minutes. The cell suspension and undigested fragments were dispersed by repeated passage through a 5-ml pipette with Dulbecco's modified Eagle medium 30% fetal calf serum. Then the cells were run through a 70-µm strainer and were washed and centrifuged. Bone marrow cells were aspirated from the tibias and femurs of mice using a 25-gauge needle. The pellets of both lung and bone marrow cells were resuspended in PBS 1% bovine serum albumin and then adjusted to 1 x 106 cells/ml and analyzed by fluorescence-activated cell sorter analysis gating on bright green cells.

Immunohistochemistry
Mice were killed and extensively perfused via the right ventricle with 6-ml sterile PBS to eliminate the blood cells. Immunohistochemical staining for GFP was performed according to the manufacturer's directions using the Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA). An anti-GFP monoclonal antibody (JL-8:#83711; Clontech, Palo Alto, CA) at a dilution of 1:500 was used as the primary antibody. The same dilution of normal serum was used for the negative control. The sections were counterstained with hematoxylin and eosin. Staining of adjacent sections was performed using the alkaline phosphatase antibody (goat anti-rabbit IgG, Poly-Alk Phos IHC amplification reagent; Chemicon, Temecula, CA) according to the manufacturer's directions. Briefly, after blocking with 10% normal goat serum (Vector), the sections were incubated with the rabbit cytokeratin antibody (Dako, Carpinteria, CA) at a dilution of 1:500 for 60 minutes. The sections were then incubated with goat anti-rabbit IgG (alkaline phosphatase antibody) for 30 minutes. After washing with PBS for three times, the sections were developed with Vector Red Alkaline Phosphatase Substrate Kit I (Vector). Cytokeratin-positive cells showed a red coloration, whereas negative cells had no reaction product.

Culture of Lung Fibroblasts
Mouse lung fibroblast cells were obtained from parabiont mice by using an outgrowth method described previously (19, 20). More than 95% of the cells were morphologically fibroblasts and stained with vimentin, and no cells were stained with CD45. The fibroblasts were used between culture passages 3 and 6.

Statistical Analysis
All data are reported as means ± SEM. The results were analyzed by Student's t test for comparison between the two groups. Values of p less than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Demonstration of Vascular Communication
To investigate the vascular communication of the parabionts, blood samples were obtained retro-orbitally 2 weeks after surgery. As shown Figure 1, these mice developed a common circulation (approximately 50% green cells in blood) by 2 weeks after surgery. The wild-type mice in parabiotic pairs that were given irradiation (9.5 Gy) after vascular communication also maintained a common circulation (approximately 60% green cells in blood) when assessed 2 weeks after irradiation. After irradiation, chimerism appeared slightly increased (approximately 60%), perhaps because irradiation increased autofluorescence (data not shown) or because of the decreased proportion of circulating wild-type cells.



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  		Figure 1. Vascular communication after parabiosis. Blood chimerism of parabionts was determined by green fluorescent protein + (GFP+) cells in the circulation. The mice were bled retro-orbitally after 2 weeks of parabiosis and 2 weeks after irradiation to wild-type mice of parabionts. Data are expressed a percentage of the GFP+ cells in the blood gating on bright green cells by fluorescence-activated cell sorter analysis. Each value represents the mean ± SE of 10 mice.

 
Flow Histogram Analysis of Cells in Parabionts
Representative flow histograms of blood, bone marrow cells, and lung cell suspensions from parabionts 4 weeks after surgery are presented in Figure 2. Flow histograms of these cells before parabiosis are also shown for comparison. In GFP mice, GFP-expressing cells were not 100% of cells in either the bone marrow or lung even before parabiosis (Figure 2A). GFP-positive cells in the lungs of GFP mice were variable between mice (average 55.9%, range 33.4–80.1%). GFP-positive cells in bone marrow were also variable and relatively low compared with lung, liver, and spleen (liver, spleen data not shown). After parabiosis, these percentages of GFP+ cells were less than 100%, reflecting either mosaic expression of GFP or engraftment of nongreen GFP-derived cells. These results were consistent with data of immunostaining for GFP. Approximately 5% of bone marrow cells and lung cell suspensions from wild-type mice were bright green cells, indicating donor derivation. Therefore, even in the steady state, cells from the circulation contributed, albeit minimally, to cells in bone marrow and lung.



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  		Figure 2. Flow cytometric analysis of bright green cells in the blood, bone marrow, and lung cells of parabionts at 4 weeks after surgery. GFP-positive cells in blood (upper panels), bone marrow (middle panels), and lung (lower panels) of parabionts were analyzed by flow cytometry. The left column shows data of wild-type mice, and the right column shows the data of GFP mice. The data of parabionts at 4 weeks after surgery are shown in Figure 2B. The data of control samples (before surgery) are also shown in Figure 2A for comparison. Each value represents the mean ± SE of GFP proportion (%). The number of independent experiments is indicated in parentheses.

 
Effect of Elastase/Irradiation on the Proportion of GFP-positive Cells in the Lung of Wild-type Mice
To investigate the contribution of blood-derived cells to the lung after injury, wild-type mice of parabiotic pairs were given elastase intratracheally (0.05 U/g) or irradiation (9.5 Gy) or the combination of irradiation (9.5 Gy) with elastase (0.05 U/g). As shown in Figure 3, elastase alone did not increase the proportion of green cells in the lungs of the wild-type mice compared with saline-treated mice. Irradiation or irradiation plus elastase significantly increased the proportion of GFP-positive cells in the lungs of wild-type mice.



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  		Figure 3. Effect of lung injury on proportions of GFP-positive cells in the lung of wild-type mice. To evaluate the effect of lung injury, lung cells from wild-type mice injected with saline/elastase intratracheally or given irradiation followed by saline/elastase injection were analyzed by fluorescence-activated cell sorter analysis. Data are expressed as percentage of GFP+ cells in the lung. Each value represents the mean ± SE of five wild-type mice. ELT = elastase; RT = radiation.

 
Immunohistochemistry for GFP/Cytokeratin
To determine which cell types were derived from the GFP donors, immunohistochemical analysis for GFP was performed. The lungs of GFP mice had a mosaic expression of GFP immunostaining (Figures 4A and 4C). Morphologically, in the wild-type parabionts, interstitial monocytes /macrophages (Figure 4E), subepithelial fibroblast-like interstitial cells (Figures 4G and 4H), and additionally type I-like alveolar cells (Figures 4I, 4J, and 4L) were immunostained for GFP. To confirm that these type I-like cells were alveolar epithelial cells, adjacent sections were immunostained with cytokeratin. As shown in Figures 4K and 4M, cells stained with GFP were also stained with cytokeratin, indicating that those cells both were donor derived and were alveolar epithelial cells. Morphologically, type II epithelial cells were not observed to be immunostained for GFP (Figures 4L and 4M). However, only one time point was evaluated.






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  		Figure 4. Immunohistochemical staining for green fluorescent protein (GFP)/cytokeratin in the lung of wild-type mice. Sections from the lungs of GFP mice were immunostained for GFP or with nonimmune serum (A and C for GFP, B and D for serum, original magnification x400 for A and B, x1,000 for C and D). The lungs of enhanced GFP mice showed a mosaic expression of GFP-positive cells (brown cells, A and C). Lung sections from saline-treated wild-type mice show that monocytes and macrophages were immunostained with GFP (E, brown cells, marked with an asterisk, original magnification x1,000). An adjacent section of E stained with nonimmune serum, showing that no reactivity was noted (F, original magnification x1,000). Lung sections from saline-treated wild-type mice showing that subepithelial fibroblast-like interstitial cells were immunostained with GFP (arrows, G and H, original magnification x1,000). A lung section from irradiated and elastase-injected wild-type mice showing that type I-like alveolar cells were immunostained with GFP (arrows, brown cells, original magnification x400 for I, x1,000 for J, same area indicated in the box in I). An adjacent section stained for cytokeratin showing the GFP+ cells in J, immunostained with cytokeratin (K, red cells, original magnification x1,000). A sample lung from elastase injected mice showing a type I-like alveolar cell also immunostained with GFP (L, arrow, brown, original magnification, x1,000) and stained with cytokeratin (M, arrow, red, original magnification, x1,000) (I: type I alveolar cell, II: type II alveolar cells). Therefore, these cells are donor-derived alveolar epithelial cells.

 
Cultured Lung Fibroblasts from Wild-type Mice
To examine whether lung fibroblasts were derived via the circulation, primary cultures of lung fibroblasts were established. As shown in Figure 5, GFP-expressing cells exhibited fibroblast-like morphology by fluorescence microscopy. More than 95% of these cells were immunostained with vimentin (data not shown). To quantify GFP-expressing lung fibroblasts, the cells were analyzed by fluorescence-activated cell sorter analysis, gating on bright green cells. Approximately 5 to 20% of cultured lung fibroblasts were GFP-expressing cells, indicating that the cells were donor origin (Figure 6). Lung injury induced by irradiation or by a combination of irradiation with elastase increased the proportion of GFP+-cultured lung fibroblasts. Double staining with CD45 by fluorescence-activated cell sorter analysis showed no contamination of lung fibroblasts with cells of hematopoietic origin (data not shown).



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  		Figure 5. Culture of lung fibroblast from wild-type mice. Lung fibroblasts were cultured from wild-type mice of parabionts. The cells exhibited fibroblast morphology by fluorescence microscopy, and more than 95% of cells were immunostained with vimentin. (A) Lung fibroblasts from wild-type C57/Bl mice were employed as negative control subjects. (B) Lung fibroblasts from GFP mice (original magnification, x100). (C) Lung fibroblasts from saline injected mice (original magnification, x100). (D) Lung fibroblasts from irradiated and saline-injected mice (original magnification, x100). Representative data from five experiments are shown.

 


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  		Figure 6. GFP+ proportion of cultured lung fibroblasts. GFP+ cells in the cultured lung fibroblasts from the wild-type mice of parabionts (n = 3–5, independent experiments) were analyzed by fluorescence activated cell sorter analysis. Lung fibroblasts were used at passage 3 or passage 4. (A) Lung fibroblasts from normal C57Bl/6 mice (negative control, n = 5). (B) Lung fibroblasts from GFP mice (n = 3). (C) Lung fibroblasts from saline-injected mice (n = 5). (D) Lung fibroblasts from elastase injected mice (n = 5). (E) Lung fibroblasts from irradiated and saline-injected mice (n = 5). (F) Lung fibroblasts from irradiated and elastase injected mice (n = 5). Each value represents the mean ± SE of the GFP+ proportion (%) of cultured lung fibroblasts. The number of independent experiments is indicated in parentheses.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated by parabiosis that type I alveolar epithelial cells and lung fibroblasts as well as interstitial monocytes /macrophages could be derived from circulating stem/progenitor cells. Lung injury induced by irradiation and the combination of irradiation with elastase increased the homing of stem/progenitor cells into the lung. Culture of lung fibroblasts from wild-type mice of parabiotic pairs confirmed that 5 to 20% of lung fibroblasts could be derived from circulating blood, although such cells could not be cultured directly from blood.

The origin of the circulating stem/progenitor cells and the way in which they generate type I alveolar epithelial cells and lung fibroblasts are unknown. It is possible that there are multiple stem/progenitor cell types in lung, some locally maintained and some derived from blood. The latter likely included CD45+ lymphohematopoietic as well as endothelial and fibroblasts precursors. The bronchial epithelial cells likely have their own primary local precursor (1–5). Although type I alveolar cells have been traditionally believed to be derived from type II alveolar cells, derivation from stem cells in the circulation is supported by this study.

Kotton and colleagues have reported on cultured, plastic adherent bone marrow cells from Rosa-26 mice transplanted to wild-type recipients treated with intratracheal bleomycin or saline (11). They observed that type I alveolar cells, but not type II cells, were donor derived 30 days after transplantation in both bleomycin and saline-treated recipients. In contrast, Krause and colleagues observed type II alveolar epithelial cells in stem cell–transplanted mice (9). They transplanted male bone marrow cells to lethally irradiated female recipients. Eleven months later, they performed fluorescent in situ hybridization and detected donor-derived type II cells. In this study, type II cells were not observed to be donor derived. The basis of these differences is currently unclear, but type II cells might be observed at later times post transplant than type I cells.

The nature of the tissue damage might be also a regulating factor (21). Tissue damage by irradiation, chemotherapy, or immunosuppression is believed to facilitate homing of blood stem cells and their differentiation into cells of organs (22). This effect may be due to direct effects on stem cells or to effects on the "niches" in which lung stem cells reside. Interestingly, the different populations of lung cells, which likely have distinct stem/progenitor cell populations residing in different niches, may be differentially sensitive to injury-induced stem cell engraftment. In this regard, donor-derived hepatocytes and epithelial cells of the skin and gastrointestinal tract have been observed without the presence of tissue damage after human blood stem cell transplantation (23).

Parabionts, generated surgically by joining two animals, have been used to demonstrate the involvement of circulating factors (16, 24, 25). Parabiotic mice have been reported to produce stable blood chimerism across major histocompatibility barriers (26). Much useful information has been obtained through parabiotic experiments evaluating hormones (24, 25), tumors (27), infections (28), and recently, stem cells (17, 29, 30). Wagers and colleagues reported in a study of parabiosis between GFP and wild-type mice that steady-state tissue maintenance derived predominantly from tissue resident progenitor cells, rather than from circulating cells (29). Very few circulation-derived cells were observed to contribute to tissue maintenance. Consistent with this, this study demonstrated relatively small contribution of circulating cells to lung in the absence of lung irradiation. After radiation, however, significant numbers of structural cells in the lung were GFP positive, indicating this derivation from the parabiotic partner via the circulation.

Fibroblasts are the major type of mesenchymal cell present in connective tissue that have the capacity to secrete matrix components and other mediators that drive tissue remodeling (31). Grimm and colleagues have suggested the existence of a circulating mesenchymal precursor cell in a human study (32). Also, Bucala and colleagues have reported a population of circulating cells that specifically enter sites of tissue injury (33). Although we cultured total blood leukocytes from wild-type mice of parabiosis in the same manner as lung fibroblasts, no fibroblasts were observed (data not shown), suggesting that such cells are rare and/or have properties different from tissue fibroblasts. Circulating mesenchymal stem/progenitor cells potentially could migrate into injured sites, differentiate into fibroblasts, and might contribute the remodeling of the lung. Ortiz and colleagues have recently reported that circulating stem cells can be recruited into lung mesenchymal cell populations and can alter sensitivity to develop bleomycin-induced fibrosis (34). The possibility that an abnormal fibrotic response is being observed cannot be excluded.

This study evaluated two forms of lung injury. Radiation greatly increased engraftment of circulation-derived cells into the lung. Elastase infusion alone had little effect, but elastase in combination with radiation showed slightly more engraftment than radiation alone. Radiation not only damages lung but has been shown to enhance engraftment of stem cells into a variety of tissues. This effect may be mediated by damage to stem cell population or to alteration of the niches in which stem cells reside.

Elastase-induced injury characteristically leads to emphysema, as was the case in this study. Elastase-induced injury is followed by initiation of repair-type responses, including synthesis of new collagen and elastin. Inhibition of repair can worsen features of emphysema. This study suggests that circulating stem/progenitor cells can participate in repair after elastase-induced emphysema, at least when the lung has been "prepared" by irradiation.

Consistent with our observations, Suratt and colleagues have reported chimerism in lungs using human lung samples after hematopoietic stem cell transplantation (14). In their report, 35.7 to 42.3% of the endothelial and 2.5 to 8.0% of epithelial cells were donor derived. However, donor-derived fibroblasts were not observed. They noted the possibility that fibroblasts might not be derived from the constituents of cord blood. However, mesenchymal precursors have been reported in umbilical cord blood (35). It is possible that timing of stem cell transplantation, particularly relative to injury of the lung, may be crucial in providing cells that differentiate into fibroblasts. The use of parabiotic animals provides a continuous source of donor-derived cells in the circulation and, therefore, may be crucial in providing fibroblast precursors. Injury of the lung is common after transplantation. Acute and organizing diffuse alveolar damage, obliterative bronchiolitis, and bronchiolitis obliterans and organizing pneumonia have been reported in transplanted patients (14). The relationship between lung damage and cell types that migrate to damaged area from blood is unknown, but donor-derived cells could contribute to these pathologic conditions.

In summary, we demonstrate that type I alveolar epithelial cells and lung fibroblasts can be derived from circulating stem/progenitor cells in a radiation/elastase-induced emphysema model. Parabiotic mice may provide appropriate models for studying pathophysiologic mechanisms that involve circulating stem/progenitor cells contributing to the repair/regeneration, or abnormal repair, of lung tissue.


   Acknowledgments
 
The authors acknowledge the extensive assistance of Dr. C Kuszynski and L. Wilke of the Cell Analysis Core Facility.


   FOOTNOTES
 
Supported by the Nebraska Research Initiative.

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

Conflict of Interest Statement: S.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; X.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; F.Q.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; Q.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; X.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.G.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.I.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 7, 2003; accepted in final form July 27, 2004


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 DISCUSSION
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