American Journal of Respiratory Cell and Molecular Biology

There is a rapid onset of organizing alveolitis/fibrosis at 120–140 d after whole lung irradiation of C57BL/6J mice. To test the hypothesis that circulating cells of bone marrow origin contribute to irradiation fibrosis, irradiated chimeric green fluorescent protein (GFP)+ C57BL/6J mice were followed for GFP+ cells in areas of lung fibrosis. In a second experimental model, C57BL/6J female mice received 20 Gy total lung irradiation, and after 60 or 80 d were intravenously injected with cells from a clonal GFP+ male bone marrow stromal cell line or male GFP+ whole bone marrow, respectively. The mice were then followed for the development of pulmonary fibrosis, and the contribution of Y-probe–positive, GFP+ cells to fibrotic areas was quantitated. Bromodeoxyuridine labeling of developing fibrotic areas showed that the cell division occurred predominantly in GFP+, Y-probe–positive, and vimentin-positive cells. Immunohistochemistry demonstrated that these cells were macrophages and fibroblasts, not endothelial cells. Mice that received manganese superoxide dismutase-plasmid/liposome intratracheal injection 24 h before total lung irradiation demonstrated a decrease in GFP+ fibroblastic cells in the lung. Thus, pulmonary irradiation fibrosis contains proliferating cells of bone marrow origin, and gene therapy prevention of this condition acts in part by decreasing the migration and proliferation of marrow origin cells.

Irradiation-induced pulmonary damage is a major complication in patients who receive total body irradiation for bone marrow transplantation and in patients receiving lung irradiation for esophagus or non–small cell lung carcinoma (NSCLC) (1, 2). In both human and animal model systems, acute pneumonitis and late organizing alveolitis/fibrosis are directly dependent upon total irradiation dose, fraction size, and lung volume irradiated (35). The pathophysiology of the late lesion termed irradiation pulmonary fibrosis or organizing alveolitis/fibrosis in the C57BL/6J mouse model is unknown. Increases in inflammatory cytokine mRNA within the lung and protein in the peripheral circulation have been detected in acute and chronic phases of lung injury (6, 7). The treatment of acute irradiation pneumonitis using steroids or nonsteriodal anti-inflammatory agents may decrease acute injury, but does not detectably prevent late histopathologic evidence of lung injury (8). It has been shown that intratracheal administration of manganese superoxide dismutase (MnSOD)-plasmid/liposomes (PL) 24 h before irradiation decreases both the acute and chronic pulmonary damaging effects of whole lung irradiation in C57BL/6J mice (9, 10), but the mechanism of protection is not known.

There is a rapid onset of fibrotic lesions in the irradiated lung, which occurs after a latent period of 6 mo to 2 yr in humans, and of 100–120 d in the C57BL/6J mouse model (1, 2, 11). The rapid fibroblast proliferation in the hemibody-irradiated mouse lung areas suggested that the source of proliferating cells might include cells arriving via circulation as well as those in the irradiated lung volume.

Bone marrow stromal cells (mesenchymal stem cells) have been shown to circulate through the peripheral blood (12, 13), and can contribute to the fibrotic lesions in chronic renal interstitial injury and to stromal elements in tumors (14, 15). There is pathologic evidence that cells of bone marrow origin can engraft the lung, forming nonhematologic cell types (1618). The present experiments were designed to determine whether cells from whole bone marrow, or from a clonal bone marrow stromal cell line derived from a green fluorescent protein (GFP)+ transgenic mouse, contributed to the histopathologic lesions in areas of organizing alveolitis/fibrosis in the irradiated GFP− mouse lung. The results demonstrate that there is a significant contribution of bone marrow–derived cells in the pathophysiology of murine lung irradiation fibrosis.

Mice

C57BL/6J GFP+ transgenic mice and GFP− littermates were obtained from Jackson Laboratories, Bar Harbor, ME. The mice were housed five per cage, fed standard laboratory chow, and managed according to the Institutional Animal Care and Use Committee. C57BL/6J GFP+ chimeric mice were established by irradiating C57BL/6J female mice to 7 Gy whole body dose, followed by an intravenous injection of 1 × 106 bone marrow cells obtained from male GFP+ mice. Chimerism was documented 30 d later by assay of > 50% GFP+ circulating nucleated white blood cells.

Cells and Cell Lines

Bone marrow was obtained from GFP+ transgenic mice by removing the femur, tibia, and humerus then flushing the bone marrow with tissue culture medium, as previously described (14, 20). The bone marrow was resuspended in phosphate-buffered saline (PBS) at a concentration of 1 × 107 cells/ml and 1 × 106 (100 μl) injected intravenously through the tail vein into each recipient C57BL/6J mouse as described (14, 20). A clonal permanent bone marrow stromal cell line was derived from a male C57BL/6J GFP+ transgenic mouse long-term bone marrow culture, as previously described (21, 22). Briefly, the marrow was flushed from the femurs and tibias of a GFP+ male mouse into a Corning plastic flask (25 mm2), passaged in Dulbecco's Modified Eagle's Medium, supplemented with 15% fetal calf serum, penicillin, and streptomycin, and maintained in a 7% CO2 high humidity incubator. After reaching confluence, the cells were passaged weekly at 1:2, 1:5, and 1:10 dilution, then cloned by limiting dilution as described (19, 20), and a clonal cell line was isolated, expanded, and frozen down in liquid nitrogen.

MnSOD-PL Administration

Mice were anesthetized using Nembutal, the trachea exposed, and mice were injected intratracheally with MnSOD-PL (500 μg of plasmid DNA) containing the human MnSOD transgene, according to previously published methods (9, 10). Control mice received either empty liposomes or no liposomes but Sham surgery (9, 10). MnSOD-PL was prepared by mixing 28 μl of lipofectant with 50 μl of pRK5 plasmid containing 500 μg of plasmid DNA (pRK5-MnSOD plasmid).

Irradiation and Cell Injections

Injection protocols are shown in Table 1

TABLE 1 Protocols for injection of GFP+ bone marrow or clonal marrow stromal cell line into irradiated C57BL/6J mice





Preparation of Mouse for GFP+ Cell Injections
Group
Source of GFP+ Cells
Day of
 20 Gy Lung Irradiation
Day of
 Total Body Irradiation
Dose of
 Whole Body Irradiation
Day of
 Cell Injection
1Bone marrow0807 Gy81
2Bone marrow stromal cell line0600 Gy60
3
Bone marrow
0
(−)60
7 Gy
(−)59

To demonstrate the role of bone marrow cells in the development of irradiation-induced fibrosis of the lungs, three protocols were used to deliver 20 Gy to the pulmonary cavity. Group 1 mice received 7 Gy whole body irradiation 80 d after 20 Gy to the lungs, and then were injected 24 h later with 1 × 106 GFP+ bone marrow cells. Other mice (Group 2) were injected on Day 60 after 20 Gy to the pulmonary cavity with 1 × 106 bone marrow stromal cells from a GFP+ bone marrow stromal cell line. In a third group, GFP+ marrow chimeras (Group 3) were established by irradiating C57BL/6J mice to 7 Gy total body irradiation 60 d before the 20 Gy lung irradiation (−)60 d. These mice were then injected with 1 × 106 GFP+ bone marrow cells at (−)59 d before the 20 Gy irradiation dose to the lungs.

. C57BL/6J female mice or C57BL/6J GFP+ chimeric female mice were irradiated using a Varian 6 MeV linear accelerator (Varian Medical Systems, Palo Alto, CA). The mice were irradiated to 20 Gy at a dose rate of 1.8 Gy per min, with the head and abdomen shielded so that only the pulmonary cavity was irradiated. C57BL/6J mice, which had been irradiated to 20 Gy to the pulmonary cavity 80 d previously, received whole body irradiation to a dose of 7 Gy. The mice were then injected with whole bone marrow obtained from a male GFP+ transgenic mouse, receiving 1 × 106 cells in PBS as described in Table 1. The bone marrow cells were injected intravenously through the tail vein in a 100 μl volume. Other groups of mice irradiated to 20 Gy 60 d previously were intravenously injected with 1 × 106 cells of the GFP+ clonal bone marrow stromal cell line. Dose–response curves documenting the optimization of total body and lung irradiation doses have been published previously (911). Protocols are compared in Table 1.

Histopathology

Control C57BL/6J mice, GFP+ marrow chimeric C57BL/6J mice, or C57BL/6J mice that had been injected with GFP+ whole bone marrow or bone marrow stromal cells were killed at 90, 100, or 120 d after pulmonary irradiation or just before the time of death due to the development of organizing alveolitis/fibrosis as indicated by a difficulty in breathing, ruffling of the fur, hunching of the back, and decreased breathing rate (911). Bromodeoxyuridine (BrdU) (50 mg/kg) was injected intraperitoneally 1 h before mice were killed. The lungs were removed, frozen in optimum cutting temperature (OCT) medium, sectioned, and co-stained with polyclonal antisera antibodies to BrdU, GFP, endothelin, vimentin (a fibroblast marker), or the macrophage marker F480, as previously described (23). The sections were fixed in methanol, incubated in 1% hydrogen peroxide for 2 min at room temperature, washed in PBS, and nonspecific binding blocked by incubating the sections in 2% goat serum for 3 min at room temperature. The primary antibodies were diluted 1:100 and placed on tissues for 2 h in a humidity box. The slides were then washed in PBS and incubated in the presence of either fluorescein isothiocynate–conjugated or phycoerythrin (PE)-conjugated secondary antibodies for 1 h at 37°C in a high humidity box as described (23). The slides were then washed in PBS, mounted with an antifade histomount, coverslipped, and examined under a fluorescent microscope. Quantitation of positive cells in single or dual antibody stained sections was performed as previously published (23).

In Situ Hybridization for Detection of the Y Chromosome

To demonstrate the presence of the Y chromosome in cells in the lungs of female C57BL/6J mice following irradiation, lung sections were removed at several time points and were stained using a murine Y chromosome hybridization kit from Cambio (Cambridge, UK). Briefly, lung sections were fixed with a 3:1 mixture of methanol:acetic acid for 30 min, incubated in proteinase K (10 μg/ml) for 45 min at 37°C, and dehydrated by serial ethanol washes for 2 min each in 70% (vol/vol) ethanol, 90%, and 5 min in 100% ethanol. The sections were air dried overnight and denatured by incubating the sections in prewarmed denaturation solution (70% deionized formamide and 30% 2× saline sodium citrate [SSC] [8.76 g NaCl, 4.41 g Na citrate, 500 ml double distilled water, pH 7.4]) at 86°C for 2 min. The denaturation was quenched by placing the slides in ice-cold 70% (vol/vol) ethanol for 4 min and dehydration by serial washing in ethanol as described above. Twenty-four hours later, the sections were hybridized overnight in the dark at 37°C with the Y probe. The sections were washed in 2× SSC buffer for 5 min, twice in stringency wash solution (50 ml deionized formamide plus 50 ml 1× SSC) for 5 min at 45°C, twice in 1× SSC at 45°C, and incubated for 4 min in detergent wash solution (500 ml of 4× SSC and 250 μl Tween-20) at 45°C. The slides were drained and mounted with 50 μl of Working Reagent B (15 μl Working reagent A [DAPI stain] plus 500 μl of mountant [antifade]). The slides were covered with glass coverslips and sealed with nail varnish and stored in the dark at 4°C.

Statistics

Data were analyzed using a Student's t test according to published methods (9, 10).

Animal Welfare

All protocols were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Veterinary care was provided by the Division of Laboratory Animal Research of the University of Pittsburgh in strict accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh guidelines.

Irradiation-Induced Organizing Alveolitis/Fibrosis Occurs at the Time of Increased Detectable BrdU Labeling of Nonendothelial, Nonmacrophage Cells in the C57BL/6J Mouse Lung

In a previous investigation, C57BL/6J female mice were irradiated to 20 Gy to both lungs, and followed for the development of organizing alveolitis/fibrosis. The percent of lung replaced by areas of fibrosis was quantitated at several time points beginning 80 d after irradiation using published methods (23). Subgroups of mice were injected with BrdU at each of several time points 1 h before killing. Increased BrdU uptake was first detected at Day 80, was significantly elevated by Day 100, and continued to increase until the time of death (23). Areas of developing fibrosis showed accumulation of BrdU-labeled positive cells in the adjacent nonfibrotic lung (23), suggesting that proliferation was a rapid and continuing process from Day 80 until death.

In the present studies, endothelial cells of the lung were carefully quantitated within fibrotic areas at each time point. In sections histopathologically co-stained for endothelin and BrdU, increased BrdU labeling (an indicator of cell division) was not detected in endothelial cells at Days 80, 90, or 100 (data not shown), or at Day 120 (Table 2

TABLE 2 Quantitation of endothelial or macrophage cell division in the histopathologic lesions in lungs of mice during time of irradiation fibrosis/organizing alveolitis



Number of Lung Cells at 120 d after Irradiation Positive for:
Group
BrdU
Endothelin
BrdU + Endothelin
F480
BrdU + F480
Nonirradiated19.7 ± 2.4 206.0 ± 7.01.0 ± 0.319.5 ± 3.70.9 ± 0.4
20 Gy
84.4 ± 10.4*
174.0 ± 25.5
2.5 ± 0.6
51.7 ± 3.9**
1.5 ± 0.6

To determine whether endothelial cells or macrophages (F480+) were dividing in areas of organizing alveolitis/fibrosis, C57BL/6J mice were irradiated to 20 Gy. The mice were irradiated to 7 Gy 80 d later and followed by a bone marrow transplant. At 120 d after irradiation, the mice (3 nonirradiated and 10 irradiated) were killed. One hour before killing, the mice were injected intraperitoneally with 50 mg/kg of BrdU. The lungs were expanded in OCT, frozen, sectioned, and co-stained with antibodies to BrdU and endothelin. Six sections from each mouse were examined under a fluorescent microscope, and the number of cells out of 1,000 scored expressing BrdU, endothelin, BrdU + endothelin, F480, or BrdU + F480 were counted. The number of BrdU cells increased at 120 d after irradiation (*P = 0.0051), whereas there was no significant change in the number of cells expressing endothelin or both BrdU and endothelin (P = 0.346 or 0.352, respectively). There was a significant increase in macrophages in the area of developing organizing alveolitis/fibrosis in irradiated mice (**P = 0.0009). However, there was no significant co-localization of BrdU+ with F480+ staining. Thus, the increased numbers of F480+ macrophages were not detectably dividing. Results are expressed as the number of cells per section counting 1,000 cells, in each of triplicate sections for each mouse per group.

and Figure 1) . Co-staining the sections at each time point with antibodies to BrdU and F480 (antimouse macrophages) demonstrated that actively dividing BrdU-positive cells were not macrophages. Results at Day 120 are shown in Table 2 and Figure 2 . Thus, the proliferating cells at the time of development of organizing alveolitis/fibrosis were neither endothelial cells nor macrophages. There was an increase in detectable accumulation of migration of macrophages, but no increase in macrophage cell division. These results are consistent with our previous publication showing that the BrdU-positive proliferating cells were not macrophages and histologically resembled fibroblasts (23).

Homing to and Proliferation of Bone Marrow–Derived Fibroblasts in Lungs at the Time of Development of Irradiation Organizing Alveolitis/Fibrosis

To determine whether proliferating fibroblast–like cells in areas of fibrosis were derived from the circulation, three experimental protocols were constructed (Table 1). In the first experiments, female C57BL/6J mice received 1 × 106 intravenously injected GFP+ male whole bone marrow cells 80 d after 20 Gy lung irradiation, and were then killed at 100 or 120 d after irradiation or at the time of moribund pulmonary injury (23). Histopathologic evaluation of the lungs was performed and demonstrated a significant contribution of GFP+ and Y-probe+ male cells in areas of developing fibrosis (Table 3

TABLE 3 Quantitation of GFP+ cells in the lungs in areas of developing irradiation pulmonary fibrosis/organizing alveolitis in marrow chimeric GFP+ or GFP+ cell injected C57BL/6J mice


C57BL/6J Group

Percent of GFP+ Cells in Areas of
 Pulmonary Irradiation-Induced
 Organizing Alveolitis/Fibrosis
20 Gy< 0.01
20 Gy + GFP+ bone marrow cells22.5 ± 13.3*
20 Gy + GFP+ bone marrow
   stromal cell line30.6 ± 4.1**
GFP+ chimeric mice + 20 Gy
47.1 ± 2.5***

Irradiated C57BL/6J mice injected with GFP+ whole bone marrow cells or cells from a GFP+ bone marrow stromal cell line, or irradiated GFP+ chimeric mice were killed at Days 120−140 after irradiation, which is the time when they die due to formation of fibrosis. The mice were killed when they became morbid as detected by increased breathing, hunching, ruffled fur, and lack of movement. This process appears within 48 h of death (9−11). The lungs were expanded with OCT, frozen in OCT, sectioned, and the percent of GFP+ cells in regions of organizing alveolitis/fibrosis was calculated. GFP+ chimeric mice had the highest percent of GFP+ cells in areas of organizing alveolitis/fibrosis, with ***47.1% compared to *22.5% and **30.6% positive cells in mice injected with either whole bone marrow cells or the bone marrow stromal cell line, respectively (P = 0.0001 and 0.0053, respectively). All three injected groups were higher than the control mice that received 7 Gy plus injection of GFP bone marrow (P < 0.0001 for all three groups compared to control irradiated mice).

and Figure 3) from Day 100 onward. We have previously published photos of hematoxylin and eosin (H&E)-stained lung sections demonstrating the appearance of organizing alveolitis/fibrosis at this time point (9, 10).

There was no significant increase in GFP+ cells detected at Day 100, but there was a significant increase in fibrotic areas comprised of GFP+, Y-probe positive cells by Day 120 in irradiated mice injected with whole bone marrow (Figure 4A)

or clonal bone marrow stromal cell line (Figure 4B), or at Day 90 in chimeric mice (Figure 4C). These marrow transplantation experiments establish that GFP+ cells of bone marrow origin home to areas of irradiated lung at the time of development of organizing alveolitis/fibrosis. Injection of MnSOD-PL 24 h before irradiation decreased the number of GFP+ cells in the lung (Figures 4A and 4B). Injection of GFP+ bone marrow cells into mice that received 700 cGy whole body irradiation showed little GFP+ cell incorporation into the lungs. At this irradiation dose, no areas of organizing alveolitis/fibrosis developed. These results confirm prior publications (911).

To determine whether endothelial cells were present in areas of developing organizing alveolitis/fibrosis, lung sections from mice irradiated 120 d previously were derived from the marrow and the sections were co-stained with antibodies to endothelin and GFP. There were few detectable endothelin-positive cells in the area of organizing alveolitis/fibrosis (Table 4)

TABLE 4 Quantitation of GFP+ endothelial marrow origin cells in regions of pulmonary fibrosis/organizing alveolitis in irradiated C57BL/6J mice


Cell Type

Number of Cells

Percent of
 Total Cells
Total308.5 ± 127.0100
GFP-positive145.0 ± 59.747.0
Endothelin-positive35.0 ± 18.211.7
GFP + endothelin-positive
1
0.33

C57BL/6J mice were irradiated to 20 Gy to the pulmonary cavity and injected at 60 or 80 d after 20 Gy with bone marrow stromal cells or whole bone marrow. The mice were killed at 120 d after irradiation and the lungs expanded in OCT, removed, frozen in OCT, and sectioned. The sections were examined for areas of organizing alveolitis/fibrosis, and the number of total cells, GFP-positive cells, and endothelin-positive cells counted. In the areas of organizing alveolitis/fibrosis, 11.7% of the cells were endothelial cells.

. There were also few detectable GFP+ and endothelin-positive cells in the areas of fibrosis. Examination of the forming fibrotic areas revealed that endothelial cells were present in areas of the lung adjacent to fibrotic areas. In areas of established fibrosis there were very few endothelial cells, and less than 1% of these endothelial cells were GFP+. The data agree with that presented in Figure 1, which demonstrated no detectable cell division of pulmonary endothelial cells in irradiated lung within areas of organizing alveolitis/fibrosis at 120 d. Furthermore, there were very few (host or recipient) endothelial cells in areas of developing organizing alveolitis/fibrosis.

Injection of Cells from a Clonal GFP+ Male Mouse Bone Marrow Stromal Cell Line Contributes to Lung Irradiation-Induced Organizing Alveolitis/Fibrosis

The above data suggested that cells of bone marrow origin contributed to areas of developing fibrosis when injected before the pulmonary stimulus for the fibrotic reaction was detectable (80 d after irradiation). The dividing cells were neither endothelin-positive nor F480-positive. To determine whether bone marrow stromal cells contributed to the pulmonary lesions, mice were injected intravenously with 1 × 106 cells of a clonal GFP+ male bone marrow stromal cell line 60 d after irradiation. Animals were killed and analyzed at Days 100, 120, or at the time of death for percent GFP+ Y-probe positive, and vimentin+ (fibroblast marker–positive) cells in the lung. There were significant numbers of vimentin-positive GFP+ cells in the lungs of stromal cell line–injected mice in areas of developing fibrosis. As shown in Table 5

TABLE 5 Quantitation of GFP-positive F480+ macrophages in areas of developing organizing alveolitis/fibrosis in GFP+ cell transplanted C57BL/6J mice



Number of Cells
Days after Irradiation
GFP-positive
Macrophage +
 GFP-positive
03 ± 20
9072.5 ± 18.569.5 ± 17.5
120
98.5 ± 23.5
65.0 ± 17.0

Chimeric GFP+ mice were irradiated to 20 Gy to the pulmonary cavity and sacrificed at 90 or 120 d after irradiation. Nonchimeric C57BL/6J mice were killed on Day 0 as controls. The lungs were expanded in OCT, excised, frozen in OCT, sectioned, and co-stained with a FITC−anti-GFP+ antibody, F480 antimurine macrophage antibody. The sections were examined under a fluorescent microscope, and the number of macrophages, GFP-positive cells, and cells that were both macrophages and GFP-positive were determined. Results are the mean in some of 1,000 cells in triplicate sections for three mice per group. At Day 90, GFP+ cells were identified as macrophages by staining with the F480 antibody. By Day 120, GFP-positive cells were both F480+ macrophages and vimentin-positive fibroblasts (Figure 5).

, at Day 120 there was a significant contribution of GFP+ cells in areas of developing organizing alveolitis/fibrosis in chimeric mice. The percent of GFP+ cells in areas of developing organizing alveolitis/fibrosis was significantly elevated at Day 120. The numbers of GFP+ cells in stromal cell line injected mice were higher 100 d after irradiation than the numbers in mice that received whole bone marrow (Figure 4). However, by 120 d there was no difference in the number of GFP+ cells in mice injected with whole bone marrow compared with the clonal bone marrow stromal cell line. Intratracheal injection of MnSOD-PL 24 h before irradiation resulted in a decrease in the percent of GFP+ clonal marrow stromal cell line cells in areas of organizing alveolitis/fibrosis at Day 120, but not at death (Figure 4B). In lung-irradiated mice that received whole bone marrow, greater than 95% of the GFP+ cells in areas of organizing alveolitis/fibrosis were vimentin+ fibroblasts (Figures 5E, 5F, and 5H) .

Fibroblastic Cells of Bone Marrow Origin Contribute to Organizing Alveolitis/Fibrosis in Pulmonary Irradiated Bone Marrow Chimeric Mice

The above experiments demonstrated that injection of either whole bone marrow or cells of a clonal bone marrow stromal cell line at the time of development of organizing alveolitis/fibrosis contributed to the cells in fibroblastic lesions. To determine whether bone marrow stromal cells were naturally recruited from the bone marrow during the development of pulmonary fibrosis, GFP+ bone marrow chimeric mice were first established, then irradiated to the lungs, and long-term migration of cells into the lungs was quantitated. For these experiments, C57BL/6J female mice received 7 Gy total body irradiation and were injected with 1 × 106 GFP+ male whole bone marrow. The animals recovered after bone marrow transplantation for 60 d. Chimeric female mice with GFP+ male bone marrow (GFP+ peripheral blood cells demonstrated to be > 80% donor origin –[data not shown]) were then irradiated to 20 Gy to both lungs, and followed for 120 d for the development of organizing alveolitis/fibrosis.

The chimeric mice were killed at several time points, including Day 90 or just before death. The percent of donor origin cells, GFP+, Y-probe+ cells in areas of developing organizing alveolitis/fibrosis was quantitated in mice from each group (Table 4 and Figures 3E and 4C). The results demonstrated a significant contribution of bone marrow–derived cells in areas of organizing alveolitis/fibrosis in irradiated mice beginning at Day 90 (Figure 4C). Marrow cells (GFP+, Y-probe+) comprised 47.1% of detectable cells in areas of fibrosis at the time of death. Sections from mice killed at Day 0, 90 d, or at the time of death were co-stained with anti-GFP+ antibody and an F480 antimacrophage antibody. In mice killed at Day 90, all GFP+ cells were identified by co-staining as macrophages in areas of developing organizing alveolitis/fibrosis (Figure 6F)

. Furthermore, at Days 120–140 or in mice killed at the time of moribund pulmonary injury, the region of alveolitis had around 60% of GFP+ cells that were co-stained as macrophages, with the remaining cells identified as vimentin+ fibroblasts (Table 5 and Figure 6I). In areas where active collagen deposition was occurring, greater than 95% of the GFP+ cells were vimentin+ fibroblasts (Figure 5). Thus, macrophages were recruited from the circulation into the area of injury, and although not themselves dividing were associated with significant fibroblast proliferation.

Intratracheal MnSOD-PL Administration before Whole Lung Irradiation Decreases the Accumulation of GFP+ Bone Marrow Cells in Areas of Developing Organizing Alveolitis/Fibrosis

Subgroups of mice injected with GFP+ whole bone marrow or bone marrow stromal cells following irradiation, as described above, received MnSOD-PL intratracheal injection 24 h before total lung irradiation. Previous studies have demonstrated that injection of antioxidant MnSOD-plasmid/liposomes decreases the magnitude of pulmonary cytokine response to total lung irradiation and the development of organizing alveolitis/fibrosis. To determine whether MnSOD-PL also decreased the homing and proliferation of circulation-derived fibroblast progenitor cells, mice in each experimental protocol were analyzed for the number and percent of GFP+ cells in areas of developing organizing alveolitis/fibrosis. In both experimental groups (Figures 4A and 4B), there were decreases in GFP+ and Y-probe+ bone marrow stromal cell–derived areas of developing organizing alveolitis/fibrosis in animals that received whole bone marrow and clonal bone marrow cell line injections, respectively, at 120 d after irradiation. However, before death there was an increase in percent of the lung expressing GFP+ cells. Injection of MnSOD-PL before irradiation delayed the recruitment of GFP+ cells to areas of organizing alveolitis/fibrosis, as well as the development of organizing alveolitis/fibrosis, as previously described.

The pathophysiology of ionizing irradiation damage to the lung is a subject of intense investigation. Acute pulmonary injury remains a major complication of total body irradiation for bone marrow transplantation, and necessitates the development of techniques for lung transmission blocking, hyperfractionated irradiation, or development of novel radioprotective compounds (1, 2). Acute pulmonary injury is associated with endothelial cell swelling, alveolar transudates at the histopathologic level, and increased mRNA and protein for acute inflammatory cytokines at the molecular biologic level (6, 7, 24). If successful, the management of acute irradiation lung injury is followed by a latent period during which histopathologic or molecular biologic evidence of irradiation damage is not readily detectable (6, 7, 24).

The late irradiation lung injury, pulmonary fibrosis, occurs with rapid and progressive onset after the latent period which follows acute irradiation injury (1, 2, 25). Dose–response curves have been published previously (911). In both the C57BL/6J and C57BL/6NHsd mouse models, it is unknown how such a rapid and significant fibroblast proliferation can occur between 120 and 140 d after irradiation (9, 23). One hypothesis is that clonal expansion of surviving fibroblast progenitor cells in the irradiated lung contributes to the proliferation of cells in fibrotic areas (26, 27). Another hypothesis suggests that turnover of a slowly proliferating cell population (with a doubling time spanning 100 d) creates late irradiation injury, a second peak of cytokine mRNA increase, and that this evokes proliferation of those surviving clones of fibroblast progenitors (28). Both of these theoretical models hypothesize that the origin of fibrotic areas in pulmonary fibrosis is attributable to cells within the lung parenchyma. A third hypothesis from our laboratory is that the rapid appearance of fibrotic areas in pulmonary fibrosis is attributable in part to circulating fibroblast progenitors which home to the irradiated lung and undergo rapid proliferation.

In the present report, we demonstrated in irradiated mice that areas of developing pulmonary organizing alveolitis/fibrosis are composed of cells arriving to the lung via circulation and from the bone marrow. Bone marrow chimeric mice receiving total lung irradiation demonstrated homing and proliferation of bone marrow–derived stromal cells to sites of developing organizing alveolitis/fibrosis.

Results from the present experiments demonstrated that the marrow–derived GFP+ positive cells were not endothelial cells. At 100 d after irradiation, GFP+ cells in the lungs included F480-positive macrophages. However, at the time of developing organizing alveolitis/fibrosis (120 d) GFP+ cells were both macrophages and vimentin+ fibroblasts. BrdU labeling and co-staining for endothelin, vimentin, or F480 of cells in irradiated lungs demonstrated that the GFP+ cells that were dividing were neither macrophages nor endothelial cells but were vimentin-positive fibroblasts. The dividing cells had the morphology of GFP+ fibroblasts. The fibroblast origin was confirmed by transplanting a clonal GFP+, Y-probe positive male bone marrow stromal cell line which produced similar results.

There was a lack of detectable increase in GFP+ endothelin-positive cells in the areas of organizing alveolitis/fibrosis of bone marrow chimeric mice under conditions in which we did detect a significant increase in dividing GFP+, vimentin-positive fibroblasts, and nondividing GFP+ F480+ macrophages. The data provide evidence that the increase in intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 upregulation in endothelial cells detected at the time of initiation of the late irradiation–induced fibrotic lesion (23) occurs in resident pulmonary endothelial cells rather than those that migrated through the circulation. A gradual accumulation of bone marrow origin GFP+ endothelial progenitors would have also been detected by our co-staining technique for endothelin and GFP, but this was not the case. We cannot rule out the possibility that a low number of endothelial progenitor cells of bone marrow origin did seed the lung at times before initiation of organizing alveolitis/fibrosis, and that these cells were either lost or pushed out of a fibrotic area by proliferating fibroblasts. Thus, the present studies confirm and extend our previous publication (20), indicating that late irradiation changes in the lung are associated with adhesion molecule upregulation in pulmonary endothelial cells present at the time of irradiation.

The present results support our hypothesis concerning the contribution of circulating cells to the development of pulmonary fibrosis. Lung irradiation results in cellular damage, which causes increased cytokine expression of tumor necrosis factor-α and transforming growth factor-β around 80–100 d after irradiation (10). This process leads to increased ICAM-1 and VCAM-1 expression in pulmonary epithelial cells in areas of lung damage (23). Increased expression of adhesion molecules attracts macrophages and progenitors from the bone marrow to sites of lung damage. The macrophages (although not dividing) reside in the lungs and recruit fibroblasts into the damaged area where fibroblasts divide and deposit collagen, contributing to nonfunctional lung and the development of pulmonary fibrosis. As collagen is deposited, endothelial cells are marginalized and replaced within the fibrotic area.

Bone marrow stromal cells have been demonstrated to circulate between bone marrow loci and other sites (1620). Intravenous injection of clonal bone marrow stromal cell lines or expanded cultures of bone marrow stromal cells has been shown to preferentially seed sites of irradiated bone marrow (19, 20). In studies of intravenous injection of bone marrow stromal cells, transient detection of cells in the lungs of recipient animals has been reported (12, 13). Kotton and coworkers demonstrated that injection of bone marrow–derived cells developed into Type I pneumocytes (17). Mesenchymal precursor cells from the marrow have been shown to serve as precursor cells for mesenchymal cells in the lungs, bone, and cartilage (16). Marrow stem cells are able to differentiate into epithelial cells in the lung, liver, GI tract, and skin (18). However, quantitation of marrow stromal cell sites of seeding to the irradiated lung in relationship to pulmonary fibrosis has not been previously reported.

The present results demonstrate that ionizing irradiation damage to the lung is a complex phenomenon, the pathophysiology of which includes contributions of both resident lung cells and cells arriving via circulation. The contribution of bone marrow stromal cells to fibrotic lesions is consistent with reports of bone marrow stromal cell involvement in the fibrotic lesions of renal allograft rejection (14) and in scirrhous tumors (15). The present results may explain published dynamic properties of scar tissue, in which treatment with agents known to block cell proliferation and homing can result in the decreased density of lesions (29, 30).

The decrease in contribution of circulating cells to regions of organizing alveolitis/fibrosis in mice receiving MnSOD-PL intratracheal injection before irradiation suggests that the mechanism of radioprotective gene therapy may be explained in part by prevention of irradiation-induced alterations of lung endothelial cells that facilitate the homing of cells from bone marrow. Previously, we have demonstrated that overexpression of MnSOD delays the onset of pulmonary fibrosis after irradiation (10, 31). Overexpression of the MnSOD transgene has also resulted in the delay of increased expression of ICAM-1 and VCAM-1 by pulmonary endothelial cells after irradiation (23). Delays in upregulation of ICAM-1 and VCAM-1 in mice injected with MnSOD-PL before irradiation may delay the recruitment of bone marrow macrophages and fibroblasts. Thus, the development of pulmonary fibrosis is delayed by injections of MnSOD-PL, indicating how MnSOD protection of the lung is manifested after irradiation. Further studies will be required to more precisely document the molecular and cellular steps involved in the circulatory/bone marrow origin of the lesions in pulmonary irradiation fibrosis.

This research was supported by the National Institute of Health, grants R01-HL 60132 and P50-CA90440.

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Address correspondence to: Joel S. Greenberger, M.D., Professor and Chairman, Department of Radiation Oncology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Room B346-PUH, Pittsburgh, PA 15213. E-mail:

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