Published ahead of print on August 2, 2007, doi:10.1165/rcmb.2007-0249OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0249OC Decreased Asbestos-Induced Lung Inflammation and Fibrosis after Radiation and Bone Marrow TransplantDepartments of 1 Pathology, 2 Medicine, and 3 Biostatistics, University of Vermont College of Medicine, Burlington, Vermont; and 4 Department of Pediatrics, University of Pittsburgh College of Medicine, Pittsburgh, Pennsylvania Correspondence and requests for reprints should be addressed to Daniel J. Weiss, M.D., Ph.D., Pulmonary and Critical Care, University of Vermont College of Medicine, Burlington, VT 05405. E-mail: dweiss{at}uvm.edu
The effect of lung irradiation on subsequent inflammatory or fibrotic lung injuries remains poorly understood. We postulated that irradiation and bone marrow transplantation might impact the development and progression of lung remodeling resulting from asbestos inhalation. Our objective was to determine whether irradiation and bone marrow transplantation affected inflammation and fibrosis associated with inhaled asbestos exposure. Inflammation, cytokine production, and fibrosis were assessed in lungs of naïve and sex-mismatched chimeric mice exposed to asbestos for 3, 9, or 40 days. Potential engraftment of donor-derived cells in recipient lungs was examined by fluorescence in situ hybridization and immunohistochemistry. Compared with asbestos-exposed naïve (nonchimeric) mice, chimeric mice exposed to asbestos for 3, 9, or 40 days demonstrated significant abrogation of acute increases in asbestos-associated inflammatory mediators and fibrosis. Donor-derived cells trafficked to lung but did not significantly engraft as phenotypic lung cells. Irradiation and bone marrow transplantation alters inflammatory and fibrotic responses to asbestos, likely through modulation of soluble inflammatory mediators.
Key Words: irradiation • asbestosis • stem cell • inflammation • fibrosis
Lung irradiation is used both for treatment of thoracic malignancies, including lung cancer, as well as part of total body irradiation in myeloablative regimens and subsequent bone marrow transplantation for hematologic and other malignancies. However, radiation can produce acute or chronic lung injury depending on dose rate, duration, pre-existing lung disease, concomitant corticosteroid use, and other factors (1–4). After nonmyeloablative radiation, acute radiation injury typically occurs 2 weeks to 3 months after treatment and is usually limited to the irradiated field. Mild injury often resolves without treatment, whereas more serious injury can result in fibrosis 6 to 12 months after treatment (1, 2). A number of mechanisms of damage by radiation in the lung, including oxidative stress, epithelial cell apoptosis, and other factors, have been reported (1–4). After myeloablative irradiation and bone marrow transplantation, additional donor-derived T lymphocyte–mediated pathologies, including idiopathic pneumonia syndrome (IPS), can occur (3, 4). In addition, recent evidence suggests that adult bone marrow–derived stem and/or progenitor cells can differentiate into structural cells, including fibroblasts (5), and that these cells can migrate to the lungs in response to fibrogenic stimuli, including lung irradiation (6–13). Although some studies have suggested amelioration of fibrotic lung injury by marrow-derived cells, a pro-fibrotic role for marrow-derived cells appears to occur in radiation-induced and other fibrotic lung injury models in mice (7–13). However, despite a substantial literature on radiation-induced lung injury, less information is available concerning inflammatory and fibrotic responses to subsequent lung injury in previously irradiated lungs. This is an important consideration for patients who have undergone therapeutic lung irradiation, and would shed further light on how radiation alters inflammatory pathways in the lung. We were particularly interested in whether bone marrow–derived stem cells might play a role in subsequent acute or chronic lung injury after lung irradiation and speculated that marrow-derived cells might be protective in this regard. To evaluate this, we used a well-characterized model of asbestos-induced acute inflammation and subsequent fibrosis (14, 15) in sex-mismatched chimeric mice created by total body myeloablative radiation and subsequent bone marrow transplantation. Notably, we found that asbestos-induced inflammation and fibrosis was reduced in chimeric versus naïve mice. Although repopulation of the lung with adult marrow-derived cells occurred, only a small proportion of marrow-derived cells acquired a lung-specific immunophenotype. This suggests that the observed effects are not likely due to structural remodeling by marrow-derived stem cells but rather a modulation of asbestos-induced lung inflammation and fibrogenesis after irradiation and transplantation.
All studies were subject to IACUC review at the University of Vermont (UVM) and conformed to institutional and American Association for Accreditation of Laboratory Animal Care (AAALAC) standards for humane treatment of laboratory animals. Before and after asbestos or sham exposure, mice were housed in pathogen-free barrier facilities in the Small Animal Facility at UVM.
Study Design and Exposures to Asbestos
Harvest, Expansion, and Administration of Total Bone Marrow Cells Whole marrow was obtained from donor adult male (8–12 wk old) transgenic mice constitutively expressing green fluorescent protein (GFP) under the control of the ubiquitin promoter (courtesy of Phillippa Marrack, Ph.D., National Jewish Medical Center, Denver, CO), washed, filtered to remove bone fragments and debris, and administered to adult female C57Bl6 recipient mice (30–32 x 106 cells/mouse) by tail vein injection 4 hours after total body irradiation (1,000 cGy administered in a single dose) using a Cesium-137 cell irradiator (ISO 1000, Model B; Nordion International, Vancouver, BC, Canada) as previously described (17, 18). Engraftment was assessed 14 days after transplantation by determining the percent of peripheral leukocytes expressing GFP using flow cytometry. Mice were exposed to asbestos or clean air (sham groups) 30 days after cell administration.
Lung Analyses Lungs were inflation fixed (20 cm) for 2 hours with ice-cold 4% paraformaldehyde, paraffin embedded, and 5 µm sections stained with hematoxylin and eosin (H&E) or Masson's trichrome stain before sections were scored using an established grading system (20) for inflammation and collagen deposition, respectively, in a blinded fashion by a board-certified pathologist (K.J.B.). To assess the presence of donor-derived cells in lungs, 5-µm sections were used for dual Y-chromosome painting and cell type–specific immunofluoresence staining using antibodies directed against CD45, Clara cell secretory protein (CCSP), or surfactant protein pro-peptide C (pro-SPC) (17, 18, 21). Secondary antibodies and fluorescence microscopy were used as previously described (17, 18).
Statistical Analyses
An Attenuated Acute Inflammatory Response in BALF and Lungs of Asbestos-Exposed Mice Occurred after Myeloablation and Reconstitution of Bone Marrow To evaluate the effects of myeloablation and reconstitution of bone marrow on asbestos-induced lung injury, adult (8–12 wk) female C57Bl6 mice were myeloablated by total body irradiation, transplanted with total bone marrow cells from adult male GFP mice, and allowed to engraft for 30 days before the initiation of asbestos exposures. At 14 days after myeloablation/reconstitution, mice were engrafted with an average of 87% of peripheral leukocytes of donor origin. As we used myeloablative irradiation and marrow rescue, an experimental group of mice that were irradiated but not transplanted could not be included.
Total cell counts in BALF were increased (P < 0.05) in sham chimeric mice compared with sham naïve mice at 40 days (Figure 2A). After 9 days of asbestos exposure, asbestos-exposed naïve mice exhibited a significant increase (P
Differential cell counts on the BALF samples showed that compared with sham naïve controls on the same day, eosinophils were elevated (P < 0.05), concomitant with decreases in the percentage of macrophages (P < 0.05), at 3 and 9 days in asbestos-exposed naïve mice (Figure 2B). Although the percentages were small in comparison, increased numbers of lymphocytes and neutrophils were also observed at 9 days in asbestos-exposed naïve mice, the time point of peak lung inflammation in this model (15, 19). Asbestos-exposed chimeric mice showed significantly increased neutrophils at 3 and 9 days, with concomitant decreases in the percentage of macrophages (P < 0.05), compared with sham-exposed chimeric mice. However, percentages of lymphocytes, eosinophils, and neutrophils were lower in asbestos-exposed chimeric mice compared with asbestos-exposed naïve mice at 9 days, and eosinophils were also lower at 3 days (P < 0.05). At 40 days, both naïve and chimeric asbestos-exposed mice had increased numbers of neutrophils, eosinophils, and lymphocytes compared with corresponding sham-exposed animals (P < 0.05).
A number of cytokines and chemokines have been implicated in asbestos-induced lung injury and fibrosis, including TGF-β, TNF-
At 9 days, asbestos exposures resulted in significant increases in IL-1β, IL-4, IL-5, IL-6, IL-12p40, MIP-1B, KC, granulocyte-CSF (G-CSF), and MCP in both naïve and chimeric mice compared with the corresponding sham-exposed group (P < 0.05, Figure 3). However, for IL-4, IL-5, IL-12p70, MIP-1β, and MCP-1, the response in asbestos-exposed chimeric mice was significantly less than in asbestos-exposed naive mice (P < 0.05). Asbestos also induced increases in RANTES in asbestos-exposed naive mice but not in asbestos-exposed chimeric mice (P < 0.05). When sham chimeric mice were compared to sham naïve mice, a significant increase in IL-12p40 was accompanied by significant decreases in IL-1a, IL-4, IL-12p70, and MIP-1β (P < 0.05, Figure 3).
At 40 days, IL-1β, G-CSF, and KC were increased (P < 0.05) in asbestos-exposed chimeric mice, but not in asbestos-exposed naive mice (Figure 3). Levels of IL-5, IL-6, IL-12p40, MIP-1β, and MCP-1 were increased in both naïve and chimeric asbestos-exposed mice compared with corresponding sham-exposed mice, but levels of IL-6, IL-12p40, and MIP-1β were significantly lower in asbestos-exposed naive mice compared with asbestos-exposed chimeric mice (P < 0.05). Compared with sham naïve mice, sham chimeric mice showed a significant increase in G-CSF, and significantly decreased levels of IL-1 Levels of acidic TGF-β1 were significantly increased in BALF of 9 and 40 day asbestos-exposed compared to sham-exposed naïve mice (P < 0.05, Figure 4). However, there was a significant decrease in acidic TGF-β1 in chimeric asbestos-exposed mice at 9 days that was not observed at 40 days (P < 0.05). Nonacidic TGF-β1 levels were comparable in all groups (data not shown).
Donor-Derived Stromal Marrow Cells Localized in Lungs of Asbestos-Exposed Chimeric Mice Large numbers of donor-derived (Y chromosome–positive) cells were identified in lungs of chimeric mice (Figures 5 and 6). The majority of these stained positively for CD45, suggesting leukocyte identity. In contrast to sham mice (Figures 5A and 5B), peribronchiolar accumulations of leukocytes were observed after exposure of chimeric mice to asbestos at 3 (Figure 5C) and 9 days (Figure 5E). At 40 days, peribronchiolar accumulation of leukocytes was noted in sham (Figures 6A and 6B) as well as asbestos-exposed animals (Figures 6C and 6D). In all groups, donor-derived cells within the airways stained positively for CD45, but did not exhibit positive staining for CCSP. However, a number of donor-derived cells localizing in the parenchyma did not stain positively for either CD45, CCSP, or pro-SPC. Based on this information, we speculate that these cells may have acquired interstitial phenotype. Table 1 shows the quantitation of donor-derived cell types in the lungs of sham and asbestos-exposed chimeric mice using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Notably, the percentage of donor-derived cells repopulating the lung increased over time in both sham and asbestos exposed mice. The average percentage of CD45–/Y+ cells ranged from 7.6 to 10% in lungs of sham mice and from 10.8 to 16.3% in lungs of asbestos exposed mice. At 3 and 40 days, significantly increased (P < 0.05) numbers of CD45–/Y+ cells were seen in the lungs of asbestos vs. sham mice (Table 1).
Lung Fibrosis Was Significantly Reduced in Chimeric Mice after 40 Days of Asbestos Exposure In both naïve and chimeric mice, asbestos exposures for 40 days resulted in significant (P  0.05) increases in fibrosis that was centered around the terminal bronchioles as determined using Masson's trichrome stain to detect collagen deposition (Figure 7). However, when compared with asbestos-exposed naïve mice, which exhibited both inflammation and increased peribronchiolar collagen deposition (Figure 7B), asbestos-exposed chimeric mice demonstrated significantly less inflammation and fibrosis (Figure 7, Table 2). Neither sham naïve nor sham chimeric animals demonstrated increased peribronchiolar fibrosis or inflammation. These latter data suggest that myeloablation and transplantation alone did not cause the inflammatory lung injury or fibrosis in this model.
A summary of the differences in inflammation and fibrosis between experimental groups is presented in Table 3.
The results of this study demonstrate that myeloablative irradiation followed by bone marrow transplantation altered inflammatory and fibrotic lung injury resulting from subsequent inhalation of chrysotile asbestos fibers. As summarized in Table 3, myeloablation and transplantation resulted in diminution of several indicators of lung inflammation after acute asbestos exposures for 3 and 9 days. Importantly, after 40 days of asbestos inhalation, chimeric mice demonstrated markedly reduced lung fibrosis when compared to naïve mice comparably exposed to asbestos. These results are less likely due to engraftment of marrow-derived cells as epithelial or interstitial cells in chimeric mouse lungs but may rather reflect alterations in lung inflammatory and immune responses after irradiation and transplantation. Rodent models of asbestos inhalation have proven useful for studying the inflammatory and subsequent fibrotic changes characteristic of clinical asbestosis. Exposure to asbestos at airborne concentrations comparable to those used in this study have resulted in prolonged inflammatory responses characterized by increased proportions of polymorphonuclear leukocytes (PMNs), both neutrophils and eosinophils, in BALF as well as inflammatory foci in terminal bronchiolar and alveolar duct regions of the lung (16, 23, 24). Transient proliferation of bronchiolar and alveolar type II epithelial cells have been observed within days after initial exposures to asbestos followed by subsequent proliferation of cells in the interstitial compartment of the lung (15, 16, 19, 23–28). The origin of the cell types involved in asbestos-induced epithelial proliferation and subsequent fibrosis has been presumed to be resident cells in the lung. However, a recent study has evaluated the role of bone marrow cells in asbestos-induced lung injury. In this study, sex-mismatched chimeric rats exposed to asbestos for 3 days demonstrated clusters of donor-derived cells in areas of bronchoalveolar duct bifurcations, the site of asbestos-induced fibrosis in this model (29). While initially the majority of cells were monocytes or macrophages, at later time points some exhibited immunohistochemical phenotype of myofibroblasts or fibroblasts and also some of the donor-derived cells appeared to be proliferating. This suggests that a population of bone marrow–derived cells, perhaps fibrocytes as has been demonstrated in other models of fibrosis (8–13), may play a pathogenic role in asbestos-induced fibrosis. In addition, rare type 2 alveolar epithelial cells that appeared to be donor-derived were identified. In parallel, our studies suggest that asbestos treatment of chimeric mice only resulted in rare airway or alveolar epithelial cells that appear to have originated from marrow-derived cells, similar to what we have observed with lung injury induced by naphthalene, endotoxin, or NO2 in chimeric mice (17, 18). The dose of radiation used in our studies was comparable to that used in other studies evaluating engraftment of lung epithelium by marrow-derived cells (30, 31). The small numbers of these cells makes it unlikely that they play a meaningful role in asbestos-induced structural remodeling. There were more numerous interstitial cells that appear to be bone marrow derived, and these were increased in asbestos versus sham exposed chimeric mice. These arguably might play a role in asbestos-induced lung injury and further characterization of these cells both as to identity and origin (i.e., derived from circulating fibrocytes or other cell populations) is required to better understand their role.
The role of specific inflammatory cells and of specific inflammatory cytokines and chemokines in development of asbestos-induced lung pathology is only partly understood (14, 15, 23). Alveolar macrophages play a prominent role in phagocytosing asbestos particles and mRNA levels of both TNF- The role of other inflammatory cells, including T lymphocytes and eosinophils, in asbestos-induced lung inflammation and fibrosis is less clear. We have recently demonstrated increased BALF levels of several pro-fibrotic Th2 cytokines, including IL-4, and the pleiotropic cytokine IL-6 after 9 days of asbestos exposure in mice (15). In the present work, levels of IL-4, IL-5, and IL-13, major effector molecules influencing Th2 inflammation and lung remodeling, as well as several IL-13–induced chemokines (MIP-1β, RANTES), were significantly elevated after 3 and/or 9 days of asbestos exposure. While eosinophils are also elevated in lungs of asbestos-exposed mice, their pathogenic role remains unclear (15). Our current studies demonstrate that BALF eosinophils were significantly decreased 3 and 9 days and lymphocytes significantly decreased 9 days after asbestos exposure in chimeric mice. Furthermore, a number of asbestos-induced increases in acute pro-inflammatory cytokines and chemokines were abrogated in chimeric versus naïve mice. Notably, mediators of Th2 inflammation, including eosinophils and Th2 cytokines (including IL-4, IL-5, and IL-13), were particularly decreased at 3 and/or 9 days. Surprisingly, the transplantation procedure itself, including total body and thus lung irradiation, had relatively little effect on basal levels of inflammatory cells or of these acute inflammatory mediators measured in BALF as determined by comparing chimeric versus naïve groups not exposed to asbestos, although several other cytokines were decreased in sham-exposed chimeric versus naïve mice. Radiation pneumonitis and subsequent lung fibrosis in mice can occur with the radiation dose used in our study (7, 30, 31). However, the effects usually manifest later, both clinically and in experimental models, than the time course we employed for evaluation of acute asbestos effects in chimeric mice, 33 and 39 days, respectively, after myeloablation and transplantation (7, 34).
Nonetheless, despite relatively minimal effects on baseline inflammatory markers by irradiation alone, asbestos inhalation after irradiation revealed a diminished capacity of the lung to mount both acute inflammatory and subsequent fibrotic responses. Surprisingly, relatively little is known about the effect of lung irradiation on the subsequent inflammatory and immune behavior of the lung in response to subsequent challenges with inflammatory or fibrotic agents. Sublethal and lethal lung irradiation can trigger a number of genetic and molecular events that can have both short-term effects and longer-term effects, often following a clinically occult latency period. Some of the more immediate effects include recruitment of inflammatory cells to lung, up-regulation of adhesion molecules, induction of oxidative injury, and generation of a number of pro-inflammatory cytokines and chemokines, notably TNF- There is also little information about inflammatory cell turnover in lung after myeloablative irradiation and transplantation or whether inflammatory cells in lung, either donor-derived or residual host-derived, function normally after myeloablative irradiation and transplantation. In our study, we allowed the recipient mice a 30-day period for engraftment before asbestos exposure, a reasonable and frequently used interval affording good peripheral engraftment and with return of normal apparent functional capacity of engrafted neutrophils and lymphocytes to respond to inflammatory stimuli. However, alveolar macrophages may require up to 3 months for full turnover after irradiation and transplantation (42). Further, little is known about eosinophil function or contribution to lung inflammation or fibrosis following irradiation and transplantation. Our results, in which levels of BALF cytokines and chemokines were mostly comparable between sham-exposed naïve and chimeric mice, suggest that inflammatory cells, a significant source of many of the measured inflammatory mediators, had appropriate basal function at the early time points assessed in this study. However, the response to asbestos exposure was significantly altered in chimeric versus naïve mice. The mechanism(s) for these effects are unclear at present, and further research is warranted concerning the function of transplanted inflammatory cells, and of residual irradiated host-derived inflammatory cells, as well as effects of irradiation on release of inflammatory mediators by lung epithelial cells, in subsequent inflammatory and fibrotic effects in the lung.
The authors thank Marilyn Wadsworth and Douglas Taatjes, Ph.D. from the Microscopy Imaging Core at UVM for help with cell imaging and photomicrographs. They acknowledge Barry Stripp, Ph.D. (University of Pittsburgh) for providing the antibody to CCSP. They also thank Maximilian MacPherson, Rob Prenovitz, Travis Beckett, and Viranuj Sueblinvong, M.D. for technical assistance, and David Hemenway, Ph.D. and Justin Robbins, Department of Civil and Environmental Engineering at UVM, for performing the inhalation experiments.
This study was supported by PO1 HL6004 from the National Heart Lung and Blood Institute (B.T.M.), and by Research Grants from the Cystic Fibrosis Foundation and American Lung Association (D.J.W.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org. Originally Published in Press as DOI: 10.1165/rcmb.2007-0249OC on August 2, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the manuscript. Received in original form June 29, 2007
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Abstract
CLINICAL RELEVANCE
, IL-1, IL-4, IL-6, IL-8, IL-13, macrophage inflammatory protein (MIP)-1
(P < 0.05). RANTES levels were higher in asbestos-exposed chimeric mice compared with both sham chimeric and asbestos-exposed naïve mice (P < 0.05). No significant differences were observed between experimental groups at any time point for BAL fluid levels of IL-10, IL-17, or eotaxin. TNF-
