Published ahead of print on March 9, 2006, doi:10.1164/rccm.200509-1381OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200509-1381OC
Fibroblasts of Recipient Origin Contribute to Bronchiolitis Obliterans in Human Lung TransplantsInstitute of Pathology; Departments of Pneumology, and Hematology, Hemostaseology, and Oncology; Division of Thoracic and Cardiovascular Surgery, Medizinische Hochschule Hannover, Hannover, Germany; and Institute of Cell and Molecular Science, Diabetes and Metabolic Medicine, Queen Mary University of London, London, United Kingdom Correspondence and requests for reprints should be addressed to Ulrich Lehmann, Ph.D., Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: lehmann.ulrich{at}mh-hannover.de
Rationale: The participation of circulating precursor cells in the development of experimental pulmonary fibrosing lesions in mice has been recently demonstrated. Objectives: This study analyzes whether circulating, bone marrow–derived, fibroblastic precursor cells contribute to the development of fibrosing lesions in human lungs, especially bronchiolitis obliterans. Methods: The occurrence of in situ microchimerism in bronchiolitis obliterans lesions of human lung allografts (n = 12) as well as of autologous lung tissue from patients post–bone marrow transplantation (n = 2) was analyzed using laser-assisted microdissection after immunohistochemical labeling of leukocytes followed by short tandem repeat–polymerase chain reaction–based genotyping. Combined immunofluorescence and fluorescence in situ hybridization for sex chromosomes was performed for independent confirmation in cases with appropriate sex mismatch (n = 2). Measurements and Main Results: The bronchiolitis obliterans lesions of all 12 lung transplant patients contained considerable numbers of recipient-derived fibroblasts (mean, 32%). The fibrosing pulmonary lesions of the two bone marrow–transplanted patients also displayed clear in situ microchimerism. The in situ detection methodology confirmed these results, although to a lower degree (6–16%). Conclusions: These data clearly demonstrate the involvement of circulating fibroblastic precursor cells in the development of human fibrosing lung lesions and provide evidence that these cells are most probably bone marrow derived. These results may open new venues regarding the prevention of fibrosis in lung transplants and potentially in other organs.
Key Words: bone marrow–derived progenitors • in situ microchimerism • lung fibrosis • transplantation Bronchiolitis obliterans syndrome (BOS) after lung transplantation remains the major cause for lung allograft dysfunction and is assumed to reflect chronic allograft rejection (1, 2). BOS is clinically defined as a substantial decrease in the forced expiratory volume. Histologically, the small airways show fibrous obliteration of the bronchiolar lumen. The generic histopathologic term "BO" has been used to describe two different pulmonary lesions: constrictive BO as the virtual hallmark of chronic rejection with fibrous effacement of the bronchial lumen (corresponding to the clinical term BOS) and classic BO with intraluminal polypoid lesions with an organizing pneumonia pattern (OP) of the surrounding parenchyma as a mostly non–rejection- mediated lesion (3). Constrictive BO (clinical BOS) is believed to be mainly triggered by repetitive episodes of acute rejection, although allogene-independent factors have been discussed (4). Both allogene-dependent and -independent mechanisms are believed to initiate a repair process with excessive proliferation of granulation tissue after epithelial damage, a concept summarized as "response to injury." Traditionally, tissue repair has been considered to be a local process involving resident cells of the involved organ only. In recent years, a paradigm shift has taken place in this field: the existence of, most probably, bone marrow–derived circulating progenitor cells that migrate to wound sites and participate in the repair process has been proposed (5–7). In line with this model, studies in experimental mouse models and the analysis of biopsies from patients after bone marrow transplantation have provided strong evidence that bone marrow–derived cells can engraft in various organs (e.g., muscle, liver, kidney, lung, and heart) and contribute to tissue repair (8–14). Subsequent studies could also demonstrate engraftment of recipient-derived cells after organ transplantation for epithelial, endothelial, and even interstitial compartments in several organs (15–21). In some instances, this could be correlated to the extent of previous organ damage (17, 22, 23). Several independent studies could show in vitro and in vivo the contribution of marrow-derived cells to epithelial, endothelial, and mesenchymal compartments in murine and human lungs (6, 10, 11, 24–28) (see also References 29 and 30 for overview and further discussion). This remarkable plasticity of circulating precursor cells offers a huge therapeutic potential (31, 32), but the identification of bone marrow–derived interstitial myofibroblasts in human renal allografts (16) and of bone marrow–derived myofibroblasts in human liver fibrosis (33) indicates that this plasticity might not always be beneficial. Therefore, the aim of this study was to analyze whether fibrotic lesions in human lung allografts contain recipient-derived cells and whether these cells are bone marrow derived, neither of which has yet been shown in humans. For this purpose, we analyzed 12 explanted lung allografts using laser microdissection of immunohistochemically stained histologic sections followed by polymerase chain reaction (PCR)–based genotyping, a sex-mismatch–independent and individual-specific method. To address the possible bone marrow origin of these cells, fibrotic lung lesions from two patients who underwent bone marrow transplantation were also microdissected and genotyped. Combined immunofluorescence and fluorescence in situ hybridization (FISH) were used for independent confirmation.
Patient Samples and Histopathology The selected patient population comprised 12 lung transplantation patients who underwent retransplantation (n = 10) or diagnostic biopsy (n = 2) procedures for clinically diagnosed and therapy-refractory BOS (see Table 1). All patients received cyclosporin A, mycophenolate mofetil, and dihydrocortisone for immunosuppression. Formalin-fixed, paraffin-embedded lung tissue was retrieved from the files of the Institute of Pathology of the Hannover Medical School following the guidelines of the local ethics committee. Constrictive BO and classic BO with an OP pattern were diagnosed by two experienced pathologists (F.L., H.K.) according to well-established histopathologic criteria (3, 34).
In addition, one lung biopsy and one lung explant from two patients after bone marrow transplantation for hematologic malignancies (see Table 1) were retrieved from the archival files. In one patient, chronic graft-versus-host reaction of the lung with the histologic pattern of constructive BO necessitated lung transplantation; in the other patient, a postinfectious inflammatory myofibroblastic tumor was resected with a prominent classic BO pattern in its vicinity.
Immunohistochemical Staining
To avoid false-positive results due to infiltrating leukocytes within BO lesions, the sections mounted on foil-coated slides for microdissection were stained with anti-CD45 antibody (clone 2B11+PD7/26, 1:100; Dako, Hamburg, Germany) and anti-CD68 antibody (clone KP1, 1:100; Dako) before laser microdissection (Figure 1) using the ready-to-use Vectastain Universal Elite ABC-Kit (Vector Laboratories, Burlingame, CA) without any pretreatment.
Laser Microdissection
Short Tandem Repeat PCR For detection of recipient-derived cells within the isolated fibrotic structures, we used a PCR assay that analyzes one highly polymorphic short tandem repeat (STR) marker, which displays a very high heterozygosity rate of up to 93% (SE33). The PCR and subsequent analysis of PCR products were performed essentially as described (22).
Quantitative Chimerism Analysis
Combined FISH and Immunofluorescence Staining Immunofluorescence staining for SMA and subsequent hybridization of X- and Y-chromosome probes were performed essentially as described (33). For details, see the online supplement.
IHC in BO after Lung Transplantation The 12 lung transplant patients included in this study underwent transplantation for pulmonary end-stage disease of different causes. All examined grafts or parts of them had to be explanted due to transplant failure. To characterize the composition of the inflammatory infiltrate in BO lesions, IHC was performed on serial sections. The inflammatory infiltrate mainly consisted of CD68+ cells (15%; range, 5–40%) and CD3+ T cells (10%; range, 0–25%). CD15+ and CD79a+ cells represented less than 10% on average. In more active BO lesions, granulocytes (CD15+), B cells (CD79a+), and T cells (CD3+) tended to be concentrated in the center of the obstructive polyp, with spindle-shaped cells in the periphery, whereas macrophages/histiocytes (CD68+) were found to be dispersed throughout the lesion. In addition, IHC for myofibroblasts (SMA) and endothelial cells (CD34) was performed. On average, half of the lesion's cells (range: 10 to 80%) showed clear positivity for SMA, whereas CD34 staining revealed only sparse endothelial cells (10%; range, 5–20%). Figure 1 shows representative photomicrographs of immunohistochemical stains of BO lesions.
In Situ Microchimerism in BO after Lung Transplantation In all 12 patients under investigation, recipient-derived mesenchymal cells could be detected unequivocally within the BO lesions (see Table 2; for further details of the PCR analyses, see Table E2). Figure 4 shows as an example the genotyping of one microdissected BO lesion from Patient 6 with clearly identifiable alleles from the donor and the recipient.
Semiquantitative Evaluation of the In Situ Microchimerism in BO in Lung Allografts For semiquantitative evaluation of the proportion of recipient-derived cells in the laser-microdissected BO lesions, a calibration plot was constructed by analyzing defined mixtures of microdissected tissue samples (see METHODS for details). The proportion of cells with the recipient's genotype was estimated to comprise, on average, approximately one-third of total cells (range, 11–59%; mean, 32%; median, 32%). This is in good agreement with the values obtained by Grimm and colleagues for the kidney interstitium (16) and Forbes and coworkers for liver fibrosis (33). It clearly indicates that a considerable proportion of the mesenchymal cells within the BO lesions in human lung allografts is recipient derived and not the product of locally proliferating donor-derived cells.
In Situ Microchimerism in Fibrotic Lung Tissue after Bone Marrow Transplantation
Because, in both cases, bone marrow donor and recipient shared a common allele, quantification of PCR results was not performed (homozygosity of recipient in these cases cannot be excluded, but is very unlikely). Peak ratio calculations were only performed if four alleles could be detected in the chimeric lesions.
In Situ Detection of Chimerism by Combined IHC/FISH
This study reports for the first time the presence of substantial numbers of recipient-derived myofibroblasts in fibroproliferative lesions in human lung allografts. In addition, the detection of donor-derived fibroblastic cells in fibrosing lung lesions from patients undergoing bone marrow transplantation provide strong evidence that these circulating fibroblastic precursor cells involved in BO are most probably bone marrow derived. These results are in good agreement with several animal studies using chimeric mice, which demonstrate the importance of the recruitment of circulating bone marrow–derived fibrocytes for the development of experimentally induced pulmonary fibrosis (6, 28, 36, 37). The findings presented here are supported by two recent publications demonstrating the involvement of mesenchymal bone marrow–derived precursor cells in the development of human kidney and liver fibrosis. Grimm and coworkers (16) reported the presence of substantial numbers of SMA-positive and Y-chromosome–positive mesenchymal cells in female kidney allografts (transplanted into males) in the neointima, the adventitia, and the interstitium (mean: 34, 38, and 30%, respectively). Similarly, Forbes and colleagues (33) found engraftment of 10 to 50% recipient-derived, circulating, bone marrow–derived myofibroblasts in cirrhosis in human liver allografts. Using a completely different methodologic approach (Y-chromosome hybridization combined with immunofluorescence analyzed by laser-scanning microscopy) and analyzing different organs (kidney or liver), these authors obtained in qualitative and quantitative terms very similar results to those we obtained for the lung: circulating myofibroblasts, most probably bone marrow derived, contribute to fibrogenesis in human allografts (on average, 10 or 30%, depending on the individual case and the detection method). In addition, earlier studies demonstrated that our newly developed approach (STR-PCR–based genotyping after laser-assisted microdissection), which is completely independent from a sex mismatch, and the more widely used Y-chromosome hybridization, which relies heavily on a specific sex constellation (i.e., female allograft and male recipient), provide concordant results analyzing the same specimens (20, 22). The values for the extent of chimerism obtained by IHC/FISH might deviate from the ones obtained by STR-PCR because the latter methodology might overestimate the extent due to plateau effects of the PCR (exaggeration of small, chimerism-indicating signals, whereas strong resident cell–derived signals might be already saturated). Also, the erroneous inclusion of small amounts of inflammatory cells leads to an overestimation of chimerism. By contrast, the in situ methodology underestimates the extent of chimerism due to signal loss (because of sectioning and/or suboptimal hybridization efficiency). In our experience, FISH detection is much more affected by suboptimal tissue fixation than PCR analyses. This might also explain some quantitative discrepancies because the specimens analyzed in this study were collected over a period of 12 yr. In only one case included in this study, the underlying disease necessitating lung transplantation was pulmonary fibrosis (Patient 9). Because chimeric fibrosis indicating extrapulmonary recruitment was found in all patients, this mechanism seems to be completely independent of fibrosing lesions as the primary disease before transplantation. Recently, Stevens and colleagues (38) and Koopmans and colleagues (39) raised the concern that in situ microchimerism detected after solid organ transplantation is due to preexisting chimerism of the allograft induced by a former pregnancy of a female donor or a blood transfusion. However, the extent of chimerism (percentage of Y-chromosome–positive cells) was in general very low (one or a few cells per tissue section or less than 5 cells per 30,000 cells). The fraction of recipient-derived cells in the laser-microdissected fibrotic lesions found in our study is well above this background chimerism (at least 8% compared with less than 0.1%). In addition, the STR-PCR identifies specifically the two recipient alleles before transplantation that can then be identified unequivocally in the laser-microdissected lesions. The extent of chimerism also excludes the few endothelial cells found in the microdissected BO lesions (see Figure 1D) as the only source of the recipient's genotype (Suratt and coworkers [40] have shown in the first study demonstrating in situ microchimerism in the human lung that the endothelial compartment can display up to 45% chimerism after bone marrow transplantation). An interesting question, which has to be addressed in future studies, is which specific subpopulation of bone marrow cells contribute to fibrosing lesions in human lung allografts. The functional importance of chemokine-mediated fibrocyte recruitment could be convincingly demonstrated by Phillips and colleagues (6) when systemic administration of anti-CXCL12 antibodies reduced accumulation of lung fibrocytes and protected mice against bleomycin-induced lung fibrosis. In future studies, it has to be addressed whether the same chemokine-based recruitment mechanisms, elegantly shown in experimental mouse models (6, 41), also take place in humans. In conclusion, the data presented in this study support the model that circulating precursor cells are intimately involved in fibrogenesis in the lung and that these fibroblastic precursor cells are most probably bone marrow derived. Therefore, aberrant pathologic fibrosis does not appear to be the result of a local activation and proliferation process alone. The fibrogenic potential of circulating precursor cells now demonstrated in three different organ systems in humans (References 16, 33, and this study) is not only contributing to a better understanding of the pathogenesis of fibrosis (one of the most common causes of organ failure in the human body) but should also lead to a more cautious approach concerning the therapeutic use of stem or progenitor cell fractions after organ damage. Currently, under modern immunosuppression, prevention of chronic allograft deterioration is the major challenge for successful organ transplantation in the long term. Therefore, these results offer new perspectives concerning the therapeutic intervention for the prevention of allograft fibrosis as the morphologic hallmark of chronic allograft dysfunction.
The authors thank Steffi Kowalczyk and Kathleen Metzig for technical assistance and Britta Hasemeier for preparation of all figures.
This study was supported by grants Deutsche Forschungsgemeinschaft KFo 119 TP2, KFo 123 TP3, and the Brauckmann Wittenberg Foundation. 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: 10.1164/rccm.200509-1381OC on March 9, 2006
Conflict of Interest Statement: V.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.G.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.M. is a patent holder and licenser of the German patent for a system of the construction of tissue microarrays, applied in the present study, and received Received in original form September 6, 2005; accepted in final form March 8, 2006
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ABSTRACT
METHODS
1,600 royalty from Zymed Laboratories in 2004. M.M. has been reimbursed by Roche, Wyeth, and Novartis for attending several conferences. He received $2,000 in 2005 for speaking at conferences sponsored by Roche. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.R.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. U.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. 
