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Stem Cells 2005;23:699-706 www.StemCells.com
© 2005 AlphaMed Press

In Vivo Characterization of Bone Marrow–Derived Fibroblasts Recruited into Fibrotic Lesions

Genichiro Ishiia, Takafumi Sangaia, Kenji Sugiyamab, Takashi Itoa,c, Takahiro Hasebea, Yasushi Endoha, Junji Magaed, Atsushi Ochiaia,c

a Pathology Division, and
b Investigative Treatment Division, National Cancer Center Research Institute East, Kashiwa, Chiba, Japan;
c Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan;
d Department of Biotechnology, Institute of Research and Innovation, Kashiwa, Chiba, Japan

Key Words. Bone marrow–derived fibroblasts • Green fluorescent protein

Correspondence: Atsushi Ochiai, M.D., Ph.D., Pathology Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa-City, Chiba 277-8577, Japan. Telephone: 81-4-7134-6855; Fax: 81-4-7134-6865; e-mail: aochiai{at}east.ncc.go.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fibroblasts, which are widely distributed and play a key part in tissue fibrosis, are phenotypically and functionally heterogeneous. Recent studies reported that bone marrow can be a source of tissue fibroblast. In the study reported here, we investigated in vivo characterization of bone marrow–derived fibroblasts recruited into various fibrotic lesions. Mice were engrafted with bone marrow isolated from transgenic mice expressing green fluorescent protein (GFP), and fibrotic lesions were induced by cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung). A small population of GFP+ fibroblast was found even in nonfibrotic skin (8.7% ± 4.6%) and lung (8.9% ± 2.5%). The proportion of GFP+ fibroblasts was significantly increased after cancer implantation(59.7%±16.3%) and excisional wounding (32.2% ± 4.8%), whereas it was not elevated after bleomycin administration (7.1% ± 2.4%). Almost all GFP+ fibroblasts in fibrotic lesions expressed type I collagen, suggesting that bone marrow–derived fibroblasts would contribute to tissue fibrosis. GFP+ fibroblasts expressed CD45, Thy-1, and {alpha}-smooth muscle actin at various proportions. Our results suggested that bone marrow–derived fibroblasts expressed several fibroblastic markers in vivo and could be efficiently recruited into fibrotic lesions in response to injurious stimuli; however, the degree of recruitment frequency might depend on the tissue microenvironment.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Injury evokes a sequence of events in tissue. Once injury occurs, the host initiates a coordinated repair response. However, if the injury is prolonged, a repeated process of repair and destruction occurs, which subsequently leads to tissue remodeling. During the process, tissue fibroblasts migrate into the injured site and produce collagens and extracellular matrix proteins in response to several extracellular stimuli. Their functions include important roles in growth and differentiation of adjacent epithelia and healing and inflammatory response. Fibroblasts represent the key source of interstitial collagens, but these cells are known to be heterogeneous with respect to a number of phenotypic and functional features [1–6]. This heterogeneity may arise not only from the activation and differentiation processes that take place in the cells but from their different cellular origins.

Stem cells in the adult have traditionally been thought to be restricted in their potential to differentiate and regenerate tissues in which they reside. Recent advances have revealed that after transplantation of bone marrow (BM), hematopoietic stem cells or nonhematopoietic mesenchymal stem cells, muscle [7–11], heart [12–14], liver [15–19], vascular cells [20, 21], and other mesenchymal cells [22–24] of donor origin have been detected. Investigators revealed that BM-derived cells can be progenitors for tissue fibroblasts that are recruited through the circulation to populate peripheral organs [25–29]. During renal fibrosis, a small number of fibroblasts were BM origin using BM chimera and transgenic reporter mice [25]. We previously reported that cancer-induced stroma generated by the human pancreatic cancer cell line consist of BM-derived fibroblasts and that BM-derived fibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development [26]. Furthermore, BM-derived fibroblasts were engrafted into multiple organs, and it was found that these cells are recruited into injured tissue [27, 28]. However, the phenotype and functional roles of BM-derived fibroblasts have not been fully understood.

In the study reported here, we investigated the possible relationship between BM-derived fibroblasts and various fibroblast phenotypes. Cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung) were used to assess whether fibroblast engraftment was modulated by tissue damage and to analyze the phenotypes of BM-derived fibroblasts. Using BM chimera mice expressing enhanced green fluorescent protein (GFP) only in BM-derived cells, we found that excisional wounding is a stimulus for the recruitment of BM-derived fibroblasts within the skin but not in the lung. Furthermore, we found that BM-derived fibroblasts expressed type I collagen, CD45, Thy-1, and -{alpha} smooth muscle actin ({alpha}-SMA).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
For cancer implantation, we generated GFP transgenic (GFP Tg) and recombination activating gene 1 knockout (RAG-1–/–) double-mutant mice. GFP Tg on C57/BL6 background and RAG-1–/– mice on B6 background [30] were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com). Briefly, the GFP Tg mice were mated with RAG-1–/– mice to generate F1 offspring that were heterogenous for both genes. The F2 offspring of the F1 interbreeding were screened by Western blot analysis for absence of serum immunoglobulin M (IgM) [19]. Mice identified as GFP+/– were screened for fluorescence of skin keratinocytes. RAG-1–/– GFP Tg mice that were identified were further intercrossed, and the F3 offspring were screened based on serum IgM level and GFP fluorescence. GFP Tg and RAG-1–/– mice were further bred. For BM transplantation (BMT), female severe combined immunodeficient (SCID) mice (C.B17 background) and C57/BL6 mice, 6 weeks of age, were purchased from Clea Japan, Inc. (Tokyo, http://www.clea-japan.co.jp) and maintained in our animal facility. All animals were maintained under specific pathogen-free and air temperature–controlled conditions throughout this study, in accordance with the institutional guidelines. Written approval of all animal experiments (K03-011) was obtained from the local Animal Experiments Committee of the National Cancer Center Research Institute.

Cell Preparation and BMT
A 21-gauge needle on a 5-ml syringe was used to flush BM from the femurs of GFP Tg RAG-1–/– mice or GFP Tg mice with RPMI-1640 medium. A single-cell suspension was prepared by repeated gentle aspirations of the marrow plug with the same syringe, and large tissue pieces were removed from the suspension by filtering them through a nylon filter. After 3.5-Gy whole-body irradiation of SCID mice or 9-Gy whole-body irradiation of B6 mice with a 50,000 Ci 60Co source in the irradiation room, 1 x 107 donor marrow cells were injected via the tail vein.

Cancer Implantation, Excisional Wounding, and Bleomycin Administration
For creating fibrotic lesions induced by cancer implantation, we transplanted marrow cells from GFP Tg RAG-1–/– mice into irradiated (3.5 Gy) SCID mice. At 4 weeks after BMT, we subcutaneously inoculated SCID recipients with a transplantable human large-cell neuroendocrine carcinoma of the lung (613LCNEC), which was established in our laboratory and propagated in SCID mice for more than 10 passages. Four weeks later, tumors that had developed were resected and examined histologically (Fig. 1Go).



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  		Figure 1. Protocol of mouse bone marrow transplantation and induction of fibrotic lesions. Marrow cells from green fluorescent protein (GFP) Tg and RAG-1–/– (GFP transgenic and recombination activating gene 1 knockout) mice were transplanted into irradiated (3.5 Gy) severe combined immunodeficient mice for cancer implantation. Marrow cells from GFP Tg mice were transplanted into irradiated (9 Gy) B6 mice for excisional wounding and bleomycin administration.

 
For creating fibrotic lesions induced by excisional wounding, we transplanted marrow cells from GFP Tg mice into irradiated (9 Gy) B6 mice. At 4 weeks after BMT, we cut the skin of the right thigh using a dispopunch (Maruho Co., Osaka, Japan). One week later, skin of the right thigh was resected and examined histologically. For control analysis, skin of the left thigh was also resected.

For creating fibrotic lesions induced by bleomycin administration, B6 mice transplanted with GFP Tg marrow cells were treated with endotracheal bleomycin (Nihon Kayaku, Tokyo, http://www.nipponkayaku.co.jp/english) 4 weeks after BMT. Briefly, bleomycin was dissolved in sterile saline at 3.3µg/ml. BMT mice were treated with 5 µg/g body weight of bleomycin or the same volume of saline only. Four weeks after treatment, the lungs were removed and histologically analyzed.

Immunohistochemical Analysis
Immunostaining was performed on 4-µm formalin-fixed, paraffin-embedded tissue sections. Sections were treated for 20 minutes using a microwave-based antigen-retrieval technique with 10 mmol/L citrate buffer, at pH 6.0 and 90°C. Endogenous peroxidases were inactivated with 3% H2O2 in methanol. Sections were incubated for 1 hour with rabbit polyclonal anti-GFP Ab (Molecular Probes, Eugene, OR, http://www.probes.com), then with the DAKO EnVision+System-HRP (Dako Cytomation, Glostrup, Denmark, http://www.dakocytomation.com). The reacted products were stained with diaminobenzidine.

Immunofluorescence and Confocal Microscopy
Paraffin-embedded specimens were cut into 4-µm thick sections. Sections were treated using either a microwave-based antigen-retrieval technique (for CD34, C-kit and {alpha}-SMA staining) or a proteinase K solution (Dako) for 10 minutes at room temperature (for collagen type I staining). When rat monoclonal antibodies were used, sections were blocked using the MOM immunodetection kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Sections were incubated with the primary antibodies, including rabbit polyclonal anti-GFP Alexa Fluor 488 (Molecular Probes) at a 1:500 dilution and rabbit polyclonal anti-collagen type I (Calbiochem, San Diego, http://www.emdbiosciences.com) at a 1:500 dilution; rat monoclonal anti-mouse CD34 (MEC14.7; HyCult Biotechnology, Uden, Netherlands, http://www.hbt.nl) at a 1:10 dilution; rabbit polyclonal anti-C-kit (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) at a 1:100 dilution; and rabbit polyclonal anti-{alpha}-SMA (Lab Vision, Fremont, CA, http://www.labvision.com) at a 1:50 dilution. After washing the sections, either Alexa Fluor 546 goat anti-rat IgG or Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes) was used as the secondary antibody. Frozen sections were cut into 5-µm thick sections and were fixed in 10% formaldehyde for 5 minutes. Endogenous peroxidases were inactivated with 3% H2O2 in methanol. Sections were incubated with the primary antibodies, including rabbit polyclonal anti-GFP Alexa Fluor 488 at a 1:500 dilution; rat monoclonal anti-mouse CD45 (30-F11; eBioscience, San Diego, http://www.ebioscience.com) at a 1:200 dilution; and rat monoclonal anti-mouse Thy 1.2 (53-2.1, BD Bioscience, Franklin Lakes, NJ, http://www.bdbioscience.com) at a 1:50 dilution. After washing the sections, Alexa Fluor 546 goat anti-rat IgG was used as the secondary antibody. Before mounting, all sections were stained with DRAQ5 (Alexis Biochemical, Lausen, Switzerland, http://www.alexis-corp.com) for the discrimination of nucleated cells.

After mounting, the sections were examined using an LSM5 Pascal confocal imaging system (Carl Zeiss, Jena, Germany, http:// www.zeiss.com). The sections were examined using an inverted microscope with an excitation wavelength of 488 nm for Alexa Fluor 488, 568 nm for Alexa Fluor 546, and 633 nm for DRAQ5. Confocal images were stored as digital files and viewed using Photoshop (Adobe, Mountain View, CA, http://www.adobe.com).

Laser Capture Microdissection and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
To confirm the specificity of immunofluorescence with collagen type I, RT-PCR was performed on fibroblasts staining positive and negative for GFP using material obtained by the laser capture microdissection system (PixCell-II; Arcturus, Mountain View, CA, http://www.arctur.com). In brief, the dehydrated 10-µm frozen tissue section was overlaid with a thermoplastic membrane mounted on optically transparent caps, and GFP+ and GFP– fibroblasts (each corresponding to 500 to 1,000 cells) were captured by focal melting of the membrane through laser activation. The captured tissues were then immersed in denaturation Trizol solution (Life Technologies, Gaithersburg, MD, http://www.lifetech.com), and total RNA was extracted. RNA was redissolved in 10 µl of oligo (dT)20 primer solution, and cDNAs were synthesized using the ThermoScript RT-PCR system (Life Technologies) in a final volume of 20 µ1. One µl of cDNA solution was subjected to 40 PCR cycles of 10 seconds at 95°C, 10 seconds at 53°C–65°C, and 5–15 seconds at 72°C in a 10-µ1 mixture containing 2.25 mM MgCl2 and 0.25 µM each of forward and reverse specific primers. The primer sequences were as follows:

Collagen type I: forward: 5'-CTACTCAGCCGTCTGTGCCT-3'; reverse: 5'-GGCAGG GCCAATGTCTAGT-3'

GFP: forward: 5'-AAGTTCATCTGCACCACCG-3'; reverse: 5'-TCCTTGAAGAAG ATGGTGCG-3'

GAPDH: forward: 5'-TTGAAGGTAGTTTCGTGGAT-3'; reverse: 5'-GAAAATCTG GCACCACACCTT-3'

Assessment of the Immunohistochemistry and Double Immunofluorescence Findings
The proportions of BM-derived fibroblasts were determined by the ratio of GFP+ fibroblasts (GFP-Fbs) to the total number of tissue fibroblasts. The fields for cell counting were randomly selected in each tissue of BMT mice (n = 3 or 4). At least 100 fibro-blasts were counted in each high-power field (x400), and each numerical value was averaged. The proportion of both GFP and each fibroblast marker (CD34 , C-kit , CD45 , Thy-1 , and {alpha}-SMA) fibroblasts to the total GFP-Fbs was analyzed by the overlay image of the three fluorescent images (GFP, fibroblast marker, and DRAQ5). At least 10 fields were randomly selected in each tissue for cell counting. At least 100 GFP-Fbs were counted in each high-power field (x400), and each numerical value was averaged. All data are presented as mean ± SEM. Comparison between groups was made using two-way analysis of variance. p < .05 was considered statistically significant.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recruitment of BM-Derived Fibroblasts into Noninjured Skin and Lung
Sublethally irradiated mice were injected with 1 x 107 GFP-labeled BM cells. GFP phenotyping of BM cells from the recipient mice demonstrated that their marrows had been reconstituted by high levels (>80%) of donor cells 4 weeks after BMT (data not shown). Since sublethal irradiation may induce sequential events of tissue damage, we first evaluated the effects of irradiation on the skin and lung of the BM chimera mice. Histological examination revealed essentially normal skin and lung architecture without fibrosis, although mild perivascular inflammatory cell infiltration was pointed out. Since we focused on fibroblasts as cells with pivotal roles in the fibrosis, BMT in the process of creating BM chimera mice had no significant effects on the morphological analysis of the fibrotic process of the skin and lung. An antibody specific for GFP was used to investigate whether fibroblasts were of BM origin. In the skin of BM chimera mice, GFP-Fbs were mainly found adjacent to striated muscle of the dermis (Fig. 2 A–CGo). The frequency of GFP-Fbs within the total skin fibroblast was 8.7% ± 4.6%. In the lung, GFP-Fbs were found located around the bronchus and the vessels within the bronchovascular bundle (Fig. 2 D–FGo). The frequency of GFP-Fbs was 8.9% ± 2.5 % (Table 1Go).



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  		Figure 2. Microscopic appearance of (A–C) skin and (D–F) lung after bone marrow transplantation. Boxes indicate magnified regions of the (A, B) skin and (D, E) lung. C and F are serial sections of B and E, respectively. Arrowhead indicates GFP+ fibroblasts. Abbreviations: GFP, green fluorescent protein; H.E., hematoxylin and eosin.

 

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  		Table 1. Population (%) of green fluorescent protein–positive (GFP+) fibroblasts
 
Increased Recruitment of BM-Derived Fibroblasts into Fibrotic Lesions Induced by Cancer Implantation and Excisional Wounding
After implantation of a transplantable human large cell neuro-endocrine carcinoma of the lung into the skin, stromal fibrosis occurred prominently (Fig. 3A–BGo). Among the fibroblasts around the cancer nests, numerous fibroblasts showed positive reaction for GFP, and the frequency of GFP-Fbs was 59.7% ± 16.3% (Fig. 3CGo; Table 2Go). Upon excisional wounding, the injured area involving the deeper structure of the dermis and tissue was replaced by granulation tissue, which is comprised of many inflammatory cells and fibroblasts. Although GFP-Fbs were observed within the whole layer of skin, these cells have a tendency to locate in the deeper layer (Fig. 2 D–FGo). The frequency of GFP-Fbs per total fibroblasts was 32.2% ± 4.8%. In contrast, within lung fibrotic lesions induced by bleomycin administration, GFP+ cells were mainly mononuclear cells without spindle cytoplasm, as shown in Figure 3IGo. The frequency of GFP-Fbs was 7.1% ± 2.4%, which was similar to the frequency found in control lung tissue.



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  		Figure 3. Microscopic appearance of skin and lung after (A–C) cancer implantation, (D–F) excisional wounding, and (G–I) bleomycin administration. Boxes indicate magnified regions of the skin after (A, B) cancer implantation, (D, E) excisional wounding, and (G, H) bleomycin administration. C, F, and I are serial sections of B, E, and H, respectively. Note that numerous GFP+ fibroblasts are found in the fibrotic lesions induced by (C) cancer implantation and (F) excisional wounding, whereas few are found in (I) bleomycin administration. Abbreviation: H.E., hematoxylin and eosin.

 

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  		Table 2. Summary of phenotypes (%) of green fluorescent protein–positive (GFP+) fibroblasts
 
BM-Derived Fibroblasts Expressed Collagen Type I
Deposition of collagen type I is a key characteristic finding in tissue fibrotic processes, and fibroblasts are a well-known major producer of this molecule. To determine whether GFP-Fbs express collagen type I and contribute to tissue fibrosis, sections from cancer implantation and excisional wounding were analyzed by confocal immunofluorescence microscopy. When germinal center B cells of the spleen in BMT mice were stained with GFP and type I collagen, numerous B cells showed positive for GFP, but type I collagen–positive cells were hardly observed (Fig. 4AGo). Any GFP+ cells could not be detected in the mice reconstituted with GFP–/– (wild-type) marrow cells (Fig. 4BGo). As shown in Figure 4C–EGo, when GFP-Fbs were intermingled with GFP– fibro-blasts within cancer implantation (Fig. 4CGo), excisional wounding (Fig. 4DGo), and noninjured skin (Fig. 4EGo), both GFP positive and negative fibroblasts expressed type I collagen. This phenomenon was further confirmed using microdissection analysis. Microdissected dermal fibroblasts of the mouse without BMT expressed type I collagen mRNA but did not express GFP mRNA (Fig. 4FGo, left lane). Microdissected GFP-Fbs around the cancer nests of BMT mice expressed both collagen type I and GFP mRNA (Fig. 4FGo, right lane). These results indicated that BM-derived fibro-blasts produce type I collagen and contribute to tissue fibrosis.



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  		Figure 4. Colocalization of green fluorescent protein (GFP) and type I collagen on fibroblasts in the fibrotic lesions induced by cancer implantation and excisional wounding. (A): Germinal center B cells of the spleen in bone marrow–transplanted (GFP Tg) mice were stained with GFP and type I collagen. (B): Fibroblasts in cancer-induced stroma in the mice reconstituted with GFP–/– (wild-type) marrow cells were stained with GFP and type I collagen. (C–E): Fibroblasts in (C) cancer implantation, (D) excisional wounding, and (E) noninjured skin were stained with GFP and type I collagen. The upper left panel shows GFP fluorescence. The upper right panel shows cells immunostained with anti-type I collagen antibody in the same area. The lower left panel shows cells stained with DRAQ5 for the discrimination of nucleated cells. The lower right panel shows a composite of both fluorophores. (F): Reverse transcription polymerase chain reaction analysis of type I collagen gene in microdissected GFP+ fibroblasts. GFP– fibroblasts also expressed type I collagen transcripts.

 
Phenotype of BM-Derived Fibroblasts Within Fibrotic Lesions in the Skin
We assessed further phenotypes of GFP-Fbs with respect to their potential identity with previously identified fibroblast phenotypes. Double immunofluorescence staining for GFP and fibroblast markers (including CD34, C-kit, CD45, Thy-1, and {alpha}-SMA) was performed with the sections from cancer implantation, excisional wounding, and noninjured skin. We could not determine any GFP+/ CD34+ or GFP+/C-kit+ fibroblasts within the fibrotic lesions of the skin. As shown in Figure 5Go, some GFP-Fbs showed CD45, and these double-positive cells were intermingled with GFP+/CD45– fibroblasts. The ratio of GFP+/CD45+ fibroblasts per GFP-Fb in the cancer-induced stroma and in the granulation tissue produced by excisional wounding was 43.1% ± 5.5% and 38.5% ± 6.3%, respectively (Fig. 5A, DGo). GFP-Fbs in noninjured skin also expressed CD45 in similar proportion (37.5% ± 3.5%) (Fig. 5GGo; Table 2Go). The cancer-induced stroma and granulation tissue induced by excisional wounding contained GFP+/Thy-1+ fibroblasts, and the frequency of double-positive cells per GFP-Fb was 49.1% ± 6.1% and 46.7% ± 2.8%, respectively (Fig. 5B, EGo). GFP+/Thy-1+ fibroblasts were also found in noninjured skin, and its frequency was 62.6% ± 15.2 % (Fig. 5HGo). The frequency of GFP+/{alpha}-SMA+ fibroblasts (BM-derived myofibroblasts) per GFP-Fb in the cancer-induced stroma was 57.4% ± 2.1%, and this frequency was significantly higher than that in noninjured skin (27.2% ± 5.2%; p = .002) (Fig. 5C, IGo; Table 2Go). To further confirm whether BM-derived fibroblasts express CD45 and Thy-1, we performed immunohistochemical staining for GFP/{alpha}-SMA/CD45, and GFP/{alpha}-SMA/Thy-1 in serial sections of cancer-induced stroma. In these sections, almost all spindle cells showed positive for both GFP and {alpha}-SMA, indicating that these cells are BM-derived myofibroblasts. Within this area, many CD45+ spindle cells (Fig. 6AGo) and Thy-1+ spindle cells (Fig. 6BGo) were observed. These results showed that BM-derived (myo) fibroblasts express CD45 and/or Thy-1.



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  		Figure 5. Colocalization of (A, D, G) green fluorescent protein (GFP)/CD45, (B, E, H) GFP/Thy-1, and (C, F, I) GFP/ {alpha}-smooth muscle actin ({alpha}-SMA) on fibroblasts in the fibrotic lesions induced by cancer implantation (A–C) and excisional wounding (D–F), as well as (G–I) fibroblasts in noninjured skin. The upper left panel shows GFP fluorescence. The upper right panel shows cells immunostained with CD45, Thy-1, or {alpha}-SMA antibody in the same area. The lower left panel shows cells stained with DRAQ5 for the discrimination of nucleated cells. The lower right panel shows a composite of both fluorophores.

 


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  		Figure 6. Immunohistochemical detection of CD45+ bone marrow (BM)–derived fibroblasts and Thy-1+ BM-derived fibroblasts. (A): Green fluorescent protein (GFP), alpha-smooth muscle actin ({alpha}-SMA), and CD45 staining of serial sections of cancer-induced stroma. (B): GFP, {alpha}-SMA, and Thy-1 staining of serial sections of cancer-induced stroma.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fibroblasts that enter and proliferate within fibrotic lesions had been thought to be of residual tissue origin; however, recent studies revealed that BM can be a source of fibroblast. The findings described by the previous reports and the current study could be a significant development toward understanding the potential effects of the BM-derived fibroblasts on the pathogenesis of tissue fibrosis. Controversy remains upon pulmonary fibrosis induced by bleomycin administration. Hashimoto et al. [29] reported that in the lung injury model induced by bleomycin, a significant number of BM-derived fibroblasts migrated into the fibrotic lesion in response to a factor released by lung injury. In contrast, we barely found a small number of GFP-Fbs (7.1% ± 2.4%), whereas many GFP+ inflammatory cells existed in the fibrotic lesion. To confirm this result, we isolated and cultured fibroblasts from bleomycin-treated lung tissues, and the population of fibroblasts expressing GFP was examined by flow cytometry. The frequency of GFP+ fibroblasts was about 7.3%, which was similar to the result found by immunohistochemical analysis. The reason for the discrepancy was unclear; however, the differences in the contribution of BM-derived fibroblasts might be caused by the different methods of irradiation or different doses of drugs.

As fibroblast is a key producer of collagen and has a central role in tissue fibrosis, we examined whether BM-derived fibroblasts also produce type I collagen. Almost all of the BM-derived fibro-blasts within a fibrotic lesion induced by cancer implantation and excisional wounding expressed type I collagen (Fig. 4A, BGo). Considering the fact that significant numbers of BM-derived fibroblasts were present within the cancer stroma (59.7% ± 16.3%) and wound healing tissue (32.2% ± 4.8%), these results argue for the major direct role for these cells in the process of pathological fibrosis.

To define the further phenotypic signatures of BM-derived fibroblasts in vivo, we performed double immunofluorescence analysis. It should be noted that GFP-Fbs within both fibrotic lesion and dermis of the noninjured skin expressed in varying degrees of CD45, Thy-1, and {alpha}-SMA, indicating BM-derived fibroblasts were heterogenous with respect to phenotypic features. Although CD45 has been considered to be a lineage-restricted panhematopoietic marker [31, 32] and CD45+ mouse marrow stromal cells could not be detected in vitro culture in our preliminary experiment (data not shown), recent study showed that human BM mesenchymal cells expressed CD45 in vivo and were dramatically downregulated by in vitro culture [33]. Singer et al. [34] reported that the BM stromal cell line generated by SV-40 transformation expressed hematopoietic markers, including CD45. In the current study, the frequency of CD45+ fibroblasts per GFP– fibroblast was 29.2% ± 0.6% (data not shown), whereas the frequency of CD45+ fibroblasts per GFP-Fb within cancer stroma and wound-healing tissue was 52.9% ± 15.9% and 66.1% ± 3.9%, respectively. Therefore, given that mesenchymal stem cells are the source of BM-derived fibroblasts, CD45 might be a candidate marker of BM-derived fibroblasts in this model. Furthermore, the presence of a significant proportion of CD45– BM-derived fibroblasts would be explained by the possibility that BM-derived fibroblasts are of heterogenous origin or that CD45 might be downregulated within fibrotic microenvironments.

Thy-1 is a cell-surface glycoprotein, whose function remains ill-defined. Human fibroblasts were heterogeneous with respect to surface Thy-1 expression [35–37]. Thy-1+ and Thy-1– subsets showed functionally distinct subpopulations in inflammatory cytokine production, prostaglandin production, CD40 expression [35, 36], and lineage differentiation [37], suggesting that the balance between these populations might contribute to tissue homeostasis. We investigated whether BM-derived fibroblasts within fibrotic lesions belong to either the Thy-1+ or the Thy-1– subpopulation. We found, however, that BM-derived fibroblasts comprised both Thy-1+ and Thy-1– subpopulations to the same degree. Therefore, BM-derived fibroblasts are phenotypically heterogeneous with respect to the Thy-1 expression, which may have important consequences in the pathogenesis of fibrotic process.

A subpopulation of fibroblasts has been reported to express {alpha}-SMA, and these cells are called myofibroblasts. Myofibroblasts have been observed in normal and pathological situations and are considered responsible for the contractile forces that close wound margins. The frequency of GFP+/{alpha}-SMA+ per GFP-Fb within cancer-induced stroma was 59.3% ± 2.0%, and this frequency was significantly higher than that found within the dermis of nonfibrotic skin (37.4% ± 8.5%). In our data, the proportion of {alpha}-SMA+ fibroblasts was variable and depended on the cancer characteristics (unpublished data). This phenomenon might be explained by the persistent provision of mediators such as transforming growth factor–beta and tumor necrosis factor-{alpha} secreted by cancer cells, which are implicated in maintaining myofibroblast formation.

Early studies described the presence of fibroblasts in normal peripheral blood, termed circulating fibrocytes. Circulating fibrocytes comprise 0.1%–0.5% of the human nonerythrocytic cell population in peripheral blood [38, 39]. Thus it is possible to speculate that BM-derived fibroblasts are identical with the population of circulating fibrocytes. However, the results obtained here revealed that BM-derived fibroblasts and circulating fibrocytes showed different phenotypes, since circulating fibrocytes express both CD45 and CD34, whereas BM-derived fibroblasts in our model express CD45 but not CD34. Furthermore, previous reports described that when using sex-mismatched BM chimera mice, circulating fibrocytes originated from the host tissue, not from transplanted marrow cells. It would be interesting to examine whether BM-derived fibroblasts and circulating fibrocytes are from the same source.

The results that GFP-Fbs could also be found in subcutaneous tissue of noninjured skin and lung suggested that BM can potentially contribute to the turnover of fibroblasts and are involved in tissue homeostasis. However, it must be kept in mind that this occurred in the context of sublethal irradiation and BMT.

Increased research on fibroblasts has made clearer the concept of subset specialization of fibroblasts. Further studies about the phenotypical and functional analyses of BM-derived fibroblasts would help us gain insight into the pathogenesis of the fibrotic process.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are grateful to Yoko Okuhara and Chie Okumura for technical support and Suzaki Motoko for help in preparing the manuscript. This work was supported in part by the Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare; the Grant for Scientific Research Expenses for Health Labour and Welfare Programs; the Foundation for the Promotion of Cancer Research, 2nd-Term Comprehensive 10-Year Strategy for Cancer Control; and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. T.S. is a recipient of Research Resident Fellowships from the Foundation for Promotion of Cancer Research.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 9, 2004; accepted for publication January 5, 2005.




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