First published online May 18, 2006
Stem Cells
Vol. 24 No.
9
September 2006, pp.
2071
-2077
doi:10.1634/stemcells.2005-0575; www.StemCells.com
© 2006 AlphaMed Press
TISSUE-SPECIFIC STEM CELLS |
Treatment and Transfer of Emphysema by a New Bone Marrow Transplantation Method from Normal Mice to Tsk Mice and Vice Versa
Yasushi Adachia,b,
Haruki Oyaizua,c,
Shigeru Taketanid,
Keizo Minaminoa,
Kazuyuki Yamaguchic,
Leonard D. Shultze,
Masayoshi Iwasakia,
Minoru Tomitaa,
Yasuhiro Suzukia,
Keiji Nakanoa,
Yasushi Koikea,
Ryoji Yasumizua,
Makoto Sataf,
Noriyuki Hiramaf,
Isao Kubotaf,
Shirou Fukuharac,
Susumu Ikeharaa,b
aFirst Department of Pathology, Kansai Medical University, Moriguchi-City, Osaka, Japan;
bTransplantation Center, Kansai Medical University;
cFirst Department of Internal Medicine, Kansai Medical University;
dDepartment of Biotechnology, Kyoto Institute of Technology, Sakyo-ku, Kyoto, Japan;
eThe Jackson Laboratory, Bar Harbor, Maine, USA;
fFirst Department of Internal Medicine, Yamagata University School of Medicine, Iida-Nishi, Yamagata, Japan
Key Words. Emphysema • Treatment • Tight-skin mice • Intrabone marrow-bone marrow transplantation
Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, Fumizono-cho, Moriguchi City, Osaka, Japan, 570-0023. Telephone: 81-6-6992-1001, ext. 2474 or 2475; Fax: 81-6-6992-1219; e-mail: ikehara{at}takii.kmu.ac.jp
Received November 22, 2005;
accepted for publication April 10, 2006.
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ABSTRACT
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We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight-skin [Tsk] mouse) to examine whether IBM-BMT could be used to treat emphysema in Tsk mice. IBM-BMT was carried out from C3H mice into Tsk mice (8–10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM-BMT [C3H
Tsk] showed similar structures to those of normal mice, whereas the [TskTsk] mice showed emphysema, as seen in age-matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM-BMT. Six months after IBM-BMT, the [TskC3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.
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INTRODUCTION
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Emphysema is one of the major factors determining morbidity and mortality in chronic obstructive pulmonary diseases, in which the destruction of the lungs’ gas exchange structures leads to inadequate oxygenation [1–3]. Emphysema is defined as the enlargement of peripheral air space in the lung (including respiratory bronchioles and alveoli), which is accompanied by the destruction of alveolar wall structures [4, 5]. The incidence of patients with emphysema has increased in the past decades as a result of the increase in environmental pollutants, cigarette smoking, and other exposure to noxious substances [6]. Furthermore, the inherited deficiency of 
-1-antitrypsin, which is the primary inhibitor of neutrophil elastase, predisposes individuals to the early onset of emphysema [7]. As it stands, only lung transplantation can provide remediation for severe emphysema.
The tight-skin (Tsk) mouse has a dominant mutation in which the heterozygote (Tsk/+) mouse shows tight skin with marked hyperplasia of the subcutaneous connective tissue (similar to scleroderma), the augmented growth of cartilage and bone, small tendons with hyperplasia of the tendon sheaths, increased thoracic size, and large distended lungs [8]. The Tsk mutation is a duplication of the mouse fibrillin one (Fbn1) gene, which results in a larger (418 kD) than normal (350 kD) protein; Tsk/Tsk mutation is embryonic-lethal, and Tsk/+ mice display increased connective tissue resulting in tight skin [9]. However, it has been suggested that not only abnormality of Fbn1 but also some cytokines are required for onset of tight skin; it has been reported that the Tsk/+ mice introduced with interleukin (IL)-4–/–, IL-4–/+, IL-4R-–/– or transforming growth factor (TGF)-ß–/+ and Tsk/+ mice treated with anti-IL-4 antibody (Ab) fail to develop cutaneous hyperplasia [10–12]. The Tsk mouse also represents an animal model for spontaneous emphysema, but deletion of IL-4 or the TGF-ß gene has no effects on emphysema [10, 11]. These results suggest that the genomic mutation by itself does not induce the cutaneous fibrosis and that there are different mechanisms underlying the development of cutaneous fibrosis and emphysema. There are several reports describing possible mechanisms underlying the development of emphysema in Tsk mice (e.g., extremely low levels of antielastase or 1-proteinase inhibitor in Tsk mice) [13, 14], but there are also some contrary data [15]. As a result, the precise mechanisms underlying the development of emphysema in Tsk mice are still unclear.
Using various autoimmune-prone mice, we have previously shown that conventional allogeneic bone marrow transplantation (BMT) can be used to prevent and treat autoimmune diseases [16–21]. Recently, we have found that intra-bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) (IBM-BMT) creates an appropriate hemopoietic environment for the early recovery of hemopoiesis and donor cell engraftment, resulting in the complete amelioration of intractable autoimmune diseases in chimeric-resistant MRL/lpr mice without using immunosuppressants [22].
In this study, we show that IBM-BMT can be used to treat emphysema in Tsk mice and that emphysema in these mice can be transferred to normal mice by IBM-BMT, suggesting that autoimmune mechanisms are involved in the development of emphysema in Tsk mice.
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MATERIALS AND METHODS
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Mouse Strains
Tsk/+ and pa/pa mutant mice on a C57BL/6 (H-2b) background were obtained from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). C3H/He (H-2k) mice (C3H mice) were obtained from Japan SLC (Hamamatsu, Sizuoka, Japan, http://www.jslc.co.jp). Because Tsk/+ mice are heterozygous of the Tsk and pa mutation, Tsk/+ mice were prepared by mating Tsk/+ male mice with pa/pa female mice. Tsk/+ progeny were identified by their black coat and eye color as well as by their characteristic loss of skin pliability. These mice were maintained in our animal facility under specific pathogen-free conditions and were used at 8–10 weeks of age.
Experimental Protocols
The Tsk/+ mice (Tsk mice) (8–10 weeks of age) with emphysema were irradiated in fractionated doses (5.5 Gy x 2 = 11 Gy; 4-hour interval) because giving fractionated doses caused less damage in the recipients and was more effective in reconstituting the hemopoietic cells than was a single dose of irradiation [23]. One day after the irradiation, the mice were transplanted with the whole BMCs (3 x 107) of C3H/He by IBM injections (C3H into Tsk) as previously described [22]. Two groups were prepared as controls. In the first group, Tsk mice were irradiated (5.5 Gy x 2) and transplanted with syngenic whole BMCs (3 x 107) by IBM (Tsk into Tsk). In the second group, C3H/He mice were irradiated (5.5 Gy x 2) and transplanted with syngeneic whole BMCs (3 x 107) by IBM (C3H into C3H). After confirming more than 90% engraftment in peripheral white blood cells, we performed morphological analyses, western blot analyses, and analyses using a confocal microscopy in (C3HTsk) mice.
For the alternative BMT experiment, we first prepared the lineage-negative (Lin–) BMCs to exclude the effects of mature lymphocytes. To prepare the Lin– BMCs, BMCs from 6-month-old mice were incubated with biotin-labeled anti-CD3, anti-B220, anti-Gr-1, anti-CD11b, anti-CD11c, and anti-NK 1.1 Abs, followed by incubation with avidin-conjugated magnet beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com/nn.404,company.html). These Abs were purchased from BD PharMingen (San Diego, http://www.bioresearchonline.com). The cells were passed through a magnetic cell sorting (MACS) midi column (Miltenyi Biotec GmbH) for negative selection of Lin– BMCs. These negatively selected BMCs were used as Lin– BMCs. Lin– BMCs (1 x 106/mouse) from Tsk/+ mice were transplanted into irradiated (6 Gy x 2) 8-week-old Tsk/+ mice and C3H mice by IBM. Lin– BMCs (1 x 106/mouse) from C3H mice were transplanted into irradiated 8-week-old C3H mice by IBM.
Fixation, Tissue Sampling, and Tissue Preparation
Animals were killed via cervical dislocation, then median sternotomy was performed. To obtain tissue for histological analyses, right heart perfusion was accomplished with calcium- and magnesium-free phosphate-buffered saline (PBS) to clear the pulmonary intravascular space; the tracheas of the mice were cannulated, and the lungs were inflated to approximately 25 cm with neutral-buffered formalin before fixation (neutral-buffered 15% formalin) overnight at 4°C as previously described [10]. Fixed samples of the lung and skin were rinsed in PBS, dehydrated, and embedded in paraffin. Sections (2 µm) were collected on microscope slides and stained with hematoxylin and eosin (H&E) and Masson Fontana for the lungs and skin, respectively. For immunofluorescence study, the lungs were frozen with OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN, http://www.informagen.com).
Morphometric Analysis
The size of alveolar airways was determined by measuring the mean chord length on H&E-stained lungs as previously described [23]. This measurement is similar to the mean linear intercept, a standard measure of air-space size. To obtain images at random for analysis, each glass slide was placed on a printed rectangular grid and a series of dots was placed on the cover glass at the intersection of the grid lines. Fields as close as possible to each dot were acquired by systematically scanning at 2-mm intervals. A minimum of six fields from each mouse lung were acquired into a Macintosh computer (Apple Computer, Inc., Cupertino, CA, http://www.apple.com). Images were acquired in 8-bit grayscale at a final magnification of 1.5 pixels per micrometer. The images were analyzed on a Macintosh G4 computer using the public domain NIH Image program written by Wayne Rasband at the National Institutes of Health. Image thresholds were manually set, and the images were then smoothed and inverted. The images were subjected to sequential logical imaging matching and operations with a horizontal and then vertical grid [24]. We analyzed at least 120 alveoli per mouse. The length of the lines overlying air space was averaged as the chord length.
Abs and Surface Marker Analysis
Fluorescein isothiocyanate (FITC)-coupled anti-H2Kb and phycoerythrin (PE)-coupled anti-H2Kk Abs from BD PharMingen were used for H-2 typing. Cells were analyzed by a FACScan (Becton Dickinson and Company, Mountain View, CA, http://www.bd.com).
Immunofluorescence Study of Lung
Frozen sections (2 µm) were stained using FITC-coupled anti-H2Kk plus PE-coupled anti-Mac-1 Abs or FITC-coupled anti-H-2Kk plus anti-cytokeratin followed by PE-coupled anti-rabbit Abs. The samples were observed using an Olympus LSM-GB200 confocal laser microscope (Olympus, Tokyo, http://www.olympus-global.com).
BMT from Aged Tsk/+ Mice to C3H Mice
Two-month-old C3H mice were irradiated with fractionated doses (6.0 Gy x 2 = 12 Gy; 4-hour interval). One day after the irradiation, Lin– BMCs were prepared, because it has been reported that mature T cells and B cells of Tsk mice induce fibrosis in normal mice [25], suggesting that contaminated mature immunological cells in the bone marrow might have some effects on emphysema, regardless of engraftment of bone marrow stem cells. The Lin– BMCs (1 x 106/mouse) of 6-month-old male Tsk/+ mice were directly injected into the tibias of C3H mice (IBM-BMT). The Lin– BMCs of Tsk/+ mice were prepared as follows: BMCs of Tsk/+ mice were incubated with biotin-labeled anti-CD3, anti-B220 Ab, anti-Gr1, anti-Mac 1, anti-NK1.1, and anti-ter119 Abs, followed by incubation with avidin-conjugated magnet beads (Miltenyi Biotec GmbH). The cells were passed through a MACS midi column (Miltenyi Biotec GmbH) for negative selection of Lin– BMCs.
Statistical Analyses
Student’s t test was used to determine statistical significance.
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RESULTS
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Amelioration of Emphysema in Tsk/+ Mice by IBM-BMT
The Tsk mouse is known as an animal model for genetically determined emphysema. Martorana et al. have reported that the enlargement of peripheral lung units in Tsk mice is evident on days 4–15 after birth and that the destruction of their lung parenchyma occurs between 15 days and 1 month, rapidly progressing by 2 months [26]. It has also been reported that the stabilization or mild progression of emphysematous lesions occurs between 2 and 8 months of age and that the further exacerbation of the parenchymal destruction develops between 8 and 16 months of age [26]. Therefore, we first examined the histology of the lungs of Tsk mice at various ages (Fig. 1). As shown in Figure 1, 8-week-old Tsk mice showed the enlargement of peripheral lung units and the destruction of lung parenchyma (Fig. 1B) in contrast to normal C3H mice (Fig. 1A). More severe lung emphysema was found in the lungs of 12-month-old Tsk mice (Fig. 1C).
We next examined whether allogeneic BMT [C3HTsk] could be used to treat emphysema by promoting new alveologenesis. To exclude the effect of irradiation on the lungs, syngeneic BMT ([TskTsk] and [C3HC3H]) was carried out. We used 8-week-old Tsk mice as recipients because 8-week-old Tsk mice already show emphysema (Fig. 1B). We first carried out conventional BMT: Tsk mice received fractionated irradiation (5.5 Gy x 2) followed by intravenous (i.v.) injection of C3H mouse BMCs 1 day after irradiation (i.v.-BMT). However, this protocol could not reconstitute the recipients with donor hemopoietic cells completely; the peripheral blood showed mixed chimerism (70%–80% donor type in the peripheral white blood cells) (Fig. 2), and emphysema in the recipients was not cured. Therefore, we carried out IBM-BMT: BMCs of donor C3H mice were directly injected into the bone marrow of Tsk mice 1 day after irradiation of 5.5 Gy x 2, as described in Materials and Methods, because we have very recently found that IBM-BMT is more effective than i.v.-BMT in treating autoimmune diseases in chimeric resistant MRL/lpr mice [22]. With this method, complete hemopoietic reconstitution with donor cells was achieved in [C3HTsk] mice observed 3 months after IBM-BMT (Fig. 2) (as we previously reported in [B6MRL/lpr] mice [22]). Six months after IBM-BMT, we killed the recipients to histologically examine the lungs. [C3HTsk] mice showed no emphysema (Fig. 1F), although [TskTsk] mice showed severe emphysema (Fig. 1E), and the lungs of [C3HC3H] mice showed a histology similar to that of non-treated C3H mice (Fig. 1D). These findings suggest that IBM-BMT can be used to not only prevent the progression of emphysema in Tsk mice but also ameliorate emphysema (Fig. 1F). It has been reported that irradiation can induce pulmonary fibrosis [27]; however, the lungs of [C3HC3H] mice showed a histology similar to that of non-treated C3H mice (Fig. 1D), suggesting that the irradiation for BMT had no harmful effects on the lungs in our experiment.
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Figure 2. The effect of IBM-BMT on the chimerism of peripheral blood. Three months after BMT, peripheral blood was obtained and stained with PE-labeled anti-H-2Kb and FITC-labeled anti-H-2Kk antibodies. Abbreviations: BMT, bone marrow transplantation; FITC, fluorescein isothiocyanate; IBM, intrabone marrow; IV, intravenous; PE, phycoerythrin; Tsk, tight-skin.
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We next quantified the alveolar changes by measuring the mean chord length. As shown in Figure 3, C3H mice did not show any changes in alveolar dimensions 6 months after syngeneic IBM-BMT [C3HC3H]. In contrast, after syngeneic BMT [TskTsk], Tsk mice showed severe enlargement of alveoli. To our surprise, [C3HTsk] mice showed a marked reduction in the size of the alveoli.
Recently, it has been reported that BMCs can differentiate into epithelial cells in the lungs [28, 29]. The phenomenon suggests that newly generated pulmonary epithelial cells from transplanted BMCs contributed to the amelioration of pulmonary structure. As shown in Figure 4A, very small numbers of pulmonary epithelial cells were detected in [C3HTsk] mice. On the other hand, we found that almost all hematopoietic cells (especially macrophages) were donor-derived (Fig. 4B), suggesting that regenerated pulmonary epithelial cells did not contribute to the improvement of the pulmonary structure in [C3HTsk] mice but that hematopoietic cells did instead.
Amelioration of Skin Structure by IBM-BMT
In general, [TskTsk] mice (8 months after birth) showed bent spine, rough skin, and disheveled hair, as seen in nontreated aged Tsk mice, whereas [C3HTsk] mice (8 months old) showed straight spine and smooth hair (data not shown). In microscopical examinations, collagen fibers were found to increase in the skin of nontreated Tsk mice (>8 months old) and [TskTsk] mice (>8 months old) in comparison with C3H mice or [C3H C3H] mice (Fig. 5). However, [C3HTsk] mice showed similar skin histology to normal C3H mice or [C3HC3H] mice (Fig. 5). These results suggest that IBM-BMT is effective in treating both scleroderma and emphysema of Tsk mice.
Transfer of Emphysema from Tsk Mice to Normal Mice by IBM-BMT
To clarify whether emphysema originates from defects of stem cells in the bone marrow, we carried out IBM-BMT from Tsk mice to normal C3H mice using immunocompetent cell-depleted BMCs (Lin– cells) of Tsk mice. Six months after IBM-BMT, the [C3HC3H] mice showed normal appearance in the lung (Fig. 6A), whereas the [TskC3H] mice showed emphysema, although not to the same degree as seen in the [TskTsk] mice (Fig. 6B).
The size of alveolar airways was determined by measuring the mean cord length on H&E-stained lungs. As shown in Figure 6D, the mean chord length in the lungs of [TskC3H] mice was significantly greater (p < .05) than that in the lungs of [C3HC3H] mice, although it was not as great as in the lungs of [TskTsk] mice, probably due to the short-term of observation (6 months after IBM-BMT).
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DISCUSSION
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The Tsk mouse was first discovered by Green et al. [8], who describe the mouse with features of established scleroderma, including dermal fibrosis. The Tsk mouse is a mutant caused by an autosomal dominant gene defect of the inbred B10.D2(58N)/SN mouse strain. The Tsk gene is located in chromosome 2, approximately 2 cm distal to the pallid locus. The Tsk gene is associated with a tandem duplication of the fibrillin (Fbn1) gene, which results in a larger-than-normal in-frame Fbn1 transcript. Because the Fbn1 protein participates in microfibril assembly and provides a structural basis for microfibril formation, it may be responsible for the connective tissue alteration [30]. The Tsk mouse also shows increased thoracic size and enlarged lungs [8]. Szapiel et al. [31] and Rossi et al. [32] made a morphological investigation of the Tsk lung. They observed a generalized enlargement of air spaces with markedly thinned or broken alveolar walls and also an increase in the size of the pores of Kohn. These morphological changes were accompanied by increases in total lung capacity and static compliance. On the basis of these findings, they proposed the Tsk mice as a model for genetically determined emphysema.
In the present study, we have examined whether allogeneic BMT could be used to treat emphysema by promoting new alveogenesis. Histological examination showed that severe emphysematous changes in Tsk mice were dramatically repaired after allogeneic BMT. Emphysema is pathologically defined as a condition of the lung characterized by the abnormal permanent enlargement of the air spaces distal to the terminal bronchiole accompanied by the destruction of alveolar walls. Both an elastolytic process and the inborn defect of connective tissue play a role in the development of emphysema in Tsk mice [31]. Barnara Gayraud et al. suggested that bone and lung abnormalities of Tsk/+ mice are due to copolymerization of mutant and wild-type Fbn1 into functionally deficient microfibrils [9]. We also carried out Western blot analyses using anti-Fin one Ab and have found that the large size of Fbl-1 (418 kD) remains even after IBM-BMT (data not shown). These results suggest that the IBM-BMT can ameliorate the morphological abnormalities of the lungs in Tsk mice regardless of the existence of abnormality of Fib 1.
It has been reported that Th2 cytokines and TGF-ß play a crucial role in the development of dermalfibrosis in mice [10]; IL-4R-–/– Tsk mice did not develop dermalfibrosis, and the deletion of one allele of TGF-ß gene of Tsk mice resulted in diminishing dermalfibrosis. Therefore, it seems likely that Th2 cytokines are related to emphysema. However, IL-4R-–/– Tsk and TGF-ß+/– Tsk mice showed emphysema, as seen in Tsk mice. These data suggest that the mechanisms underlying the induction of dermalfibrosis are different from those underlying the development of emphysema.
Recently, it has been reported that bone marrow-derived stem cells can differentiate into various cells, including epithelial cells in the lungs [28]. Therefore, using a confocal microscope, we extensively examined whether donor-derived epithelial cells can be found in the epithelial cells in the lungs of recipient [C3HTsk] chimeric mice and [GFP (green fluorescent protein)Tsk] chimeric mice. We found a very small number of donor-derived epithelial cells in the lungs of [C3HTsk] mice, and it is inconceivable that such small numbers of donor-derived epithelial cells could be associated with the amelioration of lung structure. However, we did find many donor-derived hematopoietic cells in the lung of [C3HTsk] chimeric mice (data not shown). Therefore, in the absence of immunological abnormalities, it seems likely that epithelia and/or epithelial stem cells in the recipient lungs proliferated and/or differentiated into new epithelia, resulting in regeneration of the lung. These phenomena could attribute to the facility for regrowth in the lungs in rodents [33]. Therefore, we suppose that normalization of hematopoietic stem cells, not mesenchymal stem cells, could be attributed to the improvement of pulmonary structure.
Finally, we have succeeded in transferring emphysema into normal C3H/He mice by carrying out IBM-BMT using immunocompetent cell-depleted BMCs (Lin– cells) of Tsk mice. These findings suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow and that autoimmune mechanisms are involved in the development of emphysema, as we previously reported in autoimmune-prone mice [34]. We have previously described that IBM-BMT induces tolerance easier than i.v.-BMT [22], and we have shown this phenomenon in this experiment also. The mice treated with IBM-BMT showed complete chimerism; on the other hand, the mice treated with i.v.-BMT showed mixed chimerism. Moreover, the mice treated with i.v.-BMT did not show the improvement of the pulmonary structure. It is conceivable that residual hematopoietic cells of Tsk mice disturbed the improvement of the pulmonary structure, because the hematopoietic cells of Tsk mice have some immunological abnormalities. Therefore, we think that even i.v.-BMT can improve the pulmonary structure if the complete chimerism is achieved by i.v.-BMT.
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CONCLUSION
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We have shown that allogeneic BMT can be used to not only prevent the progression of emphysema but also improve the structure of the lung.
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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H.O. and Y.A. contributed equally to this study.
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Abstract
Introduction
