Age-related thymic involution is mediated by Fas on thymic epithelial cells
Nobuyuki Yajima,
Kazuhiro Sakamaki and
Shin Yonehara
Graduate School of Biostudies and Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Correspondence to: S. Yonehara; E-mail: syonehar{at}virus.kyoto-u.ac.jp
Transmitting editor: S. Nagata
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Abstract
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Age-related thymic involution, which is linked to senescence of the immune system, was found to be mediated by the death receptor Fas. The thymus of aged Fas/ mice exhibited an intact structure and the normal differentiation of thymocytes. Both thymocytes and thymic epithelial cells (TECs) were sensitive to Fas-mediated apoptosis, and in vivo stimulation of wild-type thymocytes with anti-CD3 mAb was shown to induce apoptosis in TECs in a Fas-dependent manner. In addition, thymopoiesis continued uninterrupted in aged Fas/ mice that had been lethally irradiated and reconstituted with bone marrow cells derived from wild-type mice, while age-related thymic involution was observed in irradiated wild-type mice reconstituted with bone marrow cells from either Fas/ or wild-type mice. The results indicate that Fas on TECs plays a key role in age-related thymic involution.
Keywords: aging, apoptosis, FasL, thymocyte
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Introduction
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The thymus is a central lymphoid organ that is the site of T lymphocyte development. T cell precursors derived from bone marrow migrate into the thymus and undergo differentiation into mature T cells with the help of cytokines and cell-to-cell interactions with thymic stromal cells (1,2). During the development, immature T cell progenitors with the CD4CD8 double negative (DN) phenotype proliferate and differentiate in the outer cortex of the thymus. This maturation is accompanied by the rearrangement and expression of T cell receptor (TCR) genes (3). Then, thymocytes differentiate into CD4+CD8+ double-positive (DP) T cells, the most abundant population expressing TCR. Finally, they reach the differentiated stage, becoming CD4+CD8 or CD4CD8+ single-positive (SP) T cells (46). These mature SP T lymphocytes, located in the thymic medulla, are then released into peripheral lymphoid tissues.
In mammals from rodents to humans, age-related deterioration in the immune system has been observed to be associated with an increased susceptibility to infections, autoimmune disease and cancers. This senescence of the immune system seems to be linked to thymic involution with aging. The phenotypic process of age-related thymic involution has been well described: the thymus gradually reduces in size and weight as well as cellularity (7). The physiological changes to the thymus with aging are considered to be a consequence of alterations in bone marrow, extra- and/or intra-thymus (8). Thymic involution was reported to be associated with changes in thymocytes, such as a deficiency affecting the gene rearrangement of the TCR (9) and decrease in the number of progenitor cells derived from bone marrow (10), while changes in thymic stromal cells were demonstrated to be closely linked to the age-related thymic involution (11). The interaction between thymocyte precursors and thymic epithelial cells (TECs), a major component of thymic stromal cells, is necessary for the differentiation of not only thymocytes but also TECs. Age-related thymic involution was reported to be correlated with aging of TECs with an associated reduced expression of MHC molecules (12) and decreased production of thymopoietic cytokines (13). However, the precise molecular mechanisms of age-related thymic involution are still not well defined.
Fas (CD95/Apo-1) is a cell surface receptor belonging to the tumor necrosis factor receptor superfamily which introduces apoptosis-inducing signals into Fas-expressing cells via ligation with Fas ligand (FasL) or agonistic anti-Fas mAb (1416). Fas expression was observed in various tissues including thymus, spleen, heart, lung, liver and ovary, and high levels of Fas are expressed on T and B lymphocytes especially when these cells are activated (1719). Although previous studies have shown that the Fas/FasL system is the principal component of the peripheral immune system, the role of Fas in the central immune system is not well understood.
In this study, we analyzed the role of Fas in age-related thymic involution in mice. Aged Fas/ mice exhibit an intact thymus with an associated normal development of thymocytes, although wild-type mice having the same genetic background show thymic involution. Furthermore, Fas expressed on thymic stromal cells was shown to play a key role in age-related thymic involution.
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Methods
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Animals
C57BL/6 and Balb/c mice were purchased from CLEA Japan Inc. The Fas/ mice (20) had been maintained in our laboratory. The Fas-deficient mice on a C57BL/6 background was generated by backcrossing with C57BL/6 mice seven times. Transgenic mice expressing green fluorescent protein (GFP) under the control of CAG promoter (GFP-tg mice) (21) on the same C57BL/6 background were kindly provided by Dr S. Yamada (Institute for Virus Research, Kyoto University). All animals were housed and aged under specific pathogen-free conditions with autoclaved water and controlled feeding until used.
PCR analyses and northern hybridization
For detecting TCRß rearrangements in thymocytes, PCR analyses were carried out as described previously (22). For northern blotting, RNA was prepared from thymi of young (8-week-old) and aged (12-month-old) Fas/ and wild-type mice using Isogen (Nippongene, Tokyo, Japan). After 10 µg of total RNA was separated by agarose-gel electrophoresis and transferred to hybond-N (Amersham Bioscience), northern analysis was carried out as reported (23) using mouse IL-7 cDNA (24) as a probe for detecting IL-7 mRNA. Human elongation factor 1
(EF1
) cDNA was used as a probe for detecting the mouse control housekeeping gene EF1
(25) as previously described (16,23). Amounts of IL-7 and EF1
mRNA were quantified using ImageQuant software (Amersham Biosciences).
Flow cytometric analyses
Thymi were removed from euthanized mice, and disrupted into a single cell suspension. Isolated thymocytes were incubated on ice for 30 min with appropriately diluted antibodies in staining buffer (PBS containing 5% FBS and 0.05% NaN3). Two-color and three-color flow cytometric analyses were performed using EPICS Elite (Beckman Coulter). Antibodies used in this study were PE- and biotin-conjugated anti-CD4, FITC- and biotin-conjugated anti-CD8, PE-conjugated anti-Thy1.2, FITC- and biotin-conjugated anti-B220, and FITC-conjugated anti-mouse Fas mAb Jo2 (Pharmingen); and biotin-conjugated anti-mouse MHC class II (I-A/I-E) mAb (eBioscience).
To analyze apoptosis, thymic cells containing both thymocytes and thymic stromal cells were washed with PBS, fixed in 4% paraformaldehyde (PFA), and incubated with TUNEL reaction mixture with FITCdUTP (Boehringer Mannheim) according to the manufacturers instructions. After blocking with skim milk, cells were stained with rabbit anti-cytokeratin antibody (DAKO), and then with PEanti-rabbit IgG (Serotec).
Immunohistochemical analyses
Thymi were immersed in OCT compound (SAKURA, Tokyo, Japan), snap-frozen, and cut into 8 µm thick using a cryostat. Freshly cut frozen sections were air-dried and fixed with 4% PFA in PBS at room temperature for 20 min. Samples were blocked with PBS containing 0.1% Tween-20 and 5% skimmed milk, then incubated overnight with the cortical epithelium-reactive rat mAb 6C3 (Pharmingen) or rabbit anti-cytokeratin antibody (DAKO) in blocking solution. For analyses of the thymic cortico-medullary structure, samples were incubated with horseradish peroxidase-conjugated anti-rat Ig antibody or anti-rabbit Ig antibody (Amersham Bioscience) in PBS. Peroxidase activity was developed with 0.1% 3,3'-diaminobenzidine and 0.02% H2O2 in PBS. Sections were then counterstained with methyl green, dehydrated, and mounted with a glass coverslip for photomicroscopic analyses. For studying apoptosis, samples were incubated with rabbit anti-cleaved caspase-3 antibody (Cell Signaling) and the anti-pan cytokeratin mAb clone PCK-26 (Sigma). Alexa Fluor 594-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG (Molecular Probes) were used as the secondary antibody. Samples were observed under a confocal microscope and images were collected.
Bone marrow transplantation
Eight-week-old male mice were used as both bone marrow donors and recipients. Bone marrow cells were isolated from the femurs and tibias of donor mice. Recipient mice were lethally irradiated with 9 Gy of 137Cs gamma radiation (Gammacell Exactor 40, Nordion), and then injected with 5 x 106 nucleated cells through a fundal vein.
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Results
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Impaired age-related thymic involution in Fas/ mice
In humans and rodents, thymic involution begins early in life and gradually advances with aging. Although the largest reduction of mouse thymus was reported to be observed at the age of 9 to 12 months (9), we found apparently normal thymi in Fas/ mice over the age of 10 months. Figure 1(A) shows the appearance of the thymi of 12-month-old male wild-type and age-matched male Fas/ mice with the same C57BL/6 background. While wild-type thymus was greatly reduced in size and replaced into a considerable amount of fatty tissue, the Fas/ thymus was found to be normal in both morphology and size and we never observed accumulation of the fatty substance.

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Fig. 1. Thymus and thymocytes in aged mice. (A) Typical thymi isolated from 12-month-old wild-type and Fas/ male mice. Scale bar, 5 mm. (BG) Immunohistochemical analyses of thymi from 4-week-old Fas/ (B and C), 14-month-old Fas/ (D and E) and 14-month-old wild-type (F and G) male mice. Frozen sections were stained with anti-cytokeratin antibody (B, D and F) or thymic cortical epithelium-specific mAb 6C3 (C, E and G). Arrows indicate thymic medulla. Scale bars, 20 µm. (H and I) Analysis of thymocytes from aged Fas/ mice. Thymocytes isolated from 11-month-old male Fas/ mice were stained with PEanti-CD4 and FITCanti-CD8 mAbs and analyzed by flow cytometer (H), and expression of B220 and Thy1.2 was analyzed in CD4CD8 double-negative thymocytes by flow cytometer (I). (J) Absolute numbers of thymocytes (CD4+CD8+ DP, CD4+ SP plus CD8+ SP thymocytes) per thymus in 12-month-old wild-type (n = 7, filled circles) and Fas/ (n = 5, open circles) male mice. Lines indicate average numbers. Thymocytes numbers were calculated by multiplying the total number of mononuclear cells isolated from thymus by the percentage of thymocytes (CD4+CD8+ DP, CD4+ SP plus CD8+ SP cells) determined by flow cytometric analysis. Statistical analysis was performed using Students t-test. P = 0.04.
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We then examined the intra-thymic structure of thymi from young and aged Fas/ and wild-type male mice in immunohistochemical analyses. TECs, which can be characterized by cytoplasmic expression of cytokeratin, were highly accumulated in the medulla in not only young (4-week-old) but also aged (14-month-old) Fas-deficient thymus (Fig. 1B and D). TECs formed a reticular network in the cortex region of both mice (Fig. 1B and D). These characteristics of TECs were similarly observed in young wild-type mice (data not shown). Staining with control rabbit antibody never showed significant signals in thymi (data not shown). Staining with the cortical epithelium-reactive mAb 6C3 showed, but isotype-matched control rat antibody did not show, a similar reticular structure as staining with anti-cytokeratin antibody, while 6C3-reactive cells were observed in the medullary region of neither young nor aged Fas/ mice (Fig. 1C and E). On the other hand, thymi of 14-month-old wild-type mice had a greatly diminished cell number as well as volume, and the cortico-medullary structure was unclear (Fig. 1F and G). These results indicate that thymi of aged Fas/ mice retain a normal intra-thymic structure.
We next investigated the differentiation status of T cells in the thymus of aged Fas/ male mice, and observed thymocytes belonging to all of the differentiation stages; CD4CD8 double-negative (DN) cells (28.8%), CD4+CD8+ double-positive (DP) cells (27.1%), and CD4+CD8 single-positive (SP) cells (27.6%) or CD4CD8+ SP cells (16.5%) (Fig. 1H). The percentages of DP and DN populations, however, were less and more, respectively, than those of wild-type young mice. Because aged Fas knockout mice were reported to possess abnormal T cells known as lpr cells (surface phenotype: Thy1+B220+CD4CD8) in lungs and liver (26), we examined whether the CD4CD8 DN subset of aged Fas/ thymocytes express Thy1 and B220. Flow cytometric analyses revealed that the DN subset of aged Fas/ thymocytes contains many lpr cells (69% of DN cells) and the number of intact DN cells (Thy1+B220) is normal (7.8% of DN cells) (Fig. 1I). The increase in the DN population was shown to depend on the accumulation of lpr cells, and the increase in the population of abnormal DN T cells reduced the apparent relative ratio of DP cells, although the ratio of DP cells in aged Fas/ mice seems to be still somewhat less than that in young wild-type mice. These results suggest that the differentiation of thymocytes (CD4CD8 DN
CD4+CD8+ DP
CD4+CD8 or CD4CD8+ SP) is continuously executed in aged Fas/ mice, while the differentiation might be partly disturbed in aged Fas/ mice.
To ascertain that Fas is involved in the age-related thymic involution, we quantified the numbers of CD4+CD8+ DP, CD4+ SP and CD8+ SP thymocytes per thymus in 12-month-old wild-type and Fas/ male mice. Absolute numbers of thymocytes except for DN cells were significantly larger in Fas/ than wild-type males (Fig. 1J). Each number of DN, CD4+ SP and CD8+ SP thymocytes was also larger in Fas/ than wild-type animals (data not shown). While the number of thymocytes were quite different among Fas/ individuals (Fig. 1J), the number of DN cells, which consist of a large amount of lpr cells and a few intact thymocytes, did not show a significant difference among them (data not shown), suggesting that the intra-thymic accumulation of lpr cells did not affect the number of thymocytes in aged Fas/ mice. The results show that the differentiation and proliferation of thymocytes are normally maintained in aged Fas/ mice.
Function of thymus in aged Fas/ mice
Disruptive gene rearrangements of the T cell receptor (TCR) were reported to be associated with age-related thymic involution (9). To detect rearrangements at the TCRß locus in the thymocytes of aged Fas/ male mice, PCR analysis was performed with genomic DNA isolated from whole thymus. As shown in Fig. 2(A), similarly rearranged TCRß alleles were found in the thymi of 12-month-old Fas/ mice, 8-week-old wild-type and 8-week-old Fas/ mice. This result suggests that rearrangements of the TCRß gene in Fas/ thymi are performed in old Fas/ mice.
Several previous studies indicated that interleukin-7 (IL-7) plays a key role in the development and/or maturation of lymphocytes including T cells (13,2729). To evaluate the expression of IL-7 in thymi of Fas/ mice, northern blot analysis for IL-7 was performed with total RNA isolated from thymus (Fig. 2B). More than 7-fold higher expression of IL-7 mRNA was detected in the thymus of 12-month-old Fas/ than wild-type male mice, while almost comparable amounts of IL-7 mRNA were observed between thymus of 8-week-old wild-type and Fas/ male mice (Fig. 2B). In the thymus of 12-month-old Fas/ mice, IL-7 was shown to be produced, which might support the differentiation of T cells. Fas deficiency might prevent the loss of IL-7-producing cells in aged mice. Thus, T cell development might be continuously supported by sustained production of IL-7 in the thymus of aged Fas/ mice.
Examination of Fas on TECs and thymocytes
Fas was reported to be highly expressed on both mature and immature T lymphocytes including thymocytes in mice (19,30). In contrast, Fas was shown to be expressed in lower levels on most human thymocytes than mouse thymocytes (17,19). On the assumption that the age-related involution of both human and mouse thymus is caused by the same factor, we speculated that Fas on cells other than thymocytes might play a role. Then, we examined the expression of Fas on thymic stromal cells. Flow cytometric analysis revealed that MHC class II-positive thymic stromal cells, which include TECs, also express Fas on the surface as do MHC class II-negative thymocytes (Fig. 3A).

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Fig. 3. Expression and function of Fas in thymic stromal cells. (A) Flow cytometric analysis of the expression of MHC class II antigen and Fas in single cell suspensions containing thymocytes and thymic stromal cells prepared from thymi of wild-type and Fas/ mice. (BQ) Immunofluorescence staining of thymi with anti-cleaved caspase-3 antibody (red) and anti-keratin antibody (green). Frozen sections were prepared from thymi of Balb/c mice injected with 100 µg of anti-Fas mAb RK-8 (BE), wild-type C57BL/6 (FI) and Fas/ (JM) mice injected with 50 µg of anti-CD3 mAb 2C11 and wild-type mice injected with 50 µg of hamster IgG as isotype control (NQ). Animals were sacrificed at 16 h after the administrations. Arrows indicate that active caspase-3 co-localized with cytokeratin in TECs (yellow). E, I, M and Q show the magnified images of the rectangular regions indicated in D, H, L and P, respectively.
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Because Fas expression does not always correlate with an ability to transduce apoptosis-inducing signals, it was necessary to investigate the sensitivity of thymic stromal cells to the death signal induced by Fas crosslinking. We previously reported that administration of the anti-mouse Fas mAb RK-8 induced the apoptosis of thymic cells in vivo without killing the mouse (31). We then carefully analyzed the induction of apoptosis in thymocytes and thymic stromal cells in vivo after i.v. administration of RK-8. In this experiment, apoptotic cells were detected by staining the thymus with anti-cleaved active caspase-3 antibody. Figure 3(BE) show the accumulation of active caspase-3 in not only thymocytes but also in cytokeratin-positive cells 16 h after the administration of RK-8. To confirm DNA fragmentation-associated apoptosis in TECs, we quantified TUNEL-positive and cytokeratin-expressing cells by flow cytometry, indicating that not only keratin-negative thymocytes but also keratin-positive TECs underwent apoptosis after 24 h administration of RK-8 (Fig. 4A and B). These results clearly show that keratin-positive TECs are sensitive to Fas-mediated apoptosis in vivo.

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Fig. 4. Flow cytometric analyses of DNA-fragmentation-associated apoptosis in cytokeratin-positive TECs. Balb/c mice were injected with anti-Fas mAb RK-8 (A) or control hamster IgG (B). Wild-type C57BL/6 (C and D) and Fas/ (E and F) mice were injected with anti-CD3 mAb 2C11 (C and E) or control hamster IgG (D and F). After 24 h administration, thymic cells containing both thymocytes and thymic stromal cells were isolated and stained by TUNEL method and anti-cytokeratin antibody as described in the Methods. Percentages of TUNEL-positive and -negative cells in cytokeratin-positive cells (TECs) were indicated.
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Activated thymocyte-induced apoptosis in TECs
Next, we examined the types of cells which can supply the transmembrane death factor FasL to induce apoptosis in Fas-positive cells. To verify whether activated thymocytes, which were reported to express FasL (3234), can induce apoptosis in TECs in vivo, we injected the anti-CD3 mAb 2C11 or control IgG into wild-type and Fas/ male mice in order to activate thymocytes in vivo. After 16 h administration of 2C11, accumulation of active caspase-3 was observed in keratin-negative thymic cells of both wild-type and Fas/ mice (Fig. 3FM), while injection of control hamster IgG did not induce accumulation of active caspase-3 in thymus (Fig. 3NQ). Keratin-negative thymocytes were shown to be highly sensitive to anti-CD3 mAb-induced apoptosis in a Fas-independent manner. In contrast, activation of caspase-3 was not observed in keratin-positive cells in the Fas/ thymus (Fig. 3JM), while cleaved caspase-3 was detectable in not only keratin-negative but also keratin-positive cells in wild-type thymus (Fig. 3FI). Flow cytometric analyses also showed that the number of TUNEL-positive TECs (cytokeratin-positive) was significantly higher in wild-type than Fas/ mice after 24 h administration of 2C11 (Fig. 4CF). Thymocytes activated by stimulation with anti-CD3 in vivo were shown to induce apoptosis in keratin-positive TECs in a Fas-dependent manner. Thus, Fas expressed on TECs transduces the death signal in vivo after stimulation with FasL expressed on activated thymocytes through the TCR complex.
Thymic involution in chimeric mice
To clarify which Fas on thymocytes or thymic stromal cells is required for age-related thymic involution, we generated chimera using wild-type and Fas/ male mice. Bone marrow cells (BMCs) from wild-type and Fas/ donor mice were transplanted into lethally irradiated Fas/ and wild-type mice, respectively. In our experimental condition (8-week-old male mice were used as both donors and recipients), effects of lpr cells, which were reported to express FasL (35), were considered to be negligible because lpr cells were hardly detected in spleens of the chimeric mice at 9 months after the bone marrow transplantation (BMT): the numbers of Thy-1+ B220+ cells containing lpr cells were indicated to be 1.9% and 5.3% of total splenocytes in Fas/ BMCs-transplanted wild-type mice and wild-type BMCs-transplanted Fas/ mice, respectively, while Thy-1+ B220+ cells were shown to be 28.7% and 4.7% of total splenocytes in the similar aged Fas/ and wild-type mice, respectively.
As a control experiment, wild-type BMCs were transplanted into transgenic mice ubiquitously expressing green fluorescent protein (GFP-tg mice). The GFP-tg receiving wild-type BMCs showed thymic involution at 9 months after the transplantation while apparently normal thymi with GFP-negative thymocytes were observed at 3 months after the BMT. While the wild-type recipients of Fas/ BMCs showed apparently normal thymi without accumulation of lpr cells (<2% of Thy-1-positive thymocytes) at 3 months after the BMT, these wild-type recipients at 79 months after the BMT had thymi reduced in size similar to age-matched wild-type mice. In contrast, the Fas/ recipients of wild-type BMCs had relatively normal thymi even at 9 months after the transplantation (Fig. 5A). The numbers of DP thymocytes were shown to be greater in the wild-type BMCs-transplanted Fas/ than both Fas/ BMCs-transplanted wild-type and wild-type BMCs-transplanted GFP-tg recipients (Fig. 5B). Most of thymocytes in Fas/ recipients of wild-type BMCs were shown to express Fas as well as in wild-type mice (Fig. 5C), while most of thymocytes in wild-type recipients of Fas/ BMCs did not express Fas as in Fas/ mice (Fig. 5C), indicating that the reconstituted thymi consisted of transplanted BMCs-derived thymocytes. In addition, the reconstituted thymus in Fas/ recipients exhibited a normal T cell development with DN, DP and SP cells expressing Fas, as confirmed by flow cytometric analyses (Fig. 5D). These findings show that the intra-thymic environment surrounding thymocytes is essential for maintenance of the thymus with aging. All the results indicate that Fas on TECs but not thymocytes plays an important role in age-related thymic involution.

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Fig. 5. Age-related thymic involution in chimeric mice. (A) Typical thymi isolated from wild-type BMCs-transplanted Fas/ (left) and Fas/ BMCs-transplanted wild-type mice (right) at 9 months after the BMT. Scale bar, 3 mm. (B) Thymocytes were isolated from Fas/ BMCs-transplanted wild-type mice at 79 months after the BMT (n = 7, filled circles), wild-type BMCs-transplanted Fas/ mice at 79 months after the BMT (n = 4, open circles), and wild-type BMCs-transplanted GFP-tg mice at 9 months after the BMT (n = 2, filled square). Absolute numbers of CD4+CD8+ DP thymocytes were calculated as described in Fig. 1J. Lines indicate average numbers. Statistical analysis was performed using Students t-test. *P = 0.02. (C) Expression of Fas in thymocytes from chimeric mice. Thymocytes, isolated from Fas/ BMCs-transplanted wild-type mice (KO > WT) and wild-type BMCs-transplanted Fas/ mice (WT > KO) at 9 months after the BMT, were stained with anti-Fas mAb Jo2 and analyzed by flow cytometry. Thymocytes from two-month-old Fas/ and wild-type mice were similarly examined. (D) Analysis of thymocytes from a Fas/ recipient mouse. Thymocytes were isolated from the thymus of chimeric mice with Fas-positive thymocytes and Fas-negative TECs at 9 months after the BMT, stained with anti-CD4 and anti-CD8 mAbs, and analyzed by flow cytometry.
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Discussion
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In this study, we demonstrated that Fas deficiency impaired age-related thymic involution. In mammals, the thymus has been reported to gradually decrease in size and weight as well as cell number with aging (7). Aged Fas/ mice (12 months old), however, did not exhibit a progression of age-related thymic involution (Fig. 1A). In addition, a prominent thymic involution was observed in 14-month-old male wild-type mice (Fig. 1F and G) while apparently normal thymi were detected in age-matched Fas/ males (Fig. 1D and E). Immuno histochemical analysis revealed that the thymic cortico-medullary structure was normal in aged Fas/ mice, while the tissue structure of aged wild-type thymus was unclear (Fig. 1BG). The increase in the number of DN cells in the thymus of aged Fas/ mice was shown to mainly depend on the accumulation of lpr cells with a CD4CD8 phenotype (Fig. 1H and I). Although this accumulation lowered the percentage of CD4+CD8+ DP cells in Fas/ mice, absolute numbers of DP, CD4+ SP and CD8+ SP thymocytes were always much greater in aged Fas/ mice than age-matched wild-type mice (Fig. 1J). Thus, age-related thymic involution was shown to be controlled in a Fas-dependent manner.
Thymi of MRL-lpr/lpr mice, however, were previously reported to be atrophic (36), although our used Fas/ mice in C57BL/6 background showed normal thymus without atrophy (Fig. 1). In case of thymi of MRL-lpr/lpr mice, atrophy may be closely associated with the development of systemic autoimmune disease, which is usually observed in MRL but not in C57BL/6 genetic background (37,38), since thymi of NZB x W and BXSB mice, which also suffer from systemic autoimmune disease, were reported to be similarly atrophic as those of MRL-lpr/lpr mice (36).
We also showed the complete rearrangement of the T cell receptor gene in thymocytes from aged Fas/ mice (Fig. 2A). In addition, thymi from aged Fas/ mice maintained increased levels of IL-7 compared with wild-type thymi (Fig. 2B). IL-7 was reported to play a principal role in the development and maturation of T lymphocytes; rearrangements of T cell receptor genes are induced in lymphoid progenitors during their IL-7-dependent stage (27,28), immunodepletion of IL-7 with neutralizing anti-IL-7 antibodies causes thymic atrophy, which is reversed after termination of the administration (29), and the expression of IL-7 declines with aging (13). Thus, the expression of IL-7 in aged Fas/ mice, which can be expressed by survived TECs, might play a role in prevention of the age-related thymic involution.
IL-7 was reported to be produced by MHC class II-positive TECs (28,39), which are the main constituents of the thymic stromal cell population. MHC class II-positive epithelial cells were also reported to be required for T cell development (40). In this report, we showed that MHC-class II-positive thymic stromal cells, which contain TECs, express Fas on their surface (Fig. 3A). In addition, administration of an agonistic anti-Fas mAb induced apoptosis in TECs, which was confirmed by not only immunohistochemical analyses with double staining of cytokeratin and the cleaved form of caspase-3 (Fig. 3BE) but also flow cytometric analyses with double staining of cytokeratin and fragmented DNA (TUNEL) (Fig. 4A and B). Human TECs were also reported to express Fas on the surface and to be sensitive to apoptosis induced by agonistic anti-Fas mAb (41). Thus, TECs which produce IL-7 are sensitive to Fas-mediated apoptosis.
Injection of anti-CD3 mAb into wild-type mice also caused apoptosis not only in thymocytes in a Fas-independent manner but also in TECs in a Fas-dependent manner, because both immunohistochemical and flow cytometric analyses clearly showed that thymocytes derived from both wild-type and Fas/ mice, while TECs derived from wild-type but not from Fas/ mice, underwent apoptosis after administration of anti-CD3 mAb 2C11 (Figs 3FQ and 4CF). Since stimulation of T cells through the TCR complex was reported to induce the expression of FasL in not only peripheral organs but also thymus (3234,42), activated thymocytes might supply FasL to induce apoptosis in TECs during the normal development of thymocytes. The accumulated loss of TECs might result in a lack of stromal cell-derived cytokines such as IL-7, inducing age-related thymic involution.
Wild-type mice in MRL genetic background reconstituted with bone marrow cells from MRL-lpr/lpr mice were previously reported to die even at a young age from graft-versus-host disease (GVHD), which was described as lpr-GVHD (4345). In contrast, wild-type C57BL/6 recipient mice reconstituted with bone marrow cells from Fas/ C57BL/6 mice were shown to survive at least for 9 months after BMT (Fig. 5) as reported previously (43). Furthermore, we did not observe any thymic atrophy in our chimeric mice with Fas/ BMCs within 3 months after BMT (data not shown), indicating that lpr-GVH reaction might not be induced in thymi of our chimeric mice with C57BL/6 background. In addition, lpr cells, which were reported to express FasL (35), might be unable to induce thymic atrophy in our chimeric mice of Fas/ BMCs, because we did not observe accumulation of lpr cells in thymi (<2% of total Thy-1-positive thymocytes) as well as spleens of our wild-type recipients either 3 or 9 months after transplantation. Additionally, absolute numbers of DP thymocytes per thymus in wild-type recipients reconstituted with Fas/ BMCs were almost similar to GFP-tg recipients (C57BL/6 background) reconstituted with wild-type BMCs (Fig. 5B). Consequently, age-related thymic involution in our chimeric mice with Fas/ BMCs was suggested to be developed by similar molecular mechanisms as in wild-type C57BL/6 mice. On the contrary, aged chimeric mice, which express Fas on thymocytes but not on TECs, showed increased number of thymocytes compared with chimeric mice with Fas/ BMCs and wild-type TECs (Fig. 5B). In addition, the reconstituted thymus with Fas-negative TECs exhibited normal development of Fas-positive thymocytes even 9 months after the bone marrow transplantation (Fig. 5D). Analyses of chimeric mice with bone marrow from Fas/ and wild-type mice confirmed that Fas on TECs plays a major role in age-related thymic involution. It remains unclear, however, whether the chimeric mice with Fas-positive thymocytes and Fas-negative TECs show senescence of the immune system when they age. Further studies are necessary to clarify whether reconstituted thymus-derived Fas-positive T cells are functional in the periphery.
In conclusion, Fas on thymic stromal cells but not on thymocytes is closely involved in age-related thymic involution, while other factors would also be involved in age-related thymic involution, such as ability to regenerate TECs lost by apoptosis and to supply thymocyte progenitor cells.
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Acknowledgements
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The authors thank Drs K. K. Lee, S. Toyokuni, M. Osato, S. Yamada and R. Yasumizu for their valuable suggestions and discussions. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. N.Y. was supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Graduate school of Biostudies and Institute for Virus Research, Kyoto University.
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Abbreviations
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BMCbone marrow cell
BMTbone marrow transplantation
FasLFas ligand
GVHDgraft-versus-host disease
TECthymic epithelial cell
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