Age-associated thymic atrophy is linked to a decline in IL-7 production

Age-associated thymic atrophy is linked to a decline in IL-7 production

Experimental Gerontology 37 (2002) 455±463 www.elsevier.com/locate/expgero Age-associated thymic atrophy is linked to a decline in IL-7 production D...

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Experimental Gerontology 37 (2002) 455±463

www.elsevier.com/locate/expgero

Age-associated thymic atrophy is linked to a decline in IL-7 production Deborah Andrew*, Richard Aspinall Department of Immunology, ICSM at Chelsea & Westminster Hospital, 369 Fulham Road, London SW10 9NH, UK Received 1 June 2001; accepted 1 September 2001

Abstract Age-associated thymic atrophy results in a decline in T lymphocyte output and has been identi®ed as one of the key events that precede inef®cient functioning of the immune system in later life. Thymic atrophy is thought to result from a failure of the thymic microenvironment to support thymopoiesis in old age and recent evidence suggests that a decline in interleukin-7 (IL-7) expression may limit thymocyte development by restricting combinations of survival, proliferation and rearrangement of the TCRb chain. Using RT-PCR and the RNase protection assay, we show that the expression of IL-7 declines with age. Analysis of Connexin 43 expression, a component molecule of gap junctions, whose function is to connect epithelial cells, does not markedly decline with age. These observations suggest that a decline in IL-7 expression is not matched by a similar loss of epithelial cells. These results in conjunction with other studies lead us to speculate that IL-7 producing MHC class II positive TECs are being replaced by cells that do not have this capacity. q 2002 Published by Elsevier Science Inc. Keywords: Lymphocyte; Interleukin-7; Thymocyte; Stroma

1. Introduction The physiological ageing of the immune system in both man and mouse is associated with an accumulation of T cells showing; impaired signal transduction (Utsuyama et al., 1993), shifts in the balance of TH1 to TH2 cells (Hirokawa, 1992), alterations in the ratio of memory to naõÈve cells (Kurashima et al., 1995), the presence of memory cells which inhibit the response (Dozmorov et al., 1995), changes in the amounts of cytokines produced with age (Hobbs et al., 1993), and the presence of increasing numbers of T cells at or * Corresponding author. Tel.: 144-208-746-5979; fax: 144-208746-5997. E-mail address: [email protected] (D. Andrew). Abbreviations: IL-7, interleukin-7; SCF, stem cell factor; TN, triple negative; TEC, thymic epithelial cell

close to their replicative limit (Effros and Pawelec, 1997). One single event, which precedes all of these changes and de®ciencies, is reduction in the output of the thymus (Mackall et al., 1995; Scollay et al., 1980). The development of ab 1TCR T cells in the thymus encompasses a progressive stepwise differentiation from a multi-potent precursor, producing a mature thymocyte with de®ned potential function. The earliest precursors of the T cell pathway in the adult thymus are CD3 2CD4 loCD8 2 progenitors (Wu et al., 1991), a population further subdivided on the basis of expression of CD44 and CD25, with the most immature stage identi®ed as CD44 1CD25 2. Differentiation and commitment to the thymocyte lineage is associated with expression of CD25 and loss of CD44. The cells become CD44 1CD25 1 then CD44 2CD25 1 and during this period there is a rearrangement of the b chain of the T cell receptor (TCR)

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(Godfrey et al., 1993, 1994) and subsequent expression of this chain at the cell surface with a TCRa chain equivalent (the pre-T a chain) (Fehling and von Boehmer, 1997; von Boehmer and Fehling, 1997). There is then a loss of CD25 expression and a period of selection before the cell becomes an immature thymocyte (CD4 1CD8 1) when there is rearrangement and expression of the TCRa chain (Petrie et al., 1993). Thymocytes expressing a competent TCRab chain pair are chosen for further maturation and selection before the cell can leave the thymus to enter the periphery (Jameson et al., 1995). The microenvironment provided both by cytokines and the three-dimensional structure of the thymic stroma is crucial to the developmental process. Two of the most important cytokines in the T cell developmental pathway are stem cell factor (SCF), which binds to the c-kit receptor, and Interleukin-7 (IL-7), which binds to the IL-7 receptor. The latter contains an IL-7 speci®c a chain and a common g chain used by other cytokines including IL-2, IL-9 and IL-15. Evidence for the obligatory requirement of both SCF and IL-7 in early T cell development comes from mice doubly de®cient in both receptors. Mice, which are c-kit 2/2gc 2/2 show complete abrogation of T cell development, which is not apparent in the single de®cient mutants (Rodewald et al., 1997). The limited thymopoiesis present in IL-7 2/2, IL-7Ra 2/2, IL-7Rg 2/2 chain mice (Moore 1996; Peschon et al., 1994; Di Santo et al., 1999) and in c-kit 2/2 mice (Rodewald et al., 1995) led to the hypothesis that SCF and IL-7 may act synergistically in the early stages of the T cell development (Rodewald et al., 1997; Di Santo et al., 1999). IL-7 is produced by MHC class II positive TECs and its role in thymocyte development has been linked to assisting the survival and proliferation of thymocytes and aiding the rearrangement of TCRb chain (Candeias et al., 1997; Oosterwegel et al., 1997; Moore et al., 1993). SCF is also present in the thymus produced by stromal cells (Moore et al., 1993) as a transmembrane protein on the stromal cell surface and as a secreted soluble molecule generated by differential splicing (Majumdar et al., 1994). The contributors to the stromal elements are epithelial cells, dendritic cells, macrophages and ®broblasts.

The epithelial component is unlike that found at other sites in the body, which are characteristically in layers and situated above a basement membrane. In the thymus the epithelial cells form a network of cells with no discernable basement membrane, and communication between cells as in other epithelial cells is achieved through gap junctions. Gap junction channels are formed from proteins of the Connexin family, connecting adjacent cells and allowing the movement of molecules up to 1 kD. The Connexin proteins are named according to their molecular weights and within the murine thymus Connexin 43 is the protein, which forms the functional gap junctions (Alves, 1995). Formation of the thymus microenvironment is dependent on the interaction between epithelial cells and thymocyte progenitors (Penit et al., 1996; Shores et al., 1994). Since the latter do not change in number throughout the ageing process in the thymus, the lesion in the T cell developmental pathway, which leads to age-associated thymic atrophy must be downstream of these cells (Aspinall, 1997). The aim of this work was to follow the expression of the IL-7 gene with age and correlate this with the expression of Connexin 43 and also SCF.

2. Material and methods 2.1. Mice and derivation of tissue Normal C57BL/10 mice were obtained from Harlan Olac (Oxfordshire, UK) and were maintained in the animal house at Imperial College School of Medicine (ICSM) in accordance with the local rules and regulations. Mice were sacri®ced by CO2 asphyxiation, the thymus removed and cleaned of adipose tissue and placed into RPMI 1640 (Life Technologies, Paisley, UK) supplemented with l-glutamine (200 mM)-penicillin (100 IU/ml)-streptomycin (100 mg/ml; Sigma, Poole, UK). Cell suspensions were prepared from the thymus by pressing the tissue through a 100 mm cell strainer (Becton Dickenson, Oxford, UK) into icecold tissue culture media. Thymic stroma was retained in the cell strainer and transferred into a 1.5 ml Eppendorf (Greiner, Stonehouse, UK) containing STAT-60 (AMS Biotechnology, Witney, UK) and stored at 2408C.

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2.2. RNase protection assay The RiboQuant Multi-Probe RNase Protection Assay (Pharmingen) is a sensitive and speci®c method for the detection and quantitation of mRNA species and allows the simultaneous quanti®cation of several mRNA species in a single sample of total RNA. By incorporating probes for housekeeping gene transcripts the levels of individual mRNA species can be compared between samples. The assay was performed according to the manufacturer's instructions except 1 ml of T7 RNA polymerase (New England Biolabs (UK) Hitchin, UK) at 5000 U was used. One template set was used that identi®ed the RNA of IL-3, IL-11, IL-7, GM-CSF, M-CSF, G-CSF, LIF, IL-6 and SCF. As described in the manufacturer's instructions, total RNA from 2 to 20 mg/ lane was found to generate a suitable signal using the RPA assay and was used for each of the thymic stromas. The RNase protected probes are puri®ed and resolved on a denaturing polyacrylamide gel, and quanti®ed by autoradiography. Using a high-resolution scan of the autoradiograph (1200 dpi), the intensity of pixels in the gel band of each mRNA species in the original sample could be quantitated. The software program NIH Image 1.61 (National Institute of Health, Bethesda, Maryland, USA) was used to determine the intensity of each of the mRNA bands and so generate relative mRNA expression for each of the cytokines. The incorporation of two housekeeping gene transcripts, L32 and GAPDH, within the assay allowed the levels of individual mRNA species to be compared between young and old thymic stroma. 2.3. Measurement by RT-PCR RNA was extracted from the tissue using STAT60 (AMS Biotechnology, Witney, UK) and the protocol recommended by the manufacturers. The reverse transcription reaction was performed as follows. Approximately 2 mg of RNA, 30 units of AMV reverse transcriptase, 1 mg of oligo dT primer, dATP, dGTP, dCTP, dTTP (all at 1 mM), rRNAsin w ribonuclease inhibitor at 0.5 m in RT buffer containing 5 mM MgCl2 (all from Promega) in a total volume of 25 ml were incubated at 428C for 45 min. The cDNA generated in this way was stored at 48C and used in standard PCR reactions.

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The PCR was performed in a ®nal volume of 50 ml containing 1±4 ml of cDNA. The nucleotides, dATP, dGTP, dCTP, dTTP (Promega), were each at a ®nal concentration of 200 nM where 10% of the total dATP present was labelled with 32P (Amersham International, Slough, UK). The primers were at 200 nM each, plus 2.5 units of Taq polymerase (Advanced Biotechnologies, Epsom, UK), in PCR buffer containing 1 mM MgCl2 (Advanced Biotechnologies). The primers for glyceraldehyde phosphate dehydrogenase (GAPDH) and IL-7 have been described previously (Szabo et al., 1993; Montgomery and Dallman, 1991). The complimentary pairs of primers for the Connexin 43 gene (5 0 -GAACACGGCAAGGTGAAGAT and 5 0 -GAGCGAGAGACACCAAGGAC), which give a product of 246 bases, were designed using the Primer 3 software (Rozen and Skaletsky, 1998) and obtained from Oswel DNA Service (Southampton, UK). The PCR protocol was 1 cycle at 948C for 2 min, then 36 cycles of 948C for 45 s, 628C for 1 min (for CX43 and GAPDH, but 658C for IL-7) and 728C for 1 min. Preliminary studies revealed that these reaction conditions yielded product within the linear range. The products of the PCR were separated on a 1% agarose gel and the bands excised and placed in the wells of a T-Tray sealed with T-Tray sealer (Wallac Oi Finland) and the associated radioactivity measured on a Skatron b counter. 2.4. Limiting dilution PCR Four dilutions of cDNA were set-up for each primer set. The ®rst tube contained a ®nal volume of 50 ml containing 1 ml of cDNA. The three remaining tubes had ®vefold dilutions of cDNA also in a ®nal volume of 50 ml. Limiting Dilution PCR was then performed with primers for GAPDH, CX43 and IL-7 as described above. The products of the PCR were separated on a 1% agarose gel, viewed on a transilluminator and the image captured using the Enhanced Analysis System (Scotlab, Lanarkshire, UK). 2.5. Statistical analysis P values for the comparison of samples was carried out using a two tailed T-test for samples with unequal variance using Microsoft Excel software.

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Fig. 1. Limiting dilution PCR reveals a reduction in the levels of IL7 and CX43 with age. PCR products from GAPDH, Connexin 43 and IL-7 primer driven reactions and cDNA from mice of 2 and 24 months of age. Lane 1 shows the product of PCR with 1 ml of cDNA in 50 ml volume, and Lanes 2±4 show subsequent ®vefold dilutions from the original starting sample.

3. Results 3.1. Analysis of IL-7 and CX43 expression in young and old thymuses by limiting dilution RT-PCR Following dilutions of the cDNA made from the enriched stroma of old and young mice, and using primers for GAPDH, CX43 and IL-7, PCR was performed to quantitate these levels (Fig. 1). Comparison between the young mouse of 2 months of age and the old mouse of 24 months of age, show a similar dilution pro®le for the products of the GAPDH primers. Likewise the PCR product from the CX43 primers also showed a similar dilution pro®le with cDNA from young mouse stroma diluting in a manner comparable to the cDNA from old mouse stroma. However, differences were observed in the dilution pro®les produced in the PCR with the IL-7 primers. With the cDNA from the young mouse, a band is visible at the ®rst and second and weakly present in the third dilution. However for the cDNA from the stroma of the old mouse, bands were visible in the ®rst dilution and faintly in the second dilution but not in the third dilution. This suggested no age-related change in the amount of CX43, but a decline in the amount of IL-7. This was con®rmed with similar results obtained from thymic stromal cDNA from a separate pair of old (22 months) and young (3 months) donors.

four separate experiments using paired cDNA. In each experiment one of the cDNA pairs was from an animal of 3 months of age and the other cDNA of the pair was from an animal whose age ranged from 22 to 24 months. Fig. 2 shows the results from a representative experiment in this series and reveals that the slope of the graph for the IL-7/GAPDH ratio is steeper than the slope for the CX43/GAPDH ratio, indicating that the decline in IL-7 is greater than the decline in CX43. The ratio of IL-7/GAPDH in old animals is less than 50% of the value at 3 months of age, a reduction that is not paralleled in the CX43/ GAPDH ratio.

3.2. Analysis of IL-7 and CX43 expression in young and old thymuses by RT-PCR

3.3. Analysis of IL-7 and SCF expression in young and old thymuses by RNase protection assay

Further analysis of the amounts of CX43 and IL-7 PCR product in relation to the amount of GAPDH was calculated by radioactive labelling of the product in

To establish whether the pro®les of thymic cytokine expression change with age, the quanti®cation of IL-3, IL-6, IL-7, IL-11, GM-CSF, M-CSF, G-CSF,

Fig. 2. Reduction in levels of IL-7 and CX43 with age. This shows a representative result from four experiments demonstrating the change in ratio of IL-7 and CX43 PCR products in relation to GAPDH PCR product, using cDNA from mice of ages 3 and 22 months or 3 and 24 months. Radioactive product bands were excised from the gel, their radioactive content measured and ratios calculated.

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Fig. 3. Bands for IL-7, SCF and M-CSF mRNA are visible in the lanes containing mRNA from young (1 month) and old (22 month) male mice. The density of the bands has decreased in the lane containing the mRNA from the 22-month male mouse. Lanes contain 4 mg of young and old mRNA. A representative experiment of ®ve is shown.

Fig. 4. Cytokine mRNA expression in the thymic stroma of a male mouse decreases with age. Shown is a representative example of mRNA expression from the thymic stroma of a young and old mouse. These results were repeated ®ve times with similar results. Expression of each mRNA species, for each stroma, was determined by quantitating the intensity of pixels of each band from a scanned autoradiograph. Expression of mRNA species was standardised between each stroma by the use of the internal standard L32.

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Fig. 5. Relative cytokine mRNA expression in thymic stroma from young and old female mice. Expression of each mRNA species, from 1.5 month and 18 month old thymic stroma, was determined by quantitating the intensity of pixels of each band from a scanned autoradiograph. Expression of mRNA species was standardised between each stroma by the use of the internal standard L32. The mean relative expression is shown for young …n ˆ 3† and old …n ˆ 4† as a horizontal bar.

LIF, and SCF were performed using a Multi-Probe RNA Protection Assay (RPA) system on young (1± 1.5 months) and old (18±22 months) male and female thymic stromas. Total RNA isolated from young and old male and female thymic stroma showed mRNA expression of three of the nine cytokines assessed. The three cytokine mRNAs could be detected as bands on the gel at concentrations between 2 and 20 mg/lane of thymic stromal RNA and quantitated by measuring the pixel intensity of the gel bands from a scanned autoradiograph (Guillemin et al., 2000, 1999). Expression of mRNA species was standardised between each total RNA concentrations by the use of the internal standard L32. A representative gel demonstrating product bands for IL-7, SCF, M-CSF and the controls GAPDH and L32 is shown in Fig. 3. In this example, bands were detected using 4 mg of thymic stromal mRNA/ lane. Relative expression of each of the three cytokines expressed was greater in the young thymic stroma compared to the old for both male and female mice. Fig. 4 demonstrates the expression of cytokine mRNA in the thymic stroma from one young and one old male mouse. In this example, there was a reduction in mRNA expression of almost 7-fold, 6-fold and 3-fold for IL-7, M-CSF and SCF, respectively, in the older thymic stroma compared to the young. In

addition, the cytokine mRNA expression in the thymic stroma of three young and four old female mice decreased signi®cantly with age (Fig. 5). Comparison of stromal expression of IL-7 mRNA demonstrated a 4-fold reduction …P , 0:01†; whilst M-CSF and SCF demonstrated a 3-fold …P , 0:05† and 2-fold reduction …P , 0:01†; respectively, in old mice. 4. Discussion Several lines of evidence point to IL-7 as a factor causally linked to thymic atrophy. The ®rst is that the bottleneck in thymocyte development observed in aging occurs between the CD44 1CD25 2 and CD44 1CD25 1 triple negative (TN) stages (Aspinall, 1997), known to be a stage of development dependent on IL-7 (Kim et al., 1998). Secondly, this bottleneck is associated with an increase in apoptosis at the CD44 1CD25 1 and CD44 2CD25 1 TN developmental stages (Andrew and Aspinall, 2001), where IL-7 is known to support TCRb rearrangement (Oosterwegel et al., 1997) and apoptosis can be reversed in the presence of IL-7 (Andrew and Aspinall, 2001). The association of IL-7 with production and expression of components of the TCR has been a feature of previous work (Muegge et al., 1993) which suggested that IL-7

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was a cofactor for the V(D)J rearrangement of the TCRb chain gene, although IL-7 was later implicated as important in D-J rearrangement for the TCRb chain (Tsuda et al., 1996). A recent report, however, demonstrated that IL-7 was required to induce TCRb gene rearrangement in T lymphocyte progenitors by supporting the survival of these cells (Oosterwegel et al., 1997). Thirdly, reduction in the levels of IL-7 in vivo with anti-IL-7 antibodies, induced thymic atrophy which was reversible when the treatment was terminated, suggesting that a reduction in the available IL-7 within the thymus may account for the thymic atrophy associated with aging (Bhatia et al., 1995). Finally, IL-7Ra 2/2 mice demonstrate an arrest in T lymphocyte development prior to the expression of CD25, which is the site of the bottleneck in the aging thymus, and thymocyte differentiation could be restored in these animals by the expression of a transgenic TCR (Crompton et al., 1997). The results presented here, using PCR analysis the RPA system, reveal that the expression of intrathymic IL-7 declines with age in mice. Much of the provision of the thymic microenvironment is controlled by epithelial cells and the main source of intra-thymic IL-7 has been mapped to the MHC class II 1 cells (Oosterwegel et al., 1997; Moore et al., 1993). It remains unclear whether there is a change in the stromal cells with age (Hartwig and Steinmann, 1994; Kosco-Vilbois and Imhof, 2000) and enumeration of the total number of thymic stromal cells, particularly epithelial cells, remains complicated. Although it is possible to separate epithelial cells from the thymus (Anderson et al., 1998; Chidgey et al., 1998) it is not clear whether a procedure exists which can reliably collect all of the cells or whether it would be possible to carry out the isolation process with the same ef®ciency irrespective of the animals' age. This point is important since it makes it dif®cult to give a de®nitive answer to the question about whether there is a reduction in the number of IL-7 producing cells or a change in the level of IL-7 production per cell. The results presented here demonstrate that the decline in CX43 expression is certainly not as marked as IL-7 suggesting a reduction in IL-7 production unmatched by a similar loss in the epithelial cells. There may be some loss of epithelial cells but it is possible that epithelial cells with the capacity to produce IL-7 are being replaced by cells that do not

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have this capacity. CX43 expression has also been demonstrated by murine macrophages (Alves et al., 1996) and it is possible that an increase in the number of thymic macrophages with age may contribute to masking a decline in epithelial cell number. The RPA system also demonstrates a decline in intra-thymic SCF and M-CSF expression with age. The observation that SCF expression declines with age can be explained by the ®nding that production of SCF has been mapped to the same subset of MHC class II 1 TECs that produce IL-7 (Hofmeister et al., 1999). Therefore, these results imply that changes in epithelial cell phenotype rather than a decline in number may also be responsible for the reduction in SCF expression with age. In conclusion, the PCR and RPA experiments reported here support the hypothesis that a reduction in the overall availability of intra-thymic IL-7 reduces the ef®ciency of CD44 1CD25 2 TN cells to differentiate into their progeny, the CD44 1CD25 1 cells. Acknowledgement This work was supported by the Wellcome Trust, Grant number 051541. References Alves, L.A., Campos de Carvalho, A.C., Cirne Lima, E.O., Rocha, S.-C.M., Dardenne, M., Spray, D.C., Savino, W., 1995. Functional gap junctions in thymic epithelial cells are formed by Connexin 43. Eur. J. Immunol. 25, 431. Alves, L.A., Coutinho, S.R., Persechini, P.M., Spray, D.C., Savino, W., Campos-de, C.A., 1996. Are there functional gap junctions or junctional hemichannels in macrophages? Blood 88, 328. Anderson, G., Partington, K.M., Jenkinson, E.J., 1998. Differential effects of peptide diversity and stromal cell type in positive and negative selection in the thymus. J. Immunol. 161, 6599. Andrew, D., Aspinall, R., 2001. Interleukin-7 and not stem cell factor reverses the age-associated increase in apoptosis and decline in thymopoiesis seen with age. J. Immunol. 166, 1524±1530. Aspinall, R., 1997. Age-associated thymic atrophy in the mouse is due to a de®ciency affecting rearrangement of the TCR during intrathymic T cell development. J. Immunol. 158, 3037. Bhatia, S.K., Tygrett, L.T., Grabstein, K.H., Waldschmidt, T.J., 1995. The effect of in vivo IL-7 deprivation on T cell maturation. J. Exp. Med. 181, 1399. von Boehmer, H., Fehling, H.J., 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15, 433.

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