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

Abstract

Age-associated thymic atrophy results in a decline in T lymphocyte output and has been identified as one of the key events that precede inefficient 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 TCRβ 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.

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 naı̈ve 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 close to their replicative limit (Effros and Pawelec, 1997). One single event, which precedes all of these changes and deficiencies, is reduction in the output of the thymus (Mackall et al., 1995, Scollay et al., 1980).

The development of αβ⁺TCR T cells in the thymus encompasses a progressive stepwise differentiation from a multi-potent precursor, producing a mature thymocyte with defined potential function. The earliest precursors of the T cell pathway in the adult thymus are CD3⁻CD4^loCD8⁻ progenitors (Wu et al., 1991), a population further subdivided on the basis of expression of CD44 and CD25, with the most immature stage identified as CD44⁺CD25⁻. Differentiation and commitment to the thymocyte lineage is associated with expression of CD25 and loss of CD44. The cells become CD44⁺CD25⁺ then CD44⁻CD25⁺ and during this period there is a rearrangement of the β chain of the T cell receptor (TCR) (Godfrey et al., 1993, Godfrey et al., 1994) and subsequent expression of this chain at the cell surface with a TCRα chain equivalent (the pre-T α 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⁺CD8⁺) when there is rearrangement and expression of the TCRα chain (Petrie et al., 1993). Thymocytes expressing a competent TCRαβ 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 specific α chain and a common γ 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 deficient in both receptors. Mice, which are c-kit^−/−γc^−/− show complete abrogation of T cell development, which is not apparent in the single deficient mutants (Rodewald et al., 1997). The limited thymopoiesis present in IL-7^−/−, IL-7Rα^−/−, IL-7Rγ^−/− chain mice (Moore et al., 1996, Peschon et al., 1994, Di Santo et al., 1999) and in c-kit^−/− 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 TCRβ 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 fibroblasts. 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.