Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double‐positive thymocytes

Abstract

The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T-TG-St) was grown from transgenic mouse expressing temperature-sensitive SV40 T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP-TG-St) that binds thymocytes which are mainly CD4+8+. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4+8+ thymocytes. Both lines of double -positive cell-binding stroma exhibited strong activity of two peptidases, neutral endopeptidase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of the specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double-positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes.

Expression, regulation and functional activities of aminopeptidase N (EC 3.4.11.2; APN; CD13) on murine macrophage J774 cell line

“…Since membrane CD13 was found neither on immature thymic, nor on mature splenic T lymphocytes (Hansen et al 1993), CD13 mRNA observed in thymus in our study may be due to stromal cells. Namely, Small et al (1996) reported that thymic stromal cell lines, which bind double positive T lymphocytes, express strong APN enzyme activity. The proposed role of APN on stromal cells was contribution to local micro-environment in developing T lymphocytes.…”

Thymic aging in ICR female mice is suspended by prolonged hydrocortisone exposure

“…Here, we have studied female mice because none of 49 male mice injected by hydrocortisone acetate at the same dosage survived more than eight injections (Small et al 1989). FACS analysis revealed a trend toward decreased double positives and increased CD4+ single positive cells with aging, as others have reported in mice of more advanced age (Hirokawa et al 1990).…”

“…Thymocytes were stained with FITC-CD8 and phycoerythrin (PE)-CD4 (Pharmingen) and analyzed on a FACSort as described previously (Small et al 1996).…”

Isolation of adult murine thymic stromal cells that naturally express Notch ligands

“…Cells bearing each ligand were isolated from an immortalized thymic stromal line grown from an adult mouse (Small et al 1996). After preliminary enrichment of the cells that bind the extracellular domain of Notch, cells were treated with an antibody against the amino terminus of Jagged-1 or Delta-1 (followed by FITC-labeled reagents), and strongly fluorescent cells were sorted on the FACS.…”

“…Notch ligands could be expressed on lymphoid as well as on stromal cells, and to eliminate nonstromal elements, an immortalized stromal line was used as the source of cells (Small et al 1996). This cell line was grown from an adult (C57Bl · C3H) F 2 transgenic mouse with a temperature-sensitive SV40 T antigen under control of the mouse H-2K b promoter.…”