Murine Thymic Nurse Cells and Rosettes: Analysis of Adhesion Molecule Expression Using Confocal Microscopy and a Simplified Enrichment Method

Abstract

SUMMARY Thymic nurse cells (TNC) and T-cell stromal rosettes (ROS) are two in vivo models for stromal cell-thymocyte interactions. We describe a simplified enrichment method for TNC and ROS that overcomes the necessity for large amounts of tissue. The complexes were further analyzed with confocal microscopy, and three subunits of ROS were defined on the basis of their central cell phenotype, i.e., macrophage, dendritic, or epithelial cell rosettes. Because adhesion molecules are proposed to play a crucial role in T-cell development, we investigated CD44, LFA-1, and ICAM-1 expression in such complexes. The epithelial component of TNC expresses CD44 and ICAM-1, whereas intra-TNC thymocytes are LFA-negative. With regard to ROS, all subsets expressed CD44, and macrophage and dendritic cell ROS were also ICAM-1-positive and LFA-1-positive. The current protocol opens the possibility for further in vivo analysis of stromal cell-thymocyte interactions, e.g., for studies of scarce gene mutant mice.

Characterization of Vascular Adhesion Molecules that may Facilitate Progenitor Homing in the Post‐natal Mouse Thymus

“…We and others have previously found VCAM-1 expression by thymic stroma (Salomon et al, 1997;Prockop et al, 2002), and we have shown it to be a critical ligand for transcortical migration of intrathymic progenitors (Prockop et al, 2002). Likewise, ICAM-1 has been shown by others to be expressed on various thymocytes, including thymocytes themselves (Reiss and Engelhardt, 1999), nurse cells (Cordes et al, 1997;Oliveira-dos-Santos et al, 1997), and other stromal cells (Reiss and Engelhardt, 1999;Lucas and Germain, 2000). While we did not observe any differences in ICAM-1 expression on vascular cells with respect to the periodicity of progenitor homing, we did observe a difference in the pattern of staining on different types of vessels (see "Results" section), although the implications of this are presently unknown.…”

Glucocorticoid production in the murine thymus

“…TNC were isolated as described previously [35]. Collected supernatants were pooled, cells sedimented by a 10 min centrifugation step at 800 × g and resuspended in PBS.…”

Thymic epithelial cells synthesize a heparan sulfate with a highly sulfated region

“…This molecule is the main ligant for the CD44 molecule, a transmembrane proteoglycan expressed not only by immature differentiating thymocytes (Lynch and Ceredig, 1989), but also by TEC (Villa-Verde et al, 1994;Patel et al, 1995a;Oliveira-dos-Santos et al, 1997). Moreover, a colocalization of hyaluronic acid and CD44 was evidenced in sections of human thymuses (Patel et al, 1995b).…”