Nitric oxide cooperates with glucocorticoids in thymic epithelial cell-mediated apoptosis of double positive thymocytes

T cell development in the thymus is controlled by thymic epithelial cells (TE). While it is accepted that TE interact with maturing T cells, the mechanisms by which they trigger 'death by neglect' of double-positive (DP) thymocytes are poorly understood. We and others have demonstrated a role for TE-derived glucocorticoids (GCs) in this process. We have studied TE-induced apoptosis using an in vitro system based on co-culturing a thymic epithelial cell line (TEC) with DP thymic lymphoma cells or thymocytes (DP thymic cells). Here, we demonstrate that nitric oxide (NO·) is also involved in this death process. The inducible nitric oxide synthase (iNOS) inhibitors N^G- methyl-L-arginine and 1,4-PBIT attenuated TEC-induced apoptosis of DP thymic cells. Co-cultivation of TEC with DP thymic cells increased the expression of iNOS in TEC. A concomitant increase in NO· was detected by staining with DAF-FM diacetate. Moreover, the iNOS-regulating cytokines IL-1α, IL-1β and IFNγ were up-regulated upon interaction of TEC with DP thymic cells. Neutralizing IL-1R or IFNγ reduced TEC-induced apoptosis of DP thymic cells. Cardinally, NO· synergizes with GCs in eliciting apoptosis of DP thymic cells. Our data indicate that a cross-talk between DP thymic cells and TEC is required for proper induction of iNOS-up-regulating cytokines with a subsequent increase in iNOS expression and NO· production in TEC. NO·, in turn, cooperates with GCs in promoting death by neglect. We suggest that NO· together with GCs fine-tune the T cell selection process.