Dependence of self-tolerance on TRAF6-directed development of thymic stroma.

The microenvironments of the thymus are generated by thymic epithelial cells (TECs) and are essential for inducing immune self-tolerance or developing T cells. However, the molecular mechanisms that underlie the differentiation of TECs and thymic compartmentalization are not fully understood. Here we show that deficiency in the tumor necrosis factor receptor-associated factor (TRAF) 6 results in disorganized distribution of medullary TECs (mTECs) and the absence of mature mTECs. Engraftment of thymic stroma of TRAF6(-/-) embryos into athymic nude mice induced autoimmunity. Thus, TRAF6 directs the development of thymic stroma and represents a critical point of regulation for self-tolerance and autoimmunity.

Development of autoimmunity against transcriptionally unrepressed target antigen in the thymus of Aire-deficient mice.

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjogren's syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, alpha-fodrin. Remarkably, transcriptional expression of alpha-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.

Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels.

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.

NF-kappa B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-kappaB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

Increased insulin sensitivity and hypoinsulinemia in APS knockout mice.

A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS-deficient (APS(-/-)) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS(-/-) mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS(-/-) mice. APS(-/-) mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS(-/-) mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS(-/-) mice was increased over that of APS(+/+) mice. APS(-/-) mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes.