The present study shows the unexpected result that normal development of lymphocytes, and hence of the adaptive immune system, depends on the function of GSNOR in the regulation of endogenous SNOs. We find that genetic deletion of GSNOR causes an increase in protein S-nitrosylation and cell apoptosis in thymus and widespread lymphopenia. The defects in the immune system are largely prevented by further deletion of iNOS, indicating iNOS as the origin of immunosuppressive SNOs. Further, our results suggest that lymphocyte development may depend on functional GSNOR in hemopoietic cells.
Our study suggests that GSONR deficiency causes apoptosis through excessive protein S-nitrosylation from iNOS in thymus of immunologically unchallenged mice. Expression of iNOS has been described in dendritic cells and other stromal cells in thymus of immunologically naïve mice (8
). Thymic expression of iNOS may be increased by stimulation of TCR signaling with anti-CD3 antibody (8
) or superantigen Staphylococcal enterotoxin B (9
). Elevated iNOS activity can cause apoptosis of thymocytes (8
). We showed previously that thymocyte apoptosis caused by elevated iNOS activity in immune response was greatly increased by GSONR deficiency (15
). We find in the present study that GSONR deficiency increases apoptosis from iNOS in thymus of immunologically naïve mice. In these animals GSONR deficiency also results in increase in thymic content of total protein SNOs, the abundance of S-nitrosylated proteins, and the level of S-nitrosylation of proteins. When excessive protein S-nitrosylation is abolished by iNOS deletion, increase of thymic apoptosis is prevented, further supporting a causative role of dysregulated S-nitrosylation in apoptosis. Proteins with elevated levels of S-nitrosylation in GSNOR−/−
thymus include a few involved in the control of apoptosis.
While caspase-6 is an important execution caspase in apoptosis (24
), voltage-dependent anion channel-1 is considered important for the control of mitochondria-mediated apoptosis (25
). S-nitrosylation, which can modulate enzymatic activity, channel conductivity, and protein-protein interaction (13
), might modulate the function of caspase-6 or voltage-dependent anion channel-1 in apoptosis. S-nitrosylation of GAPDH has been showed to promote nuclear accumulation of GAPDH and to cause apoptosis in macrophages and neurons (28
). It is thus possible that the marked increase of GAPDH S-nitrosylation in GSNOR−/−
thymus may promote apoptosis of thymocytes. Because apoptosis is a key mechanism for the control of the development and number of lymphocytes in thymus (29
), increased apoptosis in thymus of GSNOR−/−
mice may cause reduced thymic output of T cells and consequently lymphopenia in periphery.
T cell lymphopenia in GSNOR−/−
mice may result largely from diminished thymic output. Reduction of CD4 and CD8 SP thymocytes in GSNOR−/−
mice is associated with reduction of CD4 and CD8 T cells in periphery. In fact the decreases of CD4 and CD8 SP cells in thymus are quantitatively comparable to those of CD4 and CD8 cells respectively in secondary lymphoid organs. Diminished thymic output typically has little effect on the number of memory T cells but often significantly decreases the number of naive T cells (30
), in part because proliferation of naive T cells induced by lymphopenia converts them to memory-like T cells (32
). Reduction of the naive T cell population with no change in the number of memory phenotype cells in GSNOR−/−
mice thus provides further evidence suggesting reduced thymic output as the major cause of T cell lymphopenia. Alternatively T cell lymphopenia in GSNOR−/−
mice might result from increase in lymphocyte death in periphery. However, apoptosis in peripheral lymphoid tissues appears unchanged from GSNOR deficiency. Nor is T cell lymphopenia likely to result from impaired proliferation of T cells in periphery, because lymphopenia-induced proliferation of T cells is unlikely to produce naive phenotype T cells (32
), and because proliferative responses of T cells from GSNOR−/−
mice are similar to those of wildtype control.
GSNOR appears to be particularly important to the development of CD4 SP thymocytes. Both CD4 and CD8 SP thymocytes develop from DP cells following positive selection. Because GSNOR deficiency causes no change in the proportions of DP and CD8 SP cells in thymocytes, but a significant reduction in the proportion of CD4 SP cells, GSNOR deficiency does not appear to affect the development of CD8 SP cells from DP cells or CD4-versus-CD8 lineage choice. Instead, GSNOR deficiency may impair the generation of CD4 SP cells from DP cells or the survival of CD4 SP lineage cells. A number of proteins important to CD4 SP lineage have been identified in thymocytes, although most of the proteins also play important roles in the development of DP or CD8 SP cells (33
). Conditional deletion of GATA-3
in DP thymocytes through CD4-Cre causes minimal changes in the percentage of DP and CD8 SP cells but significantly reduces the percentage of CD4 SP cells (35
). In addition, CD83 deficiency in thymic epithelial cells has little effect on the development of DP or CD8 SP thymocytes but significantly reduces the number of CD4 SP cells (37
). It remains to be determined if deficiency of GSNOR and GATA-3, c-Myb, or CD83 inhibits a common step or pathway of CD4 SP development.
Our results, particularly those from bone marrow chimeras, suggest that GSNOR may be important to the development of both T and B cells. GSNOR−/−
bone marrow stem cells in the competitive reconstitution experiment only gave rise to minimal DP, CD4 SP, and CD8 SP thymocytes, suggesting that GSNOR is important for T cell development not only at the stage of CD4 SP cells but also at an earlier stage. This is consistent with reduction of total thymocytes in GSNOR−/−
mice. In addition, the marked deficiency of most T and B cells, but no deficiency of granulocytes, from GSNOR−/−
bone marrow stem cells may suggest defective development of common lymphoid progenitors (38
) of GSNOR−/−
mice. The immunosuppressive effect could be a side effect of NO, or it might represent dysregulated NO signaling in lymphocyte progenitors. Alternatively, the inhibitory mechanisms in T and B lineage cells might be unrelated. We reported earlier that GSNOR, through its control of S-nitrosylation, is essential for the survival of lymphocytes in the inflammatory response (15
). Our current findings demonstrate that GSNOR is also important for the development of lymphocytes, establishing a broad function for GSNOR in the immune system.