IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3.

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.

Serum amyloid A inhibits dendritic cell apoptosis to induce glucocorticoid resistance in CD4(+) T cells.

Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4(+) T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and TH17 polarization. TH17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4(+) T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4(+) T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4(+) T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4(+) T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4(+) T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.

Conference highlight: do T cells care about the mitogen-activated protein kinase signalling pathways?

Mitogen-activated protein (MAP) kinases, which include the extracellular response kinases, p38 and c-Jun amino terminal kinases (JNK), play a significant role in mediating signals triggered by cytokines, growth factors and environmental stress. The JNK and p38 MAP kinases have been involved in growth, differentiation and cell death in different cell types. In the present paper, we describe how the JNK and p38 MAP kinase signalling pathways are regulated and their role during thymocyte development and the activation and differentiation of T cells in the peripheral immune system. The results from these studies demonstrate that the JNK and p38 MAP kinase signalling pathways regulate different aspects of T-cell mediated immune responses.

Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo.

The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is strictly regulated during the differentiation of CD4(-)CD8(-) thymocytes. Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25(+)CD44(-) stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice. Inactivation of p38 MAP kinase is required for further differentiation of these cells into CD4(+)CD8(+) thymocytes. The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation. Therefore, the p38 MAP kinase pathway is a critical regulatory element of differentiation and proliferation during the early stages of in vivo thymocyte development.

Down-modulation of CD2 delays deletion of superantigen-responsive T cells.

CD2 is a cell surface glycoprotein expressed on most T lymphocytes that is generally viewed as a cell adhesion molecule and, in this capacity, contributes to T cell receptor (TCR) signaling. CD2 has a relatively long cytoplasmic tail which associates with the src family tyrosine kinases, p56(lck) and p59(fyn), and could potentially signal directly. Down-modulation of CD2 on T cells has been shown to result in diminished proliferative capacity and interleukin (IL)-2 production. Furthermore, re-expression of CD2 can result in the restoration of these functions. This suggests that CD2 can influence the intensity of TCR signaling. As TCR signal intensity is pivotal to the induction of T cell apoptosis, we considered the hypothesis that the level of CD2 on the T cell surface may influence its propensity toward apoptosis. Using an anti-CD2 antibody, CD2 was down-modulated in vivo on mouse T lymphocytes without affecting the levels of surface CD3, TCR alphabeta, CD4 or CD8. Deletion of superantigen-responsive T cells was delayed in mice with down-modulated CD2 following the administration of staphylococcal enterotoxin B (SEB). This was paralleled by diminished apoptosis of SEB-responsive cells. The findings suggest a model whereby the level of CD2 expression influences the intensity of TCR signaling and the ability to undergo apoptosis.

JNK, but not MAPK, activation is associated with Fas-mediated apoptosis in human T cells.

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co-stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas-induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf-1/MAPK pathway versus JNK activation by Fas.

Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.