Engagement of T cells with antigenpresenting cells requires T-cell receptor (TCR) stimulation at the immune synapse.We previously reported that TCR stimulation induces the release of cellular adenosine-5-triphosphate (ATP) that regulates T-cell activation. Here we tested the roles of pannexin-1 hemichannels, which have been implicated in ATP release, and of various P2X receptors, which serve as ATP-gated Ca 2؉ channels, in events that control T-cell activation. TCR stimulation results in the translocation of P2X1 and P2X4 receptors and pannexin-1 hemichannels to the immune synapse, while P2X7 receptors remain uniformly distributed on the cell surface. Removal of extracellular ATP or inhibition, mutation, or silencing of P2X1 and P2X4 receptors inhibits Ca 2؉ entry, nuclear factors of activated T cells (NFAT) activation, and induction of interleukin-2 synthesis. Inhibition of pannexin-1 hemichannels suppresses TCR-induced ATP release, Ca 2؉ entry, and T-cell activation. We conclude that pannexin-1 hemichannels and P2X1 and P2X4 receptors facilitate ATP release and autocrine feedback mechanisms that control Ca 2؉ entry and T-cell activation at the immune synapse. (Blood. 2010; 116(18):3475-3484) IntroductionT-cell activation requires a sustained elevation of intracellular Ca 2ϩ levels, which is accomplished by Ca 2ϩ entry through calcium release-activated calcium (CRAC) channels that are composed of stromal interaction molecule 1 (STIM1) and Orai1 proteins. [1][2][3] Both proteins translocate to the immune synapse upon T-cell activation, where they mediate localized influx of extracellular Ca 2ϩ . 4 Ca 2ϩ entry contributes to the activation of nuclear factors of activated T cells (NFATs) that induce interleukin-2 (IL-2) gene expression and subsequent signaling events that lead to T-cell proliferation. [5][6][7] Recent studies have shown that extracellular adenosine-5Ј-triphosphate (ATP) regulates T-cell activation. [8][9][10] T cells release ATP in a controlled manner, as do other leukocytes, thereby facilitating intercellular communication and autocrine feedback regulation of cell function. [8][9][10][11][12][13] Stimulation of T cells by T-cell receptor (TCR) ligation, mechanical stimulation, membrane deformation, or osmotic stress induces the release of cellular ATP. 9,10,[13][14][15][16] T cells express the gap junction hemichannels pannexin-1, which can mediate ATP release and T-cell activation. 8,10 T-cell activation has been shown to involve P2X receptor subtypes. 8,10 The 7 mammalian P2X receptor family members (P2X1-7) are ATPgated ion channels. 17,18 All these receptors, with the exception of P2X5, can facilitate entry of Ca 2ϩ in response to stimulation by extracellular ATP, [18][19][20] thus suggesting that P2X receptors regulate T-cell activation by mediating Ca 2ϩ entry.T-cell activation during antigen recognition requires the formation of an immune synapse between T cells and antigen-presenting cells. The immune synapse is a complex structure with a limited number of TCRs, implying that TCR stimu...
Efficient activation of neutrophils is a key requirement for effective immune responses. We found that neutrophils released cellular adenosine triphosphate (ATP) in response to exogenous stimuli such as formylated bacterial peptides and inflammatory mediators that activated receptors for Fcγ, interleukin-8, C5a complement, and leukotriene B4. The release of ATP in response to stimulation of the formyl peptide receptor (FPR) occurred through pannexin-1 hemichannels that colocalized with FPR and P2Y2 nucleotide receptors on the cell surface to form a purinergic signaling system that facilitated the activation of neutrophils. Disruption of this purinergic signaling system by inhibiting or silencing pannexin-1 hemichannels or P2Y2 receptors blocked the activation of neutrophils and impaired innate host responses to bacterial infection. Thus, purinergic signaling is a fundamental mechanism that is required for neutrophil activation and immune defense.
Wiskott–Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP −/− mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro . Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP −/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP −/− mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.
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