Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.

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...

T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X(7) receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4(+) T cells express abundant P2X(7) receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 microM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X(7) receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X(7) receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X(7) receptors, compared to control BALB/c mice, which express fully functional P2X(7) receptors. We conclude that ATP release and autocrine, positive feedback through P2X(7) receptors is required for the effective activation of T cells.

Type 1 diabetes (T1D) may result from a breakdown in peripheral tolerance that is partially controlled by peripheral tissue antigen (PTA) expression in lymph nodes. Here we show that the transcriptional regulator deformed epidermal autoregulatory factor 1 (Deaf1) controls PTA gene expression in the pancreatic lymph nodes (PLN). The expression of canonical Deaf1 was reduced, while that of an alternatively spliced variant was increased during the onset of destructive insulitis in the PLN of NOD mice. An equivalent variant Deaf1 isoform was identified in the PLN of T1D patients. Both NOD and human Deaf1 variant isoforms suppressed PTA expression by inhibiting the transcriptional activity of canonical Deaf1. Reduced PTA expression resulting from the alternative splicing of Deaf1 may contribute to T1D pathogenesis.

Hypertonic saline (HS) resuscitation increases T cell function and inhibits posttraumatic T cell anergy, which can reduce immunosuppression and sepsis in trauma patients. We have previously shown that HS induces the release of cellular ATP and enhances T cell function. However, the mechanism by which HS induces ATP release and the subsequent regulation of T cell function by ATP remain poorly understood. In the present study, we show that inhibition of the gap junction hemichannel pannexin-1 (Panx1) blocks ATP release in response to HS, and HS exposure triggers significant changes in the expression of all P2X-type ATP receptors in Jurkat T cells. Blocking or silencing of Panx1 or of P2X1, P2X4, or P2X7 receptors blunts HS-induced p38 MAPK activation and the stimulatory effects of HS on TCR/CD28-induced IL-2 gene transcription. Moreover, treatment with HS or agonists of P2X receptors overcomes T cell suppression induced by the anti-inflammatory cytokine IL-10. These findings indicate that Panx1 hemichannels facilitate ATP release in response to hypertonic stress and that P2X1, P2X4, and P2X7 receptor activation enhances T cell function. We conclude that HS and P2 receptor agonists promote T cell function and thus, could be used to improve T cell function in trauma patients.