Injury causes a systemic inflammatory response syndrome (SIRS) clinically much like sepsis 1. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors 2. Similarly, cellular injury can release endogenous damage-associated molecular patterns (DAMPs) that activate innate immunity 3. Mitochondria are evolutionary endosymbionts that were derived from bacteria 4 and so might bear bacterial molecular motifs. We show here that injury releases mitochondrial DAMPs (MTD) into the circulation with functionally important immune consequences. MTD include formyl peptides and mitochondrial DNA. These activate human neutrophils (PMN) through formyl peptide receptor-1 and TLR9 respectively. MTD promote PMN Ca2+ flux and phosphorylation of MAP kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTD can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These can then signal through identical innate immune pathways to create a sepsis-like state. The release of such mitochondrial ‘enemies within’ by cellular injury is a key link between trauma, inflammation and SIRS.

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.