Abstract

Neutrophils constitute the first line of defense against invading pathogens, but they also are responsible for tissue destruction in pathological conditions. In a recent paper published in Science Signaling, Junger and colleagues show that adenosine 5′-triphosphate release and signaling through purinergic P2 receptors upon neutrophil activation by bacterial and inflammatory stimuli represents an important mechanism for effective neutrophil functional response and host protection.

Adenosine 5′-triphosphate (ATP) is usually confined inside the cell where it constitutes the source of chemical energy for the majority of cellular functions and serves as a substrate in signal transduction pathways. ATP is also incorporated into nucleic acids during DNA replication and transcription. However, ATP has also a less widely appreciated function as signaling molecule in the extracellular space. The extremely favorable concentration gradient between intra- and extra-cellular space makes ATP an ideal cellular resource for autocrine/paracrine signaling. In fact, ATP can be released by eukaryotic cells and activate purinergic receptors in the plasma membrane, known as P2. Two classes of P2 receptors exist in eukaryotic cells. The first is constituted by P2Y receptors, which are metabotropic, i.e. coupled to heterotrimeric guanine nucleotide-binding protein (G-protein) and modulate mainly intracellular calcium as well as cyclic AMP levels; the second is composed of P2X receptors, which are ionotropic, i.e. ligand-gated cation-permeable channels that open when bound to ATP. Both classes of receptors concur in setting the basal level of cell activation (‘the set point’) for signal transduction pathways (Corriden and Insel, 2010); apart from signaling, a wealth of functions is regulated by P2 receptors, including secretion, tissue blood flow, cell volume and inflammation. In addition, ATP is massively released upon cell death and acts as a ‘danger-associated molecular pattern’ for cells of the innate immune system. Finally, it was recently shown that release of ATP during the early stages of apoptosis induces monocyte recruitment through P2Y2 receptor thereby acting as a ‘find-me’ signal for corpse clearance (Elliott et al., 2009).

Polymorphonuclear neutrophils (PMNs) constitute the majority (>50%) of circulating white blood cells and the first line of defense against invading pathogens; however, they are also responsible for severe tissue damage in inflammatory diseases and other pathological conditions. Functional characteristics that enable PMN to protect or damage the host include migration, degranulation and release of reactive oxygen species, including superoxide radical and hydrogen peroxide, which play a crucial role in the degradation of internalized particles and bacteria. Neutrophils exploit rapid and transient release of ATP to amplify migration toward chemoattractants by autocrine signaling via P2 and adenosine (generated by cell surface ectonucleotidases, enzymes that degrade ATP to adenosine) receptors (Chen et al., 2006). Chen et al. (2010) now extend these observations by showing that the release of ATP facilitates PMN activation through P2 receptors. They analyzed PMN activation by receptors, which recognize N-formyl-Met-Leu-Phe (fMLP) bacterial peptide or mediators of innate immune system, such as interleukin-8 (IL-8), leukotriene B4 (LTB4) and the complement component C5a, all of which are G-protein-coupled receptors. As receptor unrelated to G-protein-mediated signaling, they analyzed PMN activation by Fcγ receptors (FcγRs), which elicit phagocytosis and killing of bacteria. These signaling pathways constitute fundamental mechanisms for efficient immune defense against invading pathogens.

Eukaryotic cells release ATP with different modalities. In neurons and platelets for example, ATP is stored in vesicles, which are released in response to specific stimuli (Coco et al., 2003). In other cell types ATP-containing granules are not detected and cytosolic ATP is released in response to rise in cytosolic calcium through pannexin (panx) hemichannels (Locovei et al., 2006). Alternatively, ATP can be released by membrane stress-induced opening of mechanosensitive channels, such as connexin 43 (Kang et al., 2008). Chen et al. previously observed that ATP was released predominantly from the deformed protruding region at the leading edge of the migrating neutrophil (Chen et al., 2006); now they show that panx1 hemichannels colocalize with fMLP receptors (FPRs) at the leading edge and constitute a route for ATP release in polarized neutrophils. In addition, through real-time polymerase chain reaction (RT-PCR) the human tweety homolog 3 (hTTYH3), a homolog of a gene located in Drosophila flightless encoding a maxi-anion channel, was shown to be selectively expressed in human PMN. These maxi-anion channels can also release ATP in response to fMLP and colocalized with FPRs in PMN but, differently from panx1, segregated in foci more uniformly distributed across the plasma membrane. Since it was impossible to completely inhibit ATP release by blockade of both panx1 and hTTYH3 channels the existence of other mechanisms of ATP release was also hypothesized. However, using specific inhibitors of panx1 and hTTYH3 channels the authors showed that inhibition of FPR-induced ATP release through panx1 but not hTTYH3 significantly suppressed PMN oxidative burst thereby indicating a crucial difference in the relevance of the two paths in controlling PMN functional response.

The extracellular signal-regulated kinase (ERK) pathway is activated by FPR stimulation and implicated in respiratory burst (Downey et al., 1996); inhibition of panx hemichannels by carbenoxolone or degradation of extracellular ATP by apyrase both dose dependently inhibited this signaling pathway, thus showing that ATP release was required for efficient ERK activation. Treatment of fMLP stimulated PMN with suramin, a broad spectrum antagonist of P2 receptors, reduced ERK phosphorylation, Ca2+ mobilization, degranulation, oxidative burst and chemotaxis, indicating that P2 signaling substantially contributed to FPR-mediated PMN activation. The reduction of Ca2+ signaling and oxidative burst was also obtained by knockdown of P2Y2, the most abundant P2 receptor in human PMNs and differentiated human promyelocytic leukemia HL-60 cells (dHL-60). Altogether, these results show that ATP release and P2 signaling are important elements in determining PMN responsiveness to fMLP.

Panx1 was shown to be physically associated with P2X7 receptor in macrophages (Pelegrin and Surprenant, 2006), analysis of FPR and P2Y2 in dHL-60 showed that the two receptors colocalized in the plasma membrane, thereby suggesting that FPR, panx1 and P2Y2 might co-segregate at the cell surface to integrate FPR stimulation with an autocrine purinergic feed-back loop. The relevance of this purinergic pathway was not restricted to FPR stimulation since ATP release was also observed following PMN stimulation through receptors for IL-8, C5a, LTB4 as well as Fcγ and its blockade affected ERK phosphorylation. Furthermore, pharmacological inhibition of either ATP release or P2 receptors restrained superoxide formation and phagocytosis of opsonized bacteria induced by FcγR stimulation. Thus, ATP release and P2 signaling appears as a general mechanism in the activation of PMN by infectious and inflammatory stimuli. Accordingly, P2Y2 knock-out mice were defective in up-regulating the adhesion molecule CD11b and containing bacterial infection in the cecal ligation and puncture model of peritoneal infection.

Release of chemotactic signals from inflamed or infected tissues trigger PMNs migration from the bloodstream to inflammatory sites, where they undergo receptor-mediated respiratory burst and degranulation. Tight control of neutrophil activation is required to avoid excessive tissue damage. Defining mechanisms important for PMNs activation can pave the way for novel therapeutic approaches aimed at modulating PMNs response in inflammatory conditions. The paper by Chen et al. provides compelling evidences that ATP release and autocrine purinergic loop facilitate downstream signaling events required for PMNs activation. Notably, regulation of extracellular ATP concentration by cellular ATP scavenging CD39/ENTPD1 ectonucleotidase was shown to represent an important mechanism in limiting inflammatory tissue damage (Friedman et al., 2009). The definition of panx1 hemichannels as the functionally relevant path of ATP release and of P2Y2 as trigger of the purinergic feedback loop in PMN activation warrant further investigation to understand whether panx1 and P2Y2 represent possible targets for therapeutic intervention in inflammation. Finally, autocrine signaling by ATP in PMN activation further highlights the importance of ATP as signaling molecule in enhancing immune function.

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