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Lecut, Christelle; Frederix, Kim; Johnson, Daniel M.; Deroanne, Christophe; Thiry, Marc; Faccinetto, Céline; Marée, Raphaël; Evans, Richard J.; Volders, Paul G. A.; Bours, Vincent and Oury, Cécile
(2009).
DOI: https://doi.org/10.4049/jimmunol.0804007
Abstract
ATP, released at the leading edge of migrating neutrophils, amplifies chemotactic signals. The aim of our study was to investigate whether neutrophils express ATP-gated P2X1 ion channels and whether these channels could play a role in chemotaxis. Whole-cell patch clamp experiments showed rapidly desensitizing currents in both human and mouse neutrophils stimulated with P2X1 agonists, αβ-methylene ATP (αβMeATP) and βγMeATP. These currents were strongly impaired or absent in neutrophils from P2X1 −/− mice. In Boyden chamber assays, αβMeATP provoked chemokinesis and enhanced formylated peptide- and IL-8-induced chemotaxis of human neutrophils. This agonist similarly increased W-peptide-induced chemotaxis of wild-type mouse neutrophils, whereas it had no effect on P2X1 −/− neutrophils. In human as in mouse neutrophils, αβMeATP selectively activated the small RhoGTPase RhoA that caused reversible myosin L chain phosphorylation. Moreover, the αβMeATP-elicited neutrophil movements were prevented by the two Rho kinase inhibitors, Y27632 and H1152. In a gradient of W-peptide, P2X1 −/− neutrophils migrated with reduced speed and displayed impaired trailing edge retraction. Finally, neutrophil recruitment in mouse peritoneum upon Escherichia coli injection was enhanced in wild-type mice treated with αβMeATP, whereas it was significantly impaired in the P2X1 −/− mice. Thus, activation of P2X1 ion channels by ATP promotes neutrophil chemotaxis, a process involving Rho kinase-dependent actomyosin-mediated contraction at the cell rear. These ion channels may therefore play a significant role in host defense and inflammation.
Neutrophils are key cells of the innate immune system that participate in a vast majority of inflammation-related diseases, such as coronary artery diseases, rheumatoid arthritis, and sepsis (1, 2, 3). They are programmed to exit the circulation and chemotax toward epicenters of inflammation, guided by gradients of a variety of inflammatory mediators. Neutrophils become polarized morphologically in response to chemotactic stimuli. These morphological changes are accompanied by a strongly polarized distribution of intracellular signal transduction components. The 3′-phosphoinositol lipids (PI3Ps)4 and PI3Ks; Rho GTPases, such as RhoA, Rac, and Cdc42; and the actin and microtubule cytoskeletons play key roles in signaling polarity. Attractant receptors, acting on different G proteins, trigger two divergent pathways that contribute to polarity. Frontness depends upon Gi-mediated production of PI3Ps, the activated form of Rac, and F-actin. G12 and G13 trigger backness signals, including activation of Rho, a Rho-dependent kinase, and myosin II. Functional incompatibility causes the two resulting actin assemblies to aggregate into separate domains, making the leading edge more sensitive to attractant than the back.
In addition, mechanisms of signal amplification have been proposed that contribute to maintain signaling polarity even in a shallow gradient. These mechanisms involve the release of ATP from neutrophil leading edge and its rapid metabolism to ADP, AMP, and adenosine (6). As a neutrophil moves toward an attracting stimulus, a rapid and transient release of ATP further enforces its directional movement by autocrine signaling via P2Y2 and A3 receptors. In support of this, neutrophils lacking P2Y2 receptors showed a loss in gradient sensing, whereas cells without A3 receptors showed correct directionality, but diminished speed. Although the intracellular mechanisms remain undefined, these findings demonstrate the importance of extracellular ATP in the control of neutrophil chemotaxis.
Because ATP is released by activated platelets and leukocytes, as a result of multiple stress stimuli, and upon cell lysis, its concentration can become elevated at sites of inflammation (7). By acting on neutrophils in a paracrine manner, ATP could thus help these cells to locate bacteria and tissue damage. We therefore wondered whether neutrophils expressed other ATP receptors that could contribute to chemotaxis. ATP acts on specific cell surface P2 receptors belonging to two subclasses, the G protein-coupled P2Y receptors and the ATP-gated P2X-nonselective cation channels. Eight human P2Y receptors, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–14, and seven P2X subtypes, P2X1–7, have been identified. Neutrophils express multiple P2X subtype mRNAs: P2X1,4,5,7. However, the presence of functional P2X ion channels in neutrophils has never been shown, and their contribution to neutrophil migration has never been investigated. P2X ion channels are widely distributed in various cell types and tissues (15). Only P2X7 receptors are to date established as having physiological roles in inflammation, mostly via a rapid activation of caspase-1 with subsequent release of the proinflammatory cytokine IL-1β from activated macrophages and microglia (15, 16). P2X7 is also an important modulator of T cell functions.
In this study, we demonstrate that both human and mouse neutrophils express P2X1 ion channels that play a significant role in the neutrophil response to chemotactic stimuli.
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