Endotoxin-induced autocrine ATP signaling inhibits neutrophil chemotaxis through enhancing myosin light chain phosphorylation

Abstract

Although the neutrophil recruitment cascade during inflammation has been well described, the molecular players that halt neutrophil chemotaxis remain unclear. In this study, we found that lipopolysaccharide (LPS) was a potent stop signal for chemotactic neutrophil migration. Treatment with an antagonist of the ATP receptor (P2X1) in primary human neutrophils or knockout of the P2X1 receptor in neutrophil-like differentiated HL-60 (dHL-60) cells recovered neutrophil chemotaxis. Further observations showed that LPS-induced ATP release through connexin 43 (Cx43) hemichannels was responsible for the activation of the P2X1 receptor and the subsequent calcium influx. Increased intracellular calcium stopped neutrophil chemotaxis by activating myosin light chain (MLC) through the myosin light chain kinase (MLCK)-dependent pathway. Taken together, these data identify a previously unknown function of LPS-induced autocrine ATP signaling in inhibiting neutrophil chemotaxis by enhancing MLC phosphorylation, which provides important evidence that stoppage of neutrophil chemotaxis at infectious foci plays a key role in the defense against invading pathogens.endotoxin | neutrophil | chemotaxis | ATP | myosin light chain N eutrophils are the most abundant leukocytes in humans and the first blood cells to arrive at infectious sites as part of the innate cellular immune response. Following transmigration out of the blood vessel, neutrophils migrate through interstitial tissue toward the foci of damage. Then, neutrophil recruitment mobilizes near the pathogenic sites to eliminate pathogens and necrotic tissue. Although the neutrophil recruitment cascade during inflammation has been well described, the molecules that stop neutrophil chemotaxis remain unclear.Studies in recent decades have confirmed that extracellular ATP, as a purinergic signaling molecule, participates in the pathogenesis of various inflammatory diseases such as transplantation rejection, autoimmune disease, and bacterial infection (1). As a result of ectoapyrases and ectoadenosine triphosphatases (ecto-ATPases), which hydrolyze ATP into adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO), the concentration of extracellular ATP is maintained at ∼10 nmol/L (2). However, the intracellular ATP concentration in mammalian cells is 5-8 mmol/L. Owing to the 10 6 -fold ATP gradient difference, cell necrosis or activation at inflammatory sites causes a dramatic ATP release (2). By activating the P2X (P2X1-7 subtypes) and P2Y (P2Y1, -2, -4, -6, and -11-14 subtypes) receptor families, extracellular ATP contributes to the regulation of a variety of inflammatory cell responses (3). Given the high concentration of ATP in inflammatory tissues, the effects of extracellular ATP/P2 receptors on neutrophil migration have attracted much attention. ATP released from the leading edge of the neutrophil surface amplifies chemotactic signals and directs cell orientation by feedback through the P2Y2 receptor (4). Knockout of the P2Y2 receptor decreases liver infi...

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18

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The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration

“…In principle, this suggests that both inhibition of RhoA downstream kinase (ROCK) or inability of neutrophils to engage in RhoA activity fluctuations alters chemotaxis. Although apparently conflicting, these results go hand‐in‐hand with previous observations showing that both inhibition of myosin light chain (MLC) phosphorylation cause by impaired Rac1 and RhoA activity and excesive p‐MLC phosphorylation, impair chemotaxis …”

“…Although apparently conflicting, these results go hand-in-hand with previous observations showing that both inhibition of myosin light chain (MLC) phosphorylation cause by impaired Rac1 and RhoA activity 13 and excesive p-MLC phosphorylation, impair chemotaxis. 49…”

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