NOX2 is critical for heterotypic neutrophil-platelet interactions during vascular inflammation

Abstract

Key Points• NOX2-generated ROS regulate the function of surface receptors required for platelet-neutrophil interactions during vascular inflammation.Platelet-leukocyte interactions on activated endothelial cells play an important role during microvascular occlusion under oxidative stress conditions. However, it remains poorly understood how neutrophil-platelet interactions are regulated during vascular inflammation. By using intravital microscopy with mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and their bone marrow chimera, we demonstrated that NOX2 from both hematopoietic and endothelial cells is crucial for neutrophil-platelet interactions during tumor necrosis factor alpha-induced venular inflammation. Platelet NOX2-produced reactive oxygen species (ROS) regulated P-selectin exposure upon agonist stimulation and the ligand-binding function of glycoprotein Iba. Furthermore, neutrophil NOX2-generated ROS enhanced the activation and ligand-binding activity of aMb2 integrin following N-formyl-methionyl-leucyl phenylalanine stimulation. Studies with isolated cells and a mouse model of hepatic ischemia/reperfusion injury revealed that NOX2 from both platelets and neutrophils is required for cell-cell interactions, which contribute to the pathology of hepatic ischemia/reperfusion injury. Platelet NOX2 modulated intracellular Ca 21 release but not store-operated Ca 21 entry (SOCE), whereas neutrophil NOX2 was crucial for SOCE but not intracellular Ca 21 release. Different regulation of Ca 21 signaling by platelet and neutrophil NOX2 correlated with differences in the phosphorylation of AKT, ERK, and p38MAPK. Our results indicate that platelet and neutrophil NOX2-produced ROS are critical for the function of surface receptors essential for neutrophil-platelet interactions during vascular inflammation. (Blood. 2015;126(16):1952-1964

Measuring and interpreting platelet-leukocyte aggregates

“…The development of in vivo imaging techniques, such as intravital microscopy (IVM) further allows to track cells in live animals over longer time periods in organs including cremaster muscle, liver, lung, brain and kidney (Figure 1C) (56,57), and have been greatly beneficial in analysing dynamic interaction between platelets and leukocytes (e.g. duration of interactions and behavioural changes in leukocytes and/or platelets upon contact) (10,53,55,58–61). …”

“…In this context, several markers specifically label platelets, including CD41 (GPIIb) (60), CD42 (GPIb) (61) and CD49b (α2 integrin subunit) (10,59,60,65). While the use of antibodies against these proteins enables clear identification of platelets interacting with other cells, some antibodies affect platelet function under certain conditions.…”

“…Simple epifluorescent microscopy coupled with brightfield transillumination has been applied to track platelet-leukocyte interactions over time in some settings (58,61,68). However, this approach is very limited as it offers relatively low spatial and temporal resolution of processes on a cellular level.…”

“…Similarly, platelet inhibition in clinically relevant sterile liver inflammation, such as ischemia/reperfusion injury (as observed following transplantation) reduces liver damage via blocking platelet- and P-selectin-dependent CD4 + , but not CD8 + T cell recruitment to post-ischemic sinusoids (58). Nicotinamide adenine dinucleotide phosphate oxidase 2 might also play a role in mediating platelet-neutrophil interactions in this setting (61). In another sterile injury model, following bile duct ligation-induced cholestasis, platelet aggregates support leukocyte recruitment to liver sinusoids and postsinusoidal venules involving P-selectin (63).…”

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

“…[9][10][11][12] Among several receptors and counter-receptors, the neutrophil-platelet association is primarily mediated by the interaction of neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and αMb2 integrin with platelet Pselectin and glycoprotein Ibα (GPIbα), respectively. 13 We have shown that AKT2 positively regulates the function of αMb2 integrin and P-selectin during vascular inflammation 12 and that combining hydroxyurea with AKT2 inhibition has immediate benefits in acute vaso-occlusive events and improves survival in SCD mice.…”