Neutrophil CD40 enhances platelet-mediated inflammation

Abstract 

Introduction

CD40 is a transmembrane protein expressed on monocytes, macrophages, endothelial cells, and platelets. Platelets are the richest source of soluble CD40 ligand (sCD40L) and interact with monocytes and endothelial cells via CD40. While CD40 was recently reported to be present on neutrophils, the detailed mechanism of its interaction with platelets via CD40–CD40L has not been examined.

Materials and methods

The existence of neutrophil CD40 was verified by real-time PCR and western blot. Platelet sCD40L release was measured by ELISA. Neutrophil superoxide generation was measured by chemiluminescence and confocal microscopy. The neutrophil–platelet conjugates were measured by flow cytometry.

Results and conclusion

The presence of neutrophils enhances stimulation-induced platelet release of sCD40L. The addition of platelets leads to an enhancement of neutrophil superoxide and reactive oxygen species (ROS) generation. The specificity of the CD40–CD40L pathway in the neutrophil–platelet interaction was confirmed by using recombinant soluble CD40L (rsCD40L) and an anti-CD40L antibody. The involvement of the PI3 kinase/Akt pathway in neutrophil superoxide production was revealed by using LY294002 in isolated neutrophils/platelets experiments, as well as during whole blood aggregation-mediated neutrophil–platelet conjugation. N-acetylcysteine, a scavenger of ROS, eliminates both neutrophil superoxide generation and sCD40L release from activated platelets. These data suggest that activated neutrophils release ROS in a PI3 kinase-dependent manner, contributing to platelet activation and further sCD40L release in a redox-controlled positive feed-back loop. In conclusion, our results define a new pathway by which platelets and neutrophils interact and modulate each other's function, and may be relevant in understanding acute thrombo-inflammatory processes.

Abbreviations: CD40L, CD40 ligand, sCD40L, soluble CD40 ligand, rsCD40L, recombinant soluble CD40 ligand, N-Ac-Cys, N-acetylcysteine, DCFDA, Dichlorodihydrofluorescein diacetate, ROS, reactive oxygen species, TRAP, thrombin receptor activation peptide, fMLP, formyl-Met-Leu-Phe

Keywords: Platelets, Neutrophils, CD40, ROS, Neutrophil–platelet conjugate, sCD40L