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Neutrophil CD40 enhances platelet-mediated inflammation

https://doi.org/10.1016/j.thromres.2007.12.019Get rights and content

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.

Section snippets

Chemicals and reagents

Prostaglandin E1 (PGE1), lucigenin, sodium citrate, NaH2PO4 and dextrose, fMLP (N-formyl-Met-Leu-Phe), N-acetylcysteine, wortmannin, and α-Lipoic acid were purchased from Sigma-Aldrich (St. Louis, MO). LY294002 was purchased from Cayman Chemicals (Ann Arbor, MI). Fibrinogen and human alpha thrombin were purchased from Enzyme Research Laboratories (South Bend, IN). Thrombin Receptor Activation Peptide (TRAP1–6) was purchased from Peninsula Laboratories (San Carlos, CA).

Statistics

All data are presented as mean ± standard error. Significance of difference between groups was evaluated with one-way analysis of variance (ANOVA). Statistical significance was accepted with a P value < 0.05.

CD40 is present in neutrophils

Initially, to identify CD40, neutrophil mRNA was isolated from six different donors. Real-time PCR was used to quantify the CD40 mRNA transcripts after normalization with β-actin (Fig. 1a, left panel). These data confirm the presence of CD40 in neutrophils consistent with the recent report by Khan et al. [6]. Notably, the relative abundance of CD40 in neutrophils is less than that in platelets and almost a hundred-fold less than that in the proplatelet cell line MEG01 (Fig. 1a, left panel). The

Discussion

Platelets play a significant role in the development of thrombosis and inflammation. Platelets interact with monocytes via a CD40–CD40L-mediated pathway that causes their adherence to the inflamed endothelial layer [32], [33], [34]. Neutrophils are also known to be central to many inflammatory processes and were recently reported to express CD40 [6]. Khan and colleagues [6] showed that sCD40L, which accumulates during blood component storage, has the capacity to activate adherent neutrophils

Acknowledgements

We gratefully acknowledge the help of Dr. Kahraman Tanriverdi and Kristine Morin for real-time PCR analysis. We are also thankful to Dr. Michael Kirber for his help during two-photon confocal microscopic studies.

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