Aminopeptidase N (CD13) Regulates Tumor Necrosis Factor-α-induced Apoptosis in Human Neutrophils

Andrew S Cowburn,Anastasia Sobolewski,Ben J Reed,John Deighton,Joanna Murray,Karen A Cadwallader,John R Bradley,Edwin R Chilvers

doi:10.1074/jbc.m511277200

Andrew S Cowburn, Anastasia Sobolewski + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m511277200

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Abstract

Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-alpha (TNFalpha) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFalpha in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFalpha-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFalpha-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFalpha-induced apoptosis, cell polarization, and TNFalpha-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFalpha-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFalpha-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.

Highlights

Neutrophil apoptosis is critical to the resolution of acute inflammation and the prevention of granulocytes-mediated tissue injury [1, 2]
These data suggest that neutrophil cell surface aminopeptidase N (APN)/CD13 activity is a key determinant of TNFRI expression and function and that variation in APN/CD13 activity dictates the efficacy of TNF␣-induced neutrophil apoptosis
Culturing isolated human neutrophils for 6 h with the AP inhibitors actinonin, bestatin, or the blocking peptide to CD13 alone had no effect on the basal rate of neutrophil apoptosis at any inhibitor concentration used (Fig. 2A)

Summary

Introduction

Neutrophil apoptosis is critical to the resolution of acute inflammation and the prevention of granulocytes-mediated tissue injury [1, 2]. One of the most widely studied AP is the 967-amino acid protein termed aminopeptidase N (APN) This peptidase is identical to the cell surface antigen CD13 [14] and displays activity toward an array of substrates, including the enkephalins, bradykinin, bacterial derived chemotactic peptides, tachykinins, and the cytokines granulocyte colony-stimulating factor, interferon-␥, IL-1␤, IL-2, IL-6, and IL-8 [15, 16]. APN Regulates TNF␣-induced Neutrophil Apoptosis indicated by an increase in fMLP-stimulated superoxide anion generation), and TNF␣-induced shape change. This effect is mediated via the ability of APN inhibitors to prevent the shedding of cell surface TNFRI with no effect on TNFRII or CXCR1/2 expression or cleavage. These data suggest that neutrophil cell surface APN/CD13 activity is a key determinant of TNFRI expression and function and that variation in APN/CD13 activity dictates the efficacy of TNF␣-induced neutrophil apoptosis

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