Elsevier

Cellular Immunology

Volume 239, Issue 1, January 2006, Pages 14-21
Cellular Immunology

Competitive binding of pentraxins and IgM to newly exposed epitopes on late apoptotic cells

https://doi.org/10.1016/j.cellimm.2006.02.006Get rights and content

Abstract

A random distribution of phospholipids among the inner and outer leaflet of the cell membrane occurs during apoptosis and is known as membrane flip-flop. Flip-flopped cells have binding sites for various plasma proteins, such as IgM and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP). In this study, we investigated whether pentraxins and IgM antibodies recognize the same binding sites on apoptotic cells, and whether phospholipids constitute these binding sites. Except for SAP which also bound to early apoptotic cells, pentraxins and IgM preferentially bound to late apoptotic cells. Competition experiments with different phosphatemonoesters revealed that CRP and SAP as well as part of the IgM bound to the phospholipids head groups, SAP mainly to phosphorylethanolamine, CRP to phosphorylcholine and phosphorylethanolamine and to a lesser extent to phosphorylserine, and IgM to phosphorylcholine and phosphorylserine. These results were confirmed in experiments in which proteins were adsorbed from plasma with artificial phospholipids particles. IgM and the pentraxins variably competed for the same binding sites on late apoptotic cells, SAP having the highest and CRP the lowest apparent affinity. We conclude that CRP, SAP, and part of the IgM bind to the phospholipid head groups exposed on apoptotic cells. This shared specificity as well as their shared capability to activate complement, suggest that IgM and the pentraxins CRP and SAP exert similar functions in the removal of apoptotic cells.

Introduction

Apoptosis induces a series of intracellular events leading to phenomena such as DNA fragmentation and caspase activation [1], [2]. Among the morphological changes ensuing during apoptosis, is the reorganisation of the cellular membrane leading to the exposition of anionic phospholipids in the outer leaflet of the membrane, a phenomenon known as flip-flop. Exposition of these anionic phospholipids allows receptor-specific recognition of apoptotic cells by phagocytes [3], [4]. Indeed, removal of apoptotic cells by macrophages and neighbouring cells is in part mediated by a receptor which specifically binds to the anionic phospholipid phosphatidylserine exposed on the apoptotic cell [3], [4], [5]. However, other mechanisms including binding of several plasma proteins and activation of complement [5], [6], [7], [8], are also triggered by membrane flip-flop. Among the proteins binding to apoptotic cells are pentraxins such as C-reactive protein (CRP) and serum amyloid P component (SAP) [9], [10], [11]. CRP and SAP bind to phosphatidylcholine and phosphatidylethanolamine, respectively, in a calcium-dependent fashion [12], [13], [14], [15]. CRP and SAP are supposed to participate in the clearance of these cells by phagocytes either by direct interaction with macrophage Fcγ-receptors [16], or indirectly via activation of complement [5], [6], [17]. Other proteins binding to apoptotic cells include IgM antibodies [18], [19], which also can activate complement. Most of the IgM that binds to apoptotic cells, has been claimed to bind to phosphatidylcholine[18], [20], [21]. This suggests that the pentraxins CRP, SAP, and IgM may share the same binding sites on apoptotic cells.

In the present study, we investigated the binding of IgM, CRP, and SAP to apoptotic cells, and tested the hypothesis that these proteins interact with the same binding sites.

Section snippets

Reagents

All biotinylated antibodies were monoclonal antibodies (mAbs) produced in our laboratory, including anti-CRP mAb 5G4 (IgG2a subclass) and anti-SAP mAb SAP14 (IgG1 subclass), which both have been described before [9], [22]. Anti-human IgM mAb MH-15 (IgG1 subclass), mouse IgG1 control and fluorescein isothiocyanate (FITC)-coupled rabbit anti-human IgM, F(ab′)2, were obtained from Sanquin, Business Unit Reagents (Amsterdam, The Netherlands). MAbs were biotinylated with LC-biotin-n

Binding of plasma proteins to late apoptotic cells

Jurkat cells were incubated with etoposide from 5 to 48 h to induce apoptosis. To assess binding of plasma proteins to the cells, they were incubated for another 30 min at 37 °C with 10%, v/v, recalcified plasma. Thereafter, binding of specific plasma proteins was measured by flow cytometry, in which triple staining was performed with specific, biotin-labelled antibodies, annexinV-FITC (FL 1) and PI (FL 2). Vital cells were defined as negative for both annexin V as well as PI, early apoptotic

Discussion

Several studies have shown that IgM [18], CRP [10] as well as SAP [9] can bind to apoptotic cells. Here, we demonstrate that in a plasma environment each of these proteins indeed can bind to apoptotic cells, although mainly to late apoptotic cells. This binding is proportional to the plasma concentration of these opsonins. Moreover, we show that IgM, CRP, and SAP partially compete with each other for binding to apoptotic cells suggesting they share similar binding sites.

IgM and CRP bound

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