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