Innate recognition of apoptotic cells: novel apoptotic cell-associated molecular patterns revealed by crossreactivity of anti-LPS antibodies

Cell Death and Differentiation (2013), 698–708 & 2013 Macmillan Publishers Limited All rights reserved 1350-9047/13 www.nature.com/cdd Innate recognition of apoptotic cells: novel apoptotic cell-associated molecular patterns revealed by crossreactivity of anti-LPS antibodies I Tennant1, JD Pound1, LA Marr1, JJLP Willems1, S Petrova1, CA Ford1, M Paterson1, A Devitt1,2 and CD Gregory*,1 Cells dying by apoptosis are normally cleared by phagocytes through mechanisms that can suppress inflammation and immunity. Molecules of the innate immune system, the pattern recognition receptors (PRRs), are able to interact not only with conserved structures on microbes (pathogen-associated molecular patterns, PAMPs) but also with ligands displayed by apoptotic cells. We reasoned that PRRs might therefore interact with structures on apoptotic cells – apoptotic cell-associated molecular patterns (ACAMPs) – that are analogous to PAMPs. Here we show that certain monoclonal antibodies raised against the prototypic PAMP, lipopolysaccharide (LPS), can crossreact with apoptotic cells. We demonstrate that one such antibody interacts with a constitutively expressed intracellular protein, laminin-binding protein, which translocates to the cell surface during apoptosis and can interact with cells expressing the prototypic PRR, mCD14 as well as with CD14-negative cells. Anti-LPS cross reactive epitopes on apoptotic cells colocalised with annexin V- and C1q-binding sites on vesicular regions of apoptotic cell surfaces and were released associated with apoptotic cell-derived microvesicles (MVs). These results confirm that apoptotic cells and microbes can interact with the immune system through common elements and suggest that anti-PAMP antibodies could be used strategically to characterise novel ACAMPs associated not only with apoptotic cells but also with derived MVs. Cell Death and Differentiation (2013) 20, 698–708; doi:10.1038/cdd.2012.165; published online 8 February 2013 Apoptotic cells undergo multiple surface changes, the most renowned being the externalisation of the anionic phospholipid, phosphatidylserine (PS).1 Profound alterations in plasmamembrane topology also occur as a consequence of the apoptosis programme, involving protein, carbohydrate and nucleic acid moieties in addition to lipids (reviewed in Gregory and Pound2). Gross changes in apoptotic cells include the production of plasma-membrane-bound apoptotic bodies, blebs and microvesicles (MVs),3,4 the latter serving functions in intercellular communication, including chemoattraction and activation of phagocytes.5–8 Taken together, these changes permit a multitude of molecular interactions with phagocytes triggering apoptotic cell engulfment and additional phagocyte responses including immunomodulation. Detailed activities of underlying receptor–ligand interactions participating in these responses remain to be defined but it is clear that interference with these processes can have pathological consequences (see, Savill et al.,9 Lauber et al.,10 Ravichandran and Lorenz,11 Erwig and Henson,12 Elliott and Ravichandran13 and Gregory and Pound,14 for reviews). Many of the host proteins, including CD14, C1q and collectins, that interact with apoptotic cells are molecules of the innate immune system that were first known for their involvement in host responses to microbes.15 The reverse relationship is also proven: brain angiogenesis inhibitor 1, characterised first in this context as a phagocyte receptor for apoptotic cells, was recently also shown to function as an immune receptor for bacterial binding and engulfment.16 All these molecules fall into the category of the so-called ‘pattern recognition receptors’ (PRRs) that interact with evolutionarily conserved structures – ‘pathogen-associated molecular patterns’ (PAMPs) – of microbes.17 For example, the prototypic PRR, CD14, interacts with, and regulates proinflammatory host responses to, the prototypic PAMP, lipopolysaccharide (LPS).18 Because of the involvement of PRRs in the molecular interactions of phagocytes with apoptotic cells, we and others previously suggested that apoptotic cells express host equivalents of PAMPs termed ‘apoptotic cell-associated molecular patterns’ (ACAMPs).19,20 This notion predicts that ACAMPs share spatial similarities with PAMPs that permit common PRR interactions. This led us to hypothesise that some antibodies raised against PAMPs could potentially crossreact with ACAMPs, thus providing an opportunity to use antibody approaches to probe the existence and nature of ACAMPs. Here we test this hypothesis and demonstrate that certain anti-LPS antibodies indeed show strong crossreactive binding to apoptotic cells. We characterise a host epitope specified by one of these antibodies, demonstrating (1) its association with the intracellular protein, laminin-binding protein (LBP/p40), (2) its exposure on apoptotic cells and (3) its ability to bind to cells via both plasma-membrane CD14 (mCD14)-dependent and 1 Medical Research Council (MRC) Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK and 2School of Life and Health Sciences, Aston University, Birmingham, UK *Corresponding author: CD Gregory, Medical Research Council (MRC) Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK. Tel: þ 44 (0)131 242 9170/2; Fax: þ 44 (0)131 242 9171; E-mail: Keywords: apoptosis; innate immunity; pattern recognition; LPS; antibody Abbreviations: ACAMP, apoptotic cell-associated molecular pattern; AxV, annexin V; LBP, laminin binding protein; LPS, lipopolysaccharide; mAb, monoclonal antibody; MV, microvesicle; PAMP, pathogen-associated molecular pattern; PI, propidium iodide; PRR, pattern recognition receptor; rMBL, mannan binding lectin Received 29.06.12; revised 14.11.12; accepted 20.11.12; Edited by S Nagata; published online 08.2.13 Apoptotic cell-associated molecular patterns (ACAMPs) I Tennant et al 699 -independent mechanisms. These results provide further evidence supporting the notion that apoptotic cells and microbes share certain elements of immune recognition and suggest a strategy whereby anti-PAMP antibodies can be used to probe the molecular architecture of apoptotic cells and of derived MVs. Results Certain monoclonal anti-LPS antibodies crossreact with epitopes of apoptotic cells. To test the hypothesis that certain anti-microbial antibodies can crossreact with ACAMPs, endogenous molecules exposed by cells undergoing apoptosis, a panel of monoclonal antibodies (mAbs) raised against LPS, was tested for reactivity towards apoptotic, compared with viable, cells. Initial screening was carried out by flow cytometry using a human lymphoma cell line. Antibodies were selected according to their previously characterised ability to react broadly across LPS species (Table 1). Three (out of 13 anti-LPS mAbs tested) showed clear binding to (...truncated)