Antibody-based strategies for identifying novel apoptotic-cell surface-associated molecules

The recognition and phagocytosis of apoptotic cells is not only a physiological means of waste disposal but can also result in anti-inflammatory and immunosuppressive effects. Indeed, defective clearance of apoptotic cells (ACs) is associated with autoimmune and inflammatory disease states such as systemic lupus erythematosus. To date, relatively few markers have been identified as 'eat me' signals displayed on AC surfaces despite the great potential for such molecules as diagnostic or therapeutic reagents. In this work various antibody-based strategies were employed in an attempt to identify novel AC-specific epitopes. An initial strategy utilised a phage displayed antibody library containing a repertoire of ~108 antibody fragments encoded by human germline genes as an unbiased source of binding specificity. An alternative approach was based on the knowledge that receptors used by macrophages to recognise ACs also recognise pathogen-associated molecules. By looking for the ability of antibodies raised against pathogens to crossreact with ACs the hypothesis that cells undergoing apoptosis reveal molecular patterns that resemble those on pathogen related structures was tested. Screening of phage-displayed antibodies isolated from in vitro selections performed on both whole ACs and on preparations of purified AC-associated molecules did not identify any AC-specific epitopes. However, screening of antibodies raised in vivo, that have previously been characterised as having specificity for pathogen-associated molecular patterns (PAMPs) revealed that some cross-react with cells undergoing apoptosis. One of these antibodies was found to bind an epitope found on the ubiquitously expressed ~40KDa precursor to Laminin-Binding-Protein (LBP/p40). These findings suggest that epitopes resembling PAMPs appear on the surface of mammalian cells as a result of apoptosis and that these epitopes can be found on endogenously expressed molecules which are normally excluded from the surface of viable cells. The ability of host receptors to cross-react with host and pathogenassociated epitopes in this way may lead to a greater understanding of the progression of inflammatory, infectious and tumoural processes