Structural basis for autoantibody recognition of phosphatidylserine-β2 glycoprotein I and apoptotic cells

Abstract

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by ␤2 glycoprotein I (␤2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-␤2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells. C ells undergoing apoptosis package autoantigens, including nuclear proteins and DNA, in vesicles located beneath the surface of the plasma membrane (1). The contents of such surface vesicles may serve to enforce tolerance or, under a different set of circumstances, they may induce an autoimmune response (2-4). However, it is not obvious how autoantigens from apoptotic cells might activate the adaptive immune system, given that recognition and clearance of dying cells by mononuclear scavenger cells, such as monocytes and macrophages, is rapid and noninflammatory (5).One of the earliest signals for the uptake of apoptotic cells by phagocytes is the exposure of phosphatidylserine on the outer membrane leaflet, a consequence of the loss of membrane asymmetry in apoptotic cells (6). Phagocyte recognition of apoptotic cells occurs by several alternative mechanisms, including binding via the phosphatidylserine receptor (7), class A scavenger receptors (8), vitronectin receptors (9), macrosialin (10), CD14 (11), and CD36 (12). Moreover, serum proteins such as ␤2 glycoprotein I (␤2GPI; refs. 13 and 14), and C1q, the first component of complement (15), bind apoptotic cells and enhance their uptake.An important clue linking cell death to the onset of autoimmunity is provided by autoantibodies that bind apoptotic cells (16) and recognize surface epitopes that include complexes of phospholipid and ␤2GPI (17-19). The role of such autoantibodies is not known, although it has been postulated that they may preserve tissue homeostasis by enhancing the removal of dead or dying cells (20). In autoimmunity, it is possible that autoantibodies to apoptotic cells arise secondary to an increased load of apoptotic cells (18, 21), a decreased capacity for apoptotic cell removal (22, 23), or other as yet unknow...

The evolution of human anti-double-stranded DNA autoantibodies

“…Antibody binding to dioleoyl phosphatidylserine was measured as described in ref. 15. A commercially available enzyme immunoassay was used for the detection of anti-nucleosome reactivity (Orgentec, Mainz, Germany).…”

“…Many anti-DNA antibodies show crossreactions with phospholipids (21), and murine anti-DNA antibodies have been shown to bind to apoptotic cells (10,15), most likely due to binding of phosphatidylserine exposed on the surfaces of apoptotic cells (22). We therefore tested the antibody 33.C9 and antibody revertants for binding to phosphatidylserine by ELISA.…”

Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM

“…Specific targets of anti-dsDNA Abs include blebs formed by the protrusion of nuclear fragments from the cell surface. AntidsDNA Abs may also cross-react with phospholipid structures associated with the inner plasma membrane that become externalized during apoptosis (61)(62)(63)(64)(65). As shown in Fig.…”

The consequences of apoptosis in autoimmunity

“…It has been proposed that apoptotic bodies can be targeted by autoantibodies [24] and that B cells with Ig receptors for apoptotic cells are positively selected [61]. Accordingly, B cells that react with apoptotic cells may tip the balance from tolerance to autoimmunity (Figure 2).…”