Mounting evidence suggests that systemic lupus erythematosus autoantigens are derived from apoptotic cells. To characterize the potential interactions between apoptotic cells and B cells, the D56R/S76R variant of 3H9, a murine autoantibody that binds to DNA, chromatin, and anionic phospholipids, was compared with DNA4/1, a human anti-DNA autoantibody. Flow cytometry revealed that only D56R/S76R bound to Jurkat cells treated with either of three distinct proapoptotic stimuli, Ab binding was dependent on caspase activity, and immunoreactivity developed subsequent to annexin V binding. Confocal microscopy established a structural basis for the distinct kinetics of binding. D56R/S76R preferentially bound to membrane blebs of apoptotic cells, whereas annexin V binding did not require blebs. Inhibition of ROCK I kinase, an enzyme that stimulates nuclear fragmentation and fragment distribution into blebs, significantly reduced Ab binding. Because members of the collectin and pentraxin families of serum proteins bind to blebs on apoptotic cells and assist in the clearance of cellular remains, our results suggest that Abs to blebs could affect the recognition of apoptotic cells by cells of the innate immune system and thus modify tolerance to nuclear Ags.

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...