ARTHRITIS & RHEUMATISM Vol. 60, No. 3, March 2009, pp 860–869 DOI 10.1002/art.24361 © 2009, American College of Rheumatology Ro 60 Functions as a Receptor for  2 -Glycoprotein I on Apoptotic Cells Joanne H. Reed, 1 Bill Giannakopoulos, 2 Michael W. Jackson, 1 Steven A. Krilis, 2 and Tom P. Gordon 1 Objective. The autoantigens 60-kd Ro/SSA (Ro 60) and  2 -glycoprotein I ( 2 GPI) are both displayed on the surface membrane of apoptotic cells. Epitope- spreading experiments have suggested that these au- toantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether  2 GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti–Ro 60 IgG. Methods. The interaction between soluble recom- binant Ro 60 fragments and  2 GPI was investigated in vitro by direct and saturation binding assays using native human  2 GPI and recombinant domain deletion mutants. Binding of  2 GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competi- tive inhibition with soluble recombinant Ro 60 and anti–Ro 60 IgG. Results. The Ro 60 fragment expressing a surface- exposed epitope (apotope) bound with high affinity (K d  15 nM) to domain V of  2 GPI in vitro. Beta 2 - glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies,  2 GPI blocked the binding of anti–Ro 60 auto- antibodies to apoptotic cells in a dose-dependent man- ner, and anti–Ro 60 IgG inhibited the binding of  2 GPI. Moreover,  2 GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface. Conclusion. These results demonstrate that Ro 60 functions as a novel receptor for  2 GPI on the surface of apoptotic cells. The formation of Ro 60– 2 GPI com- plexes may protect against anti–Ro 60 autoantibody– mediated tissue injury. Autoantibodies to the 60-kd Ro/SSA (Ro 60) protein are associated with primary Sjo ¨gren’s syndrome (SS), systemic lupus erythematosus (SLE), and the neonatal lupus syndrome and are the most common autoantibody of the extractable nuclear antigen family found in clinical practice (1). Ro 60 translocates to the cell surface during apoptosis, where it is thought to initiate tissue damage in congenital heart block (CHB) by binding cognate maternal antibodies (2). The finding that anti–Ro 60 IgG block the uptake of apoptotic cardiocytes by live cardiocytes suggests that this autoan- tigen may also function as a natural ligand for apoptotic cell clearance (3). We have recently identified a B cell apotope (defined as an epitope of an intracellular autoantigen that is expressed on the apoptotic cell surface) of Ro 60 within a region of -helical repeats that is recognized specifically by IgG in a subset of SLE patients who were positive for anti–Ro 60 but not anti-La (4). Since this apotope is a potential target for opsonization by trans- placental anti–Ro 60 IgG in CHB, masking of the apotope might block the formation of IgG–apoptotic cell complexes and protect the fetal heart against autoantibody-mediated tissue injury. Beta 2 -glycoprotein I ( 2 GPI) is the primary tar- get antigen recognized by autoantibodies in patients with antiphospholipid syndrome (5,6). Extensive experimen- tal evidence derived from in vivo murine models and in vitro analysis suggests that anti- 2 GPI autoantibodies in complex with  2 GPI may directly mediate a pathogenic Supported by grants from the Australian National Health and Medical Research Council. Ms Reed’s work was supported by an Australian Postgraduate award. 1 Joanne H. Reed, BSc, Michael W. Jackson, PhD, Tom P. Gordon, MD, PhD: Flinders University of South Australia, Adelaide, and Flinders Medical Centre, Bedford Park, South Australia, Austra- lia; 2 Bill Giannakopoulos, MBBS, PhD, Steven A. Krilis, MBBS, PhD, FRACP: University of New South Wales, and St. George Hospital, Sydney, New South Wales, Australia. Address correspondence and reprint requests to Tom P. Gordon, Department of Immunology, Allergy and Infectious Diseases, Flinders Medical Centre, Flinders Drive, Bedford Park, South Austra- lia 5042, Australia. E-mail: t.gordon@flinders.edu.au. Submitted for publication June 5, 2008; accepted in revised form December 5, 2008. 860