Heterogeneity and Immunochemical Properties of Anti-β₂-glycoprotein I Autoantibodies

Summary

Most anticardiolipin antibodies (ACA) associated with antiphospholipid syndrome (APS) are directed against epitopes expressed on β₂-glycoprotein I (β₂GPI). Despite a good correlation between standard ACA assays and those using purified human β₂GPI as the sole antigen, some sera from APS patients only react in the latter. This is indicative of heterogeneity in anti-β₂GPI antibodies. To characterize their reactivity profiles, human and bovine β₂GPI were immobilized on γ-irradiated plates (β₂GPI-ELISA), plain polystyrene precoated with increasing cardiolipin concentrations (CL/β₂GPI-ELISA), and affinity columns. Fluid-phase inhibition experiments were also carried out with both proteins. Of 56 selected sera, restricted recognition of bovine or human β₂GPI occurred respectively in 10/29 IgA-positive and 9/22 IgM-positive samples, and most of the latter (8/9) were missed by the standard ACA assay, as expected from a previous study. Based on species specificity and ACA results, IgG-positive samples (53/56) were categorized into three groups: antibodies reactive to bovine β₂GPI only (group I) or to bovine and human β₂GPI, group II being ACA-negative, and group III being ACA-positive. The most important group, group III (n = 33) was characterized by (i) binding when β₂GPI was immobilized on γ-irradiated polystyrene or cardiolipin at sufficient concentration (regardless of β₂GPI density, as assessed using ¹²⁵I-β₂GPI); (ii) and low avidity binding to fluid-phase β₂GPI (Kd in the range 10^–5 M). In contrast, all six group II samples showed (i) ability to bind human and bovine β₂GPI immobilized on non-irradiated plates; (ii) concentration-dependent blockade of binding by cardiolipin, suggesting epitope location in the vicinity of the phospholipid binding site on native β₂GPI; (iii) and relative avidities approximately 100-fold higher than in group III. Group I patients were heterogeneous with respect to CL/β₂GPI-ELISA and ACA results (6/14 scored negative), possibly reflecting antibody differences in terms of avidity and epitope specificity. Affinity fractionation of 23 sera showed the existence, in individual patients, of various combinations of antibody subsets solely reactive to human or bovine β₂GPI, together with cross-species reactive subsets present in all samples with dual reactivity namely groups III and II, although the latter antibodies were poorly purified on either column. Therefore, the mode of presentation of β₂GPI greatly influences its recognition by anti-β₂GPI antibodies with marked inter-individual heterogeneity, in relation to ACA quantitation and, possibly, disease presentation and pathogenesis.

• References

• 1 Roubey RAS. Immunology of the antiphospholipid syndrome. Arthritis Rheum 1996; 39: 1444-54.
• 2 Najmey SS, Keil LB, Adib DYR, DeBari VA. The association of antibodies to β₂-glycoprotein I with the antiphospholipid syndrome: a meta-analysis. Ann Clin Lab Sci 1997; 27: 41-6.
• 3 Forastiero RR, Martinuzzo ME, Cerrato GS, Kordich LC, Carreras LO. Relationship of anti β₂-glycoprotein I and anti-prothrombin antibodies to thrombosis and pregnancy loss in patients with antiphospholipid antibodies. Thromb Haemost 1997; 78: 1008-14.
• 4 Cabral AR, Amigo MC, Cabiedes J, Alarcon-Segovia D. The antiphospholipid/cofactor syndromes: a primary variant with antibodies to β₂-glycoprotein I but no antibodies detectable in standard antiphospholipid assays. Am J Med 1996; 101: 472-81.
• 5 Arvieux J, Darnige L, Hachulla E, Roussel B, Bensa JC, Colomb MG. Species specificity of anti-β₂-glycoprotein I autoantibodies and its relevance to anticardiolipin antibody quantitation. Thromb Haemost 1996; 75: 725-30.
• 6 Arvieux J, Pouzol P, Roussel B, Jacob MC, Colomb MG. Lupus-like anticoagulant properties of murine monoclonal antibodies to β₂-glycoprotein I. Br J Haematol 1992; 81: 568-73.
• 7 Benboubetra M, Gleeson A, Harris CPD, Khan J, Arrar L, Brennand D, Reid J, Reckless JD, Harrison R. Circulating anti-(xanthine oxidoreductase) antibodies in healthy human adults. Eur J Clin Invest 1997; 27: 611-9.
• 8 Pierangeli SS, Harris EN, Davis SA, DeLorenzo G. β₂-glycoprotein I (β₂GPI) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies. Br J Haematol 1992; 82: 565-70.
• 9 Matsuura E, Igarashi Y, Nagae H, Yasuda T, Triplett DA, Koike T. Anti-cardiolipin antibodies recognize β₂-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994; 179: 457-62.
• 10 Pengo V, Biasiolo A, Fior MG. Autoimmune antiphospholipid antibodies are directed against a cryptic epitope expressed when β₂-glycoprotein I is bound to a suitable surface. Thromb Haemost 1995; 73: 29-34.
• 11 Galli M, Beretta G, Daldossi M, Bevers E, Barbui T. Different anticoagulant and immunological properties of anti-prothrombin antibodies in patients with antiphospholipid antibodies. Thromb Haemost 1997; 77: 486-91.
• 12 Arvieux J, Darnige L, Caron C, Reber G, Bensa JC, Colomb MG. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb Haemost 1995; 74: 1120-5.
• 13 Roubey RAS, Eisenberg RA, Harper MF, Winfield JB. “Anticardiolipin” autoantibodies recognize β₂-glycoprotein I in the absence of phospholipid. J Immunol 1995; 154: 954-60.
• 14 Tincani A, Spatola L, Prati E, Allegri F, Ferremi P, Cattaneo R, Meroni P, Balestrieri G. The anti-β₂-glycoprotein I activity in human anti-phospholipid syndrome sera is due to monoreactive low-affinity autoantibodies directed to epitopes located on native β₂-glycoprotein I and preserved during species’ evolution. J Immunol 1996; 157: 5732-8.
• 15 Willems GM, Janssen MP, Pelsers MAL, Comfurius P, Galli M, Zwaal RFA, Bevers EM. Role of divalency in the high-affinity binding of anticardiolipin antibody-β₂-glycoprotein I complexes to lipid membranes. Biochemistry 1996; 35: 13833-42.
• 16 Wagenknecht DR, McIntyre JA. Changes in β₂-glycoprotein I antigenicity induced by phospholipid binding. Thromb Haemost 1993; 69: 361-5.
• 17 Borchman D, Harris EN, Pierangeli SS, Lamba OP. Interactions and molecular structure of anticardiolipin and β₂-glycoprotein I (β₂GPI). Clin Exp Immunol 1995; 102: 373-8.
• 18 Sanmarco M, Soler C. Heterogeneity of anti-β₂-glycoprotein I antibodies. Nouv Rev Fr Hematol 1995; 37 [Suppl II] S57-60.
• 19 Sheng Y, Sali A, Herzog H, Lahnstein J, Krilis SA. Site-directed muta-genesis of recombinant human β₂-glycoprotein I identifies a cluster of ly-sine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity. J Immunol 1996; 157: 3744-51.
• 20 Steinkasserer A, Barlow PN, Willis AC, Kertesz Z, Campbell ID, Sim RB, Norman DG. Activity, disulphide mapping and structural modelling of the fifth domain of human β₂-glycoprotein I. FEBS Lett 1992; 313: 193-7.
• 21 Shanghera DK, Wagenknecht DR, McIntyre JA, Kamboh MI. Identification of structural mutations in the fifth domain of apolipoprotein H (β_2-glycoprotein I) which affect phospholipid binding. Hum Molec Genet 1997; 6: 311-6.
• 22 Alarcon-Segovia D, Mestanza M, Cabiedes J, Cabral AR. The antiphospholipid/cofactor syndromes. I. A variant in patients systemic lupus erythematosus with antibodies to β₂-glycoprotein I but no antibodies detectable in standard antiphospholipid assays. J Rheumatol 1997; 24: 1545-51.
• 23 Barlow DJ, Edwards MS, Thornton JM. Continuous and discontinuous protein antigenic determinants. Nature 1986; 322: 747-8.
• 24 Wang M, Kandiah DA, Ichikawa K, Khamashta M, Hughes G, Koike T, Roubey R, Krilis SA. Epitope specificity of monoclonal anti-β₂-glycoprotein I antibodies derived from patients with the antiphospholipid syndrome. J Immunol 1995; 155: 1629-36.
• 25 Menon S, Isenberg DA. Fetal calf serum in growth medium obscures the detection of early anticardiolipin antibody secreting clones. J Immunol Methods 1995; 186: 65-70.
• 26 Pengo V, Biasiolo A, Fior MG. Binding of autoimmune cardiolipin-reactive antibodies to heparin: a mechanism of thrombosis?. Thromb Res 1995; 78: 371-8.