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A western blot assay detects autoantibodies to cryptic endothelial antigens in thrombotic microangiopathies

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Abstract

Autoantibodies detected by immunofluorescence, ELISA, and complement-fixation techniques have provided discriminatory markers for many human diseases. However, these commonly applied assays may fail to detect antibodies against antigenic sites which are either inaccessible or not displayed in recognizable cellular structures. Moreover, molecular identities of recognized antigen(s) are not determined with such methods. We have used Western blot analysis of cellular proteins derived from human renal microvascular endothelial cells (HRMEC) to identify autoantibodies in patients with pathological endothelial injury. Exploring the possibility that endothelial injury may expose cryptic endothelial antigens to immune recognition, we detected antibodies binding a number of distinct HRMEC proteins. Among these, antibodies recognizing specific HRMEC proteins of 43 kDa were commonly detected in plasmas from patients with thrombotic thrombocytopenic purpura (TTP) (13 of 14) and hemolytic uremic syndrome (HUS) (4 of 5) but were absent in 9 of 10 healthy subjects and 11 patients with a range of diseases not associated with endothelial injury of insult. Antibodies binding 43-kDa HRMEC antigens were detected in individual patients with systemic lupus erythematosus, anti-glomerular basement membrane nephropathy, and heparin-associated thrombocytopenia, as well as in one of three patients with immune thrombocytopenic purpura. Similar antibodies were detected in one hypercholesterolemic subject. Antibodies from four TTP patients were affinity purified and shown by two-dimensional analysis to recognize 43-kDa proteins having identical pl's (5.9, 6.0, and 6.1). Subcellular fractionation localized these antigens to cytosolic and nuclear compartments, sites presumably protected from immune recognition in the absence of endothelial injury. Western blot recognition of antiendothelial antibodies offers opportunities to define molecular characteristics and cellular distribution of antigens while generating reagents useful in their purification.

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References

  1. D'Cruz DP, Houssiau FA, Ramirez G, Baguley E, McCutcheon J, Vianna J, Haga H-J, Swana GT, Khamashta MA, Taylor JC, Davies DR, Hughes GRV: Antibodies to endothelial cells in systemic lupus erythematosus: A potential marker for nephritis and vasculitis. Clin Exp Immunol 85:254–261, 1991

    Google Scholar 

  2. Cines DB, Lyss AP, Reeber M, Dehoritias RJ: Presence of complement fixing anti-endothelial cell antibodies in systemic lupus erythematosus. J Clin Invest 73:611–625, 1984

    Google Scholar 

  3. Dillon MJ, Tizard EJ: Anti-neutrophil cytoplasmic antibodies and anti-endothelial cell antibodies. Pediat Nephrol 5:256–259, 1991

    Google Scholar 

  4. Frampton G, Jayne DR, Perry GJ, Lockwood CM, Cameron JS: Autoantibodies to endothelial cells and neutrophil cytoplasmic antigens in systemic vasculitis. Clin Exp Immunol 82:227–232, 1991

    Google Scholar 

  5. Ferraro G, Meroni PL, Tincani A, Sinico A, Barcellini W, Radice A, Gregorini G, Froldi M, Borghi MO, Balestrieri G: Anti-endothelial cell antibodies in patients with Wegener's granulomatosis and micropolyarteritis. Clin Exp Immunol 79:47–53, 1990

    Google Scholar 

  6. Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 318:1651–1657, 1988

    Google Scholar 

  7. Holt CM, Lindsey N, Moult J, Malia RG, Greaves M, Hume A, Nowell NR, Hughes P: Antibody-dependent cellular cytotoxicity of vascular endothelium: Characterization and pathogenic associations in systemic sclerosis. Clin Exp Immunol 78:359–365, 1989

    Google Scholar 

  8. Heurkens AK, Hiemstra PS, Lafeber GJ, Daha MR, Breedveld FC: Anti-endothelial cell antibodies in patients with rheumatoid arthritis complicated by vasculitis. Clin Exp Immunol 78:7–12, 1989

    Google Scholar 

  9. Tanaka Y, Tsukada N, Koh Ch-S, Yanagisawa N: Antiendothelial cell antibodies and circulating immune complexes in the sera of patients with multiple sclerosis. J Neuroimmunol 17:49–59, 1987

    Google Scholar 

  10. Yap HK, Sakai RS, Bahn L, Rappaport V, Woo KT, Ananthuraman V, Lim CH, Chiang GSC, Jordan SC: Antivascular endothelial cell antibodies in patients with IgA nephropathy: frequency and clinical significance. Clin Immunol Immunopathol 49:450–462, 1988

    Google Scholar 

  11. Savage COS, Winearls CG, Jones S, Marshall PD, Lockwood CM: Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet 1:1389–1393, 1987

    Google Scholar 

  12. Fattorossi A, Aurbach GD, Sakaguchi K, Cama A, Marx SJ, Streeten EA, Fitzpatrick LA, Brandi ML: Anti-endothelial cell antibodies: Detection and characterization in sera from patients with autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 85:4015–4019, 1988

    Google Scholar 

  13. Leung DY, Moake JL, Havens PL, Kim M, Pober JS: Lytic anti-endothelial cell antibodies in haemolytic-uraemic syndrome. Lancet 2:183–186, 1988

    Google Scholar 

  14. D'Anastasio C, Impallomeni M, McPherson GAD, Clements WG, Howells GL, Brooks PA, Batchelor JR: Antibodies against monocytes and endothelial cells in the sera of patients with atherosclerosis peripheral arterial disease. Atherosclerosis 74:99–105, 1988

    Google Scholar 

  15. Jennette JC, Falk RJ: Antineutrophil cytoplasmic autoantibodies and associated diseases: A review. Am J Kidney Dis 15:517–529, 1990

    Google Scholar 

  16. Hashemi S, Smith CD, Izaguirre CA: Anti-endothelial cell antibodies: Detection and characterization using a cellular enzyme-linked immunosorbent assay. J Lab Clin Med 109:434–440, 1987

    Google Scholar 

  17. Berzofsky JA: Intrinsic and extrinsic factors in protein antigenic structure. Science 229:932–940, 1985

    Google Scholar 

  18. Kwaan HC: Clinicopathologic features of thrombotic thrombocytopenic purpura. Semin Nephrol 24:71–81, 1987

    Google Scholar 

  19. Karmali MA: Infection by verocytotoxin-producing escherichia coli. Clin Microbiol Rev 2:15–38, 1989

    Google Scholar 

  20. Kumjian DA, Barnstein A, Rhee SG, Daniel TO: Phospholipase C-gamma complexes with ligand-activated plateletderived growth factor receptors. J Biol Chem 266:3973–3980, 1991

    Google Scholar 

  21. Wojta J, Hoover RL, Daniel TO: Vascular origin determines plasminogen activator expression in human endothelial cells. J Biol Chem 264:2846–2852, 1989

    Google Scholar 

  22. Harlow E, Lane D: Antibodies: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1988

    Google Scholar 

  23. Knowles RW: Two-dimensional gel analysis of transmembrane proteins.In Histocompatibility Testing, B Dupont (ed). New York, Springer Verlag, 1987, pp 1–45

    Google Scholar 

  24. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354, 1979

    Google Scholar 

  25. Robinson PA, Anderton BH, Loviny TLF: Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries. J Immunol Meth 108:115–122, 1988

    Google Scholar 

  26. Poskitt TR: Immunologic and electron microscopic studies in Goodpasture's syndrome. Am J Med 49:250–257, 1970

    Google Scholar 

  27. Gishman E, Porush JC, Lee SL, Churg J: Renal biopsies in lupus nephritis: Correlation of electron microscopic findings with clinical course. Nephron 10:25–36, 1973

    Google Scholar 

  28. Back H, Nilsson G, Hansson GK, Rodjer S, Tarkowski AJ: M-component with reactivity against actin associated with thrombotic thrombocytopenic purpura. Am J Med 91:429–433, 1991

    Google Scholar 

  29. Sturgeon P, Adashek EP, Self JL, Stiehm ER, Tishkoff GH: Recent developments in blood components therapy. Ann Intern Med 74:113–125, 1971

    Google Scholar 

  30. Women IP, Kovithavongs T, McConnachie PR, Dossetor JB: Immunity to tissue sensitisation, HL-A and non-HL-A, as detected by the ABCIL system. Transplantation 17:453–461, 1974

    Google Scholar 

  31. van-der-Zee JM, Heurkens AH, van-der-Voort EA, Daha MR, Breedveld FC: Characterization of anti-endothelial antibodies in patients with rheumatoid arthritis complicated by vasculitis. Clin Exp Rheumatol 9:589–594, 1991

    Google Scholar 

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  1. Division of Nephrology, Departments of Medicine and Cell Biology, Vanderbilt University, 37232, Nashville, Tennessee

    Daniel W. Koenig, Lise Barley-Maloney & Thomas O. Daniel

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  1. Daniel W. Koenig
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  2. Lise Barley-Maloney
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  3. Thomas O. Daniel
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Koenig, D.W., Barley-Maloney, L. & Daniel, T.O. A western blot assay detects autoantibodies to cryptic endothelial antigens in thrombotic microangiopathies. J Clin Immunol 13, 204–211 (1993). https://doi.org/10.1007/BF00919973

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