Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Basic and Clinical Immunology and Microbiology


College of Graduate Studies

First Advisor

H. Hugh Fudenberg

Second Advisor

R. E. Lovins

Third Advisor

R. M. Gailbraith

Fourth Advisor

P. A. Arnaud

Fifth Advisor

J. S. Graves

Sixth Advisor

H. T. Jonsson


Human erythrocytes (RBC) from whole blood were separated according to their specific densities by centrifugation on a polyvinyl-pyrrolidine-coated colloidal silica matrix (Percoll) into four major subpopulations. By indirect immunofluorescence array, the most dense RBC subpopulation with specific density >1.110 g/ml (3-5% of total RBC), was positive for membrane-bound immunoglobulin; the remaining, less dense subpopulations were negative. IgG was present on 85-95%, IgM on 28-32%, and IgA on 15-20% of the RBC in the most dense subpopulation. When these immunoglobulins were radiolabeled in situ, eluted, and used in binding studies with autologous RBC fractions subjected to thermal and/or enzymatic treatment with Vibrio cholera neuraminidase, specific binding was observed with heat-treated cells from the most dense (older), and neuraminidase-treated cells from the less dense (younger) RBC subpopulations. These results suggest that previously cryptic antigenic determinants, revealed by the activity of neuraminidase on the plasma membranes of younger RBC, are responsible for the binding of immunoglobulins to older human RBC. Autologous membrane-bound IgG was isolated from human RBC comprising a subpopulation with specific density >1.110 by affinity chromatography of purified RBC membrane glycoprotein preparations, using immobilized-wheat germ agglutinin and immobilized anti-human immunoglobulin, as immunosorbents. The immunoglobulin-containing fractions thus obtained, when further separated by chromatography on Sephadex G-200 in the presence of chaotropic agents, yielded four peaks (Ia, Ib, II, and III). Double-immunodiffusion revealed the presence of immunoglobulins in the first three peaks (IgM in peak Ia, IgA in Ib, and IgB in II), but not in peak III. Peak III was precipitated by the immunoglobulin-containing peaks (Ia, Ib, and II) in immunodiffusion assays, suggesting that the determinants responsible for the binding of autologous immunoglobulin to senescent human RBC were contained in this peak (III). Peaks Ia, Ib, and II were shown to precipitate purified asialoglycophorin, and peak III was reactive with purified autoantibodies directed against asialoglycophorin. These results suggest that the age-related antigenic determinants present on senescent human RBC are revealed by desialylation of the major RBC membrane sialoglycoprotein, and are therefore, similar or identical to the Thomsen-Friedenreich (T) antigen. Human RBC subpopulations, obtained after gradient density centrifugation and thermal and/or enzymatic treatment, were divided into three groups according to the presence of membrane-bound IgG and the expression of age-related antigenic determinants (T antigen) responsible for the binding of the IgG. The effect of membrane-bound anti-T autoantibodies on erythrophagocytosis by autologous macrophages was assessed in studies using each of the RBC subpopulations in reaction mixtures containing anti-T depleted, unfractionated (whole), or no serum. Similar phagocytosis assays were performed after pre-incubation of RBC samples with specifically labeled anti-T autoantibodies or their F(ab’)[subscript 2] fragments previously isolated from autologous senescent RBC. The results of these studies indicate that the selective phagocytosis of senescent, rather than younger RBC, but autologous macrophages is mediated by the reaction of macrophage Fc receptors with the Fc portion of anti-T autoantibodies specifically bound to age-related determinants present on the surface of senescent human RBC.


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