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Degree

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo Health Science Campus, Graduate School, 2003.

Abstract

This dissertation examines the interaction of porcine liver macrophages, Kupffer cells, and human erythrocytes. Previous research in our laboratory revealed that when porcine livers were perfused with human blood, porcine Kupffer cells bound and phagocytosed circulating human but not porcine erythrocytes. This binding was independent of antibody or complement opsonization, suggesting direct recognition of human erythrocytes by porcine Kupffer cells. There is substantial evidence that Kupffer cells from other species have germline lectin receptors that specifically recognize xenogeneic carbohydrate epitopes. Thus, our initial hypothesis was: porcine Kupffer cells recognize human erythrocytes via a lectin-carbohydrate interaction. We used a quantitative assay in which 51Cr-labeled erythrocytes bound to porcine Kupffer cells were detected. This assay allowed for the manipulation of the conditions under which the cells were incubated in order to elucidate the underlying mechanism of binding. Various techniques were used to isolate proteins and glycoproteins, such as detergent solubilization, gel chromatography, and affinity chromatography. The latest advances in mass spectrometry were utilized to reveal protein identity through tandem MS sequencing. We provide evidence that N-acetylneuraminic acid, borne on human glycophorin A, is the ligand being recognized by porcine Kupffer cells. This interaction is calcium dependent and can be inhibited with the N-acetylneuraminic acid monosaccharide or neuraminyl lacotses bearing α-2,3 and α-2,6 linkage. We also have evidence to suggest that the lectin annexin IV on the surface of porcine Kupffer cells is responsible at least in part, for the recognition of human erythrocytes. These findings support our initial hypothesis that a lectin-carbohydrate interaction is responsible for the recognition of xenogeneic human erythrocytes by porcine Kupffer cells. We propose that these data could be used to improve the performance of extracorporeal xenoperfusion. More importantly, these data support a new hypothesis that innate immune cellular recognition of xenogeneic carbohydrates by lectin receptors plays an important role in xenograft rejection.

Keywords

Xenotransplantation; Kupffer Cell; Lectin; Erythrocyte; N-Acetylneuramimic Acid

Advisor

Michael A. Rees, M.D

Pages

135p.

Document number: mco1083268448
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=mco1083268448

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