Macrophage Recognition of Xenogeneic Erythrocytes
Background: Our laboratory studies how porcine macrophages bind human erythrocytes. This recognition event is the result of the efforts contributing to the development of an extracorporeal porcine liver perfusion apparatus serving as a potential therapy for patients in end-stage liver failure. Preliminary data collected indicates the two porcine macrophage lectins Annexin IV and sialoadhesin will play a role in mediating porcine macrophage recognition of human erythrocytes. Porcine macrophage recognition of non-human primate erythrocytes will also be investigated to determine if porcine macrophages recognize all xenogeneic erythrocytes or recognition is unique to human erythrocytes perhaps providing additional information for studying porcine macrophage recognition of human erythrocytes. Methods: All porcine cells used in the discussed experiments are primary cells. Binding experiments were completed with the 51Cr binding assay. Stable cell lines expressing either sialoadhesin (pSn) or a sialoadhesin mutant (the sialic acid binding domain is disrupted, pSnRE) were tested for their ability to bind human erythrocytes. The full-length gene of Annexin IV was obtained by building a consensus sequence using porcine expressed sequence tags (EST) that have high homology with human Annexin IV. This consensus sequence containing a FLAG tag was then PCR amplified and subcloned into the pcDNA3.1(+) vector. Porcine macrophage recognition of non-human primate erythrocytes was tested using the 51Cr binding assay and immunohistochemistry. Results: Porcine sialoadhesin was identified as a macrophage lectin that binds human erythrocytes. The porcine Annexin IV gene sequence was determined and was 95.4% homologous to human Annexin IV. Porcine macrophages did not bind other non-human primate erythrocytes (gorilla, baboon, and chimpanzee). Conclusion: Progress was made in studying porcine macrophage recognition of human erythrocytes. These observations suggest the testing of the ability of the monoclonal antibody anti-porcine sialoadhesin to block porcine Kupffer cell recognition of human erythrocytes in the extracorporeal porcine liver perfusion model. Expression of the prepared plasmid pANXIVfg-pcDNA3.1+ will answer the question of whether or not porcine Annexin IV will contribute to porcine macrophage recognition of human erythrocytes. Preliminary data indicates that porcine macrophage recognition of human erythrocytes is specific to humans.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:innate immunology xenotransplantation macrophages erythrocytes lectins sialic acid
Date of Publication:01/01/2007