Differences in expression density and molecular weight of CR1-Like in erythrocytes of landrace swine

Background. Porcine erythrocytes express complement receptor 1-like (CR1-like), which is involved in immune adherence. Methods. In this study, porcine erythrocyte samples were collected from fifty-five individual Landrace swine to characterize differences in porcine CR1-like. Flow cytometry analysis was performed to examine the porcine differences in CR1-like expression density and immunoprecipitation, SDS-PAGE and Western blot were performed to detect variations in porcine CR1-like molecular weights. Results. Different mean fluorescence intensities (MFI) of porcine erythrocytes were identified in three groups as 33.016±2.889 (40.0%), 59.974±9.299 (45.5%) and 131.241±8.375 (14.5%). Under reduced condition, three porcine CR1-like molecular weight variants were identified as 85.280±0.935 kDa (9.09%), 123.939±2.752 kDa (14.55%) and 136.696±2.028 kDa (76.36%). Discussion. CR1-like was dispersed on the surface of porcine erythrocytes and promoted immune adherence. There have been no reports on whether differences in the expression levels and/or molecular weights of CR1-like in erythrocytes represent diversity in different individuals, and if so, whether this diversity influences the immune adherence of erythrocytes and/or whether the diversity is associated with CR1-like polymorphisms. At present, five candidate genes that are related to the differences above were found. Research examining erythrocyte immune adherence and CR1-like genes is under way. These results will provide theoretical data for future studies of the immunological mechanism of CR1-like in porcine erythrocytes.

Differences in expression density and molecular weight of CR1-Like in erythrocytes of landrace swine

Background. Porcine erythrocytes express complement receptor 1-like (CR1-like), which is involved in immune adherence. Methods. In this study, porcine erythrocyte samples were collected from fifty-five individual Landrace swine to characterize differences in porcine CR1-like. Flow cytometry analysis was performed to examine the porcine differences in CR1-like expression density and immunoprecipitation, SDS-PAGE and Western blot were performed to detect variations in porcine CR1-like molecular weights. Results. Different mean fluorescence intensities (MFI) of porcine erythrocytes were identified in three groups as 33.016±2.889 (40.0%), 59.974±9.299 (45.5%) and 131.241±8.375 (14.5%). Under reduced condition, three porcine CR1-like molecular weight variants were identified as 85.280±0.935 kDa (9.09%), 123.939±2.752 kDa (14.55%) and 136.696±2.028 kDa (76.36%). Discussion. CR1-like was dispersed on the surface of porcine erythrocytes and promoted immune adherence. There have been no reports on whether differences in the expression levels and/or molecular weights of CR1-like in erythrocytes represent diversity in different individuals, and if so, whether this diversity influences the immune adherence of erythrocytes and/or whether the diversity is associated with CR1-like polymorphisms. At present, five candidate genes that are related to the differences above were found. Research examining erythrocyte immune adherence and CR1-like genes is under way. These results will provide theoretical data for future studies of the immunological mechanism of CR1-like in porcine erythrocytes.

RBC Adhesive Capacity Is Essential for Efficient 'Immune Adherence Clearance' and Provide a Generic Target to Deplete Pathogens from Septic Patients

Sepsis is a life threatening organ dysfunction caused by a dysregulated host response to infection. It is a global health care problem and the leading cause of death in ICU patients. A rather unknown but effective mechanism to protect ourselves from such pathogen invasion is immune-adherence clearance (IAC). During IAC pathogens (including bacteria, fungi and viruses) can bind to red blood cells (RBCs) through complement opsonization and subsequent binding to complement receptor 1 (CR1, CD35) expressed on their cell surface. After binding of the pathogen to CR1, the RBCs deliver the pathogens to macrophages of the spleen and liver, where they are phagocytosed and degraded. To study the underlying mechanisms of IAC in more detail, we have developed an assay to monitor the transfer of opsonized pathogens bound to RBCs to human monocytes and/or macrophages under flow conditions, using confocal microscopy. The transfer of a variety of different pathogens, including S. aureus, E. coli and C. albicans, was studied in this assay. When looking at the transfer process in detail, it was noticed that RBC attach to the phagocyte prior to the transfer and remain attached shortly after the transfer, suggesting that RBC might be interacting with phagocytes through adhesion molecules such as integrins to establish firm binding between these two cell types. To test this hypothesis several adhesion molecules, on the phagocytes as well as on the RBC, were blocked by monoclonal antibodies and IAC was quantified. First, complement receptor 3 (CR3, amb2 CD11b/CD18 integrin) an important receptor for adhesion, migration and phagocytosis was blocked by monoclonal antibodies on the phagocytes, after which bacterial transfer was impaired. This indicated that CR3 is crucial for efficient IAC. To confirm the involvement of CR3 in IAC, we analyzed the monocytes of a known LAD-1 patient. This rare immunodeficiency is characterized by an inherited molecular defect of the β2integrin subunit (CD18) which results in impaired adhesion and migration of the patient's leukocytes and clinically manifests in recurrent infections. Our results showed a highly reduced interaction between the phagocytes and the RBCs, resulting in a decrease in bacterial transfer of >70%. Next, we tested the effects of blocking antibodies directed against several RBC adhesion molecules on the transfer process. When blocking CD147 (Basigin, Ok blood group) and ICAM-4 (CD242, Landsteiner-Wiener), but not Glycophorin A (GPA, CD235a) on the RBC the transfer process was largely inhibited. These findings demonstrate the involvement of direct cell-cell interactions between RBC and macrophages in IAC and provide evidence that RBC adopt a "sticky" phenotype after binding a pathogen through CR1, which enables phagocytes to bind them under flow. We anticipated that blood bank filters (currently used for leukocyte depletion) might be used to bind and filter RBC-pathogen complexes from blood due to their "sticky" phenotype. This was found to be possible in a series of experiments, not only using in vitrogenerated RBC-pathogen complexes, but also using blood from septic patients. Filtration using a standard leukocyte reduction filter showed reduction of RBC-pathogen complexes close to 100%, independent of the type of pathogen. We foresee that this knowledge can be used to develop a generic approach to deplete RBCs carrying pathogens specifically from the blood stream, and thereby filter RBC-pathogen complexes from the blood of patients suffering from sepsis. Disclosures No relevant conflicts of interest to declare.