An in vitro homologous antibody-dependent cell-mediated cytolytic (ADCC) assay system was developed using human peripheral blood mononuclear cells, blood group 0 Rhesus (D) positive red blood cells and anti-11 antibodies. Extensive studies were carried out using monocyte-depleted cultures, where the lymphoid K-cell is the putative cyglytic effector cell, and the degree of specific lysis estimated from Cr release from damaged RBC. Enhanced lysis was obtained when RBC were treated with the proteolytic enzyme papain. The coefficients of variation of replicates processed in an identical manner were less than 10%. The degree of specific lysis of anti-D sensitised D-positive RBC depended upon the number of effector cells present, the number of RBC, and the amount of antibody. Under appropriate conditions, lysis was observed with only 3 ng anti-D per culture. The specificity of lysis resided with the appropriate combination of antibody and antigen (is D-anti-D) and not with the effector cells. Donors of all ABO Rh groups were equally effective donors of K-cells and under appropriate conditions autologous RBC were lysed. The degree of specific lysis was influenced by the D-antigen dosage of RBC and the potential for determining the D-zygosity of RBC was explored. Time-course experiments showed that RBC lysis was measurable by 30 minutes incubation, and was approximately linear over 18 hrs, at which time maximum lysis was seen. Cell-to-cell contact was essential for lysis and the cytolytic process was extremely short-range with no "innocent bystander" effect being seen. Phagocytosis was not necessary for cytolysis to be observed, but effector cell mobility and intact microfilament function was required (inhibition by cytochalasin B). The divalent cations Ca and Mg were required for efficient itBC lysis. Studies with metabolic inhibitors showed that a metabolically active cell is required for K-cell activity, that both RNA and protein synthesis are necessary for maximum lysis, and that an intact microtubule system is also required. The trigger to lysis appears to be the activated Fe component of red cell-bound anti-D which interacts with the K-cell Fc receptor. Lysis was mediated by IgG antibodies and was significantly inhibited immbulins IgG. This suggests that the K-cell Fe receptor by specific for IIgG but that IgG1 and IgG3 cross-react. Application of the K-cell assay to normal individuals reveals that there was inherent biological variation between individuals and that there appeared to be 'good' and 'poor' K-cell donors in terms of cytolytic activity. K-cells were present in the cord blood of neonates and were slightly less active than adult cells. Adrenaline infusion produced a transient rise in K-cell activity at 15-30 minutes and infusion of D positive RHC into a D negative individual resulted in a transient fall at 12-14 days followed by enhanced lytic potential some weeks later. Preliminary studies into the biological activities of different sources of anti-D revealed that there was poor correlation between agglutination titre and specific RBC lysis. The potential applications of the K-cell assay system are discussed. The K-cell has the morphologic appearance of a small lymphoid cell, does not appear to have surface immunoglobulin or sheep RHC (R) receptors but does have Fe and C3 receptors. The cell is non-adherent, non-phagocytic and negative for the non-specific esterase stain. It therefore does not have any of the characteristics of a mature monocyte, but does have monocyte-specific surface antigens as demonstrated by anti-monocyte serum. It may therefore be a monocyte precursor.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663152 |
| Date | January 1978 |
| Creators | Urbaniak, S. J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/18668 |
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