Antibody-sensitized Red Blood Cells Research Articles

Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fcγ receptor (FcγR)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express FcγRs (e.g., FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA) that are involved in immune responses. The MMA exploits the mechanism of FcγR-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate FcγRs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out FcγR-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo FcγR-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of FcγR-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fcγ receptor (FcγR)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express FcγRs (e.g., FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA) that are involved in immune responses. The MMA exploits the mechanism of FcγR-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate FcγRs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out FcγR-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo FcγR-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of FcγR-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity.

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab')2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti-idiotype interactions are not required.

Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities.

Subclasses of human IgG have a range of activity levels with different effector systems but each triggers at least one mechanism of cell destruction. We are aiming to engineer non-destructive human IgG constant regions for therapeutic applications where depletion of cells bearing the target antigen is undesirable. The attributes required are a lack of killing via Fcgamma receptors (R) and complement but retention of neonatal FcR binding to maintain placental transport and the prolonged half-life of IgG. Eight variants of human IgG constant regions were made with anti-RhD and CD52 specificities. The mutations, in one or two key regions of the CH2 domain, were restricted to incorporation of motifs from other subclasses to minimize potential immunogenicity. IgG2 residues at positions 233 - 236, substituted into IgG1 and IgG4, reduced binding to FcgammaRI by 10(4)-fold and eliminated the human monocyte response to antibody-sensitized red blood cells, resulting in antibodies which blocked the functions of active antibodies. If glycine 236, which is deleted in IgG2, was restored to the IgG1 and IgG4 mutants, low levels of activity were observed. Introduction of the IgG4 residues at positions 327, 330 and 331 of IgG1 and IgG2 had no effect on FcgammaRI binding but caused a small decrease in monocyte triggering.

High doses of intravenous Ig inhibit in vitro uptake of C4 fragments onto sensitized erythrocytes

We have recently reported that intravenous Ig (IVIg) inhibits uptake of activated C3 fragments onto antibody-sensitized red blood cells (RBCs). To elucidate the mechanism by which IVIg exerts its effect on the complement system, we examined the possible interference with the C4 step of the classical complement cascade. We examined the capacity of autologous serum containing high concentrations of human IVIg to deposit C4 fragments onto model targets (guinea pig and/or human erythrocytes sensitized with rabbit anti-guinea pig/human erythrocytes IgG antibody). C4 binding was quantified with radiolabeled anti-C4. Guinea pig serum with added IVIg suppressed C4 uptake onto IgG- sensitized guinea pig erythrocytes at all time points (0, 5, 15, and 30 minutes). Using sera of guinea pigs treated with increasing doses of IVIg, this effect was shown to be dose-responsive. Serum from a patient treated with IVIg showed reduced C4 uptake onto sensitized homologous RBCs. In comparison with the serum from the same patient before IVIg therapy was administered, levels were decreased almost to background. C4 functional titers in those two samples were not different. C3 uptake was studied in parallel with C4 to compare the degree of inhibition using sera with increasing doses of IVIg in both the human and guinea pig system. C3 and C4 inhibition curves completely overlapped. Our findings suggest that IVIg is an effective inhibitor of deposition of early complement activation products (C4b, C3b) onto target surfaces and may indicate interference of IVIg with multiple sites of complement activation.

High Doses of Intravenous Ig Inhibit In Vitro Uptake of C4 Fragments Onto Sensitized Erythrocytes

We have recently reported that intravenous Ig (IVIg) inhibits uptake of activated C3 fragments onto antibody-sensitized red blood cells (RBCs). To elucidate the mechanism by which IVIg exerts its effect on the complement system, we examined the possible interference with the C4 step of the classical complement cascade. We examined the capacity of autologous serum containing high concentrations of human IVIg to deposit C4 fragments onto model targets (guinea pig and/or human erythrocytes sensitized with rabbit anti-guinea pig/human erythrocytes IgG antibody). C4 binding was quantified with radiolabeled anti-C4. Guinea pig serum with added IVIg suppressed C4 uptake onto IgG-sensitized guinea pig erythrocytes at all time points (0, 5, 15, and 30 minutes). Using sera of guinea pigs treated with increasing doses of IVIg, this effect was shown to be dose-responsive. Serum from a patient treated with IVIg showed reduced C4 uptake onto sensitized homologous RBCs. In comparison with the serum from the same patient before IVIg therapy was administered, levels were decreased almost to background. C4 functional titers in those two samples were not different. C3 uptake was studied in parallel with C4 to compare the degree of inhibition using sera with increasing doses of IVIg in both the human and guinea pig system. C3 and C4 inhibition curves completely overlapped. Our findings suggest that IVIg is an effective inhibitor of deposition of early complement activation products (C4b, C3b) onto target surfaces and may indicate interference of IVIg with multiple sites of complement activation.

Peripheral blood lymphocytes with receptors for a determinant common to B-lymphoblastoid cell lines and acute myelogenous leukemia blasts.

We investigated whether the rosetting of B-lymphoblastoid cell lines (B-LCL) by peripheral blood lymphocytes (PBLs) reflected possible interactions between lymphoid cells and immature cells of the hematopoietic system. Rosette formation could be blocked by the addition of soluble antigen extracted from B-LCL or blasts obtained from patients with acute myelogenous leukemia (AML). This inhibition was specific for AML blasts (similarly extracted material from melanoma lines had no inhibitory effect) and for the B-LCL receptor (leukemic extracts had no effect on surface receptors for sheep red blood cells (E) or antibody-sensitized red blood cells (EA)). The B-LCL receptor is present on leukemic Sezary T-cells as well as normal T-cells and its sensitivity to various enzymatic treatments is markedly different from that of E and EA receptors. In addition, B-LCLs derived from in vitro EB-viral infection of a normal donor's B lymphocytes were significantly rosetted by that donor's autologous PBLs. These data suggests the B-LCL receptor, present on mature T-cells, can recognize self determinants on myeloblasts and B-LCL. Further investigation will determine whether this interaction can affect the function of rosetted target cells.