Antibody specific for the glycophorin A complex mediates intravenous immune globulin–resistant anemia in a murine model (1)
Antibodies to red blood cell (RBC) antigens can cause immune RBC destruction, resulting in hemolytic transfusion reactions or autoimmune hemolytic disease, such as autoimmune hemolytic anemia (AHA). The classical paradigm of antibody-mediated RBC clearance in AHA involves macrophage Fcg receptor (FcgR)/complement receptor (CR)-mediated phagocytosis (extravascular hemolysis), complement-induced direct lysis (intravascular hemolysis), or a combination of both. In addition glycophorin A (GPA) antibody may have caused hemolysis via mechanism(s) potentially independent of comple- ment or macrophage FcgR. GPA is one of the most abun- dant integral proteins in the RBC membrane, present at an estimated 1 million copies per cell, and is the major con- tributor to the RBC’s negative charge due to its high sialic acid content.
TER119 (45 mg/mouse) was found to bind efficiently to circulating RBCs and cause increasing anemia with a nadir at 4 days postinjection, followed by recovery over the next 4 days. An acute episode of hemoglobinuria was observed in the first day postinjection.
The antibody induced marked splenomegaly, and the increase in spleen weight was inversely related to the decrease in RBC counts over the first 4 days. Histologic analysis confirmed the accumulation of RBCs in the spleen observable by 30 minutes after antibody injection. Interestingly, the development of anemia was partially but significantly delayed in the splenectomized mice. However, the splenectomized mice developed a similar severity of anemia on Day 4 and experienced delayed recovery of circulating RBC compared to their normal littermates.
The degree of RBC destruction in the Fcg-/- or C3-/- mice was not found to be significantly different from C57BL/6 mice, as all mice experienced anemia to a similar degree 24 hours postinjection. Because hemoglobinuria was still detected in the C3-/- mice. DBA/2 mice which is C5-deficient mice also developed a similar degree of anemia compared to C57BL/6 and BALB/c mice.
The washed RBC with the TER119 antibody were incubated at increasing concentrations in vitro for 1 hour. Flow cytometric RBC counting exhibited a dose-dependent reduction in the number of RBCs detected. Microagglutinates were clearly visible in mice 30 minutes postinjection of TER119 and persisted in these mice at 24 hours postinjection. Individual RBCs appeared to have visible variation in sizes on the first 2 days postinjection and polychromasia observed at the later stages of the anemia.
Very-high-dose Intravenous immune globulin regime was effective in significantly ameliorating the anemia caused by a mouse anti-Band 3 that has previously been shown to cause FcgR-dependent erythrophagocytosis. The same regime of IVIG treatment did not significantly affect the induction of anemia in mice treated with either high dose or low dose of the TER119 antibody, but was associated with marginally improved recovery in mice treated with low-dose TER119.
In this mouse model of IHA, that efficacy of IVIG was not apparent with an FcgR-independent GPA complex-specific antibody ( TER119) in the development of anemia. While high-dose IVIG therapy successfully improved RBC counts in mice treated with an anti-Band 3, which has been shown to cause FcgR-mediated eryth- rophagocytosis as the major pathologic mechanism. Anti-GPA cold agglutinins that also seemed to cause severe hemolysis independent of the classical macrophage and/or complement mechanisms. TER119 does not behave like a typical cold agglutinin; in fact, agglutination was more efficient at 37°C than at room temperature or at 4°C, suggesting that this antibody is more consistent with a “warm agglutinin.” In addition, unlike cold agglutinin-mediated hemolytic anemia in which the major mechanism of hemolysis is considered to be complement dependent.