Complement activation selectively potentiates the pathogenicity of the IgG2b and IgG3 isotypes of a high affinity anti-erythrocyte autoantibody

Abstract

By generating four IgG isotype-switch variants of the high affinity 34-3C anti-erythrocyte autoantibody, and comparing them to the IgG variants of the low affinity 4C8 anti-erythrocyte autoantibody that we have previously studied, we evaluated in this study how high affinity binding to erythrocytes influences the pathogenicity of each IgG isotype in relation to the respective contributions of Fcgamma receptor (FcgammaR) and complement. The 34-3C autoantibody opsonizing extensively circulating erythrocytes efficiently activated complement in vivo (IgG2a = IgG2b > IgG3), except for the IgG1 isotype, while the 4C8 IgG autoantibody failed to activate complement. The pathogenicity of the 34-3C autoantibody of IgG2b and IgG3 isotypes was dramatically higher (>200-fold) than that of the corresponding isotypes of the 4C8 antibody. This enhanced activity was highly (IgG2b) or totally (IgG3) dependent on complement. In contrast, erythrocyte-binding affinities only played a minor role in in vivo hemolytic activities of the IgG1 and IgG2a isotypes of 34-3C and 4C8 antibodies, where complement was not or only partially involved, respectively. The remarkably different capacities of four different IgG isotypes of low and high affinity anti-erythrocyte autoantibodies to activate FcgammaR-bearing effector cells and complement in vivo demonstrate the role of autoantibody affinity maturation and of IgG isotype switching in autoantibody-mediated pathology.

Figures

Figure 1.

Flow cytometric analysis of complement activation in vivo by the 34–3C and 4C8 IgG class-switch variants. Mouse RBCs were obtained 24 h after an intraperitoneal injection of 50 or 200 μg of 34–3C or 4C8 IgG variants into mice, and then stained with biotinylated goat anti–mouse C3 antibodies, followed by PE-conjugated streptavidin. Results obtained with 50 μg (thick lines) and 200 μg (thin lines) of the 34–3C IgG2a, IgG2b, and IgG3 variants in BALB/c mice, 200 μg of the 34–3C IgG3 variant in C1q−/− mice, and 200 μg of the 34–3C IgG1 or 4C8 IgG2a variant in BALB/c mice are shown. Shaded areas indicate the background staining with PE-conjugated streptavidin.

Figure 2.

Development of anemia induced by the 34–3C IgG class-switch variants in mice. BALB/c mice were injected intraperitoneally with 200 μg of purified 34–3C IgG variants (IgG1, ○; IgG2a, •; IgG2b, □; IgG3, ▴) on day 0. Results are expressed as mean Ht values of five mice.

Figure 3.

Development of anemia in WT, C3−/−, FcRγ2/−, or FcRγ/C3−/− mice after the injection of the 34–3C IgG2a mAb. Ht values of individual mice measured 4 d after the intraperitoneal injection of 50 or 200 μg of the mAb are shown.

Figure 4.

Development of anemia in WT, C3−/−, FcRγ2/−, or FcRγ/C3−/− mice after the injection of the 34–3C IgG2b variant. Ht values of individual mice measured 4 d after the intraperitoneal injection of 50 or 200 μg of the mAb are shown.

Figure 5.

Development of anemia in WT, C3−/−, or FcRγ2/− mice after the injection of 34–3C IgG1 or IgG3 variant. Ht values of individual mice measured 4 d after the intraperitoneal injection of 500 μg of 34–3C IgG1 or 200 μg of 34–3C IgG3 mAb are shown.