Relative activities of Crry and DAF on mouse erythrocytes and demonstration of the requirement of C3 in the clearance of Crry/DAF/C3^−/− erythrocytes.

(A) Percentage of transfused DAF/C3^−/− (●; n = 4), Crry/C3^−/− (○; n = 4), and Crry/DAF/C3^−/− (▴; n = 4) erythrocytes recovered from age-matched wild-type (C57Bl/6) recipient mice. Erythrocytes were pooled from 6 to 8 donor mice and labeled with biotin, and 3 × 10⁸ cells were transfused into each recipient mouse. (B) Transformation of data from panel A to show the clearance kinetics from 5 minutes (taken as 100%) to 3 days after transfusion. (C) Sensitivity of C3^−/−, DAF/C3^−/−, Crry/C3^−/−, and Crry/DAF/C3^−/− erythrocytes to antibody-induced C3 deposition in vitro (n = 8 mice in each genotype). Cells were opsonized with 34-3C (50 μg/mL) and then treated with mouse serum (1:40). MFI indicates mean fluorescent intensity. Both DAF/C3^−/− and Crry/C3^−/−cells incurred higher C3 deposition than C3^−/− cells (P < .01 and P < .001, respectively, Student t test. MFIs were as follows: for C3^−/−, 1.92 ± 0.28; for DAF/C3^−/−, 3.25 ± 0.29; for Crry/C3^−/−, 4.52 ± 0.26). The difference between Crry/C3^−/− and DAF/C3^−/−cells is also significant (P < .01, Student ttest). However, Crry/DAF/C3^−/− cells incurred much more C3 deposition than either DAF/C3^−/− or Crry/C3^−/− erythrocytes (MFI, 44.60 ± 4.82). Error bars indicate the means ± SE. (D) Elimination of Crry/DAF/C3^−/− erythrocytes in vivo was prevented by C3 deficiency (filled circles; n = 3). Open circles represent results from wild-type recipient mice (n = 4).