Figure 2.

ADCP-induced hypophagia does not reflect a global change in macrophage phagocytic capacity. (A) BMDMs were cocultured with thymocytes ± anti-CD90.2 for 2 hours. After washing to remove free thymocytes and mAb, BMDMs were rechallenged for 2 hours with apoptotic thymocytes, and the phagocytic index was calculated using void detection as in Figure 1E. Data shown are mean ± SEM (n = 5 experiments). (B) BMDM phagocytosis of mAb-opsonized and apoptotic thymocytes, as described in panel A. Data shown are mean ± SEM (n = 5; P values derived from unpaired 1-way analysis of variance [ANOVA] with multiple comparisons correction). (C) BMDMs were initially cocultured ± αCD90.2-opsonized thymocytes followed by washing and rechallenged with αCD90.2-opsonized thymocytes and total BMDM lysates analyzed for levels of phosphorylated Syk (pSyk, Y525/Y526) and total Syk by western blotting. Top panel shows representative western blot image. Lower graph shows densitometry quantification of the pSyk/Syk ratio comparing unfed and hypophagic BMDMs. Data shown are mean ± SEM (n = 4; P values derived from unpaired 2-tailed Student t test). (D) BMDMs were initially challenged with indicated mAb/target cells for 2 hours and, after washing, subsequently rechallenged with the indicated mAb/target cells for 2 hours, and relative phagocytosis was calculated. Data shown are mean ± SEM (n = 4; P values derived from unpaired 1-way ANOVA with multiple comparisons correction). (E) Relative messenger RNA (mRNA) levels for FcγRs and common γ chain (Fcer1g) were measured by quantitative reverse transcription polymerase chain reaction from BMDMs incubated with αCD90.2-opsonized targets for the indicated times. Data shown are mean ± SEM (n = 6). Unpaired 1-way ANOVA with multiple comparisons correction revealed no significant differences in Fcγ expression. *P < .05, **P < .01, ***P < .001. ns, not significant.