Figure 4.

Selective internalization and proteasomal degradation of FcγRI in hypophagic macrophages. (A) BMDMs were cocultured with thymocytes ± αCD90.2 for 2 hours to induce hypophagia and analyzed by intracellular staining and flow cytometry to measure surface and total (surface + intracellular) levels of FcγRI. Data shown are mean ± standard error of the mean (SEM; n = 3; P values derived from unpaired 1-way analysis of variance [ANOVA]). (B) BMDMs were treated as in panel A and analyzed for FcγRIIb levels. Data shown are mean ± SEM (n = 3; P values derived from unpaired 1-way ANOVA). (C) Total levels of FcγRI were measured by western blotting of total lysates from BMDMs cocultured with thymocytes ± αCD90.2 (ADCP) for the indicated times. Pretreatment with bafilomycin A1 (Baf; 100 nM) and MG132 (MG; 25 μM) for 3 hours before addition of mAb. Representative western blot image (top) and densitometry quantification of FcγRI levels (normalized to total Syk) are shown below. Data shown are mean ± SEM (n = 4; P values derived from unpaired 1-way ANOVA with multiple comparisons correction). (D) Total levels of FcγRIIb were analyzed as in panel C. Representative western blot image (top) and densitometry quantification of FcγRIIb levels (normalized to total Syk) are shown below. Data shown are mean ± SEM (n = 4; P values derived from unpaired 1-way ANOVA with multiple comparisons correction). (E) BMDMs were pretreated for 3 hours ± 25 μM of MG132 before coculture with thymocytes ± αCD90.2 for 2 hours to induce hypophagia. FcγRI was immunoprecipitated from BMDM total lysates and analyzed by western blotting with indicated Abs. Representative blot of 5 experiments is shown. Arrow indicates FcγRI band. (F) ADCP assay was performed on BMDMs pretreated ± 25 μM of MG132 for 3 hours before addition of thymocytes and αCD90.2 as described in Figure 1E. Relative phagocytosis quantification for both the initial and secondary challenges is shown in lower graphs (n = 4). Data shown are mean ± SEM (P values derived from unpaired 2-tailed Student t test [initial challenge] and unpaired 1-way ANOVA with multiple comparisons correction [secondary challenge]). (G) BMDMs were induced to undergo hypophagia for 2 hours, washed, and cultured for 24 hours ± recombinant IFN-γ (50 ng/mL) or C5a (50 ng/mL) before rechallenge with thymocytes ± αCD90.2 for 4 hours, and ADCP was measured by live-cell imaging as in Figure 1E. Representative data from 1 of 3 independent experiments are shown. (H) Quantification of ADCP rechallenge experiments in panel G. Data shown are mean ± SEM (n = 3; P values derived from unpaired 1-way ANOVA with multiple comparisons correction). (I) Surface levels of FcγRI on BMDMs from experiments in panels G and H were analyzed by flow cytometry. Data shown are mean ± SEM (n = 3; P values derived from unpaired 1-way ANOVA with multiple comparisons correction). (J) Surface levels of FcγRIIb on BMDMs from experiments in panels G and H were analyzed by flow cytometry. Data shown are mean ± SEM (n = 3; P values derived from unpaired 1-way ANOVA with multiple comparisons correction; differences observed between groups were not significant [ns]). *P < .05, **P < .01, ***P < .001, ****P < .0001. MFI, mean fluorescence intensity.