Differential membrane trafficking of FcγRI and FcγRIIa receptors. FcγRI or FcγRII were cross-linked with mouse anti–human CD64 (antibody 10.1) and/or mouse anti–human CD32 (antibody 3D3), respectively. The secondary monoclonal used for cross-linking the specific mAbs in these experiments was an Alexa Fluor 647–conjugated goat anti–mouse IgG F(ab)′2 (Invitrogen). Receptors were cross-linked for 10 minutes at 37°C to allow internalization. At this time point, cells were fixed and frozen in methanol/acetone (1:1) at −20°C for at least 24 hours before further staining. (A) In the absence of FcR cross-linking, FcγRI and FcγRIIa remain localized at the cell surface. (B) After FcR cross-linking for 10 minutes, (i) both receptors enter a compartment that stains positive for the early endosome marker EEA-1. However, whereas FcγRI enters a compartment that also stains positive for (ii) HLA-DM and (iii) LAMP-1, FcγRIIa enters a compartment that is negative for both these markers of MIIC compartments. Scale bar indicates 20 μm. Results shown are typical of at least 3 separate experiments. Anti–EEA-1 and anti-LAMP1 polyclonal antibodies were purchased from Santa Cruz. Rabbit anti–HLA-DM was provided by Dr Adrian Kelly (Immunology Unit, Department of Pathology, Cambridge University, Cambridge, United Kingdom).
