A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis

Abstract

Macrophages are specialized cells of the immune system that exhibit a prodigious capacity for phagocytosis. The ability of macrophages to internalize a substantial proportion of their plasma membrane during phagocytosis indicates that they possess a mechanism for the rapid renewal of plasma membrane. We examined the role of endocytic membrane recycling in promoting phagocytosis. In contrast to many other cell types, macrophages lack a morphologically distinct peri-centriolar recycling compartment but instead demonstrate an extensive network of transferrin receptor-positive tubules and vesicles that participated in recycling. The rate of transferrin recycling in thioglycollate-elicited murine peritoneal macrophages (thio-macrophages) was exceedingly rapid, with exocytic rate constants that were 2- to 3-fold higher than those of most other cells. Because the GTPase Rab11 has been implicated in transferrin recycling in other cells, we determined its role in transferrin recycling and phagocytosis in macrophages. Macrophages expressing epitope-tagged Rab11 demonstrated the presence of Rab11 in several intracellular membrane compartments, including endosomes and nascent phagosomes. Expression of Rab11 25N, a GTP binding-deficient allele of Rab11, led to a decreased rate of transferrin efflux and impaired Fc(gamma)R-mediated phagocytosis, where Fc(gamma)R is the receptor for the Fc portion of IgG. In contrast, expression of Rab11 70L, a GTPase-deficient allele of Rab11, led to an increased rate of transferrin efflux and enhanced phagocytosis. We conclude that macrophages have adapted a rapidly mobilizable, endocytic compartment to enhance phagocytosis. Rab11 participates in the recruitment of this compartment to the macrophage cell surface.

Figure 1

Macrophages lack a morphologically distinct peri-centriolar recycling compartment and efflux rhodamine-transferrin at a rapid rate. (A) Confocal fluorescence micrographs of the indicated cells incubated with biotin-transferrin for 1 hr at 37°C, followed by fixation and staining for biotin-transferrin (Right) using rhodamine-streptavidin, or transferrin receptor (Left) using mAb C2 followed by fluorescein-conjugated anti-rat IgG. Data are representative of three similar experiments. (Bar = 10 μm.) (B) Recycling of rhodamine-transferrin (Tfn). After acid washing to remove surface-bound rhodamine-transferrin, cell-associated rhodamine-transferrin is expressed as percent of initial rhodamine-transferrin present at time t = 0. Data points represent mean ± SEM (n = 3–4).

Figure 2

Rab11 localizes to endocytic tubules and vesicles in macrophages. Adherent RAW LR/FMLPR.2 cells were transfected with HA-Rab11 WT. After fixation, ultrathin cryosections were labeled with monoclonal anti-HA followed by rabbit anti-mouse IgG and anti-rabbit-10 nm gold. An area of a cell showing Rab11-labeled tubular cisternae and vesicles (large arrows) underneath the cell membrane; a short ruffle also is labeled (small arrows) on the membrane and in the cytosol. (Bar = 200 nm.)

Figure 3

Localization of transfected Rab11 alleles in RAW LR/FMLPR.2 cells. Adherent macrophages transfected with HA-tagged constructs as indicated were fixed and stained for transferrin (Tfn) receptor with mAb C2 followed by biotin anti-rat IgG and Alexa 532-streptavidin, and for HA with mAb HA.11 followed by Alexa 488-anti-mouse IgG. Confocal fluorescence micrographs are representative of five similar experiments. (Bar = 10 μm.)

Figure 4

Transferrin receptors are associated with nascent phagosomes. Adherent RAW LR/FMLPR.2 cells were incubated with EIgG for 15 min at 4°C to allow binding, but not ingestion, then incubated at 37°C for 2 min before fixation and staining for EIgG with Cy5-conjugated anti-rabbit IgG and for transferrin receptor with mAb C2 followed by biotin anti-rat IgG and Alexa 532-streptavidin. (A) Phase-contrast. (B) EIgG. (C) Transferrin receptor. Micrographs are representative of four similar experiments. (Bar = 10 μm.)

Figure 5

Rab11 localizes to regions of the cell engaging in phagocytosis. Adherent RAW LR/FMLPR.2 cells transfected with HA-Rab11 WT were incubated with EIgG for 4 min. After fixation, ultrathin cryosections were labeled with monoclonal anti-HA followed by rabbit anti-mouse IgG and anti-rabbit-10 nm gold. An area of a macrophage in contact with two erythrocytes (E) showing labeling on filopodia (f). Vacuoles (v) and an invagination of the plasma membrane (i) are labeled on the membrane. (Bar = 300 nm.)

Figure 6

Expression of Rab11 mutants alters the rate and extent of phagocytosis of EIgG. (A) Adherent RAW LR/FMLPR.2 cells transfected with the indicated constructs were incubated with EIgG at 4°C and the extent of binding was determined. (B) In parallel cultures, macrophages preincubated with EIgG at 4°C were incubated for varying times at 37°C to monitor the rate of phagocytosis in cells expressing either HA-Rab11 25N (■), HA-Rab11 70L (▴), or nonexpressing controls (●). Numbers in parentheses refer to percent of cell-associated EIgG ingested by 30 min. Data points represent mean ± SEM (n = 3). (C) Unsynchronized phagocytosis of EIgG was monitored in RAW LR/FMLPR.2 cells transfected with the indicated constructs. Percent phagocytosis of expressing cells compared with nonexpressing controls represents mean ± SEM (n = 3).