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Article Figures & Data

Figures

  • Figure 1.

    Expression of FcRn in human blood monocytes, NK cells, and PMNs. (A) FcRn mRNA and protein levels in freshly isolated human monocytes, NK cells, and PMNs as measured by real-time quantitative RT-PCR (AU defined as a ratio of ABL expression) and flow cytometry (geometric mean fluorescence). FcRn expression in cells was detected by mAb 22H4C11 in fixed and permeabilized cells. (B) FcRn is localized intracellularly in resting PMNs. Freshly isolated PMNs or paraformaldehyde-fixed and saponin-treated PMNs were stained with mouse anti-FcRn mAb 1G3 and measured by FACS to detect extracellular and intracellular FcRn levels, respectively. (A-B) Data are presented as means plus standard deviations. Data are representative of at least 5 experiments.

  • Figure 2.

    Localization of FcRn within PMNs. FcRn expression in human granulocytes was analyzed by costaining with (A) myeloperoxidase (10-nm gold particles) or (B) lactoferrin (15-nm gold particles) using transmission electron microscopy. FcRn is visualized as 15-nm (A) or 10-nm (B) particles (white arrows). N indicates nucleus; PM, plasma membrane.

  • Figure 3.

    Colocalization of FcRn and bacteria in PMNs. (A) Paraformaldehyde-fixed and saponin-treated PMNs after phagocytosis of Alexa488-labeled IgG-opsonized pneumococci (green) were analyzed through middle sections of cells by confocal microscopy. FcRn was visualized with a rabbit anti-FcRn antiserum (red). Bacteria, FcRn staining, and overlay are shown from left to right, respectively. (B) Fluorescence intensity (FI) of corresponding color channels shown in panel A were analyzed for whole cells showing enhanced localization of FcRn around phagosomes. In the rightmost panel, FI of FcR (red) and pneumococcal (green) are overlaid. (C) FI of pneumococci (green) and FcRn (red) across the cell along the line indicated (A, right; scale in arbitrary units). Experiments were repeated 3 times, yielding similar results.

  • Figure 4.

    FcRn facilitates PMN internalization of IgG-opsonized bacteria. (A) Antigen-binding capacity of a WT IgG1 mAb (GDob1) and its H435A IgG1 derivative to polysaccharide was indistinguishable by ELISA. IgG1 concentrations were quantified by anti-kappa capturing, and anti-Fc detection sandwich ELISA. Equal amounts of IgG were subsequently allowed to bind to pneumococcal polysaccharide 6B-coated plates, and bound IgG was detected with anti-kappa antisera. (B) Alexa488-labeled pneumococci (green) were ingested efficiently by human PMNs when incubated with WT IgG1 but not on incubation with the H435A IgG1 derivative. PMNs were visualized by red membrane staining using MAC-1 mAb (CR3/CD11b). (C) Binding (4°C) and phagocytosis (37°C) of IgG-opsonized FITC-labeled Streptococcus pneumoniae serogroup 6B measured by FACS on incubation with human PMNs in the absence (no IgG) or presence of 5 μg/mL human IgG1 (IgG1) or a mutated H435A IgG1 (H435A IgG1) variant. For evaluation of ingested bacteria (□), fluorescence of PMNs was measured after quenching of extracellular bacteria. Data are represented as phagocytic index (PI).15 (D) Ingestion of FITC-labeled pneumococci incubated at 37°C with PMNs from WT, β2M-, and FcRn-knockout mice in the presence of IgG1. Fluorescence of bound but not ingested bacteria were quenched as described in “Phagocytosis experiments.” NS indicates not significant; *P < .05 when compared with WT. Data are presented as means plus standard deviations from 2 (A) or 4 (D) experiments, respectively. Experiments (B-C) were performed 3 times, yielding similar results.

  • Figure 5.

    Effect of pH on IgG1-mediated phagocytosis. (A) The V-ATPase inhibitor chloroquine and the weak base NH4Cl inhibit phagocytosis but have no effect on association of IgG1-osonized pneumococci to PMNs. (B) Phagocytosis is increased in medium of pH 6.0 but has no effect on association of IgG1-opsonized pneumococci. **P < .01 when compared with WT by ANOVA. Data represent means plus standard deviations and are representative of at least 3 individual experiments.

  • Figure 6.

    Recycling of IgG is not responsible for enhanced phagocytosis mediated by FcRn. (A) Effect of IgG level on phagocytosis of pneumococci by wild-type and β2M-/- mouse PMNs. Note that phagocytosis by β2M-/- PMNs is more impaired at higher concentrations of human IgG1. (B) Recycling of human IgG1 by wild-type and β2M-/- mouse PMNs. Wild-type mouse PMNs and β2M-/- PMNs were allowed to take up human IgG1 antiserotype 6B mAb (10 μg/mL) for 5 minutes at 37°C in the presence of serotype 6B pneumococci. After extensive washing, cells were incubated for an additional 30 minutes in medium to allow for exocytosis/recycling of IgG, and supernatants were subsequently analyzed for the presence of human anti-6B pneumococcal antibodies by ELISA. Control samples in which mouse PMNs were incubated with IgG1 only (without bacteria) did not result in significant recycling. Experiments were repeated 4 times, with essentially identical results. Data represent means plus or minus standard deviation.

  • Figure 7.

    Effect of FcRn-TAT peptides on phagocytosis of IgG-opsonized pneumococci. (A) Sequence of the FcRn tail included in the TAT peptides. Signaling motifs of FcRn documented within the FcRn intracellular tail, the tryptophan motif, the 2 aspartic acids, and the dileucine motif are underlined (FcRn WT). Shuffled amino acids in the control peptide with otherwise identical composition are shown in bold (shuffled FcRn). (B) Both wild-type and the shuffled control peptide are translocated over the plasma membrane. Biotinylated TAT peptides were incubated with PMNs. Extracellular staining was done on fixed PMNs, and internalized TAT peptides were detected on fixed and permeabilized cells. (C) WT FcRn-TAT peptides inhibit internalization of IgG1-opsonized bacteria. (D) Control FcRnS peptides had no effect on phagocytosis of IgG1-opsonized pneumococci (experiments were carried out with 0.8 mg/mL TAT peptides). Experiments were repeated 3 times, yielding comparable results. Data in panels C and D represent means plus or minus standard deviations. *P < .05; **P < .001 when compared with WT.

  • Figure 8.

    Illustration of a proposed role for FcRn in phagocytosis. IgG-opsonized bacteria engage leukocyte FcγR (1), which initiates the phagocytic process involving FcR-ITAM signaling motifs and downstream effectors (2). Activation leads to fusion of granules containing proton-pump components and FcRn with nascent phagosomes, which lowers the pH and promotes FcRn recognition of IgG (3). This process subsequently facilitates internalization of IgG-coated targets (4). In the absence of FcRn, efficient phagocytosis does not take place (see main text).