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. 2008 Dec;19(12):5490-505.
doi: 10.1091/mbc.e07-02-0101. Epub 2008 Oct 8.

Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells

Nancy A Goebl  1 , Clifford M Babbey, Amita Datta-Mannan, Derrick R Witcher, Victor J Wroblewski, Kenneth W Dunn
Affiliations

Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells

Nancy A Goebl et al. Mol Biol Cell. 2008 Dec.

Abstract

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.

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Figures

Figure 1.
Figure 1.
Fc colocalizes with human FcRn and transferrin in recycling compartments and is not present in lysosomes during short-term labeling. Cells expressing human GFP-FcRn were labeled for 20 min with 200 μg/ml TxR-Fc (4.4 μM) at pH 7.4 and then fixed. Cells were either prelabeled with Alexa 647-dextran as a lysosomal marker (50 μg/ml overnight dextran incubation chased to lysosomes for 1 h before addition of Fcs; A–C) or labeled simultaneously with 30 μg/ml Cy5-Tf (D). Projections were made of three-dimensional (3D) image volumes for each fluor and were merged as single images in the bottom row (green, GFP-FcRn; red, Fc; blue, dextran; yellow, colocalization of GFP-FcRn and FC; white (bottom right image only), three color colocalization of GFP-FcRn, Fc, and transferrin). WT Fc (A) and the T250Q/M428L mutant Fc (B) strongly colocalize with human FcRn with prominent fluorescence in the pericentriolar recycling compartment (arrows). Very little fluorescence is observed in the dextran lysosomal compartment (arrowheads). Fluorescent Fc endosomes are very dim in cells labeled with the H435A mutant (C). Cells labeled with Fc and transferrin show extensive colocalization between Fc, transferrin and FcRn (D). Note untransfected cell in D (bottom cell), which labels with transferrin but shows very little Fc internalization. All images from a given fluor were processed and contrast-adjusted in an identical manner, with the exception of the WT Fc images, which were contrast-stretched threefold relative to T250/M428L in order to visualize the overall dimmer labeling, and the H435A images, which were likewise stretched threefold for comparability to the WT Fc images. Scale bar, 10 μm.
Figure 2.
Figure 2.
Fc internalization and distribution in cells transfected with mouse FcRn is comparable to those transfected with human FcRn. Cells expressing mouse GFP-FcRn were labeled for 20 min with 200 μg/ml TxR-Fc at pH 7.4 and then fixed. Cells were prelabeled with Alexa 647-dextran as a lysosomal marker (50 μg/ml overnight dextran incubation chased to lysosomes for 1 h before addition of Fcs). Projections were made of 3D image volumes for each fluor and were merged as single images in the bottom row (green, GFP-FcRn; red, Fc; blue, dextran; yellow, colocalization of GFP-FcRn and Fc). WT Fc (A) and the T250Q/M428L mutant Fc (B) colocalize with mouse FcRn in the PRC (arrows) and early endosomes but is not present in the lysosomes (arrowheads). Minimal internalization of the H435A mutant is observed (C). All images were contrast-adjusted in an identical manner to compare relative fluorescent intensities. All images from a given fluor were processed and contrast-adjusted in an identical manner. Note that the WT Fc image (A) is brighter and more readily visualized than the comparable image in Figure 1A of Fc internalization by a cell transfected with human FcRn, which required contrast stretching for visualization. Scale bar, 10 μm.
Figure 3.
Figure 3.
Fc recycles from cells transfected with mouse GFP-FcRn. Coverslip dishes of cells transfected with mouse GFP-FcRn were equilibrated in medium 1 with ovalbumin and secured to the microscope stage maintained at 37°C. Cells were labeled for 15 min on the microscope stage with prewarmed TxR-WT (200 μg/ml) and then rinsed in a large volume of prewarmed buffer. Live image stacks were collected at time 0 (A), 10 (B), and 30 min (C), and projections were made of 3D volumes. Rapid movement of fluorescent endosomes was observed throughout the course of the experiment, indicating adequate maintenance of appropriate temperature and pH conditions. Scale bar, 10 μm.
Figure 4.
Figure 4.
HULEC-5A (A) and MDCK (B) cells were transfected with mouse GFP-FcRn and incubated for 10 min in mixture of 200 μg/ml TxR-Fc (T250Q/M428L) and 250 μg/ml Alexa 647-dextran and then fixed. After this short incubation, Fc internalization is visible only in the transfected, but not the untransfected cells in either cell line. In contrast, dextran internalization by fluid phase pinocytosis appears comparable in both transfected and nontransfected cells. Scale bar, 10 μm. (C–E) Quantifications of Fc and dextran internalization by HULEC5A cells transfected with mouse FcRn. Cells were incubated for 10 min with 200 μg/ml TxR-WT and 250 μg/ml Alexa 647-dextran and then fixed. Total cell fluorescence was recorded for each fluor from projections of 3D volumes of 35 cells. Although dextran internalization remains constant between cells (C, ●), Fc internalization (C, ○) and the ratio of Fc/dextran (D) increase in a linear manner with increasing expression of FcRn. E. Mouse FcRn-transfected cells were labeled for 20 min with 200 μg/ml either TxR-T250Q/M428L or TxR-H435A. Cellular fluorescence of the T250Q/M428L (○, n = 15) but not the H435A mutant (●, n = 17) increases with GFP-FcRn fluorescence.
Figure 5.
Figure 5.
Internalization of fluorescently labeled Fc can be competed by a 20-fold excess of unlabeled FcRn-binding probe, but not nonbinding probe. Mouse FcRn-transfected cells were labeled for 10 min with 100 μg/ml TxR-T250Q/M428L (A–D) or TxR-WT (E–H) and then fixed. Cells in B and F were coincubated with 2 mg/ml unlabeled T250Q/M428L and in D and H were coincubated with 2 mg/ml unlabeled H435A and then fixed. In each of the four experiments, T250Q/M428L and WT controls demonstrated colocalization of the GFP-FcRn and Fc compartments (A, C, E, and G). When either Fc was coincubated with a 20-fold excess of unlabeled T250Q/M428L, Fc fluorescence levels were profoundly reduced (B and F), but little decrease in fluorescent signal was noted when a 20-fold excess of unlabeled H435A was used for the coincubation (D and H). Scale bar, 10 μm. (I–L) Quantifications of competition studies. For each of the conditions above, total cell fluorescence was recorded for each fluor from projections of 3D volumes. Cells incubated with TxR-WT (I) along with unlabeled T250Q/M428L (○, n = 20) showed a clear decrease in internalization of labeled probe relative to noncompeted cells (●, n = 23). Cells incubated with TxR-T250Q/M428L (J) along with unlabeled T250Q/M428L (○, n = 21) showed a clear decrease in internalization of labeled probe relative to noncompeted cells (●, n = 23). No differences in internalization were observed in similar cell populations incubated with TxR-WT (K) with (○, n = 28) or without (●, n = 21) unlabeled H435A. No differences in internalization were observed in similar cell populations incubated with TxR-T250Q/M428L (L) with (○, n = 25) or without (●, n = 24) unlabeled H435A.
Figure 6.
Figure 6.
HULEC-5A cells were transfected with human GFP-FcRn and incubated for 20 min in mixture of 200 μg/ml TxR-Fc and 250 μg/ml Alexa 647-dextran and then fixed. After this short incubation, internalization of TxR-T250Q/M428L (A) is visible only in the transfected, but not the untransfected cells. Despite the lower affinity of the TxR-WT Fc (B), internalization was again much more apparent in cells transfected with GFP-FcRn. In both cases, dextran internalization is comparable in transfected and nontransfected cells. Scale bar, 10 μm.
Figure 7.
Figure 7.
Fc internalization is similar at pH 6.0 and 7.4. Mouse FcRn-transfected cells were equilibrated for 10 min at pH 7.4 or 6.0 and then labeled for 10 min with 200 μg/ml TxR-WT or TxR-T250Q/M428L and 250 μg/ml Alexa 647-dextran at pH 6.0 or 7.4 and then fixed. (A and B) T250Q/M428L after incubation at pH 7.4 (A) or 6.0 (B). (C and D) WT after incubation at pH 7.4 (C) or 6.0 (D). No differences are noted in relative internalization of Fc (row 2) or dextran (row 3) at either pH. Merged images (row 4) show comparable colocalization of Fc with FcRn at either pH. Scale bar, 10 μm. (E and F) Quantification of the effect of incubation pH on Fc internalization. For each of the conditions above, total cell fluorescence was recorded for each fluor from projections of 3D volumes. Internalization of TxR-WT (○), but not dextran (●) increases proportionally to FcRn at both pH 6.0 (E, n = 26) and pH 7.4 (F, n = 21).
Figure 8.
Figure 8.
FcRn-mediated internalization of Fc is not significantly affected by endosome alkalinization. Mouse FcRn-transfected HULEC-5A endothelial cells were preincubated for 15 min with 1 μM bafilomycin and then incubated for 15 min with TxR-WT and Cy5-dextran in the continued presence of bafilomycin. Cells were then fixed for analysis. (A–C) Fluorescence micrographs of bafilomycin-treated cells. (A) Internalization of Cy5-dextran by five cells in the field, (B) Expression of GFP-FcRn by two cells in the field. (C) Internalization of TxR-WT by the two GFP-FcRn-expressing cells. (D) Quantification of internalization of Fc and dextran. For each of the conditions above, total cell fluorescence was quantified for each fluor from projections of 3D volumes. Expression of GFP-FcRn enriches internalization of Fc relative to dextran in both bafilomycin-treated (n = 30, 25) and untreated cells (n = 31, 27). (E) Internalization of Fc increases linearly with expression of GFP-FcRn in bafilomycin-treated cells (n = 23). Scale bar, 10 μm.
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