Figure 1.
Description of the transcytosis assay.
hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filter inserts (and serum starved for 1 h at 37°C before assay for experiments with 125I-transferrin) were incubated apically with the ligand or the different antibodies for 60 min at 37°C. After 1 hr, media from the apical and basolateral chambers were collected to assess paracellular flux following which the luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. The filters containing cells were transferred to a fresh plate containing pre-warmed medium and cells were chased up to the desired time points at 37°C or on ice and at these different time points, the ligand or antibody associated with cells or in the media from the apical and basolateral chambers was analyzed in a gamma counter or assessed by IgG ELISA.
Figure 2.
Apical to basolateral transport of 125I-Tfn and 125I-128.1 anti-TfR antibody in hCMEC/D3 cells.
A: Empty filters (Empty Filters) treated as described in Materials and Methods or hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filter inserts (filters with cells) (serum starved for 1 h at 37°C before assay) were incubated apically with 125I-transferrin (Tf stock 1 µg/mL) for 60 min at 37°C. After 1 hr, radioactivity associated with media from the apical (dark grey bars) and basolateral chambers (grey bars), was analyzed in a γ counter. B, C, and D: hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filter inserts were serum starved for 1 h at 37°C and then incubated apically with 1 µg/mL 125I-transferrin (in serum free medium) for 60 min at 37°C. Cells were washed to remove unbound ligand and chased up to 4 hours at 37°C (B) or on ice (D). At the end of the chase, radioactivity associated with cells (▪) and media from the apical (grey bars) and basolateral chambers (black bars), was analyzed in a gamma counter at different time points. The total amount of the radiolabelled ligand (▴) derived from combined value of radiolabel in the lysate and in the media chambers remained constant during the assay. Each data point represents values obtained from three filter membrane inserts with cells and these results are representative of three separate experiments. (C), denotes active protein (black bars), degraded protein (white bars) and free iodine (grey bars) obtained from TCA precipitation of apical or basolateral media compartments from (B). E and F: hCMEC/D3 cells grown to confluence in a contact co-culture with primary human astrocytes (E) or in a non-contact culture condition with the rat C6 cell line (F) were incubated for 1 h at 37°C in serum free media to deplete intracellular transferrin. Cultures were incubated apically with 1 µg/mL 125I-transferrin (in serum free medium) for 60 min at 37°C. Cells were washed to remove unbound ligand and cultured at 37°C. Apical to basolateral transport of the ligand (black bars) or apically recycled (grey bars) internalised ligand was determined by analyzing the whole cell lysates (▪) and the media from the apical and basolateral chambers in a gamma counter at different time points. The total amount of the radiolabelled ligand (▴) derived from combined value of radiolabel in the lysate and in the media chambers was constant during the assay. Values are means of three filters ± SEM. G and H: hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filters were incubated apically with 6.5 µg/mL 125I-128.1 (G) in a radiolabel assay format for 60 min at 37°C. Luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. Cells were chased up to 18 hrs at 37°C. At the end of the chase, radioactivity associated with cells (▪) and media from the apical (grey bars) and basolateral chambers (black bars), was analyzed in a gamma counter at different time points. The total amount of the antibody (▴) derived from combined value of radiolabel in the lysate and in the media chambers was calculated for the duration of the assay. Each data point represents values obtained from three filter membrane inserts with cells and these results are representative of more than three separate experiments. (H), denotes active protein (black bars), degraded protein (white bars) and free iodine (grey bars) obtained from TCA precipitation of apical or basolateral media compartments from (G).
Figure 3.
Uptake and fate of an antibody directed against different transcytosis receptors in hCMEC/D3 monolayers.
A–D hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filters were incubated apically with 1 µg/mL of the following antibodies: the anti-human TfR antibody, 128.1 (A), the mouse anti–human TfR antibody, MEM-189 (B), an anti-human IGF-1R antibody (C) and the mouse anti-human insulin receptor (IR) antibody, 83–14 (D) for 60 min at 37°C. Luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. Cells were chased up to 5 hrs at 37°C. At the end of the chase, antibody associated with cells (▪) and media from the apical (grey bars) and basolateral chambers (black bars), were analyzed by the sensitive IgG ELISA at different time points. The total amount of IgG (▴) derived from combined value of antibody present in the lysate and in the media chambers was calculated for the entire duration of the assay. Values are means of three filters ± SEM.
Figure 4.
Degradation of 128.1 but not MEM-189 in hCMEC/D3 cells.
hCMEC/D3 endothelial cells were pulsed with 1 µg/ml 128.1 (A) or MEM-189 (B) for 10 min at 37°CThe coverslip cultures were washed and cultured at 37°C for various time periods. Cultures were fixed in 4% PFA, permeabilised and immunostained with an antibody to the late endosomal/lysosomal marker CD63 and appropriate secondary antibodies as described in Materials and Methods and examined with a laser scanning confocal microscope. Insets: secondary antibodies only.
Figure 5.
128.1 is a high affinity antibody that competes successfully with MEM-189 for binding to TfR in ELISA and in contrast to MEM-189 shows no pH dependence.
A: Competition ELISA described in Materials and Methods showing binding of 128.1 to the extracellular subunit of TfR in the presence (▪) or absence (▴) of a pre-block by 5 µg/ml MEM-189. Similarly, binding of MEM-189 in the presence (□) or absence (▵) of 5 µg/mL 128.1. B, Binding ELISA described in Materials and Methods showing binding of the 128.1 anti-human transferrin receptor antibody (▪,□) and MEM-189 antibody (•,○) to the extracellular subunit of the human TfR at pH 7.4 (▪,•) or at pH 5.5 (□,○).
Figure 6.
pH dependence and uptake, fate of different antibodies directed against the human TfR in hCMEC/D3 monolayers.
A–D: Binding ELISA described in Materials and Methods showing binding of mouse anti-human transferrin receptor antibodies LT-71 (A), MEM-75 (B), M-A712 (C) and 13E4 (D) to the extracellular subunit of TfR at pH 7.4 (•) or at pH 5.5 (○). E–I: hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filters were incubated apically with 1 µg/mL of the following mouse anti-human TfR antibodies: LT-71 (E), MEM-75 (F), M-A712 (G), 13E4 (H) for 60 min at 37°C. Luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. Cells were chased up to 5 hrs at 37°C. At the end of the chase, antibody associated with cells (▪) and media from the apical (white bars) and basolateral chambers (grey bars), were analyzed by the sensitive IgG ELISA at different time points. The total amount of IgG (▴) derived from combined value of antibody present in the lysate and in the media chambers was calculated for the entire duration of the assay. Values are means of three filters ± SEM. (I) hCMEC/D3 cells were incubated apically with 1 µg/mL mouse anti-human TfR MEM-189 antibody as in (E–H) and cells were chased up to 5 hrs at 37°C in the presence of 50 nM Bafilomycin. At the end of the chase, antibody associated with cells (▪) and media from the apical (grey bars) and basolateral chambers (white bars), were analyzed by the sensitive IgG ELISA at different time points. The total amount of IgG (▴) derived from combined value of antibody present in the lysate and in the media chambers was calculated for the entire duration of the assay. Values are means of three filters ± SEM.