Article Figures & Data
Figures
- FIGURE 1.
Intact SLE-IgG, but not Fab and F(ab′)2, combined with apoptotic U937 cells can induce IFN-α production by PBMC in vitro. The PBMC were cultured with UV-treated U937 cells, along with intact SLE-IgG (▪) or either Fab (▦) or F(ab′) 2 (□) prepared by papain or pepsin treatment, respectively (A). As a control, the Fab (▦) and F(ab′) 2 (□) or medium (▪) were added to cultures stimulated by HSV (B). The Fab, F(ab′)2, and the intact SLE-IgG were used at a concentration of 0.25 mg/ml. After 24 h, the IFN-α levels (units per milliliter) in the culture medium were measured by DELFIA. Representative results from one of three experiments.
- FIGURE 2.
Normal IgG inhibits the IFN-α production by PBMC induced by the combination of UV-treated U937 cells and SLE-IgG or by ODN 2216. The PBMC were cultured with SLE-IgG (1 mg/ml) and UV-treated U937 cells (A) or with ODN 2216 (B) in the presence of indicated concentrations of either heat-aggregated (•) or untreated (○) normal human IgG. After 24 h, the IFN-α levels in the culture medium were measured by DELFIA. Representative results from one of three experiments.
- FIGURE 3.
Abs to FcγRII inhibit the IFN-α production by PBMC induced by the combination of UV-treated U937 cells and SLE-IgG. The PBMC were cultured together with the combination SLE-IgG and UV-treated U937 cells (A), HSV (B), or ODN 2216 (C), and the indicated concentrations of mAbs directed against FcγRI (CD64; mAb 32.2), FcγRIIa,c (CD32; mAb IV.3 or IV.3-Fab), FcγRIIa,b,c (CD32; mAb AT10), or FcγRIII (CD16; mAb 3G8). The results with the anti-FcγRIIa,b,c mAb AT10 were obtained in a separate experiment. After 24 h, the IFN-α levels (units per milliliter) in the culture medium were measured in the supernatants by DELFIA. Representative results from one of three experiments.
- FIGURE 4.
Expression of FcγRII on BDCA-2-positive PBMC and IFN-α-producing cells. A, Unstimulated PBMC were double stained with FITC-labeled anti-BDCA-2 Abs and PE-labeled FLI8.26 anti-CD32 Abs (upper dot plot) using a PE-labeled IgG2b isotype control (lower dot plot). B, The PBMC were also stimulated by HSV for 9 h in vitro, stained with FITC-labeled anti-CD32 mAb (upper dot plot) or FITC-labeled isotype control IgG2b (lower dot plot) and after permeablization with biotin-labeled anti-IFN-α mAb and PE-labeled streptavidin to detect intracellular IFN-α. Flow cytometric analysis was performed and indicated rectangular gates were used to define BDCA-2-positive PBMC (i.e., NIPC/PDC) that were either considered negative or positive for CD32. The percentage figures in the gates represent the proportion of cells in each gate of all analyzed PBMC. Representative results from the analysis of a total of eight (A) or four (B) different PBMC donors.
- FIGURE 5.
Analysis of FcγRIIa, FcγRIIb, and FcγRIIc gene expression in purified NIPC/PDC. The PBMC from six donors were enriched for BDCA-4-positive cells by magnetic bead separation, subsequently stained with FITC-conjugated anti-BDCA-2 Abs, and purified by FACS. RT-PCR was performed on mRNA prepared from the BDCA-2-positive cells (first lane) and unsorted PBMC (second lane) using specific primer pairs for FcγRIIa, FcγRIIb, FcγRIIc, and β-actin. Note that the primer pair for FcγRIIb detected FcγRIIb1 and FcγRIIb2 in unsorted PBMC, whereas the primer pairs for FcγRIIa or FcγRIIc only resulted in one band, respectively (A). The efficiency of the primers detecting the different isoforms of FcγRII was verified using as templates serial dilutions of plasmids encoding each subtype (B). For RT-PCR and FACS details, see Materials and Methods.
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