FIGURE 8.

Effects of IFN-γ stimulation on the IgG transcytosis. A, Semiquantitative RT-PCR analysis of FcRn mRNA in the human lung epithelial Calu-3 cell line. The Calu-3 cells were treated (+) with IFN-γ (25 ng/ml) (right lane) or left untreated (−) (left lane) for 24 h. Data are representative results for RT-PCR analysis of FcRn expression in Calu-3. Ratios of FcRn-GAPDH are shown as indicated. B, The pH-dependent FcRn binding of IgG. The Calu-3 cells were lysed in sodium phosphate buffer (pH 6.0 or 8.0) with 0.5% CHAPS. Approximately 1 mg of soluble proteins was incubated with human IgG-Sepharose at 4°C. The eluted proteins were subjected to 12% SDS-polyacrylamide electrophoresis and subjected to Western blot analysis. Proteins were probed with affinity-purified rabbit anti-FcRn peptide Ab and HRP-conjugated donkey anti-rabbit Ab. Immunoblots were developed with ECL. The ratio of the mock sample is assigned a value of 1.0, and the values from IFN-γ-treated sample are normalized to this value. C, Calu-3 cells (5 × 10⁵/well) were grown in a 12-well Transwell plate. When the resistance of the monolayer reached 700–1000 ohms/cm², cells were stimulated with or without IFN-γ (25 ng/ml) for 24 h. Cells were loaded with human IgG (top row) or chicken IgY (bottom row) (0.5 mg/ml) at 4°C in either the apical (lanes 2 and 3) or basolateral (lanes 4 and 5) chamber. Lane 1 represents an IgG or IgY H chain. Cells were warmed to 37°C to stimulate transcytosis, and medium was collected from the nonloading compartment 1 h later and subjected to Western blot-ECL analysis. The results are representative of at least three independent experiments. Band intensities of IgG heavy chain (HC) were compared by densitometry against IgG transported from mock-stimulated cells.