Immune complexes (IC) down-regulate the basal and interferon-gamma-induced expression of MHC class II on human monocytes

Abstract

The interaction of Fc receptors for IgG (FcgammaRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcgammaR-IC interactions inhibit the IFN-gamma-induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC-dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN-gamma are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC-induced MHC class II down-regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN-gamma. We demonstrate that IC exert a drastic inhibition of basal and IFN-gamma-induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcgammaRI and FcgammaRII. Moreover, similar results were obtained using supernatants from IC-treated PBMC. The IC-induced down-regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class II expression, antigen presentation was markedly inhibited in the presence of IC.

Fig. 1

Effect of IC on basal and IFN-γ-induced MHC class II expression. PBMC (1 × 10⁶ cells/ml) were incubated with medium (control) or IC (100 μg/ml). After 1 h at 37°C, the cells were incubated with IFN-g (240 U/ml) for 24 h as indicated in the figure. After this period, PBMC were stained with anti-HLA-DR and anti-CD14 antibodies and evaluated by flow cytometry. (a) Data are expressed as the percentage of median of fluorescence intensity (% MFI) ± s.e.m. of control cells and correspond to the CD14⁺ population of PBMC. Statistical significance was calculated using Mann–Whitney test, two-tailed, n = 17.*P < 0·0001 significantly different from control. ΩP < 0·0001 significantly different from control and IFN-γ-treated cells. ΔP < 0·01 significantly different from control and P < 0·0001 significantly different from IFN-γ-treated cells. (b) The histograms represent the CD14⁺ population of PBMC and correspond to one representative experiment of n = 17. Background fluorescence intensity (filled peak) was obtained using control isotype antibodies. x-axis: fluorescence intensity (arbitrary units); y-axis: cell number.

Fig. 2

Effect of anti-FcγRI and/or anti-FcγRII on IFN-γ-induced MHC class II expression. PBMC (1 × 10⁶ cells/ml) were incubated with either medium or anti-FcγRI (5 µg/ml) and/or anti-FcγRII (5 µg/ml) for 1 h at 4°C. Then, the cells were washed and incubated with medium or (Fab′)₂ anti‐mouse IgG (10 µg/ml) for 1 h at 37°C. After this period, the cells were washed and incubated with medium or IFN-γ (240 U/ml) for 24 h and stained with anti-HLA-DR and anti-CD14 antibodies. Data are expressed as percentage MFI of control cells and correspond to the CD14⁺ population of PBMC. The inhibition of class II antigen by IC (100 µg/ml) in the same experimental conditions was 63%. The figure shows one representative experiment of n = 5.

Fig. 3

Effect of different amounts of IC on basal and IFN-γ-induced MHC class II expression. PBMC (1 × 10⁶ cells/ml) were incubated with medium (control) or different ratios of IC as indicated in the figure (1 : 1 corresponds to the concentration 100 µg/ml). After 1 h at 37°C, the cells were incubated with (a) IFN-γ (240 U/ml) or (b) medium for 24 h. After this period, PBMC were stained with anti-HLA-DR and anti-CD14 antibodies and evaluated by flow cytometry. Data are expressed as percentage MFI ± s.e.m. of control cells and correspond to the CD14⁺ population of PBMC, n = 3.

Fig. 4

Effect of IC after IFN-γ treatment. PBMC (1 × 10⁶ cells) were incubated with medium or IFN-γ (240 U/ml) for 20 h. Then, IC (100 µg/ml) were added and incubated for a further 4 h. After this period, the cells were stained with anti-HLA-DR and anti-CD14 antibodies. Data are expressed as percentage MFI ± s.e.m. of control cells and correspond to the CD14⁺ population of PBMC. The percentage MFI of IFN-γ-treated cells at 24 or 20 h were similar and not significantly different (% MFI ± s.e.m. of control, IFN-γ 24 h: 297 ± 43; IFN-γ 20 h: 258 ± 32; n = 6). Statistical significance was calculated using Mann–Whitney test, two-tailed; n = 6. *P < 0·002 significantly different from control. #P < 0·02 significantly different from IFN-γ-treated cells.

Fig. 5

Effect of supernatants (spn) from PBMC stimulated with IC on IFN-γ-induced MHC class II expression. PBMC (1 × 10⁶ cells/ml) were incubated with IFN-γ (240 U/ml) for 24 h. Then, the cells were exposed to spn from untreated or IC-treated PBMC for 3 h. After this period, PBMC were stained with anti-HLA-DR and anti-CD14 antibodies. Spn from IC incubated with medium alone for 18 h were obtained by centrifugation and were devoid of any effects on IFN-γ-induced MHC class II expression. Data are expressed as percentage MFI ± s.e.m. of control cells and correspond to the CD14⁺ population of PBMC. Statistical significance was calculated using Mann–Whitney test, two-tailed; n = 5. *P < 0·008 significantly different from control. #P < 0·02 significantly different from IFN-γ-treated cells and P < 0·03 significantly different from spn untreated PBMC-treated cells.

Fig. 6

Effect of IC on antigen presentation. Adherent PBMC were obtained as indicated in Materials and methods and were incubated with medium or IC (100 µg/ml). After 1 h, the cells were incubated with IFN-γ (240 U/ml) for 24 h. Then, the cells were washed and incubated with non‐adherent PBMC in the absence or presence of Mycobacterium tuberculosis. The index of [³H]-thymidine incorporation was calculated as indicated in Materials and methods. Statistical significance was calculated using the Mann–Whitney test, two-tailed; n = 5. *P < 0·05 significantly different from control. ΩP < 0·03 significantly different from control and IFN-γ-treated cells. ΔP < 0·03 significantly different from IFN-γ-treated cells.