Regulation of major histocompatibility complex class II synthesis by interleukin-10

Abstract

We have shown previously that interleukin-10 (IL-10) blocks the development and T-cell stimulatory capacity of human monocyte-derived dendritic cells, without apparently down-regulating the surface expression of co-stimulatory molecules or human leucocyte antigen (HLA) molecules. In the majority of donors (60%), the cell surface levels of HLA-DR actually increased upon IL-10 treatment. Here we have shown that IL-10 does not regulate HLA-DR transcription as assessed by polymerase chain reation. Epifluorescence microscopy analysis showed that IL-10 primarily increased the intracellular pool of HLA-DR. In fact, IL-10 directly increased HLA-DR protein synthesis. However, IL-10 did not significantly alter the synthesis of invariant chain (Ii), which plays a crucial role in the assembly, transport and loading of newly formed HLA class II molecules, nor the amount of Ii reaching the cell-surface. In contrast, IL-10 increased the amount of HLA-DR-bound Iip33 shortly after the HLA-DR complex assembly. We postulate that, upon IL-10 treatment, immature Ii-associated HLA II molecules can still transit to the cell surface as they do in immature dendritic cells and recycle to the intracellular space, where they accumulate. A higher proportion of Ii-associated HLA-DR, coupled to increased membrane recycling, may contribute to the lower T-cell stimulatory capacity of IL-10-treated dendritic cells.

Figure 1

IL-10 does not affect the steady-state levels of HLA-DR mRNA. DCs were differentiated for 6 days with or without IL-10. LPS 200 ng/ml was also added to DCs for the last 2 days of culture as a positive control. Messenger RNA was reverse transcribed and the cDNA samples were normalized for β-actin content by PCR as described elsewhere (bottom panel). Normalized samples were subjected to 25 cycles of PCR using HLA-DRA-specific primers (linear phase of the PCR). Equal volumes were loaded on 1·5% agarose ethidium bromide-stained gel and observed under UV illumination. The OD of each band was measured. The graph indicates OD of the band of interest/OD of the DC band. These results are representative of three experiments.

Figure 2

Effects of IL-10 on intracellular localization of HLA-DR. DCs were differentiated for 4 days with (b,d) or without IL-10 (a,c). Cytospins were fixed in methanol: acetone and stained for HLA-DR (L243 mAb). Intracellular expression was visualized by epifluorescence microscopy under the 20 × objective using a constant exposure time (0·8 seconds, a and b). The isotype control was exposed for 2 seconds and is shown as a boxed insert within the IL-10 DC image (b). Localization of HLA-DR was further observed using the 60 × (c,d) objective. These results are representative of three experiments.

Figure 3

Both IL-10-treated and untreated DCs express high levels of Ii at both the cell surface and intracellularly. DCs were differentiated for 6 days with or without IL-10. LPS 200 ng/ml was also added to DCs for the last 2 days of culture. Ii cell-surface and intracellular expression was analysed by FACS on intact DCs or saponin-permeabilized DCs, respectively, using two independent mAb, LN2 and M-B741 (shaded histograms). Black line histograms represent stainings with the isotype control antibody for each cell type. Median fluorescence intensities are indicated in the top right corner of each FACS profile. These results are representative of seven experiments.

Figure 4

IL-10 up-regulates HLA-DR but not Ii synthesis. 4-day-old untreated DCs and IL-10 DCs were pulsed with [³⁵S]methionine for 20 min at 37°. HLA-DR or Ii complexes were immunoprecipitated from the cell lysates using the TU-36 and L243 mAb, and the LN2 mAb, respectively. Polypeptides were separated on 12·5% reducing SDS–PAGE (top panels) after incubation at 95°. The positions occupied by the free HLA-DRA, HLA-DRB and p33Ii are indicated on the left.(a) and (b) represent two different experiments. Quantification of each lane was performed using a PhosphorImager. The baseline intensity was subtracted from each profile and the radioactivity incorporated in each IL-10 DC peptide was expressed as a per cent of the corresponding DC peptide (bottom panels). These data are representative of six experiments.