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. 2008 May;38(5):1225-30.
doi: 10.1002/eji.200737902.

Interleukin-10-induced MARCH1 mediates intracellular sequestration of MHC class II in monocytes

Jacques Thibodeau  1 , Marie-Claude Bourgeois-Daigneault, Gabrielle Huppé, Jessy Tremblay, Angélique Aumont, Mathieu Houde, Eric Bartee, Alexandre Brunet, Marie-Elaine Gauvreau, Aude de Gassart, Evelina Gatti, Martin Baril, Maryse Cloutier, Séverine Bontron, Klaus Früh, Daniel Lamarre, Viktor Steimle
Affiliations

Interleukin-10-induced MARCH1 mediates intracellular sequestration of MHC class II in monocytes

Jacques Thibodeau et al. Eur J Immunol. 2008 May.

Abstract

IL-10 is a potent anti-inflammatory cytokine interfering with antigen presentation by inducing the intracellular sequestration of MHC class II (MHC-II) molecules. Here we studied the contribution of membrane-associated RING-CH (MARCH) ubiquitin ligase family members to the IL-10-induced down-regulation of MHC-II molecules. We found that MARCH1 and MARCH8 proteins are the most potent family members for the down-regulation of MHC-II surface expression in transfected cells, but only MARCH1 mRNA expression is strongly induced by IL-10 in human primary monocytes. We detected mono- and poly-ubiquitinated forms of MHC-II molecules both in IL-10-treated monocytes and in cells transfected with MARCH1. We also show direct interaction between MHC-II and MARCH1 molecules in co-immunoprecipitation assays. Finally, we found that siRNA-mediated knockdown of MARCH1 reverses IL-10-induced MHC-II down-regulation in primary monocytes. Thus, the immunosuppressive effect of IL-10 on antigen presentation is mediated through induced expression of MARCH1.

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Conflict of interest statement

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Figures

Figure 1
Figure 1
IL-10 induces MARCH1 expression. (A) Primary human monocytes were incubated with IFN-γ in the absence (panel 1) or presence (panel 2) of IL-10 for 16 h and analyzed by flow cytometry for HLA-DR and CD86 expression. (B) 293EBNA cells were transiently co-transfected with EGFP (lanes 1–8), CIITA (lanes 2–8), and with MARCH cDNA (lanes 3–8) and analyzed by flow cytometry after 48 h. Shown are mean fluorescence values (MFV) of cell surface HLA-DR expression gated on EGFP-positive cells. Values and SD are derived from duplicates of independent transfections. (C) MARCH1, −2, and −8 mRNA expression in primary human monocytes was analyzed by real-time RT-PCR. Cells were left either untreated (lane 1), or incubated for 16 h in the presence of IFN-γ (lane 2), IFN-γ and an anti-IL-10mAb (20 µg/mL; lane 3) or IFN-γ and IL-10 (lane 4). Samples were normalized for HPRT expression and results are expressed as the amount of equivalent linearized plasmid DNA standard for each MARCH. Shown is a representative example from three independent donors.
Figure 2
Figure 2
HLA-DR is ubiquitinated in MARCH1-transfected cells as well as in IL-10 treated monocytes, and interacts with MARCH1. (A) HeLa/CIITA cells were transfected with empty vector (lane 1) or an expression plasmid for FLAG-tagged MARCH1 (lane 2). At 24 h post transfection, cells were lysed and HLA-DR was immunoprecipitated using L243. Samples were separated by SDS-PAGE and analyzed on immunoblots using the ubiquitin-specific mAb P4D1 (top panel) and HLA-DRβ-specific mAb XD5.117 (middle panel). Total lysates were analyzed by immunoblotting with FLAG-specific mAb M2 (bottom panel). The arrowhead indicates the presence of mono-ubiquitinated HLA-DRβ, the bracket that of polyubiquitinated molecules. (B) Primary human monocytes were cultured for 16 h in the presence of IFN-γ (lanes 1, 3) or IFN-γ plus IL-10 (lanes 2, 4). Cells were lysed and HLA-DR molecules were immunoprecipitated with L243. Non-reduced samples were separated by SDS-PAGE and analyzed for ubiquitin by immunoblotting using P4D1 (left panel). The membrane was stripped and incubated with the HLA-DRβ-specific mAb XD5.117 (right panel). Similar results were obtained using cells from a second donor (data not shown). (C) 293T cells were transiently transfected with EYFP-MARCH1 alone (lane 1),with HLA-DRα/β (lane 2), or co-transfected with HLA-DRα/β and EYFP-MARCH1 (lane 3). At 24 h post transfection, cells were lysed and HLA-DR molecules were immunoprecipitatedwith mAb L243. Samples were separated by SDS-PAGE and analyzed on an immunoblot with a GFP-specific antiserum (top panel). The arrowhead indicates the presence of the EYFP-MARCH1 fusion protein (predicted molecular mass 58.4 kDa) and the asterisks indicate the Ig heavy and light chains. EYFP-MARCH1 expression in samples from lanes 1 and 3 was verified by flow cytometry (data not shown). The membrane was stripped and incubated with the HLA-DRβ specific mAb XD5.117 (bottom panel).
Figure 3
Figure 3
IL-10 induced down-regulation of MHC-II is dependent on MARCH1. Primary human monocytes were transfected with fluorescent-labeled (Alexa 488) control or MARCH1-specific siRNA. Cells were cultured for 16 h in the presence of IFN-γ or IFN-γ plus IL-10. (A) HLA-DR cell surface expression of siRNA-transfected cells was analyzed by flow cytometry. (B) MARCH1 mRNA expression of the cells shown in (A) was analyzed by real-time RT-PCR. Samples were normalized for HPRT expression and MARCH1 expression of the sample in lane 2 was arbitrarily set at 100. Values and SD are from duplicate PCR. Similar results were obtained on another donor.
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