FcγRIII engagement provides activating signals to NKT cells in antibody-induced joint inflammation
Hye Young Kim, Sanghee Kim, Doo Hyun Chung
Hye Young Kim, Sanghee Kim, Doo Hyun Chung
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Research Article Autoimmunity

FcγRIII engagement provides activating signals to NKT cells in antibody-induced joint inflammation

Abstract

NKT cells promote antibody-induced arthritis, but the mechanism by which NKT cells are activated in this model remains unclear. It has been proposed that Fcγ receptor (FcγR) contributes to NKT cell activation in antibody-induced arthritis. To address this issue, we explored the functions of FcγR on NKT cells in antibody-induced arthritis. RT-PCR and flow cytometric analysis demonstrated that NKT cells constitutively express surface FcγRIII but not FcγRI, -II, or -IV. FcγRIII engagement by aggregated IgG on NKT cells enhanced CD25 and CD69 expression, whereas FcγR–/– mouse NKT cells did not enhance activation. FcγRIII engagement on NKT cells enhanced the production of IL-4, IL-10, IL-13, and IFN-γ, whereas FcγR-deficient NKT cells did not alter the production of these cytokines after aggregated IgG treatment. However, FcγR-deficient NKT cells were functionally intact in terms of TCR-induced activation. Moreover, adoptive transfer of FcγR-deficient NKT cells could not restore inflammation or TGF-β production in the joint tissues of CD1d–/– mice, whereas adoptive transfer of wild-type NKT cells induced arthritis and reduced TGF-β production in joint tissues. We conclude that FcγRIII engagement by IgG in joint tissues provides activating signals to NKT cells in antibody-induced arthritis.

Authors

Hye Young Kim, Sanghee Kim, Doo Hyun Chung

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Figure 2

FcγRIII engagement activates NKT cells.

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FcγRIII engagement activates NKT cells. (A) Liver MNCs from B6 or FcγR–/...
(A) Liver MNCs from B6 or FcγR–/– mice were cultured with or without (–) aggregated IgG (10 μg/ml), and CD25 and CD69 expression was analyzed 24 hours after stimulation. Open histograms represent negative controls, in which cells were stained with anti-NK1.1 and -TCRβ mAbs and isotype-matched IgG for anti-CD25 or -CD69 mAb. Numbers indicate MFI ± SD, from 3 independent experiments. (B) DN32-D3 cells were stimulated with or without aggregated IgG for 2 hours, lysed, and immunoprecipitated with anti-Syk or -Lyn mAb followed by anti-phosphotyrosine (anti–p-Tyr). The same cell lysates were directly blotted using anti-Syk or -Lyn mAb. Sorted NKT and DN32-D3 cells were stimulated with and without aggregated IgG for 24 hours, and the total proteins were separated by SDS-PAGE and immunoblotted using anti–T-bet or –GATA-3 mAbs. (C) Sorted NKT cells from B6 or FcγR–/– mice (1 × 105 cells/well) were stimulated with aggregated IgG for 48 hours. The amounts of IL-4, IFN-γ, IL-10, and IL-13 in the culture supernatant were measured using ELISA. (D) Sorted NKT cells from B6 mice (1 × 105 cells/well) were cultured with aggregated IgG or anti-CD3 mAb at the indicated concentrations, and the amounts of IL-4, IFN-γ, IL-10, and IL-13 measured using ELISA. (E) The same cells as in D were cultured with aggregated IgG (10 μg/ml), anti-CD3 mAb (100 ng/ml), or both for 48 hours. The amounts of IL-4 and IFN-γ were measured using ELISA. Results are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.