NKT cell status and IL-10-dependent therapeutic effect of NKT cell stimulation on collagen-arthritis in DBA/1 mice

Defective NKT cell function has been linked with autoimmunity. Both the number of NKT cells and their functional capacity of releasing interferon gamma (IFN-γ) and IL-4 after TCR ligation are for instance impaired in NOD mice developing diabetes, in mice developing a model of multiple sclerosis and also in humans with autoimmune diseases. To investigate the NKT cell role in rheumatoid arthritis, we studied their quantitative and qualitative profile in collagen-induced arthritis (CIA) DBA/1 susceptible mice, and we tested whether NKT stimulation with their synthetic ligand alpha-galactosylceramide (α-GalCer) was therapeutic in CIA.

The number of NKT cells in the liver was determined by flow cytometry with an anti-TCRαβ or empty or α-GalCer-loaded CD1 tetramer. CIA was induced by immunization of DBA/1 mice with collagen II (CII) in adjuvant, and with a boost 21 days later. α-GalCer (4 μg) was administered at the same time of the first immunization. In one experiment, one group of mice was also treated with an anti-IL-10 receptor antibody. CII-specific CD4⁺ cells from lymph node (LN) response was monitored by immunizing mice with 75 μg CII in adjuvant in hind paws. Some mice were administered 4 μg α-GalCer in the CII/adjuvant mixture at the same time. Nine days after immunization, CD4⁺ cells were purified from LN and cultured with CII and antigen-presenting cells. Proliferation was measured after 3 days by measuring BrdU incorporation, and IL-4, IL-10 and IFN-γ levels were assessed by ELISA in the supernatants.

The number of NKT cells among leucocytes in the liver of DBA/1 mice was comparable to what is generally observed in C57Bl/6 and suggest a normal quantitative profile of NKT cells in DBA/1 mice. In contrast, in vivo NKT cell function was altered in DBA/1 mice since stimulation with α-GalCer (4 μg intraperitoneally) led to decreased IL-4 and IFN-γ levels in the serum 2 hours after the injection, as compared with C57Bl/6 mice (693 ± 154 pg/ml versus 1557 ± 137 g/ml IL-4 [P < 0.01] and 2077 ± 378 versus 4005 ± 581 IFN-γ [P < 0.02]). Treatment of CIA with α-GalCer at day 0 induced a clear-cut diminution of clinical (ANOVA test, P = 0.0001) and histological scores (1.19 ± 0.14 versus 1.85 ± 0.24, P < 0.05) of arthritis, as compared with the control group. Importantly, treatment of mice with an anti-IL-10 receptor abrogated the protective effect of α-GalCer. The α-GalCer-induced protection was associated with the ability of LN CD4⁺ cells from CII-immunized and α-GalCer-treated DBA/1 mice to secrete larger amounts of IL-10 upon in vitro restimulation with CII, while IL-4 and IFN-γ levels were not affected. CII-induced proliferation was slightly reduced in LN CD4 cells from CII-immunized and α-GalCer-treated DBA/1 mice as compared with controls.

These findings raise the possibility that NKT stimulation might induce a shift toward an anti-inflammatory Th2 status and could be used therapeutically to treat chronic autoimmune arthritis.