TCR Valpha14 natural killer T cells function as effector T cells in mice with collagen-induced arthritis

Abstract

Natural killer (NK) T cells are a unique, recently identified cell population and are suggested to act as regulatory cells in autoimmune disorders. In the present study, designed to investigate the role of NKT cells in arthritis development, we attempted to induce arthritis by immunization of type II collagen (CIA) in Jalpha281 knock out (NKT-KO) and CD1d knock out (CD1d-KO) mice, which are depleted of NKT cells. From the results, the incidence of arthritis (40%) and the arthritis score (1.5 +/- 2.2 and 2.0 +/- 2.7) were reduced in NKT-KO and CD1d-KO mice compared to those in respective wild type mice (90%, 5.4 +/- 3.2 and 2.0 +/- 2.7, P < 0.01). Anti-CII antibody levels in the sera of NKT-KO and CD1d-KO mice were significantly decreased compared to the controls (OD values; 0.32 +/- 0.16 and 0.29 +/- 0.06 versus 0.58 +/- 0.08 and 0.38 +/- 0.08, P < 0.01). These results suggest that NKT cells play a role as effector T cells in CIA. Although the cell proliferative response and cytokine production in NKT-KO mice after the primary immunization were comparable to those in wild type mice, the ratios of both activated T or B cells were lower in NKT-KO mice than wild type mice after secondary immunization (T cells: 9.9 +/- 1.8% versus 16.0 +/- 3.4%, P < 0.01, B cells: 4.1 +/- 0.5% versus 5.1 +/- 0.7%, P < 0.05), suggesting that inv-NKT cells contribute to the pathogenicity in the development phase of arthritis. In addition, IL-4 and IL-1beta mRNA expression levels in the spleen during the arthritis development phase were lower in NKT-KO mice, while the IFN-gamma mRNA expression level was temporarily higher. These results suggest that inv-NKT cells influence cytokine production in arthritis development. In conclusion, inv-NKT cells may promote the generation of arthritis, especially during the development rather than the initiation phase.

Fig. 1

Suppression of arthritis in NKT-KO mice and CD1d-KO mice. Ten NKT-KO (a,b) and 10 CD1d-KO mice (c,d) were immunized and boosted with chicken CII emulsified in IFA plus inactivated M. tuberculosis H37Ra. Ten C57BL/6 mice were used as the control in each examination. The incidence of CIA (a,c) and the severity of arthritis (b,d) were investigated. ▪ represents each KO mice; • represents C57BL/6 mice.

Fig. 2

Reduction of anti-CII Abs in NKT-KO and CD1d-KO mice. Twenty-eight days after the booster injection, the amount of anti-CII IgG antibody in the serum from NKT-KO (a) or CD1d-KO (b) mice and C57BL/6 mice was measured by ELISA.

Fig. 3

Cell proliferation and cytokine production stimulated by CII in NKT-KO mice compared with those in C57BL/6 mice. Five NKT-KO and five C57BL/6 mice were immunized with chicken CII emulsified in IFA plus M. tuberculosis H37Ra. Nine days after immunization, splenocytes were stimulated with CII. The degree of cell proliferation was evaluated by a BrdU ELISA method (a). The concentrations of IFN-γ (b) and L-4 (c) in the culture supernatants were measured by ELISA.

Fig. 4

Activation level of T and B cells after the booster immunization in NKT-KO mice compared with that in C57BL/6 mice. Eight NKT-KO and eight C57BL/6 mice were immunized and boosted with chicken CII emulsified in IFA plus inactivated M. tuberculosis H37Ra. Five days after the booster immunization, splenocytes were collected and stained with FITC-labelled anti-TCRβ or anti-B220, and PE-labelled anti-CD69 antibody. PI-negative cells were gated and FITC-PE double positive cells were counted (a). The proportions of CD69-positive T cells (b) or CD69-positive B cells (c) compared to the total number of T cells (TCRβ⁺ cells) or B cells (B220⁺ cells) were calculated.

Fig. 5

Cytokine mRNA expression in the spleen after the booster immunization. Total splenic RNA was collected from three male C57BL/6 mice (•) and three male NKT-KO mice (▪) 5, 10, 15, and 30 days after the booster immunization, and the relative expression levels of (a) IFN-γ, (b) IL-4 or (c) IL-1β mRNA were measured by the Taqman quantitative PCR method. *P < 0·05; **P < 0·01.