JNK1, but not JNK2, is required in two mechanistically distinct models of inflammatory arthritis

Abstract

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.

Figure 1

Jnk1^−/− mice are resistant to CIA, whereas Jnk2^−/− mice are highly susceptible. A: Clinical arthritis scores. B: Cumulative incidence of arthritis in wt, Jnk1^−/−, and Jnk2^−/− mice with CIA. The severity of arthritis was evaluated using a scoring system, as described in Materials and Methods. Results are presented as the mean ± SD. *P < 0.05 (n = 20 for Jnk1^−/−, n = 10 for Jnk2^−/−, and n = 15 for wt).

Figure 2

Histological analysis confirms the lack of inflammation in joints from Jnk1^−/− mice with CIA. A: Representative H&E-stained sections from hind paws of wt, Jnk1^−/−, and Jnk2^−/− mice obtained at day 70 after immunization. There was absent or minimal inflammation in the Jnk1^−/− section. B: The severity of arthritis was histologically evaluated using the following parameters: bone destruction, hyperplasia of the synovium, and infiltration of monomorphonuclear and polymorphonuclear cells. Each parameter was scored as described in Materials and Methods. *P < 0.05 (n = 10).

Figure 3

Altered humoral immunity in response to CII in Jnk1^−/− and Jnk2^−/− mice with CIA indicates a substantial Th1 response in the JNK2-deficient mice. A: Total CII-specific IgG serum levels in wt, Jnk1^−/−, and Jnk2^−/− mice on day 30 after immunization. IgG1 (B) and IgG2a (C) levels in wt, Jnk1^−/−, and Jnk2^−/− mice were measured on day 30 after immunization. *P < 0.05 (n = 10).

Figure 4

Altered cellular immunity in JNK-deficient mice. A: DTH response in wt, Jnk1^−/−, and Jnk2^−/− mice 24 hours after challenge with CII. B: The results at 24 hours after challenge with DNFB, suggesting a deficiency in the ability of Jnk1^−/− antigen-presenting cells in responding to certain microbial products. Antigen-induced ear swelling is presented as the percentage increase in ear thickness of the stimulated ear compared with the control ear. *P < 0.05, **P < 0.01 (n = 7 for CII and n = 6 for DNFB).

Figure 5

Failure of Jnk1^−/− macrophages to up-regulate costimulatory molecule CD86. A: FACS analysis of MHC II (I-E^k), CD86, and F4/80 expression levels (mean fluorescence intensity) in unstimulated BMMs from wt, Jnk1^−/−, and Jnk2^−/− mice (n = 6). B: Percentage increase in mean fluorescence intensity in BMMs stimulated with LPS (10 ng/mL) for 24 hours (n = 6). C: The BMMs were stimulated for 6 hours with LPS (10 ng/mL), and cytokine mRNA levels were measured by real-time PCR analysis. *P < 0.05, **P < 0.001 (n = 6). D: Protein extracts were prepared from wt and Jnk1^−/− BMMs stimulated with LPS (10 ng/mL) for the indicated time points and analyzed by immunoblotting. Data suggest that JNK1 or JNK2 is sufficient for macrophage differentiation and maturation. ERK, extracellular signal–regulated kinase. E: RAW 264.7 cells were treated with SP600125 JNK inhibitor (25 μmol/L) for 1 hour before 24-hour LPS stimulation (1 μg/mL) and co-cultured with carboxyfluorescein diacetate succinimyl ester–marked primary murine T cells for 96 hours. Although the dividing CD4⁺ cells in the control go through at least three cell divisions, most CD4⁺ cells stimulated with SP600125-treated RAW cells remain quiescent (76.8% versus 30.5% dividing cells). CFSE, carboxyfluorescein diacetate succinimyl ester.

Figure 6

Divergent roles of JNK1 and JNK2 in the development of serum-induced arthritis. Clinical arthritis in wt, Jnk1^−/−, and Jnk2^−/− mice after injection of K/BxN serum i.p., as described in Materials and Methods. A: The mice were assessed for arthritis development by clinical index score and ankle thickness. B: wt BMMs were grown in culture, as described in Materials and Methods, and i.v. injected via tail vein into Jnk1^−/− mice 1 day before and 1 and 3 days after serum transfer. The values represent the mean ± SEM. *P < 0.05 compared with A (wt mice) or B (mice not receiving BMMs under parallel conditions).