Cytokine-dependent but acquired immunity-independent arthritis caused by DNA escaped from degradation

Abstract

DNase II digests the chromosomal DNA in macrophages after apoptotic cells and nuclei from erythroid precursors are engulfed. The DNase II-null mice develop a polyarthritis that resembles rheumatoid arthritis. Here, we showed that when bone marrow cells from the DNase II-deficient mice were transferred to the wild-type mice, they developed arthritis. A deficiency of Rag2 or a lack of lymphocytes accelerated arthritis of the DNase II-null mice, suggesting that the DNase II(-/-) macrophages were responsible for triggering arthritis, and their lymphocytes worked protectively. A high level of TNFα, IL-1β, and IL-6 was found in the affected joints of the DNase II-null mice, suggesting an inflammatory-skewed cytokine storm was established in the joints. A lack of TNFα, IL-1β, or IL-6 gene blocked the expression of the other cytokine genes as well and inhibited the development of arthritis. Neutralization of TNFα, IL-1β, or IL-6 had a therapeutic effect on the developed arthritis of the DNase II-null mice, indicating that the cytokine storm was essential for the maintenance of arthritis in the DNase II-deficient mice. Methotrexate, an antimetabolite that is often used to treat patients with rheumatoid arthritis, had a therapeutic effect with the DNase II-null mice. These properties of arthritis in the DNase II-null mice were similar to those found in human systemic-onset juvenile idiopathic arthritis or Still's disease, indicating that the DNase II-null mice are a good animal model of this type of arthritis.

Fig. 1.

Activation of lymphocytes in DNase II-null mice and bone marrow transfer. (A and B) A FACS analysis of splenocytes from 1- to 2.5-mo-old DNase II^−/−IFN-IR^−/− and DNase II^+/− or ^+/+IFN-IR^−/− littermate mice (control) (n = 4 for each group). The average cell number of the indicated cell type (A), and the average percentage of CD69⁺ cells in the CD4⁺ or CD8⁺ T cell population (B) are shown with SD. (C and D) Cells from the bone marrow of DNase II^Δ/− or WT mice were transplanted into irradiated 1-mo-old WT mice. Mean arthritis scores at the indicated time after the transplantation were plotted with SD (n = 3–4 for each condition) (C). Three months after the transplantation, the serum MMP-3 was quantified by ELISA. The mean values are indicated (bars) (D). In A, B, and D, P value is shown when it is <0.2.

Fig. 2.

Development of arthritis in DNase II^−/−IFN-IR^−/−Rag2^−/− mice. (A) Arthritis scores of DNase II^−/−IFN-IR^−/−Rag2^+/+, DNase II^−/−IFN-IR^−/−Rag2^+/−, and DNase II^−/−IFN-IR^−/−Rag2^−/− mice were plotted with SD (n = 3–11 for each). P values were calculated between the DNase II^−/−IFN-IR^−/−Rag2^+/+ and DNase II^−/−IFN-IR^−/−Rag2^−/−, and when the value was <0.05, it is shown by *. (B) A section through a joint of a 12-mo-old DNase II^−/−IFN-IR^−/−Rag2^−/− mouse stained with H&E. (Scale bar, 0.1 mm.) (C) The mRNA level of the indicated genes in the joints of 11–14-mo-old WT, DNase II^−/−IFN-IR^−/−Rag2^+/−, and DNase II^−/−IFN-IR^−/−Rag2^−/− mice was quantified by real-time PCR and is expressed relative to the GAPDH mRNA level. Bars indicate the mean value. P values are shown.

Fig. 3.

TNFα-dependent arthritis. (A) Arthritis scores of the DNase II^Δ/−TNFα^+/+, DNase II^Δ/−TNFα^+/−, and DNase II^Δ/−TNFα^−/− mice (n = 6–9 for each) at the indicated time after poly(I):poly(C) injection, with SD. P values were calculated between the DNase II^Δ/−TNFα^+/+ and DNase II^Δ/−TNFα^−/−, and between the DNase II^Δ/−TNFα^+/+ and DNase II^Δ/−TNFα^+/−. *P = 0.01–0.05, **P < 0.01. (B) Section of a joint from a DNase II^Δ/−TNFα^−/− mouse 11 mo after poly(I):poly(C) injection, stained with H&E. (Scale bar, 0.1 mm.) (C) RNA from the joints of WT, DNase II^Δ/−TNFα^+/+, DNase II^Δ/−TNFα^+/−, or DNase II^Δ/-TNFα^−/− mice 9–12 mo after poly(I):poly(C) injection, was analyzed by real-time PCR for the indicated mRNA. The data are expressed relative to the GAPDH mRNA level with horizontal bars for the mean value. P values are shown.

Fig. 4.

IL-6–dependent arthritis. (A) Arthritis scores of the DNaseII^Δ/−IL-6^+/+, DNase II^Δ/−IL-6^+/−, and DNase II^Δ/−IL-6 ^−/− mice (n = 5–7 per genotype) after the injection of poly(I):poly(C), plotted with SD. P values were calculated between DNase II^Δ/−IL-6^+/+ and DNase II^Δ/−IL-6^−/− and between DNase II^Δ/−IL-6^+/+ and DNase II^Δ/−IL-6 ^+/−. *P = 0.01–0.05; **P < 0.01. (B) A section of a joint from a DNase II^Δ/−IL-6^−/− mouse 11 mo after the poly(I):poly(C) injection, stained with H&E. (Scale bar, 0.1 mm.) (C) Real-time PCR analysis of the joint RNA from WT, DNase II^Δ/−IL-6^+/+, DNase II^Δ/−IL-6^+/−, and DNase II^Δ/−IL-6^−/− mice 11–12 mo after the poly(I):poly(C) injection for the indicated mRNA. The data are expressed relative to the GAPDH mRNA level with the mean value (bars). P values are shown.

Fig. 5.

IL-1β–dependent arthritis. Rat anti-IL-1R mAb or control IgG was injected into 1-mo-old DNase II^−/−IFN-IR^−/− mice (n = 4 per group). (A) The arthritis scores were determined at the indicated time and are plotted with SD. (B) At 2.5 mo after the treatment, the serum MMP-3 level was determined. Horizontal bars indicate the mean value. The MMP-3 level of the age-matched WT littermate mice is also shown. (C) RNA from the joints of DNase II^−/−IFN-IR^−/− mice treated with rat IL-1R mAb or control IgG was analyzed by real-time PCR 2.5 mo after the treatment for the indicated mRNA. Values are expressed relative to the GADPH mRNA level with horizontal bars for the mean values. Values for the joints of the age-matched WT littermate mice are also shown. P values are shown for B and C.

Fig. 6.

Therapeutic effect of MTX, anti-IL–6R, and anti-IL-1R treatments. The DNase II^−/−IFN-IR^−/− mice (3.5–13 mo old) with affected joints were treated with MTX (n = 9–10 for each group) (A), anti-IL–6R (n = 9–10 for each group) (B and C), or anti-IL-1R mAb (n = 3–6 for each group) (D and E), and arthritis scores for the indicated mice are plotted with SD. After a 4-wk treatment with anti-IL–6R mAb (C) or anti-IL-1R mAb (E), the indicated mRNA level in the joints was quantified by real-time PCR and shown relative to the GAPDH mRNA. The mean value is indicated (bars). RNA from the age-matched littermate control mice was also analyzed. P values were calculated. In A, B, and D, *P = 0.01–0.05; **P < 0.01.