Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice

Abstract

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3^-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3^-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3^-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca²⁺ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3^-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3^-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1β, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3^-/- mice showed decreased intracellular Ca²⁺ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Keywords: B cell activation; Nkx2-3; autoimmune arthritis.

Figure A1

Comparison of the quantitative markers of bone microarchitecture in arthritic Nkx2-3^−/− and BALB/c mice based on the micro-CT scans (representative images are shown in Figure 2). We compared the bone pore number (Po.N) (A,B), volume (Po.V) (C,D) and surface (Po.S) (E,F), and the Bone Surface/Total Volume (BS/TV) values (G,H) of the metatarsal (A,C,E,G) and tarsal (B,D,F,H) regions from arthritic Nkx2-3^−/− (black bars) and BALB/c (gray bars) mice, respectively. Bars show the mean ± SEM values calculated from the data of 2 Nkx2-3^−/− and 2 BALB/c mice.

Figure 1

The comparison of the clinical parameters of recombinant human G1 (rhG1)-induced arthritis (GIA) in Nkx2-3^−/− and control BALB/c mice. Female Nkx2-3^−/− (n = 40) and control BALB/c (n = 27) mice were immunized with rhG1 and dimethyl-dioctadecyl-ammonium (DDA) adjuvant intraperitoneally three times every third week. The severity score (A) and incidence (B) of the induced arthritis is shown on the diagrams. Black arrows show the time of the third immunization (Day 42). Severity of the disease was determined every second day with the help of a scoring system ranging from 1 to 4, based on the swelling, redness and ankylosis of the joints of the paws. Clinical scores are visualized as mean ± standard error of mean (SEM). The thickness of the limbs (C) were measured with a digital caliper two weeks after the third immunization. The diagrams show the thickness values of the wrist (C/a), legs (C/b) and ankles (C/c) as mean ± SEM. Statistically significant differences (∗ p < 0.05) are indicated.

Figure 2

The comparison of the bone microarchitectural changes caused by GIA in Nkx2-3^−/− and control BALB/c mice. Arthritic Nkx2-3^−/− (n = 2) and control BALB/c (n = 2) mice were anesthetized and micro-CT scans were made from the right hind limbs. Representative images show the dorsal (A) and (B) or plantar (C,D) or side views (E,F) of the arthritic legs from BALB/c (A,C,E) and Nkx2-3^−/− (B,D,F) mice, respectively. Pseudocolored images (A/b,c, B/b,c, C/b,c, D/b,c, E,F) show the bone densities (violet and blue colors indicate low density-; yellow, red and green colors indicate high-density areas, respectively). The white dashed line-surrounded rectangular areas indicated in A/b, B/b, C/b and D/b are shown with higher magnification in A/c, B/c, C/c and D/d.

Figure 3

The comparison of the rhG1-induced immune response in Nkx2-3^−/− (n = 9) and control BALB/c (n = 9) mice. Nkx2-3^−/− mice were divided into arthritic (n = 5) and nonarthritic (n = 4) subgroups during the analysis. In all diagrams (A–F), bars show the mean ± SEM values calculated from n = 5 arthritic Nkx2-3^−/− mice (black), n = 4 nonarthritic Nkx2-3^−/− mice (gray) and n = 9 arthritic control BALB/c mice (white). Statistically significant differences (∗ p < 0.05) are indicated. (A) Proliferation of spleen cells was tested after incubation in the presence or absence of rhG1 for 5d in vitro. Bars represent the stimulation index (SI) calculated as a ratio of stimulated/nonstimulated values of the same mice. (B) In vitro cytokine production of spleen cells was tested after incubation in the presence or absence of rhG1 for 5 d. Cell culture supernatants were harvested and the specific cytokine concentrations were measured by sandwich ELISA. Note, spleen cells from the nonarthritic Nkx2-3^−/− group did not produce any measurable amount of cytokines. (C) The serum anti-rhG1-specific IgG1 antibodies were measured using indirect ELISA. Sera were diluted at 1:8000. Bars show the optical density values measured at 492 nm. (D,E) The serum anti-CCP-specific IgG1 and IgG2a antibodies were measured using indirect ELISA. Sera were not diluted. Bars show the optical density values measured at 450 nm. (F) Serum cytokine levels were measured with sandwich ELISA.

Figure 4

The comparison of the Ca²⁺-signals of B and T cells in Nkx2-3^−/− and control BALB/c mice. Cells were isolated from the inguinal (A,C,E) or mesenteric (B,D,F) lymph nodes of Nkx2-3^−/− and BALB/c mice and loaded with the Ca²⁺-specific indicator Fluo-3. Activation of B cells was induced by anti-IgM (A,B) or anti-IgG (C,D), activation of T cells was induced by anti-CD3 cross-linking (E,F). The changes in the intracellular Ca²⁺ levels of B or T cells were measured with a flow cytometer in the FL1 channel for five and a half minutes. Graphs show the time-dependent changes in the FL1 fluorescence (ratiometric with the intracellular Ca²⁺ level) as mean ± SEM values calculated from the data of n = 3 Nkx2-3^−/− and n = 3 BALB/c mice. * p < 0.05.