Involvement of IL-6, apart from its role in immunity, in mediating a chronic response during experimental arthritis

Abstract

Interleukin-6 (IL-6) is highly produced during arthritis but its exact function is still unknown. In this study we examined if IL-6, apart from its role in immunity, was involved in the local inflammatory response in experimental arthritis. IL-6 deficient (IL-6(-/-)) and wild-type mice were first compared in the antigen-induced arthritis model. IL-6 deficiency resulted in a mild, transient inflammation whereas wild-type mice developed a chronic, destructive synovitis. Wild-type mice immunized with one-tenth of the normal antigen dose still developed chronic arthritis despite low antibody levels, excluding reduced humoral immunity in IL-6(-/-) mice as a crucial phenomenon. In addition, passive immune-complex-induced arthritis did not differ between wild-type and IL-6(-/-) mice. Another option is reduced levels of Th1 cells in IL-6(-/-) mice. However, transfer of antigen-specific wild-type lymph node cells to IL-6(-/-) mice enhanced acute joint inflammation and increased cartilage damage but still could not sustain chronic inflammation, suggesting involvement of nonimmune elements of IL-6 activity in chronicity. In line with this, nonimmunologically mediated zymosan-induced arthritis developed similarly in the first week, but only wild-type mice developed chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity.

Figure 1.

Joint swelling at day 1 and 2 after mBSA injection. Immunized and naive mice were injected with 60 μg of mBSA. Joint swelling was measured as the ratio of ^99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left). ▪, IL-6^−/−; □, wild type (n = 7; *, P < 0.05; **, P < 0.005, Student’s t-test).

Figure 2.

Inflammation and cartilage damage in wild-type (A and B) and IL-6^−/− (C and D) mice on day 2 (A and C) and 7 (B and D) of the AIA. Sections were stained with safranin-O and cartilage damage was observed as loss of red staining. E: Position of patella (P), femur (F), synovium (S), growth plate (GP), and cartilage (C) is shown. Original magnification, ×200.

Figure 3.

mRNA expression in synovia from wild-type and IL-6^−/− mice on day 1 (A) and 2 (B) of the AIA. The number of PCR cycles needed to first detect the specific band on an agarose gel was compared between synovia from arthritic and contralateral nonarthritic knee joints. Increased mRNA expression for the investigated gene in arthritic synovia results in a reduced number of PCR cycles to first detect the specific band. Four mice per group were used and equal amounts of cDNA were used as assessed by PCR for glyceraldehyde-3-phosphate dehydrogenase (all appeared at cycle 14). Basal expression in the nonarthritic synovia did not differ between wild-type and IL-6^−/− mice. No signal for IL-6 was detected at the end point of the PCR with cDNA from IL-6^−/− mice. ▪, IL-6^−/−; □, wild type.

Figure 4.

A: mBSA-specific antibody subclasses before onset of AIA. Titers as depicted are the dilution of sera needed to give half the maximal optical density at 450 nm as described in Materials and Methods. ▪, IL-6^−/−; □, wild type (n = 7). B: mBSA-specific IgG2a titers in mice immunized and boostered with 100 μg of mBSA (IL-6^−/−) or with 10 or 100 μg of mBSA (wild type). Each symbol represents one mouse (P = 0.22, IL-6^−/− (100 μg) versus wild type, (10 μg) Student’s t-test). Total IgG titers also did not differ (IL-6^−/−: 1/720 ± 194, wild type: 1/734 ± 180). C: Joint swelling in wild-type mice immunized with 10, 30, or 100 μg of mBSA (n = 8). Joint swelling was measured as the ratio of ^99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left). ▪, IL-6^−/−; □, wild type (A and C: *, P < 0.05; **, P < 0.005, Student’s t-test). One of three experiments is shown.

Figure 5.

A: mBSA-specific increase in cpm (cpm mBSA minus cpm medium). Lymph nodes from seven mice per group were pooled and enriched for T cells as described in Materials and Methods. Lymph node cells (2×10⁶) together with 2×10 irradiated antigen-presenting cells were incubated for 72 hours with mBSA at 25 μg/ml. Cells were plated in sixfold. Cultures were labeled with ³H for the last 16 hours. Antigen-presenting cells were used from naive mice of the same strain. IL-6^−/− and wild-type mice did not differ in the response to concanavalin A at 1 μg/ml (IL-6^−/−, 28,002 ± 7,291 cpm; wild type, 25,436 ± 1,216 cpm) One of four experiments is shown. B: Th1/Th2 multiplex RT-PCR of IL-6^−/− (100 μg mBSA) or wild-type (10 μg mBSA) T cells stimulated for 24 hours with conA (1 μg/ml). C = positive control.

Figure 6.

Joint swelling on day 2 of AIA in IL-6^−/− mice after transfer of 4×10 lymph node cells derived from mBSA (wild-type/mBSA) or ovalbumin (wild-type/OVA) immunized wild-type mice (A) or of mBSA (IL-6^−/−/mBSA) immunized IL-6^−/− mice (B). NT = normal AIA in wild-type mice immunized with 10 μg of mBSA. Joint swelling was measured as the ratio of ^99mTc uptake in the arthritic knee (right) over the nonarthritic knee (left) (n = 6; *, P < 0.05, Student’s t-test, n.s. = not significant). ▪, IL-6^−/−, □, wild type. One of three experiments is shown.