Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

Abstract

During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcgamma receptors (FcgammaRs) (mainly FcgammaRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcgammaRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.

Figure 1

Adenoviral-vector-mediated IL-13 expression in knee joints of C57Bl/6 mice. (a) Naive knee joints and (b) total knee joint sections 24 hours after injection of AxCANI (adenovirus encoding no gene) or of (c) AxCAhIL-13 (adenovirus encoding interleukin-13). Injection of AxCAhIL-13 resulted in 0.4 ng/ml IL-13 at day 1, which increased to 5.5 ng/ml by day 7 (a). Injection of AxCANI resulted in a mild thickening of the synovial lining (S) and some invading inflammatory cells in the joint cavity (JC) (b), whereas no inflammation was observed after AxCAhIL-13 injection (c). Plotted values are means ± SEM of data from 5 mice. *P < 0.05. Original magnification 200×. F, femur; P, patella.

Figure 2

Joint inflammation in arthritic knee joints of C57Bl/6 mice injected with AxCANI (adenovirus encoding no gene) or AxCAhIL-13 (adenovirus encoding interleukin-13). At (a) day 3 and (b) day 7 after the onset of immune-complex-mediated arthritis. The inflammatory cell mass was significantly enhanced by IL-13 in both the joint cavity and the synovium 3 days after arthritis induction. Bars show the means ± SEM for 10 mice. Significance was evaluated using the Mann–Whitney U test. *P < 0.05.

Figure 3

Immunohistochemical detection of inflammatory cells in knee joints of mice with immune-complex-mediated arthritis (ICA). (a) Polymorphonuclear neutrophils and (b) macrophages in synovium 3 and 7 days after injection of AxCANI (adenovirus encoding no gene) or AxCAhIL-13 (adenovirus encoding interleukin-13). Polymorphonuclear neutrophils were detected using the specific rat anti-mouse monoclonal antibody NIMPR14, and macrophages were detected using an antibody against the membrane marker F4/80. At day 7, the amount of NIMPR14-positive features was significantly higher in the synovium of AxCAhIL-13-injected arthritic knee joints, while the amount of F4/80-positive features was significantly lower. The bars represent means ± SEM for 10 mice. Data were evaluated using the Mann–Whitney U test. *P < 0.05.

Figure 4

Chondrocyte death in the knee joints of mice with immune-complex-mediated arthritis (ICA). (a) At day 3 and 7 in arthritic knee joints injected with injected with AxCANI (adenovirus encoding no gene) or AxCAhIL-13 (adenovirus encoding interleukin-13) and (b) expression profiles of Fcγ receptor I (FcγRI), II, and III mRNA levels induced by IL-13 in synovium. IL-13 significantly decreased chondrocyte death, both at day 3 and at day 7 (a). Cycle threshold (Ct) values of FcγRI, II, and III in arthritic knee joints injected with AxCANI were subtracted from the Ct values for FcγRs after injection of AxCAhIL-13. Ct values were corrected for glyceraldehyde-3-phosphate dehydrogenase content for each individual sample. (b) FcγRI mRNA level was down-regulated by IL-13, whereas an up-regulation was observed for both FcγRII and III. Bars represent means ± SEM for 10 mice. Mann–Whitney U test. *P < 0.05. D, Δ.

Figure 5

Matrix-metalloproteinase-mediated aggrecan damage in knee joints of mice with immune-complex-mediated arthritis. VDIPEN expression at day 3 and 7 after the induction of immune-complex-mediated arthritis in knee joints injected with AxCANI or AxCAhIL-13. Note that VDIPEN expression was reduced by IL-13 both at day 3 and day 7. Values represent the mean ± SEM for 10 mice. *P < 0.05, Mann–Whitney U test. AxCAhIL-13 = adenovirus encoding interleukin-13; AxCANI = adenovirus encoding no gene.