Oxygen radical production determines chondrocyte death and regulates matrix metalloproteinase-mediated matrix degradation during interferon gamma-accelerated immune complex arthritis

Objective

In earlier studies we found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during interferon gamma (IFN-γ)-regulated immune complex (IC)-mediated arthritis [1]. As binding of ICs to FcγRI leads to oxygen radical production, we now investigate the contribution of oxygen radicals to induction of both parameters of cartilage destruction using P47phox knockout mice. These mice have a defect in NADPH-oxidase activation and fail to produce oxygen radicals.

Methods

IFN-γ was expressed in knee joints of P47phox^-/- and their wildtype (WT) controls by local injection of adenoviral IFN-γ construct. One day thereafter, a passive IC-mediated arthritis was induced. Chondrocyte death and MMP-mediated cartilage destruction were measured in various layers of the knee joint using histology of total knee joints. Neoepitopes induced by MMPs were detected using immunolocalisation and anti-VDIPEN antibodies. Synovium was isolated and mRNA levels (MMPs/ tissue inhibitors of metalloproteinases [TIMPs]) were determined using quantitative RT-PCR.

Results

High levels of IFN-γ were found 1 day after injection of the IFN-γ adenoviral construct in knee joints of P47^-/- and WT controls, and resulted in a high and comparable upregulation of FcγRI up to day 7 in both groups. Induction of IC-mediated arthritis in IFN-γ-treated knee joints resulted in prominent but comparable joint inflammation both at day 3 and day 7. At day 7, macrophages formed the dominant cell type. When compared with WT controls, RNA levels of MMP-3 and MMP-13, which are crucial in cartilage destruction within this model, were elevated in inflamed synovia of P47phox^-/- whereas TIMP-3, a crucial inhibitor of MMP-3, was completely absent. Nevertheless, MMP-mediated cartilage destruction was significantly lower in P47phox^-/- at day 7 (between 30–60%). Moreover chondrocyte death that was evident in arthritic WT controls (20–60% at day 3 and 30–80% at day 7) was completely blocked in P47phox^-/-.

Conclusion

During IFN-γ-regulated IC-mediated arthritis, oxygen radicals, which are strongly induced after FcγRI stimulation, determine chondrocyte death and largely mediate MMP-mediated cartilage destruction.