NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-gamma-stimulated immune complex arthritis.

In previous studies we have found that FcgammaRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-gamma-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcgammaRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-gamma was overexpressed by injection of adenoviral IFN-gamma in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-gamma-stimulated arthritic p47phox-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox-/- mice at both time points. FcgammaRI mRNA expression was significantly lower, and FcgammaRII and FcgammaRIII were higher, in p47phox-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-gamma-stimulated IC-mediated arthritis. Upregulation of FcgammaRI by oxygen radicals may contribute to cartilage destruction.

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

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.

Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis.

OBJECTIVE: In human osteoarthritis (OA), various forms of pathology are observed. Besides cartilage damage and fibrosis, neogenesis of bone, osteophyte formation, also occurs. Osteophytes are thought to limit joint movement and cause pain. The objective of this study was to investigate whether synovial macrophages are involved in osteophyte formation in experimental OA, and if they are, to study which mechanism may be involved. DESIGN: Experimental OA was induced by two intra-articular injections of collagenase on alternate days into murine knee joints. The role of synovial lining macrophages in this model was studied by selective removal of these cells prior to OA induction using clodronate liposomes. After 1 and 2 weeks, knee joints were dissected and examined (immuno)histologically. RESULTS: At days 7 and 14 after induction of OA, osteophyte formation and fibrosis were observed. Depletion of synovial macrophages resulted in spectacular reduction of osteophyte formation, 84% and 66%, respectively, at days 7 and 14. Fibrosis and synovial activation, measured by MRP8/14 expression, were also ameliorated (40-60%). In addition, production of growth factors (TGFbeta, BMP-2 and BMP-4) in the lining was largely prevented but production of these growth factors in deeper layers of the synovium and the periosteum did not differ from controls. CONCLUSIONS: These results indicate the synovial macrophage to be a pivotal cell in the synovium mediating osteophyte formation and other OA-related pathology, like fibrosis, during experimental OA. Production of growth factors and induction of synovial activation by these cells may play a crucial role in this event.

Increased expression of Fcgamma receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor alpha and matrix metalloproteinase.

OBJECTIVE: To evaluate Fcgamma receptor (FcgammaR) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), IL-12, and matrix metalloproteinase 1 (MMP-1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of FcgammaRI, FcgammaRII, and FcgammaRIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation. METHODS: Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. FcgammaR I, II, and III were detected, as well as the proinflammatory mediators IL-1, TNFalpha, IL-12, and MMP-1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony-stimulating factor to obtain macrophages. Using fluorescence-activated cell sorting, the expression of FcgammaRI, FcgammaRII, and FcgammaRIII was determined. On day 7, macrophages were stimulated with heat-aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme-linked immunosorbent assay, and production of gelatinases/collagenases was measured by degradation of fluorescent gelatin. RESULTS: Immunohistochemistry showed higher FcgammaRII and FcgammaRIII expression in RA synovium than in controls. FcgammaRII and FcgammaRIII, but not FcgammaRI, were highly correlated with the number of synovial macrophages. Consistent with this, TNFalpha expression correlated positively with FcgammaRIII expression. Moreover, MMP-1 expression strongly correlated with FcgammaR I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of FcgammaRII and FcgammaRIII compared with controls. Twenty-four hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNFalpha and gelatinase/collagenase was measured. CONCLUSION: RA synovium and mature RA macrophages express significantly elevated levels of FcgammaRII and FcgammaRIII, resulting in much higher production of TNFalpha and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of FcgammaR on mature synovial macrophages is involved in the pathology of RA.