LIGHT is involved in the pathogenesis of rheumatoid arthritis by inducing the expression of pro-inflammatory cytokines and MMP-9 in macrophages

Abstract

Macrophages play a crucial role in the perpetuation of inflammation and irreversible cartilage damage during the development of rheumatoid arthritis (RA). LIGHT (TNFSF14) and its receptor TR2 (TNFRSF14) are known to have pro-inflammatory activities in foam cells of atherosclerotic plaques. We tested a hypothesis that LIGHT and TR2 are involved in activation of monocyte/macrophages in RA synovium. Immunohistochemical analysis of RA synovial tissue samples revealed that both LIGHT and TR2 are expressed in CD68 positive macrophages. In contrast, synovial tissue samples from osteoarthritis (OA) patients failed to reveal the expression of LIGHT. Expression of TR2 in RA synovial macrophages was also detected using flow cytometry analysis. To identify the role of LIGHT in the functioning of macrophages in RA, we isolated macrophage enriched cells from RA synovial fluid and stimulated them with LIGHT. LIGHT induced expression of matrix metalloproteinase-9 and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8. These data indicate that LIGHT and TR2 expressed in macrophages are involved in the pathogenesis of RA by inducing the expression pro-inflammatory cytokines and matrix degrading enzymes.

Figure 1

Expression patterns of LIGHT and TR2 in RA synovium. Synovial tissue sections from RA (a) and OA patients (b) were analyzed for the expression of vWF (the marker for endothelial cells), LIGHT, TR2, and CD68 (the marker for macrophages) using immunohistochemical analysis as described in materials and methods. Sublining areas around the newly formed microvessels are magnified in the lower panel. The box in the low magnification picture (40X, upper panel) indicates the area magnified in high magnification pictures (400X, lower panel). Note that microvessels (arrows) are stained with antivWF antibodies but not with other antibodies. The pictures are representatives from analysis using 7 different RA and 5 different OA synovial tissue specimens.

Figure 2

Expression of LIGHT and TR2 in macrophages in the RA synovium. Synovial tissue sections were analyzed for the expression of LIGHT, TR2, and CD68 using double immunohistochemical analysis as described in materials and methods. CD68 was stained in red, LIGHT or TR2 in brown, and nuclei were stained with hematoxylin (H) in blue. Box in the low magnification pictures (100X, upper panel) indicate the area magnified in high magnification pictures (400X, lower panel). Note that areas heavily infiltrated with lymphocytes (white arrows) are not stained with anti-LIGHT nor anti-TR2 monoclonal antibodies. The analysis was performed on samples obtained from two different RA patients with essentially the same results.

Figure 3

Expression of TR2 is increased in RA synovial macrophages. White blood cells obtained from peripheral blood (PB) or synovial fluid (SF) from RA patients were stained as described in materials and methods. Mean fluorescence intensity (MFI) values of TR2 and CD11b in granulocytes and macrophages were compared. Open, grey, and black bars represent MFI obtained from background staining, specific staining of peripheral blood cells, and specific staining of synovial fluid cells, respectively. In the insets, histograms from specific staining (filled area) and background staining (open area) of synovial fluid cells were compared. The experiments were repeated three times in different RA patients with essentially the same results.

Figure 4

LIGHT treatment causes induction of pro-inflammatory cytokines in macrophage enriched cells isolated from RA synovium. Adherent mononuclear cells and granulocytes isolated from RA synovial fluids were stimulated with recombinant human LIGHT in the presence or absence of 100 U/ml IFN-γ. Culture supernatants were collected 24 h after activation and cytokine concentrations were measured using ELISA. Error bars represent standard deviation of triplicate measurements. C, no treatment control; L, treatment with 1 µg/ml LPS as a positive control. The experiments were repeated three times with cells isolated from different RA patients with essentially the same results. *P < 0·01 and **P < 0·001 vs. no treatment control.

Figure 5

LIGHT treatment causes induction of MMP-9 in macrophage enriched cells isolated from RA synovium. Adherent mononuclear cells isolated from synovial fluid of RA patients were stimulated with recombinant human LIGHT in the presence or absence of 100 U/ml IFN-γ. Culture supernatants were collected 24 h after activation and MMP-9 expression levels were measured using gelatin-zymogram. As a positive control, cells were treated with 1 µg/ml LPS.