Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1ß and tumour necrosis factor α

Abstract

Objective: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1ß and TNFα.

Methods: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 µg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), IFNγ, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay.

Results: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1ß, or TNFα increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1ß, and TNFα did not induce M-CSF, GM-CSF, IFNγ, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1ß, but less potent than that of TNFα. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1ß, and greatly inhibited induction by TNFα.

Conclusion: IL18, IL1ß, or TNFα can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1ß, but less potent than TNFα.

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Figure 1.

 Soluble RANKL production in cultured human T cells in the presence of IL18, IL1ß, or TNFα. (A) T cells were isolated from the peripheral blood of healthy donors. (B) T cells were isolated from the peripheral blood of healthy donors, and then simulated with 2 µg/ml PHA and 0.5 ng/ml IL2 for 24 hours. (C) T cells were isolated from the synovium of patients with RA. Cells were cultured in quadruplicate. The supernatants were harvested after 48 hours' incubation. RANKL levels were determined by ELISA. Data represent mean (SD). *p<0.05, **p<0.01 compared with control group. Results shown are representative of four independent experiments.

Figure 2.

 Membrane bound RANKL expression in cultured human T cells in the presence of IL18, IL1ß, or TNFα. RANKL expression on T cells was analysed by flow cytometry. A representative histogram overlay of 10 ng/ml IL18 (a), 10 ng/ml IL1ß (b), or 10 ng/ml TNFα (c) stimulated (solid line) and unstimulated (broken line) cells. Changes in ΔMFI (difference between the mean fluorescence intensity (MFI) of positive staining and the MFI of the isotype matched negative control) are also shown (d). *p<0.05, **p<0.01 compared with the control group. See fig 1 for other information.

Figure 3.

 Osteoclast formation induced from the precursor cells in different conditions. All the cultures were stimulated with 33 ng/ml M-CSF. With the indicated conditions, the cultures were further stimulated with 30 ng/ml soluble RANKL or 10 ng/ml TNFα, or cocultured with PHA prestimulated T cells in the presence of 10 ng/ml IL18, IL1ß, or TNFα. After 7–10 days of culture, cells were fixed and stained for TRAP. TRAP positive giant cells with numerous (⩾3) unstained nuclei were considered as mature osteoclasts. See scale bar in each picture. In (B) T indicates PHA prestimulated T cells. *p<0.05, **p<0.01 compared with the group with T cells only.

Figure 4.

 Resorption activity of induced osteoclasts on dentine slices. The osteoclasts were induced as in fig 3. Dentine slices were stained in Mayer's haematoxylin solution, and pits were photographed under a reflected light microscope (A). Scale bar 100 µm. The total pit areas were measured in four randomly selected areas of each dentine slice using an image analysis system (B). In (B) T indicates PHA prestimulated T cells. **p<0.01 compared with the group with T cells only.

Figure 5.

 Osteoclast formation induced from the precursor cells cocultured with RA synovial T cells. All the cultures were stimulated with 33 ng/ml M-CSF. With the indicated conditions, the cultures were further stimulated with 10 ng/ml IL18, IL1ß, or TNFα. Scale bar 200 µm. In (B) T indicates RA synovial T cells. **p<0.01 compared with the group with T cells only.

Figure 6.

 Bone resorption activity of osteoclasts induced by RA synovial T cells. The osteoclasts were induced as in fig 5. The total pit areas were measured in four randomly selected areas of each dentine slice using an image analysis system. T indicates RA synovial T cells. **p<0.01 compared with the group with T cells only.

Figure 7.

 A schematic diagram of IL18, IL1ß, or TNFα indirectly stimulating osteoclastogenesis through T cells in synovitis. In RA synovium, accumulated monocytes/macrophages (and synovial fibroblasts) produce proinflammatory cytokines, such as TNFα, IL1ß, and IL18. These cytokines enhance RANKL production from activated T cells, and then induce osteoclast precursors to differentiate into mature osteoclasts.