Exposure to receptor-activator of NFkappaB ligand renders pre-osteoclasts resistant to IFN-gamma by inducing terminal differentiation - PubMed (original) (raw)

Exposure to receptor-activator of NFkappaB ligand renders pre-osteoclasts resistant to IFN-gamma by inducing terminal differentiation

Willis Huang et al. Arthritis Res Ther. 2003.

Abstract

While it has been established that IFN-gamma is a strong activator of macrophages and a potent inhibitor of osteoclastogenesis in vitro, it is also known that this cytokine is produced in particular settings of inflammatory bone loss, such as infection and psoriatic arthritis. Because of the different kinetics between rapid IFN-gamma macrophage activation (<24 hours) and the slower receptor-activator of NFkappaB ligand (RANKL) osteoclast differentiation (7 days), we postulated that IFN-gamma would have different effects on early-stage and late-stage osteoclast precursors. In RAW264.7 cells and primary splenocyte cultures, pretreatment with RANKL rendered these cells resistant to maximally anti-osteoclastogenic doses of IFN-gamma. These cells were also resistant to IFN-gamma-induced nitric oxide production, morphological change, and surface upregulation of CD11b and receptor-activator of NFkappaB, suggesting that early exposure of osteoclast precursors to RANKL induces a broad resistance to the cellular effects of IFN-gamma. Changes in STAT1 activation did not correlate with this resistance, as IFN-gamma activated STAT1 equally in both early-stage and late-stage pre-osteoclasts. Furthermore, we failed to observe changes in TRAF6 expression following IFN-gamma treatment in pre-osteoclasts. Together these data support a model of inflammatory bone loss in which early exposure to RANKL can prime osteoclast precursors to form in the presence of high levels of IFN-gamma using mechanisms independent of the signal molecules STAT1 and TRAF6.

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Figures

Figure 1

Figure 1

Receptor-activator of NFκB ligand (RANKL) induces osteoclastogenesis in RAW264 cells. (a) Bacterially produced GST-RANKL protein was purified and visualized with SDS-PAGE/Coomassie Blue against 99% pure BSA standards. (b) RAW cells were cultured with the indicated doses of GST-RANKL for 4 days and then stained for tartrate-resistant acid phosphatase.

Figure 2

Figure 2

IFN-γ dominantly inhibits receptor-activator of NFκB ligand (RANKL)-mediated osteoclastogenesis. (a) RAW cells received the indicated cytokine treatments of IFN-γ (10 ng/ml) and/or RANKL (200 ng/ml). Cells were stained for the osteoclast marker tartrate-resistant acid phosphatase (TRAP) on day 5 and osteoclast numbers were counted; a 1× micrograph of a representative well is shown above its corresponding column. TRAP positivity and osteoclast formation are seen only in the absence of IFN-γ. (b)-(e) Micrographs (10×) of the same groups show that IFN-γ stimulates a stellate morphology and inhibits osteoclastogenesis. All panels represent continuous cytokine treatment, except for the IFN-γ washout group (e). OC, osteoclast.

Figure 3

Figure 3

Pretreatment with receptor-activator of NFκB ligand (RANKL) permits osteoclastogenesis in the presence of maximally inhibitory IFN-γ in a dose-dependent and time-dependent manner. RAW cells were grown in the presence of continuous RANKL (100 ng/ml) for 4 days, in the absence or presence of IFN-γ as indicated on either day 0 or 2 of culture. (a) Representative photographs from each group are shown at 10× magnification, and (b) the average number of osteoclasts per well ± SEM is presented. (c), (d) The experiments were repeated with primary osteoclast precursors in splenocyte cultures. For both RAW cells and primary osteoclast precursors, RANKL pretreatment allowed for osteoclastogenesis despite the presence of a maximally inhibitory dose of IFN-γ (10 ng/ml). *P < 0.05 compared with either untreated controls or cells treated with IFN-γ for the whole culture period.

Figure 4

Figure 4

Osteoclast formation in the presence of IFN-γ depends upon receptor-activator of NFκB ligand (RANKL) pretreatment in a dose-dependent and time-dependent manner. (a) RAW cells were pretreated with the indicated doses of RANKL for 2 days. On day 3, cells were switched to media with RANKL (200 ng/ml GST-RANKL) and various doses of IFN-γ, were then fixed and tartrate-resistant acid phosphatase (TRAP) stained after day 4. (b) The mean ± SEM of three independent experiments containing the highest dose of IFN-γ in (a) with statistics. *P = 0.05 compared with RANKL pretreatment at 0 ng/ml. (c) RAW cells treated continuously with a suboptimal dose of GST-RANKL (50 ng/ml), to slow osteoclastogenesis, were given IFN-γ (10 ng/ml) at the indicated time points. All cells were fixed and stained for TRAP on day 6. TRAP staining was quantitated densitometrically as described in Materials and methods. RANKL-treated RAW cells (no IFN-γ) stained on day 2 were only 10–20% TRAP-positive. All cultures with RANKL pretreatment ≥ 24 hours underwent osteoclastogenesis. *P = 0.05 compared with the IFN-γ day 0 group.

Figure 5

Figure 5

TRAF6 levels and STAT1 signaling are intact in late pre-osteoclasts. (a) Untreated RAW cells were stimulated with IFN-γ for 5, 15 and 60 min, and then protein extracts were analyzed by immunoblotting as described in Materials and methods. Multiple signals were achieved by stripping and reprobing the same blot. TRAF6 expression increased with IFN-γ signaling. (b) RAW cells were cultured for 4 days in the presence of the indicated cytokines. Osteoclasts were observed in the receptor-activator of NFκB ligand (RANKL) group, while activated macrophages were observed in the RANKL + IFN-γ group similar to that described in the previous experiments. TRAF6 expression was remarkably consistent in these cells. (c) Nuclear extracts were prepared from untreated RAW cells and late pre-osteoclasts (treated with RANKL for 2 days) and immunoblotted with antiphospho-Stat1 antibodies to assess nuclear translocation in extracts obtained at the indicated time points following IFN-γ treatment. Late pre-osteoclasts translocated phospho (p)-Stat1 as well as untreated RAW cells.

Figure 6

Figure 6

Receptor-activator of NFκB ligand (RANKL) pretreatment impairs IFN-γ-induced nitric oxide (NO) production in a dose-dependent manner. (a) RAW cells were treated as in Figure 2a, and supernatants assayed for NO production on day 4. NO production was significantly inhibited by pretreatment with RANKL (*P < 0.01 versus the IFN-γ only group). (b) RAW cells were grown in the presence of various doses of RANKL, then supplemented with various doses of IFN-γ and maximal GST-RANKL (200 ng/ml) on day 2. NO was assayed on day4. Black bars indicate cells treated on day 2 with 100 ng/ml IFN-γ. (c) This group (indicated by black bars) with statistics. Inhibition of NO production by RANKL pretreatment is dose dependent, with more inhibition with higher doses of RANKL (*P < 0.05 versus 0 ng/ml RANKL).

Figure 7

Figure 7

Late pre-osteoclasts are resistant to IFN-γ-induced surface expression of CD11b and receptor-activator of NFκB (RANK). (a) Cells were treated with the indicated cytokines for 4 days, then analyzed for CD11b expression by FACS. Late pre-osteoclasts (RAW cells pretreated with 200 ng/ml receptor-activator of NFκB ligand [RANKL] for 2 days prior to IFN-γ treatment) expressed CD11b at levels nearly identical to RANKL-only treated cells, suggesting that late pre-osteoclasts are resistant to IFN-γ-induced CD11b upregulation. (b) Identically treated cells were analyzed for RANK expression by FACS. The proportion of RANKhi cells was nearly identical in RANKL-only treated cells and late pre-osteoclasts, suggesting that late pre-osteoclasts are resistant to IFN-γ-induced upregulation of RANK. MFI, mean fluorescence intensity.

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