Dose effect of cytokines on c-Fos, c-Jun, Egr-1 and NF-κB nuclear translocation
We first tested the effect of IL-1β, TNF-α and IL-17 alone on RA synoviocytes to define the minimal and optimal concentrations able to induce nuclear translocation of the major TFs. A time course was performed and 30 min after the initiation of stimulation appeared to be a time point where most of the TFs were observed in the nucleus (data not shown). After 30 min of stimulation with increasing cytokine concentrations, cells were fixed and stained. The dose effect of each cytokine was evaluated by the measurement of the mean green intensity of the nuclear staining with image analysis software. The quantitative data are presented in Table . Results are compared with the condition using medium alone.
Dose effect of individual cytokines on TF nuclear translocation
RA synoviocytes did not show any TF translocation without cytokine. With increasing concentrations of IL-1β, ranging from 0 to 500 pg/ml, C-Fos and C-Jun were found in the nucleus with concentrations from 100 to 500 pg/ml (P < 0.001 versus medium alone). NF-κB activation was more sensitive since a concentration as low as 10 pg/ml was sufficient (P < 0.01 versus medium). For IL-1β concentrations ranging from 0 to 500 pg/ml, no translocation was observed for Egr-1. Accordingly, an optimal concentration of 100 pg/ml IL-1β was chosen as a positive control for further experiments, while a suboptimal concentration of 10 pg/ml IL-1β had a weak effect only on NF-κB translocation.
Similarly, with concentrations of TNF-α ranging from 0 to 500 pg/ml, C-Fos and C-Jun translocation was observed in the nucleus (P < 0.01 and P < 0.001, respectively). NF-κB nuclear localization was observed for the same concentrations (P < 0.001) while no Egr-1 translocation was observed. Accordingly, the optimal concentration of TNF-α was defined as 100 pg/ml.
IL-17 activation was clearly less potent since concentrations of IL-17 as high as 100 ng/ml did not have an effect on C-Fos, C-Jun or Egr-1 nuclear translocation. These concentrations did, however, induce NF-κB translocation (P < 0.01 versus medium).
Effect of individual cytokines on other TF translocation
Initial experiments were performed to establish the optimal conditions under which a first set of factors was activated. Additional experiments were performed to study the other members of the AP-1 complex. Fra-1, Fra-2, FosB, JunB and JunD nuclear translocation was evaluated after 30 min of cytokine addition at low and optimal concentrations (Table ).
As already observed for C-Fos, C-Jun, and NF-κB, 30 min of treatment with IL-1β (100 pg/ml) induced a nuclear translocation of FosB, Fra-1, Fra-2 and JunD (P < 0.001 versus medium alone), but not of JunB. Conversely, a concentration of 10 pg/ml IL-1β had no effect except on Fra-1 (P < 0.01) and on NF-κB (P < 0.01).
TNF-α (100 pg/ml) induced a nuclear localization of FosB, Fra-1, JunB and JunD (P < 0.001 versus medium), but not of Fra-2. FosB activation was more sensitive to the effect of TNF-α, since a concentration as low as 10 pg/ml was sufficient to induce its translocation (P < 0.001 versus medium).
IL-17 (100 ng/ml) had a weak effect on Fra-1 (P < 0.05) but not on FosB, on Fra-2, on JunB and on JunD. A concentration of 50 ng/ml IL-17 had no effect.
Effect of cytokine combination on TF nuclear translocation
IL-1β and TNF-α are mainly produced by monocytes while IL-17 is produced by T cells. To reproduce the interactions observed in the synovium, these three cytokines were combined at low concentrations, which may reflect the in vivo situation.
Combined treatment with low concentrations of IL-1β and of TNF-α induced a nuclear translocation of all TFs except Egr-1 (Fig. ). A synergistic effect was observed since the same low concentrations of IL-1β or TNF-α used alone had no effect. C-Jun was particularly sensitive to the effect of the combined treatment of IL-1β and TNF-α. Furthermore, an enhanced stimulation was observed for Fra-1 and NF-κB when compared with the effect observed with individual cytokines at low concentration. Figure shows a representative experiment in which a nuclear localization of C-Jun was observed in most cells after stimulation with the combination of low concentrations of cytokines.
Figure 1 Quantification of the effects of individual cytokines and their combinations on TF nuclear translocation. After 12 hours of culture without FCS, rheumatoid arthritis synoviocytes (104 cells/cm2) were stimulated for 30 min with cytokines at the cited concentrations. (more ...)
Figure 2 Effect of IL-1β, tumor necrosis factor alpha (TNF-α) and IL-17 used alone and in combination on C-Jun translocation. Rheumatoid arthritis synoviocytes were stimulated for 30 min with cytokines at the cited concentrations. Cells were fixed (more ...)
IL-17 treatment in combination with TNF-α further induced the nuclear translocation of C-Fos, Fra-1, C-Jun and NF-κB. In the same way, combined treatment with low concentrations of IL-1β and of IL-17 showed a synergistic effect on the nuclear translocation of C-Fos, of FosB, of Fra-1, of Fra-2, of C-Jun, of JunD and of NF-κB. This combination had a particular effect on Fra-1 and NF-κB activation.
The combination of the three cytokines was even more potent, leading to a synergistic effect on all of the TF translocations except for Egr-1. This IL-1β, TNF-α and IL-17 combination of cytokines had a particularly strong synergistic effect on the nuclear translocation of Fra-1, of C-Jun and of NF-κB.
Effect of proinflammatory cytokines on mRNA expression
To determine whether these effects were related to an increase in translocation or in de novo synthesis we examined the mRNA expression, looking at whether it was particularly sensitive to a synergistic increase. The results for c-jun are shown as an example before and 20 min after cytokine treatment. At a suboptimal concentration of IL-1 (10 pg/ml) or TNF-α (10 pg/ml) c-jun mRNA expression was observed, whereas IL-17 (50 ng/ml) had no effect.
These cytokines were then combined at these low concentrations (IL-1, 10 pg/ml; TNF-α, 10 pg/ml; IL-17, 50 ng/ml) with no effect or a low effect when used alone. Treatment with the combination of low concentrations of IL-1 and TNF-α induced an additive effect on mRNA expression of c-jun, whereas the combination of low concentrations of IL-17 with TNF-α had no effect on the TNF-α -induced expression of c-jun. In contrast, the combination of low concentrations of IL-1 with IL-17 induced a synergistic effect on IL-1 and induced c-jun mRNA expression. This picture was similar to the effect seen with the combination of IL-1, TNF-α and IL-17 (Fig. ).
Figure 3 Effects of IL-1β, tumor necrosis factor alpha (TNF-α) and IL-17 used alone and in combination on c-jun mRNA expression. After 12 hours of culture without FCS, rheumatoid arthritis fibroblast-like synoviocytes were stimulated for 20 min (more ...)
Effect of proinflammatory cytokines used alone or in combination on OPG levels
We selected OPG as a marker of effects at the protein level to look at a functional consequence of these interactions of cytokines. Supernatants from RA fibroblast-like synoviocytes were collected after 72 hours of culture following cytokine addition. Concentrations of OPG were measured by ELISA.
In medium alone, RA synoviocytes produced 1 ng/ml OPG. The addition of IL-1 (10 pg/ml) doubled that production, whereas TNF-α (10 pg/ml) or IL-17 (50 ng/ml) had no effect (Fig. ). The combination of IL-17 with IL-1 and, moreover, with TNF-α further increased that production, the effect being maximal when the three cytokines were combined.
Figure 4 Determination of osteoprotegerin (OPG) levels in supernatants obtained from rheumatoid arthritis (RA) synoviocytes stimulated by proinflammatory cytokines alone or in combination. RA synoviocytes were cultured with cytokines at the cited concentrations. (more ...)