Fig 1.
IL-1β synergizes with IL-17 to produce IL-6, independently from Cd exposure.
Time course of IL-6 production in supernatants of synoviocytes exposed to cytokine combinations. Red lines indicate the chosen condition to perform the next experiments. Data are represented as mean ± SEM of at least three independent experiments; * shows differences between non-inflammatory and inflammatory conditions, # shows differences due to Cd addition; */# p<0.05, **/##p<0.01.
Fig 2.
Cd slightly accumulates in synoviocytes exposed to IL-17/IL-1β, through the reduced expression of ZIP-8 and MT-1F.
A. Cd concentration in medium as function of time and after 48-hour exposure. Gray dashed line: control experiment with no cytokine addition. Vertical dashed line at 48 hours: wash. B. Cd concentration in synoviocytes after 48-hour exposure to cytokines followed by wash and 72 hours in normal medium. Measured performed at 120 hours. C. Gene expression of Cd transporters in comparison to GAPDH. Black histograms: exposure to Cd. Data are represented as mean ± SEM of at least three independent experiments; * shows differences between non-inflammatory and inflammatory conditions, *p<0.05.
Fig 3.
Cd shifts the monocyte-derived cytokine production through the TNF pathway.
IL-1β and TNF-α production were measured after 5 days of exposure to Cd A. In single-cultured monocytes and PBMCs, activated or not with PHA B. In co-cultures of RA synoviocytes and PBMCs activated or not with PHA. Data are represented as mean ± SEM of at least three independent experiments; * shows differences between non-inflammatory and inflammatory conditions, *p<0.05. Linked points represent cells from the same subject.
Fig 4.
Cd addition in the presence of IL-1β increases IL-6 production.
A. Annexin V staining of Cd synoviocytes stimulated or not with IL-17 TNF-α and IL-1β and their combination in the presence or not of Cd after 5 days. Dot plot of morphology and histrograms of annexin V staining representative of the experimental conditions (without stain, Cd 0.1 ppm, positive control, 0.1 ppm +IL-17/IL-1 and 0.1 ppm +IL-17/TNF) and graph representing all experimental conditions. B. Neutral red assay of synoviocytes stimulated or not with IL-17 TNF-α and IL-1β and their combination in the presence or not of Cd after 5 days. Dashed line in line with 100% viability. C. IL-6 production in supernatants of synoviocytes previously exposed to cytokine combinations and to 0.1 ppm of Cd (0.89 μM) for 5 days. Data are represented as mean ± SEM of at least three independent experiments; * shows differences between non-inflammatory and inflammatory conditions, # shows differences due to Cd addition; */# p<0.05.