Fig 1.
TNF-α was produced by PBMCs in an IL-1ß-dependent fashion.
IL-1ß(A) and TNF-α(B) concentrations were measured in PBMC SN treated without or with IL-1ra (250 and 1000 ng/ml; 15 and 59 nM). IL-1ra was added at time 0 and SN collected at times 2, 4, 6,10, 16 and 24h. The relationship between TNF-α and IL-1ß concentrations were measured in the same samples obtained at the different times in absence or presence of IL-1ra. Data from 250 and 1000 ng/ml IL-1ra-treated samples were pooled (Fig 1C). Data are expressed as pg/ml from 2x104 cells in 96 well plates. This experiment has been repeated twice.
Fig 2.
IL-6 and MCP-1 production as a function of IL-1ß and TNF-α concentrations in absence or presence of IFN-γ.
IL-6 (A) or MCP-1 (B) concentration were measured after MSCs were treated with different concentration of TNF-α or IL-1β with or without IFNγ (50ng/ml). To precisely analyze the stimulating activities, basal Il-6 and MCP-1 expression (without treatment) have been subtracted from the concentrations measured under TNF-α or IL-1β with or without IFNγ stimulation. These basal concentrations were in the range of 10 to 20% of the maximal stimulating effect for IL-6 and in the range of 33% for MCP-1. The data are representative of 2 separate experiments. One over 3 measurements have been plotted for the clarity of the figures. The concentrations measured under 20 ng/ml concentration are represented as a single point (± SEM) as they did not fit with the one binding hyperbola which fitted all other concentrations obtained at different TNF-α concentrations.
Fig 3.
TNF-α-induced hUC-MSC death amplified by IFN-γ.
hUC-MSCs of two different clones (Clones 63 and 69, 2x104 in 96-well plates) were stimulated with TNF-α (20 ng/ml, 1.2 nM) or TNF-α associated with IFN-γ (50 ng/ml, 3 nM) for 48h. Then, cell death was scored by CellTiter-Glo Luminescent Cell Viability Assay (mean±SEM of triplicate ATP measurements expressed as % of control untreated cells; * p<0.001 when compared with untreated cells). Representative of 3 different experiments.
Fig 4.
TNF-α-induced hUC-MSC death amplified by TRAIL.
hUC-MSCs of two different clones (Clones 63 and 69, 2x104 in 96-well plates) were stimulated with TRAIL (500 ng/ml, 28 nM) alone or in association with TNF-α (20 ng/ml, 1.2 nM) and IFN-γ (50 ng/ml, 3 nM) for 48h. Then, cell death was scored by CellTiter-Glo Luminescent Cell Viability Assay. Data from the two clones were pooled (mean±SEM of six ATP measurements; *** p<0.0001 when compared with untreated cells; °p<0.05, °°p<0.01 when compared with TRAIL alone-treated cells). Representative of 3 different experiments.
Fig 5.
Mechanisms involved in cell death.
hUC-MSCs of clone 63 (2x104 in 96-well plates) were pretreated for 2h with zVAD-fmk (V, 20 μM) or necrostatin-1 (N, 50 μM) then stimulated with TRAIL (500 ng/ml, 28 nM) alone or in association with TNF-α (20 ng/ml, 1.2 nM) and IFN-γ (50 ng/ml, 3 nM) for 24h. Then, cell death was scored by CellTiter-Glo Luminescent Cell Viability Assay. Data are presented as mean±SEM of three ATP measurements; *** p<0.0001, **p<0.01 when compared with untreated cells; °p<0.05, °°p<0.01 when compared with TRAIL-treated, TRAIL-associated with TNF-α or TNF-α associated with IFN-γ-treated cells). Representative of 2 different experiments using alternatively hUC-MSCs clone 63 and 69 with the same results.
Fig 6.
Relationship between cell death and cytokine concentrations.
hUC-MSCs of different reactive clones (2x104 in 96-well plates) were stimulated with TNF-α (20 ng/ml, 1.1 nM), TRAIL (500 ng/ml, 28 nM) alone or in association with TNF-α or TNF-α associated with IFN-γ (50 ng/ml, 3 nM) for 48h. Then, cell death was scored by CellTiter-Glo Luminescent Cell Viability Assay and expressed as % of control untreated cells. A statistically significant one-phase decay was observed for both IL-6 and MCP-1 with r2 = 0.74 and 0.92 respectively.
Fig 7.
Inhibitor of Apoptosis Proteins (IAPs) were involved in MCP-1/IL-6 production under high dose TNF-α stimulation.
A. hUC-MSCs (2x104 in 96-well plates) were pretreated for 2h with the IAP inhibitor GDC-0152 at increasing concentrations (0–1000 nM), then stimulated with TNF-α (20 ng/ml, 1.2 nM). After a further 24h, trypan blue was used to exclude cell toxicity. SN was collected and IL-6 and MCP-1 concentrations were measured by ELISA. B. hUC-MSCs(5×105 in T25 bottle) were pretreated with GDC-0152(1000nM) for 2 h, then stimulated with TNF-α (20 ng/ml, 1.2 nM). 24 hours later, protein from nucleus and cytoplasma were extracted separately and the amount of NF-kB were detected by Western blot. Data are as mean±SEM of triplicate measurements; *p<0.05, **p<0.01, ***p<0.001 when compared to untreated cells. These experiments were repeated 3 times with the same results, using clone 69 and another TNF-α sensitive clone (clone 120003).
Fig 8.
Scheme of interactions between hUC-MSCs and PBMCs.
Cytokine receptors are not presented, LC for Low Concentration, HC for High Concentration. Thick full lines are for cytokine production. Dashed line are for cell stimulation.