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MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2

Grazia Maria Spaggiari, Heba Abdelrazik, Flavio Becchetti and Lorenzo Moretta

Article Figures & Data

Figures

  • Figure 1

    MSC inhibit monocyte progression to immature DCs. A representative experiment. Expression of CD14, CD1a, CD80, CD86, and CD83 on monocytes (at day 0) and after induction of DC differentiation to immature DCs (day 5) or full maturation (day 7). Phenotypic analysis of monocytes was performed on PBMCs by gating on the leukocyte subset according to the forward scatter and side scatter scatter, whereas iDC surface phenotype was analyzed in CD14+ cells cultured for 5 days with GM-CSF and IL-4 either in the absence or in the presence of MSCs. Mature DC phenotype was analyzed after LPS-induced maturation of cells cultured alone or with MSCs either starting at day 0 or at day 5. Numbers indicate percentages of positive cells for CD14 and CD1a expression, whereas they represent mean fluorescence intensity for the surface density of CD80, CD86, and CD83 markers.

  • Figure 2

    MSC-induced inhibition of monocyte differentiation into mDCs. Freshly purified CD14+ cells were cultured with GM-CSF and IL-4 for 5 days, LPS was added for 2 additional days to induce DC maturation. Cultures were set either in the presence (■) or in the absence (Embedded Image) of MSCs. (A) Mean of percentages of CD14 and CD1a positive cells. (B) Mean of the surface density of CD80, CD86, and CD83 evaluated as MRFI (“Cytofluorimetric analysis”). Results were obtained from 12 independent experiments.

  • Figure 3

    MSCs inhibit the production of IL-12 and the capability of stimulating T-cell response in MLR of monocyte-derived cells. (A) Il-12p70 production was measured in culture SN after 48-hour stimulation with LPS of monocyte-derived cells cultured for 5 days with GM-CSF and IL-4 under different culture conditions. Standard (ctr), with the addition of MSCs or of MSCs and the PGE2 inhibitor NS-398 or of PGE2 (1 μM) added at day 0, with MSCs added at day 5. Results are expressed as pg/mL. (B) In MLR experiments, monocyte-derived cells were irradiated and used in graded doses to stimulate allogeneic T cells (105 responder cells/well). After culture for 5 days, T-cell proliferation was evaluated by incubating cells with 3H-thymidine for additional 16 hours. Cells were then harvested and 3H-thymidine uptake was measured. Because of the variable degree of proliferation of different T-cell populations used as responder cells, data are not expressed as cpm values but as percentages. We considered 100% the maximal proliferation of T cells stimulated with DCs (5 × 102 cells/well) obtained under standard conditions, and relative percentage the proliferation of T cells stimulated by cells obtained under the other culture conditions. Bars represent mean ± SD from 5 independent experiments. *P < .05; **P < .01.

  • Figure 4

    PGE2 production by MSCs is up-regulated in monocyte-MSC cocultures. PGE2 levels were measured in culture SN of MSCs cultured alone (■) or with monocytes (Embedded Image) induced to differentiate into DCs. PGE2 was evaluated by ELISA assay performed on SN collected at 24, 48, and 72 hours of culture. Results are expressed as mean of PGE2 levels measured in 7 independent experiments performed using different MSC populations. Data are expressed in pg/mL. **P < .01.

  • Figure 5

    Involvement of MSC-derived PGE2 in the inhibition of DC differentiation: analysis of CD1a and CD14 surface expression. Purified CD14+ cells were cultured with GM-CSF and IL-4 to induce differentiation into DCs. Cultures were performed either in the absence (■) or in the presence (▧) of MSCs. In addition, the PGE2 inhibitor NS-398 (5 μM) was added to monocyte-MSC cocultures (Embedded Image) or 1 μM PGE2 was added to monocytes cultured alone (Embedded Image). After 5 days, phenotypic analysis was performed to check DC differentiation. (A) A representative experiment. Numbers represent percentages of positive cells. (B) Expression of CD14 and CD1a in cells cultured under the described culture conditions. Results are expressed as mean ± SD of the percentages of marker-positive cells obtained from the analysis of 11 independent experiments performed. ***P < .001.

  • Figure 6

    IL-6 production by MSCs under different culture conditions. IL-6 production was evaluated in MSCs cultured alone or in combination with monocytes (either in the absence or in the presence of the PGE2 inhibitor NS-398) with GM-CSF and IL-4. After 5 days, culture SNs were collected and IL-6 was measured by ELISA assay. Data are represented as mean ± SD of IL-6 levels (expressed as ng/mL) evaluated in 6 independent experiments performed. **P < .01.