Gingival fibroblasts (GFs) or conditioned medium from GFs modify the morphology of monocyte-derived dendritic cells.

<p>Giemsa staining on cytospin slides of cells prepared from cultures of A. Monocytes (day 0). B. Monocyte-derived dendritic cells after 7 days in culture with medium for differentiation (MD, containing rGM-CSF and rIL-4); these cells present the typical characteristics of DC with clearly visible dendrites. C. Monocyte-derived cells co-cultured for 7 days with GFs in a Transwell® system with MD. D. Monocyte-derived cells cultured for 7 days in the presence of conditioned medium from GFs with MD. Monocyte-derived cells (C and D) and monocyte-dendritic cells (B) show morphological differences, in particular the absence of typical dendritesfrom monocyte-derived cells. The results are representative of three independent experiments. Scale bar: 20 µm, original magnification ×400.</p

Mature monocyte-derived DCs obtained with (CM+) or without (CM-) conditioned medium from GFs were used to stimulate allogeneic CD4+ T cells purified from the PBMCs from two donors (PBMC1 and PBMC2); thymidine incorporation is expressed in counts per minute (cpm).

<p>Mature monocyte-derived DCs obtained in the presence of CM from GFs displayed a significantly capacity than controls to stimulate the proliferation of allogeneic CD4+T cells. Mean ± SD of triplicates are shown and results are representative of two independent experiments. *Significant difference (P = 0.002) and **Significant difference (P<0.001).</p

Detection of IL-6, VEGF and TGFβ1 by ELISA in CM and supernatant from cell culture (pg/mL).

<p>Results are representative of 4 independent experiments realized in duplicate.</p><p>IL-13 and IL-10 were not detected in CM and supernatant from cell culture.</p><p>MD: medium for differentiation (rGM-CSF+rIL-4).</p><p>CM: conditioned medium from gingival fibroblasts.</p><p>D7: after 7 days in culture.</p><p>ND: not detected.</p

Percentage of the differentiation of CD1a+ monocyte-derived dendritic cells using conditioned media from 4 independent gingival fibroblasts donors.

<p>MD: medium for differentiation (rGM-CSF+rIL-4).</p><p>CM1, CM2, CM3 and CM4: conditioned media from independent gingival fibroblasts donors 1 to 4.</p><p>For each CM, results are shown as mean ± SD of at least 3 independent experiments in duplicate.</p>*<p>P<0.0001.</p

Antibodies against VEGF and IL-6 reduce the inhibitory effect of conditioned medium (CM) from GFs.

<p>A. Neutralizing antibodies against VEGF (10 µg/ml) and IL-6 (10 µg/ml) significantly reduced (P<0.05) the inhibition of CD1a+ monocytes-derived cells by CM from GFs. In contrast, neutralizing antibodies against TGFβ1 significantly increased (P<0.05) this inhibitory effect. B. The effect of the neutralizing antibodies against VEGF and IL-6 was dose-dependent. The results reported are means ±SD for four independent experiments in duplicate. *Significant difference to the value for MD+CM (P<0.05).</p

Soluble factors from gingival fibroblasts (GFs) inhibit the differentiation of CD1a⁺ dendritic cells.

<p>A. Analysis of the induction of the differentiation of monocyte-derived DCs by MD (rGM-CSF and rIL-4) after 7 days co co-culture of GFs (lower compartment) with monocytes (upper compartment) in a Transwell® chamber system. The effect of several GF/monocyte ratios (1∶25 to 1∶5) on CD1a and DC-SIGN expression was analyzed by flow cytometry. Results are expressed as percentages ± SD of CD1a⁺ DC-SIGN⁺ positive cells in the upper right part of each figure. Percentages of CD1a⁺ DC-SIGN⁺ dendritic cells decreased with increasing GF/monocyte ratio. Percentages are means ± SD of three independent experiments. B. Monocytes cultured with conditioned medium (CM) from GFs in the presence of MD for 7 days. Results are representative of four separate experiments and results are expressed as percentages ± SD of CD1a⁺ DC-SIGN⁺ positive cells in the upper right part of each figure. C. The differentiation of monocyte-derived DCs was also monitored by assessing the expression of CD1a and CD14 on day 7. The percentage of CD1a⁺ dendritic cells was significantly lower (P<0.0001) and that of CD14⁺ cells significantly higher (P<0.0001) in the presence of CM than in controls. Results are expressed as means ± SD and the histogram reports the results of 12 independent experiments using monocytes from 12 different donors. CM: conditioned medium. * Significant difference (P<0.0001).</p

Co-localization of CD90⁺ gingival fibroblasts (GFs) and CD14⁺ monocytes in superficial gingival connective tissue.

<p>A. Numerous GFs are revealed as red fluorescence by PE-conjugated secondary antibody (magnification ×40). B. CD14⁺ monocytes are revealed as green fluorescence by FITC-conjugated secondary antibody (magnification ×40). C. DAPI staining with superposition of red and green fluorescence showing the close proximity of the two cell populations in the gingival tissue, consistent with communication between these cell types (magnification, ×40). D. Isotype-matched antibodies as negative controls (magnification ×40), scale bar: 50 µm.</p