Abstract
We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.
Highlights
Fibroblasts, the most abundant cells of the stroma, are characterized by their morphology, ability to adhere, their production and degradation of the extracellular matrix (ECM) and the absence of epithelial, vascular and leukocyte lineage markers
Monocyte-derived Cell Morphology is Affected by the Presence of Gingival Fibroblasts (GFs) or Conditioned Medium from GFs
Monocytes cultured in the TranswellH apparatus (Fig. 1C) with gingival fibroblasts (GFs), such that the two cell types were separated by a porous membrane, or in presence of conditioned medium (CM) (Fig. 1D), were round, and did not develop the veiled appearance or other morphological characteristics of dendritic cells (DCs)
Summary
Fibroblasts, the most abundant cells of the stroma, are characterized by their morphology, ability to adhere, their production and degradation of the extracellular matrix (ECM) and the absence of epithelial, vascular and leukocyte lineage markers. Fibroblasts have a direct role in suppressing immune responses in the spleen, where they drive the development of regulatory dendritic cells (DCs), following their activation by infectious agents [4]. Dermal fibroblasts release IL-6, which up-regulates the expression of functional M-CSF receptors on monocytes, allowing the monocytes to bind autocrine M-CSF [5,6,7] which switches monocyte differentiation to macrophages rather than DCs. A recent study showed that human cytomegalovirus induced production of IL-6 by infected cells leading to the inhibition of DC differentiation [8]. Fibroblasts may be a major source of anti-inflammatory mediators and are thought to be involved in the regulation of DC functions: they synthesize factors modulating DC functions, such as chemokines and other cytokines (including IL-6 and TGFb), matrix components and matrix-degrading enzymes [13]. Therapeutic utilization of fibroblasts and their biologically active products is an emerging approach for the control of chronic inflammatory diseases [14]
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