Murine corneal stroma cells inhibit LPS-induced dendritic cell maturation partially through TGF-β2 secretion in vitro

Abstract

Purpose: The peripheral cornea contains mature and immature resident dendritic cells (DCs) while the central cornea is exclusively equipped with immature DCs. There must be some factors that cause immature DCs. This study investigated whether corneal stroma cells (CSCs) inhibit DC maturation by secreting cytokines.

Methods: The messenger ribonucleic acid (mRNA) and protein level of transforming growth factor beta 2 (TGF-β(2)) was analyzed using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Immature DCs were induced to mature in the presence of lipopolysaccharide (LPS) and with concentrations of CSC culture supernatant (containing and not containing neutralizing TGF-β(2) antibodies). Then, the DC phenotypic and functional maturation were analyzed.

Results: CSCs exhibited positive expressions of TGF-β(2) mRNA and secreted high concentrations of TGF-β(2) protein. In the presence of LPS, DCs, which were treated with a CSC culture supernatant, displayed reduced expressions of cluster of differentiation 80 (CD80), CD86, and major histocompatibility complex II (MHC II) in a dose-dependent manner. Moreover, treated DCs showed lower T-cell stimulation capacity and a higher endocytosis function. However, these phenotypic and functional modifications were partially reversed after the application of neutralizing TGF-β(2) antibodies.

Conclusions: This study demonstrates that CSCs can partially inhibit LPS-induced DC maturation through TGF-β(2) secretion in vitro.

Figure 1

Isolated murine CSCs. CSCs exhibited condensed cell bodies with numerous processes (A), positive immunofluorescence staining of keratocan protein (B), positive expression of ALDH and keratocan genes. Bar=35 μm.

Figure 2

The phenotypic characterization of cultured cells by flow cytometry analysis. Data demonstrated that T cells were positive for CD3 (A), and DCs were positive for CD11c (B). Isotype control (open histogram) and stain with relevant antibody (filled histogram) are shown.

Figure 3

Flow cytometry analysis demonstrated that CSC culture supernatant suppressed the expression of costimulatory molecules, CD80 and CD86, and MHC II in a dose-dependent manner during DC maturation. The expression of each surface molecule (filled) compared to the respective isotype control (empty). Representative example of three experiments.

Figure 4

Flow cytometric analysis of endocytic activity of DCs. Mature DCs (A), DCs treated with 50% CSC culture supernatant (B), and DCs treated with 50% CSC culture supernatant in presence of neutralizing TGF-β2 antibody (C). The empty histograms represent the uptake of FITC-dextran at 4 °C. The histogram is from one representative experiment out of three performed.

Figure 5

RT–PCR analysis of immunomodulatory cytokines in CSCs. CSCs showed positive expression of TGF-β₂, PTGS₂, and M-CSF, but negative expression of IL-10.

Figure 6

The phenotypic characterization of DCs treated with 50% CSC culture supernatant in presence of neutralizing TGF-β₂ antibody, measured by flow cytometry. The expression of each surface molecule (filled) compared to the respective isotype control (empty). Representative example of three experiments.