Role of cell-cell communication in inhibiting butyric acid-induced T-cell apoptosis

Abstract

We have previously demonstrated that human gingival fibroblasts rescue butyric acid-induced T-cell apoptosis via proinflammatory cytokines such as interleukin 6 (IL-6) and IL-11, which are produced by fibroblasts stimulated with butyric acid. In this study, we determined if T-cell adhesion to human gingival fibroblasts influenced the susceptibility of T cells to butyric acid-induced apoptosis. We have shown that the number of Jurkat T cells adherent to gingival fibroblasts (Gin-1 cells) was significantly increased by the addition of butyric acid. All Jurkat cells that adhered to Gin-1 cells remained viable, while the nonadherent Jurkat cells dropped into apoptosis. The increase in T-cell adhesion to fibroblasts was also observed when Jurkat cells, but not Gin-1 cells, were pretreated with butyric acid. The expression levels of CD44, very late antigen 2 (VLA-2) and VLA-5 but not of leukocyte function-associated antigen 1 (LFA-1) and VLA-4 on Jurkat cells were increased following treatment with butyric acid. Furthermore, pretreatment of butyric acid-sensitized Jurkat cells with monoclonal antibodies against CD44, VLA-2, and VLA-5, but not LFA-1 and VLA-4, followed by coculture with Gin-1 cells inhibited T-cell adhesion to fibroblasts and increased apoptosis of nonadherent T cells after coculture of gingival fibroblasts and Jurkat cells. These results indicate that T-cell adherence to fibroblasts is enhanced by butyric acid and that butyric acid-induced T-cell apoptosis is down-regulated by T-cell adhesion to gingival fibroblasts through an interaction with the adhesion molecules CD44, VLA-2, and VLA-5 expressed on T cells stimulated with butyric acid.

FIG. 1.

Effect of butyric acid on Jurkat cell adhesion to the Gin-1 cell monolayer. Jurkat cells were directly cocultured with Gin-1 cells in the presence or absence of 5 mM butyric acid for 30, 60, and 120 min. The numbers of Jurkat cells adherent to Gin-1 cells were counted by using a phase-contrast microscope (A and B). In other experiments, Jurkat or Gin-1 cells were pretreated with 5 mM butyric acid for 2 h and then cocultured with Gin-1 or Jurkat cells, respectively, for 1 h. The numbers of Jurkat cells adherent to Gin-1 cells were counted by using a phase-contrast microscope. The results are expressed as the means ± standard errors of the means (error bars) of three different experiments with triplicate cultures. Values that were significantly different from those of corresponding negative controls at P < 0.01 are indicated by asterisks.

FIG. 2.

Analysis of Jurkat cells that adhere to Gin-1 cells. Jurkat cells were directly cocultured with Gin-1 cells in the presence of 5 mM butyric acid. The viability of Jurkat cells that were adherent or nonadherent to Gin-1 cells was examined by DePsipher assay (A) or SYTOX green nucleic acid staining (B), followed by confocal laser scanning microscopy. The results are expressed as the means ± standard errors of the means (error bars) of three different experiments with triplicate cultures. Values that were significantly different from those of corresponding negative controls at P < 0.01 are indicated by asterisks. The viability of Jurkat cells that were adherent or nonadherent to Gin-1 cells was also examined by agarose gel electrophoresis of DNA extracted from Jurkat cells (C). Lane U, unadhered T cells; lane A, adhered T cells.

FIG. 3.

Effect of butyric acid on the expression of adhesion molecules on Jurkat cells. Jurkat cells were stained with FITC-labeled anti-CD44, anti-VLA-2, and anti-VLA-5 Abs and isotype control Abs after treatment with 5 mM butyric acid for 16 h and analyzed by using a FACSCalibur. Similar results were obtained in five independent experiments.

FIG. 4.

MAbs against adhesion molecules inhibit adhesion of Gin-1 cells to Jurkat cells and increase apoptosis. Jurkat cells were pretreated with 5 mM butyric acid for 2 h and then treated with MAbs against CD44, VLA-2, and VLA-5 for 30 min. The Jurkat cells were then added to a monolayer of Gin-1 cells and incubated for 1 h (A) or 21 h with 5 mM butyric acid (B). In panel A, the numbers of Jurkat cells adherent to Gin-1 cells were counted by using a phase-contrast microscope. The results are expressed as the means ± standard errors of the means (error bars) of three different experiments with triplicate cultures. Values that were significantly different from those of corresponding negative controls at P < 0.01 are indicated by asterisks. In panel B the viability of Jurkat cells nonadherent to Gin-1 cells was examined by surface binding of annexin V. Results shown are representative of three independent experiments.