Lipopolysaccharide stimulates butyric acid-induced apoptosis in human peripheral blood mononuclear cells

Abstract

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3(+)-T-cell apoptosis followed by similar levels of both CD4(+)- and CD8(+)-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3(+) PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4(+) and CD8(+) cells.

FIG. 1

(A) Dose-dependent effects of butyric acids on cell proliferation. PBMC were cultured with the indicated concentration of butyric acid in the presence of solid-phase anti-CD3 MAb (○) or ConA (•) for 48 h. Cellular proliferation was determined by an MTT assay and is expressed as the percentage of the absorbance obtained without butyric acid. The results are expressed as the mean ± SE of three different experiments with triplicate cultures. Values significantly different from the corresponding negative controls without butyric acid at P < 0.05 are indicated by asterisks. (B) Agarose gel electrophoresis of DNA extracted from PBMC treated with butyric acid for 16 h. Lanes: M, molecular weight markers (HaeIII-digested φX174 DNA); 1, untreated control cells; 2 to 6, cells treated with 5, 2.5, 1.25, 0.62, and 0.31 mM butyric acid, respectively. (C) Activities of caspase-1 and caspase-3 in butyric acid-treated PBMC. PBMC were cultured with or without 5 mM butyric acid for the indicated times. Cell extracts were prepared and caspase activities were measured as described in Materials and Methods. The results are expressed as the mean ± SE of three different experiments with triplicate cultures. Values significantly different from the corresponding negative controls without butyric acid at P < 0.05 are indicated by asterisks.

FIG. 2

Effect of LPS on butyric acid-induced apoptosis in PBMC. (A) PBMC were treated with the indicated concentration of butyric acid in the presence (•) or absence (○) of LPS (100 μg/ml) for 21 h. (B) PBMC were treated with the indicated concentration of LPS (micrograms per milliliter) in the presence of 5 mM butyric acid (BA) for 21 h. Harvested cells were assayed by the DPA assay. The results are expressed as the mean ± SE of three different experiments with triplicate cultures. Values significantly different from corresponding LPS-free butyric acid values at P < 0.05 are indicated by asterisks.

FIG. 3

Effect of LPS on sub-G₁ accumulation in butyric acid-treated PBMC. PBMC were treated with the indicated concentrations of LPS (micrograms per milliliter) in the presence of 5 mM butyric acid (BA) for 16 h. The DNA content was analyzed by PI staining. The results are expressed as the mean ± SE of three different experiments with triplicate cultures. Values significantly different from corresponding LPS-free butyric acid values at P < 0.05 are indicated by asterisks.

FIG. 4

LPS increases annexin V-FITC staining of apoptotic cells in butyric acid-treated PBMC. PBMC were double-stained with annexin V-FITC and PI after treatment with 5 mM butyric acid (BA) in the presence or absence of LPS (concentrations given in micrograms per milliliter) for 21 h and analyzed by FACScan analysis. Annexin V⁺, PI⁻ cells are the early apoptotic cells, and annexin V⁺, PI⁺ cells are the late apoptotic cells. The figure is representative of five experiments with similar results.

FIG. 5

Cytofluorimetric analysis of annexin V on CD4⁺-cell populations. PBMC were double stained with FITC-labeled annexin V and PE-labeled anti-CD4 MAb after treatment with 5 mM butyric acid (BA) in the presence or absence of LPS (concentrations given in micrograms per milliliter) for 21 h and analyzed by FACScan analysis. Data show the expression of CD4 on apoptotic cells. The figure is representative of five experiments with similar results.

FIG. 6

Cytofluorometric analysis of annexin V on CD8⁺-cell populations. PBMC were double stained with FITC-labeled annexin V and PE-labeled anti-CD8 MAb after treatment with 5 mM butyric acid (BA) in the presence or absence of LPS (concentrations given in micrograms per milliliter) for 21 h and analyzed by FACScan analysis. Data show the expression of CD8 on apoptotic cells. The figure is representative of five experiments with similar results.