Tumor necrosis factor alpha enhances Actinobacillus actinomycetemcomitans leukotoxin-induced HL-60 cell apoptosis by stimulating lymphocyte function-associated antigen 1 expression

Abstract

We demonstrated previously that Actinobacillus actinomycetemcomitans leukotoxin (Ltx) is greatly able to induce apoptotic signaling in cells that are positive for lymphocyte function-associated antigen 1 (LFA-1), a cell receptor of Ltx. We investigated in this study whether inflammatory cytokines can regulate apoptosis of human leukemic HL-60 cells induced by Ltx. Of the cytokines tested, tumor necrosis factor alpha (TNF-alpha) significantly enhanced the Ltx-induced cell apoptosis. Northern and Western blotting analyses showed that TNF-alpha enhanced the expression of CD11a in the cells at both the mRNA and protein levels but did not do so for CD18 expression. TNF-alpha also enhanced the binding of Ltx to the cells. We also observed by measuring the mitochondrial transmembrane potential and the generation of superoxide anion that the cytokine enhanced Ltx-induced apoptosis in HL-60 cells. In addition, interleukin-1beta significantly enhanced Ltx-induced cell apoptosis, although the enhancing activity was lower than that of TNF-alpha. These stimulatory effects of both cytokines were also observed for human polymorphonuclear leukocytes. The ability of TNF-alpha to increase cell susceptibility to Ltx could be inhibited by preincubation of the cells with a monoclonal antibody against TNF receptor 1 but not by preincubation of the cells with a monoclonal antibody against anti-TNF receptor 2. Furthermore, the results of an assay of caspase 3 intracellular activity (PhiPhiLuxG1D2) showed that Ltx-induced caspase 3 activation was completely neutralized by CD18 antibody treatment, although significant neutralization was also observed with anti-CD11a antibody. Taken together, the results of the present study indicate that TNF-alpha acts as a potent stimulator of Ltx-induced HL-60 cell apoptosis via TNF receptor 1-mediated upregulation of LFA-1 expression.

FIG. 1.

TNF-α enhances human LFA-1 (CD11a/CD18) mRNA expression in and protein production by HL-60 cells in a time- and dose-dependent fashion. (A) TNF-α enhanced the human CD11a mRNA level in a time-dependent manner, but human CD18 mRNA was expressed constitutively over the same time course. (B) HL-60 cells (10⁶) were treated with TNF-α at concentrations ranging from 10 to 500 U/ml for 30 min and lysed with detergent (1% Triton X-100 in 0.15 M NaCl), and the proteins from each cell lysate were electrophoresed on 7.5% SDS gels. Gels were blotted onto nitrocellulose membranes, and fluorographs were developed after incubation (overnight at 4°C) with either anti-human CD11a MAb or anti-human CD18 MAb. Relative band intensities were determined by using NIH Image version 1.62 software. The values below the bands are the mean fold increases in the expression levels in HL-60 cells incubated with TNF-α compared with the expression by unstimulated HL-60 cells.

FIG. 2.

Time course of binding of purified Ltx to HL-60 cells treated with TNF-α (•) or ΔI TNF-α (○). HL-60 cells (10⁶) were incubated with TNF-α or ΔI TNF-α for 0, 15, 30, or 60 min at 37°C. The cells were then incubated with 3 × 10⁻¹⁰ M biotinylated purified Ltx for 10 min at 4°C. The cells were then washed with HBSS, stained with streptavidin-phycoerythrin for 30 min at 4°C, and analyzed by flow cytometry. Data are shown as the means ± standard errors of the means for five different experiments. Values significantly different from the value for Ltx binding at each time point, as determined by using Student's t test (*, P < 0.01; **, P < 0.001), are indicated.

FIG. 3.

TNF-α enhances Ltx-induced changes in ΔΨ_m and superoxide anion generation. Ltx (3 × 10⁻¹⁰ M)-induced alterations in DiOC₆(3) and HE staining of HL-60 cells were evaluated for 1 h at 37°C (B). As a control, cells were incubated with ΔI Ltx (A) for 1 h. Cells were pretreated with 500 U of TNF-α per ml for 30 min and then incubated with ΔI Ltx (C) or 3 × 10⁻¹⁰ M Ltx (D) for 1 h. Subsequently, cells were stained with DiOC₆(3) and HE and analyzed by flow cytometry.

FIG. 4.

TNF-α enhances Ltx-induced activation of caspase 3 in HL-60 cells. A rhodamine-derivatized, cell-permeable, caspase 3-specific substrate (PhiPhiLuxG₁D₂) was used to evaluate whether activated caspase 3 was present in Ltx-treated cells. HL-60 cells were incubated with ΔI Ltx (A) or 3 × 10⁻¹⁰ M Ltx (B) for 1 h and then examined for cleavage of the substrate as determined by flow cytometry. Other cells were pretreated with 500 U of TNF-α per ml for 30 min and incubated with ΔI Ltx (C) or 3 × 10⁻¹⁰ M Ltx (D) for 1 h. These results are representative of three independent experiments; at least 5,000 cells were analyzed per experiment.

FIG. 5.

Effect of β2 integrin antibodies on Ltx-induced caspase 3 activation in HL-60 cells. (A) HL-60 cells were incubated with (b, d, f) or without (a, c, e) TNF-α (500 U/ml) for 30 min at 37°C and then pretreated with normal mouse IgG (10 μg/ml; a, b), anti-human CD11a antibody (10 μg/ml; c, d), or anti-human CD18 antibody (10 μg/ml; e, f) for 30 min at 4°C. After having been washed with HBSS, the cells were stimulated with 3 × 10⁻¹⁰ M Ltx for 1 h at 37°C. Caspase 3 activity was measured by using a PhiPhiLuxG₁D₂ kit and a flow cytometer and is expressed as the percentage of rhodamine-stained cells. (B) The results are expressed as the mean ± standard deviation percent inhibition of the value obtained for cells in the presence of normal mouse IgG. An identical experiment performed independently gave similar results.

FIG. 6.

Inhibitory effect of anti-human TNF-R1 MAb on Ltx-induced activation of caspase 3 after treatment of HL-60 cells with TNF-α. HL-60 cells (10⁶) were pretreated with 10 μg of normal mouse IgG per ml, 10 μg of anti-human TNF-R1 MAb per ml, 10 μg of anti-human TNF-R2 MAb per ml, or 10 μg of anti-human TNF-R1 MAb per ml, and 10 μg of anti-human TNF-R2 MAb per ml for 1 h at 37°C and then incubated with 500 U of TNF-α per ml for 30 min. After having been washed with HBSS, the cells were stimulated with 3 × 10⁻¹⁰ M Ltx for 1 h at 37°C. Caspase 3 activity was measured by using a PhiPhiLuxG₁D₂ kit and a flow cytometer and was evaluated as the percentage of rhodamine-stained cells. The percentage of inhibition of caspase 3 activity was calculated by the following formula: percent inhibition = 100 × (percentage of Ltx-treated cells rhodamine positive after treatment with TNF-α − percentage of Ltx-treated cells rhodamine positive after treatment with MAb and TNF-α)/(percentage of Ltx-treated cells rhodamine positive after treatment with TNF-α − percentage of cells rhodamine positive after treatment with Ltx only). Data are shown as the means ± standard errors of the means for three experiments performed in duplicate.

FIG. 7.

Effects of three other inflammatory cytokines on CD11a/CD18 mRNA expression, Ltx-induced alterations in ΔΨ_m and superoxide anion generation, and Ltx-induced caspase 3 activation of HL-60 cells. (A) The cells were incubated with medium alone, IL-1β, IL-1α, TNF-α, IL-4, or IL-6 for 30 min, and total RNA was then analyzed by Northern blotting with DIG-labeled CD11a, CD18, and GAPDH PCR probes. Relative band intensities were determined by using NIH Image version 1.62 software. The values below the bands are the mean fold increases in the expression levels in HL-60 cells incubated with the cytokines compared with the expression by unstimulated HL-60 cells. (B) HL-60 cells were pretreated with medium alone (a, b), IL-1β (c, d), IL-4 (e), or IL-6 (f) for 30 min and then incubated with ΔI Ltx (a, c) or 3 × 10⁻¹⁰ M Ltx (b, d, e, f) for 1 h. Cells were stained with DiOC₆(3) and HE to measure ΔΨ_m and superoxide anion production, respectively, and analyzed by flow cytometry. Data are plotted as DiOC₆(3) fluorescence versus ethidium fluorescence. Bars indicate the setting for quadrant analysis; values represent the percentage of cells in each quadrant. Results are representative of three experiments. (C) HL-60 cells were pretreated with medium alone (a, b), IL-1β (c, d), IL-4 (e), or IL-6 (f) for 30 min and then incubated with ΔI Ltx (a, c) or 3 × 10⁻¹⁰ M Ltx (b, d, e, f) for 1 h. The cells were then assessed for caspase 3 activation by using a PhiPhiLuxG₁D₂ kit. Stained cells were analyzed by flow cytometry. Bars indicate region of positive fluorescence; the percentage of positive cells is presented in each panel. Results are representative of three experiments.

FIG. 8.

Effects of TNF-α or IL-1β on upregulation of CD11a and Ltx-induced caspase 3 activation of HPMNs. (A) Freshly isolated HPMNs (10⁶/ml) were incubated with TNF-α (•) or IL-1β (○) at concentrations ranging from 100 to 1,000 U/ml or with medium (control) for 30 min at 37°C. The cells were then washed and incubated with fluorescein isothiocyanate-labeled anti-human CD11a MAb (30 min at 4°C) and analyzed by flow cytometry (5,000 cells were scored for green fluorescence). Data are shown as the mean percentages of positive cells ± standard deviations for duplicate tubes. The data for stimulated cells significantly different from the data for control cells, as determined by using Student's t test (*, P < 0.05; **, P < 0.01), are indicated. The experiments were performed three times, and similar results were obtained in each experiment. (B) Freshly isolated HPMNs were incubated with ΔI Ltx (a) or Ltx (b) for 1 h and then examined for cleavage of the substrate as determined by flow cytometry. Other cells were pretreated with TNF-α (c) or IL-1β (d) at 500 U/ml for 30 min and incubated with Ltx for 1 h. These results are representative of three independent experiments; at least 5,000 cells were analyzed per experiment.