Tumour necrosis factor-α regulates human eosinophil apoptosis via ligation of TNF-receptor 1 and balance between NF-κB and AP-1

Abstract

Eosinophils play a central role in asthma. The present study was performed to investigate the effect of tumour necrosis factor-α (TNF-α) on longevity of isolated human eosinophils. In contrast to Fas, TNF-α inhibited eosinophil apoptosis as evidenced by a combination of flow cytometry, DNA fragmentation assay and morphological analyses. The effect of TNF-α on eosinophil apoptosis was reversed by a TNF-α neutralising antibody. The anti-apoptotic effect of TNF-α was not due to autocrine release of known survival-prolonging cytokines interleukins 3 and 5 or granulocyte-macrophage-colony-stimulating factor as their neutralisation did not affect the effect of TNF-α. The anti-apoptotic signal was mediated mainly by the TNF-receptor 1. TNF-α induced phosphorylation and degradation of IκB and an increase in NF-κB DNA-binding activity. The survival-prolonging effect of TNF-α was reversed by inhibitors of NF-κB pyrrolidinedithiocarbamate and gliotoxin and by an inhibitor of IκB kinase, BMS-345541. TNF-α induced also an increase in AP-1 DNA-binding activity and the antiapoptotic effect of TNF-α was potentiated by inhibitors of AP-1, SR 11302 and tanshinone IIA and by an inhibitor of c-jun-N-terminal kinase, SP600125, which is an upstream kinase activating AP-1. Our results thus suggest that TNF-α delays human eosinophil apoptosis via TNF-receptor 1 and the resulting changes in longevity depend on yin-yang balance between activation of NF-κB and AP-1.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effect of TNF-α on apoptosis in human eosinophils.

Effect of TNF-α on apoptosis in culture for 40 h (A). Representative graphs from relative DNA fragmentation assay of propidium iodide-stained eosinophils are shown in (B and C). In (B and C) figures in top right corner represent percentage of cells showing decreased DNA content. In (D) is shown the DNA fragmentation in eosinophils cultured without (lane 1) and with 10 ng/ml TNF-α (lane 2). The typical apoptotic morphology (cell shrinkage and condensation of nuclear chromatin) of May-Grünwald-Giemsa-stained cytokine-deprived eosinophils is shown in (E) and the reduction in the number of cells showing apoptotic morphology when cultured with TNF-α (10 ng/ml) for 40 h is shown in (F). Scale bar (E and F) is 10 µm. In (G and H) representative graphs from Annexin-V FITC (FL1-H) and uptake of propidium iodide (FL2-H) analysis of eosinophils are shown. In (G and H) figures in top right corner represent percentages of Annexin-V positive cells (FITC+/PI- and FITC+/PI+). In (I) is shown the effect of neutralising TNF-α (A-TNF-α) antibody (5 µg/ml) on the inhibition of apoptosis induced by TNF-α. The isotype control was IgG1 (5 µg/ml) and had no effect on eosinophil apoptosis during the 40 h culture. In A and I apoptosis was assessed by flow cytometric measurement of relative DNA content. Each datapoint represents the mean ± SEM of 4–6 independent determinations using eosinophils from different donors. * indicates P<0.05, **P<0.01 and ***P<0.001 as compared with the respective control without TNF-α and # P<0.05 as compared with the respective control without TNF-α neutralising antibody.

Figure 2. Effect of Fas on apoptosis in human eosinophils.

The effect of Fas mAb CH-11 (A) and soluble RhFasL (C) on apoptosis during culture for 40 h. In (B) is shown the effect of isotype control antibody (lane 1; IgM 100 ng/ml) and Fas mAb CH-11 (lane 2; 100 ng/ml) on DNA fragmentation in human eosinophils after culture for 24 h. In (D) is shown the effect of antagonistic Fas mAb ZB4 on human eosinophil apoptosis induced by soluble RhFasL (100 ng/ml) or Fas mAb CH-11 (100 ng/ml). The isotype control for Fas mAb ZB4 was IgG1 (5 µg/ml) and it did not affect eosinophil apoptosis. In A, C and D apoptosis was analysed by flow cytometric measurement of relative DNA content. Mean ± SEM, n = 4–5. **indicates P<0.01 and ***P<0.001 as compared with the respective control.

Figure 3. Effect of TNF-receptor antibodies on apoptosis of human eosinophils in the presence of TNF-α.

The effect of TNF-R1 mAb (10 µg/ml; ▪) and TNF-R2 mAb (10 µg/ml; ▴) on the anti-apoptotic effect of TNF-α in isolated human eosinophils cultured for 40 hours. The control TNF-α concentration-response curve (•) was made in the presence of IgG1 isotype control (10 µg/ml). Apoptosis was assessed by flow cytometry measuring the relative DNA content of propidium iodide-stained eosinophils. Each data point represents the mean ± SEM of 6–8 independent determinations using eosinophils from different donors. Results are expressed as percentage of control. Solvent control in the absence of TNF-α is set as 100%.

Figure 4. Effect of TNF-α on IκBα phosphorylation.

The effect of TNF-α (100 ng/ml) in the absence and presence of IKK-1/IKK-2 inhibitor BMS-345541 (10 µM) on the phosphorylation of IκBα (p-IκB) in isolated human eosinophils. Incubations were terminated at the indicated time-points after addition of TNF-α or medium. In lower panel is shown the ratio of optical density (OD; arbitrary units) values of p-IκB and actin bands (mean ± SEM) from seven experiments using eosinophils isolated from different donors. * indicates P

Figure 5. Effect of TNF-α on IκB expression and NF-κB DNA binding.

In (A) is shown the effect of TNF-α (100 ng/ml) on the expression of IκBα in isolated human eosinophils. Incubations were terminated at the indicated time-points after addition of TNF-α (top panel) or medium in the simultaneously prepared control cells isolated from the same donor (lower panel). The data are representative of three separate experiments using eosinophils isolated from different donors. In (B) is shown the effect of TNF-α on NF-κB DNA binding in isolated human eosinophils. Incubations were terminated at the indicated time-points after addition of TNF-α (100 ng/ml). NF-κB DNA-binding activity was analyzed by electrophoretic mobility shift assay. Arrowheads indicate the different specific bands. In (C) is shown the total optical density (OD; arbitrary units) values of the abovementioned three specific bands (mean ± SEM) from five experiments using eosinophils isolated from different donors. * indicates P

Figure 6. Effect of NF-κB inhibitors on apoptosis of human eosinophils in the presence of TNF-α.

The effects of NF-κB inhibitors A. PDTC (▪ 1 µM; ▴ 10 µM; ▾100 µM), B. gliotoxin (▪ 0.9 µg/ml) and the inactive methylgliotoxin (▴ 0.9 µg/ml) and C. an inhibitor of IκB kinases-1 and -2, BMS-345541 (▴ 10 µM) on TNF-α-induced inhibition of apoptosis in isolated human eosinophils after 40 h culture. In each figure, the concentration-response curve of TNF-α in the absence of inhibitors (solvent control) is indicated by (•). Apoptosis was assessed by flow cytometry measuring the relative DNA content of propidium iodide-stained eosinophils. Each data point represents the mean ± SEM of 6-9 independent determinations using eosinophils from different donors. In A, the percentage of apoptotic eosinophils in the absence of TNF-α and PDTC was 57.4±5.5 and in the presence of PDTC it was 63.6±11.0 (1 µM; P>0.05), 49.1±8.0 (10 µM; P>0.05) and 33.1±2.7 (100 µM; P

Figure 7. Effect of TNF-α on AP-1 activation in human eosinophils.

The effect of TNF-α (100 ng/ml) on AP-1 activation in isolated human eosinophils. Incubations were terminated at the indicated time-points after addition of TNF-α. AP-1 DNA-binding activity was analyzed by electrophoretic mobility shift assay (A). Arrowheads indicate the different specific bands. The data are representative of three experiments. In (B) is shown the total optical density (OD; arbitrary units) values of the abovementioned specific bands (mean ± SEM) from 3 experiments using eosinophils isolated from different donors.

Figure 8. Effect of inhibitors of c-jun-N-terminal kinase and AP-1 on apoptosis of human eosinophils in the presence of TNF-α.

The effects of c-jun-N-terminal kinase inhibitor SP 600125 (A: ▪ 10 µM) and inhibitors of AP-1, SR 11302 (B; ▪ 1 µM) and tanshinone IIA (C; ▪ 1 µg/ml) on TNF-α-induced inhibition of apoptosis in isolated human eosinophils after 40 h culture. In each figure, the concentration-response curve of TNF-α in the absence of inhibitors (solvent control) is indicated by (•). Apoptosis was assessed by flow cytometry measuring the relative DNA content of propidium iodide-stained eosinophils. Each data point represents the mean ± SEM of 8-14 independent determinations using eosinophils from different donors. * indicates P