p38 Mitogen-Activated Protein Kinase in beryllium-induced dendritic cell activation

Abstract

Dendritic cells (DC) play a role in the regulation of immune responses to haptens, which in turn impact DC maturation. Whether beryllium (Be) is able to induce DC maturation and if this occurs via the MAPK pathway is not known. Primary monocyte-derived DCs (moDCs) models were generated from Be non-exposed healthy volunteers as a non-sensitized cell model, while PBMCs from BeS (Be sensitized) and CBD (chronic beryllium disease) were used as disease models. The response of these cells to Be was evaluated. The expression of CD40 was increased significantly (p<0.05) on HLA-DP Glu69+ moDCs after 100 μM BeSO₄-stimulation. BeSO₄ induced p38MAPK phosphorylation, while IκB-α was degraded in Be-stimulated moDCs. The p38 MAPK inhibitor SB203580 blocked Be-induced NF-κB activation in moDCs, suggesting that p38MAPK and NF-κB are dependently activated by BeSO₄. Furthermore, in BeS and CBD subjects, SB203580 downregulated Be-stimulated proliferation in a dose-dependent manner, and decreased Be-stimulated TNF-α and IFNγ cytokine production. Taken together, this study suggests that Be-induces non-sensitized Glu69+ DCs maturation, and that p38MAPK signaling is important in the Be-stimulated DCs activation as well as subsequent T cell proliferation and cytokine production in BeS and CBD. In total, the MAPK pathway may serve as a potential therapeutic target for human granulomatous lung diseases.

Keywords: CBD; DCs; Lymphocyte proliferation; NF-κB; p38MAPK.

Fig 1

The effect of BeSO₄ on the phenotype of moDCs with HLA-DPβ1 Glu69+ genotype. At day 7, moDC were treated with 10 or 100μM BeSO₄ for 24 h. Cell surface markers were measured using flow cytometry. Numbers represent the percentage of positive cells after Be treatment. The expression of (1a) CD40 was increased significantly from moDCs in Glu69+ subjects after after 100μM BeSO₄ stimulation for 24h on day 7 (* p<0.05, n=4). There was no significant difference on the expression of (1b) CD80, (1c) CD86 and (1d) HLA-DR after Be-stimulation.

Fig 2

Both NF-κB and p38MAPK were stimulated by Be in the monocyte DC Glu69+ cell models. At day 7, moDC were treated for different time periods (0, 30 min, 1h and 2h) with 10μM or 100μM BeSO₄. The levels of IKB and the phosphorylation of p38MAPK were evaluated by Western blotting. Total p38MAPK levels were determined as a loading control. Results of one representative subject out of four are shown, with similar results noted for other subjects. The conditions presented by lane are as follows: 1. DC Control, 2. DC with 10 μM BeSO₄ for 30 min, 3. DC with 100 μM BeSO₄ for 30 min, 4. DC with 10 μM BeSO₄ for 1h, 5. DC with 100 μM BeSO₄ for 1h, 6. DC with 10 μM BeSO₄ for 2h, 7. DC with 100 μM BeSO₄ for 2h.

Fig 3

The p38 Mitogen-Activated Protein Kinase Inhibitor SB203580 regulates Be-induced NF-κB activation in Glu69+ subjects. At day 7, moDCs were treated for different time periods with 10μM or 100μM BeSO₄ in the presence or absence of 10μM SB203580. The nuclear level of NF–κB p50 and the actin were evaluated by Western blotting. actin levels were determined as a loading control and results of one representative subject are shown, with similar results noted for the other subjects. The conditions presented by lane are as follows: 1. DC Control, 2. DC with TNF for 30 min; 3. DC with 10μM SB203580, 4. DC with 10 μM BeSO₄ for 1h, 5. DC with 10μM SB203580 and 10μM BeSO₄ for 1h, 6. DC with 100 μM BeSO₄ for 1h, 7. DC with 10μM SB203580 and 100μM BeSO₄ for 1h.

Figure 4

The p38 Mitogen-Activated Protein Kinase Inhibitor SB203580 inhibits the Be-stimulated (a) CBD and (b) BeS PBMCs proliferation. The stimulation index (SI) of Be-stimulated (a) CBD PBMCs (n=8) and (b) BeS PBMCs (n=8) treated with 10μM or 100μM BeSO₄ and 1μM or 10μM SB203580. Values are represented as mean stimulation index (SI). **p<0.01.

Figure 5

The p38 Mitogen-Activated Protein Kinase Inhibitor SB203580 treatment inhibits Be-stimulated CBD PBMC cytokines (a) TNFα and (b) IFNγ production (n=3). PBMCs were treated with 10μM or 100μM BeSO₄ and 1μM or 10uM SB203580. The data values were normalized according to each subject’s 10μM Be-stimulated TNFα and IFN γ levels. *p<0.05, **p<0.01.