Beryllium-induced TNF-alpha production is transcription-dependent in chronic beryllium disease

Abstract

Beryllium (Be)-antigen presentation to Be-specific CD4(+) T cells from the lungs of patients with chronic beryllium disease (CBD) results in T cell proliferation and TNF-alpha secretion. We tested the hypothesis that Be-induced, CBD bronchoalveolar lavage (BAL) T cell, transcription-dependent, TNF-alpha secretion was accompanied by specific transcription factor upregulation. After 6 h of Be stimulation, CBD BAL cells produced a median of 883 pg/ml TNF-alpha (range, 608-1,275 pg/ml) versus 198 pg/ml (range, 116-245 pg/ml) by unstimulated cells. After 12 h CBD BAL cells produced a median of 2,963 pg/ml (range, 99-9,424 pg/ml) TNF-alpha versus 55 pg/ml (range, 0-454) by unstimulated cells. Using real-time RT-PCR, Be-stimulated TNF-alpha production at 6 h was preceded by a 5-fold increase in TNF-alpha pre-mRNA copy number:beta-actin copy number (Be median ratio 0.21; unstimulated median ratio 0.04). The median ratio of mature TNF-alpha mRNA:beta-actin mRNA was upregulated 1.4-fold (Be median ratio 0.17; unstimulated median ratio 0.12). Be exposure in the presence of the transcription inhibitor pentoxifylline (PTX) decreased CBD BAL cell TNF-alpha pre-mRNA levels > 60%, whereas treatment with the mRNA splicing inhibitor 2-aminopurine (2AP) decreased levels 40% relative to Be exposure alone. PTX treatment decreased mature TNF-alpha mRNA levels 50% while 2AP decreased levels > 80%, relative to Be exposure alone. Beryllium exposure specifically upregulated transcription factors AP-1 and NF-kappaB. The data suggest that Be exposure induces transcription-dependent TNF-alpha production, potentially due to upregulation of specific transcription factors.

Figure 1.

(a) A representative example of intracellular TNF-α expression by CD4⁺ BAL T cells from a patient with CBD after short-term culture with medium alone, 10 μM BeSO₄, or 10 ng/ml SEB. The percentage of CD4⁺ CBD BAL T cells expressing intracellular TNF-α is shown in the upper right quadrant of each density plot. (b) The percentage (%) of CD4⁺ BAL T cells expressing intracellular TNF-α in response to either medium alone, 10 μM BeSO₄, or SEB is shown. The bar indicates the median value for the four subjects with CBD tested.

Figure 2.

The kinetics of TNF-α mRNA upregulation in Be-stimulated CBD BAL cells (n = 4). (a) The median ratio of TNF-α pre-mRNA copy number to β-actin mature mRNA copy number at 0, 0.5, and 1 h in unstimulated (open bars) and Be-stimulated (filled bars; 100 μM BeSO₄) CBD BAL cells. (b) The median ratio of TNF-α mature-mRNA copy number to β-actin mature mRNA copy number at 0, 0.5, and 1 h in unstimulated (open bars) and Be-stimulated (filled bars; 100 μM BeSO₄) CBD BAL cells. *P < 0.05, Wilcoxon matched pairs.

Figure 3.

Transcription-mediated TNF-α pre-mRNA copy number and mature mRNA copy number:β-actin mature mRNA copy number in CBD BAL cells (n = 4) after 6 h of Be stimulation (100 μM BeSO₄). (a) The median ratio of TNF-α pre-mRNA to β-actin after exposure to 100 μM BeSO₄ alone (solid bars) versus Be + PTX (open bars) or Be + 2AP (striped bars). (b) The median ratio of TNF-α mature mRNA to β-actin after exposure to 100 μM BeSO₄ alone (solid bars) versus Be + PTX (open bars) or Be + 2AP (striped bars). (c) The median ratio of TNF-α pre-mRNA to β-actin after culture in medium alone (solid bars) or medium alone + PTX (open bars) or + 2AP (striped bars). (d) The median levels of TNF-α mature mRNA to β-actin after culture in medium alone (solid bars) or medium alone + PTX (open bars) or + 2AP (striped bars). (e) The median levels of TNF-α pre-mRNA to β-actin after exposure to 1 μg/ml LPS alone (solid bars) versus LPS + PTX (open bars) or LPS + 2AP (striped bars). (f) The median levels of TNF-α mature mRNA to β-actin after exposure to 1 μg/ml LPS alone (solid bars) versus LPS + PTX (open bars) or LPS + 2AP (striped bars).

Figure 3.

Transcription-mediated TNF-α pre-mRNA copy number and mature mRNA copy number:β-actin mature mRNA copy number in CBD BAL cells (n = 4) after 6 h of Be stimulation (100 μM BeSO₄). (a) The median ratio of TNF-α pre-mRNA to β-actin after exposure to 100 μM BeSO₄ alone (solid bars) versus Be + PTX (open bars) or Be + 2AP (striped bars). (b) The median ratio of TNF-α mature mRNA to β-actin after exposure to 100 μM BeSO₄ alone (solid bars) versus Be + PTX (open bars) or Be + 2AP (striped bars). (c) The median ratio of TNF-α pre-mRNA to β-actin after culture in medium alone (solid bars) or medium alone + PTX (open bars) or + 2AP (striped bars). (d) The median levels of TNF-α mature mRNA to β-actin after culture in medium alone (solid bars) or medium alone + PTX (open bars) or + 2AP (striped bars). (e) The median levels of TNF-α pre-mRNA to β-actin after exposure to 1 μg/ml LPS alone (solid bars) versus LPS + PTX (open bars) or LPS + 2AP (striped bars). (f) The median levels of TNF-α mature mRNA to β-actin after exposure to 1 μg/ml LPS alone (solid bars) versus LPS + PTX (open bars) or LPS + 2AP (striped bars).

Figure 3.

Transcription-mediated TNF-α pre-mRNA copy number and mature mRNA copy number:β-actin mature mRNA copy number in CBD BAL cells (n = 4) after 6 h of Be stimulation (100 μM BeSO₄). (a) The median ratio of TNF-α pre-mRNA to β-actin after exposure to 100 μM BeSO₄ alone (solid bars) versus Be + PTX (open bars) or Be + 2AP (striped bars). (b) The median ratio of TNF-α mature mRNA to β-actin after exposure to 100 μM BeSO₄ alone (solid bars) versus Be + PTX (open bars) or Be + 2AP (striped bars). (c) The median ratio of TNF-α pre-mRNA to β-actin after culture in medium alone (solid bars) or medium alone + PTX (open bars) or + 2AP (striped bars). (d) The median levels of TNF-α mature mRNA to β-actin after culture in medium alone (solid bars) or medium alone + PTX (open bars) or + 2AP (striped bars). (e) The median levels of TNF-α pre-mRNA to β-actin after exposure to 1 μg/ml LPS alone (solid bars) versus LPS + PTX (open bars) or LPS + 2AP (striped bars). (f) The median levels of TNF-α mature mRNA to β-actin after exposure to 1 μg/ml LPS alone (solid bars) versus LPS + PTX (open bars) or LPS + 2AP (striped bars).

Figure 4.

The levels of transcription factors NF-κB and AP-1 as determined by EMSA, in the nuclei of isolated CBD BAL cells from two subjects, that were unstimulated (−) or exposed to 100 μM BeSO₄ (+). Constitutive levels of nuclear transcription factors were determined for NF-κB and AP-1 by EMSA in unstimulated, isolated CBD BAL macrophages (MO), and isolated CBD BAL T cells (T cell) at 0 time and are compared with the changes in transcription factor levels after 24 h in unstimulated isolated T cells (−) or isolated T cells exposed to 100 μM BeSO₄ (+). Cold oligonucleotide competition (CC) controls (absence of a band), and EMSA antibody supershift (SS) assay controls (arrows on right) for isolated T cells after 24 h of exposure to 100 μM BeSO₄ (+) indicated the specificity of the transcription factor bands.