High beryllium-stimulated TNF-alpha is associated with the -308 TNF-alpha promoter polymorphism and with clinical severity in chronic beryllium disease.

Beryllium (Be)-antigen stimulates tumor necrosis factor-alpha (TNF-alpha) from bronchoalveolar lavage (BAL) cells in chronic beryllium disease (CBD). This study tested the hypothesis that high concentrations of Be-stimulated TNF-alpha are related to polymorphisms in the TNF-alpha promoter and clinical markers of disease severity in CBD. Demographic and clinical information was obtained from patients with CBD (n = 20). TNF-alpha concentrations were measured in BAL cell culture supernatant by ELISA. A priori, we categorized CBD subjects as either high or low TNF-alpha producers using a cutoff of 1,500 pg/ml. The TNF-alpha promoter sequence, +64 to -1045, was determined by direct sequencing. Human leukocyte-associated antigen (HLA)-DPB1 and -DRB1 genotyping was determined by polymerase chain reaction (PCR). High Be-stimulated TNF-alpha was associated with TNF2 alleles, Hispanic ethnicity, presence of HLA-DPB1 Glu69, and absence of HLA-DR4. Be-stimulated TNF-alpha concentrations correlated with markers of disease severity, including chest radiograph, beryllium lymphocyte proliferation, and spirometry. We found no novel TNF-alpha promoter polymorphisms. These data suggest that the TNF2 A allele at -308 in the TNF-alpha promoter region is a functional polymorphism, associated with a high level of Be-antigen-stimulated TNF-alpha and that these high TNF-alpha levels indicate disease severity in CBD.

IL-4 fails to regulate in vitro beryllium-induced cytokines in berylliosis.

Bronchoalveolar lavage (BAL) cells from patients with chronic beryllium disease (CBD) have been used to evaluate the beryllium-specific immune response and potential immunotherapeutics. Beryllium induces interferon-gamma (IFN-gamma), interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-10 (IL-10) from BAL cells. An antibody to IL-2 and recombinant human (rHu) IL-10 is able to partially suppress the beryllium-stimulated immune response. To obtain BAL cells, bronchoscopy is required, providing risk to the patient and a limited number of cells to study the immune response. As a result, the objectives of the study were to determine 1) whether CBD peripheral blood mononuclear cells (PBMNs) stimulated with beryllium would produce a similar cytokine pattern as BAL cells, and 2) whether this response could be modulated by interleukin-4 (IL-4), an immunomodulatory cytokine. CBD and normal individuals' PBMN and BAL cells were stimulated with and without beryllium sulfate. To modulate this antigen-stimulated response, we added rHu IL-4 to the unstimulated and beryllium-stimulated cells. IFN-gamma, IL-2, TNF-alpha, IL-6 and IL-10 cytokine concentrations were determined from cell supernatants by enzyme-linked immunosorbent assays (ELISA), while IL-4 messenger ribonucleic acid (mRNA) was assessed using polymerase chain reaction (PCR). Beryllium did not stimulate any of these cytokines from normal PBMNs. Increasing levels of IL-6 and TNF-alpha were produced constituitively by CBD PBMNs over time. Compared to the unstimulated CBD PBMNs, beryllium stimulated significant IFN-gamma, TNF-alpha, IL-2, IL-6 and IL-10 production. This response was similar to that stimulated from CBD BAL cells, although of a much lower magnitude. Low levels of IL-4 mRNA were found in CBD and control PBMNs, which were not increased with beryllium stimulation. The beryllium-stimulated cytokine levels were not decreased by the addition of IL-4. IL-4 was unable to downregulate any of these beryllium-stimulated cytokines from CBD BAL cells or increase IL-4 mRNA from either CBD PBMN or BAL cells, and thus is an unlikely immunomodulatory agent in CBD. From the data, it was concluded that chronic beryllium disease peripheral blood mononuclear cells provide a model to study the beryllium-stimulated immune response. Interleukin-4's inability to downregulate any of the beryllium-stimulated cytokines makes it an unlikely therapeutic candidate in chronic beryllium disease.

Beryllium-stimulated apoptosis in macrophage cell lines.

In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

Beryllium-stimulated production of tumor necrosis factor-alpha by a mouse hybrid macrophage cell line.

Chronic beryllium disease (CBD) results from exposure to the light-weight metal beryllium (Be). In vitro stimulation of bronchoalveolar lavage cells from CBD subjects causes the production of high levels of TNF-alpha, IFN-gamma and IL-6. We tested the hypothesis that Be-stimulation might induce the production of TNF-alpha by macrophage cell lines. We observed that H36.12j cells (12j), a mouse hybrid macrophage cell line, but not other mouse and human macrophage cell lines, produced TNF-alpha upon Be-stimulation. The response was maximal at 100 microM BeSO4 and did not occur when 12j cells were stimulated with either aluminum sulfate or cobalt sulfate. Beryllium-stimulated the production of 725+/-25 pg/ml (mean +/- SEM) TNF-alpha protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-alpha protein production was accompanied by an increased intracellular TNF-alpha mRNA at 3 and 24 h. The addition of 10U or 100U of rMu-IFN-gamma to Be-stimulated 12j cells further increased TNF-alpha production 1.5-4 fold (1.6+/-0.1 ng/ml) respectively. Bacterial lipopolysaccharide (LPS, 1 microg/ml) stimulated production of TNF-alpha in 12j culture supernatants after 6 h (515+/-151 pg/ml). This early versus late TNF-alpha production suggests that LPS and Be both stimulate 12j cell TNF-alpha synthesis, but through different pathways. We report for the first time, the direct effects of Be stimulation on the ability of 12j cells to produce TNF-alpha. The 12j cell line, contrasted with other macrophage hybrids that do not respond to Be-stimulation, may provide a useful tool to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-gamma amplifies TNF-alpha production in granulomatous diseases.

Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line.

We tested the hypothesis that beryllium (Be) could stimulate H36.12j cell (12j) TNF-alpha production by transcription factor-mediated pathways similar to those induced by either LPS- or IFN-gamma stimulation. Unstimulated 12j cells produce constitutive levels of TNF-alpha (175+/-18 pg/ml, mean +/- SEM) detected by ELISA of culture supernatants after 24 h. Beryllium-stimulated (100 microM BeSO4) 12j cell TNF-alpha (724+/-47 pg/ml) was observed after 24 h while LPS-stimulated (1 microg/ml) TNF-alpha (515+/-151 pg/ml) after 6 h. Recombinant-Mu-IFN-gamma (10 U) stimulated 12j cell TNF-alpha at lower levels (284+/-31 pg/ml) while rMu-IFN-gamma + Be-stimulated 12j cells produced 1195+/-225 pg/ml TNF-alpha. Constitutive levels of transcription factors were observed in unstimulated 12j cell nuclei. In LPS-stimulated 12j cells IkappaBalpha was degraded in the cytoplasm and increased levels of NF-kappaB were found in nuclei after 30 min. After 3 h there were increased levels of AP-1 and CREB, with increased amounts of Fos family, Jun B and Jun D transcription factors. In contrast, Be-stimulation failed to increase the levels of any transcription factor tested, NF-kappaB, AP-1, AP-2, CREB, C/EBP, Sp-1, Egr-1, Ets, NF-Y or Oct-1, in 12j cells. A pattern of increased transcription factors, similar to that observed for LPS-stimulation, was found in 12j cell nuclei after stimulation with rMu-IFN-gamma. However, NF-kappaB was increased at 3 h while AP-1 (Jun B and Jun D) and CREB were increased at 15 h. Co-stimulation of 12j cells with rMu-IFN-gamma + Be increased the levels of NF-KB in 12j cell nuclei at 3 h, and the levels of AP-1 and CREB at 15 h, however, only Jun B was increased. Our data show 12j cell TNF-alpha production was associated with increased levels of transcription factors present in nuclei with disparate kinetics and patterns of expression depending on the trigger. We reject our initial hypothesis and conclude that Be-stimulation signals 12j cell TNF-alpha synthesis via a transcription factor-independent pathway. Beryllium may induce novel pathways of macrophage cytokine gene regulation.

Partial IL-10 inhibition of the cell-mediated immune response in chronic beryllium disease.

Chronic beryllium disease (CBD) provides a human disorder in which to study the delayed type IV hypersensitivity response to persistent Ag that leads to noncaseating pulmonary granuloma formation. We hypothesized that, in CBD, failure of IL-10 to modulate the beryllium-specific, cell-mediated immune response would result in persistent, maximal cytokine production and T lymphocyte proliferation, thus contributing to the development of granulomatous lung disease. To test this hypothesis, we used bronchoalveolar lavage cells from control and CBD subjects to evaluate the beryllium salt-specific production of endogenous IL-10 and the effects of exogenous human rIL-10 (rhIL-10) on HLA expression, on the production of IL-2, IFN-gamma, and TNF-alpha, and on T lymphocyte proliferation. Our data demonstrate that beryllium-stimulated bronchoalveolar lavage cells produce IL-10, and the neutralization of endogenous IL-10 does not increase significantly cytokine production, HLA expression, or T lymphocyte proliferation. Second, the addition of excess exogenous rhIL-10 partially inhibited the beryllium-stimulated production of IL-2, IFN-gamma, and TNF-alpha; however, we measured no change in T lymphocyte proliferation or in the percentage of alveolar macrophages expressing HLA-DP. Interestingly, beryllium salts interfered with an IL-10-stimulated decrease in the percentage of alveolar macrophages expressing HLA-DR. We conclude that, in the CBD-derived, beryllium-stimulated cell-mediated immune response, low levels of endogenous IL-10 have no appreciable effect; exogenous rhIL-10 has a limited effect on cytokine production and no effect on T lymphocyte proliferation or HLA expression.

Beryllium induces IL-2 and IFN-gamma in berylliosis.

Chronic beryllium disease (CBD) provides a model for study of the Ag-stimulated, cell-mediated immune response that, over time, progresses to granulomatous lung disease. Using cells obtained with bronchoalveolar lavage from patients with CBD and normal individuals, we evaluated beryllium salt-stimulated T lymphocyte proliferation and production of proinflammatory cytokines. Our findings demonstrate that beryllium sulfate stimulates production of both IL-2 and IFN-gamma, not IL-4 and IL-7. We observed a brief time course for IL-2 protein (6-48 h after BeSO4 stimulation) and mRNA production (3-6 h) and a protracted time course for IFN-gamma protein (24-168 h) and mRNA (0.25-168 h). Beryllium-stimulated T lymphocyte proliferation and IFN-gamma release were only partially inhibited by neutralization of IL-2. On the basis of these findings, we hypothesized that IFN-gamma and the IL-2/IFN-gamma-inducible alpha subunit of the soluble IL-2 receptor were elevated in serum and bronchoalveolar lavage fluid of individuals with disease and were molecular markers of granulomatous disease. The data demonstrate that levels of the alpha subunit of the soluble IL-2 receptor, but not IFN-gamma, are elevated in the serum (median = 1428 U/ml; interquartile range = 823-2137 U/ml) and bronchoalveolar lavage fluid (median = 1.56 U/ml, interquartile range = 1.04-4.22 U/ml) of patients with CBD and correlate with the degree of pulmonary lymphocytosis and clinical measures of disease severity. We conclude that IL-2 and IFN-gamma are produced in the beryllium-stimulated, cell-mediated immune response with different time courses and that the alpha subunit of the soluble IL-2 receptor may serve as a biomarker of disease progression.

Chronic respiratory disease associated with long-term ambient concentrations of sulfates and other air pollutants.

Seventh-day Adventist (SDA) non-smokers who had resided since 1966 within five miles of their 1977 residence (n = 3914) completed a standardized respiratory symptoms questionnaire in 1977 and again in 1987. For each participant, cumulative ambient concentrations from 1977 to 1987 of suspended sulfates (SO4) in excess of several cutoffs as well as mean concentrations were estimated by interpolating monthly ambient concentration statistics from state air monitoring stations to the individual's residential and workplace zip codes. There were significant associations between ambient concentrations of suspended sulfates and development of new cases of asthma, but not new cases of overall airway obstructive disease (AOD) or chronic bronchitis. Comparison of previous analyses, in this population, of respiratory disease symptoms and total suspended particulates (TSP), ozone, and sulfur dioxide (SO2), and multipollutant analyses of these pollutants with SO4, indicated these results were not due to a surrogate relationship with other air pollutants. Development of definite symptoms of AOD and chronic bronchitis was most strongly related to TSP.