Research Symposium: Basic Mechanisms and Human Health
Session 2. Pulmonary Signaling Pathways Activated by Beryllium
Beryllium Stimulates a Local Angiotensin System in Chronic Beryllium Disease
T. Hendry-Hofer1, A.F. Fontenot2, E.A.
Barker1, M. Boguniewicz1, L.S. Newman1,
and L.A. Maier2
Background: Increased serum angiotensin converting enzyme (ACE) is associated with granulomatous lung diseases such as sarcoidosis and chronic beryllium disease (CBD). CBD is marked by a beryllium specific cellular immune response, which results in granuloma formation and may progress to fibrosis. High serum ACE has been associated with disease severity in CBD, although the source and function of a local angiotensin system is not known. Mast cells found in fibrosis surrounding granulomas are a source of basic fibroblast growth factor (bFGF). Angiotensin II (ATII), an enzymatic product of ACE, stimulates bFGF production.
Objective: This study tested the hypothesis that ACE and its enzymatic product angiotensin II (ATII) are stimulated by beryllium during granuloma formation in individuals with CBD.
Methods: Following informed consent, 25 subjects with CBD and 5 with sarcoidosis were enrolled in this study. Of the 25 with CBD, 5 underwent beryllium (Be) skin patch testing, using aluminum as a negative control. Skin patch biopsies were collected at various intervals between 0 and 35 days. Immunohistochemistry was used to evaluate CD3, CD4, CD8, CD68, ACE and ATII in skin patch biopsies and morphometric analysis was used for quantification. The remaining 20 subjects with CBD underwent bronchoscopy with bronchoalveolar lavage (BAL). Five subjects with sarcoidosis were used as BAL controls. BAL cells were stimulated with and without beryllium sulfate at 10-4M and 10-5M for 0, 24, 72, and 120 hours. Aluminum sulfate at 10-4M was used as a negative control. ACE and ATII were determined in cell supernatants by ELISA and RIA respectively. Immunohistochemistry was performed on unstimulated cells to localize CD3, CD4, CD68, ACE and ATII in BAL cells.
Results: CD3, CD4, CD68, ACE and ATII were present by 48 hours in Be skin patch biopsies. ATII continued to increase over time, while ACE staining peaked at 96 hours. Double immunohistochemistry revealed predominant colocalization of CD4 with ACE and ATII with CD68. CBD BAL cells produced constitutive levels of ACE (median 12 ng/ml) and ATII (median 42 pg/ml). Sarcoidosis BAL cells also produced constitutive levels of ACE (median 15 ng/ml) and ATII (median 36 pg/ml). Be stimulated CBD BAL cells produced significantly more ACE compared to unstimulated cells (median 20 ng/ml vs. 12 ng/ml). ACE production did not increased in response to Be in sarcoidosis BAL cells (median 13 ng/ml vs. 15 ng/ml). Be did not stimulate ATII in CBD BAL cells (median 34 pg/ml vs. 42 pg/ml). In sarcoidosis BAL cells, ATII was not stimulated by Be (median 33 pg/ml vs. 36 pg/ml). Immunohisto-chemistry of BAL cells revealed colocalization of ACE with CD4 and ATII with CD68.
Conclusion: Be stimulates ACE production by T cells and ATII by monocytes in the skin during granuloma formation. Significant ACE is stimulated in BAL cells in response to Be, with T cells producing ACE and monocytes producing ATII. This suggests that Be stimulates a local angiotensin system in CBD It is possible that the angiotensin system is important in the immune response to beryllium and/or progression to fibrosis, which will be the topic of future studies.
K08 HL03887, R01 ES06358-06, M01 RR00051
High Levels of Nitric Oxide (NO) in Exhaled Breath in Patients with Chronic Beryllium Disease
R.A. Dweik, D. Laskowski, and S.C. Erzurum
Chronic Beryllium Disease (CBD) is an occupationally-acquired lung disease that begins as a sensitizing T cell-mediated immune response to beryllium antigen which develops into non-caseating granulomatous lung inflammation. Nitric oxide (NO) measurement in exhaled gases has been proposed as a noninvasive marker of lung inflammation. In this context, we hypothesized that NO may be high in patients with CBD due to active granulomatous lung inflammation. To evaluate this hypothesis, exhaled NO was measured in beryllium-exposed workers who were referred for evaluation of CBD after a positive blood lymphocyte proliferation test to beryllium. Following a 15 sec breath hold at total lung capacity, individuals exhaled against 10 cm of water pressure into a Mylar balloon while wearing a nose clip. Measures of NO in the exhalate were determined in duplicate using a chemiluminescent analyzer (NOA 280). All individuals had lung biopsies to determine granulomatous lung disease. Granuloma-positive individuals had higher exhaled NO levels than granuloma-negative individuals [CBD 12±2 ppb (n=8) vs granuloma-negative 7±1 ppb (n=8); p=0.04]. These results provide further support for an association between active lung inflammation and increased NO in exhaled breath.
Nitric Oxide (NO) Attenuation of Interferon Gamma (IFN-Gamma) Responses in Chronic Beryllium Disease (CBD): Evidence for Mechanisms Independent of Interleukin (IL)-18
M.J. Thomassen, C. Farver, R.A. Dweik, D. Culver, B. Yen-Lieberman,
M. Kavuru, and B.P. Barna
Mechanisms regulating bronchoalveolar lavage cell (BAL) IFN-gamma responses to beryllium salts (BE) were investigated in CBD, and BE-hypersensitive subjects without CBD. BE exposure (24h) elicited IFN-gamma (746 +/- 245 SEM pg/ml) in CBD BAL but none in untreated cells (<25.6 pg/ml, p<0.001,n=14) or in BE-treated BAL from BE-hypersensitive Individuals without lung disease (p<0.001, n=15). Because NO is elevated in CBD airways (Dweik et al AJRCCM, 161,A731, 2000), we examined effects of DETA NONOate (DNO), an NO generator, on BAL IFN-gamma. DNO reduced BE-stimulated IFN-gamma levels by 74% (p<0.01; n=3) vs. untreated. Analysis of IL-18 and IL-12 (cytokines known to augment IFN-gamma) indicated that BE elevated BAL IL-18 (229 +/- 62 vs. control 76 +/- 31 pg/ml, p<0.05, n=7) but IL-12 was undetectable (<15.4 pg/ml, n=6). Since DNO also reduced BE-stimulated IL-18 (55.7%, p<0.01, n=4), the role of IL-18 in BAL IFN-gamma was examined. Antibody to IL-18 drastically reduced IL-18 levels (95%, p<0.01, n=2) but IFN-gamma was only marginally affected (19%). Results suggest that NO represents a potent blocker of BE-stimulated IFN-gamma but mechanisms may involve sites other than IL-18 or IL-12.
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