1. Beryllium Stimulates a Local Angiotensin System in Chronic Beryllium Disease T Hendry-Hofer* AP Fontenot¶, EA Barker*, M Boguniewicz*, LS Newman* and LA Maier

2. Background • Angiotensin Converting Enzyme (ACE) implicated in the immune response in granulomatous disease • Present on monocytes, macrophages, T cells • Reflects granulomatous burden • ACE inhibitor treatment of mice with S. mansoni decreases granulomatous response • ATII is produced from ATI, by ACE • Acts on two receptors, AT1 and AT2 • Proliferation of fibroblasts, production of collagen • Stimulates a number of growth factors • Block AT1 receptor, reduce fibrosis

3. Background (cont.) • Serum ACE levels are associated with disease severity in CBD • Does not discriminate CBD from BeS or Be Exposed • Serum ACE activity correlated with granulomatous inflammation (Newman LS, et al. Am Rev Respir Dis. 1992; 146: 39-42) • ACE polymorphism has been associated with increased serum ACE in CBD • 287 bp insertion (I) or deletion (D) at intron 16 • Highest serum ACE activity in CBD subjects with DD genotype (Maier LA, et al. Am J Respir Crit Care Med. 1999; 159: 1342-1350)

4. Hypothesis ACE and ATII will be stimulated in the skin and BAL fluid of individuals with CBD during granuloma formation

5. Methods • Study Subjects: • CBD was defined by: • A positive blood and or BAL lymphocyte proliferation test or positive skin patch test and • The presence of non-caseating granulomas on biopsy • ________________________________________________ • Sarcoidosis was defined by: (ATS, Statement on Sarcoidosis,1999) • A compatible case history and chest x-ray • The presence of non-caseating granulomas • Negative BAL stains and culture for acid fast bacteria, fungi or parasites • No history of beryllium exposure

6. Beryllium Skin Patch Testing and Immunohistochemistry • Patch Testing – Aqueous solution of 1% BeSO4 for 48 hours • – Punch biopsies at 0, 2, 3, 4, 14 and 35 days • Unstimulated CBD lavage cells were attached to slides by cytospin followed by methanol fixation • Immunohistochemistry was used to localize: CD3, CD4, CD8, CD68, ACE and ATII • Morphometric analysis used for quantification

7. ACE and ATII Protein • Bronchoscopy: BAL cells were obtained by routine lavage from 20 CBD and 5 sarcoidosis cases • BAL Cell culture:. Cells were cultured at 1 x 106 cells/ml in the presence and absence of 10-4 M BeSO4 • Protein Analysis: • ATII was measured by RIA • ACE was measured by ELISA

8. Summary • ACE colocalized with CD4+ T cells • ATII colocalized with CD68+ macrophages • Be stimulates: – ACE and ATII in skin patch granulomas – increased ACE production by CBD BAL cells • Be does not stimulate: – ACE production by sarcoidosis BAL cells – ATII production by CBD or Sarcoidosis BAL Cells

9. Conclusions • During beryllium stimulated granuloma formation • ACE is produced by CD4+ T cells • ATII by CD 68+ macrophages • Beryllium stimulates ACE production in the skin and lung of individuals with CBD • A local angiotensin system is stimulated by Be during granuloma formation in CBD subjects

10. May Gillespie Eric Wilcox Richard Sawyer Karen Dockstader Lynn Jui Michele Bausch This grant was funded by: NIH K08 HL-03887 NIH R01ES06538 NIH M01 RR-00051 Acknowledgments