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, AT 1 and AT 2 –Proliferation of fibroblasts, production of collagen –Stimulates a number of growth factors –Block AT 1 receptor, reduce fibrosis

3. 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) Background (cont.)

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% BeSO 4 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 10 6 cells/ml in the presence and absence of 10 -4 M BeSO 4 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. Acknowledgments 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