Beryllium Stimulates a Local Angiotensin System in Chronic Beryllium Disease

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
• ________________________________________________
  
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• 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
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