# Presentation: 845961

## Beryllium Stimulates a Local Angiotensin System in Chronic Beryllium Disease

- T Hendry-Hofer* AP Fontenot¶, EA Barker*, M Boguniewicz*, LS Newman* and LA Maier

## 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

**Notes:**
The local angiotensin system has been studied extensively in cardiovascular disease. It has also been studied in granulomatous diseases such as sarcoidosis and CBD.

ACE has been implicated the inflammatory immune response in granulomatous disease. It has been found present on monocytes macrophages and T cells. 

Studies using an animal model have found ACE mRNA expression reflects the granulomatous burden

Treatment with an ACE inhibitor of mice infected with a parasite that causes liver granulomas decreases granulomatous inflammation.

ATII is the enzymatic product of ACE.

ATII acts on two receptors AT1R which seems to be primarily involved in the growth promoting effects of ATII and the AT2R. The role of the AT2R is widely unknown, but it has been implicated in some studies with apoptosis.

ATII has also been implicated in the proliferation of fibroblasts as well as the production of collagen. This proliferation has been linked to some of the growth factors that  ATII stimulates, such as bFGF, PDGF, TGFB and others.
Studies have linked the fibrotic effects of ATII to the AT1R. When the AT1R is blocked, fibrosis if reduced.

## 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)

- 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.)**_

**Notes:**
Studies have found increased ACE activity in sarcoidosis and specifically studies in our laboratory studies examining the angiotensin system have found increased serum ACE is associated with disease severity in CBD. 

Though serum ACE does not discriminate CBD from BES or BeExp, serum ACE does correlate with the granulomatous inflammation in CBD

A polymorphism within the ACE gene has also been associated with increased sACE in CBD 
A 287bp  insertion or deletion occurs within the intron of the ACE gene.
Highest serum ACE activity has been found in CBD subjects homozygous for the deletion

## Hypothesis

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

**Notes:**
From this we hypothesize that ACE and ATII will increase in response to beryllium in the Skin and BAL during granuloma formation in CBD subjects.

Tcell and mac responding to beryllium by increasing ACE production and as a result the enzymatic product of ACE ATII also increases.

## 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

## 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**

## 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

## 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

## 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**

## 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