Beryllium-Induced Macrophage Apoptosis in Chronic Beryllium Disease

Richard T. SawyerValerie A. Fadok

Lisa A. MaierLori A. Kittle

Jack M. RoutesLee S. Newman

Departments of Medicine and PediatricsNational Jewish Medical and Research CenterUniversity of Colorado Health Sciences Center

Denver, Colorado

Background

• Be-stimulated H36.12j cells do not activate

NF-B/IB (Hamada et al. Toxicol. 2000;143:249)

• Be-induces H36.12j and P388D.1 mouse macrophage apoptosis(Sawyer et al. Toxicol. 2000;149:129)

• Be-stimulated CBD and BeS BAL cells down regulate surface CD-14 expression

(Kittle, Sarc. Vasc. Diff Lung Dis. 2002; 19: in press)

SIMS detects beryllium inside CBD granulomas

0 50 100 150 200 250 300

BERYLLIUM CPS

p = 0.028

p = 0.027

INSIDE THEGRANULOMA

(n = 5)

OUTSIDE THEGRANULOMA

(n = 5)

Open bar = average + SE Be cpsBlack bar = average maximum + SE Be cps

Latency12 + 2 yr

Abraham, 2002:Low Be levels in the lungs of BeS (n = 7) or Be-exposed/fibrosis (n = 4)

Beryllium-stimulates TNF- production by CBD BAL cells

Be-stimulated BeS/BAL cells produce constitutive TNF-.

48h24h

Be+

48h24h

Be-

0

500

1000

1500

2000CBD

0

500

1000

1500

2000BeS

48h24h

Be+

48h24h

Be-

* *

Beryllium stimulates apoptosis in CBD lung granulomas

Kittle et al. Sarc. Vasc. Diff. Lung Dis. 2002; 19: (in press)

BeS H&E

CBD H&E CBD TUNEL

BeS TUNEL

Hypothesis

• Beryllium stimulation could induce apoptosis

in CBD BAL and BeS BAL macrophages

• Beryllium-simulated human lung macrophage

apoptosis could be triggered by beryllium-

stimulated TNF-

Methods

UnstimulatedAl-Stimulated

Be-Stimulated

TEM/DiffQuick

Propidium Iodide

TUNEL Staining

* BeS and CBD BAL cells* Isolated BAL macrophages and lymphocytes

UnstimulatedBe-stimulated (100, 10 , 1 M BeSO4)Al-stimulated [100 M Al2(SO4)3]

NUCLEAR FRAGMENTATION:• Transmission electron microscopy• Propidium iodide stain• TUNEL stain

CBD bronchoalveolar lavage cells

Be-stimulated CBD BAL macrophageswith nuclear fragmentation

Be-stimulated CBD BAL macrophages (NS-ES+)with nuclear fragmentation

Beryllium-stimulates CBD BALmacrophage apoptosis

PI staining and TEM

Beryllium-stimulates CBD BALmacrophage apoptosis

CBD

BD-FMK +

24 HOURSBE - BE +

BD-FMK -

0

20

40

60

80

100

24 HOURSBE - BE +

48 HOURSBE - BE +

(Kittle; Sarc. Vasc. Diff Lung Dis. 2002; in press)

CONCLUSIONS

Beryllium induced CBD and BeS BAL macrophages apoptosis:

* independent of granulomatous inflammation

* caspase-mediated

QUESTION:

Could Be-induced macrophage apoptosis due to the up-regulation of Be-stimulated TNF-?

1. BeS BAL macrophages

2. Control and TNF- (-/-) bone marrow derived MO

TUNEL staining of BeS BAL macrophages

UNSTIMULATED 10 M BeSO4

Beryllium induces BeS BAL macrophage apoptosis based on PI and TUNEL staining

*p < 0.05 versus unstimulated control; n = 4

0 10 20 30 40

% APOPTOSIS - TUNEL+

% APOPTOSIS - PI STAIN

UNSTIMULATED

100 M BeSO4

100 M Al2(SO4)3

% APOPTOSIS

**

Beryllium induces apoptosis of control and TNF- (-/-) bone marrow derived macrophages

* p < 0.05 versus unstimulated controls; n = 4

0 5 10 15 20 25

TNF- -/-

C57Bl/6N

% TUNEL POSITIVE

UNSTIMULATED

100 M Al2(SO4)3

100 M BeSO4

10 M BeSO4

1 M BeSO4

*

*

*

*

*

CONCLUSION

Beryllium-induced human lung macrophage

apoptosis is independent of constitutive or

Be-stimulated TNF-

QUESTION:

Do all BAL cells undergo Be-induced apoptosis?

86% of adherent CBD BALcells are esterase positivemacrophages

86% of non-adherent CBD BAL cells are esterase negativesmall lymphocytes

Bost et al., AJRCMB 1994; 10:506

A Unstimulated Macrophage C Unstimulated Lymphocyte

D Lymphocyte + Be 100 MB Macrophage + Be 10 M

CONCLUSIONS

Beryllium-induced human lung macrophage apoptosis:

• Occurs in vivo

• Occurs with and without granulomatous inflammation

• Is caspase-mediated

• Independent of constitutive or Be-stimulated TNF-• Occurs in macrophages - in general

(Sawyer et al. Toxicol. 2000;149:129)

Sawyer et al., Internatl. Immunopharm. 2002; 2:249

What to do with the bodies?

Fadok, 2002

“The usual suspects”