1. Beryllium-Induced Macrophage Apoptosis in Chronic Beryllium Disease Richard T. Sawyer Valerie A. Fadok Lisa A. Maier Lori A. Kittle Jack M. Routes Lee S. Newman Departments of Medicine and Pediatrics National Jewish Medical and Research Center University of Colorado Health Sciences Center Denver, Colorado

2. Background Be-stimulated H36.12j cells do not activate NF-  B/I  B  (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)

3. SIMS detects beryllium inside CBD granulomas Open bar = average + SE Be cps Black bar = average maximum + SE Be cps Latency 12 + 2 yr Abraham, 2002: Low Be levels in the lungs of BeS (n = 7) or Be-exposed/fibrosis (n = 4)

4. Beryllium-stimulates TNF-  production by CBD BAL cells Be-stimulated BeS/BAL cells produce constitutive TNF- .

5. Beryllium stimulates apoptosis in CBD lung granulomas Kittle et al. Sarc. Vasc. Diff. Lung Dis. 2002; 19: (in press) BeS H&E CBD H&ECBD TUNEL BeS TUNEL

6. 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- 

7. Methods * BeS and CBD BAL cells * Isolated BAL macrophages and lymphocytes Unstimulated Be-stimulated (100, 10, 1  M BeSO 4 ) Al-stimulated [100  M Al 2 (SO 4 ) 3 ] NUCLEAR FRAGMENTATION: Transmission electron microscopy Propidium iodide stain TUNEL stain

8. CBD bronchoalveolar lavage cells

9. Be-stimulated CBD BAL macrophages with nuclear fragmentation

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

11. Beryllium-stimulates CBD BAL macrophage apoptosis PI staining and TEM

12. Beryllium-stimulates CBD BAL macrophage apoptosis (Kittle; Sarc. Vasc. Diff Lung Dis. 2002; in press)

13. 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

14. TUNEL staining of BeS BAL macrophages UNSTIMULATED 10  M BeSO 4

15. Beryllium induces BeS BAL macrophage apoptosis based on PI and TUNEL staining *p < 0.05 versus unstimulated control; n = 4

16. Beryllium induces apoptosis of control and TNF-  (-/-) bone marrow derived macrophages * p < 0.05 versus unstimulated controls; n = 4

17. CONCLUSION Beryllium-induced human lung macrophage apoptosis is independent of constitutive or Be-stimulated TNF-  QUESTION: Do all BAL cells undergo Be-induced apoptosis?

18. 86% of adherent CBD BAL cells are esterase positive macrophages 86% of non-adherent CBD BAL cells are esterase negative small lymphocytes Bost et al., AJRCMB 1994; 10:506

19. A Unstimulated MacrophageC Unstimulated Lymphocyte D Lymphocyte + Be 100  MB Macrophage + Be 10  M

20. 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)

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

22. What to do with the bodies? Fadok, 2002

23. “The usual suspects”