1) The document examines how the inflammatory protein serum amyloid A (SAA) delays apoptosis in human neutrophils through activation of the ALXR receptor and downstream signaling pathways involving Akt and ERK kinases. 2) SAA prevents mitochondrial dysfunction and caspase activation, thereby inhibiting neutrophil apoptosis. 3) The lipoxin 15-epi-LXA4 is able to override the anti-apoptotic effects of SAA, directing neutrophils back towards apoptosis and facilitating resolution of inflammation.

1. Aspirin-triggered lipoxins override the apoptosis
delaying action of serum amyloid A in human
neutrophils: A novel mechanism for resolution of
inflammation
János G. Filep,1
Levente Jozsef,1
Tarek Khreiss,1
Wanling Pan,1
Nicos A. Petasis,2
Charles N. Serhan,3
and Driss El Kebir1
1
Research Center, Maisonneuve-Rosemont Hospital,
University of Montreal, Montreal, QC, Canada H1T 2M4
2
Department of Chemistry, University of Southern California, Los Angeles, CA 90089
3
Center for Experimental Therapeutics and Reperfusion Injury,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02155

2. Serum amyloid A
• Marker of inflammation
• Classical acute-phase protein
• Serum concentration: <1 µg/mL to 1000 µg/mL
• Prognostic factor in rheumatoid arthritis and unstable angina
• Can be detected in inflamed tissues
• Activates neutrophils
• Undergo constitutive apoptosis
• Can be rescued from cell death by pro-inflammatory mediators
• Suppressed neutrophil apoptosis is a characteristic feature of
rheumatoid arthritis and unstable angina
Neutrophils

3. Aims
• whether SAA modulates constitutive neutrophil apoptosis
• what signaling events are associated with this event
• whether such actions can be influenced by aspirin-
triggered 15-epi-LXA4

4. SAA delays neutrophil apoptosis
El Kebir et al., J. Immunol. 179:616-622, 2007

5. SAA signals through ALXRSAA signals through ALXR
SAA
ALXR
** P<0.01
n = 5
24 h culture
N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc2):
antagonist of the fMLP receptor and ALXR

6. Activation of Akt and ERKActivation of Akt and ERK
AktERK
PI-3kMEK
SAA
ALXR
SAA: 10 µg/ml

7. Blockade of Akt and ERK reversesBlockade of Akt and ERK reverses
the SAA actionsthe SAA actions
AktERK
PI-3kMEK
SAA
ALXR
n = 5-7
** P<0.01 vs. untreated
††
P<0.01 vs. SAA
24 h culture

8. p p
BAD
p p
Mcl-1
BAD Mcl-1
AktERK
PI 3kMEK
SAA
ALX
Phosphorylation of BAD
Challenge: SAA, 10 µg/ml for 30 min

9. Aging
?
Mitochondrial Δψm ↓
SAA prevents disruption ofSAA prevents disruption of Δψm
n = 6
* P<0.05
** P<0.01

10. Mitochondrial Δψm ↓
Cytochrome c
Aging
?
Cytochrome c releaseCytochrome c release
*
**
P<0.05
P<0.01
n = 6

11. Mitochondrial Δψm ↓
Cytochrome c
Activation of
caspase-3
Aging
?
SAA attenuates caspase-3 activationSAA attenuates caspase-3 activation
n = 5
*P<0.05
**P<0.01

12. Mitochondrial Δψm ↓
Cytochrome c
Activation of
caspase-3
Aging
?
SAA attenuates caspase-3 activationSAA attenuates caspase-3 activation
n=5
*P<0.05
**P<0.01

13. Lipoxins do not affect neutrophil apoptosis
0
20
40
60
80
+
Untreated 15-epi-LXA4 ATLa
24 h culture
%Viableorapoptoticcells
n = 5
Jozsef et al., PNAS 99:13266, 2002
Viable cells
Annexin-V positive cells

14. 15-epi-LXA4 reverses SAA suppression of apoptosis
Pretreatment with 15-epi-LXA4
n = 5
*P<0.05
**P<0.01

15. 15-epi-LXA4 inhibits SAA-induced signaling

16. Treatment with 15-epi-LXA4 at 60 min post-SAA
n = 6

17. Treatment with 15-epi-LXA4 at 15 or 60 min post-SAA

18. SAA
Annexin-1
LXA4
ATL
Apoptosis
ALXR: A pleiotropic receptor

19. Conclusions
• Our results provide evidence for a mechanism by which SAA may
contribute to inflammation by rescuing neutrophils from apoptosis.
• SAA suppresses neutrophil apoptosis by preventing mitochondrial
dysfunction and consequently inhibiting cytochrome c release and
caspase-3 activation.
• 15-epi-LXA4 overrides the apoptosis delaying signals activated by
SAA and thus redirecting neutrophils to apoptosis, consistent with
facilitation of the resolution of inflammation.

20. Filep lab
Levente József
Tarek Khreiss
Driss El Kebir
Wanling Pan
Janos G. Filep
Flow cytometry lab
Sophie Ouellette
Chantal Baron
Blood donors
Collaborators
Lawrence A. Potempa (Immtech International)
John S.D. Chan (CR-CHUM)
Charles N. Serhan (Harvard University)
Nicos A. Petasis (U. of Southern California)
Acknowledgements

22. ALXR: A pleiotropic receptor
Chiang et al, Pharmacol. Rev. 2006

23. SAA-induced phosphorylation of BAD through
activation of Akt and ERK1/2

24. SAA signals through ERK1/2 and Akt
n=5-7
** P<0.01vs untreated
††
P<0.01vs. SAA
24 h culture

25. SAA prevents disruption of ∆Ψm
n=6
* P<0.05
** P<0.01

26. SAA inhibition of mitochondrial cytochrome c release
and caspase-3 activation

27. SAA, caspase inhibition and neutrophil apoptosis
n=5
* P<0.05
** P<0.01
24 h culture

28. SAA-induced activation of Akt and ERK

29. Biosynthesis of lipoxins

30. Formyl-peptide receptors
• FPR: high affinity receptor for fMLP
• FPLR1/ALXR: low affinity receptor for
fMLP plus many other ligands
• FPLR2: does not bind fMLP, ligands?

31. Survival signals for neutrophils

33. 15-epi-LXA4 overcomes SAA suppression of apoptosis
C SAA 15-epi-LXA4