bioactive lipids, montreal sept 2007
TRANSCRIPT
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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,1Levente Jozsef,1 Tarek Khreiss,1 Wanling Pan,1 Nicos A. Petasis,2 Charles N. Serhan,3 and Driss El Kebir1
1Research Center, Maisonneuve-Rosemont Hospital, University of Montreal, Montreal, QC, Canada H1T 2M4
2Department of Chemistry, University of Southern California, Los Angeles, CA 90089 3Center for Experimental Therapeutics and Reperfusion Injury,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02155
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Serum amyloid A
• Marker of inflammation• Classical acute-phase protein• Serum concentration: <1 g/mL to 1000 g/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
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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
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SAA delays neutrophil apoptosis
El Kebir et al., J. Immunol. 179:616-622, 2007
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SAA signals through ALXRSAA signals through ALXRSAA
ALXR
** P<0.01n = 5
24 h culture
N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc2): antagonist of the fMLP receptor and ALXR
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Activation of Akt and ERKActivation of Akt and ERK
AktERK
PI-3kMEK
SAA
ALXR
SAA: 10 µg/ml
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Blockade of Akt and ERK reverses Blockade 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
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p p
BADp p
Mcl-1
BAD Mcl-1
AktERK
PI 3kMEK
SAA
ALXPhosphorylation of BAD
Challenge: SAA, 10 µg/ml for 30 min
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Aging
?
Mitochondrial Δψm ↓
SAA prevents disruption of SAA prevents disruption of Δψm
n = 6* P<0.05** P<0.01
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Mitochondrial Δψm ↓
Cytochrome c
Aging
?
Cytochrome c releaseCytochrome c release
***
P<0.05P<0.01
n = 6
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Mitochondrial Δψm ↓
Cytochrome c
Activation ofcaspase-3
Aging
?
SAA attenuates caspase-3 activationSAA attenuates caspase-3 activation
n = 5*P<0.05**P<0.01
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Mitochondrial Δψm ↓
Cytochrome c
Activation ofcaspase-3
Aging
?
SAA attenuates caspase-3 activationSAA attenuates caspase-3 activation
n=5*P<0.05**P<0.01
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Lipoxins do not affect neutrophil apoptosis
0
20
40
60
80
+
Untreated 15-epi-LXA4 ATLa
24 h culture
% V
iabl
e or
apo
ptot
ic c
ells
n = 5
Jozsef et al., PNAS 99:13266, 2002
Viable cells
Annexin-V positive cells
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15-epi-LXA4 reverses SAA suppression of apoptosis
Pretreatment with 15-epi-LXA4
n = 5*P<0.05**P<0.01
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15-epi-LXA4 inhibits SAA-induced signaling
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Treatment with 15-epi-LXA4 at 60 min post-SAA
n = 6
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Treatment with 15-epi-LXA4 at 15 or 60 min post-SAA
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SAA
Annexin-1
LXA4
ATL
Apoptosis
ALXR: A pleiotropic receptor
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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.
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Filep labLevente JózsefTarek KhreissDriss El KebirWanling PanJanos G. Filep
Flow cytometry labSophie OuelletteChantal Baron
Blood donors
CollaboratorsLawrence A. Potempa (Immtech International)John S.D. Chan (CR-CHUM)Charles N. Serhan (Harvard University)Nicos A. Petasis (U. of Southern California)
Acknowledgements
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ALXR: A pleiotropic receptor
Chiang et al, Pharmacol. Rev. 2006
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SAA-induced phosphorylation of BAD through activation of Akt and ERK1/2
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SAA signals through ERK1/2 and Akt
n=5-7** P<0.01vs untreated†† P<0.01vs. SAA
24 h culture
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SAA prevents disruption of ∆Ψm
n=6* P<0.05** P<0.01
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SAA inhibition of mitochondrial cytochrome c release and caspase-3 activation
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SAA, caspase inhibition and neutrophil apoptosis
n=5 * P<0.05** P<0.01
24 h culture
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SAA-induced activation of Akt and ERK
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Biosynthesis of lipoxins
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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?
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Survival signals for neutrophils
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15-epi-LXA4 overcomes SAA suppression of apoptosis
C SAA 15-epi-LXA4