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

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

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

SAA delays neutrophil apoptosis

El Kebir et al., J. Immunol. 179:616-622, 2007

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

Activation of Akt and ERKActivation of Akt and ERK

AktERK

PI-3kMEK

SAA

ALXR

SAA: 10 µg/ml

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

p p

BADp p

Mcl-1

BAD Mcl-1

AktERK

PI 3kMEK

SAA

ALXPhosphorylation of BAD

Challenge: SAA, 10 µg/ml for 30 min

Aging

?

Mitochondrial Δψm ↓

SAA prevents disruption of SAA prevents disruption of Δψm

n = 6* P<0.05** P<0.01

Mitochondrial Δψm ↓

Cytochrome c

Aging

?

Cytochrome c releaseCytochrome c release

***

P<0.05P<0.01

n = 6

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

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

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

15-epi-LXA4 reverses SAA suppression of apoptosis

Pretreatment with 15-epi-LXA4

n = 5*P<0.05**P<0.01

15-epi-LXA4 inhibits SAA-induced signaling

Treatment with 15-epi-LXA4 at 60 min post-SAA

n = 6

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

SAA

Annexin-1

LXA4

ATL

Apoptosis

ALXR: A pleiotropic receptor

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.

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

ALXR: A pleiotropic receptor

Chiang et al, Pharmacol. Rev. 2006

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

SAA signals through ERK1/2 and Akt

n=5-7** P<0.01vs untreated†† P<0.01vs. SAA

24 h culture

SAA prevents disruption of ∆Ψm

n=6* P<0.05** P<0.01

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

SAA, caspase inhibition and neutrophil apoptosis

n=5 * P<0.05** P<0.01

24 h culture

SAA-induced activation of Akt and ERK

Biosynthesis of lipoxins

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?

Survival signals for neutrophils

15-epi-LXA4 overcomes SAA suppression of apoptosis

C SAA 15-epi-LXA4