Neutrophils in the

Pathogenesis of Sepsis

John C. Marshall, MD FRCSC

Critical Care Canada Forum

2012

Toronto, Canada

October 29, 2012St. Michael’s Hospital University of Toronto

Songhui Jia

Maria Jimenez

Yue Li

Zeenat Malam

Jean Parodo

Ravi Taneja

Jennifer Tsang

Bill Watson

Thanks to …

Neutrophils are a Dynamic Population

• Constitutively apoptotic

• 6 – 8 Hours In vivo

• 24 – 36 Hours In vitro

• 1011 PMN released and die by

apoptosis each day

Neutrophils kill

bacteria and phagocytose

damaged tissue

Activated

neutrophils also damage

adjacent normal tissues

Neutrophil Antimicrobial Defenses

Production of reactive

oxygen intermediates

Antimicrobial peptides

• BPI

• Defensins

• Alkaline phosphatase

• Lysozyme

• Lactoferrin

• Elastase

• Cathepsin G

Neutrophil

extracellular

traps (NETs)

Time-dependent

Effects of Neutrophil

Depletion in CLP

- Hoesel et al Shock

24:40, 2005

- 24 Hours + 12 Hours

Lo

g C

FU

/ml B

loo

d

0

2

4

6

8Non-specific IgG

Anti-PMN

Time Relative to CLP

*

**

Duration of In Vitro Culture

0 Hours 18 Hours 24 Hours

Perc

en

t A

po

pto

sis

0

10

20

30

40

50

60

Control

Sepsis

- Taneja, Crit Care Med 32:1460,2004

PMN Apoptosis is Delayed in Sepsis

LPS

*

**

Microbial Products Host-derived Mediators

Endotoxin Interleukin (IL)-1β

Lipoteichoic acid IL-2

Mannan IL-3

Modulins from CONS IL-4

E. coli verotoxin IL-6

H. pylori surface proteins IL-8

Butyric acid Tumor Necrosis Factor

Propionic acid Interferons

G-CSF, GM-CSF

Respiratory syncytial virus Leptin

PBEF

C5a

Cathelicidins

Physiologic ProcessesTransendothelial migration

Factors Inhibiting Neutrophil Apoptosis

% A

po

pto

sis

0

10

20

30

40

*

‡‡ ‡

‡ ‡‡

* Control

Herb A

PDTC

CHX

Control LPS

(1mg/ml)GM-CSF

(100U/ml)

Delayed Apoptosis Requires Cellular

Activation and Protein Synthesis- Watson, J.Immunol. 161:957, 1998

Control Anti-sense Sense Anti-sense

+ IL-1

Perc

en

t A

po

pto

sis

0

10

20

30

40

50

Control

LPS (1ug/ml)

GM-CSF (100U/ml)

Effects of Anti-sense to IL-1 on

LPS and GM-CSF Inhibition of PMN Apoptosis

- Watson, J.Immunol. 161:957, 1998.

Pre-B Cell Colony

Enhancing

Factor (PBEF)

• 52 kDa secreted protein

• Cytokine-like TATT motifs in mRNA

• Synergizes with IL-7 to induce

pre-B cell colony formation

- Samal; Mol.Cell Biol. 14:1431, 1994

ConLps

Sepsis Patients

1 2 3 4 5 6 7 8

PBEF mRNA is Highly Expressed

in Sepsis PMN

PBEF

G3PDH

- Jia et al J Clin Invest 113:1318, 2004

No Oligo Sense Non-sense Anti-sense

Perc

en

t A

po

pto

sis

0

5

10

15

20

25

30

*

PBEF Anti-sense Partially Restores

PMN Apoptosis in Sepsis

- Jia et al J Clin Invest 113:1318:2004

0

10

20

30

40

50

60

70

Control PBEF S199A S200A

% a

po

pto

sis

Anti-apoptotic effect of Nampt active PBEF on

trauma PMN is inhbited by prior addition of

insulin (20 µU/mL) N=6

no insulin

add ins 2nd

add ins 1st

Hotchkiss et al Crit.Care Med.

28:3207, 2000

- Imai et al, JAMA 289:2104, 2003

Trauma and Sepsis

Result in Systemic

Epithelial Cell

Apoptosis

Cytochrome C

CD95/TNFR

Caspase 8

Procaspase 9

APAF-1

Caspase 3Bcl2

Mcl1

Inhibitors of Apoptosis:

Survivin

CIAP1,2

XIAP1, NAIP

DNA, Protein cleavage

SMAC/Diablo

MDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIK

DALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYL

NTRKEEMERELQTPGRAQISAYRFHFCRMSWAEANSQ

CQTQSVPFWRRVDHLLIRVMLYQISEEVSRSELRSFKF

LLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKR

VCAQINKSLLKIINDYEEFSKGEELCGVMTISDSPREQD

SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKL

HSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIY

EILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPI

YELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPV

ETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNC

VSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNY

EVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD

Caspase-8

A549 HEK293

Perc

en

t H

yp

od

iplo

id D

NA

0

10

20

30

40

50

* *

** **

DPI - - + - -Catalase - - - + -

GSH - - - - +

No PMN + PMNWestern:

~ Caspase-8

~pY

Trauma Neutrophils

Induce Caspase-8

Dephosphorylation and

Apoptosis of A549

Lung Epithelial Cells

pY

pY

SHP-1

Cleavage of caspase-3 and

progression of apoptosis

lyn

PI3 Kinase Activation

PBEF Transcription

IAP Synthesis

CD95/TNFR

FADD

Pro-caspase-8

Caspase-8 Phosphorylation

Caspase-8

Dephosphorylation

p10

p18

The Caspase-8 Survivalsome

IL-1R

Candida : Neutrophil Ratio

Control 1 : 1 5 : 1 10 : 1

Perc

en

tag

e A

po

pto

sis

0

10

20

30

50

60

70

80

90

Phagocytosis of Candida albicans Induces

Neutrophil Apoptosis In Vitro

40

*

- Rotstein, Shock 2000; 14:278

Ischemia/reperfusion I/R + E. coli

Intratracheal E. coli Attenuates

Pulmonary Neutrophilia

- Sookhai, Ann Surg 2002; 235:285

Time (Hours)

0 10 20 30 40 50

Perc

en

t S

urv

ival

0

20

40

60

80

10

0

Sham

I/R + E.coli

I/R + Saline

- Sookhai Ann Surg 2002; 235:285

Intratracheal Heat-killed E. coli Attenuates

Mortality Following Ischemia/Reperfusion

Conclusions

• Functional neutrophils are critical to

the early response against infection

• Their prolonged activity contributes to

inadvertent organ injury

• Accelerating their involution through

apoptosis may limit this delayed

harm

Thank you!!