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Cytokine Balance: The main effect of cytokine network

Cem Gabay, MDDivision of Rheumatology

Geneva, Switzerland

ANKARA ROMATOLOJİ SEMPOZYUMU “Sitokinler: Sağlıkta ve Romatizmal Hastalıklarda”06 Nisan 2007 Sheraton Otel, Ankara

CytokinesMicrobial productsDegraded EC matrixC5a

Cytokine

Cytokine mRNA

Cytokine promoter

Transcription factorProtein kinases

13

4

Cytokine2

R

Regulation of Cytokine Production


Cytokine CascadeThe example of turpentin injury-induced acute-phase response

IL-1β

IL-6

Liver

Serum amyloid A

Tissue injury

Turpentine injection

Circulation

TurpentineSaline

GAPDH mRNA

SAA mRNA

IL-1β

IL-6

Liver

Serum amyloid A

Tissue injury

Turpentine injection

Circulation

TurpentineSaline

GAPDH mRNA

SAA mRNA

Gabay et al. Eur J Immunol 2001Fantuzzi et al. J Immunol 1997


Cytokine Network

Cytokines with additive or synergistic effects

Cytokines with inhibitory effects

Receptor antagonists (IL-1Ra, IL-1F5)

Soluble receptors (sIL-1R, sTNF-R)

Intracellular signal regulators


Soluble receptor can enhance the effect of their ligand


Signaling Inhibitors

degradation

NF-κB

IKK

p50p65

TNF-α, IL-1, LPS

IκB-αgene

IκB-α

PP

P PIκB-α

proteasome

NUCLEUS


The IL-1 SystemAn example of agonist/antagonist balance


IL-1 : A Pleiotropic Cytokine

acute phaseprotein production

procoagulant state

collagenaseand stromelysin

bone resorption

fibroblast and epithelial cell proliferation

feversleeploss of appetitesocial withdrawal

vasodilationadhesion molecules chemotaxisincreased permeability

immune activation

stimulation ofhematopoiesis

Prostaglandin E2


The IL-1 Family

Signal PeptidaseIL-1Ra

IL-1Ra Receptor antagonist

IL-1βIL-1α agonists

IL-1β

IL-1α ?

ICE= Caspase 1


IL-1Ra IsoformsIL-1RN gene

IL-1Ra mRNAs

IL-1Ra proteins

transcription: alternative promoters

alternative splicing

translation: alternative initiation

ic1 ic2 1 2 3 4

sIL-1RaicIL-1Ra1

icIL-1Ra2

sIL-1RaicIL-1Ra1

icIL-1Ra2 icIL-1Ra3


The IL-1 receptor is a heterodimer

IL-1

IL-1R Type I

+ +

IL-1RAcP


Regulation of IL-1 by IL-1Ra

IL-1Ra

+ +

IL-1R AcP

IL-1R Type I

IL-1Ra does not signalANKARA ROMATOLOJİ SEMPOZYUMU “Sitokinler: Sağlıkta ve Romatizmal Hastalıklarda”

06 Nisan 2007 Sheraton Otel, Ankara

Both sIL-1Ra and icIL-1Ra1 Can Inhibit IL-1Thymocyte Co-Stimulation Assay

0

2

4

6

8

10

12

14

IL-1Ra (ng/ml) 0 1 1 1 1 0 0 0 1 10 100 100

[3H

]thym

idin

e up

take

(cpm

x 1

03)

sIL-1RaicIL-1Ra1

IL-1β (ng/ml)

Gabay et al. J Immunol 1997


IL-1 ReceptorsIL-1R Type I

IL-1αIL-1βIL-1Ra

bindsbindsIL-1βbindsbinds

IL-1R Type II(shed)

IL-1R Type II

IL-1R AcP(soluble)

•

IL-1R AcP

Does not Does not bind ILbind IL--1 1 on its ownon its own


ILIL--1Ra1Ra

IL-1 Receptor Type II and IL-1Ra reinforce rather than cancel each other’s actions

ILIL--11ββ

IL-1R Type I+ IL-1Ra

SolubleIL-1R Type II


Collagen Induced Arthritis

5 days 60 days


Collagen-induced arthritisKinetics of IL-1β and IL-1Ra Proteins

% o

f pos

itive

ly st

aini

ng c

ells

0

2

4

6

8

10

12

14

16

18

20

days of arthritis5 10 15 20 30 60

IL-1β/IL-1Ra ratio: 1.4 0.97 0.76 0.23 0.07 0.035

% o

f pos

itive

ly st

aini

ng c

ells

*

*

**

**

**

**

IL-1β

IL-1Ra

Gabay et al. Arthritis Rheum 2001


days

0

25

50

75

100

arth

ritic

mic

e (%

)

0 20 40 60 80

wild-typesIL-1Ra tg

0

2

4

6

clin

ical

sco

re

0 20 40 60 80

wild-type

sIL-1Ra tg

Palmer et al. Eur J Immunol 2003

Collagen-Induced arthritisOverexpression of IL-1Ra blocks the development of arthritis

Wild-type Transgenic

IL-1Ra deficient mice

Early onset and more severe collagen-induced arthritis

Ma et al. Arthritis Rheum 1998

Spontaneous polyarthritis when bred in the BALB/Ca background

Horai et al. J Exp Med 2001


The role of IL-1/IL-1Ra balance in vascular inflammation and atherosclerosis

IL-1Ra polymorphism (IL-1RN*2) with coronary and carotid atherosclerosisCirculation 1999

Decreased production of icIL-1Ra1 in human umbilicalvein endothelial cells (HUVEC) from homozygousIL-1RN*2 individualsArterioscler Thromb Vasc Biol 2000

IL-1β, sIL-1Ra and icIL-1Ra1 are expressed in aorticarches of ApoE -/- mice with atherosclerotic lesionsCardiovasc Res 2005


IL-1Ra Transgenic MiceGabay et al. Eur J Immunol 2001

Transgenic mice overexpressing human icIL-1Ra1 or sIL-1Ra from a strong ubiquitous promoter (CMV/β-actin)

Serum hIL-1Ra (ng/ml)

icIL-1Ra1 tg 883 + 395sIL-1Ra tg 14’397 + 1235wild-type not detectable


IL-1 in the Pathogenesis of Atherosclerosis

ApoE -/-

ApoE -/-sIL-1Ra Tg

ApoE -/-icIL-1Ra1 Tg

0

2

4

6

8

10

12

14

16

ApoE -/- ApoE -/-icIL-1Ra1 Tg

*

Lipi

dst

aini

ngar

ea in

the

thor

aco-

abdo

min

al a

orta

(%)

0

5

10

15

20

25

ApoE -/- ApoE -/-sIL-1Ra Tg

*

Lipi

dst

aini

ngar

ea in

the

thor

aco-

abdo

min

al a

orta

(%)

Merhi-Soussi et al. Cardiovasc Res 2005

ApoE -/- ApoE -/- icIL-1Ra1 Tg

ApoE-/- IL-1Ra +/+ ApoE-/- IL-1Ra -/-

Oil red O

Anti-Mac1


Vascular inflammation is modulatedby endogenous IL-1Ra

ApoE -/-IL-1Ra +/+

ApoE +/+IL-1Ra -/-

ApoE -/-IL-1Ra -/-


Role of IL-1/IL-1Ra Balance in ApoE -/- mice

Atherosclerosis is partly mediated by an inflammatory reaction with macrophages and presence of IL-1

IL-1 blockade by IL-1Ra decreases the development of vascular inflammation and atherosclerosis

IL-1Ra deficiency is associated with uncontrolled vascular inflammation and tissue damage resembling some of the pathological features of severe vasculitis


Are the biologic functions of intracellular cytokines

dependent or independent of their receptor ?


Cellular and extracellularlocalization of IL-1α

Nuclear localization sequence


Cellular localization of preIL-1αin SaOS cells

preIL-1α ∆NLS preIL-1α

Palmer et al. Cytokine 2005


PreIL-1α Exerts Inhibitory Effecton SaOS cell Proliferation

0

5

10

15

20

cell

num

ber(

ratio

/ ba

selin

e)

*

1 2 3

SaOS cells transfectedwith empty vector (1)

preIL-1α (2)

∆NLS preIL-1α (3)


Effect of preIL-1α on Cell Proliferation is Independent of IL-1R

3H-th

ymid

ine

inco

rpor

atio

n (c

pm)

*

0

2500

5000

7500

10000

pcDNA preIL-1α

*

pcD

NA

preI

L-1α

+IL-1α

+IL-1β

pcD

NA

pcD

NA

3H-th

ymid

ine

inco

rpor

atio

n (c

pm)

0

2500

5000

7500

10000

+IL-1Ra


PreIL-1α and icIL-1Ra1 Double Transfections in SaOS Cells

PreIL-1α or icIL-1Ra1 PreIL-1α and icIL-1Ra1 Empty vectors

Anti-IL-1Ra

Anti-IL-1α


Intracellular IL-1Ra1 does not inhibit the effect of preIL-1α

0

2,5

5

7,5

10

12,5

cell

num

ber(

ratio

/ ba

selin

e)

**

Em

pty

vect

ors

Pre

IL-1

α

icIL

-1R

a1P

reIL

-1α

icIL

-1R

a1

Concentrations of IL-1RaCell lysates1678 to 4413 ng/mlCell supernatants53 to 218 ng/ml

IL-1Ra/IL-1α ratioLysates: 36 to 111Supernatants: 24 to 42


To examine the role of icIL-1Ra1 in skin inflammation using knock-out and transgenic mice


keratinocytes high IL-1α expressionconstitutive icIL-1Ra1 expressionboth increased by inflammatory stimuli

IL-1/IL-1Ra system in the skin

leukocytes IL-1β IL-1α, sIL-1Ra, icIL-1Ra1

dermal fibroblasts constitutive icIL-1Ra1 expressionfurther increased by inflammatory stimuliIL-1αIL-1β, sIL-1Ra inducible

To examine the role of IL-1/IL-1Ra in skin inflammation

Phorbol ester (PMA) skin painting(Oberyszyn, 1993, Mol. Carcinogen; Lee, 1994, Faseb J)


PMA painting in WT miceSkin histology

WT PMA

WT acetone

Histological scoring

0

50

100

150

200

250

0

0.05

0.1

0.15

0.2

0.25

acetone (n=5)

PMA (n=8)

*p<0.05 vs acetone

*

*

epid

erm

al th

ickn

ess

(mm

)

infla

mm

atio

n (c

ells

/fiel

d)ANKARA ROMATOLOJİ SEMPOZYUMU “Sitokinler: Sağlıkta ve Romatizmal Hastalıklarda”

06 Nisan 2007 Sheraton Otel, Ankara

PMA painting in WT mice

Skin cytokine expression

0

50

100

150

200

250

300

cyto

kine

/L32

(AU

)

IL-1α IL-1β IL-1Ra IL-18 IL-6

*

*

SAA

(µg/

ml)

020406080

100120140160180200

•

*

Serum SAA

*p<0.05 vs acetone

acetone (n=5)

PMA (n=8)


PMA painting in absence of IL-1 activity

Serum SAASA

A (µ

g/m

l)

02040

6080

100120140

160180200

*

PMA (n= 4-8)

*p<0.05 vs WT

WT

icIL-1Ra1 tg

IL-1RIKO0

0.05

0.1

0.15

0.2

0.25

epid

erm

al th

ickn

ess

(mm

)

WTIL-1RI KO

icIL-1Ra1 tg


0

50

100

150

200

250

300IL

-1•

/L32

(A.U

.)

* p<0.05 vs WT

IL-1Ra

icIL-1Ra1 tg

IL-1RI +/+ +/+-/-

no yesno

+/+ +/++/+

*

+/+ -/- -/-

yes no yes

-/- -/- -/-

Intracellular IL-1Ra regulates Skin IL-1α expression


Local and systemic inflammation is mediated by IL-1 following phorbol ester (PMA) application on the skin

Keratinocyte proliferation in response to PMA is not mediated by IL-1

The effects of icIL-1Ra1 in skin inflammation and cytokine production are dependent on the presence of IL-1 receptor

Palmer et al. J Invest Dermatol 2007


The Expanded IL-1 Family

signal peptidaseIL-1Ra

IL-1βcaspase-1

IL-1αcalpain?

all chromosome 2q

chromosome 11q

IL-18caspase-1

IL-1F7?

IL-1F5

IL-1F6

IL-1F8

IL-1F9

IL-1F10

IL-33

chromosome 9p

caspase-1 ?

NF-HEV


ConclusionsThe balance between cytokines and cytokine modulatorsplay a major role in the regulation of the inflammatoryresponses

Changes in this balance in favor of inhibitors may lead to minor responses or rapid resolution of inflammation, whereas the absence (or low levels) of inhibitors will favorexagerated or sustained inflammation with subsequentmajor tissue damage

Intracellular cytokine exert their effect inside cells withoutinteraction with cell surface receptors or after their release outside cells as secreted cytokines


AcknowledgementsDivision of RheumatologyGaby Palmer, Faten Merhi-Soussi, David Magne, Dominique Talabot, Céline Lamacchia, David Moulin, Françoise Mezin

Division of CardiologyBrenda KwakChristos ChadjichristosFrançois Mach

University of Sheffield, UKMartin NicklinJenny Barton

Amgen Inc, Seattle, USAJohn SimsJennifer Towne

Apotech, LausanneOlivier Donzé


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