Cytokine Balance: The main effect of cytokine network. Cem Gabay, MD Division of Rheumatology Geneva, Switzerland

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1 Cytokine Balance: The main effect of cytokine network Cem Gabay, MD Division of Rheumatology Geneva, Switzerland

3 Regulation of Cytokine Production Cytokines Microbial products Degraded EC matrix C5a Protein kinases Transcription factor R Cytokine Cytokine 4 Cytokine mrna Cytokine promoter

4 Cytokine Cascade The example of turpentin injury-induced acute-phase response Tissue injury Turpentine injection Liver Serum amyloid A IL-1β Saline Turpentine IL-6 Circulation SAA mrna Gabay et al. Eur J Immunol 2001 Fantuzzi et al. J Immunol 1997 GAPDH mrna

Serum amyloid A IL-1β Saline Turpentine IL-6 Circulation SAA

5 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

6 Soluble receptor can enhance the effect of their ligand

7 Signaling TNF-a, IL-1, LPS Inhibitors IKK NF-kB P P P P IkB-a proteasome p50p65 degradation IkB-a IkB-a gene NUCLEUS

8 The IL-1 System An example of agonist/antagonist balance

9 IL-1 : A Pleiotropic Cytokine procoagulant state fever sleep loss of appetite social withdrawal acute phase protein production collagenase and stromelysin bone resorption fibroblast and epithelial cell proliferation vasodilation adhesion molecules chemotaxis increased permeability immune activation stimulation of hematopoiesis Prostaglandin E 2

fibroblast and epithelial cell proliferation vasodilation adhesion molecules

10 The IL-1 Family IL-1a? IL-1b ICE = Caspase 1 IL-1Ra Signal Peptidase IL-1a IL-1b IL-1Ra agonists Receptor antagonist

11 IL-1RN gene IL-1Ra Isoforms ic1 ic IL-1Ra mrnas icil-1ra1 icil-1ra2 transcription: alternative promoters sil-1ra alternative splicing IL-1Ra proteins icil-1ra1 sil-1ra translation: alternative initiation icil-1ra2 icil-1ra3

sil-1ra alternative splicing IL-1Ra proteins icil-1ra1

12 The IL-1 receptor is a heterodimer IL-1R Type I IL-1R AcP IL-1 + +

13 Regulation of IL-1 by IL-1Ra IL-1R Type I IL-1R AcP IL-1Ra + + IL-1Ra does not signal

14 Both sil-1ra and icil-1ra1 Can Inhibit IL-1 ThymocyteCo-Stimulation Assay [ 3 H]thymidine uptake (cpm x 10 3 ) IL-1β (ng/ml) IL-1Ra (ng/ml) sil-1ra icil-1ra1 Gabayet al. J Immunol 1997

x 10 3 ) 14 12 10 8 6 4 2 0 IL-1β (ng/ml) IL-1Ra (ng/ml)

15 IL-1 Receptors IL-1R Type I IL-1R Type II IL-1R Type II (shed) IL-1R AcP IL-1R AcP (soluble) binds IL-1a IL-1b IL-1Ra binds IL-1b Does not bind IL-1 on its own

16 IL-1 Receptor Type II and IL-1Ra reinforce rather than cancel each other s actions Soluble IL-1R Type II IL-1R Type I + IL-1Ra IL-1b IL-1Ra

17 Collagen Induced Arthritis 5 days 60 days

18 % of positively staining cells % of positively staining cells Collagen-induced arthritis Kinetics of IL-1β and IL-1Ra Proteins ** * ** IL-1β IL-1Ra ** * ** days of arthritis IL-1β/IL-1Ra ratio: Gabay et al. Arthritis Rheum 2001

16 14 * 12 10 8 6 4 2 0 ** IL-1β IL-1Ra ** * ** 5 10 15 20 30 60 days of

19 Collagen-Induced arthritis Overexpression of IL-1Ra blocks the development of arthritis arthritic mice (%) wild-type sil-1ra tg wild-type clinical score sil-1ra tg 0 Wild-type days Transgenic Palmer et al. Eur J Immunol 2003

sil-1ra tg wild-type clinical score 0 6 4 2 0 20 40 60 80 sil-1ra tg

20 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

21 The role of IL-1/IL-1Ra balance in vascular inflammation and atherosclerosis IL-1Ra polymorphism (IL-1RN*2) with coronary and carotid atherosclerosis Circulation 1999 Decreased production of icil-1ra1 in human umbilical vein endothelial cells (HUVEC) from homozygous IL-1RN*2 individuals Arterioscler Thromb Vasc Biol 2000 IL-1β, sil-1ra and icil-1ra1 are expressed in aortic arches of ApoE -/- mice with atherosclerotic lesions Cardiovasc Res 2005

22 IL-1Ra Transgenic Mice Gabay 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 sil-1ra tg wild-type not detectable

23 IL-1 in the Pathogenesis of Atherosclerosis ApoE -/- ApoE -/- sil-1ra Tg ApoE -/- icil-1ra1 Tg ApoE -/- Lipid staining area in the thoraco-abdominal aorta (%) Lipid staining area in the thoraco-abdominal aorta (%) ApoE -/- icil-1ra1 Tg ApoE -/- ApoE -/- sil-1ra Tg * * Merhi-Soussi et al. Cardiovasc Res 2005 ApoE -/- ApoE -/- icil-1ra1 Tg

24 ApoE-/- IL-1Ra +/+ ApoE-/- IL-1Ra -/- Oil red O Anti-Mac1

25 Vascular inflammation is modulated by endogenous IL-1Ra ApoE -/- IL-1Ra +/+ ApoE +/+ IL-1Ra -/- ApoE -/- IL-1Ra -/-

26 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

27 Are the biologic functions of intracellular cytokines dependent or independent of their receptor?

28 Cellular and extracellular localization of IL-1α Nuclear localization sequence

29 Cellular localization of preil-1α in SaOS cells preil-1α NLS preil-1α Palmer et al. Cytokine 2005

30 PreIL-1α Exerts Inhibitory Effect on SaOS cell Proliferation SaOS cells transfected with empty vector (1) preil-1α (2) NLS preil-1α (3) cell number (ratio / baseline) *

31 pcdna pcdna pcdna Effect of preil-1α on Cell Proliferation is Independent of IL-1R 3H-thymidine incorporation (cpm) preil-1a * +IL-1a +IL-1b 3H-thymidine incorporation (cpm) IL-1Ra pcdna preil-1a *

32 PreIL-1α and icil-1ra1 Double Transfections in SaOS Cells Anti-IL-1a Anti-IL-1Ra PreIL-1α or icil-1ra1 PreIL-1α and icil-1ra1 Empty vectors

33 Intracellular IL-1Ra1 does not inhibit the effect of preil-1α Concentrations of IL-1Ra Cell lysates 1678 to 4413 ng/ml Cell supernatants 53 to 218 ng/ml IL-1Ra/IL-1a ratio Lysates: 36 to 111 Supernatants: 24 to 42 cell number (ratio / baseline) 12,5 10 7,5 5 2,5 0 Empty vectors * PreIL-1α icil-1ra1 * PreIL-1α icil-1ra1

34 To examine the role of icil-1ra1 in skin inflammation using knock-out and transgenic mice

35 IL-1/IL-1Ra system in the skin keratinocytes high IL-1α expression constitutive icil-1ra1 expression both increased by inflammatory stimuli dermal fibroblasts constitutive icil-1ra1 expression further increased by inflammatory stimuli IL-1α IL-1β, sil-1ra inducible leukocytes IL-1β IL-1α, sil-1ra, icil-1ra1 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)

36 Skin histology PMA painting in WT mice Histological scoring WT acetone acetone (n=5) PMA (n=8) WT PMA epidermal thickness (mm) * inflammation (cells/field) * 0 0 *p<0.05 vs acetone

37 PMA painting in WT mice Skin cytokine expression Serum SAA acetone (n=5) cytokine/l32 (AU) PMA (n=8) * * IL-1a IL-1b IL-1Ra IL-18 IL-6 SAA (µg/ml) * *p<0.05 vs acetone

38 PMA painting in absence of IL-1 activity Serum SAA PMA (n= 4-8) SAA (µg/ml) WT IL-1RIKO * icil-1ra1 tg epidermal thickness (mm) WT IL-1RI KO icil-1ra1 tg *p<0.05 vs WT

39 Intracellular IL-1Ra regulates Skin IL-1α expression * 300 * p<0.05 vs WT IL-1 /L32 (A.U.) IL-1RI +/+ -/- +/+ +/+ -/- -/- icil-1ra1 tg no no yes yes no yes IL-1Ra +/+ +/+ +/+ -/- -/- -/-

40 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

41 The Expanded IL-1 Family IL-1a calpain? IL-1F5 IL-1b caspase-1 IL-1F6 IL-1Ra IL-1F7 signal peptidase? all chromosome 2q IL-1F8 IL-1F9 IL-1F10 IL-18 caspase-1 chromosome 11q IL-33 NF-HEV caspase-1? chromosome 9p

42 Conclusions The balance between cytokines and cytokine modulators play a major role in the regulation of the inflammatory responses 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 favor exagerated or sustained inflammation with subsequent major tissue damage Intracellular cytokine exert their effect inside cells without interaction with cell surface receptors or after their release outside cells as secreted cytokines

43 Acknowledgements Division of Rheumatology Gaby Palmer, Faten Merhi-Soussi, David Magne, Dominique Talabot, Céline Lamacchia, David Moulin, Françoise Mezin Division of Cardiology Brenda Kwak Christos Chadjichristos François Mach University of Sheffield, UK Martin Nicklin Jenny Barton Amgen Inc, Seattle, USA John Sims Jennifer Towne Apotech, Lausanne Olivier Donzé

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