Chapter 2 The role of interleukine-6 (IL-6) in the pathophysiology of RA

1. Chapter 2The role of interleukine-6 (IL-6) in the pathophysiology of RA BR 8657 – 03/2009

2. Content • Interleukine-6 (IL-6) • IL-6 structure and source • IL-6 and cell signalling • IL-6 immune effects • IL-6 articular effects • IL-6 systemic effects • Summary of IL-6 effects in RA

3. IL-6 structure • Member of IL-6-type cytokine family1 • Activates target genes involved in differentiation, survival, apoptosis and proliferation • Small polypeptide cytokine2 • Four α-helix bundle • Structure stabilisationby intramolecular disulphide bridges Adapted from Heinrich PC, 20031 1Heinrich PC, et al. Biochem J 2003;374:1-20 2Heinrich PC, et al. Biochem J 1998;334:297-314

4. IL-6 is produced by various cell types IL-6 mediates a multitude of processes Naka T, et al. Arthritis Res 2002;4(Suppl 3):S233-S242

5. IL-6 and cell signalling • IL-6 receptor: 2 types1,2 • mIL-6R = membrane-bound IL-6 receptor  conventional signalling • Found on limited cell types: hepatocytes, monocytes, macrophages and some lymphocytes • sIL-6R = soluble IL-6 receptor  transsignalling • Found in most body fluids • IL-6 binding to its receptor does not convey signalling • Glycoprotein 130 (gp130), a transmembrane protein, is required to associate with the IL-6/IL-6R complex and is responsible for transducing the signal across cellular membranes • gp130 is expressed ubiquitously IL-6R gp130 1Plushner SL. Ann Pharmacother 2008;42:1660-1668 2Heinrich PC, et al. Biochem J2003;374:1–20

6. IL-6 binds to a membrane-bound receptor or a soluble receptor IL-6 membrane receptor IL-6 receptor complex IL-6 IL-6 and IL-6 soluble receptor

7. IL-6 conventional signalling via mIL-6R IL-6/mIL-6R complexes engage gp130 molecules IL-6 binds to the membrane-bound IL-6 receptor mIL-6R mIL-6R gp130 molecules interact to initiate conventional IL-6/IL-6R signalling Conventional signalling is limited to cells expressing mIL-6R gp130 Adapted from Jones SA, et al. J Interferon Cytokine Res 2005;25:241-253 Adapted from Scheller J, Rose-John S. Med Microbiol Immunol 2006;195:173-183

8. IL-6 transsignalling via sIL-6R sIL-6R sIL-6R Not all cells express mIL-6Rs, but gp130 is widely expressed Transsignalling occurs in cells expressing only gp130 gp130 But the signalling complex uses soluble IL-6R Adapted from Jones SA, et al. J Interferon Cytokine Res 2005;25:241-253 Adapted from Scheller J, Rose-John S. Med Microbiol Immunol 2006;195:173-183

9. gp130 Tocilizumab inhibits conventional signalling and transsignalling IL-6 cannot bind sIL-6R mIL-6R Tocilizumab binds mIL-6R and sIL-6R gp130 Membrane-bound signalling Transsignalling Adapted from Jones SA, et al. J Interferon Cytokine Res 2005;25:241-253 Adapted from Scheller J, Rose-John S. Med Microbiol Immunol 2006;195:173-183 Adapted from Mihara M, et al. Int Immunopharmacol 2005;5:1731-1740

10. IL-6 effects Hematopoietic stem cell Megakaryocyte Platelets • Immune response • B cells • Differentiation, antibody formation • T cells • Proliferation, differentiation, survival • Bone marrow • Neutrophils  leucocytosis • Megakaryocytes  thrombocytosis • Liver • Synthesis of acute phase reactants • Hepcidin  anaemia of chronic disease • Anti-apoptotic • Bone tissue • Osteoclasts  bone absorption • Differentiation and activation Endothelial cell Hepatocyte IL-6 Antibodies B cell Keratinocyte T cell Osteoclast Fibroblast Matrix Metalloproteinases Differentiation Proliferation Activation Scheinecker, Smolen & Weyand, Essentials of the Pathogenesis of Rheumatoid Arthritis, 2008

11. IL-6: overview of activities • Presence of RF • Hypergammaglobulinemia Production of acute-phase proteins (e.g. CRP) and hepcidin B cell Hepatocytes Maturation of megakaryocytes T cell activation IL-6 Thrombocytosis Induction of adhesion molecules Activation of osteoclasts Differentiation of monocytes to macrophages Bone absorption Adapted from Yoshizaki K, et al. Springer Semin Immunopathol 1998;20:247–259 Jones SA, et al. J Interferon Cytokine Res 2005;25:241-253

12. IL-6 immune effects • Interaction with B cells • IL-6 was originally identified as a B cell differentiating factor (BCDF) or B cell stimulatory factor-2 (BSF-2) • Stimulates B-cell differentiation into mature plasma cells and thus increases immunoglobulin secretion • Stimulates the production of autoantibodies such as Rheumatoid Factor (RF) • Interaction with T cells • Regulatory role in T cell proliferation, differentiation and survival • Drives the differentiation of naïve T cells into cytotoxic cells • Promotes the development of Th17 cells, driving autoimmunity (animal models) • Stimulates IL-2 receptor expression and IL-2 production • Activated T cells contribute to the production of cytokines and proteases that degrade connective tissue and promote osteoclast formation Scheinecker C, Smolen JS and Weynand CM. 2008 Kishimoto T. Arthritis Res Ther 2006;8(Suppl 2):S2 Yoshizaki K, et al. Springer Semin Immunopathol 1998;20:247–259 Smolen J and Aletaha D. Eur J Health Econ 2008;8(Suppl 2):S39-S47

13. TH1 IL-6 immune effects: Interaction with B cells Dendritic cells Range of cytokinesincluding IL-6 TH0 Produce cytokines IL-6 Ig production Plasma cell B cell ActivatedB cell Differentiation/Proliferation Model with human dendritic cells Jego G, et al. Curr Dir Autoimmun 2005;8:124–139 Scheinecker C, Smolen JS and Weynand CM. 2008

14. TH17 TH2 Treg TH1 TH IL-6 immune effects: The role of IL-6 in T helper cell differentiation IFN-γ IL-12 Pathogen clearance IL-4 Protection against tissue injury TGF-β TGF-β IL-6 Autoimmune disease In vitro and in vivo investigation TH=T helper cell; Treg=regulatory T cell; TGF-β=transforming growth factor-β; IFN-γ=interferon-γ Mangan PR, et al. Nature 2006;441:231–234 Bettelli E, et al. Nature 2006;441:235–238 Scheinecker C, Smolen JS and Weynand CM. 2008

15. – + + + – – TH17 TH0 Treg IL-6 immune effects: IL-6 promotes the generation of TH17 cells at the expense of Treg cells IL-6 IL-6 TGF-β TGF-β IFN-γ Inhibition of autoimmunity and protection against tissue injury Induction of autoimmune tissue injury Research in mice and in vitro T cell differentiation research Bettelli E, et al. Nature 2006;441:235–238 Scheinecker C, Smolen JS and Weynand CM. 2008

16. IL-6 articular effects Synoviocytes VEGF Endothelial cells Pannus formation Osteoclast activation  bone resorption IL-6 Leukocyte recruitment Joint destruction MMP's Cartilage destruction Local inflammation Lipsky PE. Arthritis Res Ther 2006;8(Suppl 2):S4

17. Leucocyte trafficking = hallmark of inflammation r2=0.774 4,000 p<0.01 • IL-6 contributes to leukocyte recruitment at inflammatory sites • IL-6/sIL-6R increases leukocyte recruitment by: • Activating production of a subset of chemokines by endothelial cells1 • Upregulating expression of adhesion molecules1 • IL-6 supports neutrophil recruitment2 • Significant correlation between IL-6 levels and levels of neutrophil adhesion to endothelial cells 2,000 IL-6 (pg/ml) 0 0 20 40 60 80 Adherent neutrophils 1Romano M, et al. Immunity 1997;6:315–325 2Lally F, et al. Arthritis Rheum 2005;52:3460–3469

18. IL-6 increases neutrophil survival * * * Polymorphonuclear leukocytes (PMN) incubated with IL-6 had enhanced survival vs untreated controls (*p<0.05 vs control) PMN survival at 24h as function of PMN concentration and IL-6 dose Culture system: PMNs isolated from healthy human donors cultured and incubated with IL-6 doses Biffl WL, et al. J Leukoc Biol 1995;58:582-584

19. Reduction of IL-6 levels correlate with improvement of disease activity • Overproduction of IL-6 in RA1 • High levels of IL-6 and sIL-6R are found in serum and in the synovial fluid • IL-6 levels in RA patients are higher than in patients with osteoarthritis • IL-6 levels were found to correlate with markers of disease activity, incl. ESR and CRP • IL-6 levels correlate with clinical signs of active disease such as morning stiffness and swollen joints1,2 • Early morning stiffness or joint count correlate with CRP levels but the correlation with IL-6 levels is even higher than with changes in CRP • Decrease of IL-6 concentration is a good prognostic marker for clinical outcome under treatment Correlation of ∆laboratory measures with ∆outcome measures 1Scheinecker C, Smolen JS and Weynand C. 2008. 2Straub RH, et al. Br J Rheumatol 1997;36:1298-1303

20. * * * * 0 5 10 20 40 IL-6 articular effects: Joint destruction induced by excessive IL-6 200 • Local bone destruction in RA is mediated by osteoclasts within the synovial tissue1 • Formation of osteoclast-like cells in vitro is induced by addition of IL-6 and sIL-6R2 • Cooperative effects of IL-6 and sIL-6R on osteoclast-like cell formation • Synovial fluid from patients with RA can induce the formation of osteoclast-like cells in vitro2 • Effect is blocked by neutralisation of IL-6R signalling *p<0.05 150 Osteoclast-like cells/well 100 50 0 human IL-6 (ng/ml) 1Schett G. Arthritis Res Ther 2007;9:203 (doi:10.1186/ar2110) 2Kotake S, et al. J Bone Miner Res 1996;11:88–95

21. IL-6 directly induces osteoclast formation: central role in mediating bone destruction in RA IL-6, TNF, IL-1, IL-17 Synovial fibroblast RANKL expression IL-6/sIL-6R Activation RANKL RANK M-CSF Differentiation Osteoclast Monocyte Cathepsin K Bone destruction Bone M-CSF = Macrophage Colony Stimulating Factor; RANKL = Receptor Activator of Nuclear factor κβ Ligand Schett G. Arthritis Res Ther 2007;9:203 (doi:10.1186/ar2110). Kudo O, et al. Bone 2003;32:1-7

22. IL-6 induces synoviocyte expression of VEGF: central role in pannus formation Angiogenesis is a key process in the formation and maintenance of the pannus as invasion of cartilage and bone requires an increased blood supply Vascular Endothelial Growth Factor (VEGF) has been identified as a key element in the development of new blood vessels IL-6 Synovial fibroblasts ( synoviocyte) VEGF Endothelial cells Pannus formation Paleolog EM. Arthritis Res 2002;4(Suppl 3):S81–S90

23. IL-6/sIL-6R – – + + + + IL-1 – + + + + + TNF- – + + + + + IL-6 may contribute to pannus formation by stimulating VEGF production • IL-6, in synergy with IL-1 or TNF-α, induces VEGF production from synovial cells • Elevated serum levels of VEGF in RA are normalised by IL-6R inhibition 16 12 8 VEGF (x102 pg/ml) 4 0 anti-TNF- anti-IL-6R IL-1R antagonist – – – Inhibitors Nakahara H, et al. Arthritis Rheum 2003;48(6):1521–1529

24. IL-6 systemic effects Liver The acute-phase response Acute-phase proteins IL-6 Hepcidin production Anaemia Osteoclast activation Inflammation Systemic osteoporosis Cardiovascular risk↑ Fatigue and mood HPA axis HPA axis = Hypothalamic-Pituitary-Adrenal axis Nishimoto N and Kishimoto T. Nat Clin Pract Rheumatol 2006;2(11):619-626; 2Lipsky PE. Arthritis Res Ther 2006;8(Suppl 2):S4 (doi:10.1186/ar1918); Chrousos GP. N Engl J Med 1995;332(20):1351-1362; Sattar N and McInnes IB. Curr Opin Rheumatol 2005;17:286-292

25. IL-6 is the major cytokine influencing the hepatic synthesis of acute phase proteins Acute-phase response CRP ↑ SAA ↑ Fibroblasts Haptoglobin ↑ Fibrinogen ↑ IL-6 Liver Ceruloplasmin ↑ C3, C4 ↑ Albumin ↓ Transferrin ↓ Hepcidin CRP = C-reactive protein; SAA = serum amyloid A Nishimoto N and Kishimoto T. Nat Clin Pract Rheumatol 2006;2(11):619-626 Panichi V, et al. Kidney Int 2000;58(Suppl 76):S96-S103

26. 30,100 30,000 700 600 C-reactive protein 500 Serum amyloid A 400 Change in plasma concentration (%) Haptoglobin 300 Fibrinogen 200 100 C3 0 Transferrin Albumin 0 7 14 21 Timeafter inflammatory stimulus (days) IL-6 is the chief stimulator of acute-phase protein production • Inflammation is generally accompanied by a systemic reaction known as the acute-phase response, which is characterised by a rapid alteration in the levels of several plasma proteins • IL-6 is the chief stimulator of the production of most acute-phase proteins, including CRP Gabay C and Kushner I. N Engl J Med 1999;340(6):448–454

27. IL-6 induces hepcidin production by hepatocytes: cause of anaemia of chronic inflammation Hepcidin inhibits: Release of iron from macrophages Absorption of dietary iron (iron deficiency) Inflammation Macrophage iron release IL-6 Hepcidin Intestinal iron absorption Macrophage Hepatocytes Andrews NC. J Clin Invest 2004;113(9):1251–1253 Nemeth E, et al. J Clin Invest 2004;113(9):1271–1276

28. Overproduction of IL-6 causes anaemia • Increased serum IL-6 concentrations correlate with anaemia in RA patients1 • IL-6 infusions induced anaemia in a rat model2 8 Control 10.5 6.8 7 IL-6 p=0.0001 10.0 6 9.5 5 3.9 9.0 Hb (mmol/l) Median serum IL-6 (pg/ml) 4 8.5 3 * * * * 8.0 *p<0.01 2 * * * 7.5 1 * Treatment 0 7.0 Anaemic Non-anaemic 0 5 10 15 20 25 Treatment Time (days) 1Voulgari PV, et al. Clin Immunol 1999;92(2):153–160 2Jongen-lavrencic M, et al. Clin Exp Immunol 1996;103:328–334

29. Excessive IL-6 contributes to osteoporosis in RA • Patients affected by RA often have systemic osteoporosis, independent of corticosteroid treatment1 • Osteoclast activation by excessive levels of IL-6 leads to bone resorption2 • IL-6 deficient mice are protected from bone loss caused by oestrogen depletion3 • Selective overexpression of IL-6 also inhibits osteoblast function4 • Reduction in enzymatic activity • Lower osteoblast surface per bone surface 1Lipsky PE. Arthritis Res Ther 2006;8(Suppl 2):S4 (doi:10.1186/ar1918) 2Kotake S, et al. J Bone Miner Res 1996;11:88-95 3Poli V, et al. EMBO 1994;13(5):1189-1196 4De Benedetti F, et al. Arthritis Rheum 2006;54(11):3551-3563

30. IL-6 is a potent stimulator of the HTA axis Cytokines influencing HPA axis: IFN-α, IFN-γ, IL-2 TNF-α, IL-1 IL-6 Fatigue and mood Chrousos GP, et al. N Engl J Med 1995;332:1351-1362

31. Increased incidence of cardiovascular events in RA Consecutive patients with RA assessed for 1-year occurrence of CV-related hospitalisations (n = 236) • Patients with rheumatoid arthritis have a threefold increase in cardiovascular events compared with the general population. • Increased incidence of CV events in RA patients is independent of traditional CV risk factors • Additional mechanisms are responsible for CV disease in RA CV = cardiovascular *age, sex, smoking, diabetes, hypercholesterolemia, systolic blood pressure, body mass index Del Rincón I, et al. Arthritis Rheum 2001; 44:2737–2745

32. 7 Apparently healthy women (n = 27 939) 6 5 Relative risk of future cardiovascular events 4 3 2 1 0 <0.5 0.5–1.0 1.0–2.0 2.0–3.0 3.0–4.0 4.0–5.0 5.0–10.0 10.0– 20.0 >20.0 “low risk” “moderate risk” “high risk” C-reactive protein is an independent predictor of cardiovascular risk • Cardiovascular risk increased linearly from the very lowest to the very highest levels of high sensitivity CRP 8 hsCRP (mg/l) Ridker PM and Cook N. Circulation 2004;109:1955–1959

33. Increasing risk of future myocardial infarction with increasing IL-6 concentration Apparently healthy men (n = 14 916) 3.0 p = 0.001 p = 0.005 2.0 p = 0.3 Relative risk of MI 1.0 1.0 1.4 2.8 2.3 0 1 2 3 4 <1.04 1.04–1.46 1.47–2.28 >2.28 Quartile of IL-6 (pg/ml) MI = myocardial infarction Ridker PM, et al. Circulation 2000;101:1767–1772

34. Mechanisms linking RA and increased vascular risk Synovitis IL-6, IL-1 TNF-α Adipose tissue lipolysis Insulin resistance Skeletal muscle FFAs HDL↓TG↑ LDL↑ CRP Fibrinogen ICAM 1 vWf, tPA Endothelial activation Oxidative stress↑ Foam Cell Macrophage Accelerated atherogenesis vWf = von Willebrand factor; tPA = tissue plasminogen activator; FFAs = free fatty acids; TG = triglyceride; ICAM = intracellular adhesion molecule Sattar N, et al. Circulation 2003;108:2957–2963

35. Summary of IL-6 effects in RA • Immune system • B cells • T cells • Th17 • Systemic effects • Acute phase protein production • Anaemia via hepcidin production • Increased CV risk • Osteoporosis • HPA-axis – fatigue and mood • Articular effects • Osteoclast activation • Neutrophil recruitment and survival • Pannus formation IL-6