Objectives

1. Cytokines and Cytokines ReceptorsDr. Abdelilah Soussi GounniDepartment of ImmunologyEmail: gounni@cc.umanitoba.ca

2. Objectives • Overview, brief historical review, current nomenclature systems. • Overview of cytokine receptor construction, signaling and function • Describe human chemokine and chemokine receptor subfamilies, the cellular sources, and distribution of chemokine receptor expression. • Discuss the functions of chemokines: chemotaxis and other chemokine-mediated mechanisms.

3. Cytokines • “Immunocytokines”,“Immunotransmitters”“Lymphokines, Monokines; Interleukins” • IL-2 : Morgan et al 1976 • IFN-a and b. Tanuguchi et al , 1980 • IFN-g and lymphotoxin. Gray et al, 1982 • TNF. Aggarwal BB , 1984, Pennica D and Goeddel, 1984

4. Cytokines • Most of cytokines are glycoproteins : peptide signal ( IL-1, TNF-a). • Others are not. • Some cytokines behave like classical hormone : act at systemic level. • Produced by a wide variety of cells: not by specialized organs in contrast to hormones. • Secreted proteins : difficult to predict their site at which they exert their biological function • Work at very low concentration: sometimes fentomolar range

5. Cytokines Families • Hematopoeitin: IL-2, IL-4… • Interferon: IFN-a, g, b • IL-10: IL-10, IL-19, IL-20, IL-24, IL-26 • Tumor necrosis: TNF, LT-B, Trail, ..... • IL-12: IL-12, IL-23, IL-27. • IL-17: IL-17A, IL-25,.. • Unassigned : IL-16, MIF, TGF-b, ..

6. Biological Activity • Hematopoiesis. • Innate immunity. • Adaptive immunity. • Inflammation.

7. IL-1/IL-6 IL-11/IL-4 TNF IL-1/IL-6 IL-11/IL-4 TNF SCF IL-3 GM-CSF G-CSF IL-5? SCF IL-3 GM-CSF G-CSF IL-5? Cytokines & Hematopoeisis Stem cell Natural killer cell SCF T lymphocytes IL-7 +others ? IL-7 +others Lymphoid progenitor B lymphocytes Pluripotent cells (CD34+) CD34 CD33 IL-11 CFU-GEMM Megakaryocyte progenitor Erythrocyte progenitor Granulocyte Macrophage Progenitor CFU-GM

8. Cytokines & Hematopoeisis • Expansion. • Maturation. • Differentiation.

9. Cytokines and Innate Immunity • Macrophage ontogeny and function. Trapnell et al, 2002

10. Th cell differentiation Gaffen SL, 2011

11. Cytokines and adaptive Immunity

12. 1 Evidence of expression (+) or increased ( ) or decreased ( ) expression in the airways of asthmatic subjects or following antigen challenge in murine models of allergic inflammation. 2 No effect (NE) or stimulation (+) of eosinophil recruitment to the airways of mice overexpressing cytokine gene or following exogenous administration of cytokines. 3 The effect of "inhibitory" cytokines on Th2-mediated eosinophilia. NE, no effect; , augmentation; , inhibition. 4 The effect of monoclonal antibodies to various cytokines on allergen-induced eosinophilia. NE, no effect; , inhibition. 5 The effect of cytokine gene disruption on eosinophilia induced by allergen. NE, no effect; , inhibition. Cytokines in inflammation (e.g eosinophilia) 

13. Cytokines complexity : double edged sword O’shea et al, 2002

14. Cytokines receptors family • Immunoglobulin superfamily: IL-1, M-CSF, C-kit. • Class I cytokine receptors: IL-2, IL-3, IL-5, GM-CSF.. • Class II cytokine receptors: IFNsR, IL-10. • TNF receptors: TNFa, b, NGF, FAS.

15. Subfamily of Class I Cytokine receptor • IL-2 receptor subfamily: common g subunit. Liao W et al, 2011

16. IL-2R forms g-b: intermediate affinity Nk cells, resting T cells g-b-a: high affinity a: low affinity Activated CD4+ and CD8+ cells The schematic structure of the IL-2 receptor subunits, IL-2Rß and IL-2R. The numbers are amino acid positions from the amino terminals. The solid lines indicate the conserved cysteine residues. WSXWS indicates an amino acid sequence of tryptophan—serine—unconserved amino acid—tryptophan—serine. Box 1, Serine, Acidic, and Proline in the cytoplasmic domain of IL-2Rß represent the box 1, serine-rich, acidic, and proline-rich regions, respectively. SH2 and C30 in the cytoplasmic domain of IL-2R   represent the SH2 subdomains and carboxyl terminal 30 amino acid residues, respectively.

17. IL-2R signalling Growth, Survival, Transcriptional regulation, Effector differentiation Liao W et al, 2011

18. IL-2 and CD4 + T cell differentiation Liao W et al, 2011

19. Subfamily of Class I Cytokine receptor • GM-CSF receptor subfamily: common b subunit. Target cells Eosinophils Macrophages Neutrophils Basophils Mast cells

20. Mechanism of Activation of the GM‐CSF, IL‐3, and IL‐5 Family of Receptors STEM CELLSVolume 16, Issue 5, pages 301-313, 1 SEP 1998 DOI: 10.1002/stem.160301http://onlinelibrary.wiley.com/doi/10.1002/stem.160301/full#fig1

21. Subfamily of Class I Cytokine receptor • IL-6 receptor subfamily: common gp130 subunit.

22. Class II Cytokines Receptors Renauld JC, 2003

23. Class II Cytokine Receptor (CRF2) family Renauld JC, 2003

24. 12 members of the class II cytokine receptor family. • Majority are composed of classic trans-membrane proteins • Functional receptor complexes composed of heterodimere subunits that allow high-affinity binding sites for the ligand. • One particular receptor complex can bind several cytokines.

25. TNF super family and receptors 19 members 29 Receptors Aggarwal BB, 2003

26. Aggarwal BB, 2003

27. TNF super family and receptors • 19 members that signal through 29 receptors. • Regulate normal functions • Hematopoeisis • Morphogenesis • Immune response • Dark side : • Tumorigenesis • Transplant rejection • Septic shock • Bone resorption • RA • Diabetes

28. The main physiological and pathological effects linked to members of the TNF superfamily. Aggarwal BB, 2003

29. Decoy receptors in the interleukin-1 (IL-1)/Toll and tumor necrosis factor receptor (TNFR) families Each receptor is encoded by a different gene, with the exception of Fas decoy receptor (FDR), which is generated from the Fas gene by differential splicing. Symbols: triangles, IL-1 family ligands; trimeric sticks, TNFR agonists; pink circles, Ig domains; red oval, single Ig domain IL-18-binding protein (BP); yellow boxes, Toll/IL-1R (TIR) domains; green circles, cysteine-rich domains (CRDs); green semi-circles, short CRDs; blue DD boxes, death domains; blue D box, incomplete death domain. Abbreviations: AcP, accessory protein; AcPL, accessory protein-like chain; DcR, decoy receptor; DR, death receptor; GPI, glycosylphosphatidylinositol; IL-1Rrp, IL-1 receptor related protein; L, ligand; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor  B; TRAIL, tumor necrosis factor  -related apoptosis-inducing ligand.

30. The type II interleukin-1 receptor (IL-1RII) as a decoy

31. Summary • Cytokines are a family of protein mediators that are important in transducing information between various cell types. • Cellular sources of cytokines include those cell types considered to play pivotal roles in the immune system as well as in inflammatory responses. • Non-immune cells can also produce cytokines. Epithelial cells, smooth muscle cells and fibroblasts. • Cytokines fulfill a number of roles during immune and inflammatory reactions, and may display overlapping or redundant functions.

32. Summary • Many cytokine receptors have a subunit structure, with common subunits shared between receptors, and serving as affinity modifiers/signal transducers. • Cytokines exert their effects on target cells by activating intracellular signaling mechanisms. • Cytokines activate the cells through many pathways: • Classical signal transduction pathways. • Novel signaling mechanisms, including the Jak-STAT pathways of transcriptional regulation. • Pathways involving lipid second messenger.

33. Chemokines and Chemokines Receptors Describe human chemokine and chemokine receptor subfamilies, the cellular sources of chemokines and distribution of chemokine receptor expression. Discuss the functions of chemokines: chemotaxis and other chemokine-mediated mechanisms that contribute to immune responses, independent of chemotaxis.

34. Chemokines • Single polypeptide chains :70-125 aa (7-17KDa), highly basic (heparin binding proteins). • 20 to 95% sequence similarity to each other: duplication from ancestral gene. • More than 45 chemokines, and 20 receptors so far identified. • Contain between 1 and 3 disulfide bonds (except fractalkine/CX3CL1 and CXCL16)

35. Based on structural criteria, cysteine residues in the ligand, 4 subclasses have been defined. Arrangement of cysteine residues located in N terminal region allows to define: C family: XCL1 ( lymphotactin) CC family:CCL11(Eotaxin) CXC family : CXCL8(IL-8)…. CX3C family: CX3CL1 (fractalkine) Chemokines Bacon et al, 2002

36. Chemokines Receptors • Chemokine receptors belongs to Class A family of G protein coupled receptors (GPCR), 7 Trans-Membrane (TM) domain. • Highly homolog to rhodopsin. • Signal mainly through Gi type proteins. • Single polypeptide chain. • 25 to 80% sequence similarity to each other.

37. Structure of Chemokine Receptors Onuffer JJ et al, 2002

38. Molecular structure of chemokine receptors Onuffer JJ et al, 2002

39. Genomic mapping • CXC chemokines : human chr 4 (5 in mice). • CC chemokines : human chr 17 (11 in mice). • CXC chemokine receptors : human chromosome 2. • CC chemokine receptors : human chromosome 3.

40. Human chemokines and chemokine receptors Recep Ligands • CXC chemokines are presented as hexagons and CC chemokines as circles. • Circles are also used for lymphotactins and symbols with a bar represent membrane-anchored chemokines. • The numbers within the chemokine symbols correspond to the chemokine numbers in the systematic nomenclature. • Within the four groups, colors are usedto indicate chemokineswith structural similarities. Baggiolini M, 2002

41. Chemokine receptors, ligands and cellular distribution Modified from Onuffer J et al, 2002

42. Chemokine receptors distribution in Th-1 and Th-2 cells Selective recruitment to sites of inflammation and secondary lymphoid tissues. Chensue SW, 2001

43. Chemokine receptor expression in human leucocytes Neutrophil: CXCR1; CXCR2; CxCR4 Monocytes : CCR1; CCR2, CCR5 Eosinophil : CCR3; CCR1 Basophils : CCR1, CCR2, CCR3 But can change with environment eg, inflammation, …

44. Chemokines and chemokines receptors: Agonist and antagonist activity • CXCR3 agonists (Th-1 response): CXCL-11/I-TAC; CXCL-9/MIG; CXCL-10/IP-10 are antagonists of CCR3 (Th-2 response). • CCR1/CCR2/CCR3 agonist CCL-7/MCP3 is an antagonist for CCR5. • N terminal portion of the chemokine plays an important role in both activities. • CCL-5/RANTES: n terminal modification by adding of: • a methionine results in an antagonist. • amino-oxypentane group: agonist for CCR3, partial agonist for CCR5 and to lesser extent CCR1

45. Role of chemokines • Cellular activation. • Eotaxin/CCL-11, RANTES/CCL-5, … , • Inflammatory mediator release. MCP-3/CCL-2, MCP-4/CCL-13 • Cell trafficking and homing. CCR-3 • Promotion of Th2 inflammatory responses.TARC/CCL-17 and CCR4 • Hematopoiesis. eg, SDF-1/CXCL-12 • Regulation of IgE synthesis • Homeostasis. Eotaxin/CCL-11 • Neural communication

46. Chemokine mediated chemotaxis: in vivo and in vitro system • GAGs are important under the influence of shear flow • Some chemokines requires also heparan sulfate binding Johnson Z et al, 2004

47. Chemokines trigger firm leukocyte adhesion and trans-endothelial migration. Sequential steps model: Tethering and rolling. Activation an firm adhesion Diapedesis GAGs EC Nickel R, 1999

48. The traffic of dendritic cells and its dependence on changing chemokine receptor expression

49. Viral chemokine, chemokine binding factor and chemokine receptors Chensue SW, 2001

50. Gamma herpesvirus chemokine:M3 Transgenic expression of M3 in pancreatic islets CCL19 CCL21 Jensen KK et al, 2003