This document summarizes various inflammatory mediators including cytokines, chemokines, lipid mediators, and neuropeptides. It describes the sources and functions of different cytokines such as IL-1, TNF-α, IFN-γ, and chemokines such as IL-8 and RANTES. It also discusses the roles of lipid mediators like leukotrienes and prostaglandins in inflammation.

1. Inflammatory mediators Dr Chris Corrigan Reader/Consultant GKT School of Medicine

2. Mediators of Inflammation Cytokines Chemokines Lipid mediators (leukotrienes, prostaglandins) Neuropeptides Endothelin and NO

3. Cytokines

4. Cytokines (1) Soluble (glyco)proteins produced by leucocytes and other cell types Chemical communicators between cells, but usually not end effector molecules Bind to specific receptors on the surface of target cells Most are growth/differentiation factors for haematopoietic cells; some have systemic effects

5. Cytokines (2) Pleiotropicitiy and redundancy of functional effects Tend to mediate localised effects (very short half-life in circulation), although some act systemically Receptors typically comprise two or more polypeptide chains which can be grouped into families

6. Cytokines (3) Act not singly but in patterns Typically , any one cytokine will affect the expression of other cytokines and/or their receptors Problem of implicating particular cytokines in disease processes: Correlation with disease severity Use of agonists and antagonists

7. Regulation of cytokine expression Constitutive (steady state haematopoiesis, e.g. M-CSF, G-CSF, SCF, IL-6) Stored pre-synthesised: In granules (e.g. GM-CSF, TGF-  On membranes (e.g. TNF-  ) Complexed to extracellular matrix (e.g. TGF-  ) Most are not constitutively expressed but synthesised rapidly in response to stimulation and/or other cytokines

8. Other cytokine control mechanisms Processing (cleavage, precursors) Sequestration Soluble binding proteins (including soluble receptors) Naturally occurring antagonists (IL-1RA) Modulation of receptors

9. Cytokine receptor superfamilies Ig constant region-like domains (C1, C2) Complement control protein domains Fibronectin type III domains (FNIII) Cytokine receptor domains (CK)

10. Cytokine receptor superfamilies Superfamily Receptors for Common structure Cytokine receptor IL-2 (  ), IL-4, Single spanning membrane IL-3/5 (  ), IL-6, glycoporteins with CK, IL-9, IL-12, etc. FNIII and C2 Ig domains Interferon receptor IFN-  , IL-10 SSMG, FNIII domains Immunoglobulin IL-1, IL-6, FGF, Domains containing the M-CSF, SCF “Ig fold” Protein tyrosine EGF, PDGF, M-CSF Intracellular PTK domains kinase receptor SCF, FGF NGF receptor NGF, TNF-I/II 3-4 cysteine-rich repeats in extracellular molecule G-protein coupled C5a, PAF, 7 transmembrane domains 7 transmembrane IL-8 and chemokines with 3 extra- and 4 intra- spanning receptor cellular loops Complement protein IL-2(  ), complement Multiple repeats short domains

11. “Pro-inflammatory” cytokines: IL-1, TNF-  , IL-6 Broad range of pro-inflammatory properties locally and systemically Cachexia, fever, neutrophilia, acute-phase response

12. Interleukin-1 (1) Two forms, IL-1  with only 20% homology but very similar activity Pro-peptides cleaved extracellularly by neutral proteases Natural inhibitor IL-1RA Monocyte/macrophages, keratinocytes, epithelium, T cells, LGL, B cells, endothelium, Langerhans, dendritic, fibroblasts, neutrophils, eosinophils, smooth muscle, etc.

13. Interleukin-1 (2) Stimuli: toxins, haemaggluitinins, C5a, TNF-  , antigen-specific T cell contact Two receptors IL-RI/II (Ig superfamily) on leucocytes, dendritic, fibroblast, neural, endothelium, etc.

14. Interleukin-1 (3) Effects: Haematopoiesis Cytostatic to tumour cells; enhances cell-mediated tumour killing Other cytokines: IL-6, IL-8, TNF-  , etc. Fever, acute phase response T cell proliferation following antigen-specific activation Development of pre-B cells

15. TNF-  and IL-6 TNF-  IL-6 Source Many Many Structure Pro-peptide; trimerises Single peptide Tumour suppression +++ + Acute phase response +++ ++ Receptor TNFRI/II (CD120a/b) Heterodimer  -chain CD126  -chain CD130 Receptor distribution Widspread Widespread Endothelium T cells, B cells, T cells, APC plasma cells

16. IL-2 and IL-15 IL-2 IL-15 Source Activated T cells Multiple (T cell, (B cells) placenta, muscle, epithelium, monocytes) Actions T cell autocrine/paracrine growth factor B cell differentiation and Ig secretion Monocytes: IL-1 secretion, cytotoxicity, phagocytosis NK cells, LAK cells Receptor  subunits  chains of IL-2R Receptor subunits  other names CD25, Tac, p55 CD122, p75 Kd (nM) 10 1000 none signal transduction no yes yes

17. IL-4 and IL-13 IL-4 IL-13 Source “Th2-type” CD4 T cells “Th1/0/2”-type T cells Some CD8 T cells Some CD8 T cells Thymocytes, eosinophils, mast cells, basophils Actions B cells: class II MHC, CD40, CD23, Ig synthesis IgE class switching (not in the mouse!) Growth and proliferation No effect on T cells of T cells “ Th2-type” T cells Monocytes: complex effects: MHC class II, antigen presentation, cytokine release

18. IL-10 Homologous to BCRF1 (EBV genome) T cells, B cells, monocyte/macrophages Inhibits cytokine synthesis by “Th1-like” T cells and NK cells, and antigen presentation and cytokine (TNF-  , IL-6, IL-8) production by macrophages Enhances activation and maturation of B cells (Ig synthesis), thymocytes, mast cells

19. IL-3, IL-5 and GM-CSF Common signal-transducing  -chain in heterodimeric receptors Clustered on chromosome 5 (with IL-4, M-CSF) Activated T cells, mast cells, eosinophils IL-5 is uniquely eosinophil-specific IL-3: pluripotential stem cells, granulocyte/macrophage/erythroid/platelet precursors GM-CSF: mature granulocytes and monocytes

20. IL-12 Heterodimer 35/40 kDa Induces IFN-  synthesis, NK and LAK activity independently of IL-2 (CMI) Induces “Th1-type”, but inhibits “Th2-type” T cell responses Inhibits IgE synthesis by B cells

21. Interferon-  T cells and NK cells Antiparallel homodimer Receptor: single transmembrane protein with accessory protein. Expressed on T/B cells, APC, granulocytes, epithelial cells, endothelial cells, tumour cells Inhibits viral replication Activates macrophages: tumoricidal, microbicidal, IL-1, IL-6, TNF-  Class II MHC expression Prototype “Th1-type” cytokine

22. Th1 Th2 IL-2 IFN-  IL-4 IL-5 TNF-  IL-6 IL-3 IL-10 GM-CSF IL-13 IL-9? Cytolysis Help for IgE synthesis Help for IgG/A/M synthesis Th0 TNF-  Cytokines

23. IFN-  IL-4 IL-13 Th1 Th2 Th0 IL-12 IFN-  IFN-  NK IL-4 IL-13 Mast IL-4 - - M  + - + +

24. Interleukin-18 Actions Growth of T cells and NK cells with production of IFN-  , TNF-  Synergises with IL-12 to induce IL-18R on T cells and activated B cells causing IFN-  release and inhibition of IgE synthesis Sources Monocyte/macrophages, intestinal epithelial cells, osteoblasts

25. Interleukin-18 (2) pro-IL-18 18 kDa caspase 1 (IL-1  CE) Structure: similar to IL-1  Receptor: IL-1R related protein

26. Cytokines and Th1/2 T cell development Pro-Th1 cytokines Pro-Th2 cytokines IFN-  IL-4 IL-12 IL-18 IFN- 

27. Interleukin-16 (1) Actions CD4 ligand, causing activation and chemotaxis of CD4 bearing cells (CD4 T cells, monocytes, eosinophils) (“lymphocyte chemotactic factor”) Resistance to HIV-1 replication (2,4,9 kb transcripts), but not cellular penetration Sources Epithelium, T cells, mast cells, macrophages, eosinophils

28. Interleukin-16 (2) 20 kDa 30 kDa pro-IL-16 caspase 3 121 HIV-1 tetramers CD4 ligand

29. IL-16 and allergic inflammation mast cell IL-5, GM-CSF Rantes, MIP-1  eotaxin IL-16 histamine IL-5 LTB 4 + eosinophil CD4 T cell CD4 CD4

30. Interleukin-17 T cell regulation of haematopoiesis Enables fibroblasts to sustain CD34+ precursor cells and direct maturation towards neutrophils ? Via IL-6, IL-8, G-CSF

31. IL-10 family:  -helices IL-19: monocytes, B cells IL-20: monocytes (keratinocytes) IL-22/TIF: NK, CD4+ Th1 cells IL-24/mda-7: T cells, melanocytes, monocytes, NK cells, B cells IL-26/AK155: CD4 T memory cells, NK cells Receptors: class II (CRF2):  and  chains

32. Interleukin-21 4  -helix cytokine most closely related to IL-15 Secreted by CD4 T cells in response to antigen stimulation Differentiation of NK cells (CD16, IFN-  ) Blocks further recruitment of activated NK cells

33. Interleukin-23 2 subunits p19 and p40 p19 4  -helix cytokine related to p35 subunit of IL-12 p40 is subunit of IL-12 Engages IL-12R  2 Memory cell proliferation and IFN-  production Dendritic cells

34. Chemokines

35. Chemokines Small MW (8-10 kDa) peptides: mediators of acute inflammatory responses Chemotactic cytokines Chemoattraction, activation, diapedesis of granulocytes (also T cells/monocytes) Angiogenic Bind to heparin and stromal proteoglycans IgE-independent basophil degranulation

36. Chemokine families CXCL 1-15 Two cysteines separated by one amino acid Chromosome 4q12-q21, 10 24-84% homology N-terminal Glu-Leu-Arg (ELR) (neutrophil active) or non-ELR (not neutrophil active) CCL 1-28 Two adjacent cysteines Chromosome 17 25-71% homology CL 1/2 One cysteine Chromosome 1 CX3CL 1

37. Chemokine receptors Seven transmembrane spanning G-protein coupled receptor superfamily 6 CXCR chemokine, 10 CCR chemokine receptors cloned, more to come CXCR 4, CCR 5, CCR 3 mediate entry of HIV into T cells/monocytes Most chemokines bind to several receptors CCR 3: confined to eosinophils and Th2-type T cells: binds to eotaxin, RANTES, MCP-2/3/4

38. Chemokine receptors G-protein coupled, 7 transmembrane spanning Chemokine selectivity and range of expression on leukocytes overlap extensively Some constitutive, some inducible, some susceptible to down-regulation

39. CXCL chemokines Neutrophil chemotaxis (IL-8 most potent) T cell chemotaxis (e.g. IL-8 CD8+ T cells) Neutrophil activation Suppress (IL-8, GRO-  , PF4) or stimulate (SDF-1) myeloid colony formation Stimulate (ELR subgroup) or suppress (non-ELR subgroup) angiogenesis Fibroblast collagen synthesis (CTAP III) Post-translational modification, e.g. PBP and NAP-2

40. Some CXCL chemokines CXCR 3 IP-10 CXCL 10 CXCR 3 MIG CXCL 9 CXCR 1/2 IL-8 CXCL 8 CXCR 2 NAP-2 CXCL 7 CXCR 2 ENA-78 CXCL 5 PF 4 CXCL 4 CXCR 2 GRO  CXCL 1-3

41. CCL chemokines Monocyte/lymphocyte chemotaxis/activation: MIP-1  (CD4+), RANTES (CD4/CD45RO+) Eosinophil chemotaxis/activation: eotaxin, eotaxin 2 (eosinophils only), RANTES, MCP-3, MCP-4, (MCP-2, MIP-1  ) Histamine releasing activity: MCP-1, MCP-3, MIP-1  , RANTES, MCP-2 HIV anti-viral effects: RANTES, MIP-1  , MIP-1  Post-translational modification, e.g. CD26 and RANTES, eotaxin

42. Some CCL chemokines CCR 3 Eotaxin 2/3 CCL 24/26 CCR 4 TARC CCL 17 CCR 2/3 MCP-4 CCL 13 CCR 3/5 Eotaxin CCL 11 CCR 1/2/3 MCP-3 CCL 7 CCR 1/3/5 RANTES CCL 5 CCR 1/5 MIP-1  CCL 3/4 CCR 2 MCP-1 CCL 2 CCR 8 I-309 CCL 1

43. Chemokine receptor expression by T cells Th1 CXCR 3 Th2 CCR 3 CCR 4 CCR 8

44. Cooperation between eotaxin and IL-5 in eosinophil accumulation eotaxin IL-5 eosinophils & precursors eosinophils Eotaxin mediated but IL-5 dependent Bone marrow Tissues

45. Eotaxin, eosinophils and “Th2-type” T cells Th2 T cell Eosinophil CCR3 CCR3 eotaxin eotaxin IL-5 GM-CSF

46. Sources of eotaxin Epithelial cells Endothelial cells T cells Eosinophils Fibroblasts

47. Lipid mediators (leukotrienes, prostaglandins)

48. Leukotrienes and prostanoids Ubiquitous oxygenated fatty acids Physiological functions: temperature regulation, coagulation, parturition, blood pressure Newly formed mediators

49. 5(S)-HETE Prostaglandins Thromboxanes LTB 4 Nuclear membrane phospholipids 5(S)-HPETE LTA 4 LTC 4 LTD 4 Arachidonic acid LTE 4 Phospholipase A 2 5-lipoxygenase 5-lipoxygenase LTA 4 hydrolase  -glutamyl transpeptidase LTC 4 synthase Dipeptidases Cyclooxygenase

50. Cellular sources of leukotrienes Predominantly LTB 4 Predominantly LTC 4 /D 4 /E 4 Monocytes Monocytes Eosinophils Macrophages Macrophages Mast cells Neutrophils Basophils (B cells) Reflects restricted expression of 5-lipoxygenase LTC 4 synthase and LTA 4 hydrolase are widely expressed

51. Biological activities of leukotrienes LTB 4 LTC 4 /D 4 /E 4 Increase leucocyte adhesion to endothelium Increase microvascular permeability Neutrophil (eosinophil) chemotaxis Vasoconstriction Neutrophil lysosomal release Smooth muscle contraction and superoxide generation Increase mucus secretion Bronchoconstriction (via TXA 2 ) CD8 T cell cytotoxicity IgG production Augment IL-2, IFN-  production

52. Leukotriene degradation LTB 4 20-  hydroxylation Further oxidation to aldehyde and acid LTC 4 Conversion to LTD 4 /E 4 Extracellular metabolism (H 2 O 2 ) (sulphoxides)  -oxidation

53. Leukotriene receptors LTB 4 LTB 4 (B-LT) receptor LTC 4 /D 4 /E 4 CysLT1: inflammatory cells, smooth muscle CysLT2: inflammatory cells, vascular smooth muscle Others?

54. Leukotrienes in disease Bronchial asthma and allergic rhinitis Fibrosing lung disease Infant/adult respiratory distress syndrome Rheumatoid arthritis Psoriasis Inflammatory bowel disease Myocardial infarction

55. Leukotriene inhibitors LTD 4 receptor antagonists Zafirlukast Pranlukast Montelukast 5-lipoxygenase inhibitors Zileuton

56. Prostanoids Arachidonic acid 15-keto-PGE 2 PGG 2 PGH 2 PGE 2 PGH synthase (cyclooxygenase) 15-keto-PGF 2  PGF 2  6-keto-PGF 1  PGI 2 (prostacyclin) TXB 2 TXA 2 (thromboxane) 9  ,11  PGF 2  PGD 2

57. Biosynthesis and cellular sources PGH synthase 2 (COX) isoforms: COX-1 (ubiquitous, constitutive) COX-2 (inducible in inflammation) Major products characteristic of different cells: Mast cells PGD 2 Platelets TXA 2 Endothelium PGI 2 Smooth muscle PGI 2 Epithelium PGE 2 Macrophages PGF 2  , PGE 2 , TXA 2

58. Prostanoid receptors PGD 2 DP PGE 2 EP-1(smooth muscle contraction)/2(relaxation)/3/4 PGF 2  FP PGI 2 IP TXA 2 TP-1/2 (more?) G-protein linked 7 transmembrane domain proteins Considerable cross-reactivity between receptors

59. Prostanoids: pro-inflammatory actions Pain Swelling Vascular engorgement Smooth muscle contraction/relaxation

60. Neuropeptides

61. Tachykinins Substance P (SP), neurokinin A/B Neuropeptide K (NPK), NP-  Found in neurons in the CNS only (NKB), or in the CNS and peripheral nerves (SP, NKA) Somatic sensory (C-fibre) nerves Sensitive to capsaicin

62. Tachykinins: receptors and metabolism Receptors NK 1 NK 2 NK 3 Affinity SP>NKA=NKB NKA>NKB>>SP NKB>NKA>>SP Distribution Wide Wide Brain & spinal cord Metabolism Angiotensin converting enzyme (ACE): endothelium Neutral endopeptidase (NEP): epithelium

63. Tachykinins: inflammatory efects Non-adrenergic, non-cholinergic (NANC) effects of peripheral nerve stimulation Direct or via axon reflexes: “neurogenic inflammation” Smooth muscle contraction, plasma leakage, mucus secretion, vasodilatation Mast cell degranulation (SP) Priming and adherence of granulocytes

64. Calcitonin gene-related peptide (CGRP)  -CGRP (separate gene)  -CGRP (alternative splicing of calcitonin gene) Widely distributed in peripheral neurones Metabolised by mast cell tryptase and chymase (also NEP) High-affinity binding sites in vascular smooth muscle

65. CGRP: pro-inflammatory effects Relaxation of smooth muscle, particularly large and small blood vessels, mediated via NO release Enhances, but does not cause vascular leakage Increases neutrophil adherence, inhibits T cell proliferation

66. Other neuropeptides Vasoactive intestinal peptide (VIP) VIP-related peptides (PHM, PHV, helodermin, helospectins) Neuropeptide tyrosine Opioids Gastrin-releasing peptide Galanin

67. Other possible effects of neuropeptides Pain and hyperalgesia Smooth muscle contraction Vascular leakage Arthritis: pain, fibroblast and synoviocyte proliferation

68. Endothelin (ET) and nitrous oxide (NO) ET (vasoconstriction) NO (vasodilatation) Vascular endothelium and endocardium Epithelial cells Peripheral NANC nerves CNS Placenta Inflammatory leucocytes

69. Endothelin (ET) and nitrous oxide (NO): synthesis ET NO pro-ET ET 1-3 L-arginine L-citrulline + NO N G -hydroxy-L-arginine big ET NADPH NADP O 2 H 2 O NADPH NADP O 2 H 2 O

70. NO synthase: isoforms Isoform Expression Distribution nNOS constitutive CNS, peripheral NANC nerves, epithelium eNOS constitutive endothelium, placenta (membrane anchored) iNOS inducible many cells IL-1, TNF-  , IFN-  , LPS stimulatory TGF-  , corticosteroids inhibitory

71. ET and NO: signalling ET 7 transmembrane domain receptor coupled to G-protein Activates PLC and PKC, AP-1, gene expression NO Acts by local diffusion Binds to haem moiety of guanylyl cyclase, elevating cGMP Non-specific cytotoxic/cytostatic effects: forms peroxynitrate which binds to Fe 2+

72. Mediators of Inflammation Cytokines Chemokines Lipid mediators (leukotrienes, prostaglandins) Neuropeptides Endothelin and NO