Inflammatory Mediators Corrigan

KING’SCollege

LONDONFounded 1829Respiratory Medicine & Allergy

Inflammatory mediators

Dr Chris Corrigan

Reader/Consultant

GKT School of Medicine

KING’SCollege


Mediators of Inflammation

• Cytokines

• Chemokines

• Lipid mediators (leukotrienes, prostaglandins)

• Neuropeptides

• Endothelin and NO

KING’SCollege


Cytokines

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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 severityUse of agonists and antagonists

KING’SCollege


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

KING’SCollege


Other cytokine control mechanisms

• Processing (cleavage, precursors)

• Sequestration

• Soluble binding proteins (including soluble receptors)

• Naturally occurring antagonists (IL-1RA)

• Modulation of receptors

KING’SCollege


Cytokine receptor superfamilies

• Ig constant region-like domains (C1, C2)

• Complement control protein domains

• Fibronectin type III domains (FNIII)

• Cytokine receptor domains (CK)

KING’SCollege


Cytokine receptor superfamiliesSuperfamily Receptors for Common structureCytokine 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 domainsImmunoglobulin IL-1, IL-6, FGF, Domains containing the

M-CSF, SCF “Ig fold”Protein tyrosine EGF, PDGF, M-CSF Intracellular PTK domainskinase receptor SCF, FGFNGF receptor NGF, TNF-I/II 3-4 cysteine-rich repeats in

extracellular moleculeG-protein coupled C5a, PAF, 7 transmembrane domains7 transmembrane IL-8 and chemokines with 3 extra- and 4 intra-spanning receptor cellular loopsComplement protein IL-2(), complement Multiple repeats short

domains

KING’SCollege


“Pro-inflammatory” cytokines: IL-1, TNF-, IL-6

• Broad range of pro-inflammatory properties locally and systemically

• Cachexia, fever, neutrophilia, acute-phase response

KING’SCollege


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.

KING’SCollege


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.

KING’SCollege


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

KING’SCollege


TNF- and IL-6

TNF- IL-6Source Many ManyStructure Pro-peptide; trimerises Single peptideTumour suppression +++ +Acute phase response +++ ++Receptor TNFRI/II (CD120a/b) Heterodimer

-chain CD126-chain CD130

Receptor distribution Widspread WidespreadEndothelium T cells, B cells,T cells, APC plasma cells

KING’SCollege


IL-2 and IL-15IL-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 secretionMonocytes: IL-1 secretion, cytotoxicity, phagocytosisNK cells, LAK cells

Receptor subunits chains of IL-2R

Receptor subunits other names CD25, Tac, p55 CD122, p75Kd (nM) 10 1000 nonesignal transduction no yes yes

KING’SCollege


IL-4 and IL-13

IL-4 IL-13Source “Th2-type” CD4 T cells “Th1/0/2”-type T cells

Some CD8 T cells Some CD8 T cellsThymocytes, eosinophils, mast cells, basophils

Actions B cells: class II MHC, CD40, CD23, Ig synthesisIgE class switching (not in the mouse!)Growth and proliferation No effect on T cellsof T cells“Th2-type” T cellsMonocytes: complex effects: MHC class II, antigenpresentation, cytokine release

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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

KING’SCollege


Th1 Th2

IL-2IFN-

IL-4IL-5

TNF-

IL-6

IL-3IL-10GM-CSF

IL-13IL-9?

Cytolysis

Help for IgE synthesisHelp for IgG/A/M synthesis

Th0

TNF-Cytokines

KING’SCollege


IFN- IL-4IL-13

Th1

Th2

-

-Th0

IL-12IFN-

M +

IFN-

NK

IL-4IL-13

-

Mast

+

IL-4

+

KING’SCollege


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

KING’SCollege


Interleukin-18 (2)pro-IL-18

18 kDa

caspase 1(IL-1 CE)

Structure: similar to IL-1Receptor: IL-1R related protein

KING’SCollege


Cytokines and Th1/2 T cell development

Pro-Th1 cytokines Pro-Th2 cytokines

IFN- IL-4IL-12IL-18IFN-

KING’SCollege


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

KING’SCollege


Interleukin-16 (2)

20 kDa 30 kDapro-IL-16

caspase 3

121

HIV-1

tetramers

CD4 ligand

KING’SCollege


IL-16 and allergic inflammation

mast cell

IL-5, GM-CSFRantes, MIP-1eotaxin

IL-16

histamineIL-5LTB4

+

eosinophil

CD4

CD4 T cell

CD4

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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-12R2• Memory cell proliferation

and IFN- production• Dendritic cells

KING’SCollege


Chemokines

KING’SCollege


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

KING’SCollege


Chemokine familiesCXCL 1-15Two cysteines separated by one amino acidChromosome 4q12-q21, 1024-84% homologyN-terminal Glu-Leu-Arg (ELR) (neutrophil active) or non-ELR(not neutrophil active)CCL 1-28Two adjacent cysteinesChromosome 1725-71% homologyCL 1/2One cysteineChromosome 1CX3CL 1

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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

KING’SCollege


Some CXCL chemokines

CXCL 1-3 GRO CXCR 2

CXCL 4 PF 4

CXCL 5 ENA-78 CXCR 2

CXCL 7 NAP-2 CXCR 2

CXCL 8 IL-8 CXCR 1/2

CXCL 9 MIG CXCR 3

CXCL 10 IP-10 CXCR 3

KING’SCollege


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

KING’SCollege


Some CCL chemokinesCCL 1 I-309 CCR 8

CCL 2 MCP-1 CCR 2

CCL 3/4 MIP-1 CCR 1/5

CCL 5 RANTES CCR 1/3/5

CCL 7 MCP-3 CCR 1/2/3

CCL 11 Eotaxin CCR 3/5

CCL 13 MCP-4 CCR 2/3

CCL 17 TARC CCR 4

CCL 24/26 Eotaxin 2/3 CCR 3

KING’SCollege


Chemokine receptor expression by T cells

Th1• CXCR 3

Th2• CCR 3• CCR 4• CCR 8

KING’SCollege


Cooperation between eotaxin and IL-5 in eosinophil accumulation

Bone marrow

eotaxin IL-5

eosinophils& precursors

eosinophils

Tissues Eotaxin mediatedbut IL-5 dependent

KING’SCollege


Eotaxin, eosinophils and “Th2-type” T cells

Th2 T cell Eosinophil

CCR3 CCR3

eotaxin

eotaxin

IL-5GM-CSF

KING’SCollege


Sources of eotaxin

• Epithelial cells• Endothelial cells• T cells• Eosinophils• Fibroblasts

KING’SCollege


Lipid mediators (leukotrienes, prostaglandins)

KING’SCollege


Leukotrienes and prostanoids

• Ubiquitous oxygenated fatty acids

• Physiological functions: temperature regulation, coagulation, parturition, blood pressure

• Newly formed mediators

KING’SCollege


5(S)-HETE ProstaglandinsThromboxanes

LTB4

Nuclear membrane phospholipids

5(S)-HPETE

LTA4

LTC4

LTD4

Arachidonic acid

LTE4

Phospholipase A2

5-lipoxygenase

5-lipoxygenase

LTA4 hydrolase

-glutamyl transpeptidase

LTC4 synthase

Dipeptidases

Cyclooxygenase

KING’SCollege


Cellular sources of leukotrienes

Predominantly LTB4 Predominantly LTC4/D4/E4

Monocytes Monocytes EosinophilsMacrophages Macrophages Mast cellsNeutrophils Basophils(B cells)

Reflects restricted expression of 5-lipoxygenaseLTC4 synthase and LTA4 hydrolase are widely expressed

KING’SCollege


Biological activities of leukotrienes

LTB4 LTC4/D4/E4

Increase leucocyte adhesion to endotheliumIncrease microvascular permeability

Neutrophil (eosinophil) chemotaxis VasoconstrictionNeutrophil lysosomal release Smooth muscle contractionand superoxide generation Increase mucus secretionBronchoconstriction (via TXA2)CD8 T cell cytotoxicityIgG productionAugment IL-2, IFN- production

KING’SCollege


Leukotriene degradation

LTB4

• 20- hydroxylation• Further oxidation to aldehyde and acid

LTC4

• Conversion to LTD4/E4

• Extracellular metabolism (H2O2) (sulphoxides)

-oxidation

KING’SCollege


Leukotriene receptors

LTB4

• LTB4 (B-LT) receptor

LTC4 /D4/E4

• CysLT1: inflammatory cells, smooth muscle

• CysLT2: inflammatory cells, vascular smooth muscle

• Others?

KING’SCollege


Leukotrienes in disease

• Bronchial asthma and allergic rhinitis

• Fibrosing lung disease

• Infant/adult respiratory distress syndrome

• Rheumatoid arthritis

• Psoriasis

• Inflammatory bowel disease

• Myocardial infarction

KING’SCollege


Leukotriene inhibitors

LTD4 receptor antagonists

• Zafirlukast

• Pranlukast

• Montelukast

5-lipoxygenase inhibitors

• Zileuton

KING’SCollege


ProstanoidsArachidonic acid

15-keto-PGE2

PGG2

PGH2

PGE2

PGH synthase (cyclooxygenase)15-keto-PGF2

PGF2

6-keto-PGF1

PGI2

(prostacyclin)

TXB2

TXA2

(thromboxane)

9,11PGF2

PGD2

KING’SCollege


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 PGD2 Platelets TXA2

Endothelium PGI2 Smooth muscle PGI2

Epithelium PGE2 Macrophages PGF2, PGE2, TXA2

KING’SCollege


Prostanoid receptors

PGD2 DPPGE2 EP-1(smooth muscle contraction)/2(relaxation)/3/4PGF2 FPPGI2 IPTXA2 TP-1/2 (more?)

G-protein linked 7 transmembrane domain proteinsConsiderable cross-reactivity between receptors

KING’SCollege


Prostanoids: pro-inflammatory actions

• Pain

• Swelling

• Vascular engorgement

• Smooth muscle contraction/relaxation

KING’SCollege


Neuropeptides

KING’SCollege


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

KING’SCollege


Tachykinins: receptors and metabolism

ReceptorsNK1 NK2 NK3

Affinity SP>NKA=NKB NKA>NKB>>SP NKB>NKA>>SPDistribution Wide Wide Brain & spinal cord

Metabolism

Angiotensin converting enzyme (ACE): endotheliumNeutral endopeptidase (NEP): epithelium

KING’SCollege


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

KING’SCollege


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

KING’SCollege


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

KING’SCollege


Other neuropeptides

• Vasoactive intestinal peptide (VIP)

• VIP-related peptides (PHM, PHV, helodermin, helospectins)

• Neuropeptide tyrosine

• Opioids

• Gastrin-releasing peptide

• Galanin

KING’SCollege


Other possible effects of neuropeptides

• Pain and hyperalgesia

• Smooth muscle contraction

• Vascular leakage

• Arthritis: pain, fibroblast and synoviocyte proliferation

KING’SCollege


Endothelin (ET) and nitrous oxide (NO)

ET (vasoconstriction) NO (vasodilatation)

Vascular endothelium and endocardiumEpithelial cells

Peripheral NANC nervesCNSPlacentaInflammatory leucocytes

KING’SCollege


Endothelin (ET) and nitrous oxide (NO): synthesis

ET NO

pro-ET

ET1-3

L-arginine

L-citrulline + NO

NG-hydroxy-L-argininebig ET

NADPH NADPO2 H2O

NADPH NADPO2 H2O

KING’SCollege


NO synthase: isoforms

Isoform Expression Distribution

nNOSconstitutive CNS, peripheral NANC nerves, epitheliumeNOS constitutive endothelium, placenta (membrane anchored)iNOS inducible many cells

IL-1, TNF-, IFN-, LPS stimulatoryTGF-, corticosteroids inhibitory

KING’SCollege


ET and NO: signalling

ET7 transmembrane domain receptor coupled to G-proteinActivates PLC and PKC, AP-1, gene expression

NOActs by local diffusionBinds to haem moiety of guanylyl cyclase, elevating cGMPNon-specific cytotoxic/cytostatic effects: forms peroxynitrate whichbinds to Fe2+

KING’SCollege


Mediators of Inflammation

• Cytokines

• Chemokines

• Lipid mediators (leukotrienes, prostaglandins)

• Neuropeptides

• Endothelin and NO

Inflammatory Mediators Corrigan

Entertainment & Humor

Transcript of Inflammatory Mediators Corrigan