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Chapter 4 Part 1 Sept. 11, 2014 Sharon S. Evans, Ph.D. Department of Immunology 845-3421 [email protected] Cytokines, Cytokine Receptors and Chemokines Membrane-bound IL-6 receptor The Role of IL-6 in Inflammatory Diseases Dr. Bernard Rubin

Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

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Page 1: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Chapter 4 Part 1

Sept. 11, 2014

Sharon S. Evans, Ph.D. Department of Immunology

845-3421 [email protected]

Cytokines, Cytokine Receptors and Chemokines

Membrane-bound IL-6 receptor The Role of IL-6 in Inflammatory Diseases Dr. Bernard Rubin

Page 2: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Macrophage Cytokine Release

During Immune Response

Page 3: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Macrophage Cytokine Release

During Immune Response

What triggers cytokine release?

What is the downstream action of cytokine?

Page 4: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Objectives

• Be able to read and understand cytokine

field .

• Have general understanding of current

status of cytokine-driven Th cell subset

differentiation.

• Understand molecular basis of positive

and negative regulation by cytokines.

Page 5: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline Cytokines – Part 1

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

Cytokines – Part 2

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 6: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 7: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine – General Characteristics

(Greek) Cyto = cell Kinein = to move

> 200 cytokines

• Contribute to cell-to-cell communication; rarely act alone

• Inducible by discrete stimuli • Produced by many cells; bind to many cells • Low molecular weight (20 – 30 kDa) regulatory

proteins; some direct effector activity • Typically soluble but can be membrane bound • Secreted by lymphocytes, monocytes, other cells

Page 8: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Hormones Growth Factors Cytokines

• Systemic

• Inducible

• Produced by specialized glands

• Act on unique cell type

• Local

• Constitutive

• Produced by multiple cell types

• Act on multiple cells

• Local (although can act systemically)-act short distances; short T1/2 in blood

• Inducible

• Produced by multiple cell types

• Act on multiple cells

Page 9: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine
Page 10: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokines:

• Interleukins, IL (~35) • Known by common/functional

names (tumor necrosis factor,TNF; interferon, IFN)

• Chemokines (~50); smaller

MW (8-10 kDa)

• Bind high affinity receptors (Kd = 10 -8 – 10 -12M)

• Sensitivity in pM range

• Local reactivity

Hallmarks:

Page 11: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine release

T lymphocyte

Immunological

synapse

Dendritic cell

T lymphocyte

High cytokine

concentration at

site of cell-cell contact

Page 12: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Susceptibility determined by receptor expression

Increase cytokine receptors

Increase cytokine production

Act on multiple cell types

Mobilize network of interacting cells

Page 13: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

General Characteristics

• Mediate and regulate the immune system

• Secretion is brief and self-limited

• Individual cytokines are produced by multiple cell types

• Act on multiple cell types-pleiotrophic

Page 14: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Pleiotrophy – Different biological effects on different cell targets

Page 15: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

General Characteristics

• Mediate and regulate the immune system

• Secretion is brief and self-limited

• Individual cytokines are produced by multiple cell types

• Act of multiple cell types-pleiotrophic

• Actions are redundant

Page 16: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Redundancy – > 2 cytokines with similar function

Page 17: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

General Characteristics

• Mediate and regulate the immune system

• Secretion is brief and self-limited • Individual cytokines are produced by

multiple cell types • Act of multiple cell types-pleiotrophic • Actions are redundant • Synergistic and antagonistic actions of

cytokines

Page 18: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Synergy – combined effects of 2 cytokines > the effect of individual cytokines

Antagonism – effect of one cytokine blocked by another

Page 19: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine cascade – activation of one cytokine produced by one cell type induces cytokine production by other cell types

Page 20: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 21: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokines discovered in 1960s

Antigen stimulation Mitogens

Leukocytes

Supernatant (conditioned medium)

Immune cells

Proliferation Differentiation

Maturation Effector function

Page 22: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokines discovered in 1960s

Antigen stimulation Mitogens

Leukocytes

Supernatant (conditioned medium)

Immune cells

Proliferation Differentiation

Maturation Effector function

Initially analysis difficult: Low amounts (high activity) Difficult to purify Lack assays systems (mainly depend

on bioassays)

Page 23: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Growth Study of IFN Activity Addition of growth factors

(fetal calf serum / nutrients)

IFN-sensitive Daudi B cells

IFN-resistant Daudi B cells

Assay growth

+ IFN

Page 24: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Growth Inhibition of Daudi B cells by IFN-α (Measure Day 3)

Cell N

umber

0

50

100

150

200

250

0 1 10 100

IFN-resistant

IFN-sensitive

IFN-α Concentration (U/ml)

Scarozza et al., J Interferon Res, 1992

Appenheimer et al, Am J Health Syst Pharm, 1998

Page 25: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Progress in Cytokine Studies • 1970-1980s – gene cloning

– Produce large amounts of recombinant cytokines

• 1980s – Monoclonal antibody technology

– Develop enzyme-linked immunosorbant assays

(ELISA); highly sensitive measurement of

cytokine concentration

Page 26: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Principle of ELISA Assay

Adapted from R&D Systems Inc., Minneapolis, Minnesota

See also Fig. 20-7 in Kuby 7th edition Chapter 20

Plate pre-coated

with antibody

specific for cytokine

Standards or samples

added

Enzyme-linked antibody

specific for cytokine added Substrate solution added to

generate color

Cytokine (IL-12)

Wash Wash

Intensity of substrate

color depends on

cytokine concentration

Page 27: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

ELISA Assay

Page 28: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Progress in Cytokine Studies • 1970-1980s – gene cloning

– Produce large amounts of recombinant cytokines

• 1980s – Monoclonal antibody technology

– Develop enzyme-linked immunosorbant assays

(ELISA); highly sensitive measurement of

cytokine concentration

• 1990s – present – other cytokine assays

developed

– Luminex

– Elispot

– Intracellular cytokine assays

Page 29: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

SA/Biotin Kd~10-14 M

Page 30: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Image from eBioscience

See also Fig. 20-9 in Kuby 7th edition Chapter 20

Human IL-17A ELISPOT:

Human PBMCs No mitogen

Human PBMCs PMA/Ionomycin

24 hours

Quantify spots

Page 31: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Progress in Cytokine Studies • 1970-1980s – gene cloning

– Produce large amounts of recombinant cytokines

• 1980s – Monoclonal antibody technology

– Develop enzyme-linked immunosorbant assays

(ELISA); highly sensitive measurement of

cytokine concentration

• 1990s – present – other cytokine assays

developed

– Luminex

– Elispot

– Intracellular cytokine assays

Page 32: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Leukocyte

Flow Cytometric Approach to Evaluate Intracellular Cytokine (IL-6)

IL-6

IL-6

IL-6

IL-6

IL-6

Page 33: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Leukocyte

Brefeldin A

IL-6

Blocks protein transport from ER to golgi cytokines accumulate in ER

IL-6 IL-6

Flow Cytometric Approach to Evaluate Intracellular Cytokine (IL-6)

Page 34: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Leukocyte

PE-IL-6 mAb

FITC-CD14 mAb

Fix

Permeabilize

Stain

Chen, Immunity 2004

IL-6 IL-6

IL-6

Cell n

umber

Intracellular IL-6

Control Ab IL-6 Ab

Flow Cytometric Approach to Evaluate Intracellular Cytokine (IL-6)

Page 35: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 36: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine Groups (6)

• IL-1 family

• Hematopoietin family

• Interferon family

• Tumor necrosis family

• IL-17 family

• Chemokine family

Page 37: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

α helix β sheets

Derived from x-ray

crystallographic

analysis

• Amino acid sequences

vary

• Similar polypeptide fold,

four α-helical regions (A-

D) roughly parallel to

each other, connected

by loops-unique to

cytokines

Common Structure Shared by Cytokines

Page 38: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

General Functions

• Promote proinflammatory responses – IL-1ß, IL-18, IL-33

– IL-17

• Mediators of natural/innate immunity – Type I IFN

– TNF-a

– IL-17

Page 39: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

General Function

• Regulators of lymphocytic growth, activation and differentiation – IL-2, IL-4, IL-5, IL-12, IL-15

• Activators of inflammatory cells – Type II IFN

• IFN-g

• Stimulators of hematopoiesis – IL-3, IL-5, IL-7, GM-CSF

Page 40: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 41: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Analysis of Cytokine Receptors

IFN-γRβ / Glial cell nucleus

• Biochemical analysis of receptors hampered by

– low amount of receptors

on cell surface – multiple receptor

subunits

Page 42: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Ab detection of cell surface receptors

IL-2 receptor (CD25)-

expressing cell

Fluorescently-labeled

anti-CD25 antibody

Control

Activated

Cel

l Nu

mb

er

IL-2Rα (CD25)

Autofluorescence

CD25 Ab

eBioscience CD25 = α chain of IL-2 receptor

Page 43: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IL-6

B

IL-6

B

IL-6

B

IL-6

B

IL-6

B IL-6

B

SA

IL-6

B SA

SA

SA

IL-6

B

IL-6

B

IL-6

B

SA

IL-6

B

Biotinylated ligand

SA Fluorescently-labeled

streptavidin

Receptor-expressing cell

Fluorokine Analysis of Cytokine Cell Surface Receptor

IL-6

Unlabeled ligand

(competitor)

or IL-6R blocking Ab

Page 44: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cell

Nu

mb

er

Fluorescence Intensity

IL-6-Biotin

Irrelevant

Protein-Biotin

10%

IL-6

Blocking Ab

44%

5%

Appenheimer et al., Eur J Immunol, 2007

Fluorokine Analysis of IL-6 Cell Surface Receptor

Page 45: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 46: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Families of Cytokine Receptors

• Six classes of receptors

– IL-1 family of cytokine receptors; part of the immunoglobulin superfamily receptors

– Hematopoietin receptor family (Class I cytokine receptor family); multiple receptor subunits

– Interferon receptor family (Class II cytokine receptor); multiple receptor subunits

– TNF receptor family

– IL-17 receptor family

– Chemokine receptor family

Page 47: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Receptor Family Ligands

IL-1 IL-18 IL-33 M-CSF C-Kit

Immunoglobulin superfamily receptors

Page 48: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IL-1 Family of Cytokine Receptors

Page 49: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Receptor Family Ligands Hematopoietin receptor Family (Class I)

W - tryptophan

S - serine P Y-

IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-9 IL-11 IL-12 IL-13 IL-15

IL-21 IL-23 IL-27 GM-CSF G-CSF OSM LIF CNTF GH Prolactin TGF-ß

Page 50: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Subfamilies of hematopoietin receptors have identical signaling subunits

• GM-CSF receptor subfamily (common beta chain)

• IL-6 receptor subfamily (common gp130 subunit)

• IL-2 receptor subfamily (common γ subunit)

Page 51: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokines bind α chain with low affinity; αβ dimer

has high affinity for cytokine transduce signal

Page 52: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Multiple subunits – noncovalent association

α chain binds cytokine

β chain – signal transducing chain

Page 53: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

• IL-6 family cytokines compete for

limited # of β chains

• Overlapping biological activities

Page 54: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine
Page 55: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

(CD25)

& Treg

cells

5x103 R/cell

Page 56: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

• β γ chains form pocket

• α chain completes pocket, forms lid-accounts for high

affinity binding of trimeric receptor

• Signal transduction mediated by β and γ chains but all 3

chains required for high affinity binding of IL-2

Page 57: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Lack WSXWS

P Y-

Receptor Family Ligands Interferon receptor Family (Class II)

IFN-α IFN-ß IFN-γ IL-10 IL-19 IL-20 IL-22 IL-24 IL-26 IL-28 IL-29

Page 58: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Receptor Family Ligands TNF receptor superfamily

TNF-α TNF-ß CD27L CD30L CD40L FasL APRIL BAFF Lymphotoxin-ß TRAIL

Page 59: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Receptor Family Ligands IL-17 Receptor Family

IL-17A (CTL-8) IL-17B IL-17C IL-17D IL-17E (IL-25) IL-17F vIL-17 (ORF13)

Page 60: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IL-17 Receptor Family

• Composed of 5 protein chains: IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE

• Contain SEF/IL-17R (similar expression to fibroblast growth factor/IL-17 receptor) domains

Page 61: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Receptor Family Ligands Chemokine receptors CCL21 (SLC)

IL-8 RANTES MIP-1 PF4 MCAF NAP-2

Page 62: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Summary PART 1

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

PART 2 (Tuesday/September 16, 2014)

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 63: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

End Part 1

Page 64: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Chapter 4 Part 2

Sept. 16, 2014

Sharon S. Evans, Ph.D. Department of Immunology

845-3421 [email protected]

Cytokines, Cytokine Receptors and Chemokines

Membrane-bound IL-6 receptor The Role of IL-6 in Inflammatory Diseases Dr. Bernard Rubin

Page 65: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Macrophage Cytokine Release

During Immune Response

Page 66: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline PART 1

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

PART 2 (Thursday/September 16, 2014)

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 67: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Keys to Cytokine Specificity

• Cytokine • Cytokine receptor • Type of pathway activated

– NFκB (e.g., IL-1, IL-17, and TNF) – JAK/STAT (e.g., hematopoietin and interferon) – G protein (e.g., chemokines)

• Sequences activated in promoter of target genes

• Particular cell type – e.g., IL-4 induces different gene expression in T cells vs B cells

Page 68: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Common features of IL-1, IL-17 and TNF family signaling: NFκB pathway

Specificity dictated by: • receptor • signaling mediators • promoter sequence of

target gene • cell type

But the receptors, signaling mediators, and response are different.

Page 69: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Common features of IL-1, IL-17 and TNF family signaling: NFκB pathway

Specificity dictated by: • receptor • signaling mediators • promoter sequence of

target gene • cell type

But the receptors, signaling mediators, and response are different.

Page 70: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Common features of IL-1, IL-17 and TNF family signaling: NFκB pathway

Specificity dictated by: • receptor • signaling mediators • promoter sequence of

target gene • cell type

But the receptors, signaling mediators, and response are different.

Page 71: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Unifying cytokine signaling model – Class I & Class II cytokine receptors

Specificity dictated by: • receptor • signaling mediators • promoter sequence of

target gene • cell type

Page 72: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

α subunit

JAK2

TYK2 JAK1

IFN-α or IFN-γ

STAT1

STAT1

STAT1 STAT1

STAT1 STAT1

Untx

IFN-α

IFN Activation of JAK/STAT Signaling Pathway

Dunn et al, Cancer Res 65:3447, 2005

STAT1

β subunit

Page 73: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine
Page 74: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors (II)

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 75: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

TH subsets

• TH1 subset – Viral infection, intracellular pathogen – Bacterial cell infection – Cell-mediated response – IgG2a-Complement fixation, phagocytosis

• TH2 subset – Soluble bacterial products, heminthic (roundworm) infection – Ab/humoral response – IgMIgE – Allergic responses – IgG1 –does not activate C’

Page 76: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Signal 2

Costimulation

(LFA-1/ICAM-1)

Signal 3

Cytokines

IL-12, IFN-γ

Cytokines Program T Cell Activation

and Differentiation

IFNγR

IL-12R

Th1

NK NK/T

Th1 cells IL-12

IFNγ

Page 77: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IFN-γ also produced by NK cells, DC

IFN-γ stimulates IL-12 secretion and

upregulates IL12R on T cells

Cytokine-mediated generation and cross-regulation of TH subsets

α

Eosinophils, T cells,

mast cells, basophils,

produce IL-4

OX40L

Page 78: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

*Need to know example of cytokines

produced by TH subsets

α

Page 79: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Tuberculoid type of leprosy – involves cell- mediated destruction of

mycobacteria

Lepromatous leprosy – primarily Ab response

Leprosy - chronic infection caused by bacteria Mycobacterium leprae

Figure 11-12

Kuby Immunology, Seventh Edition

© 2013 W.H. Freeman and Company

Page 80: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Stat4

IL-12

Cross-regulation at the intracellular level

Similar figure found

in Fig 11-10 but

lacks IFN-γ

Page 81: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IL-10

IL-10 Acts Indirectly via Control of Monocytes/Macrophage/DC to

Inhibit TH1 Response

• Downregulate MHC class II on APC • Block IL-12 secretion • Inhibit nitric oxide (NO)

production • Inhibit production of inflammatory

cytokines (IL-1, IL-6, IL-8, GM-CSF, G-CSF, TNF-α)

Th2

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Th17 and Treg Cells

• Th17 (CD4+, FoxP3-) – IL-17 is a pro-

inflammatory cytokine

– Promotes secretion of pro-inflammatory cytokines (IL-6) from fibroblasts, epithelial and endothelial cells.

– Th17 cells are critical to anti-bacterial immunity.

– Overexpression of IL-17 is associated with rheumatoid arthritis, SLE, MS and asthma

• Treg (CD4+, Foxp3+)

– Natural (develop in the thymus)

• Prevent effector T cell development in LN

• Produce IL-4, IL-10, TGFβ

– Induced (develop in the periphery)

• Develop under the influence of TGF-ß

• Inhibit effector T cell function in periphery.

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Revised Th Differentiation

Dong, C., Nature Rev. Immunol. 6: 329, 2006

IL-6

TGFβ

STAT3

(STAT3)

(Smad3)

Alveolar MΦ = source IL-23 RORγt

Page 84: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Development of Th17 vs Treg Cells

Weaver, C.T. et al, Immunity 24:677, 2006

STAT3/IL-17

IL-23

Smad3

Smad3

Foxp3/IL-10

Page 85: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

(RORgt)

Th17 IL-17A/F, IL-21, IL-22

(GATA-3)

Th2 IL-4, IL-5, IL-13

(T-bet)

Th1 IFN-γ

(Bcl6)

Tfh IL-21

CD4

Thp MHC II

a-CD3 a-CD28

IL-12, IFN-γ

IL-4

IL-21, IL-6

APC

(Stat4, STAT1)

(Stat6)

(Stat3)

IL-9 Th9

(PU.1)

Adapted from Mark Kaplan, Indiana U.

TGF-β, IL-2

TGF-β+IL-6, IL-23 (SMAD3, Stat3)

TGF-β+IL-4 (SMAD3, Stat6)

Foxp3

iTreg TGFβ IL-10

Current model of Th differentiation

(SMAD3, Stat5)

Page 86: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

(T-bet)

Th1

Protective Pathogenic Cell-mediated immunity to intracellular pathogens; immunity to protozoa (Leishmania); immunity to fungi (Candida); immunity to bacteria (Mycobacteria)

Autoimmunity – Multiple sclerosis, arthritis, colitis

(GATA-3)

Th2

Humoral immunity; immunity to helminthic parasites (Nippostrongylus, Schistosoma, Trichuris); Immunity to viruses (measles); Immunity to bacteria (Borrelia)

Allergy and atopic responses, cancer

(RORgt)

Th17

Immunity to extracellular infectious disease; Immunity to bacteria (Klebsiella), Bacteroides (Citrobacter); Immunity to fungi (candida)

Autoimmunity- Multiple sclerosis, arthritis, colitis, psoriasis

(Bcl6)

Tfh Provide B cell help leading to antibody production GC formation; class switching

Autoimmunity- systemic lupus erythematosus

Immunity to extracellular parasites Immunity to helminthic parasites - Trichuris

Allergy and atopic responses Th9 (PU.1)

Foxp3

iTre

g

Immunoregulation, suppress immunity (Th1, Th17)

Cancer

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Regulation of Cytokines

• Chromatin Structure

– Regulation of expression

Page 88: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Alterations in Chromatin Structure

Ifng

TH1 conditions: Ifng activation

1. Transcription factors direct chromatin

remodeling activity (recruit histone

acetyl transferase (HAT), etc)

2. Chromatin remodeling changes the

accessibility of cis-regulatory regions

(e.g., histone acetylation (Ac) and DNA

demethylation generally associated

with transcriptional activation at

cytokine gene locus).

• T-bet induces hyperacetylation of

histones in Ifng locus

• GATA3 induces hyperacetylation of

histones in Il4 locus; STAT6 necessary

for histone acetylation and DNA

demethylation

3. Gene silencing achieved by negative

regulatory factors (e.g., GATA-3

antagonizes T-bet activity)

T-bet- histone acetylation

STAT4-necessary for histone acetylation

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Regulation of Cytokines

• Chromatin Structure

– Regulation of expression

• Post-translational processing

– Inflammasome (processing of pro-IL-1β)

– Shedding by ectoenzymes (TNF, TNFR)

Page 90: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IL-1β Processing in Inflammasome

Nature Reviews/ Molecular Cell Biology 4, 95, 2003

Nature Reviews/Immunology 7,33, 2007

Nature Clinical Practice/Rheumatology 4,34, 2008

Procaspase-1

Caspase-1

IL-18

IL-18 also

processed in

inflammasome

NFκB

Page 91: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Regulation of Cytokines

• Chromatin Structure

– Regulation of expression

• Post-translational processing

– Inflammasome

– Shedding by ectoenzymes

• SOCS/CIS Family

– Regulation of activity

Page 92: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Activity of Suppressor of Cytokine Signaling (SOCS/CIS)

• SOCS compete with STATs for cytokine receptor binding

• SOCS inhibit JAK activity and target JAKs for proteosome-mediated degradation

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Role of SOCS in Th1/2 Development

Page 94: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Outline

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

• Signal transduction by cytokine receptors (II)

• T cell differentiation

• Cytokines and disease pathogenesis

• Role of cytokines in inflammation (Chapt. 4 and 14)

Page 95: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine-Related Diseases

• Septic shock – systemic

bacterial infections,

trauma, injury, ischemia

• Cytokine activity implicated

in lymphoid, myeloid, and

epithelial cancers

• Cytokines drive

autoimmunity Septic shock

(TNF, IL-1β)

Page 96: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokines Drive Epithelial Cancers (Colon, Liver cancer)

Michael Karin & Florian R. Greten

Nature Reviews/ Immunology 5, 749-759, 2005

Page 97: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine Therapies

Target CD25 (IL-2Rα) - Kidney, heart transplant (Host vs graft) – also proposed to target Treg in cancer

Page 98: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine
Page 99: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

Cytokine Targets in Rheumatoid Arthritis

The Scientist Tocilizumab

Page 100: Cytokines, Cytokine Receptors and Chemokines · • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine

IL-6 Targeting in Rheumatoid Arthritis

Inflammation

Humanized

Ab to IL-6R

Concept of blocking IL-6 signaling by Tocilizumab

STAT3

JAK1/2

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Summary Cytokines - PART 1

• Biological activity of cytokines

• Methods to analyze cytokines

• Structure of cytokines

• Detection/structure of cytokine receptors

Cytokines - PART 2

• Signal transduction by cytokine receptors

• T cell differentiation

• Cytokines and disease pathogenesis

CHAPTER 4 and 14 (Sept. 18 & 23, 2014)

• Role of cytokines/chemokines in lymphocyte

trafficking and inflammation