Chapter 4 Part 1

Sept. 15 & 20, 2016

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

845-3421 sharon.evans@roswellpark.org

Cytokines, Cytokine Receptors and Chemokines

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

Macrophage Cytokine Release During Immune Response

Macrophage Cytokine Release During Immune Response

What triggers cytokine release? What is the downstream action 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.

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)

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)

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

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

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:

Cytokine release

T lymphocyte Immunological synapse

Dendritic cell

T lymphocyte

High cytokine concentration at site of cell-cell contact

Susceptibility determined by receptor expression

Increase cytokine receptors Increase cytokine production Act on multiple cell types Mobilize network of interacting cells

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

Pleiotrophy – Different biological effects on different cell targets

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

Redundancy – > 2 cytokines with similar function

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

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

Antagonism – effect of one cytokine blocked by another

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

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)

Cytokines discovered in 1960s

Antigen stimulation Mitogens

Leukocytes

Supernatant (conditioned medium)

Immune cells

Proliferation Differentiation

Maturation Effector function

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)

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

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

Cell

Num

ber

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

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

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

ELISA Assay

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

SA/Biotin Kd~10-14 M

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

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

Leukocyte

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

IL-6

IL-6

IL-6

IL-6

IL-6

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)

Leukocyte PE-IL-6 mAb

FITC-CD14 mAb

Fix Permeabilize

Stain

Chen, Immunity 2004

IL-6 IL-6

IL-6

Cell

numbe

r Intracellular IL-6

Control Ab IL-6 Ab

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

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)

Cytokine Groups (6) • IL-1 family

• Hematopoietin family

• Interferon family

• Tumor necrosis family

• IL-17 family

• Chemokine family

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

General Functions • Promote proinflammatory responses

– IL-1ß, IL-18, IL-33 – IL-17

• Mediators of natural/innate immunity – Type I IFN – TNF-α – IL-17

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-γ

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

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)

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

Ab detection of cell surface receptors

IL-2 receptor (CD25)- expressing cell

Fluorescently-labeled anti-CD25 antibody

Control

Activated

Cell N

umbe

r

IL-2Rα (CD25)

Autofluorescence

CD25 Ab

eBioscience CD25 = α chain of IL-2 receptor

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

Cel

l Num

ber

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

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)

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

Receptor Family Ligands IL-1 IL-18 IL-33 M-CSF C-Kit

Immunoglobulin superfamily receptors

IL-1 Family of Cytokine Receptors

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

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)

Cytokines bind α chain with low affinity; αβ dimer has high affinity for cytokine → transduce signal

Multiple subunits – noncovalent association α chain binds cytokine β chain – signal transducing chain

• IL-6 family cytokines compete for limited # of β chains

• Overlapping biological activities

(CD25)

& Treg cells

5x103 R/cell

CD25 Not normally expressed alone

• 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

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

Receptor Family Ligands TNF receptor superfamily

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

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)

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

Receptor Family Ligands Chemokine receptors CCL21 (SLC)

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

Summary PART 1 • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine receptors

PART 2 (Tuesday/September 22, 2015) • Signal transduction by cytokine receptors • T cell differentiation • Cytokines and disease pathogenesis • Role of cytokines in inflammation (Chapt. 4 and 14)

End Part 1

Chapter 4 Part 2

Sept. 15 & 20, 2016

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

845-3421 sharon.evans@roswellpark.org

Cytokines, Cytokine Receptors and Chemokines

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

Macrophage Cytokine Release During Immune Response

Outline PART 1 • Biological activity of cytokines • Methods to analyze cytokines • Structure of cytokines • Detection/structure of cytokine receptors

PART 2 (Thursday/September 22, 2015) • Signal transduction by cytokine receptors • T cell differentiation • Cytokines and disease pathogenesis • Role of cytokines in inflammation (Chapt. 4 and 14)

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

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.

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.

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.

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

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

target gene • cell type

*STAT, signal transducer and activator of transcription

*

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

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)

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 – IgM→IgE – Allergic responses – IgG1 –does not activate C’

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

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 +

*Need to know example of cytokines produced by TH subsets

α

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

Stat4

IL-12

Cross-regulation at the intracellular level

Similar figure found in Fig 11-10 but lacks IFN-γ

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

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.

Revised Th Differentiation

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

IL-6 TGFβ

STAT3

(STAT3) (Smad3)

Alveolar MΦ = source IL-23 RORγt

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

(RORγt) 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

α-CD3 α-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)

(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

(RORγt) 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 iTreg

Immunoregulation, suppress immunity (Th1, Th17)

Cancer

Regulation of Cytokines

• Chromatin Structure – Regulation of expression

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

Regulation of Cytokines

• Chromatin Structure – Regulation of expression

• Post-translational processing – Inflammasome (processing of pro-IL-1β)

– Shedding by ectoenzymes (TNF, TNFR)

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

Regulation of Cytokines

• Chromatin Structure – Regulation of expression

• Post-translational processing – Inflammasome – Shedding by ectoenzymes

• SOCS/CIS Family – Regulation of activity

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

Role of SOCS in Th1/2 Development

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)

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)

Cytokines Drive Epithelial Cancers (Colon, Liver cancer)

Michael Karin & Florian R. Greten Nature Reviews/ Immunology 5, 749-759, 2005

Cytokine Therapies

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

Cytokine Targets in Rheumatoid Arthritis

The Scientist Tocilizumab

IL-6 Targeting in Rheumatoid Arthritis

Inflammation

Humanized Ab to IL-6R

Concept of blocking IL-6 signaling by Tocilizumab

STAT3 JAK1/2

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. 24 & 29, 2015) • Role of cytokines/chemokines in lymphocyte

trafficking and inflammation