Cytokines, Cytokine Receptors and Chemokines

Home , Chemokine, Cytokine, Cytokine receptor

Chapter 4 Part 1 Sept. 15 & 20, 2016

Sharon S. Evans, Ph.D. Department of Immunology 845-3421 [email protected] 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 • Local • Local (although can act systemically)-act short distances;

short T1/2 in blood

• Inducible • Constitutive • Inducible

• Produced by • Produced by • Produced by specialized multiple cell multiple cell glands types types

• Act on unique • Act on multiple • Act on multiple cell type cells cells

• Interleukins, IL (~35)

• Known by common/functional names (tumor necrosis factor,TNF; interferon, IFN) Cytokines: • Chemokines (~50); smaller MW (8-10 kDa)

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

Hallmarks: • Sensitivity in pM range

• Local reactivity T lymphocyte

T lymphocyte Immunological synapse Cytokine release

Dendritic cell

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

Cytokines discovered in 1960s Supernatant (conditioned medium) Antigen stimulation Mitogens

Leukocytes Immune cells

Proliferation Differentiation Maturation Effector function Cytokines discovered in 1960s Supernatant (conditioned medium) Antigen stimulation Mitogens

Leukocytes Immune cells

Initially analysis difficult:  Low amounts (high activity) Proliferation  Difficult to purify Differentiation  Lack assays systems (mainly depend Maturation on bioassays) Effector function Growth Study of IFN Activity

Addition of growth factors (fetal calf serum / nutrients)

IFN-sensitive IFN-resistant Daudi B cells Daudi B cells

+ IFN

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

250

200 IFN- resistant 150

CellNumber 100

50 IFN- sensitive

0 0 1 10 100 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

Intensity of substrate color depends on cytokine concentration

Cytokine (IL-12)

Plate pre-coated Standards or samples Enzyme-linked antibody Substrate solution added to with antibody added specific for cytokine added generate color specific for cytokine

Wash Wash

Adapted from R&D Systems Inc., Minneapolis, Minnesota See also Fig. 20-7 in Kuby 7th edition Chapter 20 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 Human IL-17A ELISPOT:

Human PBMCs Human PBMCs No mitogen PMA/Ionomycin

24 hours

Quantify spots Image from eBioscience See also Fig. 20-9 in Kuby 7th edition Chapter 20 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 Flow Cytometric Approach to Evaluate Intracellular Cytokine (IL-6)

Leukocyte

IL-6

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

Leukocyte

IL-6 IL-6 IL-6

Brefeldin A

Blocks protein transport from ER to golgi → cytokines accumulate in ER Flow Cytometric Approach to Evaluate Intracellular Cytokine (IL-6)

FITC-CD14 mAb Control Ab IL-6 Ab

Leukocyte PE-IL-6 mAb

IL-6 IL-6

IL-6 Cell number Cell Fix Permeabilize Stain Intracellular IL-6 Chen, Immunity 2004 Outline

Cytokine Groups (6)

• IL-1 family

• Hematopoietin family

• Interferon family

• Tumor necrosis family

• IL-17 family

• Chemokine family Common Structure Shared by Cytokines

β sheets α helix

• Amino acid sequences vary • Similar polypeptide fold, four α-helical regions (A- D) roughly parallel to each other, connected Derived from x-ray by loops-unique to crystallographic cytokines analysis 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

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

Control IL-2 receptor (CD25)- Autofluorescence expressing cell CD25 Ab

Fluorescently-labeled Activated anti-CD25 antibody

Cell Number

IL-2Rα (CD25)

CD25 = α chain of IL-2 receptor eBioscience Fluorokine Analysis of Cytokine Cell Surface Receptor

Receptor-expressing cell Unlabeled ligand (competitor)

B B IL-6 IL-6 B Biotinylated ligand B IL-6 IL-6 IL-6 or IL-6R blocking Ab

B B IL-6 IL-6 SA B SA SA IL-6 B Fluorescently-labeled B SA IL-6 streptavidin IL-6

SA SA B B IL-6 IL-6 Fluorokine Analysis of -6 of IL Analysis Fluorokine

Cell Number Cell Surface Receptor Surface Cell - Protein Blocking Ab Irrelevant IL - IL 6- - Biotin Biotin Biotin Fluorescence Intensity Fluorescence 6

Appenheimer et al., Eur J Immunol, 2007 Immunol, J Eur al., et Appenheimer 10% 44% 5%

Outline

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 Immunoglobulin IL-1 superfamily receptors IL-18 IL-33 M-CSF C-Kit IL-1 Family of Cytokine Receptors Receptor Family Ligands Hematopoietin receptor IL-2 IL-21 Family (Class I) IL-3 IL-23 IL-4 IL-27 IL-5 GM-CSF IL-6 G-CSF IL-7 OSM IL-9 LIF IL-11 CNTF W - tryptophan IL-12 GH P Y- S - serine IL-13 Prolactin IL-15 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 Not normally expressed alone

(CD25)

& Treg cells

3 5x10 R/cell • 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 Receptor Family Ligands Interferon receptor IFN-α Family (Class II) IFN-ß IFN-γ IL-10 IL-19 IL-20 IL-22 IL-24 IL-26 P Y- Lack WSXWS IL-28 IL-29

Receptor Family Ligands TNF receptor TNF-α superfamily TNF-ß CD27L CD30L CD40L FasL APRIL BAFF Lymphotoxin-ß TRAIL

Receptor Family Ligands IL-17 Receptor IL-17A (CTL-8) Family 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 Cytokines, Cytokine Receptors and Chemokines

Chapter 4 Part 2 Sept. 15 & 20, 2016

Sharon S. Evans, Ph.D. Department of Immunology 845-3421 [email protected] 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

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

Specificity dictated by: • receptor • signaling mediators • promoter sequence of target gene • cell type Common features of IL-1, IL-17 and TNF family signaling: NFκB pathway

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

Specificity dictated by: • receptor • signaling mediators • promoter sequence of target gene • cell type Common features of IL-1, IL-17 and TNF family signaling: NFκB pathway

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

Specificity dictated by: • receptor • signaling mediators • promoter sequence of target gene • cell type 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 IFN Activation of JAK/STAT Signaling Pathway

IFN-α or IFN-γ

Untx JAK2 JAK1 TYK2

IFN-α α subunit β subunit STAT1 STAT1

STAT1 STAT1 STAT1

STAT1 STAT1

Dunn et al, Cancer Res 65:3447, 2005

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’

Cytokines Program T Cell Activation and Differentiation NK IFNγ IL-12 NK/T Th1 cells

Signal 3 Cytokines Signal 2 Costimulation IL-12R IL-12, IFN-γ (LFA-1/ICAM-1)

IFNγR Th1 IFN-γ also produced by NK cells, DC IFN-γ stimulates IL-12 secretion and upregulates IL12R on T cells α

OX40L +

Eosinophils, T cells, mast cells, basophils, produce IL-4 Cytokine-mediated generation and cross-

regulation of TH subsets α

*Need to know example of cytokines

produced by TH subsets Leprosy - chronic infection caused by bacteria Mycobacterium leprae

Tuberculoid type of leprosy – involves cell- mediated destruction of mycobacteria Lepromatous leprosy – primarily Ab response

Stat4

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

Cross-regulation at the intracellular level IL-10 Acts Indirectly via Control of Monocytes/Macrophage/DC to

Inhibit TH1 Response

• Downregulate MHC class II on APC • Block IL-12 secretion IL-10 • Inhibit nitric oxide (NO) production • Inhibit production of inflammatory cytokines (IL-1, IL-6, IL-8, GM- Th2 CSF, G-CSF, TNF-α)

Th17 and Treg Cells

• Th17 (CD4+, FoxP3-) • Treg (CD4+, Foxp3+) – IL-17 is a pro- – Natural (develop in inflammatory cytokine the thymus) – Promotes secretion of pro-inflammatory • Prevent effector T cytokines (IL-6) from cell development in LN fibroblasts, epithelial and • Produce IL-4, IL-10, endothelial cells. TGFβ – Th17 cells are critical to – Induced (develop in anti-bacterial immunity. the periphery) – Overexpression of IL-17 is associated with • Develop under the rheumatoid arthritis, influence of TGF-ß SLE, MS and asthma • Inhibit effector T cell function in periphery.

Revised Th Differentiation

STAT3 Alveolar MΦ = source IL-23 RORγt

IL-6 (STAT3) TGFβ (Smad3) Dong, C., Nature Rev. Immunol. 6: 329, 2006 Development of Th17 vs Treg Cells

Foxp3/IL-10

Smad3

IL-23 STAT3/IL-17

Weaver, C.T. et al, Immunity 24:677, 2006 Current model of Th differentiation Th1 IFN-γ (T-bet)

Th2 IL-4, IL-5, IL-12, IFN-γ (GATA-3) IL-13 α-CD3 (Stat4, STAT1) α-CD28 IL-4 (Stat6) Th9 IL-9 CD4 TGF-β+IL-4 (PU.1) MHC II APC Thp (SMAD3, Stat6)

IL-21, IL-6 Tfh (Stat3) IL-21 (Bcl6) TGF-β+IL-6, IL-23 (SMAD3, Stat3) Th17 IL-17A/F, IL- (RORγt) 21, IL-22 TGF-β, IL-2 (SMAD3, Stat5) iTreg TGFβ Adapted from Mark Kaplan, Indiana U. Foxp3 IL-10 Protective Pathogenic Cell-mediated immunity to intracellular Th1 pathogens; immunity to protozoa Autoimmunity – Multiple sclerosis, (T-bet) (Leishmania); immunity to fungi (Candida); arthritis, colitis immunity to bacteria (Mycobacteria) Humoral immunity; immunity to helminthic Th2 parasites (Nippostrongylus, Schistosoma, Allergy and atopic responses, cancer (GATA-3) Trichuris); Immunity to viruses (measles); Immunity to bacteria (Borrelia)

Th9 Immunity to extracellular parasites Allergy and atopic responses (PU.1) Immunity to helminthic parasites - Trichuris

Provide B cell help leading to Tfh antibody production Autoimmunity- systemic lupus (Bcl6) GC formation; class switching erythematosus

Immunity to extracellular infectious Th17 disease; Immunity to bacteria (Klebsiella), Autoimmunity- Multiple sclerosis, (RORγt) Bacteroides (Citrobacter); Immunity to arthritis, colitis, psoriasis fungi (candida)

Immunoregulation, suppress iTre Cancer immunity (Th1, Th17) Foxp3g Regulation of Cytokines

• Chromatin Structure – Regulation of expression

Alterations in Chromatin Structure 1. Transcription factors direct chromatin remodeling activity (recruit histone TH1 conditions: Ifng activation 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 T-bet- histone acetylation histones in Ifng locus STAT4-necessary for histone acetylation

• GATA3 induces hyperacetylation of histones in Il4 locus; STAT6 necessary Ifng for histone acetylation and DNA demethylation

3. Gene silencing achieved by negative regulatory factors (e.g., GATA-3 antagonizes T-bet activity)

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

NFκB

Caspase-1 IL-18 also Procaspase-1 processed in inflammasome

IL-18

Nature Reviews/ Molecular Cell Biology 4, 95, 2003 Nature Reviews/Immunology 7,33, 2007 Nature Clinical Practice/Rheumatology 4,34, 2008 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 Septic shock autoimmunity (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

Tocilizumab The Scientist IL-6 Targeting in Rheumatoid Arthritis

Concept of blocking IL-6 signaling by Tocilizumab

Humanized Ab to IL-6R

JAK1/2 STAT3

Inflammation 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