Cytokines, Cytokine Receptors and Chemokines

Cytokines, Cytokine Receptors and Chemokines Chapter 4 Part 1 Sept. 11, 2014 Sharon S. Evans, Ph.D. Department of Immunology 845-3421 sharon.evans@roswellpark.org 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 T h 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 T 1/2 in blood Inducible Produced by specialized glands Act on unique cell type Constitutive Produced by multiple cell types Act on multiple cells Inducible Produced by multiple cell types Act on multiple cells

Interleukins, IL (~35) Cytokines: Known by common/functional names (tumor necrosis factor,tnf; interferon, IFN) Chemokines (~50); smaller MW (8-10 kda) Hallmarks: Bind high affinity receptors (K d = 10-8 10-12 M) 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

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

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

Cytokines discovered in 1960s Antigen stimulation Mitogens Supernatant (conditioned medium) Leukocytes Immune cells Initially analysis difficult: Low amounts (high activity) Difficult to purify Lack assays systems (mainly depend on bioassays) Proliferation Differentiation Maturation Effector function

Growth Study of IFN Activity Addition of growth factors (fetal calf serum / nutrients) IFN-sensitive Daudi B cells IFN-resistant Daudi B cells + IFN Assay growth

Cell Number Growth Inhibition of Daudi B cells by IFN-α (Measure Day 3) 250 200 150 IFN-resistant 100 50 0 IFN-sensitive 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 Cytokine (IL-12) Intensity of substrate color depends on cytokine concentration 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 Wash Wash Adapted from R&D Systems Inc., Minneapolis, Minnesota See also Fig. 20-7 in Kuby 7 th edition Chapter 20

ELISA Assay

SA/Biotin Kd~10-14 M

Human IL-17A ELISPOT: Human PBMCs No mitogen Human PBMCs PMA/Ionomycin 24 hours Image from ebioscience See also Fig. 20-9 in Kuby 7 th edition Chapter 20 Quantify spots

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

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

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 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

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

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

Analysis of Cytokine Receptors Biochemical analysis of receptors hampered by IFN-γRβ / Glial cell nucleus low amount of receptors on cell surface multiple receptor subunits

Cell Number Ab detection of cell surface receptors IL-2 receptor (CD25)- expressing cell Control Autofluorescence CD25 Ab Fluorescently-labeled anti-cd25 antibody Activated 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 IL-6 B IL-6 B IL-6 B IL-6 Biotinylated ligand IL-6 or IL-6R blocking Ab B IL-6 B IL-6 SA B IL-6 SA B SA IL-6 B IL-6 SA Fluorescently-labeled streptavidin SA B IL-6 SA B IL-6

Cell Number Fluorokine Analysis of IL-6 Cell Surface Receptor Irrelevant Protein-Biotin 10% IL-6-Biotin 44% IL-6 Blocking Ab 5% Fluorescence Intensity Appenheimer et al., Eur J Immunol, 2007

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 Immunoglobulin superfamily receptors Ligands IL-1 IL-18 IL-33 M-CSF C-Kit

IL-1 Family of Cytokine Receptors

Receptor Family Hematopoietin receptor Family (Class I) P Y- W - tryptophan S - serine Ligands 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) & T reg cells 5x10 3 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 Interferon receptor Family (Class II) P Y- Lack WSXWS Ligands IFN-α IFN-ß IFN-γ IL-10 IL-19 IL-20 IL-22 IL-24 IL-26 IL-28 IL-29

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

Receptor Family IL-17 Receptor Family Ligands 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 Chemokine receptors Ligands 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 16, 2014) 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. 16, 2014 Sharon S. Evans, Ph.D. Department of Immunology 845-3421 sharon.evans@roswellpark.org 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 16, 2014) 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

Unifying cytokine signaling model Class I & Class II cytokine receptors Specificity dictated by: receptor signaling mediators promoter sequence of target gene cell type

IFN Activation of JAK/STAT Signaling Pathway IFN-α or IFN-γ JAK1 JAK2 TYK2 Untx α subunit β subunit STAT1 IFN-α 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)

T H subsets T H 1 subset Viral infection, intracellular pathogen Bacterial cell infection Cell-mediated response IgG2a-Complement fixation, phagocytosis T H 2 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 IL-12 IFNγ NK NK/T Th1 cells Signal 2 Costimulation (LFA-1/ICAM-1) IL-12R Signal 3 Cytokines IL-12, IFN-γ Th1 IFNγR

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 crossregulation of T H subsets

α *Need to know example of cytokines produced by T H subsets

Leprosy - chronic infection caused by bacteria Mycobacterium leprae Tuberculoid type of leprosy involves cell- mediated destruction of mycobacteria Lepromatous leprosy primarily Ab response Figure 11-12 Kuby Immunology, Seventh Edition 2013 W.H. Freeman and Company

IL-12 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 T H 1 Response Th2 IL-10 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)

Th17 and Treg Cells Th17 (CD4 +, FoxP3 -) IL-17 is a proinflammatory 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 Alveolar MΦ = source IL-23 STAT3 RORγt Dong, C., Nature Rev. Immunol. 6: 329, 2006 IL-6 (STAT3) TGFβ (Smad3)

Development of Th17 vs Treg Cells Foxp3/IL-10 Smad3 Smad3 IL-23 STAT3/IL-17 Weaver, C.T. et al, Immunity 24:677, 2006

Current model of Th differentiation Th1 (T-bet) IFN-γ APC a-cd3 a-cd28 MHC II CD4 Thp IL-12, IFN-γ (Stat4, STAT1) IL-4 (Stat6) TGF-β+IL-4 (SMAD3, Stat6) Th2 (GATA-3) Th9 (PU.1) IL-4, IL-5, IL-13 IL-9 Adapted from Mark Kaplan, Indiana U. IL-21, IL-6 (Stat3) TGF-β+IL-6, IL-23 (SMAD3, Stat3) TGF-β, IL-2 (SMAD3, Stat5) Tfh (Bcl6) Th17 IL-17A/F, IL- 21, IL-22 (RORgt) itreg Foxp3 IL-21 TGFβ IL-10

Th1 (T-bet) Th2 (GATA-3) Protective Cell-mediated immunity to intracellular pathogens; immunity to protozoa (Leishmania); immunity to fungi (Candida); immunity to bacteria (Mycobacteria) Humoral immunity; immunity to helminthic parasites (Nippostrongylus, Schistosoma, Trichuris); Immunity to viruses (measles); Immunity to bacteria (Borrelia) Pathogenic Autoimmunity Multiple sclerosis, arthritis, colitis Allergy and atopic responses, cancer Th9 (PU.1) Tfh (Bcl6) Th17 (RORgt) itre Foxp3 g Immunity to extracellular parasites Immunity to helminthic parasites - Trichuris Provide B cell help leading to antibody production GC formation; class switching Immunity to extracellular infectious disease; Immunity to bacteria (Klebsiella), Bacteroides (Citrobacter); Immunity to fungi (candida) Immunoregulation, suppress immunity (Th1, Th17) Allergy and atopic responses Autoimmunity- systemic lupus erythematosus Autoimmunity- Multiple sclerosis, arthritis, colitis, psoriasis Cancer

Regulation of Cytokines Chromatin Structure Regulation of expression

Alterations in Chromatin Structure T H1 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- histone acetylation STAT4-necessary for histone acetylation Ifng 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)

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 Procaspase-1 Caspase-1 IL-18 also 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 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 T reg in cancer

Cytokine Targets in Rheumatoid Arthritis The Scientist Tocilizumab

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. 18 & 23, 2014) Role of cytokines/chemokines in lymphocyte trafficking and inflammation