Lecture 17 Cytokines

What are cytokines? A collection of polypeptides used for communications between cells Play role similar to hormones (messengers of the endocrine system) Hormones usually act at a distance Cytokines act locally Differ from growth factors that are produced constitutively, while cytokine production is carefully regulated Play an important role in both innate and adaptive immunity

Cytokine nomenclature Interleukins (1-18) Interferons (a,b,g) Others (common names)

Cytokine -mediated effects Cell growth Cell differentiation Cell death Induce non-responsiveness to other cytokines/cells Induce responsiveness to other cytokines/cells Induce secretion of other cytokines

How do cytokines tell cells what to do? Produced by cells as part of normal cellular activity and/or the result of environmental trigger Bind to receptors on cells Trigger signal transduction pathways Initiate synthesis of new proteins

Properties of cytokines Proteins Low molecular weight Bind to receptor on either cell which produced it or another cell Receptor binding triggers a signal Signal results in altered pattern of gene expression

Cytokines can act in three different manners Autocrine Cytokine binds to receptor on cell that secreted it Paracrine Cytokine binds to receptors on near by cells Endocrine Cytokine binds cells in distant parts of the body

Cytokine Actions Pleiotropy Redundancy Synergy Antagonism Act on more than one cell type (INFa/b) Redundancy More than one cytokine can do the same thing (IFNa/b and IFN) Synergy Two or more cytokines cooperate to produce an effect that is different or greater than the combined effect of the two cytokines when functioning separately (IL-12 and IL-8) Antagonism Two or more cytokines work against each other (IL-4 and IL-12)

How can non-specific cytokines act specifically? Only cells expressing receptors for specific cytokines can be activated by them Many cytokines have very short half-lives Only cells in close proximity will be activated High concentrations of cytokines are needed for activation May require cell-to cell contact

Five cytokine receptor families Immunoglobulin superfamily receptors Class I cytokine receptor family (hematopoietin receptors) Binds most of the cytokines in the immune and hematopoietin systems Class II cytokine receptor family TNF receptor family Chemokine receptor family

Cytokines regulate the immune response Cells with the appropriate receptors become activated To differentiate To express receptors which will make them receptive to other cytokines To secrete other cytokines

Signal Transduction by cytokine receptors Cytokine receptors on different cell types trigger different events How do you get the message from the outside of the cell to the machinery inside?

Cytokines, growth factors and hormone signal transduction pathways Cytokines, growth factors (GF), and hormones are all chemical messengers that mediate intercellular communication. The regulation of cellular and nuclear functions by cytokines, growth factors, and peptide or protein hormones is initiated through the activation of cell surface receptors (Rc). All receptors have two main components: 1) a ligand-binding domain that ensures ligand specificity and 2) an effector domain that initiates the generation of the biological response upon ligand binding. The activated receptor may then interact with other cellular components to complete the signal transduction process. Many growth factors bind to receptors that are linked through G-proteins to membrane-bound phospholipase C (PLC). Activation of PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerols (DAG) and D-myo-inositol- 1, 4, 5-triphosphate (IP3). IP3 regulates intracellular Ca2+ by binding to the IP3 receptor on the endoplasmic reticulum (ER) and stimulating Ca2+ release from the ER. Free intracellular Ca2+ can bind to calmodulin, and this Ca2+ -calmodulin complex, in the presence of cyclic-AMP (cAMP), activates protein kinase A (PKA) by binding to the regulatory subunit of the enzyme. DAG binds to and activates protein kinase C (PKC). Other hormone receptors may be linked through G-proteins to adenylate cyclase (AC) instead of PLC. Activation of AC increases the cellular levels of cAMP and, in the presence of the Ca2+-calmodulin complex, will activate PKA. Additionally, some growth factor and cytokine receptors are protein tyrosine kinases (PTK) that are directly activated by ligand-receptor interaction. Activation of any of the protein kinases, PKA, PKC, or PTK, catalyzes the phosphorylation of other proteins within the cell. Enzymes that are activated or inhibited by phosphorylation may mediate functional processes within the cell, while others may be one step in a protein kinase cascade that regulates nuclear events. Steroid hormones (i.e. estrogen, glucocorticoids), thyroid hormone, vitamin D3, and retinoids are all small lipophilic molecules that easily penetrate both the cellular and nuclear membranes to enter the nucleus where they bind to their respective receptors that are ligand-dependent transcription factors. These ligand-receptor complexes bind to specific DNA response elements in the promoter region and regulate gene expression. References Luttrell, L.M., et al., G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors. Adv. Second Messenger Phosphoprotein Res. 31, 263-277 (1997). Marshall, C.J., Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 80,179-185 (1995). Kumar, R., Thompson, E.B., The structure of the nuclear hormone receptors. Steroids 64, 310-319 (1999)

The Jak/Stat Signaling Pathway A wide variety of extracellular signals activate the Stat (signal transducers and activators of transcription) class of transcription factors. Cytokines, lymphokines, and growth factors all signal through a related superfamily of cell surface receptors that are associated with and activate Janus kinases (Jaks). Ligand-induced dimerization of the receptor induces the reciprocal tyrosine phosphorylation of the associated Jaks, which, in turn, phosphorylates tyrosine residues on the cytoplasmic tail of the receptor. These phosphorylated tyrosines serve as docking sites for the Src Homology-2 (SH-2) domain of the Stat protein, and Jak catalyzes the tyrosine phosphorylation of the receptor-bound Stat. Phosphorylation of Stat at a conserved tyrosine residue induces SH-2-mediated homo- or heterodimerization, followed by translocation of the Stat dimer to the nucleus. Stat dimers bind to specific DNA response elements in the promoter region of target genes to activate gene expression. APS (adaptor molecule containing pleckstrin homology and SH-2 domains) can inhibit the Jak-Stat pathway by binding to the cytoplasmic domain of the receptor where it is phosphorylated (activated) by Jak. Activated APS binds to c-Cbl and blocks Stat activation. References Wakioka, T., et al., APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia 13, 760-767 (1999). Schindler, C., Cytokines and JAK-STAT signaling. Exp. Cell Res. 253, 7-14 (1999).

Involvement of cytokines in the immune response Alert to infection.tumor/etc. Recruit cells to site Specify type of immune response Immune effector phase Immune down-regulation Immune memory and resetting the system Early mediators (IFNa/b) Chemokines (MIP-1a) Early & late mediators (IL-2, IFNg, IL-4, IL-5) Down-regulators (IL-10, TNFg) Maintenance of cytokines, etc. (GM-CSF, IL-3, IL-7, etc.)

Early mediators Interferons a/b Induced by dsRNA, etc. Induced by CD40/CD40L pathway IFNs can induce more of themselves Directly interferes with viral replication Activation of T and NK cells

Chemokines Recruit to sites of infection MIP-1a (NK and T cells) MIG, RANTES (CD4+T cells) IL-8 (neutrophils) Eotaxin (eosinophils)

Early mediators IL-12, IL-15, 1l-18, IFN-g (from NK cells), IL-10 Proinflammatory mediators Produced by cell associated with innate immunity (macrophages, NK, etc.) Mediate direct effects Promote inflammation Shape downstream responses

Late mediators IL-2, IL-4, IL-5, IFN-g, TNF, IL-6, IL-10 Produced by cells of the adaptive immune response (T and B cells) Direct effects More immunoregulatory functions

Cytokine secretion and biological activities of TH1 and TH2 Subsets Type 1 Type 2 Cell-mediated Immune response (intracellular Organisms) Humoral response (parasites) T cell IL-2 IFN-g TNF IL-4 IL-5

Down regulators IL-10, IL-11, TGF-b Inhibit proliferation, cytokine production Produced by both innate and adaptive cells

Maintenance cytokines GM-CSF, IL-3, IL-7, IL-9, etc. Induce cell differentiation, cell growth

Cytokine cross-regulation In a a given immune response, either TH1 or TH2 response dominates Cytokines of one response tend to down-regulate the other type of response Example: TH1 cells secrete IFN-g, which inhibits proliferation of TH2 subset

Role of TH1/TH2 balance in determining disease outcomes Balance of two subset determines response to disease Leprosy Tuberculoid (TH1, CMI response, patient lives) Lepromatous (TH2, humoral response, patient dies)

Cytokine-related diseases Bacterial septic shock Blood pressure drops, clots form, hypoglycemia ensues, patient dies LPS triggers results in TNF release TNF induces IL-1 which induces IL-6 and IL-8 Bacterial toxic shock and related diseases Superantigens trigger large numbers of T cells which release massive amounts of cytokines (Super antigens are bacterial toxins that bridge CD4 T cell receptors and the MHC class II molecules on APC’s, bypassing the need for antigen) Lymphoid and myeloid cancers Some cancer cells secrete cytokines Chagas’ disease Trypanosoma cruzi infection results in sever immune suppression Depression of IL-2 receptor production

Components of the immune system Help  T cell CD8  T cell CD4  T cell B cell Inflammatory cytokines Cytotoxic T cells ? Antibody Intra- and Extracellular Inflammatory Mechanisms to Destroy or Inactivate Pathogens Macro- phages Interferon & Non-lymphoid Cytokines Complement Granulo- cytes Adapted from Marrack and Kappler, 1994

Infectious agents that target cytokines Epstein-Barr virus foster the generation of T helper cells that do not produce IL-2. EBV produces an analog of IL-10 that favors TH2 cells, rather than TH1. Parasites such as tape worms induce high levels of IgE, an immunoglobulin induced by TH2 cells. Since TH1 cells mediate inflammation, this may be a protective ploy to avoid destructive inflammatory processes.

Immunosuppressive effects of oral bacteria on immune function Impairment of B and T cell function (P. intermedia, P. asaccharolytica, P. endodontalis, P. melaninogenica) Production of specific toxins that kill monocytes (A. actinomycetemcomitans) Provoke the release of peroxide, prostaglandins and other mediators capable of inhibiting lymphocyte function (T. denticola) Modulate expression of cytokines

Cytokine-inducing components of Periodontopathogens Taken from Wilson, M., Reddi, K., Henderson, B. 1996. Cytokine-inducing components of periodontopathogenic bacteria. J. Periodont. Res. 31:393-407. Pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF) are believed to be the major pathological mediators of inflammatory diseases ranging from arthritis to periodontal diseases. It is believed that components of microorganisms have the capacity to induce cytokine synthesis in host cells.

Cytokine-inducing components of Gram-positive bacteria

Cytokine-inducing components of Gram-negative bacteria

Cytokine-induction by LPS from periodontopathogens other than P Cytokine-induction by LPS from periodontopathogens other than P. gingivalis

Cytokines produced by host cells in response to components/products from periodontopathogens

Interferon Action Viral replication stimulates the infected host cell to produce interferon. Interferon induces uninfected cells to produce antiviral proteins that prevent translation of viral mRNA degrade viral nucleic acid Viral replication is blocked in uninfected cells

Therapeutic uses of cytokines Modulation of TH activation Interfere with receptor function Interfere with cytokine Make it unable to bind to receptor Make it unable to act

Examples of therapeutic uses Soluble T-cell receptor Anti-IL-2R Interleukin analogs which bind receptor, but do not trigger activation (ties up receptor) Toxins conjugated to cytokines which kill activated T-cells Administration of cytokines to enhance immunity (side effects/ short half lives) Allergies