1. Lecture 17 Cytokines

2. 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

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

4. 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

5. 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

6. 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

7. 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

8. 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)

9. 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

10. 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

11. 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

12. 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?

13. 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, (1997).
Marshall, C.J., Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 80, (1995).
Kumar, R., Thompson, E.B., The structure of the nuclear hormone receptors. Steroids 64, (1999)

14. 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, (1999).
Schindler, C., Cytokines and JAK-STAT signaling. Exp. Cell Res. 253, 7-14 (1999).

15. 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.)

16. 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

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

18. 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

19. 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

20. 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

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

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

23. 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

24. 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)

25. 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

26. 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

27. 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.

28. 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

29. Cytokine-inducing components of Periodontopathogens
Taken from Wilson, M., Reddi, K., Henderson, B Cytokine-inducing components of periodontopathogenic bacteria. J. Periodont. Res. 31:
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.

30. Cytokine-inducing components of Gram-positive bacteria

31. Cytokine-inducing components of Gram-negative bacteria

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

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

34. 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

35. 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

36. 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