1. Lecturer: Ge Jin, Ph.D., ge.jin@case.edu, 3683791 Learning Objectives: 1. describe basic properties of cytokines 2. describe how cytokines regulate immunity (e.g. increase or decrease MHC expression and T cell activation…) 3. cytokine and oral diseases NOTE: you need to download the PowerPoint file to your computer to read notes. Slides containing research results from my lab will be added during the lecture.

2. Cytokines properties, categories, signaling, function Body as Host: Immune Molecules

3. Cytokines  small, secreted, non-antibody proteins  produced by cells involved in both innate & adaptive immunity  mediate and regulate immunity, inflammation, and hematopoiesis

4. Cytokines  Lymphokine: made by activated lymphocytes, especially T H cells, e.g. IL-2  Monokine: made by mononuclear phagocytes, e.g. Mig/CXCL9  Chemokine: chemotactic activity, e.g. IL-8, CXCL12  Interleukin: interaction between leukocytes IL-1, IL-8, IL-10, IL-13……  named by activity: T umor N ecrosis F actor  (TNF  ), C olony S timulation F actor (CSF), T ransforming G rowth F actor  (TGF  )….

5. Cytokines  grouped by structures into families  interferons (IFN): type I (IFN  and IFN  ), type II (IFN  )  Interleukin: IL-1, IL-2  chemokine: CXCL and CCL chemokines  TNF  : TNF , FasL, CD40L…  hematopoietin: erythropoietin (EPO), colony- stimulation factors (CSF)

6. Properties of Cytokines  produced in response to immune stimuli -- not store pre-formed -- synthesis: DNA  mRNA  protein  secretion -- slow cellular response  can act on the cells that produce them (autocrine action)  can act on nearby cells (paracrine action)  can act on distance cells (endocrine action)

7. Properties of Cytokines  can be produced by many cell types and act on many cell types (pleiotropic)  different cytokines can have similar actions (redundant)

8. -- share receptors -- defect in a unique cytokine have little effect -- defect in a share component (common receptor) can have profound effects e.g. IL-2R  defect X- linked SCID (Severe Combined Immunodeficiency)

9. Properties of Cytokines  can modulate synthesis of other cytokines - cascades: e.g. TNF   IL-1  IL-6, IL-8… - enhance or suppress production of other cytokines: positive or negative  influence the action of other cytokines - antagonistic - additive - synergistic  short half life, low plasma concentration, bind to receptor with high affinity

10. Immunoglobulin superfamily: IL1R, TLRs… Cytokine Receptors (grouped by structures into families) Toll-IL-1 Receptor domain (TIR) Toll-like receptors (TLRs) leucine-rich repeats immunoglobulin domain IL-1 receptor cell membrane

11. TNFR family: TNF , FasL, CD40L… Cytokine Receptors

12. GM-CSFR  IL-3R IL-5R    GM-CSF IL-2 IL-3 IL-5 IL-15IL-7IL-9   IL-2R  IL-2  IL-15R  IL-2  IL-7RIL-9R class I receptor family: hematopoietin family gp130 CNTFR cell membrane GM-CSF: Granulocyte macrophage colony-stimulating factor IL: interleukin gp130: glycoprotein 130 (m.w. 130 kDa) IL-6IL-11 CNTF LIF/OSM

13. class II receptor family: Interferon , , and  Cytokine Receptors IFNARI IFNAR2c IFNAR2b IFN  R  IFN  R  type I IFN receptor type II IFN receptor cell membrane type I IFN: IFN  and IFN , type II: IFN 

14. chemokine receptor family: CCR1-5, CXCR1-4 NH 3 COOH binding of a ligand to the receptor Cytokine Receptors cell membrane

15. Cytokine Receptors TGF  receptors TGF  receptors have intrinsic kinase activity when interacting with ligands.

16. Cytokine Signaling cytokines membrane receptors phosphorylation cascades transcription, Ca++… signal transduction binding activation

17. Cytokine Signaling  NF  B signaling pathways  JAK/STAT signaling pathways  Chemokine signaling pathways  Cross-talk between pathways

18. NF  B Signaling NF  B signaling inducers:  cytokines: IL-1, TNF ,…  microbial cell components: Fn cell wall, lipopolysacchrides (LPS),…  virus: retroviruses, double-stranded RNA (dsRNA),… Receptors:  IL-1 receptors  Toll-like receptors (TLRs, innate immunity)  TNF  receptors Nuclear Factor  B (NF  B):  dimeric transcription factors: p65/RelA, c-Rel, RelB, p105/p50, p100/p52  sequestered by inhibitory I  B proteins and retained in cytosol in resting cells

19. NF  B IRAK TRAF6 p p IRAK4 pellino1 IBIB IBIB p p TF IRAK p p p p u u degradation u TAK1 activation u u u u p p IBIB IBIB ubiquitination& degradation u u TRAF6 p TAK1 TAB1 TAB3 TAB2 Ubc13/Uev1A complex III IRAK TRAF6 p p IRAK4 pellino1 TAK1 TAB1 TAB3 TAB2 p complex II NEMO IKK  IKK  p IKK activation p u ubiquitination phosphorylation IL-1 receptor IRAK TollipMyD88 IRAK4 TRAF6 p p complex I IL-1 nucleus cell membrane IL-6 IL-8 hBD-2 …. innate & adaptive immunity p NF  B activation NF  B p nuclear translocation p TF NF  B p

20. NF  B Signaling Alternative NF  B activation pathways of immunity CD40L, LT-B, BAFF NIK I  B degradation NF  B activation CD4/TCR PKC UV-light p38-CKII

21. JAK/STAT Signaling Pathway  JAK (Janus Kinases) : a family of tyrosine kinases, JAK1-3, Tyk2  STAT (Signal Transducers and Activators of Transcription) : transcription factors, STAT1-6,…  Cytokines: IFN  / , IFN- , Epo, GM-CSF, IL-6, IL-13…  in resting cells, non-phosphorylated, monomeric STATs reside in cytosol

22. SJ Baker et al, Oncogene (2007) 26, 6724–6737.

23. IFN  /  IFN  receptor binding phospho- Receptor JAKs Receptor JAKs STATs DNA STAT dimers target gene transcription phospho- translocation nucleus binding cdc25a, cyclin D1~3, c-myc, cyclin E, MMP9 p21, caspase 3, 1, 8…

24. Chemokine Signaling  2 major families: the CCL family and CXCL family  CCL1~27, CXCL1~14  receptors: G-protein coupled 7 transmembrane

25.    GiGi SDF    PI-3KMAPKPLC target genes Adhesion, polarization, chemotaxis CCL12 (SDF) signaling p p GRK  -arrestin recycling degradation SDF

26. Cross-talk between Cytokine Signaling Pathways chemokine receptor interferon receptor IL, TLR, TNF  receptors STATs NF  B PI-3K TAK1 TGF  receptor

27. Cytokines and Immunoregulation Mediators of Innate Immunity Mediators of adaptive immunity Stimulators of hematpoiesis

28. Cytokines and Immunoregulation Mediators of Innate Immunity  TNF   IL-1  IL-10  IL-12  IFN , IFN   IFN   Chemokines Mediators of adaptive immunity Stimulators of hematpoiesis

29. Tumor Necrosis Factor  (TNF  )  Produced by activated macrophages and T cells  Most important mediator of acute inflammation in response to microbes, such as LPS  Induces production of myeloid CSFs, IFN- , IL-6, IL-8 and other chemokines  Mediate recruitment of neutrophils and microphages to site of inflammation by stimulating cells to produce adhesion molecules (e.g. ICAM-1)  Stimulates endothelial cells and macrophages to produce chemokines  A potent pyrogen causing fever by direct action or via IL-1  Promotes production of acute phase proteins, such as CRP  Roles in rheumatoid arthritis, psoriasis, tuberculosis, …

30. prothrombotic macrophage activation hematopoiesis lymphocyte development

31. Interleukin 1 (IL-1)  Produced by activated macrophages, stimulated lymphocytes, keratinocytes, fibroblasts  Activates the NF  B signaling pathway (similar effects to TNF)  Helps activate T cells  Can be induced by inflammation, injury, and infection

32. Interleukin 10 (IL-10)  Produced by macrophages, B cells, Th2 cells  Originally identified as cytokine synthesis inhibitory factor  Suppresses inflammatory responses  Inhibits production of IFN- , IL-2, IL-3, TNF , GM-CSF  Stimulate thymocytes, mast cells, B cells  Limits Th1 response, promotes Th2 cell development, shifts response to Th2 type (phagocytosis  Ab production)  Inhibits expression of class II MHC and co-stimulatory molecules on macrophages

33. Interleukin 12 (IL-12)  Produced by macrophages, dendritic cells, Tc cells, NK cells  Belongs to the IL-6 cytokine family  Has immunoregulatory effect on NK cells and T cells  Stimulates production of IFN-   Promotes Th cells  Th1  Enhances differentiation of C ytotoxic T L ymphocytes (with IL-2)  Enhances cytolytic functions of T cells and NK cells

34. Type I Interferon (IFN- , IFN-  )  Produced by macrophages and virus-infected cells  Inhibits viral replication in cells via PKR and RNaseL  Increases expression of MHC I and Tc mobilization  Stimulates production of IFN-  by activated T cells  Activate NK cells

35. Type II Interferon (IFN-  )  Produced primarily by Th1  Induce ICAM production in endothelial cells  Activate NK cells  Increase MHC I and MHC II expression to help Th cell and APC interaction  Promotes B cell differentiation to plasma cell  Promotes cytotoxic T cell differentiation

36. Chemokines  Produced by many leukocytes and other types of cells  Large family of molecules (over 50)  Have significant structural homology and overlapping functions  Chemotactic for leukocytes, such as PMN, T and B cells  Recruit leukocytes to sites of infection and inflammation  Involved in lymphocytes trafficking, wound healing, metastasis, angiogenesis, lymphoid organ development….

37. Cytokine in Immunregulation Mediators of Innate Immunity Mediators of adaptive immunity  IL-2  IL-4  IL-5  TGF   IL-10  IL-12  IFN-  Stimulators of hemotopoesis

38. Interleukin 2 (IL-2)  Produced by lectin- or antigen-activated Th cells  Powerfully immunoregulatory lymphokine  Main growth factor for both T and B lymphocytes  Activates NK cells and monocytes CTLA-4: C ytotoxic T-L ymphocyte A ntigen 4, inhibits T cell function.

39. Interleukin 4 (IL-4)  Produced by macrophages, Th2 cells, activated B cells  Has complex biological actions via cytokine production  Enhances antigen-presenting activity of B to T cells  Stimulates development of Th2 cells from naïve Th cell  Stimulates Ig class switch from IgG1 to IgE (allergy)

40. Interleukin 5 (IL-5)  Produced by Th2 cells  Originally identified as a B cell differentiation factor  Aids in the growth and differentiation of eosinophils and late-developing B cells to plasma cells

41. Transforming growth factor  (TGF  )  Produced by T cells, macrophages, other cell types  30 members  Have effect on many cell types  Have pro- and anti-inflammatory effect  Inhibits proliferation of T cells and activation of B cells  Acts on PMNs and endothelial cells to block the effects of pro-inflammatory cytokines

42. Cytokine and Immunoregulation Mediators of Innate Immunity Mediators of adaptive immunity Stimulators of hematopoiesis (Colony Stimulating Factors)  GM-CSF: promotes differentiation of bone marrow progenitors  M-CSF: promotes growth and differentiation of monocytes and macrophages  G-CSF: promotes production of PMNs

43. emdbiosciences.com

44. Regulation of Immune Responses regulatory mechanisms: recognition phase

45. Regulation of Immune Responses regulatory mechanisms: antibody antibody competes with B cells for antigen antigen/antibody complexes binding to Fc receptors sends an inhibitory signal to B cells

46. Regulation of Immune Responses regulatory mechanisms: Tregs  Regulatory T cells (Tregs) do not inhibit initial T cell activation and proliferation  They are not Th1 or Th2 cells  They can suppress both Th1 and Th2 responses  They inhibit a sustained response and prevent chronic and potentially damaging responses

47. Regulation of Immune Responses regulatory mechanisms: Tregs Naturally occurring Tregs CD4+CD25+Foxp3+ cells derived from thymus IL-2, cell contact dependent inhibition Foxp3 is required for Treg development Foxp3 CD25 (part of IL-2R) CD4

48. Regulation of Immune Responses regulatory mechanisms: Tregs Induced Tregs CD4+CD25+Foxp3+ cells induced by antigen CD25 (part of IL-2R)CD4 T cells antige+IL-10 TGF  Foxp3 Treg cells

49. Cytokines and Oral Diseases bacterial colonization periodontal disease (PD) bacterial invasion osteoclast formation/bone loss host responses chemokines: bone resorption, osteoclast survival IL-1: up-regulated, stimulates bone loss IL-6: proinflammatory to bone resorption TNF  : induces cytokine production, stimulate inflammation and bone loss RNAKL: inducer of osteoclast formation and activity, bone loss

50. Colony stimulating factors (CSFs) hematological disorders associated with cancer therapy Erythropoietin (EPO) anemia associated with kidney disease IFN-  multiple sclerosis IFN-  chronic granulomatous disease (GCD) IL-2 kidney cancer, melanoma IL-11 thrombocytopenia following high dose chemotherapy TNF  mAb Infliximab for rheumatoid arthritis and Crohn’s disease Cytokines and Clinical Applications

51. Cytokines properties, categories, signaling, function What are cytokine? Interleukines, chemokines, monokines, … cytokine receptors NFkB, JAK/STATA, chemokine signaling pathways role of cytokines in immunomodulation cytokines and oral diseases (PD)