1. A visual review….

3. physiologic responses that require cytokines development of cellular and humoral immune responses, induction of the inflammatory response, regulation of hematopoiesis, control of cellular proliferation and differentiation, the healing of wounds. act in an antigen-nonspecific manner.

4. Cytokines: Mediators “low” molecular weight proteins Less than 30 kDa Can be classified by recurrent architecture

5. Functional features Potent –Some function at 10 -15 Molar Local –autocrine –paracrine –(sometimes) endocrine Highly interactive –pleiotropic –redundant –synergistic / antagonistic

6. Functional features Potent –Some function at 10 -15 Molar

7. Functional features Local –autocrine –paracrine –(sometimes) endocrine

8. Cytokine Actions Pleiotropy –Act on more than one cell type (INF  ) Redundancy –More than one cytokine can do the same thing (IFN  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. Functional features Highly interactive pleiotropic redundant synergistic antagonistic

10. 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 –Only cells in close proximity will be activated –May require cell-to cell contact

11. Cytokine families I.Hematopoietic family II.Interferon family III.Tumor necrosis factor family IV.Chemokine family I, II, and III elicit physiological responses. IV serves as a chemoattractant.

12. Hematopoietic family Defined by architecture…

13. Interferon family…. http://www.hon.ch/Library/Theme/Allergy/Glossary/ ifn.html http://www.ncbi.nlm.nih.gov/mapview/map_search.c gi?chr=hum_chr.inf&query=Interferon&qchr=&strai n=&neighb=off&advsrch=off http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id =147570

14. Tumor necrosis factor… Time to be critical… Question: What does the text say? Answer: Not a lot. –More specifically, there is no text. –There is an entry in Table 12.1 (which stretches over four pages!) What does it say? –“Has toxic effects. Induces cytokine secretion…”

15. So, something’s amiss… Try another approach. Cytokines are soluble immunomodulators. That means that they are ligands. Question: Can they be characterized in terms of their receptors? Answer: Yes.

16. Cytokine Receptors Expression of cytokine receptors controls the ability of a cytokine to act on a cell. Cell activation increases cytokine receptor expression.

17. Cytokine Receptor Families 5 different families of receptors based on common structural motifs. --> see book for more details

18. IL-2 Receptor Subfamily Shared common  subunit Only IL-2 and IL-15 have unique alpha subunit

19. GM-CSF Receptor Subfamily Hematopoietin Receptors IL-3, IL-5 GM-CSF activate common  subunit. Cytokine Receptor Signal through JAKs and STATs

20. The receptor families:

21. Of receptors and signal transduction… Receptors for cytokines have surface component, single transmembrane component, and cytoplasmic tails. Commonly multimeric. The protomers can associate in different groupings; the various associations have different affinities for cytokines; hence, different intensities of signaling. The different protomers also have different signaling functions, e. g. phosphorylation sites.

22. 01/23/0822 Signal Transductions often involves phosphorylation: Kinase- enzyme that places phosphate groups on proteins. –Tyrosine kinase-* –Serine-threonine kinase- –Histidine kinase Either receptor is a kinase OR associates with one. Phosphatase- enzyme that removes phosphates –Regulatory- OFF

23. 01/23/0823

24. 01/23/0824 Tyrosine kinase associated receptors

25. 01/23/0825 Cytokine signal transduction depends upon a cytoplasmic protein tyrosine kinase: Janus Kinase Family (JAKs): –“2 heads”- 2 symmetrical kinase domains. –Cytoplasmic tyrosine kinases Associate with cytoplasmic tails of the receptors. Function-

26. 01/23/0826 Who are the activated Transcription Factors? STATs- Signal Transducer & Activator of Txn: –Contain SH2 domains: Binds phosphorylated tyr. Permits dimer formation. Activated dimers function as txn activators (DNA binding domain).

27. 01/23/0827

28. 01/23/0828 SH2 domain SH2 domains creates a hole so that the tyr-P from other proteins can plug in!

29. 01/23/0829 Fig 6-25, p. 165, Parham.

30. 01/23/0830

31. 01/23/0831

32. 01/23/0832

33. 01/23/0833

34. 01/23/0834 Model applies to other cytokine receptors: (IL2) http://binfo.ym.edu.tw/mb/imag es/stat_dimer.gif

35. 01/23/0835 How do cytokines mediate specific results in different cell types? 1. Use of individual receptors or different subfamily of receptors. 2. There are different STAT proteins & different JAKs. –Recognize different genes! 3. Same STATs may recognize different genes in different cell types.

36. What are the principal signaling systems? JAK’s & STAT’s

37. OK, let’s look at a complex image

38. And, an important example…

39. And, an update…

40. VI. CD4 + T helper Subsets Th1/Th2 Cytokine Bias CD4+ T helper cells can be divided into subsets based on their cytokine production. T h 1 cells produce IL-2, IFN- , TNF-  CKs which activate cell mediated immunity T h 2 cells activate IL-4, IL-6, IL-10 CKs that activate humoral immunity These Th subsets were originally identified using mouse T cell clones.

41. Mouse Th Subset Cytokine Th1 Th2 Table 12-4 from Goldsby

42. Th0 ---> Th1 or Th2 Original mouse experiments on Th cells (Mosmann et al (DNAX) 1986 J Immunol) Antigen specific T cells placed in culture with antigen and APCs to make T cell lines. Spleen cells (T h 0) add IL-12  T h 1 cells  IL-2, IFN- , TNF-  Spleen cells (T h 0) add IL-4  T h 2 cells  IL-4, IL-6,IL-10 (T h 0 --precursor cell that produces IL-2, IL-4, and IFN- .)

43. Th1/Th2 Naïve T h 0 IL-2, IL-4, IFN-  IL-4 IL-12 Effector T h 1 cell IL-2, IFN-  Effector T h 2 cell IL-4, IL-6, IL-10

44. Th1/Th2 Antagonism IL-4 blocks Th1 IFN-  blocks Th2 IL-4 IL-12 T h 1 cell IL-2, IFN-  T h 2 cell IL-4, IL-6, IL-10 IFN-  IL-4

45. Th1/Th2 Regulation T-bet is a transcription factor that is required for Th1 specific genes such as IL-12R  IL-12 T h 1 cell IL-2, IFN-  T h 2 cell IL-4, IL-6, IL-10 IFN-  Enhances T-bet IL-4 blocks T-bet

46. IL-4 vs IFN-  T-bet (Th1 associated) activated by IFN-  and turned off by IL-4. Conversely in Th2 transcription factor GATA-3 activated by IL- 4 turned off by IFN- .

47. Role for Th1 vs Th2 in Immune Response Both subsets activated in lymph nodes (LN) immune responses to complex antigens. Th1 cells leave LN to find activated endothelium tissue to activate macrophages. Th2 cells can stay in LN to activate B cells.

48. What controls Th1 vs Th2? 1) Amount of antigen. Mouse experiments originally showed high dose for Th1. 2) MHC and TCR affinity. High affinity TCR = Th1. 3) Dendritic cell subsets during activation. APC subsets activate Th1 or Th2 preferentially. 4) Toll-like receptor activation.

49. Influence of APC Subsets on Th1/ Th2 Dendritic cell Myeloid-like dendritic cells produce abundant IL-12 and drive Th1. Lymphoid-like dendritic cells produce low levels of IL-12 are permissive for Th2.

50. Toll-like receptors (TLRs) Influence of APCs on Th1/ Th2 Evidence for TLR activation influencing Dendritic cell maturation. –TLR9 binds bacterial CpG DNA –TLR4 binding to bacterial heat shock proteins TLR activation induces APC expression of IL-12, IL-23, IL-27 Th1

51. TLR vs IL-12 in Th1/ Th2 development New evidence suggests that TLR activation influencing Th1 outcome through initiation of TLR adapter molecule MyD88. May be more important than IL-12 for Th1.

52. Th Cytokine Bias in Disease Examples Leishmania in mice (Richard Locksley) C57Bl.6 mice mice have Th1 immune response and resolve infection. BALB/c mice produce Th2 cytokines unable to control Leishmania lesions. Leprosy in Humans (Robert Modlin) Tuberculoid form has Th1 response and limits disease (healing). Lepromatous form has Th2 response and uncontrolled disease (leprosy).

53. Th Cytokine Bias in Disease: Leprosy Skin disease caused by Mycobacterium leprae Lepromatous: has Th2 response and uncontrolled disease (leprosy). Tuberculoid: has Th1 response and limits disease (healing).

54. Cytokine Bias in Leprosy RNA from skin lesions of patients

55. X-Linked SCID Common  chain Deficiency Mutation in  chain so unable to signal through IL-2, IL-4, IL-7, IL-9, IL-15. No T cells abnormal thymus. Immunocompromised susceptible to infections. SCID Patient with severe Candida in mouth.

56. Cytokine Receptor Signaling JAKs and STATs (Model of Signal) Binding of cytokine ligand brings together receptor subunits JAKs (Janus Associated Kinases) are tyrosine kinases that phosphorylate tyrosines. STAT (Signal Transducers and Activators of Transcription ) dimerize for function.

57. JAKs and STATs Usage by CK Receptors Overlapping and Unique JAK1 is commonly used by CKs from completely different CKR families STAT6 is ONLY used IL-4.

58. The Yin and Yang of Th1 and Th2 Immune Responses

59. Measuring Cytokines Protein amount by ELISA. Good for in vitro experiments. Protein amount by bioactivity assay using CK dependent cell lines. RNA message by PCR.

60. Antigen Capture ELISA for IL-2