1. Institute of Immunology Zhejiang University
Cytokines
Xiaojian Wang
Institute of Immunology Zhejiang University

2. Introduction to cytokines
Cytokines are small soluble proteins that mediate immune and inflammatory reactions and are responsible for communications between leukocytes or between leukocytes and other cells via binding the receptor.
是除免疫球蛋白和补体的另一类特异性免疫效应物质

3. The cytokine needs to be present there
Cytokines work by binding to a target cell. Therefore, for a cytokine to have an effect, two things have to occur:
The cytokine needs to be present there
The target cell needs to have a receptor on its surface
Therefore, regulation can occur at either step.
The research of CK can help us to clarify the mechanism of immune responses at the molecular level and to understand the pathogenesis of some diseases.
Many CKs and their antagonists can be used in the treatment of many diseases (such as cancer, infection, autoimmune diseases) and to promote hemopoiesis, etc

4. Nomenclature According to their source 1) Lymphokine
2) Monokine
According to their function
1) Colony stimulating factor
2) Interleukin
3) Interferon
4) Tumor necrosis factor
5) Growth factor
6) Chemokine
Cytokines nomenclature has little to do with structural relationships among molecules. Cytokines produced by lymphocytes are also called lymphokines. Whereas those produced by monocytes or macrophages are called monokines.

5. Manner of action 1) Autocrine: IL-2 Th Th
2) Paracrine : IL DC Th
3) Endocrine: IL-1, TNF-
Under certain circumstances (e.g. septic shock), large amounts of CKs (such as TNF-) are produced, they may be active distant from their site of secretion.
Most of the cytokines, act only locally, over short dsitances, in either an autocrine manner ( on the producting cell itself)
Or a paracrine manner (on adjacent cells)

6. Manner of action Act on the producing cell itself
Act on the adjacent cells

7. Cytokines 1. General Properties of Cytokines
2. Functional Categories of cytokines
3. Cytokine receptors
4. Biologic function of Cytokines
5. Cytokine related diseases
6. Therapeutic Use of cytokines

8. 1. General Properties
1.1. The actions of cytokines are often pleiotropic and redundant;
1.2. Cytokines often influence the synthesis and actions of other cytokines ;
1.3. Cytokine actions may be local and systemic ;
1.4. Cytokines initiate their actions by binding to specific membrane receptors on target cells;
1.5. The cellular responses to most cytokines consist of changes in gene expression in target cells, resulting in the expression of new functional protein in the target cells.
Cytokines all share some similar properties. They were summarized here. I will not read you through this but show you one by one.

9. 1.1 The actions of cytokines are often pleiotropic and redundant
One cytokine can target different target cells

10. 1.1 The actions of cytokines are often pleiotropic and redundant
Different cytokines can exert similar effect

11. Cytokine Network The cytokine network:
Many cytokines have multiple functions and apparent redundancy of actions, these two properties forming a complex communication network.
Cytokine
Network

12. 1.2 Cytokines often influence the actions of and synthesis of other cytokines;
Cytokines can work together or against each other

13. One cytokine can induce another cytokine and so on…….
1.2 Cytokines often influence the actions and the synthesis of other cytokines (Cascade effect)
One cytokine can induce another cytokine and so on…….
Boost the immune response

14. 1.3 Cytokine actions may be local and/or systemic
Most other cytokines , act only locally over short sidtances, in either a paracrine manner (on adjacent cells) or an autocrine manner ( on the producting cell itself)
Under certain circumstances (e.g. septic shock), large amounts of CKs (such as TNF-) are produced, they may be active distant from their site of secretion.

15. 1.4 Cytokines initiate their actions by binding to specific membrane receptors on target cells.
1.5 The cellular responses to most cytokines consist of changes in gene expression in target cells, resulting in the expression of new functional protein in the target cells

16. General Properties
Pleiotropism
Redundancy
Synergy
Antagonism
Network

17. 2. Functional Categories of cytokines
CKs are classified into 6 functional categories
2.1 Interleukines
2.2 Interferons
2.3 Colony Stimulating Factors
2.4 TNF family
2.5 Chemokines
2.6 Transforming Growth Factors

18. 2.1 Interleukins (IL)
1.Cytokines produced by leukocytes and act on leukocytes
The term interleukine, (inter-) as a means of communication,
(-leukin) deriving from the fact that many of these proteins are produced by leukocytes and act on leukocytes.
2. The interleukins are produced by a wide variety of body cells. The function of the immune system depends in a large part on interleukins,
The majority of interleukins are synthesized by helper CD4+ T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T, B, and hematopoietic cells. And rare deficiencies of a number of them have been described, all featuring autoimmune diseases or immune deficiency.
The majority of interleukins are synthesized by helper CD4+ T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T, B, and hematopoietic cells. And rare deficiencies of a number of them have been described, all featuring autoimmune diseases or immune deficiency.

19. Interleukin, IL IL 1～38 IL-2（T cell growth factor）
IL-4（ B cell growth factor ,Th2 type）
IL-6（ Th2 type ）
IL-8（belongs to chemokine）
IL-10（Th2 type）
IL-11（stimulator of platelet）
IL-12（Th2 type）

20. IL-2

22. IL-4

24. 2.2 Interferons (IFNs)
Interferons (IFNs) are natural cell-signaling proteins produced by the cells of the immune system to challenges such as viruses, parasites and tumor cells.
Groups
Type I IFN: IFN- and IFN-
The major source is leukocytes (PDC), fibroblasts and virus infected cells.
Help prevent the spread of virus to uninfected cells.
Type II IFN: IFN-
IFN- is produced by activated T cells and NK cells. Its main function is immune regulation.
Interferons assist the immune response by inhibiting viral replication within host cells, activating immune cells (natural killer cell and macrophages), increasing antigen presentation to T Lymphocytes, and increasing the resistance of host cells to viral infection.
There are 3 known classes of interferons; type I, type II and type III. All classes are very important in fighting viral infections. Their presence also accounts for some of the host symptoms to infections, such as sore muscles and fever.

25. IFN-a 的 抗 病 毒 作 用 病毒
抑制病 毒复制
病毒复制
IFN-诱导蛋白
诱导刺激
信号转导 胞核 胞核
IFN-a

28. 2.3 Colony-stimulating factor
Colony-stimulating factors (CSFs) are secreted glycoproteins which bind to receptor proteins on the surfaces of hemopoietic stem cells and thereby activate intracellular signaling pathways which can cause the cells to proliferate and differentiate into a specific kind of blood cell (usually white blood cells）。

29. 2.3 Colony-stimulating factor
The name “colony-stimulating factors” comes from the method by which they were discovered. Hemopoietic stem cells were cultured in semi solid matrix which prevents cells from moving around, so that if a single cell starts proliferating, all of the cells derived from it will remain clustered around the spot in the matrix where the first cell was originally located, and these are referred to as "colonies." It was therefore possible to add various substances to cultures of hemopoietic stem cells and then examine which kinds of colonies (if any) were "stimulated" by them.
The substance which was found to stimulate formation of colonies of macrophages, for instance, was called macrophage colony-stimulating factor, for granulocytes, granulocyte colony-stimulating factor, and so on.

30. 2.4 Tumor necrosis factors
TNF was originally identified (and was so named) as a substance that can cause the necrosis of tumors in vivo.
TNF- and TNF-
TNF- is produced by LPS-stimulated mononuclear phagocytes and activated T cells. Involved in inflammation.
TNF- is also termed lymphotoxin (LT), and is produced mainly by activated T cells.

33. 2.5 Chemokines
Chemokines are a family of small cytokines, or proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines.
Chemokines share structural characteristics such as small size (all of them are all about 8-10 kilodaltons in size), and all of them have four cysteine residues in conserved locations that are key to forming their 3-dimensional shape.

34. 2.5 Chemokines About 50 different members. 2 lymphotactin 17 28 1
Subfamilies: CXC (), CC (), C (), CX3C (),
based on their amino acid sequence . Most chemkines contain two or three pairs of cysteine(C)
One unusual human chemokine , called lymphotactin , has only a single cysteine pair, C subfamily.
if there is no intervening amino acid between these two cysteine, called CC, If the cysteine are separated by a single amino acid , these are termed the CXC,
(cysteine residues).The lagrest chemokine, called fractalkine, has three residues interposed between C1 and C2. 1

35. 2.6 Transforming growth factor (TGF)
Transforming growth factor (sometimes referred to as Tumor growth factor, or TGF) is used to describe two classes of polypeptide growth factors, TGFα and TGFβ.
TGFα is upregulated in some human cancers. It is produced in macrophages, brain cells, and induces epithelial development.
TGFβ exists in three known subtypes in humans, TGFβ1, TGFβ2, and TGFβ3. These are upregulated in Marfan's syndrome and some human cancers, and play crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system.

36. Pleiotropic effects of TGF-β on leukocytes
Pleiotropic effects of TGF-β on leukocytes. All leukocytes produce and respond to TGF-β. The yin-yang symbol illustrates the fact that TGF-β exerts both stimulatory and inhibitory effects on immune cells

37. 3. Cytokine Receptors
3.1 Five families

38. Each contain an immunoglobulin-like domain in their extracellular regions
Immunoglobin domain Receptors in this family has the immunoglobin domain named after the immunoglobulin molecules

39. This receptor family have conserved amino acid sequence motifs in the extracellular domain consisting of four positionally conserved cysteine residues (CCCC) and a conserved sequence of tryptophan-serine-( any amino acid)- tryptophan-serine (WSXWS,where X is the nonconserved amino acid). The receptors for all the cytokines classified as hematopoietins belong to the class I cytokine receptor family, which also is called the hematopoietin receptor family.

40. The class II cytokine receptors possess the conserved CCCC motifs, but lack the WSXWS motif which is present in class I cytokine receptors. Initially only the three interferons, were thought to be ligands for these receptors. However, recent work has shown that receptors for the IL-10 ,IL19 are also members of this group.

41. Tumor necrosis factor receptors (TNFRs) are cytokine receptors that binds TNFs (TNF).
They were also called death receptors since they can transduce signals lead to cell death,

42. 19 functionalchemokine receptor have so far been identified.
Chemokine receptors are receptors of chemokines. There have been 19 distinct chemokine receptors described in mammals. They each have a 7 transmembrane (7TM) structure and couple to G-Protein for signal transduction within a cell, making this kind of receptor belong to a large protein family of G protein-coupled receptors. Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux in intracellular calcium (Ca2+) ions (calcium signaling). This causes cell responses, including the onset of a process known as chemotaxis that traffic the cell to a desired location within the organism.

43. Immunoglobulin gene superfamily

44. 3.2 Multi-subunit receptor and common receptor subunit
one for cytokine binding
another for cytokine signaling
Common receptor subunit
There is same receptor subunit for cytokine signaling among the different cytokine receptors. e.g. IL-2R

45. There is same receptor subunit for cytokine signalling among the different cytokine receptors. e.g. IL-2R
The same receptor subunit for cytokine signaling among the different cytokine receptors. e.g. IL-2R

46. Another feature common to most of the hematopoietin (class I cytokine) is multiple subunits, often including one subunit that binds specific cytokine molecules and another that mediates signal transduction.

50. 3.3 Soluble cytokine receptors
Is the extracellular part of the receptor
Functions
1) antagonist of CKs: competitively bind to CK
2) delivery tools of CKs
3) index of diagnosis and prognosis of diseases: sIL-2R

52. 4. Cytokines have numerous functions.
Mediators and regulators of Innate Immunity
Mediators and regulators of adaptive immunity
Stimulators of hematopoiesis
Now , we have learned about the definition of CK. General Properties of Cytokines, Functional Categories of cytokines, Cytokine receptors

53. The innate immunity comprises the cells and mechanisms that defend the host from infection, in a non-specific manner. Innate immune systems provide immediate defense against infection, but does not confer long-lasting or protective immunity to the host.
The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic challenges. It can recognize and remember specific pathogens and confer long-lasting or protective immunity to the host. It is adaptive immunity because the body's immune system prepares itself for future challenges.
Innate immune response refers to any inborn resistance to an invaded pathogen,
24 hours

54. 4.1 Main Cytokines in innate immunity
抗细菌作用： TNF, IL-1,IL-6, IL-8
抗病毒作用： IFNa/b IFN-Y, IL-12, MCP
TNF
Interleukin-1
Interleukin-12
Type I interferons
Interleukin-10
Interferon-γ (IFN-γ):

55. TNF
Mediator of acute inflammatory response to gram- negative bacteria and other infectious microbes.
Mainly produced by mononuclear phagocytes (Macrophage stimulated with LPS);
Can be membrane protein or
released (trimer)

56. Biological Actions of TNF
In the low quantities, TNF recruit neutrophils and monocytes to sites of infection and activate these cells to eradicate microbes;
Large amount of TNF can cause systemic clinical and pathological abnormalities such as Septic shock (endotoxin shock): vascular collapse.

57. Interleukin-1 (IL-1):
Mediator of inflammatory response to infectious microbes.
Mainly produced by activated mononuclear phagocytes;
Two forms IL-1α and IL-1β
(30% homologous), IL-1 β
need to be cleaved to be
active
Similar effect as TNF

58. Interleukin-12
Produced by activated mononuclear phagocytes and dendritic cells
Stimulate IFN production by NK and T lymphoctyes.
Enhance cytolytic functions of activated NK and CTL cells.
Is a link between innate and adaptive immunity.

59. Type I interferons: IFN-α (20) and IFN-β
Stimulated by viral infection, mediate early innate immune response to viral infections- establishment of “antiviral state”
Biological Actions:
Inhibits viral replication
Stimulate the development of Th1 cells

60. Interleukin-10:
IL10: produced by activated macrophages, is an inhibitor of activated macrophages and dendritic cells and inhibits the production of IL12;
IL10 is thus involved in the control of innate immune reactions and cell- mediated immunity.
IL-10 inhibitroy factor .

61. Interferon-γ (IFN-γ):
IFN-γ is the principal macrophage-activating cytokine, T lymphocytes and NK cells produced the IFN-γ and activated macrophages to kill phagocytosed microbes.

62. 4.2 Cytokines mediate and regulate adaptive immunity
Interleukin-2
Interleukin-4
Interferon-γ
Transforming Growth Factor-β

64. Interleukin-2:
IL-2 is a growth factor for antigen-stimulated T-lymphocytes and is responsible for T cell clonal expansion after antigen recognition.
Promote proliferation of antigen-specific T cells;
Promote the proliferation and differentiation of other immune cells;

65. Interleukin-4 (IL4):
IL4 is the major stimulus for the production of IgE antibodies and for the development of Th2 cells from naïve CD4+ helper T cells.
Biological Actions:
Stimulate B cell Ig heavy chain class switching to IgE isotype
Stimulate Th2 cells
Inhibit Th1 differentiation and cell-mediate immune response

66. Interferon-γ (IFN-γ):
The role of IFN-γ in adaptive immunity is to promote the differentiation of Th1 helper cell and cell-mediated immune reaction.
IFN- γ can also inhibit the development of Th2 helper cells
IFN-γ stimulates the expression of MHC I and MHC II molecules and costimulators on APCs.

67. Transforming Growth Factor-β (TGF-β):
Produced by Ts, macrophages, and other cell types
It inhibits the proliferation of T cells and the activation of macrophages.
Acts on PMNs and endothelial cells to block pro-inflammatory cytokine effects
Inhibitory factor: IL-10 ,TGF-β

68. 4.3 Cytokines in
hematopoiesis

69. 5.Cytokine and disease

70. Cytokine-related diseases
1. 细胞因子风暴： Bacterial septic shock
endotoxins (cell walls) stimulate production
of IL-1 and TNF-
2. 致热与炎症病理损害： IL-1, IL-6
3. 肿瘤的发生与逃逸
Cancers of lymphoid system- overproduction
of cytokines such as IL-6 (B-cell) or IL-5
(Hodgkin’s disease) or IL-2 (adult T-cell
leukemia). IL-10, TGF-B 抑制机体的免疫功能。
4. 免疫系统相关疾病
1.）超敏反应 IL-4。 2.)自身免疫病：类风湿关节炎，TNF-a
3.） 免疫缺陷病 Chagas ’ disease (parasitic)- suppression of
-subunit of IL-2 receptor 4.）器官移植排斥反应：
IL-1, IL-2,TNF-a

71. 6. Cytokine therapies Interferons IFN-- certain types of tumors
IFN-- multiple sclerosis
these are antiviral
IFN-- chronic granulomatous disease
IL-2- certain types of cancer
infusion
LAK (lymphokine-activated killer) cells
TILs (tumor-infiltrating lymphocytes)
GM-CSF- immune deficiencies
Alternately, one might wish to enhance immunity (immunodeficiency or cancer)
Administration of various cytokines or combinations of cytokines (for example, IL-2, IFN-g)
Or, one can take NK or TC cells out of the patient, activate the cells with cytokines such as IL-2, then transfer them back into the patient (LAK, lymphokine-activated cells)

72. TNF (tumor necrosis factors)
TNF- and 
In some cases inhibit proliferation of tumor
cells but not normal cells
May damage vascular endothelial cells in
tumors, thus inhibiting blood supply
to the tumor
Anti-TNF-a antibody

74. Considerations when Considering Therapeutic Applications
Normally, cytokines are transferred from one cell to another over a very short distance.
Probably yields very high doses in that local environment that are difficult to mimic clinically
Interactions with other cytokines are undoubtedly important.
Cytokines have a short half-life and are degraded quickly. Requires continual administration.
Cytokines have many different effects (pleiotropy); therefore, often see unexpected and undesirable side-effects.

75. Sample question
Which of the following cytokines plays an inhibitory effect on macrophages and dendritic cells terminating inflammatory response?
IL-2
IL-4
IFN-
IL-10
Answer=D

76. Sample question
Which of the following cytokines promote the differentiation of Th1 helper and inhibit the development of Th2 helper cells ?
IFN-γ
IL-4
IFN-
IL-2
Answer=A
Characteristic of cytokine to perform their biological functions?

77. Thanks for your attention!