CYTOKINES AND IMMUNE RESPONSES

1. CYTOKINES AND IMMUNE RESPONSES Beverly E. Barton, Ph.D. Assistant Professor Department of Surgery/Division of Urology UMDNJ-NJMS MSB F672 185 S. Orange Avenue Newark, New Jersey 07103 Telephone 973-972-0662 E-mail bartonbe@umdnj.edu Telefacsimile 973-972-3892

2. CYTOKINES Cytokines are a group of low molecular weight regulatory proteins secreted by leukocytes as well as a variety of other cells in the body in response to stimuli. Membrane-bound forms of some of the secreted cytokines are known as well. Cytokines regulate the intensity and duration of the immune response by stimulating or inhibiting the activation, proliferation, and/or differentiation of various cells, and by regulating the secretion of antibodies or other cytokines or mediators. Almost ALL cells secrete at least a few cytokines.

3. MODES OF CYTOKINE TRAVEL INTRACRINE JUXTACRINE PARACRINE AUTOCRINE ENDOCRINE BLOOD VESSEL from Bafico & Aaronson, Cancer Med, 2002

4. Autocrine e.g. IL-2 on T cells - clonal expansion Paracrine e.g. T cell cytokines act on B cells - isotype switching Endocrine e.g., IL-1 & fever Blood or lymph vessel 3 MAJOR ROUTES OF CYTOKINE TRAVEL STIMULUS

5. 10 Rx: NONE OVA TOXOID 20 Rx: NONE OVA TOXOID MIF is here A (VERY) SHORT HISTORY OF CYTOKINES 1957-- Isaacs & Lindemann described IF- in virally-infected chick chorioallantoic membranes 1965 -- Wheelock described IF- in PHA-treated leukocytes 1966 -- MIF described by David & Bloom in sups of Ag-stimulated lymphocytes Photomicrograph from original paper depicting Ag-specific MIF production by guinea pig peritoneal cells.

6. IL-6R Lacks Tyr kinase domain JAK (a kinase) docks instead IGF-1R Has Tyr kinase domain Cytokines differ from growth factors in the structure of the receptor.

7. MOST CYTOKINE RECEPTORS ARE IN 2 CLASSES CLASS I CYTOKINE RECEPTORS Most of the cytokine-binding receptors that function in the immune and hematopoietic systems belong to this receptor family. There are conserved amino acid sequence motifs in the extracellular domain - 4 positionally conserved Cys residues (CCCC) and a conserved sequence of Trp-Ser-X-Trp-Ser (WSXWS) where X is a nonconserved amino acid.  The receptors consist of 2 polypeptide chains: a cytokine-specific subunit and a signal-transducing subunit, which is usually not specific for the cytokine.  In a few cases these receptors are trimers.  The signal transducing subunit is required for high affinity binding of the cytokine. CLASS II CYTOKINE RECEPTORS These receptors possess the conserved cysteine motifs, but lack the WSXWS motif present in class I cytokine receptors. The IFs bind to Class II receptors.

8. CYTOKINES BY FUNCTION CYTOKINES THAT MEDIATE AND REGULATE INNATE IMMUNITY Type I interferons— Dr. Pestka’s lecture Tumor necrosis factor-a Interleukins 1, 6, 10, 12, 15, 18, and more Chemokines—a special subclass; won’t deal with here CYTOKINES THAT MEDIATE AND REGULATE SPECIFIC IMMUNITY Interleukins 2, 4, 5, 13, 16, 17, and more Interferon-g (Type II interferon; Dr. Pestka’s lecture) Lymphotoxin-a (Tumor necrosis factor-b) CYTOKINES THAT STIMULATE HEMATOPOIESIS c-kit ligand Interleukins 3, 7, 9, 11, more Colony stimulating factors— M-CSF, G-CSF, GM-CSF, EPO, TPO Overlapping functions are common.

9. IL-1 FAMILY Potent inflammatory effects, especially IL-1 & IL-1. IL-18 induces IF-, bridging Inflammatory & innate/adaptive immune responses. IL-1 induces TNF, IL-6 as well as Inflammatory mediators from various cells, notably cells at the interface of the environment. IL-1 self-regulates through induction of IL-1Ra & IL-10. IL-1 is an endogenous pyrogen (fever inducer). IL-33 induces TH2 cytokine synthesis, polarizing to TH2 (Ab) responses. IL-1 Family Receptors Two receptors (RI and RII), both members of Ig gene superfamily. IL-1RI found on T and B cells, fibroblasts, keratinocytes, endothelial cells, chondrocytes, hepatocytes, and synovial lining cells; has a MW of 80 kDa. IL-1RII found on B cells, neutrophils, & bone-marrow cells; has a MW of 60 kDa. Difference in MW arises from IL-1RII having shorter cytosolic region. Some evidence that 2 receptors mediate different biological events even though they both bind IL-1 & IL-18. A 3rd non-signaling receptor is found as well, possibly with regulatory function. IL-33 signals only through RII. RI

10. IL-18 Signal (bacterial, etc.) to monocytes IL-18 IF- MHC Class I & II, IL-18 Enhanced Ag pres. to T cells T cell effector functions: TH1 TH2----B cells make Ab TH17 CTL IL-1 Signal (bacterial, etc.) to monocytes, epithelium, etc. IL-1 IL-6 TNF- Req. for Ig class Dendritic cell maturation switching Very enhanced Ag pres. to T cells Higher avidity Ab response T cell effector functions: TH1 TH2----B cells make Ab  TH17 CTL (Monocytes can be macrophages in blood or tissue.)

11. a virus (not to scale) IL-1 THIS WAY TO THE BRAIN! Blood vessel ENDOCRINE IL-1 PRODUCES FEVER

12. IL-2-LIKE CYTOKINES The common  subunit associates with each specific cytokine receptor to form the high affinity cytokine receptor. The  subunit has a very short cytoplasmic tail; it probably does not signal. IL-2 secreted primarily by T helper lymphocytes activated by stimulation with T cell mitogens, or by interaction of T cell receptor complex with Ag/MHC complexes on surfaces of antigen-presenting cells. The response of TH cells to activation is induction of IL-2 and high affinity receptors for IL-2 and, subsequently, clonal expansion of antigen-specific T cells. Here IL-2 is an autocrine factor, driving the expansion of the Ag-specific T cells. IL-2 also acts as a paracrine factor, influencing the activity of other cells, both within & outside of the immune system. B cells and natural killer (NK) cells respond, when properly activated, to IL-2. The so-called lymphokine activated killer,or LAK cells, appear to be derived from NK cells under the influence of IL-2.

13. AUTOCRINE IL-2 DRIVES CLONAL EXPANSION OF T CELLS IL-2 Ag-specific stimulus T cell IL-4 PARACRINE IL-4 DRIVES CLONAL EXPANSION OF B CELLS B cell IgG1 AUTOCRINE OR PARACRINE IL-4 OR IL-13 DRIVES Ig CLASS SWITCHING TO IgE IgE

14. IL-4 • Stimulates production of Ab-producing B cells, leading to the production of Ig • & class-switching to IgE. It also promotes CD8+ cell growth and TH2 cell differentiation. • On macrophages, IL-4 induces MHC class II expression, but inhibits production of pro- • inflammatory cytokines (IL-1, TNF). • Induction of IL-4 secretion is Ag-specific and MHC restricted. Antigenic stimulation also results • in increased responsiveness of TH cells to IL-4. Ag-induced proliferation of TH cells is inhibited by anti-IL-4. • Thus, IL-4 mediates the proliferation of TH cells by an Ag-induced autocrine mechanism. • During T cell--B cell interactions involving soluble protein antigens, IL-4 and not IL-2 • is the critical cytokine for activating resting B cells and inducing proliferation of the TH cells. • IL-9 • Up-regulates TH1 responses by inhibiting T cell apoptosis. • IL-9 is preferentially produced by TH2 cells and is active on various cell types such as T and B cells, mast cells • and hemopoietic progenitors. • IL-13 • Has structural and functional similarities to IL-4 and promotes B cell differentiation. • Also promotes Ig class switching to IgE. • IL-15 • Shares several activities with IL-2 and is produced by epithelial cells and monocytes. • IL-15 induces T cell proliferation, enhances NK cell cytotoxicity and stimulates B cells to • proliferate and secrete Ig.

15. IL-7 • Is a T cell growth and activation factor, and a macrophage activating factor. • Interleukin-7 exerts pleiotropic effects on the immune system; it affects pre-B cells, • thymocytes, mature T cells, lymphokine activated killer cells (LAK), monocytes, and • macrophages.  • IL-7 stimulates the proliferation of pre-B cells harvested from bone marrow.  IL-7 activates these • cells in vitro.  • IL-7 restores V(D)J rearrangement to developing T cells outside of the thymic environment.  • Normal maintenance and proliferation of thymic progenitor cells requires the presence of IL-7.  • In combination with phorbol 12-myristate 13-acetate, IL-7 drives the activation of resting • CD4+ and CD8+ T-cells along a pathway that is independent of IL-2. • IL-7 maintains naïve CD4+ T-cells in vitro for up to 15 days, which suggests that it is a survival • factor for these cells. • Peripheral blood monocytes are activated by IL-7: lyse tumor cells, secrete IL-6, IL-1, IL-1, • and TNF-.  • Cyclosporine A (CsA), an immunosuppressive drug, inhibits the transcription of many cytokines • (for example,  IL-2 and IL-4), but does not affect IL-7.

16. IL-3 IL-5 GM-CSF IL-3, IL-5, GM-CSF IL-3 Produced by activated T cells. Stimulates the proliferation of all hematopoietic precursors. Stimulates differentiation of osteoclasts from bone marrow. IL-5 Is chiefly a growth and activation factor for eosinophils and B cells. Eosinophils in particular are noted for their contribution to late phase allergic-type disorders. Eosinophils make up less than 10% of the circulating leukocyte population, yet are extremely important in the inflammatory response to allergic and parasitic challenges. Eos are the cells of the Ab-dependent cell-mediated cytotoxicity responses (ADCC-- tumors, parasites). Cells known to express IL-5 include eosinophils, NK cells, CD8+ T cells, mast cells, CD4+ T cells,  T cells, and IL-1-activated endothelial cells. IL-5 is best known for its activity on B cells and eosinophils. IL-5 appears to induce the differentiation of activated conventional B-2 cells into Ig-secreting cells. In addition, it induces the growth of B-1 progenitors as well as IgM production by B-1 cells. In mice, IL-5 promotes production of IgA, IgE and IgG1. GM-CSF Is chiefly a differentiation factor for myeloid cells -- neutrophils, macrophages. G = granulocyte.

17. a gp130 a gp190 IL-6-LIKE CYTOKINES IL-6 IL-11 leptin IL-6 Stimulates many types of cells. Endocrine response by liver to it results in the acute phase response. Required for Ig class switching and for B cells to differentiate into plasma cells. Endogenous pyrogen. While a number of Interleukins such as IL-1 and IL-10 are seemingly pleiotrophic in their effects, IL-6 may be considered the prototypic pleiotrophic cytokine. This is reflected in the variety of names originally assigned to IL-6 based on function, including Interferon 2, IL-1-inducible 26 kD Protein, Hepatocyte Stimulating Factor, Cytotoxic T-cell Differentiation Factor, B cell Differentiation Factor (BCDF) and/or B cell Stimulatory Factor 2. Once all the activities associated with the names for IL-6 became connected with one common gene, the name IL-6 was proposed. A number of cytokines make up an IL-6 cytokine family. Membership in this family is based on a helical cytokine structure and receptor subunit makeup. Just about every cell type seems to express IL-6; expression of the others more limited. IL-6 production is correlated with 10 cell activation but others with 20. IL-6 has been described as both a pro-inflammatory and anti-inflammatory molecule, a modulator of bone resorption, a promoter of hematopoiesis, and an inducer of plasma cell development. IL-6 also has been shown to influence IL-4 production. It has been suggested that antigen-driven, APC-derived IL-6 may influence naive CD4+ T cells to produce IL-4 and express the IL-4 receptor. Thus, given the close approximation of APC and T cell, transient APC-derived IL-4 could initiate a T cell autocrine loop whereby naive T cells direct their own phenotype commitment. CNTF CT-1 LIF NNT-1 OSM IL-31

18. AUTOCRINE OR PARACRINE IL-4 OR IL-13 DRIVES Ig CLASS SWITCHING TO IgE IgG1 AUTOCRINE OR PARACRINE IL-6 DRIVES Ig CLASS SWITCHING IgG2A IgM IgG1 IgE IL-6 PRODUCED BY MACROPHAGES, T CELLS, AND B CELLS DURING RESPONSES TO Ag DRIVES B CELL EXPANSION AND Ig CLASS SWITCHING IL-6 IS REQUIRED FOR B CELL DIFFERENTIATION INTO PLASMA CELLS.

19. 2 types of receptors TNF FAMILY The TNF family includes cell surface proteins, such as CD40. TNF isthe principle cytokine thatmediates acute inflammation. In excessive amounts it also is the principal cause ofsystemic complicationssuch as the shock cascade. Major functions of TNF: acts on endothelial cells to stimulate inflammation and coagulation stimulates endothelial cells to produce selectins and ligands for leukocyte integrins during diapedesis stimulates endothelial cells and macrophages to produce chemokines stimulates macrophages to secrete IL-1 activates neutrophils induces acute phase response by liver cachectic factor (muscle proteolysis and fat catabolism) cytotoxic for some tumor cells interacts with hypothalamus to induce fever and sleep stimulates the synthesis of collagen in wound healing and tissue repair activates macrophages and dendritic cells is required by thymocytes for normal T cell differentiation TNF is produced by monocytes, macrophages, dendritic cells, TH1 cells, and others. TNF is secreted and also active in membrane-bound form.

20. TNF Receptors 2 types of TNF receptors: one has a ”Death Domain” & is responsible for cytotoxicity. They are type II membrane proteins. The other signals through NF-B & is responsible for the activation/differentiation properties. Ligand/receptor complexes trimerize to transduce either signal. Lymphotoxin- (LT) = TNF LT plays a role in the recruitment and activation of neutrophils and in lymphoid organogenesis. Produced by T cells, including Tc; plays a role in Tc function. Also produced by NK cells and B cells. Lymphotoxin- (LT) Is not secreted, although a specific receptor exists. Active as membrane-bound cytokine. Aside from cytotoxic properties, it is essential for the formation of germinal centers.

21. IL-10 FAMILY Recently a family of related cytokines has emerged, comprising a series of herpesviral and poxviral members and several cellular sequence homologs, including IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26. Although the predicted structure of these molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. IL-10 was discovered initially as an inhibitory factor for the production of TH1 cytokines. Subsequently, pleiotropic inhibitory and stimulatory effects on various types of blood cells were described for IL-10, including its role as a survival and differentiation factor for B cells. IL-10, which is produced by activated monocytes and T cells, as well as other cell types, such as keratinocytes, appears to be a crucial factor for at least some forms of peripheral tolerance and a major suppressor of the immune response and inflammation. The inhibitory function of IL-10 is mediated by the induction of regulatory T cells (CD4+ CD25+). IL-10 is also a survival and differentiation factor for B cells.

22. IL-22 mediates acute-phase response signals in hepatocytes and IL-20 induces the hyper- proliferation of keratinocytes; proposed as a pathogenic mechanism of psoriasis. Many immunosuppressive and immunostimulatory functions described for IL-10; biological activities of the others are distinct from those of IL-10, at least as far as is known for IL-20, IL-22/IL-TIF and IL-24/MDA-7. Moreover, the site of expression is distinct for each. IL-10 is produced by TH2 cells, macrophages, keratinocytes. The expression patterns are more restricted for the others. IL-26/AK155 - monocytes and various types of T cells IL-22/IL-TIF - CD4+ T cells IL-24 - melanocytes, LPS-stimulated monos, TH2 cells after stimulation with anti-CD3 + IL-4 IL-19 - LPS-treated monos, B cells. IL-20 - unclear-- low levels only in some tissue samples from skin and trachea .

23. IL-12 FAMILY IL-12 Heterodimer: p35 and p40 Receptor: 1 and 2 subunits Acts in a contrasting manner to IL-4; promotes TH1 differentiation; induces IF- production Primary mediator of early innate immune responses to intracellular microbes Inducer of cell-mediated immunity Stimulates synthesis of IF- by T cells & NK cells Increases the killing activity of CTLs & NK cells Stimulates differentiation of naive CD4+ T cells into IF-producing TH1 cells. Produced mainly by macrophages and dendritic cells. IL-23 Heterodimer: p19 and p40 (same p40 as IL-12) Receptor: 1 and IL-23-specific subunits Produced by activated DCs Induces synthesis of pro-inflammatory cytokines (eg, IL-17) by CD4+ TH1 cells Does NOT induce IF- production Implicated in many autoimmune disorders IL-23-specific subunit of receptor homologous to IL-12 2 subunit May play a key role in innate immunity to microbial pathogens

24. Collison & Vignali. IL-35: Odd one out or part of the family? Immunol. Rev. 226(1): 248-262, 2009. IL-27 is related to both IL-12 and IL-6 cytokine families.

25. IL-23 required for terminal differentiation of TH17 cells Nature Immunology 10, 236 - 238 (2009) Don't leave home without it: the IL-23 visa to TH17 cells Yeonseok Chung & Chen Dong Interleukin 23 is tightly associated with TH17 cell–mediated inflammation and autoimmunity. A new study of mice deficient in its receptor shows that interleukin 23 is required for the terminal differentiation of TH17 cells in vivo. Nature Immunology 10, 314 - 324 (2009) The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17–producing effector T helper cells in vivo Mandy J McGeachy Whether interleukin 23 (IL-23) affects the differentiation of or simply the maintenance of TH17 cells is unclear. The data in this paper by McGeachy and colleagues show that IL-23 is required for the full differentiation and proliferation of effector TH17 cells in vivo.

26. IL-35 Heterodimer: p35 and EBI3* Receptor: UNKNOWN Inhibits T cell differentiation; expressed by Tregs Expands Tregs; inhibits TH17cells; has therapeutic effects in collagen arthritis model. Tregs from IL-35 KO mice fail to ameliorate inflammatory bowel disease in mouse model. *EBI3 = Epstein-Barr virus Induced gene 3

27. IL-14 Induces B-cell proliferation, inhibits Ig secretion, and selectively expands memory B cells Produced by T cells Has homology with complement factor Bb IL-16 Soluble ligand to the CD4 molecule (its receptor) Chemotactic properties for CD4+ cells Growth factor for CD4+ T cells, upregulating MHC class II and the IL-2 receptor CD25. Possible sources include CD8+ T cells, eosinophils, macrophages, mast cells

28. IL-17 FAMILY IL-17 (IL-17A) - secreted exclusively by a subset of T cells -- TH17 cells Human IL-17A gene product - 150 amino acids with a MW of 15 kDa, secreted as disulfide- linked homodimer of 30–35 kDa 5 related cytokines share 20–50% homology to IL-17 IL-17 - designated IL-17A to indicate that it is the founding member of the IL-17 cytokine family IL-17F - produced primarily by activated memory T cells IL-17B, IL-17C, IL-17D, and IL-17E - secreted by a wider assortment of cells IL-17R - transmembrane proteins of ~130 kDa; expressed on nearly all cells (IL-17RA-D) IL-17A induced by IL-23; plays proinflammatory role in joint destruction of RA Signaling by IL-17 via IL17 receptor plays an important role in host defense against pathogens (J. Exp. Med. Published online Feb. 9, 2009 doi:10.1084/jem.20081463) IL-25 (aka IL-17E) Involved in development of TH2 responses Involved in expulsion of helminthic parasites Amplifies/exacerbates allergic responses

29. Taken from Gaffen. An Overview of IL-17 function & signaling. Cytokine. 43(3): 402-407, 2008. IL-17 family ligands Produced by Binding receptor(s) IL-17 (IL-17A, CTLA-8) T cells (TH17) IL-17RA, IL-17RC (IL-17RL) IL-17A/F heterodimer T cells IL-17RA, IL-17RC IL-17B Numerous tissues IL-17RB? IL-17C Prostate, fetal kidney IL-17D Numerous tissues IL-17E/IL-25 Numerous tissues IL-17RB (IL-25R) IL-17F T cells IL-17RA, IL-17RC vIL-17 (ORF13) H. Saimiri IL-17RA, IL-17RC? Unknown IL-17RD Unknown IL-17RE It is currently believed that IL-17 regulates innate immunity. Activates PMNs and expression of anti-microbial genes.

30. Gaffen. An Overview of IL-17 function & signaling. Cytokine. 43(3): 402-407, 2008.

31. IL-27 & IL-30 IL-27 - related to both IL-6 and IL-12 families (which are related to each other by receptor homology) IL-30 is p28 subunit of IL-27 IL-27 opposes actions of IL-23, thus has potent anti-inflammatory activity Predict that IL-27 inhibits TH17 differentiation & maintenance. Receptor is believed to consist of 2 peptides, gp130 and WSX-1 (aka IL-27R) (For this reason, one might include these 2 in the IL-6 family)

32. IL-28 & IL-29 IL-28 and IL-29 are a recently discovered family of novel class II cytokines distantly related to IF- and IL-10. IL-29 aka IF-1 & discovered by UMDNJ faculty member Serge Kotenko IL-28 aka IF-3 Both have antiviral activity against encephalomyocarditis and other viruses. Receptor is composed of two chains, IL-28R and IL-10R

33. IL-35 AND COUNTING “IL-32 is the name given to the NK4 transcript first reported in IL-2 activated T lymphocytes and natural killer cells 13 years ago without known function. The novel cytokine has six isoforms.” Dinarello CA & Kim SH. IL-32, a novel cytokine with a possible role in disease. Ann Rheum Dis. 2006. Nov;65 Suppl 3:iii61-iii64. no homology with other cytokines induced by IL-2 and IL-18 induces IF-, IL-8, MIP-1 induces IL-1 and IL-6 through a caspase-dependent mechanism IL-33: see IL-1 IL-34: next slide IL-35: see IL-12

34. IL-34 IL-34 is known also as FPT025. The protein does not show sequence homology with known cytokines or growth factors. It is expressed in spleen, skin, brain, and other tissues. IL-34 human primary monocyte proliferation and/or survival. Human IL-34 specifically binds to human primary monocytes and activates ERK1/2 phosphorylation in a human monocytic cell line, THP-1. IL-34 regulates myeloid lineage differentiation, proliferation, and survival; promotes CFU-GM and CFU-M colony formation from human bone marrow cells in a colony formation assay; stimulates expression of myeloid cell surface markers. IL-34 appears to act via the receptor for M-CSF. Lin H et al Discovery of a cytokine and its receptor by functional screening of the extracellular proteome. Science 320(5877): 807-811 (2008).

35. DANGER SIGNAL MICROORGANISM NEUTROPHIL MONOCYTE, MACROPHAGE IL-1, TNF IL-18 ANTI-MICROBIAL MECHANISMS (ROI, RNI, etc.) B CELLS DENDRITIC CELLS IL-12 IL-6 IL-4 Ab: Enhanced Phagocytosis Activation of Complement ADCC T CELLS IL-2 TH1, TC CYTOKINE CASCADE LINKS INFLAMMATORY AND IMMMUNE RESPONSES INFLAMMATORY IMMUNE

36. IL-12, IF- TH1: IL-2, TNF, IF- TH0 TH1 APC TH2 IL-4 IL-4 TH2 TH2: IL-4, IL-5, IL-6, IL-13 eosinophil basophil CYTOKINES DETERMINE NATURE OF ADAPTIVE IMMUNE RESPONSE