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`Cytokines, Chemokines and Their Receptors
`Mark J. Cameron and David J. Kelvin.
`
`Introduction
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`Cytokines, their Receptors and their Genes
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`Chemokines, their Receptors and their Genes
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`The immune system is skilled in communication and designed to respond quickly,
`specifically and globally to protect an organism against foreign invaders and
`disease. The cytokine superfamily of proteins is an integral part of the signaling
`network between cells and is essential in generating and regulating the immune
`system. Much progress has been made recently in interpreting how the immune
`system communicates with, or is mediated by, cytokines and chemotactic cytokines
`(chemokines). These interacting biological signals have remarkable capabilities,
`such as influencing growth and development, hematopoiesis, lymphocyte
`recruitment, T cell subset differentiation and inflammation. This chapter provides
`brief synopses for a comprehensive list of immune-related cytokines and
`chemokines. Information such as gene cloning and mapping details, protein
`characteristics and expression, receptor usage, source and target cells, major
`biological functions and knockout phenotype is described for each cytokine and
`chemokine. With an approach that organizes cytokines and chemokines into
`interacting groups with related physical and/or functional properties, this chapter
`aims to highlight the capability of this system to maintain widespread impact and
`functional complementation while not sacrificing regulation and specificity of action.
`A more complete understanding of these properties may lead to more advanced
`means of correcting improper cytokine- or chemokine-mediated immune
`responses, such as those causing autoimmune disease.
`
`Detailed and reliable communication must occur through a complex system of
`network connections to accomplish a task at a modern workstation. In parallel, the
`immune system is an interdependent biological network charged with
`developmental tasks and the responsibility of protecting its host against injury and
`infection. An immune cell within a given microenvironment can respond to signals
`received through its receptors with its own protein-based language that will
`influence the cell itself (autocrine effect) or other cells throughout the organism
`(paracrine effect). The language of cytokines is critical in this communication.
`Cytokines are small soluble factors with pleiotropic functions that are produced by
`many cell types as part of a gene expression pattern that can influence and
`regulate the function of the immune system.
`
`1
`The term cytokine was proposed by Cohen et al in 1974 to replace lymphokine, a
`term coined in the late 1960's to denote lymphocyte-derived soluble proteins that
`2
`possess immunological effects. Since the latter designation misleadingly
`suggested that lymphocytes were the only source for these secreted proteins, the
`term cytokine slowly became preferred. Following the introduction of this general
`term, the Second International Lymphokine Workshop held in 1979 proposed the
`interleukin (IL) system of nomenclature to simplify the growing list of identified
`cytokines. Ironically, this partially adopted system introduced confusion in that the
`interleukins, presently numbering at least 23, affect many cell types but their name
`implies that they act only among leukocytes. As a result, modern cytokine
`nomenclature is a mix of the widely accepted, but slightly misleading, interleukin
`designations and other proteins still known by their original names. A good
`example of these potential points of confusion is the chemotactic cytokine
`(chemokine) IL-8, which is produced by and targets a wide variety of cell types
`
`Apotex Ex. 1017, p.1
`
`

`

`including leukocytes and nonleukocytes.
`
`As this chapter unfolds, repeated mention of a number of cytokines and
`chemokines will make it clear that these proteins can be part of a bigger immune
`program, e.g., T cell subset differentiation. Mature CD4 and CD8 T cells leave the
`thymus with a naive phenotype and produce a variety of cytokines. In the
`periphery, these T cells encounter antigen presenting cells (APCs) displaying either
`major histocompatibility complex (MHC) class I molecules (present peptides
`generated in the cytosol to CD8 T cells) or MHC class II molecules (present
`peptides degraded in intracellular vesicles to CD4 T cells). Following activation,
`characteristic cytokine and chemokine secretion profiles allow the classification of
`3-7
`CD4 T helper (Th) cells into two major subpopulations in mice and humans.
`Th1
`cells secrete mainly IL-2, interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β),
`whereas Th2 cells secrete mainly IL-4, IL-5, IL-6, IL-10 and IL-13. Th1 cells support
`cell-mediated immunity and as a consequence promote inflammation, cytotoxicity
`and delayed-type hypersensitivity (DTH). Th2 cells support humoral immunity and
`serve to downregulate the inflammatory actions of Th1 cells. This paradigm is a
`great example of an integrated biological network and is very useful in simplifying
`our understanding of typical immune responses and those that turn pathogenic. For
`example, the failure to communicate “self” can lead to a loss of tolerance to our
`own antigens and prompt destructive immune responses to self-tissues and
`autoimmune disease. Autoimmunity, the major focus of this book, is the underlying
`mechanism of a set of conditions, such as type 1 diabetes mellitus, multiple
`sclerosis and rheumatoid arthritis. Autoimmune diseases may be caused in part by
`cytokine- and chemokine-mediated dysregulation of Th cell subset differentiation.
`The main factors affecting the development of Th subsets, aside from the context in
`which the antigen and costimulatory signals are presented, are the cytokines and
`chemokines in the stimulatory milieu. A better understanding of the properties and
`interactions of the individual cytokines and chemokines that play a role in Th cell
`activation may lead to more advanced treatments for autoimmune disease.
`
`The proceeding sections will introduce many of the currently identified cytokines
`and chemokines, along with their receptors. You will find that cytokines and
`chemokines with related structure and/or function are clustered into groups of
`interdependent homologues, e.g., the IL-1-like cytokines. A particular group of
`cytokines or chemokines can exhibit functional redundancy with, and widespread
`impact on, other groups of cytokines or chemokines, e.g., IL-1-like cytokines and
`IL-6-like cytokines. Interestingly, this can occur while maintaining several regulatory
`features, such as internal checkpoints and specificity of action. It is therefore hoped
`that this chapter may serve as more than a brief catalogue of the field of cytokines,
`chemokines and their receptors, but may also highlight the remarkable capabilities
`of this interacting network of biological signals.
`
`Cytokines, their Receptors and their Genes
`
`Go to:
`
`Table 1 introduces the human cytokines and lists some of their properties, such as
`receptor usage and physical characteristics. Each human cytokine described in
`Table 1 has a murine counterpart so the basic list can be used interchangeably in
`regards to terminology. Hundreds of cytokines have been identified. In the interest
`of conciseness the table includes only common cytokines with recognized immune
`function, many of which are discussed in more detail below. Excluded are the
`‘growth factors’, neurobiological proteins and ‘trophins’, for example. It is also
`beyond the scope of this chapter to describe how cytokines signal through their
`receptors in any detail. One popular cytokine signaling mechanism used by
`cytokines such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15 and the interferons,
`however, begins with dimerization of the appropriate receptor chains upon ligand
`binding. Following this, different types of receptor-associated Janus family tyrosine
`kinases (Jak) are activated which phosphorylate the receptor chains and allow the
`recruitment and activation of other kinases and transcription factors, such as those
`of the signal transducer and activator of transcription (Stat) family. This promotes
`the rapid translocation of these proteins to the nucleus and stimulation of target
`gene transcription (see references 8 and 9 for more details on cytokine signaling).
`
`Apotex Ex. 1017, p.2
`
`

`

`Table 1
`
`Common human cytokines and their
`1
`receptors .
`
`IL-1-Like Cytokines
`
`Firstly, the interleukins are comprised mostly of hematopoietic growth factors and
`can be further divided into groups of proteins as shown in Table 1. The IL-1-related
`group of pro-inflammatory cytokines consists of IL-1α, IL-1β, IL-1 receptor
`antagonist (IL-1RA) and IL-18. IL-1α and IL-1βare produced mainly by mononuclear
`10
`and epithelial cells upon inflammation, injury and infection.
` These two proteins
`are of primary importance to the outcome of these challenges to the immune
`system in that they trigger fever, induce a wide variety of acute phase response
`10
`(APR) genes and activate lymphocytes.
` IL-1α and IL-1β arise from two closely
`linked genes that, along with the IL-1RA gene, lie on human (and mouse)
`10,11
`chromosome 2.
`The two forms of IL-1 are quite similar in function since they
`12
`both signal through the IL-1 type 1 receptor (IL-1-R1/CD121a).
` Both proteins can
`also bind to the IL-1 type 2 receptor (IL-1-R2/CDw121b) which does not appear to
`13
`be involved in signaling, except as a possible decoy.
` The IL-1 receptor genes are
`located on human chromosome 2 along with their ligands, albeit at a distance.
`
`Murine knockout studies confirm the importance of IL-1 in fever responses and the
`APR. While at least three studies involving the IL-1β knockout mouse demonstrate
`that fever development is suppressed upon turpentine or lipopolysaccharide (LPS)
`14-16
`challenge,
`one study demonstrates that the role of IL-1β as a pyrogen is not
`17
`obligatory and that its absence can in fact exacerbate an induced fever response.
`The latter conflicting result may stem from differences in experimental protocol or
`14
`reagents.
` Knockout studies also show that while both forms of IL-1 can induce
`fever responses, fever induction is not reduced in IL-1α knockout mice, indicating
`14
`that IL-1βcan compensate for IL-1α but not vice versa.
` The role for IL-1 in the
`APR (a series of cellular and cytokine cascades in reaction to trauma or infection
`that help limit damage) was confirmed in a localized tissue damage model of
`turpentine injection where challenged IL-1β-deficient mice did not develop an
`18
`APR.
` Accordingly, IL-1R1 knockout mice are irresponsive to IL-1 in the induction
`18
`of IL-6, E-selectin and fever.
` These mice also have a reduced APR to
`19
`turpentine.
`
`IL-1RA is produced by virtually any cell that can produce IL-1 and is similar in
`20
`structure to IL-1β but lacks its agonist activity.
` The different species of IL-1RA, a
`secreted form with a signal peptide and at least two intracellular forms, arise from
`20,21
`alternative splicing of different first exons on chromosome 2.
` IL-1RA
`represents an intriguing example of a naturally occurring cytokine receptor
`antagonist. IL-1RA may be an acute phase protein that may serve to regulate the
`agonist effects of IL-1 during chronic inflammatory and infectious disease because
`its expression is influenced by cytokines, viral and bacterial products, bound
`20
`antibody and acute phase proteins, such as IL-1, IL-4, IFN-γ and LPS.
` Consistent
`with this notion are two studies of IL-1RA-deficient mice which exhibit growth
`retardation, an exacerbated fever response to turpentine injection, increased
`lethality following LPS injection and decreased susceptibility to Listeria
`14,22
`monocytogenes.
`These observations verify the importance of balance in the IL-
`1 system in mediating these immune challenges.
`
`IL-18, initially termed interferon-γ inducing factor (IGIF), is a pro-inflammatory
`23
`cytokine that is encoded on human chromosome 11 and mouse chromosome 9.
`IL-18 has been placed in the IL-1 group of interleukins because it bears structural
`homology to IL-1α and β, is converted into a mature form by IL-1β converting
`enzyme (ICE) along with IL-1β and binds to the IL-18 receptor (IL-18R or IL-1R
`23
`related protein).
` The IL-18R resembles the IL-1R and transduces IL-1R
`23
`signaling.
` IL-18 shares biological function with IL-12 in that it induces IFN-γ
`secretion (in synergy with IL-12), enhances natural killer (NK) cell activity and
`23
`24
`promotes inflammatory Th1 cell responses.
` Accordingly, when IL-18 or its
`25
`receptor
` is knocked out, mice exhibit defective NK cell activity and Th1
`responses. More recently, however, the role of IL-18 as a pro-inflammatory
`cytokine has been questioned because IL-18 can also potentiate regulatory Th2
`
`Apotex Ex. 1017, p.3
`
`

`

`responses, perhaps by inducing IL-4 production by natural killer T (NKT) cells in
`26-28
`certain situations.
`
`Common γChain Cytokines
`
`Cytokines that utilize the common γ chain (γc/CD132) in their receptor comprise the
`next group of interleukins, namely IL-2, IL-4, IL-7, IL-9, IL-13 and IL-15. These
`diverse cytokines invoke lymphocyte activation and differentiation (the outcome of
`which can vary) and possess some redundancy in biological function because of
`29
`their common receptor subunit.
` The γc itself cannot bind cytokines; however, new
`30
`evidence suggests that it can be shed as a soluble negative modulator.
` Indeed,
`γc-deficient mice are severely immunocompromised, as are humans with γc
`31,32
`defects.
`
`IL-2 is expressed from a gene on human chromosome 4 or mouse chromosome 3
`and is mainly secreted by activated T cells. IL-2 and the heteromultimeric IL-2
`receptor (IL-2R) complex (combinations of IL-2Rα/CD25, IL-2Rβ/CD122 and γc)
`are upregulated on T cells following antigenic or mitogenic stimulation leading to
`clonal expansion. As such, IL-2 is commonly regarded as an autocrine or paracrine
`T cell growth factor but it actually has effects on many cell types, such as B cells,
`29,33,34
`NK cells, macrophages and neutrophils.
`The IL-2 knockout mouse exhibits
`immune dysregulation caused by defects in T cell responsiveness in vitro; however,
`35,36
`only delays in normal T cell functionality were found in vivo.
` Interestingly, IL-
`37
`38
`2Rα-
` and IL-2Rβ-deficient
` mice exhibit loss of T cell regulation and
`autoimmunity, indicating that proper IL-2 signaling may be required to induce
`regulatory T cells and/or eliminate abnormally activated T cells via the reversal of T
`39
`cell anergy or apoptosis (programmed cell death) induction, respectively.
`
`The IL-4 gene is located on human chromosome 5 (along with the IL-3, IL-5, IL-9,
`IL-13 and granulocyte macrophage colony stimulating factor (GM-CSF) genes) and
`murine chromosome 11 (along with the IL-3, IL-5, IL-13 and GM-CSF genes). Short
`40
`or long isoforms of IL-4 can exist arising from alternative splicing.
` IL-4 is
`produced by activated T cells, mast cells, basophils and NKT cells and targets
`many cell types, including B cells, T cells, macrophages and a wide variety of
`29,41
`hematopoietic and nonhematopoietic cells.
` Physiologic signal transduction via
`IL-4 depends on heterodimerization of the IL-4 receptor α chain (IL-4Ra/CD124),
`42
`with γc and possibly the IL-13 receptor α chain (IL-13Ra/CD213a1).
` IL-4 is the
`principal cytokine required by B cells to switch to the production of immunoglobulin
`(Ig)E antibodies, which mediate immediate hypersensitivity (allergic) reactions and
`41
`help defend against helminth infections.
` IL-4 also inhibits macrophage activation
`and most of the effects of IFN-γ on macrophages. However, the most important
`biological effect of IL-4 with respect to immune modulation is the growth and
`differentiation of Th2 cells. As described earlier, Th2 cells support humoral
`immunity and serve to downregulate the inflammatory actions of Th1 cells.
`Moreover, stimuli that favour IL-4 production early after antigen exposure favour the
`3
`43
`development of Th2 cells. IL-13 is also associated with this subset of T cells.
`Like IL-4, and along with the fact that it maps closely to IL-4 and shares receptor α
`subunits with IL-4, IL-13 is expressed by activated T cells, induces IgE production
`44
`by B cells and inhibits inflammatory cytokine production.
` These properties of IL-4
`and IL-13 have been convincingly demonstrated in mice lacking the IL-4 or IL-13
`45-48
`gene.
` These mice are deficient in the development and maintenance of Th2
`cells.
`
`The remaining γc cytokines, IL-7, IL-9 and IL-15, are potent hematopoietic factors
`expressed from genes on human chromosome 8 and mouse chromosome 3,
`human chromosome 5 and mouse chromosome 13, and human chromosome 4 and
`mouse chromosome 8, respectively. IL-7, expressed by stromal and epithelial cells,
`stimulates immature B cells, thymocytes and mature T cells via its receptor
`49-51
`consisting of the IL-7 receptor α chain (IL-7Rα/CD127) and the γc.
` Knocking
`out IL-7 or IL-7Rα/CD127 causes severe defects in thymic T cell and B cell
`development consistent with the critical roles that IL-7 and its receptor play in
`51-56
`maturation of the immune system.
` IL-9 promotes the growth of mast cells, B
`cells and other T cells and is mainly expressed by activated T cells, especially Th2
`29,43,57
`cells.
` Confirming only the role of IL-9 in enhancing mast cells, the recently
`
`Apotex Ex. 1017, p.4
`
`

`

`generated IL-9 knockout mouse exhibits normal T cell (Th2) responses but not
`58
`characteristic mast cell expansion upon lung challenge.
` IL-15, produced by
`activated monocytes, epithelial cells, and a variety of tissues, shares biological
`activities with IL-2 in that it stimulates NK cells, B cells and activated T
`29,59-61
`cells.
` The IL-15 receptor (IL-15R) consists of combinations of IL-15Rα, IL-
`2Rβ/CD122 and γc. Similarities in function between IL-2 and IL-15 are partially due
`to receptor subunit sharing. A recent study, however, provides evidence that IL-2
`and IL-15 control different aspects of primary T-cell expansion in vivo. IL-15 is
`critical for initiating T cell divisions, whereas IL-2 can limit T cell expansion by
`62
`decreasing γc expression and rendering cells susceptible to apoptosis.
` The α
`chain ligand specificity and broad cellular expression range of IL-15 allows for
`29
`differential activity even outside of the immune system.
` IL-15- and IL15Ra-
`deficient mice were recently generated. Initial studies confirm the role of IL-15 in
`NK cell stimulation and indicate a role for IL-15 in peripheral CD8 T cell
`63,64
`maintenance upon immune challenge.
`
`Common β Chain Cytokines
`
`Cytokines that utilize the common β chain (βc/CDw131) in their receptor comprise
`the next group of interleukins, namely IL-3, IL-5 and GM-CSF. The genes for IL-3,
`IL-5 and GM-CSF are closely linked and lie on human chromosome 5 and mouse
`65
`chromosome 11.
` Like the γc cytokines, these associated (but not particularly
`homologous at the amino acid sequence level) βc cytokines overlap in biological
`65
`function because of their common receptor subunit.
` When the βc is mutated,
`normal hematopoiesis is noted but impaired immune responses can be observed
`that are most likely due to a loss of responsiveness to IL-5 and GM-CSF, rather
`66,67
`than IL-3.
`
`IL-3, originally termed multicolony stimulating factor (multi-CSF), is produced by
`activated T cells and stimulates both multipotential hematopoietic cells (stem cells)
`and developmentally committed cells such as granulocytes, macrophages, mast
`68-70
`cells, erythroid cells, eosinophils, basophils and megakaryocytes.
`The human
`IL-3 receptor consists of CD123 and βc/CDw131. The mouse IL-3 receptor has an
`additional β chain called β
`, the function of which can be compensated for by
`IL-3
`67
`CD123 if knocked out.
` Knocking out CD123 itself also has little effect on
`71
`hematopoiesis.
` On the other hand, if IL-3 is knocked out, mast cell and basophil
`66
`72
`development upon challenge is affected,
` as well as some forms of DTH,
`confirming a role for IL-3 in host defense and expanding hematopoietic effector
`cells.
`
`IL-5, originally identified as a B cell differentiation factor, is produced mainly by
`activated T cells (especially Th2 cells) and aids in the growth and differentiation of
`73-75
`eosinophils and late-developing B cells.
`When IL-5 or CDw125 is absent, mice
`exhibit developmental defects in certain B cells (CD5/B-1 B cells) and a lack of
`76,77
`eosinophilia upon parasite challenge.
`
`Lastly, GM-CSF, as its name suggests, was originally found to stimulate
`granulocytes and macrophages. GM-CSF has since been found to be expressed by
`many cell types, including macrophages and T cells, and shares many of the
`functions of IL-3 in stimulating a variety of precursor cells, including macrophages,
`78-80
`neutrophils and eosinophils.
`Interestingly, GM-CSF-deficient mice have normal
`hematopoietic development but suffer from pulmonary disease perhaps caused by
`81
`a lack of lung surfactant clearance by alveolar epithelial cells or macrophages.
`
`IL-6-Like Cytokines
`
`IL-6 is the prototype cytokine representing the next group of interleukins. Most of
`the members of this group utilize the glycoprotein 130 (gp130) or CD130 receptor.
`IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte
`colony-stimulating factor (G-CSF) and IL-12 have partially overlapping functions
`and are key mediators in various immune processes including hematopoiesis and
`the APR. CD130-deficient mice exhibit embryonic lethality, a finding that appears to
`82
`be linked to a significant role for CD130-dependent signaling in homeostasis.
`
`IL-6, with its gene situated on human chromosome 7 and mouse chromosome 5,
`
`Apotex Ex. 1017, p.5
`
`

`

`utilizes the CD130 receptor and the IL-6 receptor α chain (IL-6Ra/CD126). The IL-
`6Rα/CD126 can exist in a soluble form and serves as an important cofactor by
`83
`extending the cytokine's half-life.
` IL-6 was originally characterized as a
`84,85
`differentiation factor of B cell hybridomas.
`Producers of IL-6 include fibroblasts,
`endothelial cells, macrophages, T cells (Th1) and B cells. IL-6 is a primary inducer
`of fever, hormones, acute phase proteins and T and B cell expansion upon injury
`86
`and infection.
` It can also act as a cofactor in hematopoiesis by increasing GM-
`87
`CSF and macrophage colony stimulating factor (M-CSF) expression.
` IL-6-
`deficent mice exhibit a severely blunted APR following infection or
`88,89
`injury,
`problems in early hematopoiesis and T and B cell function and Th1
`90
`development.
` Interestingly, IL-6 can nonetheless act as an anti-inflammatory
`91
`agent in some instances.
`
`IL-11, originally identified as a pleiotropic stromal cell-derived cytokine, is encoded
`92,93
`on chromosome 19 in humans and chromosome 7 in mice.
`IL-11 also utilizes
`the CD130 receptor along with the IL-11 receptor α chain (IL-11Rα). IL-11 is
`produced by, and has effects on, many hematopoietic and nonhematopoietic cell
`94,95
`types.
`IL-11, like IL-6, is known to stimulate acute phase protein synthesis in
`94,95
`the liver.
`IL-11 also collaborates with other cytokines we have already
`discussed, such as IL-3, IL-4, IL-7, IL-13 and GM-CSF, to stimulate (by shortening
`cell-cycle time) the proliferation of hematopoietic stem cells and progenitor cells and
`94,95
`induce the differentiation of megakaryocytes.
`The collaborative nature of IL-11
`in vivo may explain why knockout studies have yet to identify a defective phenotype
`(at least in the hematopoietic compartment) associated with a lack of IL-11
`96
`signaling.
` Interestingly, IL-11 could also be an anti-inflammatory mediator as it
`inhibits macrophage pro-inflammatory cytokine production and can exert protective
`91
`effects in several disease models.
`
`LIF is a ligand for CD130 and the LIF receptor (LIFR). LIF is associated with the
`91,97,98
`differentiation of many cell types.
` In this regard, LIF can both inhibit the
`differentiation of embryonic stem cells and promote the survival of hematopoietic
`precursors. LIF can stimulate inflammatory cytokine production. Its expression can
`be upregulated or downregulated in response to inflammatory cytokines such as IL-
`1 and TNF or regulatory cytokines such as IL-4, respectively. LIF is therefore often
`classified as a pro-inflammatory cytokine; however, recent evidence may suggest
`91
`otherwise in some situations.
` LIF knockout mice display several phenotypes
`91
`depending on the disease model.
` This may be due to the observation that loss of
`LIF expression perturbs the establishment of a normal pool of stem cells, but not
`99
`the terminal differentiation of these cells.
` Unlike IL-6, LIF can also stimulate the
`hypothalamic-pituitary-adrenal axis in response to stress and disease. This
`property has been elegantly demonstrated in a recent study of the LIF knockout
`mouse where mice did not respond to immobilization-induced stress with the
`100
`normal indicators.
` It is also interesting to note that the genes for LIF and OSM
`lie in tandem on human chromosome 22 and mouse chromosome 11 and are
`101,102
`transcribed in the same orientation.
`OSM is a very similar cytokine produced
`mainly by activated macrophages and T cells with inflammatory and growth factor
`101,102
`properties.
`
`G-CSF (or colony stimulating factor-3) is produced by fibroblasts and monocytes
`103-105
`and stimulates granulocyte progenitor cells and neutrophils.
`The G-CSF
`gene is located on human chromosome 17 and mouse chromosome 11 and
`creates two active polypeptides (differing by only three amino acids) by differential
`103
`mRNA splicing.
` The G-CSF receptor (G-CSFR) is expressed on multipotential
`104
`hematopoietic progenitor cells and in cells of the myeloid lineage.
` The
`importance of G-CSF in granulocyte differentiation and neutrophil development has
`been verified in G-CSF- and G-CSFR-deficient mice. These mice have lower
`numbers of circulating neutrophils, a decrease in granulocytic precursors and
`106,107
`impaired terminal differentation of granulocytes.
`
`In discussing the IL-6-like cytokines, it bears to mention the heterodimeric cytokine
`IL-12. IL-12 was originally called NK cell stimulatory factor and can be regarded as
`108-110
`a cytokine and soluble receptor complex.
`The “cytokine” subunit, commonly
`known as IL-12α or p35, is coded for on human and mouse chromosome 3, shows
`homology with the IL-6-like cytokines and is not active on its own. The “soluble
`
`Apotex Ex. 1017, p.6
`
`

`

`receptor” subunit, called IL-12β or p40, is coded for on human chromosome 5 and
`mouse chromosome 11, is a member of the cytokine receptor superfamily with
`homology to IL-6Ra/CD126 and has activity via the IL-12 receptor (IL-12R/CD212)
`when partnered with IL-12a. While both soluble subunits are required for biological
`111
`activity, the two components are differentially regulated.
` IL-12 is produced by
`APCs and has immunoregulatory effects on NK cells and T cells, two cell types that
`112
`express the IL-12R.
` IL-12 plays a critical role in cell-mediated immunity by
`acting as a requisite cytokine in pushing the balance between Th1 cells and Th2
`cells towards Th1-type predominance. It is therefore no surprise that IL-12-deficient
`mice are defective in mounting an IFN-γ- or Th1-mediated immune response and/or
`respond with default Th2 responses when stimulated with antigen or infected with
`113-115
`parasites or bacteria.
`An interesting note on IL-12 is that a new composite
`cytokine has been described in mice and humans that consists of a novel a subunit,
`116
`p19, that combines with IL-12β to form a unique cytokine called IL-23.
` IL-23 has
`similar biological functions to IL-12 in that it can induce IFN-γ expression by T cells
`for example, yet it can act distinctly through an unidentified novel receptor
`116
`subunit.
`
`IL-10-Like Cytokines
`
`IL-10, IL-19 and IL-20 are members of the next related group of interleukins, those
`with homology to IL-10. The genes for these cytokines are closely linked on human
`117,118
`and mouse chromosome 1.
`Originally identified as human cytokine synthesis
`inhibitory factor (CSIF), IL-10 plays a major role in suppressing inflammatory
`responses. It does this by inhibiting the synthesis of IFN-γ, IL-2, IL-3, TNF-α and
`119,120
`GM-CSF by cells such as macrophages and Th1 cells.
` However, there is
`also evidence that IL-10 can act as a stimulator of thymocytes, mast cells and B
`120
`cells.
` Monocytes and T cells (Th2 cells) are considered to be the main sources
`of IL-10, although many other cell types can be made to produce IL-10 including B
`120
`cells, mast cells and keratinocytes.
` The participation of IL-10 in limiting Th1 cell
`responses and favoring Th2 cell development has been explored in IL-10 knockout
`mice. Mice that are deficient in IL-10 spontaneously develop chronic intestinal
`inflammation caused by uncontrolled cytokine production from dysregulated
`121,122
`macrophages and Th1 cells.
` IL-19 and IL-20 have been recently identified
`as IL-10 homologues. IL-19 is under patent application and not yet described while
`118
`IL-20 appears to stimulate keratinocytes via its unique receptor.
`
`Interferons
`
`The interferons are a family of cytokines that play a pivotal role in pathogen
`resistance. There are two types of interferons, type I and II, that signal through
`123
`different receptors to produce distinct, but overlapping, cellular effects.
` The
`pleiotropic cytokines IFN-α, originally referred to as leukocyte interferon, and IFN-β,
`originally referred to as fibroblast interferon, are type I interferons that are secreted
`124-128
`by virus-infected cells.
`Infection by most viruses causes a reaction in the
`host that includes innate and adaptive immune responses, such as the production
`of cytokines, increased expression of MHC class I and cytotoxic T cell mobilization.
`IFN-αand IFN-β, coded for by genes on human chromosome 9 and mouse
`128
`chromosome 4, appear to be central players in innate immune responses.
` IFN-
`αand IFN-β also have the unique ability to regulate adaptive T cell responses,
`129
`perhaps directly by stimulating production of IFN-γ by activated T cells
` or
`130
`indirectly by inhibiting IL-4-inducible gene expression in monocytes.
` These
`properties have been verified in knockout mice. Mice lacking the type I IFN receptor
`(CD118) exhibit impaired antiviral defenses and are deficient in promoting IFN-γ
`129,131
`production by T cells.
`
`IFN-γ, also known as immune interferon or type II interferon, is secreted by
`123
`activated T cells (Th1 cells) and NK cells.
` It was originally identified as an
`antiviral agent and its gene was mapped to human chromosome 12 and mouse
`123,132,133
`chromosome 10.
` IFN-γ signals through its own CDw119 receptor and
`has many biological functions. For example, IFN-γ can stimulate macrophages,
`increase antigen processing and expression of MHC molecules, promote an Ig
`class switch to IgG2a antibody secretion, and control the proliferation of
`
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`123
`transformed cells.
` The immunomodulatory function of IFN-γ, however, has
`become a major research focus for this cytokine. IFN-γ secretion is the hallmark of
`proinflammatory Th1 cells but its exact role in T cell subset differentiation remains
`unclear. Th1 responses are associated with cell-mediated immunity and can best
`134
`deal with intracellular invaders. Mice with mutations in IFN-γ
` or IFN-γ
`135
`receptor
` expression show decreased macrophage and NK cell activity and
`increased susceptibility to many intracellular pathogens and viruses. Cell-mediated
`immune responses can still develop in IFN-γ knockout mice even though
`136,137
`enhancements in Th2-type responses can be observed.
`As discussed above,
`IL-12 plays a critical role in eliciting Th1 responses. IFN-γ may act in synergy with
`IL-12 to accelerate development of the Th1 cell subset and also repress Th2 cells
`123
`either directly or indirectly.
`
`Tumor Necrosis Factors
`
`The TNF family is another example of a large group of interrelated cytokines that
`138,139
`has stimulated a vast amount of scientific study.
`Most of this work has
`140
`centered on the TNF family members' shared properties as cell death effectors.
`The TNF family has been expanding a great deal recently so we have chosen five
`representative proteins, TNF-α, TNF-β, lymphotoxin (LT)-β, LIGHT (an acronym for
`homologous to lymphotoxins, exhibits inducible expression, and competes with
`HSV glycoprotein D f