Type I Cytokines and Interferons, and Their Receptors

Warren J. Leonard

OVERVIEW AND ISSUES OF NOMENCLATURE

Cytokines are proteins that are secreted by cells and transduce signals via specific cell surface receptors on either the cytokine-producing cell (autocrine actions) or on other target cells (paracrine actions). From this operational type of description, it is clear that the distinction between cytokines, growth factors, and hormones may be imprecise. In general, cytokines and growth factors are similar, except that the term cytokine most often refers to molecules involved in host defense that have actions on leukocytes, whereas the term growth factor more often refers to molecules acting on other somatic cell types. Cytokines most often act locally. For example, in the interaction between a T cell and an antigen-presenting cell, cytokines are produced and usually exert potent actions locally, with rather limited biologic halflives in the circulation. In contrast, hormones are released and then disseminated via the bloodstream throughout the body, with actions on distal target organs. Nevertheless, this distinction between cytokines and hormones is not absolute, with certain cytokines acting at longer distances as well.

In the immune system, terms such as monokines and lymphokines were originally devised to identify the cellular source for cytokines.¹ Specifically, monokines included molecules such as interleukin (IL)-1, which was first recognized to be produced by monocytes, and lymphokines included molecules such as IL-2, which was first described as a T-cell growth factor produced by T lymphocytes. The monokine/lymphokine nomenclature can be problematic; however, when a cytokine is synthesized by more than one type of cell. This resulted in the adoption of the term cytokine, as proposed by Stanley Cohen in 1974.²^,³ The term cytokine refers to a protein made by a cell (“cyto”) that acts (“kine”) on target cells. Cytokines can have very broad ranges of actions, including on cell development, differentiation, growth, cytolytic activity of effector cells, survival, apoptosis, and chemotaxis.

Many cytokines are referred to as interleukins to indicate molecules that are produced by one leukocyte and act on another.⁴ However, this term is also problematic as some interleukins (eg, IL-1 and IL-6) are additionally produced by cells other than leukocytes and/or exert actions on cell types beyond the immune system. For example, IL-7 is produced by stromal and epithelial cells rather than by typical leukocytes. Furthermore, nomenclature can be inconsistent. For example, IL-7 is highly related to another cytokine that is denoted as thymic stromal lymphopoietin (TSLP) rather than as an interleukin (see section on IL-7 and Thymic Stromal Lymphopoietin), even though both can be produced by stromal and epithelial cells and share a receptor component as well as select actions on lymphocytes. The more descriptive name of TSLP correctly describes its production by thymic stroma but obscures its production by skin epithelial cells, and that major target cells include dendritic cells (DCs) and cluster of differentiation (CD)4+ T cells.

Among the many classes of cytokines, the “type I” cytokines are distinctive in their sharing a similar four &agr;-helical bundle structure, as detailed in the section on Type I Cytokines—Structural Considerations, and correspondingly, their receptors share characteristic features that have led to their description as the cytokine receptor superfamily, hematopoietin receptors, or type I cytokine receptors.⁵^,⁶^,⁷^,⁸ Although many interleukins are type I cytokines, some are not. For example, two of the major “proinflammatory cytokines,” IL-1 and IL-6, are interleukins, but IL-6 is a type I cytokine, whereas IL-1 is not. One interleukin, IL-8, is a CXC family chemokine, an entirely different type of molecule involved in chemotaxis. Moreover, IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29 are more similar to interferons (IFNs) and are denoted as type II cytokines.

In summary, the term inter leuk in indicates a relationship to leukocytes, whereas the identification of a cytokine as a type I or type II cytokine indicates general properties of its three-dimensional structure. Knowing that a molecule is a type I cytokine is instructive as it indicates a likely general structure for the cytokine receptor as well as the mechanism of signal transduction. In contrast, the identification of a molecule as an interleukin provides little information other than that it often, but not always, is a type I or type II cytokine of immunologic interest.

In addition to molecules of primarily immunologic interest, other important proteins, such as growth hormone, prolactin, erythropoietin (Epo), thrombopoietin, and leptin, are type I cytokines and their receptors are type I cytokine receptors. Despite having their major actions outside the immune system, these cytokines nevertheless share important signal transduction pathways with type I cytokines of immunologic interest. By focusing on type I cytokines and IFNs and their receptors, this chapter necessarily focuses on cytokines that are evolutionarily related and share common signaling pathways, instead of focusing on common functions per se. For example, although IL-6 has overlapping actions with IL-1 and tumor necrosis factor (TNF)-&agr;, these latter proinflammatory cytokines are not discussed in this chapter
because they are not type I cytokines, and the signaling pathways they use are distinct from those used by IL-6. This illustrates the important concept that similar end functions can be mediated via more than one type of signaling pathway. This is not to minimize the observation that many type I cytokines in fact do have similar/overlapping functions, as detailed in the section on “cytokine redundancy.”

The field of IFN research is older than the cytokine field, but both fields more recently have developed in parallel. In fact, one can consider the IFNs to be the first cytokines that were identified. IFN was discovered as an antiviral activity in 1957. This turned out to be type I IFN (IFN&agr;/&bgr;). Type II IFN (IFN&ggr;) was discovered in 1965. Over time, it was recognized that type I cytokines and IFNs/type II cytokines share a number of common features, including signaling pathways.

An unfortunate nomenclature issue exists that should be noted. There are specialized popuplations of T cells that include T helper 1 (Th1) and T helper 2 (Th2) cells (see following discussion), which produced specialized sets of cytokines, such as IFN&ggr; by Th1 and IL-4 by Th2 cells. These are sometimes called type 1 and type 2 cytokines (for Th1 and Th2 cytokines); unfortunately, this jargon can result in confusion as IL-4 is a type I (ie, four &agr;-helical bundle) cytokine that is functionally a type 2 cytokine (in that it is produced by Th2 cells), where IFN&ggr; is a type II structure cytokine produced by Th1 cells.

FIG. 25.1. Schematic of Four &agr;-Helical Bundle Cytokines. Schematic drawing showing typical short-chain and long-chain four helical bundle cytokines. Although these both exhibit an “up-up-down-down” topology to their four &agr; helices, note that in the short-chain cytokines, the AB loop is in behind the CD loop, whereas in the long-chain cytokines the situation is reversed. See text. The figure was provided by Dr. Alex Wlodawer, National Cancer Institute.

TYPE I CYTOKINES AND THEIR RECEPTORS

Type I Cytokines—Structural Considerations

Type I cytokines typically share only limited amino acid sequence identity, but strikingly, all type I cytokines whose structures have been solved by nuclear magnetic resonance and/or x-ray crystallographic methods achieve similar three-dimensional structures,⁵^,⁶^,⁷^,⁸^,⁹ and those whose structures have not yet been solved are believed to share similar three-dimensional structures. Type I cytokines contain four &agr; helices and thus are designated as four &agr;-helical bundle cytokines (Fig. 25.1). Within their structures, the first two and last two &agr; helices are each connected by long overhand loops, resulting in an “up-up-down-down” topologic structure, as the first two helices (A and B) can be oriented in an “up” orientation and the last two helices (C and D) can be oriented in a “down” orientation, as viewed from the N- to C-terminal direction. As shown in Figure 25.1, the N- and C-termini of the cytokines are positioned on the same part of the molecule.

Type I cytokines are either “short chain” or “long chain” four &agr;-helical bundle cytokines based on the lengths of the &agr; helices.⁸ Some of the short-chain cytokines include IL-2,
IL-3, IL-4, IL-5, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-7, IL-9, IL-13, IL-15, IL-21, macrophagecolony stimulating factor (M-CSF), stem cell factor (SCF), and TSLP, whereas long-chain cytokines include growth hormone, prolactin, Epo, thrombopoietin, leptin, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), novel neurotrophin-1 (NNT-1)/B cell-stimulating factor-3 (BSF-3)/cardiotrophin-like factor (CLC), and granulocytecolony stimulating factor (G-CSF) (Table 25.1).⁸^,¹⁰^,¹¹ The &agr; helices are approximately 15 amino acids long in short-chain helical cytokines and 25 amino acids long in long-chain cytokines. Additional differences include differences in the angles between the pairs of helices, and the AB loop is “under” the CD loop in the short cytokines but “over” the CD loop in the long cytokines (see Fig. 25.1).⁷^,⁸^,¹² Moreover, short-chain cytokines have &bgr; structures in the AB and CD loops, whereas long-chain cytokines do not.

TABLE 25.1 Four Helical Bundle Cytokines

Short-Chain Cytokines	Long-Chain Cytokines
IL-2	IL-6
IL-4	IL-11
IL-7	Oncostatin M
IL-9	Leukemia inhibitory factor
IL-13	CNTF
IL-15	Cardiotropin-1
IL-21	NNT-1/BSF-3
TSLP
IL-3	Growth hormone
IL-5^a	Prolactin
GM-CSF	Erythropoietin
	Thrombopoietin
M-CSF^a,^b	Leptin
SCF^b	G-CSF
BSF-3, B cell-stimulating factor-3; CNTF, ciliary neurotrophic factor; G-CSF, granulocytecolony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; M-CSF, macrophage-colony stimulating factor; NNT-1, novel neurotrophin-1; SCF, stem cell factor; TSLP, thymic stromal lymphopoietin.
^aDimers. ^bDifferent from the other four helical bundle cytokines in that the M-CSF and SCF receptors (CSF-1R and c-kit, respectively) have intrinsic tyrosine kinase activity and are not type I cytokine receptors.

The division of type I cytokines into short-chain and longchain cytokines has evolutionary considerations and correlates with grouping of their receptor chains for these two subfamilies of type I cytokines. An analysis of short-chain cytokines has revealed that 61 residues comprise the family framework, including most of the 31 residues that contribute to the buried inner core. The similarities and differences in the structures of IL-2, IL-4, and GM-CSF have been analyzed. 5 Among these cytokines, there is considerable variation in the intrachain disulfide bonds that stabilize the structures. For example, IL-4 has three intrachain disulfide bonds, GM-CSF has two, and IL-2 has only one. In IL-4, the first disulfide bond (between Cys 24 and Cys 65) connects loop AB to BC, the second disulfide bond (between Cys 46 and Cys 99) connects helix B and loop CD, and the third disulfide bond (between Cys 3 and Cys 127) connects the residue preceding helix B with helix D. In GM-CSF, the N-terminus of helix B and the N-terminus of &bgr; strand CD are connected by one disulfide bond, whereas the other disulfide bond connects the C-terminus of helix C and a strand following helix D. In IL-2, a single essential disulfide bond between Cys 58 and Cys 105 connects helix B to strand CD. Thus, each cytokine has evolved distinctive disulfide bonds to stabilize its structure, although it is typical that helix B is connected to the loop between helices C and D. The structures formed by helices A and D are more conserved than those formed by helices B and C, primarily due to the interhelical angles; helix D and the connecting region are the most highly conserved elements among the three cytokines.⁵ This is of particular interest, as the regions of type I cytokines that are most important for cytokine receptor interactions (based on analogy to the growth hormone receptor structure, see subsequent discussion) include helices A and D and residues in the AB and CD loops, whereas helices B and C do not form direct contacts.⁵

Variations on these typical four &agr;-helical bundle structures can occur. For example, IL-5 is unusual in that it is a dimer, wherein the ends containing the N- and C-termini are juxtaposed, and helix D is “exchanged” between the two covalently attached monomers so that helix D of each molecule actually forms part of the four helix bundle of the other.¹³ M-CSF is also a dimer, but no exchange of helix D occurs.¹²

The IFNs form related albeit distinctive structures from type I cytokines and are designated as type II cytokines.⁸ IFN&bgr; has an extra helix that is positioned in place of the CD strand.¹⁴ IFN&ggr; is a dimer, each of which consists of six &agr; helices¹⁵ (Fig. 25.2). Two of these helices are interchanged, including one from each four &agr;-helical bundle.¹²^,¹⁵ IL-10, which is closely related to IFN&ggr;, has a similar structure,¹⁶ as
presumably do the more recently identified IL-10-like molecules, including IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29. Interestingly, although not universally the case, most four &agr;-helical bundle cytokines have four exons, with helix A in exon 1, helices B and C in exon 3, and helix D in exon 4.⁷ A related organization is found for IFN&ggr;, as well as for the long-chain helical cytokines, growth hormone, and G-CSF. However, there are exceptions, for example, IL-15 is divided into nine exons, whereas the IFN&agr;s and IFN&bgr; are encoded by single exons.

FIG. 25.2. Structure of the Interferon &ggr;Receptor (IFN&ggr;R). Shown are ribbon diagrams of the structures of the IFN&ggr;/IFN&ggr;R complex as an example of a type II cytokine/cytokine receptor. In the IFN&ggr;R, only IFNGR-1 complexed to the IFN&ggr; dimer is shown, as the full structure with IFNGR-2 is not available. See text for discussion of the structure. The IFN&ggr;-IFNGR-1 structure is from Walter et al.⁴⁰¹ The figure was provided by Dr. Alex Wlodawer, National Cancer Institute.

Receptors for Type I Cytokines

The first report suggesting that type I cytokines interacted with receptors with similar features identified similarities in the sequences of the erythropoietin receptor and the IL-2 receptor &bgr; chain,¹⁷ and subsequent analysis of a large number of type I cytokine receptors established these receptors as a superfamily.¹⁸ Type I cytokine receptors are generally type I membrane-spanning glycoproteins (N-terminus extracellular, C-terminus intracellular). The only exceptions are proteins such as the CNTF receptor &agr; chain (see the following discussion), which lacks a cytoplasmic domain and instead has a glycosylphosphatidylinositol (GPI) anchor; however, the orientation of this protein is otherwise similar to that of a type I membrane protein. In their extracellular domains, a number of conserved features have been noted (Table 25.2). These include four conserved cysteine residues that are involved in intrachain disulfide bonding, and a tryptophan residue, located two amino acids C-terminal to the second conserved cysteine. In addition, a membrane proximal WSXWS (Trp-Ser-X-Trp-Ser) motif is generally conserved, although again exceptions exist, for example, in the growth hormone receptor, the motif is a substantially different YGEFS (Tyr-Gly-Glu-Phe-Ser) sequence, and in the IL-23R, it is WQPWS (Trp-Gln-Pro-Trp-Ser). In some cases, such as the common cytokine receptor &bgr; chain, &bgr;_c, shared by the IL-3, IL-5, and GM-CSF receptors (see subsequent discussion), the extracellular domain is extended, with a duplication of the domains containing the four conserved cysteines and the WSXWS motif. Another shared feature of type I cytokine receptors is the presence of fibronectin type III domains.

The two pairs of conserved cysteine residues are typically encoded in two adjacent exons, and the exon containing the WSXWS motif is typically just 5′ to the exon encoding the transmembrane domain. Although serines can be encoded by six different codons (ie, sixfold degeneracy in codon usage), only two of these (AGC and AGT) dominate as the codons used for the serines in WSXWS. All of these features indicate a common ancestral type I cytokine receptor.

TABLE 25.2 Features Common to Type I Cytokine Receptors

Extracellular domain
1.	Four conserved cysteine residues, involved in intrachain disulfide bonds
2.	WSXWS motif
3.	Fibronectin type III modules
Cytoplasmic domain
1.	Box 1/Box 2 regions—The Box 1 region is a proline-rich region that is involved in the interaction of Janus family tyrosine kinases.

Overall, analogous to limited sequence identity between type I cytokines, there is only limited sequence identity among type I receptor molecules. Nevertheless, they appear to form similar structures, based on the known structures for the receptors for growth hormone, prolactin, erythropoietin, IL-4, IL-13, IL-6, and IL-2⁹^,¹⁹^,²⁰^,²¹^,²²^,²³^,²⁴^,²⁵ as well as the modeling of other cytokine receptor molecules based on the known structures. The cytokines and their receptors have presumably coevolved, with the differences in amino acid sequences between different cytokines allowing for their distinctive interactions with their cognate receptor chains. Despite amino acid differences, there are also several sets of cytokines that coevolved to interact with shared receptor chains, which form type I cytokine and cytokine receptor subfamilies.⁸^,¹¹

In addition to these noted similarities in the extracellular domains, there are sequence similarities that are conserved in the cytoplasmic domain of cytokine receptors. In particular, membrane proximal “Box 1/Box 2” regions are conserved (see Table 25.2), with the proline-rich Box 1 region being the most conserved.²⁶ This will be discussed in greater detail related to its role in the binding of JAK kinases.

Type I Cytokine Receptors Are Homodimers, Heterodimers, or Higher Order Receptor Oligomers

The first cytokine receptor structure solved was that for growth hormone (Fig. 25.3).¹⁹ Prior to x-ray crystallographic analysis, it was believed that growth hormone bound to its
receptor with a stoichiometry of 1:1, but x-ray crystal solution structure revealed that a single growth hormone molecule interacts with a dimer of the growth hormone receptor, in which each receptor monomer contributes a total of seven &bgr; strands. Perhaps the most striking finding is that totally different parts of growth hormone interact with the same general region of each receptor monomer. The three-dimensional structure for the growth hormone/growth hormone receptor complex is shown in Figure 25.3. Solving the structure also clarified the basis for the assembly of the growth hormone receptor complex.¹⁹ Growth hormone first interacts with one receptor monomer via a relatively large and high-affinity interaction surface (site I), spanning ˜1230 Å². A second receptor monomer then interacts with the growth hormone/growth hormone receptor complex via two contact points: one on growth hormone (spanning ˜900 Å²) (site II) and the other on the first receptor monomer (spanning ˜500 Å²) (site III), located much more proximal to the cell membrane. Thus, three extracellular interactions are responsible for the formation and stabilization of the growth hormone/growth hormone receptor complex. Mutations in critical residues in site I should prevent growth hormone binding to its receptor, whereas inactivating mutations in site II can be predicted to prevent dimerization and signal transduction, suggesting a basis by which different classes of antagonists might be identified.

FIG. 25.3. Structure of the Growth Hormone Receptor. Shown are ribbon diagrams of the structure of the growth hormone receptor as an example of a type I cytokine receptor. For growth hormone, both growth hormone receptor monomers are shown. See text for discussion of the structures. The growth hormone/growth hormone receptor structure is from de Vos et al.¹⁹ The figure was provided by Dr. Alex Wlodawer, National Cancer Institute.

The growth hormone/growth hormone receptor structure revealed that the growth hormone receptor extracellular domain is composed of two fibronectin type III modules, each of which is approximately 100 amino acids long and contains seven &bgr; strands, resulting in the formation of an immunoglobulin (Ig)-like structure. The contact surface between ligand and receptor occurs in the hinge region that separates these two fibronectin type III modules. Analysis of a growth hormone-prolactin receptor complex revealed a similar structure for the prolactin receptor.²⁰

The growth hormone/growth hormone receptor structure served as a paradigm for the structures of other type I cytokine receptors. As a receptor homodimer, it immediately served as a model for other homodimers, such as the Epo receptor, whose structure was solved²¹ using a small protein mimetic (20 amino acid long peptide) of Epo.²⁷ The Epo receptor structure is similar to that of the growth hormone receptor, although the “site III” stem region interaction surface in the Epo receptor is much smaller than that in the growth hormone receptor, comprising only 75 Å².²¹

In addition to the structural similarities for the growth hormone and Epo receptors and perhaps other homodimeric type I cytokine receptors, a similar structure was achieved by the heterodimeric growth hormone/growth hormone receptor/prolactin receptor structure,²⁰ in which one of the growth hormone receptor monomers is replaced by a prolactin receptor molecule. Thus, the two surfaces of growth hormone interact either with two identical monomers of growth hormone receptor or with two nonidentical monomers in the case of the growth hormone receptor/prolactin receptor heterodimer.

It seems reasonable that cytokine-receptor systems with a homodimeric receptor are evolutionarily older than those with heterodimeric receptors, and that the coordination of two different receptor chains in heterodimeric receptors would have evolved in order to allow higher levels of specialization. In this regard, it is interesting that growth hormone and Epo, whose actions are vital for growth and erythropoiesis, bind to receptor homodimers, whereas the heterodimeric structures that typify the immune system are perhaps more “specialized” functions that arose later in evolution.

Interestingly, all type I cytokines known to interact with homodimers (growth hormone, prolactin, Epo, and G-CSF) are long-chain helical cytokines, although other long-chain helical cytokines (eg, cytokines whose receptors contain gp130; see following discussion) interact with heteromeric receptors. The short-chain cytokines that signal through homodimers are SCF and M-CSF, but in these cases, the receptors (c-kit and CSF-1R, respectively) are different from type I cytokine receptors in that they contain intrinsic tyrosine kinase domains. Thus, SCF and M-CSF are not typical type I cytokines and all other short-chain cytokines signal through heterodimers or more complex receptor structures (eg, IL-2 and IL-15 receptors have three components).

Heterodimeric receptors are involved when site II on a cytokine has evolved to a point where it interacts with a different receptor molecule than site I does. This latter situation is the case for many cytokines, including all short-chain type I cytokines except for SCF and M-CSF. Overall, several sets of type I cytokines fall into distinct groups, wherein each group shares at least one common receptor component. This phenomenon is observed for certain sets of both shortchain and long-chain four &agr;-helical bundle cytokines, and depending on the set of cytokines, the shared chain interacts either with site I or site II.

The structures of the low- and high-affinity forms of the IL-2 receptor have now been solved⁹^,²³^,²⁸; these are the first complete structures for a short-chain cytokine/receptor complex.²⁹ Moreover, they are of added interest in that the low-affinity receptor involves the interaction of IL-2 with IL-2R&agr;, which is not a type I cytokine receptor protein but instead is a distinctive sushi-domain containing protein, whereas the high-affinity receptor includes not only IL-2R&agr; but also IL-2R&bgr; and &ggr;_c, which are both type I cytokine receptor proteins that are shared either with IL-15 (IL-2R&bgr;) or with IL-4, IL-7, IL-9, IL-15, and IL-21 (&ggr;_c),³⁰ as discussed in the following text. In the structure of IL-2 bound to its high-affinity receptor (Fig. 25.4), there is a long peptide connecting the IL-2R&agr; globular head and transmembrane segment, allowing the binding site on this protein to extend relatively far from the cell surface in order to bind the dorsal surface of IL-2. Both the IL-2/IL-2R&agr; and IL-2/IL-2R&bgr; contacts are independent, and IL-2R&agr; does not appear to contact either IL-2R&bgr; or &ggr;_c; however, IL-2/IL-2R&bgr; forms a composite surface with &ggr;_c, somewhat analogous to the composite surface formed by growth hormone and one growth hormone receptor monomer for binding to a second monomer. As anticipated, the surface interaction between IL-2 and &ggr;_c is smaller than that between IL-2 and either of the other chains. In addition to the heterodimeric IL-2 receptor structure,²³ the IL-6³¹^,³² and LIF³³ receptor complexes,
the IL-4/IL-13 receptor³⁴ complexes, and GM-CSF receptor structure³⁵ have been solved.

FIG. 25.4. Structure of the High-Affinity Interleukin (IL)-2 Receptor. This is the first structure for a short-chain type I cytokine complexed to its complete receptor. It is particularly interesting in that it includes the sushi-domain containing IL-2R&agr; chain as well. Reprinted from Wang et al.,²³ with permission of Dr. Garcia and Science magazine.

TYPE I CYTOKINE RECEPTOR FAMILIES AND THEIR RELATIONS

Cytokines that Share the Common Cytokine Receptor &ggr;Chain (Interleukin-2, Interleukin-4, Interleukin-7, Interleukin-9, Interleukin-15, and Interleukin-21)

The receptors for six different immunologically important cytokines, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, share the common cytokine receptor &ggr; chain, &ggr;_c (CD132).¹¹^,³⁰^,³⁶^,³⁷^,³⁸^,³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³^,⁴⁴^,⁴⁵ These cytokines are all short-chain four &agr;-helical bundle cytokines; basic features of these cytokines are summarized in Table 25.3. The properties of these cytokines and their distinctive receptor chains are summarized in the following text, followed by a discussion of the discovery that they share a common receptor component and the implications thereof.

IL-2 is important not only for its function but also historically, as it was the first type I cytokine that was cloned,⁴⁶ the first type I cytokine for which a receptor component was cloned,⁴⁷^,⁴⁸ and the first short-chain type I cytokine whose receptor structure was solved.²³ Many general principles have derived from studies of this cytokine, including its being the first cytokine that was demonstrated to act in a growth factor-like fashion through specific high-affinity receptors, analogous to the growth factors being studied by endocrinologists and biochemists.⁴⁹^,⁵⁰

Mature IL-2 is a 133 amino acid long peptide that can act as a major T-cell growth factor, in keeping with its original discovery as a T-cell growth factor.⁵¹ Although IL-2 is not produced by resting T cells, it is rapidly and potently induced following antigen encounter with resting CD4+ T cells, and transcription and synthesis of IL-2 are often used as indicators of T-cell receptor (TCR)-mediated cellular activation. Although the antigen determines the specificity of the T-cell immune response, the interaction of IL-2 with high-affinity IL-2 receptors regulates the magnitude and duration of the subsequent response, based on the amount of IL-2 produced, the levels of high-affinity receptors expressed, and the duration of IL-2 production and receptor expression. IL-2 can act in either an autocrine or paracrine fashion, depending on whether the producing cell is also the responding cell or whether the responding cell is a nonproducing cell. The gene encoding IL-2 is located on chromosome 4,⁵² and like many other helical cytokines, its gene consists of four exons.⁷ IL-2 binds to three different classes of receptors. These are formed by different combinations of three different chains, IL-2R&agr;,⁴⁷^,⁴⁸^,⁵³ IL-2R&bgr;,⁵⁴^,⁵⁵^,⁵⁶^,⁵⁷ and a protein initially called IL-2R&ggr;³⁶ but now known as the common cytokine receptor &ggr; chain, &ggr;_c.¹¹^,³⁰^,³⁷ These different classes of IL-2 receptors are discussed in the following text.

In addition to its being a T-cell growth factor, IL-2 has other important actions as well (see Table 25.3).²⁹ For example, it can increase Ig synthesis and J chain transcription in B cells,⁵⁰^,⁵⁸^,⁵⁹ potently augment the cytolytic activity of

natural killer (NK) cells,⁶⁰^,⁶¹^,⁶² induce the cytolytic activity of lymphokine activated killer cells, promote the elimination of autoreactive cells in a process known as antigen-induced (or activation-induced) cell death,²⁹^,⁵⁰^,⁶³ and promote the differentiation of regulatory T (T_reg) cells,⁶⁴^,⁶⁵ cells that suppress inappropriate responses and are important for immunologic tolerance. Low-dose IL-2 is sufficient to promote T_reg-cell survival, and thereby, for example, can protect mice from developing autoimmune diabetes.⁶⁶ Interestingly, IL-2 can also prime CD8+ T cells during a primary response to undergo enhanced proliferation in vivo during a secondary response,⁶⁷^,⁶⁸ and autocrine IL-2 is believed to be required for secondary expansion of CD8+ memory T cells.⁶⁹ There appears to be a complex interplay between IL-2 and inflammatory signals to regulate effector and memory cytolytic T-lymphocytes generation in lymphocytic choriomeningitis virus infection, with persistent IL-2 promoting effector rather than memory cytotoxic T-lymphocyte development.⁷⁰^,⁷¹ In Listeria monocytogenesis infection, Th1 effector memory cells highly express the transcription factor T-Bet and IL-2R&agr;, and IL-2R&agr; appears to be critical for the development of these cells.⁷² Presumably because of its critical role in T_reg development, the absence of IL-2, IL-2R&agr;, or IL-2R&bgr; leads to autoimmunity. Interestingly, the production of IL-2 by mast cells has been reported to contribute to the suppression of chronic allergic dermatitis by its increasing the relative number of T_reg cells at the site of inflammation.⁷³

TABLE 25.3 Features of Cytokines Whose Receptors Share &ggr;_c

Cytokine	Major Source	Size^a	Actions	Chromosome Location (h/m)	Genomic Org
IL-2	Activated T cells (Th1 cells)	h153 aa/20aa m169aa/20aa 15.5 kDa	T-cell growth factor B-cell growth, Ig production, J chain expression Induces LAK activity Induces tumor infiltrating lymphocyte activity Augments NK activity Critical roles in antigen-induced cell death Stimulates macrophage/monocyte Antitumor effects Promotes T_reg development Promotes Th1 and Th2 differentiation Inhibits Th17 differentiation	4q26-27/3	Four exons
IL-4	Activated T cells (Th2 cells) CD4+NK1.1+ natural T cells	h153/24 aa m140 aa/20 aa 18 kDa	B-cell proliferation Ig class switch: IgG1, IgE production Augment MHC II, Fc&egr; receptors, IL-4R&agr;, and IL-2R&bgr; expression Th2 cell differentiation Antitumor effects	5q31.1/11	Four exons
IL-7	Stromal cells	h177 aa/25aa m154aa/25aa 17-25 kDa	Thymocyte growth T-cell growth Pre-B-cell growth in mice but not human Survival and growth of peripheral T cells CD4+ and CD8+ T-cell homeostasis	8q12-13/3	Six exons
IL-9	Activated Th cells	h144aa/18aa m144aa/18aa 14 kDa	Th helper clones Erythroid progenitors B cells Mast cells/allergic responses Fetal thymocytes	5q31-35/13	Five exons
IL-15	Monocytes and many cells outside the immune system^b	h162aa/48aa m162aa/48aa 14-15 kDa	Mast cell growth NK cell development and activity T-cell proliferation CD8+ T-cell homeostasis	4q31/8	Nine exons
IL-21	Activated CD4+ T cells	h162aa/31 aa m146aa/24 aa	Comitogen for T-cell proliferation Inhibits B-cell proliferation to anti-IgM + IL-4 Augments B-cell proliferation to anti-CD40 Conflicting reports related to NK cells Cooperates with IL-7 and IL-15 to expand CD8 cells Antitumor effects Drives terminal B-cell differentiation to plasma cells Proapoptotic for B and NK cells Promotes Tfh differentiation Promotes Th17 differentiation	4q67-27/3	Five exons
CD, cluster of differentiation; Ig, immunoglobulin; IL, interleukin; LAK, lymphokine activated killer; MHC, major histocompatibility complex; NK, natural killer; Tfh, T follicular helper; Th, T helper; T_reg, regulatory T.
^ah and m refer to human and mouse, respectively. The number of amino acids refers to the length of the open reading frame/length of signal peptide. The number of amino acids in the mature protein is therefore the difference between these numbers. Note that for IL-15, residues 1 to 29 have been identified as a signal peptide and 30 to 48 as a propeptide. ^bMore IL-15 messenger ribonucleic acid is produced in skeletal muscle, kidney, placenta, and lung than in thymus or spleen. It is important to note, however, that IL-15 messenger ribonucleic acid is widely expressed without concomitant production of IL-15 protein so that the source of biologically meaningful IL-15 may be more limited.

IL-2 is also important in inducing or inhibiting Th differentiation. It is required for efficient Th1 74 and Th2 differentiation,⁷⁵^,⁷⁶^,⁷⁷^,⁷⁸ which are populations of T helper cells first described based on patterns of cytokine production by mouse T cells.⁷⁹ This theme was then extended to human cells as well,⁸⁰^,⁸¹^,⁸²^,⁸³^,⁸⁴ although there are some variations in humans and mice in terms of the degree of how tightly restricted cytokine expression is. IFN&ggr; is the cytokine most reliably produced by Th1 cells; IL-2 is also produced by these cells, although without as rigorous an association. In contrast, IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 are produced by Th2 cells. In both species, certain type I cytokines (eg, IL-3 and GM-CSF) are produced by both Th1 and Th2 cells. IL-12 and the transcription factors signal transducer and activator of transcription (STAT)4 and T-Bet are the major transcription factors promoting Th1 differentiation, whereas IL-4 and the transcription factor STAT6 drive Th2 differentiation. For Th1 cells, IL-2 acts by STAT5-dependent induction and expression of IL-12R&bgr;1/IL-12R&bgr;2 and T-Bet,⁷⁴ whereas for Th2 differentiation, IL-2 via STAT5 promotes IL-4⁷⁵^,⁷⁶ and IL-4R&agr;⁷⁷ expression. In addition, IL-2 also affects chromatin remodeling at the Ifng locus during Th1 differentiation.⁸⁵ IL-2 also inhibits Th17 differentiation,⁸⁶ in part via downregulation of IL-6R&agr;/gp130 expression⁷⁴ and by inducing T-bet,⁷⁴^,⁸⁷ which prevents Runx1-mediated activation of ROR&ggr;t, a transcription factor that drives Th17 differentiation.⁸⁷

Like IL-2, IL-4 is produced primarily by activated CD4+ T cells.⁸⁸^,⁸⁹ IL-4 is also produced by CD4+NK1.1+ “natural” T cells, denoted as natural killer T (NKT) cells,⁹⁰ and by mast cells and basophils.⁸⁸ IL-4 is the major B-cell growth factor, and it promotes Ig class switch, enhancing the production and secretion of mouse IgG1 (human IgG4) and being essential for the production of IgE.⁸⁸ IL-4 is involved in the physiologic response to parasites, including helminths, and for allergen sensitization. IL-4 induces expression of class II major histocompatibility complex molecules and increases cell surface expression of the CD23 (the low-affinity IgE receptor) on B cells. In addition to its actions on B cells, IL-4 can also act as a T-cell growth factor, inducing proliferation in both human and mouse T cells, and is critical for normal differentiation of Th2 cells.⁸⁹ Moreover, when IL-4 and transforming growth factor (TGF)-&bgr; are combined, cells that produce IL-9 (Th9) cells are induced (see following discussion). When combined with phorbol 2-myristate 3-acetate, IL-4 is also a potent comitogen for thymocytes. Importantly, IL-4 can inhibit certain responses of cells to IL-2.⁹¹ Moreover, IL-4 can exert actions on macrophages, hematopoietic precursor cells, stromal cells, and fibroblasts.⁹² The gene encoding IL-4 is located on human chromosome 5 (5q23.3-31.2) and mouse chromosome 11,⁹²^,⁹³ in the same region as IL-3, IL-5, IL-13, and GM-CSF. The type I IL-4 receptor is expressed on T cells and other hematopoietic cells consists of the 140 kDa IL-4R&agr; protein⁸⁸^,⁹⁴^,⁹⁵^,⁹⁶ and &ggr;_c.³⁸^,³⁹ Expression of IL-4R&agr; tends to be quite low, and cells that potently respond to IL-4 often express only a few hundred receptors per cell. In addition to the type I IL-4 receptor, an alternate form of the receptor (the type II IL-4 receptor), containing IL-4R&agr; and IL-13R&agr;1, although not expressed on mature T cells, is expressed on many other cell types and can transduce IL-4 signals into these cells. For example, IL-13R&agr;1 is expressed on neonatal Th1 cells and the type II IL-4 receptor has been implicated as mediating apoptosis of these cells.⁹⁷

IL-7 is not produced by lymphocytes but instead is a 152 amino acid long cytokine that is produced by stromal cells and certain other cells.⁹⁸^,⁹⁹^,¹⁰⁰ Based on the analysis of patients with X-linked severe combined immunodeficiency (SCID), JAK3-deficient SCID, and IL-7R&agr;-deficient SCID, IL-7 receptor-dependent signaling is essential for T-cell development in humans (discussed subsequently). The major role of IL-7 is to enhance thymocyte survival, growth, and differentiation¹⁰¹^,¹⁰²^,¹⁰³^,¹⁰⁴^,¹⁰⁵ as well as low-affinity peptide-induced proliferation, and thus it promotes homeostatic proliferation of naive and memory CD8+ T cells.¹⁰⁰^,¹⁰⁵^,¹⁰⁶^,¹⁰⁷ Additionally, IL-7 can regulate the homeostasis of CD4+ memory T cells¹⁰⁸^,¹⁰⁹ and also can stimulate the growth of mature T cells.³⁰^,¹⁰⁵^,¹¹⁰^,¹¹¹ Although IL-7, as noted previously, is believed to primarily be a stromal factor, IL-7 has also been noted to be produced by the liver and kidney but not spleen or lymph nodes after toll-like receptor (TLR) signaling. Although basal liver production is low, after TLR signaling, hepatic IL-7 can promote CD4 and CD8 T-cell survival and promotes antigen-specific T-cell responses.¹¹² In addition, in the mouse, IL-7 is vital for the growth of mouse pre-B cells,⁹⁶^,⁹⁸^,¹⁰³^,¹⁰⁴^,¹¹¹ and transient IL-7 signaling can inhibit Ig heavy chain gene rearrangements.¹¹³ IL-7 via STAT5 has also been shown to inhibit Ig-&kgr; recombination in pro-B cells.¹¹⁴ In contrast to its requirement for B-cell development in the mouse, there is normal B-cell development in patients with defective IL-7 signaling, as is found in patients with X-linked SCID, JAK3-deficient
SCID, and IL-7R&agr;-deficient SCID (see following discussion). Thus, human B cells can develop normally in the absence of IL-7 responsiveness, demonstrating that in humans, IL-7 is not vital for the growth of human pre-B cells,³⁰^,¹¹⁵ and it remains unknown whether IL-7 plays important roles in human B-cell biology. Interestingly, IL-7 also acts on DCs, and this appears to limit the homeostatic proliferation of T cells under lymphopenic conditions.¹⁰⁰^,¹¹⁶ Clinically, IL-7 has been of interest in terms of its ability to induce T-cell proliferation and to thereby increase T-cell numbers, with a greater effect on CD8+ than on CD4+ T cells, as well as to augment the diversity of the TCR repertoire¹¹⁷; IL-7 also can increase antigen-specific responses following vaccination¹⁰⁰ and can promote antiviral immunity, apparently in part due to its induction of IL-22 and repression of SOCS3.¹¹⁸ The gene encoding IL-7 is located on human chromosome 8q12 to 8q13¹¹⁹ and mouse chromosome 3. The functional IL-7 receptor contains the 75 kDa IL-7R&agr;¹²⁰ and &ggr;_c.³⁷^,⁴⁰ Interestingly, signaling via the TCR, IL-2 or IL-7 can downregulate IL-7R&agr; expression,¹²¹^,¹²² with PI 3-kinase/AKT and GFI1B being implicated in its downregulation in T cells.¹²²^,¹²³^,¹²⁴ The downregulation of IL-7R&agr; not only can both decrease responsiveness of cells but can also increase the availability of the cytokine for other cells that are poised to respond.¹¹ Induction of IL-7R&agr; requires PU.1 in B cells¹²⁵ and another Ets family protein, GABP, in T cells.¹²³

IL-9 was originally described as a mouse T-cell growth factor¹²⁶ that is produced by activated T cells and can support the growth of T-helper clones but not of cytolytic clones.¹²⁷ In contrast to IL-2, its production is delayed, suggesting its involvement in later, perhaps secondary signals. In the mouse, IL-9 can exert proliferative effects on erythroid progenitors, B cells, B-1 cells, mast cells, and fetal thymocytes. IL-9 is identical to mast cell growth-enhancing activity, a factor present in conditioned medium from splenocytes,¹²⁸ and synergizes with IL-3 for maximal mast cell proliferation. IL-9 is highly expressed in the lung of patients with asthma,¹²⁹ and overexpression leads to airway inflammation and Th2 cytokine production.¹³⁰ Interestingly, IL-9 is substantially made in the lung by innate lymphoid cells, and in these cells neutralizing antibodies to IL-9 lowered expression of IL-13 and IL-5, supporting a link between IL-9 and the regulation of the Th2 response.¹³¹ Consistent with the action of IL-9 on thymocytes in vitro, IL-9 transgenic mice develop thymic lymphomas, and IL-9 is a major antiapoptotic factor for such tumors.¹³² Nevertheless, IL-9 knockout mice have normal T-cell development¹³³; instead, they exhibit a defect in pulmonary goblet cell hyperplasia and mastocytosis following challenge with Schistosoma mansoni eggs, a synchronous pulmonary granuloma formation model. However, there was no defect in eosinophilia or granuloma formation.¹³³ Mice expressing an IL-9 transgene in the lung exhibit airway inflammation and bronchial hyperresponsiveness; nevertheless, IL-9-/- mice exhibit normal eosinophilia and airway hyperreactivity in an ovalbumin-induced inflammatory model.¹³⁴^,¹³⁵^,¹³⁶ Thus, although IL-9 can contribute to allergic/pulmonary responses, there are compensatory cytokines that substitute for IL-9 in at least certain settings. IL-9 can be produced by the Th9 populations of cells, which can be induced by IL-4 plus TGF-&bgr; in a fashion that is dependent on PU.1 and interferon regulatory factor (IRF)-4.¹³⁷ Interestingly, the addition of IL-25 further enhances the production of IL-9.¹³⁸ IL-9-producing cells are typically IL-9+IL-10+Foxp3-effector T cells and can be derived from Th2 cells.¹³⁹^,¹⁴⁰ Overall, IL-9 is believed to play a key role in allergic responses; additionally, given the ability of both Th17 and T_reg cells to produce IL-9 during autoimmunity and transplantation, it will be interesting to further clarify the role of IL-9 in autoimmunity.¹⁴¹ Currently, there is some confusion, as one study showed that antibody blockade of IL-9 or IL-9R&agr; blocks experimental autoimmune encephalomyelitis (EAE) disease progression,¹⁴² whereas another study showed that IL-9R&agr; knockout mice develop more severe EAE.¹³⁸ While mouse IL-9 is active on human cells, human IL-9 is not biologically active on mouse cells (the opposite situation from that for IL-2). Human IL-9 is located on chromosome 5q31 to 5q35,¹⁴³ which is also the location for the genes encoding IL-3, IL-4, IL-5, IL-13, and GM-CSF. In contrast, mouse IL-9 is “isolated” on chromosome 13, while IL-3, IL-4, IL-5, IL-13, and GM-CSF are clustered on chromosome 11. IL-9 binds to the 64 kDa IL-9R&agr; binding protein, which is similar in size to &ggr;_c,¹⁴⁴ and the functional IL-9 receptor consists of IL-9R&agr; plus &ggr;_c.³⁰^,⁴¹^,⁴²

IL-15 was identified as a T-cell growth factor that also unexpectedly was expressed in the supernatant of a human T-lymphotropic virus type I (HTLV-I)-transformed T-cell line.¹⁴⁵^,¹⁴⁶ Although IL-15 messenger ribonucleic acid (RNA) is produced by a range of nonlymphocytic cell types, it is difficult to detect physiologic levels of IL-15 protein.¹⁴⁷ Its main site of synthesis appears to be DCs and monocytes, and unlike IL-2, IL-15 is not produced by activated T cells.¹⁴⁸ IL-15 receptors are widely expressed; although IL-15 is perhaps most important for the development of NK cells¹⁴⁹^,¹⁵⁰^,¹⁵¹ and CD8+ memory T cells,¹⁵⁰^,¹⁵¹ it also has both paracrine and autocrine actions on DCs, including promoting the survival of these cells.¹¹ Interestingly, it also regulates the TCR repertoire of &ggr;&dgr; intraepithelial lymphocytes¹⁵² and cross talk between different types of DCs.¹⁵³ The receptor for IL-15 on T cells contains IL-2R&bgr;,⁴³^,¹⁴⁷^,¹⁵⁴ &ggr;_c,⁴³ and an IL-15-specific protein, IL-15R&agr;. IL-15R&agr; shares a number of structural similarities with IL-2R&agr;, including that it is a sushi domaincontaining protein (IL-2R&agr; has two sushi domains, whereas IL-15R&agr; has one),¹⁵⁵ and the IL2RA and IL15RA genes are closely positioned on human chromosome 10p14.¹⁵⁶ A distinctive feature of IL-15 signaling is that it signals substantially by a process called transpresentation, wherein IL-15R&agr; on the surface of dendritic cells or monocytes will transpresent IL-15 to responding cells such as CD8+ T cells or NK cells that express IL-2R&bgr; + &ggr;_c.¹⁴⁸^,¹⁵⁷ IL-15R&agr; has a longer cytoplasmic domain than IL-2R&agr; and appears to be critical for IL-15R&agr; function, although potentially not for transpresentation.¹⁵⁸ Interestingly, transpresentation can be utilized by IL-2 as well.¹⁵⁹ The very high affinity of IL-15R&agr; for IL-15 is explained by a large number of ionic interactions mediated by the sushi domain.¹⁶⁰ Note that these responding cells can also express IL-15R&agr;. In contrast to IL-2, which is a growth factor as well as a mediator of antigen-induced (or activationinduced) cell death and promoter of T_reg differentiation, the
role of IL-15 appears to be more focused on growth of CD8+ T cells,¹⁶¹ maintaining long-lasting, high-avidity T-cell responses to foreign pathogens (ie, CD8+ T-cell memory).¹⁴⁸^,¹⁶² This ability has suggested it could have potential as a vaccine adjuvant.¹⁶³ Overall, the fact that IL-15 promotes the proliferation and differentiation of B, T, and NK cells; the cytolytic activity of CD8 T cells; and the maturation of dendritic cells, yet, unlike IL-2, fails to stimulate immunosuppressive T_reg cells indicates that it has an array of properties that make it potentially promising as an anticancer therapeutic agent; as a result, a number of ongoing clinical trials are now in progress.¹⁶⁴

IL-21 is the most recently identified member of the IL-2 family of cytokines. IL-21 can bind to specific receptors and exert actions on T, B, NK cells, DCs, and macrophages.¹⁶⁵ It augments T-cell proliferation as a comitogen,¹⁶⁶ can cooperate with IL-7 or IL-15 to drive the expansion of freshly isolated mouse CD8+ T cells, and it can augment the antitumor activity of CD8+ T cells.¹⁶⁷ IL-21 promotes the differentiation of Th17 cells¹⁶⁸^,¹⁶⁹ as well as the generation of T follicular helper cells.¹⁷⁰ In the case of Th17 differentiation, IL-21 is part of an IL-6 to IL-21 to IL-23 signaling cascade¹⁶⁹^,¹⁷¹^,¹⁷² to drive the differentiation of these cells in an ROR&ggr;-dependent manner.¹⁷³ Polarization of Th17 cells is dependent on TCR stimulation, TGF-&bgr;, and IL-6 but is independent of IL-23, which instead may be required for maintaining/expanding these cells.¹⁷⁴^,¹⁷⁵^,¹⁷⁶^,¹⁷⁷

The actions of IL-21 on B cells are particularly complex. It augments B-cell proliferation when combined with anti-CD40 or lipopolysaccharide (LPS) but inhibits proliferation in response to anti-IgM + IL-4¹⁶⁶; this inhibition is reversed if anti-CD40 is additionally provided. It induces apoptosis of incompletely activated B cells, perhaps serving a role analogous to that of IL-2 in activation-induced cell death of T cells to eliminate incompletely activated cells.¹⁶⁵ In contrast, IL-21 drives terminal differentiation to plasma cells of more fully activated cells. Strikingly, IL-21 can drive plasma cell differentiation of both peripheral memory B cells and cord blood B cells, at least in part explained by its ability to induce expression of BLIMP1.¹⁷⁸^,¹⁷⁹ Strikingly, IL-21 regulates not only BLIMP-1 but also a broad range of genes via a functional cooperation between STAT3 and IRF4.¹⁸⁰ In IL-21R knockout mice, following immunization, IgG1 is diminished related to a role for IL-21 in class switching to IgG1 and IgG3, whereas IgE is elevated, related to the ability of IL-21 to inhibit C&egr; transcription¹⁸¹ and/or to its ability to augment the apoptosis of IgE-producing B cells. Analysis of IL-21R/IL-4 double knockout mice has revealed that IL-21 cooperates with IL-4 to globally regulate Ig production in that these mice exhibit a panhypogammaglobulinemia, mimicking the T-cell phenotype in humans with X-linked SCID.¹⁸² IL-21 can also cooperate with IL-15 and Flt-3 ligand to increase development of NK cells¹⁶⁶ and augment antitumor activity¹⁸³; however, it was also reported to oppose the actions of IL-15¹⁸⁴ and can also direct NK cell apoptosis. Interestingly, IL-21 exerts potent antitumor effects, including against large established solid tumors.¹¹^,¹⁶⁵ In the pMEL-1 CD8 TCR transgenic system, the effector cells recognize a melanoma antigen. When cells are adoptively transferred into tumor-bearing mice and animals are vaccinated with the cognate antigen, the addition of IL-21 or IL-21 plus IL-15 has potent antitumor effects.¹⁶⁷ Moreover, treatment of cells with IL-21 in vitro prior to adoptive transfer results in markedly enhanced antitumor effects in vivo, conferring a distinctive differentiation program from that conferred by IL-2.¹⁸⁵ IL-21 is now in phase II clinical trials for cancer. In addition to its anticancer activity, IL-21 can also promote autoimmunity. Elevated IL-21 levels have been reported in the BXSB-Yaa mouse model of systemic lupus erythematosus,¹⁷⁸ and elevated IL-21 levels have been found in a subset of humans with systemic lupus erythematosus (P. Lipsky, personal communication). Elevated IL-21 has also been associated with other autoimmune processes, including in the non-obese diabetic (NOD) mouse.¹⁸¹ More importantly, in mouse models of type 1 diabetes, lupus, and experimental allergic uveitis, no autoimmune disease develops on an IL-21R knockout background.¹⁸⁶^,¹⁸⁷^,¹⁸⁸ IL-21 unexpectedly can also be immunosuppressive via its induction of IL-10.¹⁸⁹^,¹⁹⁰ Consistent with this, an IL-21/IL-10/STAT3 pathway is required for normal development of memory CD8+ T cells after lymphocytic choriomeningitis virus (LCMV) infection¹⁹¹; similarly, STAT3, presumably by an overlapping mechanism is important for human T-cell memory development, as evidenced by studies in patients with autosomal dominant hyper-IgE syndrome, which is caused by dominant negative mutations in STAT3.¹⁹² IL-21 has complex roles in viral responses. For LCMV, IL-21 is required to control chronic infection,¹⁹³ whereas IL-21 mediates the pathogenic response after infection with pneumonia virus of mice.¹⁹⁴ Interestingly, for hepatitis B virus, IL-21 appears to be critical in determining the age-dependent effectiveness of immune responses, wherein decreased IL-21 production in younger individuals hinders the generation of critical lymphoid responses to the virus, whereas more robust IL-21 production in adults is associated with viral clearance that occurs in 95% of patients.¹⁹⁵

The receptor for IL-21 consists of IL-21R plus &ggr;_c.³⁰^,⁴⁴ IL-21R is most related to IL-2R&bgr;, and like IL-2R&bgr;, its expression is induced following cellular stimulation with anti-CD3 or phytohemagglutinin, and in addition, its expression is augmented in T cells following transformation with HTLV-I.¹⁹⁶ Both human and mouse IL-21 can act on cells of the other species. IL-21 is on human chromosome 4q26 to 4q27, while its receptor is on chromosome 16p11, immediately downstream of the IL4R gene.

Thus, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 collectively exhibit partially overlapping roles related to T cells, NK cells, B cells, and mast cells, and together would be expected to play vital roles for normal development and/or function of these cellular lineages. As discussed in the following, IL-2, IL-7, IL-9, and IL-15 utilize primarily STAT5A and STAT5B; IL-4 activates primarily STAT6; and IL-21 activates STAT1, STAT3, STAT5A, and STAT5B, with STAT3 being the dominant STAT for this cytokine.¹¹ The fact that these six cytokines share &ggr;_c is of particular interest, especially as the gene encoding &ggr;_c is mutated in patients with the most common form of severe combined immunodeficiency in humans.

X-Linked Severe Combined Immunodeficiency Disease Results from Mutations in the Gene Encoding &ggr;_c

The &ggr; chain was originally identified as a third component of the IL-2 receptor³⁶ after it became clear that IL-2 receptor &agr; and &bgr; chains alone were not sufficient to transduce an IL-2 signal. The hypothesis that the &ggr; chain was a shared component of receptors for cytokines in addition to IL-2 was motivated from a comparison of the clinical phenotypes in humans that result from defective expression of IL-2 versus. the &ggr; chain. In 1993, it was discovered that mutations in the gene encoding the &ggr; chain resulted in X-linked SCID (the disease is also designated as SCIDX1).³⁰^,¹⁹⁷ X-linked SCID is characterized by profoundly diminished numbers of T cells and NK cells³⁰^,⁴⁵^,¹⁹⁷^,¹⁹⁸^,¹⁹⁹^,²⁰⁰^,²⁰¹ (Table 25.4). Although the B cells are normal in number, they are nonfunctional, apparently due to a lack of T-cell help as well as an intrinsic B-cell defect.⁴⁵^,²⁰⁰^,²⁰² In contrast to the profoundly decreased number of T cells in patients with X-linked SCID, IL-2-deficient patients²⁰³^,²⁰⁴ and mice²⁰⁵ have normal numbers of T cells (the phenotypes of mice deficient in type I and type II cytokines and their receptor, JAK kinases, and STAT proteins are summarized in Table 25.15). This observation indicated that defective IL-2 signaling was unlikely to be responsible for X-linked SCID, making the finding that the gene encoding the &ggr; chain was mutated in X-linked SCID all the more unexpected. Thus, the conundrum was why a defect in a component of a receptor would cause a more severe than a defect in the corresponding cytokine. This led to the hypothesis that the &ggr; chain was critical for other cytokine receptors as well.¹⁹⁷ In this model, defective IL-2 signaling either did not contribute to the defects in X-linked SCID or these defects were explained by the simultaneous inactivation of multiple signaling pathways.⁴⁵^,¹⁹⁷ Indeed, it was found that the &ggr; chain was also an essential component of both the IL-4 and IL-7 receptors on T cells,³⁷^,³⁸^,³⁹^,⁴⁰ leading to it being renamed as the common cytokine receptor &ggr; chain, &ggr;_c.³⁷^,³⁸ IL-9, IL-15, and IL-21 were subsequently also shown to share &ggr;_c³⁰ (Fig. 25.5).

The sharing of &ggr;_c by six different cytokine receptors revealed that X-linked SCID is a disease of defective cytokine signaling. The major deficiencies in X-linked SCID can be attributed to defects related to different cytokines. Based on the dramatically diminished T-cell development not only in IL-7-deficient¹⁰⁴ or IL-7R&agr;-deficient¹⁰³ mice but also in IL-7R-deficient humans with T-B+NK+ SCID,²⁰⁶^,²⁰⁷ yet normal T-cell development in mice deficient in IL-2,²⁰⁵ IL-4,²⁰⁸^,²⁰⁹ both IL-2 and IL-4,²¹⁰ IL-9,¹³³ IL-15,¹⁵¹ IL-15R&agr;,¹⁵⁰ IL-21R,¹⁸²^,¹⁸⁴ and most if not all of the defect in T-cell development in patients with X-linked SCID can be attributed to defective IL-7 signaling.³⁰ In addition to profoundly diminished numbers of T cells, humans with X-linked SCID lack NK cells. As discussed previously, NK-cell development is defective in IL-15- and IL-15R&agr;-deficient mice, indicating that it is defective IL-15 signaling that is responsible for the defective NK-cell development in X-linked SCID.³⁰

TABLE 25.4 Features of X-Linked Severe Combined Immunodeficiency

1.	Absent or profoundly diminished numbers of T cells and mitogen responses
2.	Absence of NK cells
3.	Normal numbers of B cells, but defective B-cell responses
4.	IgM can be normal but greatly diminished Igs of other classes
5.	XSCID carrier females exhibit nonrandom X-inactivation patterns in their T cells and NK cells; the X-inactivation pattern is random in surface IgM-positive B cells but nonrandom in more terminally differentiated B cells.
Ig, immunoglobulin; NK, natural killer; XSCID, X-linked severe combined immunodeficiency.

FIG. 25.5. Schematic of the Receptors for Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21, Showing Interactions with JAK1 and JAK3. The figure shows that IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 all share &ggr;_c. IL-2R&agr; and IL-15R&agr; are not shown. Whereas the distinctive chains associate with JAK1, &ggr;_c associates with JAK3. Mutations in &ggr;_c cause X-linked severe combined immunodeficiency or more moderate forms of X-linked immunodeficiency. Mutations in JAK3 cause an autosomal recessive form of severe combined immunodeficiency (see text).

In contrast to the greatly diminished number of T cells and absent NK cells in patients with X-linked SCID, B-cell numbers are normal. This is in contrast to the greatly diminished numbers of B cells in &ggr;_c-deficient mice²¹¹^,²¹² as well as mice deficient in either IL-7 or IL-7R&agr;, indicating that IL-7 is not required for pre-B-cell development in humans and underscoring a major difference for IL-7 in human versus mouse biology. Indeed, patients with IL-7R-deficient SCID have a T-B+NK+ form of SCID.²⁰⁶^,²⁰⁷ Although B cells develop in patients with X-linked SCID, they are nonfunctional. This is due in part to a lack of T-cell help (given the near absence of T cells in X-linked SCID), but a variety of data indicate an intrinsic B-cell defect as well.³⁰ As discussed
previously, analysis of IL-4/IL-21R double knockout mice indicate that defective signaling by IL-4 and IL-21 appear to explain the intrinsic B-cell defect in X-linked SCID.¹⁸²

Rationale for the Sharing of &ggr;_c

Why should there have been evolutionary pressure to maintain the sharing of &ggr;_c, given the obvious increased risk associated with sharing a receptor component when it is mutated? There are at least two different types of models.⁴⁵^,²¹³ First, the sharing of &ggr;_c could be a basis for shared actions. For example, given that IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 can each act as T-cell growth factors, at least in vitro, it is possible that &ggr;_c might couple to signal transducing molecule(s) that promote T-cell growth. Second, the sharing of &ggr;_c might represent a means by which one cytokine using &ggr;_c can modulate the signals of the others. To understand this model, it is important to recognize that, in contrast to antigen receptor complexes, cytokine receptor components individually are targeted to the cell surface, and the formation/stability of receptor complexes is promoted or stabilized by ligand binding, as noted in the discussion of growth hormone, wherein the second receptor monomer recognizes the combined surface of growth hormone and the first growth hormone receptor monomer¹⁹ or for the IL-2 receptor where &ggr;_c “sees” a combined IL-2/IL-2R&bgr; surface.²³ This latter finding is consistent with &ggr;_c originally having been coprecipitated with IL-2R&bgr; in the presence but not in the absence of IL-2,²¹⁴ and dimerization of IL-2R&bgr; and &ggr;_c is known to be required for efficient signaling.²¹⁵^,²¹⁶ Thus, receptor heterodimerization at a minimum is stabilized by the cytokine and physiologically may be absolutely dependent on the presence of the cytokine. In the absence of stable preformed cytokine receptor complexes between &ggr;_c and the other receptor chains, one can envision that &ggr;_c might be differentially recruited to different receptors based on the relative amount of a cytokine or its binding efficiency. In a situation where &ggr;_c is limiting, a cytokine might then not only induce its own action but could also simultaneously inhibit the action of another cytokine that was less efficient at recruiting &ggr;_c to its cognate receptor complex.

An analysis of mice deficient in IL-2,²⁰⁵ IL-2R&agr;,²¹⁷ IL-2R&bgr;,²¹⁸ and &ggr;_c²¹¹^,²¹² provides the interesting observation that although the mice lacking &ggr;_c have defective signaling in six different cytokine pathways, mice deficient in IL-2, IL-2R&agr;, and IL-2R&bgr; appear to be less healthy than the &ggr;_c-knockout mice with more markedly activated T cells and autoimmunity that is not evident in &ggr;_c-deficient mice. This is at least in part explained by the fact that IL-2-deficient mice lack T_reg cells and thus develop autoimmune disease,⁶⁵ whereas &ggr;_c-deficient mice lack T cells due to defective IL-7 signaling and thus lack effector T cells. These and other data indicate that &ggr;_c plays a major role in regulating lymphoid homeostasis, as originally indicated.²¹⁹

TABLE 25.5 Features of Cytokines whose Receptors Share the Common &bgr; Chain, &bgr;_c

Cytokine	Major Source	Size	Cellular Targets	Chromosome Location (h/m)	Exons
IL-3	T cells	h152/19aa m166/26 aa 22-34 kDa	Multiple lineages	5q31.1/11	5
IL-5	T cells	h139/22aa m133/21 45 kDa dimer	Eosinophils B cells (?)	5 q31.1/11	4
GM-CSF	T cells	h144/17aa m141/17aa 23 kDa	Granulocytes Macrophages	5q31.1/11	4
GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin.

Cytokines whose Receptors Share the Common &bgr; Chain, &bgr;_c (Interleukin-3, Interleukin-5, and Granulocyte Macrophage-Colony Stimulating Factor)

The hematopoietic cytokines, IL-3, IL-5, and GM-CSF (Table 25.5) are all synthesized by T cells and exert effects on cells of hematopoietic lineage.²²⁰^,²²¹^,²²² These cytokines are vital for proliferation as well as differentiation of myeloid precursor cells. Of these three cytokines, IL-3 is the most pluripotent²²¹ and historically was also called multi-CSF, reflecting the large number of lineages on which it can act. It can act to promote proliferation, survival, and development of multipotent hematopoietic progenitor cells and of cells that have become dedicated to a range of different lineages, including granulocyte, macrophage, eosinophil, mast cell, basophil, megakaryocyte, and erythroid lineages. It induces a range of effects, including, for example, inducing the production off IL-4 by basophils.²²³ IL-3 also can exert end-function effects, such as enhancing phagocytosis and cytotoxicity. GM-CSF is mainly restricted to the granulocyte and monocyte/macrophage lineages, but its actions are nevertheless still quite broad.²²⁰ It is both a growth and survival factor. In addition, it can expand the number of antigen-presenting cells, such as DCs, and thereby may greatly expand the ability of the host to respond to antigen. IL-23 and ROR&ggr;t can augment GM-CSF production in Th cells, whereas IL-12, IFN&ggr;, and IL-27 negatively regulate its expression, and GM-CSF is now known to be required for the initiation of autoimmune neuroinflammation in experimental autoimmune encephalitis. Collectively, these results suggest that GM-CSF is important for encephalitogenic actions of both Th1 and Th17 cells.²²⁴^,²²⁵ GM-CSF mediates autoimmune effects at least in part by enhancing IL-6-dependent
Th17 cell development and survival.²²⁶ Whereas IL-3 and GM-CSF can act on eosinophils, they act at much earlier stages than IL-5, presumably expanding the number of eosinophil-committed precursors cells. IL-5 stimulates the eosinophilic lineage and eosinophil release from the bone marrow and is essential for expanding eosinophils after helminth infections,²²⁷^,²²⁸ and can mediate the killing of S. mansoni. IL-5 can also induce Ig production in B cells activated by contact with activated Th cells in mouse systems, and IL-5 and IL-5R&agr; knockout mice have diminished CD5+ B1 cells and decreased thymocytes until approximately 6 weeks of age.²²⁷^,²²⁸ Interestingly, the production of IL-5 by memory Th2 cells is driven by GATA3 but downregulated by expression of eomesodermin, which interacts with GATA3. Most memory Th2 cells produce the transcription factor Eomes, but the CD62^loCXCR3^lo population has decreased Eomes and is the population of cells that produces IL-5.²²⁹

On cells that express receptors for more than one of these cytokines, such as eosinophilic progenitors that express receptors for IL-3, IL-5, and GM-CSF, or on mouse pre-B cells, which express receptors for IL-3 and IL-5, the signals induced are indistinguishable.²²¹^,²²² Thus, the differential lineage specificities of these cytokines are determined by the cellular distribution of their receptors rather than by fundamental differences in the signals that are induced by each cytokine. These observations are explained by studies demonstrating that each of these three cytokines has its own unique 60 to 80 kDa &agr; chain (ie, IL-3R&agr;, IL-5R&agr;, and GM-CSFR&agr;),²³⁰^,²³¹^,²³²^,²³³^,²³⁴^,²³⁵^,²³⁶ but that they share a common 120 to 130 kDa &bgr; chain, &bgr;_c.²²²^,²³⁷^,²³⁸^,²³⁹ The &agr; chains are the principal binding proteins for the cytokines, whereas the shared &bgr;_c subunit augments binding affinity but does not exhibit binding activity in the absence of the proper &agr; chain. The &agr; chains have relatively short cytoplasmic domains (approximately 55 amino acids long for IL-3R&agr;, IL-5R&agr;, and GM-CSFR&agr;) and are not believed to play major roles in signaling function, whereas &bgr;_c, with its cytoplasmic domain of 432 amino acids, is the primary determinant of the signal. As a result, there is a relative compartmentalization of binding and signaling function for these cytokines, although the cytoplasmic domains of the GM-CSFR&agr; and IL-5R&agr; chains (and by analogy, perhaps the IL-3R&agr; chain), as well as &bgr;_c, appear to be capable of at least modulating the growth signals in transfected cells.²²²^,²⁴⁰^,²⁴¹^,²⁴² In any case, the sharing of &bgr;_c helps to explain why the signals induced by IL-3, IL-5, and GM-CSF are similar on cells that can respond to more than one of these cytokines. The situation for the &bgr;_c family of cytokines is therefore quite different from the receptors for &ggr;_c family cytokines, wherein the chains with the largest cytoplasmic domains (IL-2R&bgr;, IL-4R&agr;, IL-7R&agr;, IL-9R&agr;, and IL-21R) not only contribute most to signaling specificity but also are the proteins principally involved in ligand binding (note that for IL-2 and IL-15, IL-2R&agr; and IL-15R&agr;, respectively, serve important roles as well). The shared chain, &ggr;_c, serves a vital accessory function (hence the development of X-linked SCID when the IL2RG gene is mutated), but it contributes less to cytokine binding and does not provide an obvious basis for signaling specificity (see subsequent discussion).

An interesting feature of the &bgr;_c family of hematopoietic cytokines is that there appears to be considerable redundancy of function so that knockout mice that lack the ability to respond to all three cytokines (GM-CSF, IL-3, and IL-5) due to deletion of &bgr;_c as well as the mouse IL-3-specific &bgr;_c-like protein (discussed in the following text) nevertheless exhibit relatively normal hematopoiesis. These observations do not minimize the potency of these particular cytokines but instead underscore a substantial redundancy for a particularly important set of functions.²²¹^,²²²^,²⁴³ It is also noteworthy that &bgr;_c-deficient mice exhibit defective host responses to infectious challenge, suggesting that these hematopoietic cytokines play a vital role in promoting immune function.

Cytokines whose Receptors Share gp130 (Interleukin-6, Interleukin-11, Oncostatin M, Ciliary Neurotropic Factor, Leukemia Inhibitory Factor, Cardiotrophin-1, Novel Neurotrophin-1/B Cell-Stimulating Factor-3/Cardiotrophin-Like Factor, and Interleukin-27)

There are now eight cytokines that are known to utilize gp130 as a signal transducing molecule.²⁴⁴^,²⁴⁵^,²⁴⁶^,²⁴⁷^,²⁴⁸^,²⁴⁹^,²⁵⁰^,²⁵¹^,²⁵²^,²⁵³^,²⁵⁴^,²⁵⁵ Some of the properties of these cytokines are summarized in Table 25.6. This family is often referred to as the IL-6 family of cytokines and includes IL-6, IL-11, OSM, LIF, CNTF, CT-1, NNT/BSF-3/CLC, and IL-27. This group of cytokines comprises molecules with a diverse range of actions, ranging beyond the hematopoietic and
immune systems to the central nervous and cardiovascular systems, making them even more “multifunctional” than the &ggr;_c and &bgr;_c families of cytokines, whose actions are more restricted to the lymphoid and hematopoietic systems.

TABLE 25.6 Cytokines whose Receptors Share gp130^a

Cytokine	Chromosome Location (h/m)
IL-6	7p21/5
IL-11	19q13.3-13.4/7
LIF	22q12.1-12.2/11
OSM	22q12.1-12.2/11
CNTF	11q12.2/19
CT-1	16p11.1-11.2/7
NNT-1/BSF-3/CLC	11q13/19
IL-27
Overlapping actions of several gp130 cytokines
	IL-6	IL-11	LIF	OSM	CNTF	CT-1
Growth of myeloma cells	+	–	+	+	+	?
Maintenance of embryonic stem cell pluripotency	–	–	+	+	+	+
Induction of hepatic acute phase proteins	+	+	+	+	+	+
Induction of cardiac hypertrophy	–	+	+	+	+/-	+
Induction of osteoclast formation	–	+	+	+	?	?
Enhanced neuronal survival/differentiaion	+	+	+	+	+	+
Inhibit adipogenesis	?	+	+	?	?	?
BSF-3, B cell-stimulating factor-3; CLC, cardiotrophin-like factor; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin-1; IL, interleukin; LIF, leukemia inhibitory factor; NNT-1, novel neurotrophin-1; OSM, oncostatin M.
^aMost of the data in this table are derived from Yin et al.248
Note that NNT-1 can support survival of chicken embryonic and sympathetic neurons, can induce amyloid A, and analogous to IL-6 can induce B-cell hyperplasia.

IL-6 was originally identified and then cloned as a B-cell differentiation factor that stimulated terminal differentiation/maturation of B cells into antibody-producing plasma cells.²⁵⁶^,²⁵⁷^,²⁵⁸ However, IL-6 also can exert effects for T-cell growth and differentiation (and thus is a thymocyte “comitogen”), induce myeloid differentiation into macrophages, induce acute-phase protein synthesis of hepatocytes, and exert actions on keratinocytes, mesangial cells, hematopoietic stem cells, the development of osteoclasts, and neural differentiation of PC12 cells.²⁵⁰ IL-6 is also a mediator of amplified lymphocyte trafficking during febrile inflammatory responses, which is dependent on IL-6-mediated activation of L-selectin.²⁵⁹ IL-6 binds to an 80 kDa IL-6 binding protein, denoted IL-6R&agr;, which has a comparatively short 82 amino acid long cytoplasmic domain.²⁵⁷^,²⁶⁰ This IL-6-IL-6R&agr; complex then interacts with and recruits the 130 kDa signal transducing molecule, gp130, which together with IL-6R&agr; can form a functional IL-6 receptor.²⁵⁰ From a structural perspective, gp130 contains a total of six fibronectin type III modules, with the four conserved cysteine residues and the WSXWS motif being located in the second and third of these modules, starting from the N-terminus. As such, these regions are topologically positioned at greater distance external to the cell membrane than is the case for the other type I cytokine receptors discussed previously.

The IL-6 system illustrates a novel twist related to the properties of their principal binding proteins: The cytoplasmic domain of IL-6R&agr; is superfluous for signaling; a soluble form of the IL-6R&agr; extracellular domain is in fact sufficient for ligand binding and coordination with gp130, a process termed IL-6 transsignaling.²⁶¹ Thus, in the presence of soluble IL-6R&agr; and IL-6, many cell types that express gp130 but not IL-6R&agr; are capable of signaling in response to IL-6. It was observed that IL-6 signaling requires the dimerization of gp130 and that the overall complex containing two molecules each of IL-6, IL-6R&agr;, and gp130 (a dimer of a trimer or a hexamer),³²^,²⁶²^,²⁶³^,²⁶⁴^,²⁶⁵ providing a possible paradigm for the stoichiometry of subunits for other members of the IL-6 family of cytokines.²⁴

IL-11 was identified as a factor produced by a stromal cell line in response to stimulation with IL-1.²⁶⁶^,²⁶⁷ It has a number of effects on hematopoiesis, particularly in combination with IL-3 and SCF. Because IL-11 exhibited “IL-6-like activities,” a complementary deoxyribonucleic acid (DNA) was isolated based on the presence of IL-6-like activity in the presence of antibodies to IL-6.²⁶⁸ Other actions of IL-11 include the ability to stimulate the proliferation of lymphoid and hematopoietic progenitor cells, stimulate megakaryocytic progenitors and megakaryocyte maturation, and stimulate erythroid progenitors (an action not shared by IL-6).²⁶⁶^,²⁶⁷ Overall, it acts on T cells, B cells, hematopoietic cells, epithelium, endothelial cells, and osteoclasts.²⁶⁹ Like IL-6, IL-11 induces acute-phase proteins and augments antigen-specific B-cell responses, but it does not stimulate human myeloma cells.²⁶⁶^,²⁶⁷^,²⁶⁸^,²⁷⁰ Subsequently, adipogenesis inhibitory factor was cloned and found to be identical to IL-11,²⁷¹ revealing another action of IL-11. IL-11 is also produced by lung eosinophils and various structural cells in the lung and is expressed in patients with modest-to-severe asthma.²⁷² IL-11 signals via a receptor complex containing both IL-11R&agr;²⁷³ and gp130.²⁴⁴ Interestingly, IL-11R&agr; messenger RNA can be alternatively spliced to yield a form lacking the cytoplasmic domain, and like IL-6R&agr;, a soluble form of IL-11R&agr; can coordinate with IL-11 to transsignal in cells expressing gp130.²⁷⁴ Studies on the stoichiometry of the IL-11 receptor complex failed to reveal dimerization of gp130 to itself or LIFR&bgr;. Thus, assuming that it forms a hexameric receptor complex, only five of the members are known: two molecules of IL-11 and IL-11R&agr;, respectively, and one of gp130, suggesting that another component may still be found.²⁷⁵ IL-11 and STAT3 are expressed in gastrointestinal cancers associated with inflammation, suggesting a possible link to cancer.²⁶⁹

LIF is another multifunctional cytokine originally cloned based on the activity associated with its name.²⁷⁶ LIF can suppress the differentiation of pluripotent embryonic stem cells, inhibit adipogenesis (like IL-11), and induce monocyte differentiation of the M1 murine leukemia cell line, thus mimicking a number of the actions of IL-6.²⁷⁷ In addition, it exerts a number of actions in the central nervous system, and LIF was shown to be identical to cholinergic neural differentiation factor.²⁷⁸ and can induce acetylcholine synthesis while simultaneously suppressing catecholamine production, thereby inducing cholinergic function while suppressing noradrenergic function.²⁷⁸ LIF has been shown to be essential for embryo implantation.²⁷⁹ LIF binds to a receptor (LIFR&bgr;) that is structurally related to gp130,²⁸⁰ but the functional LIF receptor (LIFR) receptor requires the heterodimerization of LIFR&bgr; and gp130 as well.²⁴⁶ Interestingly, whereas IL-6 promotes Th17 differentiation, this is inhibited by LIF, apparently based on LIF’s ability to activate ERKs and to augment the expression of SOCS-3, which can inhibit STAT3 activation.²⁸¹ Interestingly, LIF can also promote the differentiation of T_reg cells and may exhibit therapeutic potential in multiple sclerosis.²⁸² LIF maintains mouse embryonic stem cells, but LIF is not capable of maintaining human embryonic stem cells or mouse epiblast stem cells in the pluripotent state.²⁸³

CNTF was discovered based on its ability to promote neuronal survival.²⁸⁴^,²⁸⁵ CNTF signals through a receptor comprising LIFR&bgr; and gp130 but requires a specific binding protein,²⁸⁶^,²⁸⁷ now denoted CNTF receptor (CNTFR)&agr;. Interestingly, CNTFR&agr; lacks transmembrane and cytoplasmic domains and instead is a GPI-linked receptor molecule. CNTFR&agr; appears to provide a receptor-cytokine surface with which gp130 and LIFR&bgr; can interact. Thus, CNTF is like IL-6 in that each requires initial binding to a receptor component (CNTFR&agr; or IL-6R&agr;) that does not require its own cytoplasmic domain for signaling. Whereas IL-6 signaling involves homodimerization of gp130, CNTF signaling involves the heterodimerization of LIFR&bgr; and gp130. In fact, the functional CNTFR appears to be a hexameric structure containing two molecules of CNTF, two of CNTFR&agr;, and one each of gp130 and LIFR&bgr;.²⁸⁸ The receptor is expressed largely within the nervous system and in skeletal muscle, accounting for largely restricted actions of CNTF.²⁸⁶

OSM is a growth regulator that was originally identified based on its ability to inhibit the growth of A375 human melanoma cells.²⁸⁹^,²⁹⁰ OSM is a potent growth factor for Kaposi sarcoma in patients with acquired immunodeficiency syndrome.²⁹¹^,²⁹² Not only OSM can bind directly to gp130 and signals through a receptor combination of gp130 and LIFR&bgr;²⁴⁶ but also has an alternative receptor comprising a specific OSM receptor subunit (OSMR&bgr;) and gp130.²⁹³ These are now known as the type I and type II OSM receptors (OSMRs), respectively. OSM can enhance the development of both endothelial cells and hematopoietic cells, possibly by increasing hamangioblasts, a common precursor for endothelial and hematopoietic cells.²⁵¹

CT-1 was initially isolated based on its actions on cardiac muscle cells.²⁹⁴ However, it is now clear that it is a multifunctional cytokine with hematopoietic, neuronal, and developmental effects, in addition to its effects on cardiac development and hypertrophy.²⁹⁵^,²⁹⁶ Like OSM and LIF, CT-1 can also signal through a heterodimer of LIFR&bgr; and gp130.²⁴⁹ Interestingly, the CT-1 receptor on motor neurons may involve a third receptor component, possibly GPI linked.²⁹⁷^,²⁹⁸

NNT-1/BSF-3, like CNTF, can also support the survival of chicken embryonic sympathetic and motor neurons.²⁵³ Interestingly, in mice, NNT-1/BSF-3 can augment the effects of IL-1 and IL-6 and is a B-cell-stimulating factor (hence the term BSF-3). The NNT-1 receptor contains LIFR&bgr; and gp130.²⁵³ NNT-1 is also known as CLC, and this forms a complex with a soluble receptor protein known as cytokine-like factor-1. Together, this complex is a second ligand for CNTFR.²⁹⁹ It appears that CLC can also interact directly with soluble CNTFR to form a related cytokine.²⁵⁴

IL-27 is an IL-6-related cytokine³⁰⁰ that represents a dimer of the p28 protein (also known as IL-30) and EB13 (Epstein-Barr virus-induced gene 3) (also known as IL-27B), which can induce proliferation of naive CD4+ T cells. p28 can exist by itself independently of EB13 and is capable of antagonizing gp130-mediated signaling by IL-6, and indeed, overexpression of p28 in transgenic mice prevents normal development of germinal centers and antibody production.³⁰¹ Together with IL-12, IL-23, CLC/cytokine-like factor-1, and CLC/soluble CNTFR, this is one of five cytokines that represent dimers including a type I cytokine and a soluble receptor-like protein. IL-27 signals via gp130 and the WSX-1/TCCR receptor,³⁰⁰ which is discussed in the section on diseases of cytokine receptors and related molecules. IL-27 has proinflammatory and anti-inflammatory effects. Although it was suggested to promote Th1 responses, it is clear that it can also antagonize such responses, and IL-27 can promote effector responses of both CD4+ and CD8+ T cells, of NK cells, and it additionally stimulates mast cells. The anti-inflammatory effects of IL-27 are indicated by the absence of WSX-1 resulting in increased mast cell and macrophage responses.³⁰²^,³⁰³

Thus, eight cytokines (IL-6, IL-11, LIF, CNTF, OSM, CT-1, NNT-1/BSF-3, and IL-27) all have receptors that are dependent on gp130.³⁰⁴ These can be divided into two sets of cytokines: those known to not require LIFR&bgr; (IL-6, IL-11, and IL-27) and those that use both gp130 and LIFR&bgr; (LIF, CT-1, OSM, CNTF, and NNT-1/BSF-3) (Table 25.7), with OSM having two forms of receptors, each of which contains gp130 but only one of which contains LIFR&bgr;. When cytokines share essentially the same receptor, one can hypothesize that two cytokines might exert identical actions on cells that can respond to both cytokines. It is clear that the presence of IL-6R&agr;, IL-11R&agr;, and CNTFR&agr; (either on the cell surface or as a soluble receptor form, discussed subsequently) determines whether a cell can respond to IL-6, IL-11, and CNTF. This raises the interesting question as to whether functional homologues of these proteins will also exist for LIF, OSM, CT-1, NNT-1/BSF-3, and IL-27.

TABLE 25.7 Composition of Receptors for the Interleukin-6 Family of Cytokines

Cytokines whose receptors do not contain LIFR&bgr;
IL-6	IL-6R&agr; + gp130
IL-11	IL-11R&agr; + gp130
OSM	OSMR&bgr; + gp130
Cytokines whose receptors contain LIFR&bgr;
LIF	LIFR&bgr; + gp130
OSM	LIFR&bgr; + gp130
CNTF	CNTFR&agr; + LIFR&bgr; + gp130
CT-1	LIFR&bgr; + gp130 + ?CT1R&agr;
NNT-1/BSF-3 /CLC + CLF-1	CNTFR&agr; + LIFR&bgr; + gp130
CLC + soluble CNTFR&agr;	LIFR&bgr; + gp130
Cytokines whose receptors contain OSMR&bgr;
OSM	OSMR&bgr; + gp130
IL-31	IL-31R + OSMR&bgr;
BSF-3, B cell-stimulating factor-3; CLC, cardiotrophin-like factor; CLF-1, cytokinelike factor-1; CNTF, ciliary neurotrophic factor; CNTFR, CNTF receptor; CT-1, cardiotrophin-1; IL, interleukin; LIF, leukemia inhibitory factor; NNT-1, novel neurotrophin-1; OSM, oncostatin M.
Note that the sharing of CNTFR&agr; by CNTF and the dimeric NNT-1/BSF-3/CLC – CLF-1 ligand helps to explain why the phenotype in CNTF-/- mice is less severe than that found in CNTFR&agr;-/- mice.

Significance of the Sharing of Receptor Chains

Interestingly, &ggr;_c, &bgr;_c, and gp130 all contribute to signaling, but none of these shared cytokine receptor proteins has primary binding activity for any known cytokine. Instead, they each increase binding affinity in the context of the primary binding protein for each cytokine. Consequently, the capacity of a cell to respond to a given cytokine is determined by the unique binding chain(s), but signaling pathways can be shared.

Other Receptors with Similarities to gp130 (Granulocyte-Colony Stimulating Factor Receptor, OB-R, Interleukin-12R&bgr;1, Interleukin-12R&bgr;2, and Interleukin-31R)

As noted previously, LIFR&bgr; and OSMR&bgr; bear some similarities to gp130.²⁹³ In addition, the G-CSF receptor, the leptin receptor (also denoted OB-R, for obesity receptor), IL-12/IL-23 receptor components, and IL-31R all resemble gp130. The amino acid identity among these different receptors, compared pairwise, ranges from 18% to 32%, with LIFR&bgr; and OSMR&bgr; being the most similar. Although G-CSF is granulocyte-colony stimulating factor, it may have
a broader role. For example, G-CSF has been shown to stimulate myoblast proliferation and thereby to affect skeletal muscle formation,³⁰⁵ indicating broad pleiotropic actions by this cytokine.

Leptin

Leptin is the product of the obesity (ob) gene, an adipose tissue-derived cytokine that plays a role in body weight homeostasis.³⁰⁶^,³⁰⁷^,³⁰⁸ Additionally, leptin affects thymic homeostasis, increasing thymocyte number and having antiapoptotic effects in the thymus and periphery. It is proinflammatory, promoting Th1 differentiation, and augmenting the production of TNF-&agr;, IL-1, and IL-6 by monocytes/macrophages.³⁰⁸ The leptin receptor, OB-R, was cloned and found to be most closely related to the gp130 signal transducer, G-CSF receptor, and LIFR&bgr;.³⁰⁹ Interestingly, this receptor is encoded by the “diabetes gene,” which is mutated in db/db mice.³¹⁰

Interleukin-12, IL-23, and IL-35

As noted previously, IL-12 is the major inducer of Th1 cells. IL-12 is primarily produced by phagocytic cells in response to bacterial and intracellular parasites, such as Toxoplasma gondii, but it is also produced by other antigen-presenting cells, such as B cells.³¹¹ IL-12 potently induces the production of IFN&ggr; by NK cells and T cells and is also a growth factor for preactivated but not resting NK and T cells. IL-12 was originally discovered as NK cell stimulatory factor.³¹² IL-12 can also induce the production of IL-2, IL-3, GM-CSF, IL-9, TNF-&agr;, and M-CSF, although inducing IFN&ggr; is perhaps its most important known action.³¹¹^,³¹³^,³¹⁴ As is discussed in the section on immunodeficiency diseases, IL-12 is essential for the proper clearing of mycobacterial infections. It is interesting that Th2 cells do not respond to IL-12; the lack of responsiveness of these cells to IL-12 results from their loss of expression of the IL-12R&bgr;2 subunit of the IL-12 receptor.³¹⁵ Apparently, IL-4 inhibits IL-12R&bgr;2 expression, whereas IL-2⁷⁴ and IFN&ggr;³¹⁵ induce its expression. The abilities of mice to survive infections is critically linked to the Th patterns of cytokines. For example, the ability to survive T. gondii infection is dependent on IFN&ggr;/IL-12 production (a Th1 pattern).⁸³

IL-12 can be thought of as having vital roles in both innate immunity and acquired immune responses. It is rapidly produced by NK cells and then T cells in response to antigens or foreign pathogens. This rapid response facilitates the activation of first-line defense against infections. In addition, however, IL-12 is also required for the subsequent differentiation of specialized T-cell populations, including its STAT4-dependent priming of Th1 cells for optimal production of IFN&ggr; and IL-2. IL-12 can act synergistically with hematopoietic growth factors, such as IL-3 and SCF, to support the proliferation and survival of hematpoietic stem cells.³¹¹ Structurally, IL-12 is a covalently linked dimer of 35 (IL-12A) and 40 (IL-12B) kDa peptides³¹¹; thus, successful production of IL-12 requires that a cell transcribe both the p35 and p40 genes.³¹⁶ Interestingly, whereas p35 bears sequence similarity to IL-6 and G-CSF, p40 is homologous to the extracellular domains of IL-6R&agr;, CNTFR&agr;, and G-CSF receptor and bears some of the features typical of type I receptors, including four conserved cysteines, a conserved tryptophan, and a WSEWAS motif, which has similarity to the typical WSXWS motif.³¹¹ Moreover, as both IL-12 receptor (IL-12R&bgr;1 and IL-12R&bgr;2) chains bear some similarity to gp130,³¹²^,³¹⁶^,³¹⁷ one can think of p40 as a functional homologue of the soluble p80 IL-6R&agr; chain. Thus, for this cytokine, part of the “receptor” has become part of the cytokine. Interestingly, all cells that produce IL-12 synthesize much more p40 than p35, suggesting that the careful control of signaling is at the level of the “primordial” p35 cytokine part of IL-12. p40 is on human chromosome 5q31 to 5q33 while p35 is on 3p12 tp 3p13.2.³¹¹ As noted previously, it is interesting that IL-2 induces expression of IL-12R&bgr;1 and IL-12R&bgr;2.

IL-23 is similar to IL-12 in that it also contains p40.³¹⁸ However, rather than also containing p35, IL-23 is a dimer of a p40 and p19 (IL-23A). IL-23 signals via a receptor that contains IL-12R&bgr;1 but not IL-12R&bgr;2.³¹⁹ Instead, another receptor chain, denoted IL-23R, is the second component of the IL-23R³²⁰; both IL-12R&bgr;2 and IL-23R are located on chromosome 1 within 150 kb of each other.³²⁰ Both IL-12 and IL-23 activate JAK2 and TYK2. IL-12 and IL-23 can activate STAT1, STAT3, STAT4, and STAT5. STAT4 is the dominant STAT protein activated by IL-12.³²⁰ Interestingly, as compared to human IL-23R, mouse IL-23R contains a 20 amino acid duplicated region that spans the WQPWS motif. IL-23 appears to play a vital role in the maintenance, rather than in the initial differentiation of Th17 cells (IL-17-producing cells), and IL-23 is essential for resistance to certain diseases, such as EAE, which were originally believed to be Th1-mediated diseases, as elimination of IL-23 by eliminating either the p40 subunit shared by IL-12 and IL-23 or by eliminating p19 confers resistance to EAE.¹⁷⁵^,³²¹ Whereas IL-23 promotes Th17 cell numbers, IL-27 can negatively regulate their development.³²¹ In the CD40-mediated model of inflammatory bowel disease, IL-12 p35 secretion controlled wasting disease and serum cytokine production but did not affect mucosal pathology, which instead was associated with IL-23/p19.³²² Interestingly, IL-23R has been identified as an inflammatory bowel disease gene,³²³ and IL-23 can drive intestinal inflammation via direct actions on T cells.³²⁴ Separate from Th17 cells, a population of innate lymphoid cells that produce IL-17, IL-22, and IFN&ggr; in response to IL-23 have been identified as the mediators of inflammatory bowel disease.³²⁵ IL-23 and its receptor are nevertheless critical for the terminal differentiation of Th17 cells.¹⁶⁹^,³²⁶ IL-27 is a related cytokine (discussed previously), but it can act as an inhibitor of Th1 responses associated with intracellular infections such as T. gondii. Whereas host defense to T. gondii is dependent on IL-12 and IFN&ggr;, IL-27-deficient mice control the parasite replication but develop a lethal inflammatory disease that is dependent on CD4+ T cells. Additionally, IL-27 can inhibit Th2 actions.³⁰³

IL-35 is produced by T_reg cells and plays a role in immunosuppression. It is a heterodimer of IL-12A (p35) and IL-27B (EBI3).³²⁷ IL-35 treatment of naive mouse or human T cells induces regulatory cells that have been called iTR35 cells³²⁸ and promote tolerance and tumor progression.

Interleukin-31

IL-31 is a four &agr;-helical bundle cytokine that fits into the greater IL-6 extended family in that its receptor consists of IL-31RA and the OSMR&bgr;.³²⁹ IL-31R is a gp130-like type 1 receptor, with four splice variants. IL-31 is produced primarily by activated T cells and when overexpressed results in pruritis, alopecia, and skin lesions, indicating a potential role in dermatitis.³²⁹^,³³⁰

Other Examples of Shared Receptor Molecules

Interleukin-7 and Thymic Stromal Lymphopoietin Share Interleukin-7 Receptor &agr; Chain

In addition to IL-7, a second stromal factor, TSLP has been identified that shares at least some actions with IL-7.¹¹^,³³¹^,³³²^,³³³ The TSLP receptor is a heterodimer of TSLPR and IL-7R&agr;.³³⁴^,³³⁵ Interestingly, TSLPR is 24% identical to &ggr;_c, making it the cytokine receptor most like &ggr;_c.³³⁴ Human and mouse TSLP share only 43% amino acid identity, and human and mouse TSLP receptors share only 39% identity.³³⁶ This is very low for human and mouse orthologues of cytokine receptors, for example, human and mouse &ggr;_c are 70% identical.³³⁷ In addition to their wide sequence divergence, mouse and human TSLP were originally suggested to substantially differ in their actions. Mouse TSLP was first reported to be a B-cell differentiation factor that is important for the development of IgM+ immature B cells from pre-B cells and to also be a weak thymic comitogen. In contrast, human TSLP appeared not to exert effects on these lineages but was found to be an epithelial-derived cytokine that potently activated DCs related to Th2 allergic responses, an action not then known to be shared by mouse TSLP.³³⁸^,³³⁹^,³⁴⁰ However, it is now clear that mouse TSLP can also activate DCs and that it plays a critical role in both atopic disease and asthma in mouse models, and that these effects of TSLP are mediated via actions on both DCs and CD4+ T cells.³⁴¹^,³⁴²^,³⁴³^,

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