Biological functions and therapeutic opportunities of soluble cytokine receptors

Cytokines control the immune system by regulating the proliferation, differentiation and function of immune cells. They activate their target cells through binding to specific receptors, which either are transmembrane proteins or attached to the cell-surface via a GPI-anchor. Different tissues and individual cell types have unique expression profiles of cytokine receptors, and consequently this expression pattern dictates to which cytokines a given cell can respond. Furthermore, soluble variants of several cytokine receptors exist, which are generated by different molecular mechanisms, namely differential mRNA splicing, proteolytic cleavage of the membrane-tethered precursors, and release on extracellular vesicles. These soluble receptors shape the function of cytokines in different ways: they can serve as antagonistic decoy receptors which compete with their membrane-bound counterparts for the ligand, or they can form functional receptor/cytokine complexes which act as agonists and can even activate cells that would usually not respond to the ligand alone. In this review, we focus on the IL-2 and IL-6 families of cytokines and the so-called Th2 cytokines. We summarize for each cytokine which soluble receptors exist, were they originate from, how they are generated, and what their biological functions are. Furthermore, we give an outlook on how these soluble receptors can be exploited for therapeutic purposes.     

Replacing factor-dependency with that for lysozyme: affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor.

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.     

Gene therapy of autoimmune diseases with vectors encoding regulatory cytokines or inflammatory cytokine inhibitors

Gene therapy offers advantages for the immunotherapeutic delivery of cytokines or their inhibitors. After gene transfer, these mediators are produced at relatively constant, non-toxic levels and sometimes in a tissue-specific manner, obviating limitations of protein administration. Therapy with viral or nonviral vectors is effective in several animal models of autoimmunity including Type 1 diabetes mellitus (DM), experimental allergic encephalomyelitis (EAE), systemic lupus erythematosus (SLE), colitis, thyroiditis and various forms of arthritis. Genes encoding transforming growth factor beta, interleukin-4 (IL-4) and IL-10 are most frequently protective. Autoimmune/ inflammatory diseases are associated with excessive production of inflammatory cytokines such as IL-1, IL-12, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma). Vectors encoding inhibitors of these cytokines, such as IL-1 receptor antagonist, soluble IL-1 receptors, IL-12p40, soluble TNFalpha receptors or IFNgamma-receptor/IgG-Fc fusion proteins are protective in models of either arthritis, Type 1 DM, SLE or EAE. We use intramuscular injection of naked plasmid DNA for cytokine or anticytokine therapy. Muscle tissue is accessible, expression is usually more persistent than elsewhere, transfection efficiency can be increased by low-voltage in vivo electroporation, vector administration is simple and the method is inexpensive. Plasmids do not induce neutralizing immunity allowing repeated administration, and are suitable for the treatment of chronic immunological diseases.     

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y₆ receptor mediated impact on interleukin (IL)-8 production, (2) P2Y₁₁ receptor-mediated affects on IL-12/23 output, and (3) P2X₇ receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.     

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y₆ receptor mediated impact on interleukin (IL)-8 production, (2) P2Y₁₁ receptor-mediated affects on IL-12/23 output, and (3) P2X₇ receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.     

Cytokine Actions in the Central Nervous System

Cytokines and chemokines have been implicated in contributing to the initiation, propagation and regulation of immune and inflammatory responses. Also, these soluble mediators have important roles in contributing to a wide array of neurological diseases such as multiple sclerosis, AIDS Dementia Complex, stroke and Alzheimers disease. Cytokines and chemokines are synthesized within the central nervous system by glial cells and neurons, and have modulatory functions on these same cells via interactions with specific cell-surface receptors. In this article, I will discuss the ability of glial cells and neurons to both respond to, and synthesize, a variety of cytokines. The emphasize will be on three select cytokines; interferon-gamma (IFN-γ), a cytokine with predominantly proinflammatory effects; interleukin-6 (IL-6), a cytokine with both pro- and anti-inflammatory properties; and transforming growth factor-beta (TGF-β), a cytokine with predominantly immunosuppressive actions. The significance of these cytokines to neurological diseases with an immunological component will be discussed.     

Interleukin-6 biology is coordinated by membrane bound and soluble receptors

Cytokine receptors exist in membrane bound and soluble form. Both forms bind their ligands with comparable affinity. While most soluble receptors are antagonists in that they compete for the ligands with their membrane counterparts, some soluble receptors are agonists. In this case, the complex of ligand and soluble receptor binds on target cells to a second receptor subunit and initiates signal transduction. Soluble receptors of the IL-6 family of cytokines are agonists. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. This process has been named transsignaling. We have shown that in several chronic inflammatory diseases like chronic inflammatory bowl disease, peritonitis and rheumatoid arthritis, transsignaling via the soluble IL-6R complexed to IL-6 is a crucial point in the transition from the acute to the chronic state of the disease. The mechanism by which the IL-6/ soluble IL-6R complex regulates the inflammatory state is discussed.     

Identification of the IL-17 receptor related molecule IL-17RC as the receptor for IL-17F

The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.     

A fusion protein of the gp130 and interleukin-6Ralpha ligand-binding domains acts as a potent interleukin-6 inhibitor

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an alpha-subunit (IL-6Ralpha) that binds IL-6 with low affinity and a beta-subunit (gp130) that binds the IL-6.IL-6Ralpha complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6.soluble IL-6Ralpha complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Ralpha (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies.     

VSTM1-v2, a novel soluble glycoprotein, promotes the differentiation and activation of Th17 cells

Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.     

IL-6 type cytokine receptor complexes: hexamer, tetramer or both?

The typical protein fold of most cytokines is a bundle of four antiparallel helices. This 'four-helical bundle fold' seems to be unique to cytokines and has not been detected in other proteins. Cytokine receptors, however, can be classified as a subfamily of the immunoglobulin superfamily. Cytokines using the same receptor subunits are grouped into cytokine families. The interleukin-6 (IL-6) type cytokine family comprises six members. IL-6 type cytokines may interact with three receptor subunits instead of the usual two subunits. A tetramer would be the simplest model to describe such a receptor complex, but present orthodoxy describes the active complexes of IL-6 and ciliary neurotrophic factor (CNTF) as hexamers. Here, we summarize the structural and biochemical information on IL-6 type cytokines and discuss interactions between cytokine and individual receptor subunits at alternative positions. Contradictory results regarding the stoichiometry and assembly of signaling receptor complexes are rationalized by a new, unique model. The model stipulates that a ligand-induced transition from an active tetrameric to an inactive hexameric complex serves as a molecular switch that turns off cytokine signals in the presence of supraoptimal cytokine concentrations.     

The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex

IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.     

Protection against Th17 Cells Differentiation by an Interleukin-23 Receptor Cytokine-Binding Homology Region

Th17 cells have been reported to produce proinflammatory cytokines like Interleukin-17, IL-22, and regarded as important players in various inflammatory diseases. One of the IL-12 cytokine family cytokines, IL-23, composed of p19 and p40 subunit, is known for its potential to promote Th17 development and IL-17 producing, and the IL-23/IL-17 pathway is considered to be potential therapeutic target for autoimmune inflammation responses. Knockout mice deficient in either IL-23 or IL-17 related genes can suppress the allergic responses. Several IL-23 or IL-17 neutralizing agents are being evaluated in vitro or in vivo to disrupt the IL-23/IL-17 axis. Herein, we report that prokaryotically expressed soluble IL-23 receptor cytokine-binding homology region as an endogenous extracellular receptor analogue could be a natural antagonist against IL-23/IL-17 axis. We provide evidence that IL23R-CHR can bind to IL-23 in a dose-dependent manner in vitro, and block IL-23 signal by IL23R-CHR reducing the RORγt expression, which in turn lowers the expression of IL-17/IL-22, thus protecting naive CD4+ T cells against Th17 development. Together, this study indicates the importance of IL-23 pathway in Th17 development and the negative regulation of Th17 development by IL23R-CHR, and highlights the important roles of the soluble receptor extracellular region in the therapeutic strategy of neutralizing IL-23.     

Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions

Cytokines that signal through Class II receptors form a distinct family that includes the interferons and interleukin 10 (IL-10). Recent identification of several IL-10 homologs has defined a cytokine subfamily that includes AK155, IL-19, IL-20, IL-22, and IL-24. Within this subfamily, IL-19, IL-20, and IL-24 exhibit substantial sharing of receptor complexes; all three are capable of signaling through IL-20RA/IL-20RB, and IL-20 and IL-24 both can also use IL-22R/IL-20RB. However, the biological effects of these three cytokines appear quite distinct: immune activity with IL-19, skin biology with IL-20, and tumor apoptosis with IL-24. To more fully elucidate their interactions with the receptor complexes, we have performed a series of in vitro assays. Reporter, proliferation, and direct STAT activation assays using cell lines expressing transfected receptors revealed differences between the receptor complexes. IL-19 and IL-24 also exhibited growth inhibition on a cell line endogenously expressing all three receptor subunits, an effect that was seen at cytokine levels two orders of magnitude above those required for STAT activation or proliferation. These results demonstrate that, although this subclass exhibits receptor complex redundancy, there are differences in ligand/receptor interactions and in signal transduction that may lead to specificity and a distinct biology for each cytokine.     

Poxviruses: Capturing Cytokines and Chemokines

Cytokines play a critical role in the regulation of immune responses and constitute important targets for virus immune evasion mechanisms. One strategy used by large DNA viruses is to encode proteins that mimic cytokines or cytokine receptors, which modulate the activity of cytokines during infection. Poxviruses encode a unique set of proteins that are secreted from the infected cell and function as soluble cytokine receptors or binding proteins and sequester tumor necrosis factor, interleukin-1β, or chemokines. Characterization of these poxvirus proteins is providing information on virus pathogenesis, the function of cytokines, and new strategies for immune modulation and therapeutic intervention.     

Cytokines in chronic rheumatic diseases: is everything lack of homeostatic balance?

Biological systems have powerful inbuilt mechanisms of control intended to maintain homeostasis. Cytokines are no exception to this rule, and imbalance in cytokine activities may lead to inflammation with subsequent tissue and organ damage, altered function, and death. Balance is achieved through multiple, not mutually exclusive, mechanisms including the simultaneous production of agonist and antagonistic cytokines, expression of soluble receptors or membrane-bound nonsignaling receptors, priming and/or reprogramming of signaling, and uncoupling of ligand/receptor pairing from signal transduction. Insight into cytokine balance is leading to novel therapeutic approaches particularly in autoimmune conditions, which are intimately linked to a dysregulated cytokine production.     

Evaluation of the role of cytokines in autoimmune disease: The importance of TNFα in rheumatoid arthritis

Cytokines and growth factors are involved in all important biological processes. Hence it is anticipated that they will be of importance in autoimmune disease. The pathogenesis of autoimmune diseases involves a number of stages, initiation, perpetuation and tissue damage, each of which involves different cell and molecular interactions. In this review, we will discuss an outline of the cytokine involvement in the various stages of autoimmune development, prior to focusing on the analysis of cytokines in rheumatoid arthritis. Cytokines exert their effect via high affinity cell surface receptors. Thus an understanding of cytokines involves the analysis of receptor expression, and also of cytokine inhibitors. Currently there is only adequate knowledge of these aspects in rheumatoid arthritis (RA), and as such the emphasis of this review is on RA. One of the major reasons for being interested in the role of cytokines in autoimmunity is to define possible therapeutic targets. There is now considerable evidence that TNFα is such a target in RA, and the effect of anti TNFα monoclonal antibody therapy in RA is discussed.     

Discovery and mechanism of ustekinumab

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, “humanized” and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor β1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.