Cytokines

Abstract

Cytokines designate a broad category of factors that are produced by immune cells and/or act on immune cells. They are small proteins or peptides, mostly glycosylated, that regulate cellular growth, differentiation, and/or activity in a para- or autocrine manner. Those peptides, which are involved in growth and differentiation of target cells, are also named growth factors. Others regulate immune or inflammatory reactions and, in order to differentiate them from other factors, can be called mediators. However, there are examples for cytokines able to do both, and also factors other than cytokines can be assorted into these groups. The majority of cytokines are not exclusively synthesized by a single specific cell type. Moreover, certain cytokines are produced by both immune and nonimmune cells. On the other hand, a given cytokine can act on several cell types. And again, both immune and nonimmune cells may be affected by the same cytokine. Generally, cytokines are most important in regulatory processes of the immune system and can be classified into five subgroups: interleukins, tumor necrosis factors, interferons, chemokines, and colony-stimulating factors. This, however, is not an unambiguous and broadly accepted definition or classification of cytokines, but it is rather used to give this chapter a structure.

Final manuscript submitted on July 04, 2016.

1 Introduction

CYTOKINES designate a broad category of factors that are produced by immune cells and/or act on immune cells. They are small proteins or peptides, mostly glycosylated, that regulate cellular growth, differentiation, and/or activity in a para- or autocrine manner. Those peptides, which are involved in growth and differentiation of target cells, are also named GROWTH FACTORS. Others regulate immune or inflammatory reactions and, in order to differentiate them from other factors, can be called MEDIATORS. However, there are examples for cytokines able to do both, and also factors other than cytokines can be assorted into these groups. The majority of cytokines are not exclusively synthesized by a single specific cell type. Moreover, certain cytokines are produced by both immune and nonimmune cells. On the other hand, a given cytokine can act on several cell types. And again, both immune and nonimmune cells may be affected by the same cytokine. Generally, cytokines are most important in regulatory processes of the immune system and can be classified into five subgroups: interleukins, tumor necrosis factors, interferons, chemokines, and colony-stimulating factors. This, however, is not an unambiguous and broadly accepted definition or classification of cytokines, but it is rather used to give this chapter a structure.

In the 1960s it was demonstrated that supernatants of stimulated immune cells could regulate function and growth of leukocytes. Out of those supernatants, factors were partially purified and designated by function-related names such as lymphocyte activation factor (LAF) or T-cell growth factor (TCGF). These were grouped according to their major producing cells: monokines or lymphokines. In the 1970s Cohen suggested [1] the use of the term cytokines as a more general denomination. Since it became apparent that identical molecules had been described by different groups giving them different names, the term INTERLEUKINS (IL) was proposed at the second International Lymphokine Workshop held in 1979 “as a system of nomenclature ... based on the ability to act as communication signals between different populations of leukocytes.” Concomitantly, the names IL-1 for LAF and IL-2 for TCGF were introduced.

Also in the 1960s, a cytotoxic activity produced by lymphocytes in response to mitogen or antigen was described and called lymphotoxin (LT). About 10 years later, another cytotoxic activity able to kill certain transplantable tumor cells was detected. The respective proposed factor was named TUMOR NECROSIS FACTOR (TNF) [2]. Again 10 years later, the two, indeed different, corresponding proteins were identified: TNF from macrophages and LT from lymphocytes [3]. Confusingly, TNF was renamed TNF-α and LT TNFβ. When in 1993 LTβ (and the LTαβ complex) was identified, along with the realization that its biologic effect differs from that of TNF-α, the nomenclature was again revised, and the former TNFβ is now called LTα, while there is no further need for indexing TNF by an α.

INTERFERONS (IFNs) have been originally described in 1957 by the virologists A. Isaacs and J. Lindemann. IFNs are classified as factors produced by virus-infected cells capable of conferring resistance to infection with homologous or heterologous viruses [4]. However, soon it was recognized that they also regulate immune responses, and, thus, they became a subgroup of the cytokines. The regulation of immune and inflammatory reactions is the predominant function of IFNγ, also called type 2 or immune interferon. Structurally different from IFNγ are members belonging to the subgroups IFNα and IFNβ, which, both composed of series of members, make up the large family of type 1 interferons.

In 1987 a gene encoding a neutrophil-attractive protein, named IL-8, was cloned. The structure of this protein, today designated CXCL8, indicated that other structurally similar chemoattractive proteins have been identified earlier. This family of structurally related cytokines involved in migration and activation of immune cells was designated CHEMOKINES at the Third International Symposium of Chemotactic Cytokines in 1992 [5]. Chemokines, small molecular weight proteins ranging in size from approx. 8 to 15 kDa, are characterized by the position of the first two of four cysteine residues in highly conserved positions within their amino-terminal protein sequence [6]. Chemokines were divided into four major groups, designated CXC, CC, C, and CX₃C, where X denotes amino acids positioned between the two characterizing cysteine residues. The addition of L followed by a number to this nomenclature indicates a specific chemokine ligand, while the addition of R and a number indicates a chemokine receptor.

COLONY-STIMULATING FACTORS (CSFs) were first described in 1966 by Metcalf, reflecting the observation that they upon addition to bone marrow cells in semisolid medium promote the formation of granulocyte or monocyte colonies [7]. CSFs predominantly function as inducers of growth and differentiation of hematopoietic stem cells but can also activate fully differentiated immune cells, thus belonging to the group of cytokines.

Since hematopoiesis, immune cell development and maturation (Fig. 6.1), and activation of an immune response, including inflammation, are covered by other chapters of this textbook, here we will just very briefly introduce these issues and focus on the functions of cytokines in the diverse biological processes and how they can be targeted to obtain a therapeutic benefit.

Fig. 6.1

Cytokines involved in the differentiation of cells of the immune system. CSF colony-stimulating factor, EPO erythropoetin, G granulocyte, IL interleukin, M monocyte, SCF stem cell factor, TPO thrombopoietin

To this end, cytokines themselves can be exploited as drugs. Examples for this are CSFs and IFNs as described in the following. However, from a quantitative point of view, much more fruitful proved to be the possibility of developing inhibitors of cytokine activities. Their therapeutic potential is largely demonstrated in the groups of interleukins, TNFs, and chemokines.

A major breakthrough in the understanding of an immune reaction was the recognition that different immune mechanisms are executed by different cells, which are, in turn, regulated by again different cell populations. In this regard, an important finding was the identification of several subsets of CD4⁺ T helper cells (Th cells), which develop, i.e., polarize, from naïve Th cells upon activation with the cognate antigen [8]. These Th cell subsets are characterized by the specific cytokine profile they secrete. As such, Th1 cells mainly produce IFNγ; Th2 cells IL-4, IL-5, and IL-13; Th17 cells IL-17; and regulatory T cells (Treg) IL-10 and TGF-β. These cytokines are produced exclusively by the respective subset and not only promote specific types of immune effector functions but also cross-inhibit the polarization of other Th cell subsets.

More recently, an additional family of lymphoid cells was identified, which, however, differ from “conventional” lymphoid cells, T cells, and B cells, inasmuch as they do not rearrange DNA segments to develop a huge repertoire of receptors with different specificities [9]. Thus, the newly discovered lymphoid cells were named innate lymphoid cells (ILCs). In analogy to the Th cell subsets, also ILCs are subgrouped according to specific cytokines they exclusively produce. Thus, group 1 ILC (ILC1) are identified by production of IFNγ, while ILC2 produce IL-5 and IL-13, and ILC3 release their key cytokine IL-22 (Fig. 6.2).

Fig. 6.2

Activation of T and B lymphocytes. B B lymphocyte, B₀ virgin B lymphocyte, B′, B″ activated B lymphocytes, GM CSF granulocyte/monocyte colony-stimulating factor, IFN interferon, IL interleukin, PC plasma cell, Th T helper lymphocyte, Tc cytotoxic T lymphocyte, TNF tumor necrosis factor, I MHC class I molecule, II MHC class II molecule

2 Interleukins

Interleukins mediate signals from one leukocyte to the other, from any other cell to a leukocyte, or vice versa. Interleukins have been numbered consecutively upon their characterization/identification. Thus, this numbering does not contribute to their systematic classification. Today (April 2016), the list of interleukins comprises 39 entries (Table 6.1); however, several of them appear with a number of isoforms, indicated by an index (Greek or Arabic letter) after the number.

Table 6.1 Interleukins

2.1 The IL-1 Family

Already the first interleukin in that list

, IL-1, exists in two isoforms, IL-1α and IL-1β, and is eponymous for a large family of cytokines acting on structurally related receptors [10]. The family of IL-1 cytokines consists of IL-1, IL-18, IL-33, IL-36, IL-37, and IL-38. IL-36 exists in different isoforms, too: IL-36α, IL-36β, and IL-36γ. All these IL-1 family ligands are agonistic at their respective receptors but exert different functions: while IL-1, IL-18, IL-33, and IL-36 are pro-inflammatory, IL-37 delivers an anti-inflammatory signal at the IL-18 receptor. The mechanism how IL-38 exerts its anti-inflammatory function is not well elaborated yet. Moreover, for IL-1 and IL-36, isoforms with antagonistic, thus competitive binding activity at the respective receptor exist, called IL-1RA and IL-36RA, where RA denotes “receptor antagonist” [10,11,12].

2.2 IL-1

IL-1 is produced upon infection mainly by macrophages, endothelial cells, and fibroblasts and acts on virtually all cells and each organ. Generally, IL-1 together with TNF serves as the major mediator of inflammatory diseases, including autoimmune, infectious, and degenerative ones. In the central nervous system (CNS), it acts as endogenous pyrogen inducing fever, and systemically, it induces the acute phase response, either directly or indirectly via the induction of IL-6 production. Both central and systemic effects are executed in order to initially promote and then orchestrate the innate and the adaptive immune reaction.

IL-1α and IL-1β are distinct gene products but bind to the same receptor, the type I IL-1 receptor (IL-1RI), and therefore bear similar, but not identical, biological activities. IL-1RA as well binds IL-1RI, but with a higher affinity than IL-1α or IL-1β do. Thus, the agonists IL-1α/IL-1β and the antagonist IL-1RA compete for IL-1RI binding. The agonist complex composed of IL-1α or IL-1β and the IL-1RI recruits the IL-1 receptor accessory protein (IL-1RAcP), resulting in a ternary complex able to induce signal transduction, thus to elucidate a biological effect, i.e., inflammation. The complex of IL-1RA and IL-1RI, in contrast, due to steric hindrance is unable to recruit the IL-1RAcP and, therefore, to generate a biological response. Thus, IL-1RA is an endogenous regulator of IL-1 activity, essentially necessary to limit the acute inflammatory response, as demonstrated by patients born with inactive IL1RA, who suffer from severe systemic and local inflammation.

IL-36 proteins are produced by epithelial cells and immune cells and induce the production of cytokines promoting Th1 and Th17 cell polarization. IL-36 acts via the receptor called IL-1R-related protein 2 (IL-1Rrp2), which, when ligand-bound, also recruits IL-1RAcP. Biologically, IL-36 is highly similar to the IL-1 and thus reflects most of the aspects depicted there, including the existence of an endogenous receptor antagonist, IL-36RA. This, however, gave no rise for effective therapeutic developments so far [13].

2.3 Pharmacological Implications for IL-1

Human IL-1RA has been cloned and is now being produced commercially as recombinant, N-terminally methionylated, and non-glycosylated therapeutic protein (ANAKINRA) in an E. coli expression system. ANAKINRA is approved by the European Medicines Agency (EMA) for the therapy of rheumatoid arthritis (RA) and cryopyrin-associated periodic syndromes (CAPS). CAPS are rare diseases resulting from a gain-of-function mutation in the cryopyrin gene (CIAS1/NALP3) which is part of the inflammasome. The activated inflammasome induces the proteolytic maturation and the secretion of IL-1β [14]. Thus, CAPS patients show elevated systemic concentrations of IL-1β and then subsequently produced IL-6, as well as some other acute phase (AP) proteins. In synovial fluid and plasma of patients with RA, elevated concentrations of IL-1β can be found, too, and IL-1β is thought to be a central mediator of the synovitis characteristic for RA. ANAKINRA reduces the biological activity of IL-1β by competitive antagonism at the IL-1RI and decreases the systemic concentrations of IL-6 and AP proteins. Consequently, RA or CAPS patients treated with ANAKINRA exhibit significantly ameliorated inflammatory symptoms [15].

Another strategy for blockade of IL-1 signaling is the use of monoclonal antibodies against the cytokine itself. Indeed, the fully human IgG_1,κ monoclonal IL-1β antibody CANAKINUMAB, which is produced in murine hybridoma cells, has been approved for the treatment of systemic juvenile idiopathic arthritis (SJIA), gout arthritis, and CAPS. CANAKINUMAB significantly reduces IL-1β activity by establishing high-affinity IL-1β/IL-1β antibody complexes, thus leading to less IL-1RI receptor activation and less inflammatory symptoms in these diseases [16].

A third way to block IL-1 signaling is the neutralization of the circulating interleukin by binding to soluble IL-1R. Therefore, a dimeric fusion protein of the ligand binding part of the IL-1RI and the IL-1RAcP to the human Fc-domain of IgG1 (RILONACEPT) has been constructed and was approved in Europe as orphan drug for the treatment of CAPS (see above), but the marketing authorization has been voluntarily withdrawn by the producing company due to commercial reasons.

2.4 IL-18 and IL-33

IL-18 and IL-33 are mainly involved in Th1- and Th2-directed adaptive immune reactions, respectively, such as autoimmune diseases, metabolic syndrome, or inflammatory bowel disease and response to parasites, lung inflammation, or fibrosis. Both interleukins are produced by a variety of cell types, including epithelial cells and monocytic cells. While IL-18 promotes the production of IFNγ (originally IL-18 was named IFNγ-inducing factor) in polarized Th1 cells, IL-33 drives the synthesis of Th2-type cytokines, IL-4, IL-5, and IL-13, in Th2 cells and ILC2. IL-18 and IL-33 are recognized by receptor complexes similar to the IL-1 receptor composed of a binding protein and an accessory protein, called, in the case of IL-18, IL-18Rα and IL-18Rβ [17] and, for IL-33, ST2, which associates upon ligand binding with the IL-1RAcP [18]. Interestingly, IL-18Rα can also bind IL-37, which, however, does not act as a classical competitive antagonist. It seems that the complex of IL-37 and IL-18Rα recruits another membrane molecule, TIR8, which together induce anti-inflammatory signaling. Consequently, mice, upon transgenic overexpression of IL-37, are protected against experimentally induced colitis and ischemic diseases. IL-37, since it induces dendritic cells (DC) to promote regulatory T-cell (Treg) activation, seems to be a regulator of the adaptive immune response.

2.5 Pharmacological Implications for IL-18

Because IL-18 has shown antitumor effects in preclinical animal models, the therapeutic potential of recombinant IL-18 in treating solid tumors, metastatic melanoma, lymphomas, or ovarian cancer is currently evaluated in clinical trials, and preliminary results point to no clear-cut but more complex role of IL-18 in several cancers [19, 20]. The benefit of ANTAGONIZING IL-18 in treating chronic inflammatory conditions is the subject of ongoing trials with monoclonal IL-18 antibodies (targeting inflammation in type II diabetes mellitus) or with recombinant IL-18BP (targeting inflammation in Still’s disease). To date, no final results of these studies have been published.

2.6 The IL-2 or Common Cytokine Receptor γ-Chain Family

The pleiotropic IL-2 mainly induces proliferation of both CD4 (helper) and CD8 (cytotoxic) T-cell and natural killer (NK)-cell activities. IL-2 is primarily produced by activated T cells themselves and thus provides a prototypic auto- or paracrine growth factor. It is necessary for the clonal expansion of activated T cells, a process by which a single, antigen-specific T cell gives rise to up to 10⁷ descendants. Since this is a central step in the activation of adaptive immunity, nature has created a backup system, IL-15, which can restore inadequate IL-2 activity [21, 22]. The receptors of these two cytokines use an identical component, the common cytokine receptor γ-chain (γ_c), a feature they share also with IL-4, IL-7, IL-9, and IL-21. Therefore, they are summarized to the γ_c family of cytokines. γ_c is essential for the receptor functions, and, thus, mutations in this protein affect the biological activity of all IL-2 family cytokines, which are essential for the normal development and activity of lymphoid cells. Indeed, such mutations are found in humans with X-linked severe combined immunodeficiency (XSCID).

Besides its main biological targets, T and NK cells, IL-2 also act on B cells, ILC, and neutrophils, in which it promotes proliferation and augments cytokine production. Resting T cells express the IL-2 receptor (IL-2R) β-chain together with γ_c forming a receptor dimer with intermediate affinity for IL-2. Upon activation of T cells, expression of IL-2 and of the IL-2α chain is induced, the latter giving rise to a trimeric high-affinity IL-2R, composed of IL-2Rα, IL-2Rβ, and IL-2Rγ_c. Thus, activated T cells are maximally responsive to auto- or paracrine stimulation by IL-2 [23].

During a primary immune response, antigen-specific naïve T cells are activated, expand clonally due to the activity of IL-2, and differentiate into effector T cells. The orientation of effector T-cell differentiation, called polarization, is instructed by means of key cytokines and transcription factors, such as IL-4 and GATA3, which promote the polarization of Th2 cells. Th2 cells control humoral immunity and are mainly involved in the reaction against extracellular antigens but also in allergic inflammation. IL-21, together with IL-23, and RORγt induce polarization of Th17 cells. Th17-type cytokines, IL-17A, L-17F, and IL-22, are involved in the immune response to bacterial infections and also in autoimmune diseases. Finally, IL-2 in combination with TGF-β promotes the differentiation of Treg, necessary for maintaining self-tolerance.

2.7 Pharmacological Implications for IL-2

This responsiveness of T cells can impede the success of organ transplantation, where it can ultimately lead to transplant rejection. The murine/human chimeric monoclonal IgG_1,κ antibody BASILIXIMAB binds with high affinity to the IL-2Rα chain, thus preventing the binding of the key T-cell activator IL-2. BASILIXIMAB is approved in conjunction with other immunosuppressants for the prophylaxis of acute rejection of renal transplants, where it dampens the inadequate cellular immune response.

The immunoregulatory function of IL-2 is harnessed in cancer treatment, too. A modified form of the human IL-2 gene, recombinantly expressed in E. coli (ALDESLEUKIN), shows immunostimulatory and antitumorous activity in vivo. ALDESLEUKIN is approved for the treatment of metastatic renal cell carcinoma [24]. Together with histamine dihydrochloride, ALDESLEUKIN is used to treat acute myeloid leukemia (AML). It is hypothesized that histamine via the H₂-receptor inhibits generation of reactive oxygen species (ROS) by neutrophilic granulocytes. ROS are increased in the tumor environment, thereby inhibiting cytokine action on NK and T cells. Thus, IL-2 stimulation of NK and T cells is more effective with co-administered histamine [25, 26].

2.8 Th2-Type Cytokines

Structurally, IL-4 is a member of the above discussed IL-2 cytokine family. However, functionally it is grouped together with IL-5 and IL-13 into the Th2-type cytokines cells. Of these, IL-5 is the key factor regulating activation, recruitment, differentiation, and proliferation of eosinophils. The receptor for IL-5 (IL-5R), which is composed of two chains, α and β, belongs to the group of type 1 cytokine receptors. The IL-5Rα chain which virtually is exclusively expressed on eosinophils specifically binds IL-5 but with low affinity. The IL-5-bound IL-5Rα chain does not generate a cellular signal, but upon polymerization with the β-chain, resulting in a high-affinity IL-5R complex, the cell is adequately activated. The β-chain, also called common β-chain, is not an exclusive component of the IL-5R but is also shared by the receptors for IL-3 and GM-CSF. This chain, essential for the signal transduction, associates with the protein tyrosine kinases JAK2 and Lyn. Of note, by alternative splicing a soluble IL-5Rα chain can be formed. This soluble receptor chain binds IL-5 as well as does the membrane-inserted chain, but is unable to form a signal-transducing complex. Thus, it sequesters free IL-5 and functionally acts as antagonist.

In lung biopsies of a subgroup of asthmatic patients, IL-5 mRNA expression was found to be enhanced as compared to controls. Moreover, the quantity of IL-5 mRNA in those samples correlated with the clinical severity of the disease. In mouse models of asthma, the inhalative provocation enhanced IL-5 expression and blockade of IL-5 activity resulted in the reduction of eosinophil numbers in the lung.

Also IL-31 can be regarded a Th2-type cytokine, an atypical one, since it is not exclusively produced by Th2 cells but also by mast cells, macrophages/monocytes, and DC. Its synthesis, however, seems to be regulated by IL-4, and functionally it is clearly associated with allergic diseases, and it is a major factor in the generation of pruritus. Therefore, in addition to IL-5, it provides a highly attractive drug target.

2.9 Pharmacological Implications for IL-5 and IL-13

Since 2016, patients with severe, refractory eosinophilic asthma can be treated with the humanized monoclonal IgG_1,κ IL-5-antibody MEPOLIZUMAB. This antibody binds IL-5 with high affinity and specificity and thereby decreases the IL-5-initiated signal transduction leading to growth, differentiation, recruitment, and survival of eosinophilic granulocytes. MEPOLIZUMAB thereby significantly lowers the rate of asthmatic exacerbations and the dosage of oral glucocorticoids needed for the effective control of asthma activity [27, 28]. Other possible indications for MEPOLIZUMAB currently under investigation are COPD with eosinophilic bronchitis, hypereosinophilic syndrome, and eosinophilic esophagitis.

Two phase III clinical trials have investigated the clinical efficacy of the IL-13 antibody LEBRIKIZUMAB in patients with asthma (LAVOLTA I/II). However, the results of the studies are inconsistent so far [29, 30].

2.10 The IL-6 Family

IL-6 is eponymous for a family of cytokines that originally were characterized by their helical structure and by sharing a common receptor component, the glycoprotein 130 (gp130). Besides IL-6, the family includes IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine (CLC). As for its structure, the above discussed IL-31 is also a member of the IL-6 family of cytokines. It, however, does not signal via the gp130, but via a complex composed of the gp130-like IL-31 receptor A (IL-31RA) chain and the OSM receptor ß subunit.

IL-6 is one of the key mediators regulating inflammation. Upon activation by infectious agents, IL-6 is produced by macrophages, endothelial cells, and T cells and activates many tissue cells including endothelial cells and parenchymal cells to produce effector molecules of inflammation. In the liver, IL-6 together with IL-1 and TNF induced the secretion of acute phase proteins, and in the CNS, they are pyrogenic. The regulation of IL-6 activity is complex. The IL-6-specififc IL-6 receptor (IL-6R) is expressed in its membrane-bound form only on liver cells and leukocytes. After ligand binding the receptor associates with gp130 and activates target cells. gp130 is expressed on a large variety of cells, which do not express the IL-6-specific receptor chain but nevertheless can respond to IL-6. This responsiveness is due to the generation of a soluble form of the IL-6R (sIL-6R), able to bind IL-6 as well and then to associate with membranous gp130 (mgp130). This complex of IL-6, sIL-6R, and mgp130 is able to generate signal transduction, called IL-6 trans-signaling. IL-6 trans-signaling is inhibited by a soluble form of gp130 (sgp130) that sequesters the IL-6/sIL-6R complex, thereby avoiding the association with mgp130. Notably, the interaction between IL-6 and the membrane-bound IL-6R is not affected by the latter mechanism. Thus, sgp130 focuses IL-6 activity on cells expressing the IL-6R.

2.11 Pharmacological Implications for IL-6 Family Members

Soluble and membrane-bound IL-6 receptors can be blocked pharmacologically by monoclonal antibodies, thereby preventing pro-inflammatory IL-6 signal transduction. For the treatment of the chronic inflammatory rheumatoid arthritis, the recombinant humanized monoclonal IgG1 IL-6 antibody TOCILIZUMAB is approved in Europe [31]. Alternatively, not the IL-6 receptor but the circulating cytokine itself can be bound by antibodies. SILTUXIMAB is a chimeric (human/murine) IgG_1,k antibody against IL-6, interfering with the binding of IL-6 to both soluble and membranous IL-6R. However, although SILTUXIMAB blocks the very same signal-transduction pathway as TOCILIZUMAB does, it is currently approved only for the treatment of multicentric Castleman’s disease (MCD). MCD is a rare, aggressive lymphoproliferative disease, which is partly caused by an overproduction of systemic IL-6, leading to the pathologic expansion of lymphoid cells, especially B cells [32].

The IL-6 protein has been fused recombinantly to the extracellular domain of its cognate receptor IL-6R creating the superagonistic protein “hyper-IL-6” capable of activating even cells containing only gp130. Thus, hyper-IL-6 is more than 100-fold as potent as IL-6 alone, and the fusion protein can be efficiently used for in vitro expansion of hematopoietic stem cells [33]. In principle, the resultant stem cells could be used for transplantation, but currently there are no convincing data proving a superiority of ex vivo expanded stem cells over unmanipulated stem cells.

Since IL-11 acts as platelet growth factor, inducing megakaryopoiesis in vivo, the therapeutic effect of recombinant human IL-11 (rhIL-11) in thrombocytopenias is currently being investigated [34].

CNTF has been proven effective in rescuing retinal ganglion cells in preclinical animal models of, e.g., retinitis pigmentosa [35]. Thus, CNTF-secreting eye implants for the treatment of retinitis pigmentosa are now being tested in clinical trials for their clinical efficacy.

2.12 The IL-10 Family

The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29 [36]. Based on their biological functions, three subgroups can be defined. The first consists of only IL-10 itself, which mainly reduces excessive inflammatory responses. The second subgroup is composed of IL-19, IL-20, IL-22, IL-24, and IL-26. These cytokines primarily protect epithelial cells from damage by extracellular pathogens and promote tissue remodeling and wound healing. Members of the third subgroup, IL-28A, IL-28B, and IL-29, are also called type III IFNs (IFN λs). Similar to type I and type II IFNs, type III IFNs induce antiviral responses, however, primarily on epithelial cells.

IL-10 itself is mainly produced by monocytes and T cells, i.e., Th2 cells and induced Treg. It reduces inflammatory processes and thus limits collateral inflammation-induced tissue damage. In fact, together with TGF-β, which is produced by iTreg as well, IL-10 is the most potent anti-inflammatory cytokine.

The receptor for IL-10 is a tetrameric complex, composed of the two IL-10 receptor(R)1 chains and IL-10R2 chains, the latter being structurally similar to the IFN receptor. While IL-10R1 mediates specific ligand binding, IL10R2 is essential for initiating signal transduction, which involves Tyk2- and JAK1-mediated STAT3 phosphorylation. Phosphorylated STAT3 molecules form homodimers and translocate into the nucleus to promote expression of gene products that limit inflammation.

The most prominent phenotype of mice deficient in IL-10 or IL-10R expression is the spontaneous development of chronic enterocolitis associated with an increased prevalence for colorectal carcinomas. Thus, these mice are a relevant model for human inflammatory bowel diseases (IBD), and indeed, in genome-wide association studies, polymorphisms in the genes encoding IL-10 or the IL-10R have been identified as susceptibility loci with association to IBD [37, 38].

2.13 Pharmacological Implications for IL-10

The role of IL-10 in immune responses is largely an anti-inflammatory one. Therefore, recombinant human IL-10 (rhIL-10) has been tested for the treatment of Wegener granulomatosis, psoriasis, IBD, and RA [28]. Most interestingly, there are innovative approaches for targeted delivery of IL-10 to inflamed tissues, e.g., by fusing the cytokine to the antibody fragment F8 (targeting vessels in inflamed tissue) or by using genetically modified organisms producing rhIL-10 for local gastrointestinal delivery [39, 40]. On the other hand, since IL-10 is involved in the development of pathogenic B cells in patients suffering from systemic lupus erythematosus (SLE) [41], an IL-10 antibody (BT-063) is currently being tested for clinical efficacy in treating SLE.

2.14 The IL-12 Family

A unique feature of the family of IL-12 cytokines, which comprises besides IL-12 itself cytokines IL-23, IL-27, and IL-35, is their composition as heterodimeric proteins. Individual pairs of an α-chain (p19, p28, or p35) with a β-chain (p40 or Ebi3) constitute the individual cytokine. Thus, IL-12 is composed of p40 and p35, while IL-23 consists of p40 combined with p19. The β chain of IL-27 and IL-35 is Ebi3, which pairs with p28 or p35, respectively. The remaining two combinations, p28/p40 and p19/Ebi3, are poorly characterized so far [42, 43].

The main function of IL-12, produced mainly by microbe-activated DCs, macrophages, and B cells, is the induction of IFNγ expression by T cells and NK cells, thereby promoting Th1 polarization. IL-23 has pro-inflammatory properties as well but plays a role in the Th17-type immune response. The role of IL-27 and IL-35 has not been ultimately defined; however, they seem to be rather anti-inflammatory.

The receptors for IL-12 cytokine family members are dimeric protein complexes, which, common with the cytokines, share subunits. Pro-inflammatory IL-12 and IL-23 share the IL-12Rβ1 chain that pairs with the IL-12Rβ2 chain and the IL-23R chain, respectively [44]. The probably anti-inflammatory IL-27 and IL-35, in contrast, share with each other and with IL-6 family members the gp130 chain, which associates with WSX-3 (IL-27 receptor) or IL-12Rβ2 (IL-35).

Moreover, some of the IL-12 cytokine chains are functional as monomers or homodimers. Homodimers of p40 antagonize IL-12 by competing for receptor binding. Interestingly, they still are able to activate DCs. Monomers of p28, also referred to as IL-30, probably can inhibit IL-6- and IL-27-induced signaling by sequestering gp130.

2.15 Pharmacological Implications for IL-12 Family Members

A dysregulation of IL-12/IL-23 signaling has been associated with the pathology of psoriasis/psoriasis arthritis and Th1/Th17 inflammatory diseases like rheumatoid arthritis and Crohn’s disease. Since IL-12 and IL-23 share the p40 subunit, both cytokines can be targeted by a single monoclonal p40 antibody. Indeed, the recombinantly produced human monoclonal IgG_1,κ antibody USTEKINUMAB reduces the bioactivity of IL-12/IL-23 in vivo. It is efficient and approved for the treatment of psoriasis/psoriasis arthritis and is evaluated in clinical trials for its efficacy in other Th1-type inflammatory diseases such as multiple sclerosis, RA, and Crohn’s disease [45].

An alternative approach to inhibit IL-12/IL-23 signaling is the use of inhibitors of cytokine synthesis. The small molecule STA-5326 mesylate, which can be administered orally, downregulates IL-12p35 and IL-12/IL-23p40 synthesis on the transcriptional level and is currently being tested in clinical trials for its use in the treatment of rheumatoid arthritis and Crohn’s disease [46].

Monoclonal antibodies targeting solely IL-23p19 are being evaluated for their efficacy in the treatment of RA, CD, and psoriasis/psoriasis arthritis at the moment. The IL-23p19 antibody TILDRAKIZUMAB, for example, has proven effective and superior to placebo in a phase IIb clinical trial [47].

Instead of inhibiting IL-12 bioactivity, clinical trials regarding antitumor effects of IL-12 focus on the use of recombinant human IL-12 (rhIL-12) or plasmids encoding IL-12 (pIL-12). Subcutaneously administered rhIL-12 promotes activation of NK cells and of the cytotoxic T-cell response and is thus thought to boost the antitumor activity of the immune system. pIL-12 can be directly injected into tumorous tissue and subsequently be electroporated into the local cells. This leads to a targeted expression of IL-12 in the tumor. Both approaches are evaluated in the treatment of some cancers like lymphomas, prostate cancer, and melanoma. Very recently, pIL-12 has been started to be tested as “boosting agent” for DNA vaccination, e.g., against HIV-1 or some cancers [48].

2.16 The IL-17 Family

Human IL-17A was originally described in 1993 (initially named CTLA8) and gave rise to the identification of five homologous proteins (IL-17B–IL-17F), which together are grouped to the IL-17 family of cytokines. Notably, the IL-17 proteins occur as homodimers, and only one heterodimer, composed of IL-17A and IL-17F (IL-17A/IL-17F), has been identified [49]. The main IL-17-producing cell type is a subpopulation of effector T cells, referred to as Th17 cells, which are generated from naïve CD4⁺ T cells due to the activity of a series of cytokines including TGF-β, IL-21, IL-1β, IL-6, and IL-23. Besides this, IL-17 is also produced by CD8⁺ T cells, γδ-T cells, NK cells, lymphoid tissue inducer cells, macrophages, neutrophils, and group 3 innate lymphoid cells.

So far, five IL-17 receptor (IL-17R) subunits, called IL-17RA–IL-17RE, have been identified, which pair to generate IL-17 isoform-specific heterodimeric receptors. Thus, IL-17A, IL-17F, and IL-17A/IL-17F, the most abundant and best described IL-17 isoforms, are recognized by the IL-17RA/IL-17RC dimer, while IL-17RA/IL-17RB binds IL-17E, and IL-17RA/IL-17RE binds IL-17C. IL-17B and IL-17D bind to IL-17RB and IL-17RD, respectively, both paired with a so far not identified second receptor chain.

IL-17 is a pleiotropic cytokine; thus, its receptors are expressed on a wide variety of cell types. The lack of IL-17 or IL-17R expression in mice enhances susceptibility to infections with extracellular bacteria and fungi, indicating a central role of IL-17 in host defense. This effect is mediated, at least in part, by the IL-17-induced expression of other mediators by, e.g., epithelial cells or macrophages. These mediators include growth factors such as G-CSF and chemokines such as CXCL8 (IL-8) or CCL20, which activate granulopoiesis and attract neutrophils or lymphocytes to the site of infection, respectively. IL-17-induced mediators also include pro-inflammatory cytokines (IL-1, IL-6, TNF), linking the activity of IL-17 with inflammatory diseases. Indeed, in patients suffering from rheumatoid arthritis or psoriasis enhanced IL-17 concentrations or Th17 cell numbers have been detected, indicating a beneficial effect of drugs targeting IL-17 in these diseases.

2.17 Pharmacological Implications for IL-17

Currently, two recombinant monoclonal anti-IL-17A antibodies (SECUKINUMAB and IXEKIZUMAB) are approved for the therapy of severe plaque psoriasis. In clinical studies, patients receiving SECUKINUMAB or IXEKIZUMAB had significantly reduced “psoriasis area and severity indices” (PASI) as compared to patients receiving placebo. Even in comparison to those patients receiving the comparator anti-psoriatic therapy ETANERCEPT (see below, TNF), the IL-17A antibodies showed a significantly better improvement of PASI scores. SECUKINUMAB has been proven effective in the treatment of arthritic manifestations of psoriasis and Morbus Bechterew, too, and is therefore also approved for these indications. Both SECUKINUMAB and IXEKIZUMAB are currently being evaluated for their efficacy in the treatment of rheumatoid arthritis, and preliminary results point to a possible approval for treating RA, too [50,51,52]. Interestingly, a clinical trial investigating the efficacy of SECUKINUMAB in the treatment of active Crohn’s disease had to be terminated, because the placebo arm of the study exhibited higher reductions of disease activity as compared to the patients treated with IL-17 antibody [53].

3 Tumor Necrosis Factors (TNF)

TNF, together with IL-1 and IL-6, are the main mediators of induction and orchestration of an inflammatory reaction, the so-called master cytokines of inflammation (Fig. 6.3). As such, TNF is involved in virtually all diseases with a contribution of inflammation. TNF, a homo-trimer in its active form, is synthesized mainly by macrophages either in a membrane-bound or in a secreted form. As suggested by its name, it possesses antitumor activity but is also involved in the induction of fever and acute phase protein; in cell proliferation, differentiation, migration, and survival; and in the induction of apoptosis. TNF was assigned to a large superfamily of distantly related proteins, the TNF superfamily (TNFSF) with more than 20 members (Table 6.2) [54]. Most of them are type 2 transmembrane proteins and only TNFSFL1 (LTβ) and TNFSFL2 (TNF) represent true cytokines. TNF effectively activates many tissue cells, including endothelial cells and parenchymal cells, to secrete effector molecules of inflammation, thereby contributing to the inflammatory process. In addition, TNF also enhances the activities of mononuclear phagocytes and other leukocytes, in an autocrine and paracrine manner, thereby amplifying the inflammatory reaction in an autoregulatory loop.

Fig. 6.3

Immune reactions and inflammation. Ag antigen, Ab antibody, B B lymphocyte, C complement, C activated complement components, IFN interferon, IL interleukin, M macrophage, TC tissue cells, TNF tumor necrosis factor

Table 6.2 Tumor necrosis factor superfamily (TNFSF) (selected members)

Two types of TNF receptors (TNFR) have been identified: TNFR1 (p55) and TNFR2 (p75). In analogy to their corresponding ligands, their active form is trimeric, and together with related receptors, they are grouped into a family, the TNFR superfamily (TNFRSF). TNFR1, but not TNFR2, contains a so-called death domain (DD), able to activate caspase cascades resulting in apoptosis. Both TNFR1 and TNFR2 are able to recruit TRAF proteins leading to activation of NF-κB (nuclear factor κB), a central transcription factor associated with inflammation, i.e., cell death/survival, activation, differentiation, and cytokine production. TNFR1 is believed to transmit the majority of TNF-induced biological responses; however, whether TNF leads to NF-κB activation and inflammation or to caspase activation and apoptosis depends on the cellular context.

3.1 Pharmacological Implications for TNF

A chimeric (mouse/human) antibody directed to TNF (INFLIXIMAB) represented the first example of a specific cytokine-blocking antibody and has since proven efficacious in the treatment of psoriasis, psoriasis-arthritis/rheumatoid arthritis, or inflammatory bowel diseases (IBD). INFLIXIMAB—in combination with METHOTREXATE— significantly reduced the clinical severity of arthritis as well as the progression of tissue destructions as apparent by X-ray measurements in clinical studies. Regarding IBD patients, INFLIXIMAB has proven effective for the induction and perpetuation of clinical remission—a subgroup of patients even showed persistent, steroid-free remission under INFLIXIMAB therapy. The successful drug INFLIXIMAB was followed up by further antibodies including completely human antibodies with similar properties and indications (ADALIMUMAB, GOLIMUMAB) and a construct of the F_ab fragment of a humanized antibody coupled to polyethylene glycol (PEG), which has been approved for the therapy of rheumatoid/psoriasis arthritis or axial spondyloarthritis (CERTOLIZUMAB-PEGOL) [55, 56]. The coupling of peptides or proteins to PEG is called pegylation and serves the purpose of altering the pharmacokinetics of the primary substance: Pegylated proteins are taken up more slowly from subcutaneous depots and show delayed excretion, too. Illustrating the principle of self-limitation, the extracellular domains of TNFR can be released during immune or inflammatory reactions. As these soluble receptor fragments contain the full cytokine binding site, they bind their cognate cytokine TNF and thereby dampen its biological effect. A dimeric TNF-receptor construct (ETANERCEPT)—in which the ligand binding domain of the human TNFR2/p75 was fused to the Fc-domain of human IgG1 to increase affinity and half-life in vivo—has been approved with similar indications, effectiveness, and side effects as INFLIXIMAB [57]. The major side effect of the therapy with anti-TNF drugs is an increased risk of infections—as would be expected!—including recurrence of tuberculosis and occasionally septic shock [58].

4 Interferons (IFNs)

IFNγ is the lead cytokine produced by Th1 cells, which develop due to the activity of IL-12. During polarization of the Th cell immune reaction, Th1-derived IFNγ suppresses the generation of Th2 cells, which are involved in the defense against extracellular pathogens and in allergic reactions. Other cells producing IFNγ include CD8⁺ cytotoxic T cells, NK cells, and type 1 innate lymphoid cells. The main function of IFNγ is to activate macrophages, e.g., in case of bacterial infection. Indeed, together with LTβ, IFNγ constitutes the most important macrophage-activating factors, which promote the immune reaction by enhancing the expression of antigen-presenting MHCII molecules as well as the production of cytokines and the execution of macrophage effector mechanisms. In addition, IFNγ participates in NK-cell activation and affects B-cell differentiation. Thus, IFNγ mediates adaptive immunity-driven effector mechanisms against bacterial infection and, likewise, may provide also antiviral and antitumor activities.

Type 1 IFNs , IFNα and IFNβ, can be secreted by almost all cell types; however, some cells likely are specialized for this function. Thus, a dendritic cell (DC) subset, plasmacytoid DC, and fibroblasts produce abundant amounts of IFNα and IFNβ, respectively, upon stimulation by, e.g., viral nucleic acids or proteins. Thirteen isoforms of IFNα are known so far (Table 6.3), each encoded by a single gene. These genes, Ifna1, Ifna2, Ifna4, Ifna5, Ifna6, Ifna7, Ifna8, Ifna10, Ifna13, Ifna14, Ifna16, Ifna17, and Ifna21, are located together in a single gene cluster. Two genes encode the IFNβ isoforms IFNβ1 and IFNβ3. The major function of type 1 IFNs is to induce an antiviral status in cells neighboring the virus-infected cells and to activate innate immune mechanisms, i.e., macrophages and granulocytes, combatting viral infections [59].

Table 6.3 Interferons

All type 1 IFNs bind to the same receptor, type 1 IFN receptor (IFN1R). Consequently, mice devoid of IFN1R expression are highly susceptible to virus infection. Ligand-activated IFN1R is a heterodimer consisting of the chain IFNαR1 and IFNαR2, which initiate signaling via a Tyk-JAK-STAT pathway [60]. Activated STAT molecules eventually bind to specific DNA sequences, IFN-stimulated response elements (IRSE), which regulate the expression of a wide range of genes involved in the antiviral response.

4.1 Pharmacological Implications for IFNs

Recombinant human IFNγ is used therapeutically for the prevention of severe infections in patients with septic granulomatosis or malignant osteopetrosis. The exact mechanism of how IFNγ exactly improves the immune competence of these patients is unknown, but it is hypothesized that IFNγ induces a respiratory burst and an upregulation of HLA-DR/Fc receptors on macrophages, thereby increasing antibody-/cell-dependent cytotoxicity [61].

IFNα (IFNα-2a and IFNα-2b) is indicated in chronic hepatitis B and hepatitis C as well as in the treatment of some malignant tumors, including hairy cell leukemia or chronic myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell leukemia, malignant melanoma, hypernephroma, or bladder carcinoma [62]. Unfortunately, it is ineffective against the majority of carcinomas. Covalent conjugates of IFNα-2a or IFNα-2b with monomethoxy-polyethylene glycol (Peginterferon α-2a or α-2b) have been introduced for the treatment of hepatitis B and C.

Approved indications for recombinant IFNβ (IFNβ-1a and IFNβ-1b) are relapsing remitting forms of multiple sclerosis and first manifestations of an inflammatory demyelinating process [63]. Antitumor effects of IFNβ in humans are uncertain.

Clinical side effects of IFNs include the common “flu-like” syndrome (fever, fatigue, shivering, muscle and joint pain), paresthesias, disturbances of the central nervous system, major depression, gastrointestinal disturbances, cardiac symptoms, granulocytopenia, thrombocytopenia, or anemia.

5 Chemokines

Inflammatory—including allergic—diseases usually are confined to certain organs. This implies that all cells of the immune system which participate in the underlying pathomechanisms must emigrate from the blood vessels and invade into the perspective tissue. On the other hand, antigenic material of infective agents penetrating into the body must be taken up by antigen-presenting dendritic cells in order to be carried to the adjacent lymphoid organs such as the regional lymph nodes to initiate an effective immune response. The very complex migration of leukocytes which proceeds in several subsequent defined steps and, similarly, the migration of dendritic cells are controlled by various cytokines. Among these the family of chemokines plays a pivotal role [64].

More than 40 chemokines have been cloned in human beings (Table 6.4). According to the number and position of cysteine residues in the N-terminus, four groups can be distinguished, which are also represented at the genomic level by gene clusters. Those chemokines, in which the two characteristic cytokines C are separated by an arbitrary amino acid X which are denominated as CXCL1 to CXCL15, are predominantly chemotactic for neutrophils and some—forming a small separate subcluster—for T-lymphocyte subsets. Chemokines with directly adjacent cysteins, CCL1 to CCL27, mostly attract monocytes/macrophages (and again a subset of lymphocytes) and XC chemokines and XCL1 and XCL2 (or CL1 and CL2) lymphocytes. The CX₃CL family contains only one member.

Table 6.4 Chemokines (some selected)

Chemokine receptors all belong to the G-protein-coupled receptors. The 18 receptors are subclassified into 4 groups according to their binding specifications. Thus, CXC chemokine binds to CXC receptors (CXCR), CC chemokines to CCR, XC chemokines to XCR, and CX3C chemokines to CX₃CR. So far, six CXCR, ten CCR, one XCR, and one CX₃CR have been identified [65]. Within these groups, selectivity of single ligands/receptors is overlapping, but not between the groups.

Chemokines have fundamental roles in development, homeostasis, and function of the immune system. Within the immune system, they can be divided into the homeostatic and the inflammatory category. The homeostatic chemokines are constitutively expressed regulating the structural organization and cellular composition of peripheral lymphoid organs such as lymph nodes. They also govern the recirculation of lymphocytes. The expression of inflammatory chemokines is strongly induced by pro-inflammatory stimuli, predominantly in cells of the immune system such as macrophages and T lymphocytes but also in fibroblasts. Inflammatory chemokines participate in the development of inflammatory and immune reactions not only by their chemotactic properties but also as potent activators of their target cells.

5.1 Pharmacological Implications for Chemokines

The chemokine receptors CCR5 (and to a lesser extent CXCR4) have raised great interest as co-receptors (in addition to CD4) for the entry of human immunodeficiency virus (HIV) into macrophages and T lymphocytes. Selective blockade of the human CCR5 leads to an inhibition of the entry of HIV-1 with CCR5 tropism. Thus, the low molecular weight CCR5 antagonist MARAVIROC is approved for the treatment of HIV infections and AIDS [66].

During a screening for HIV-inhibiting drugs in the early 1990s, the selective CXCR4 antagonist PLERIXAFOR was discovered. One could speculate that this drug would be another possible inhibitor of viral entry, but since PLERIXAFOR has very limited oral bioavailability, the drug has never been explored and marketed for HIV therapy. Instead, it has gained approval for mobilization of hematopoietic stem cells into the peripheral blood prior to autologous transplantation (in combination with G-CSF). It is hypothesized that PLERIXAFOR leads to leukocytosis and release of hematopoietic stem cells by preventing the cognate ligand CXCL12 from binding to CXCR4 [67].

6 Colony-Stimulating Factors (CSFs)

The major differentiation factors of the myelomonocytic cell lineage are well known. Several of them are named colony-stimulating factors (CSF) according to the observation which led to their discovery, to stimulate outgrowth of colonies from bone marrow cell cultures. Some of these factors—e.g., stem cell factor (SCF) and multi-CSF (synonymous with IL-3)—regulate early differentiation steps. Others, such as granulocyte/monocyte (GM) CSF, control intermediate steps or selectively induce the final differentiation into mature (neutrophilic) granulocytes (G-CSF) or monocytes (M-CSF). Similarly, erythropoietin, synthesized in the kidney, promotes generation of erythrocytes, and thrombopoietin, which is synthesized in the liver and spleen, promotes formation of platelets (Table 6.5).

Table 6.5 Myeloid differentiation factors, erythropoietin, and thrombopoietin

6.1 Pharmacological Implications for CSFs

As gene technology has facilitated the production of sufficient amounts, CSFs were the first cytokines exploited as drugs. Erythropoietin has become established as the drug of choice for the treatment of severe anemias during terminal renal diseases or due to cytostatic therapy, and thrombopoietin has been applied successfully in clinical trials for the treatment of thrombocytopenias [68]. A relatively new class of agents is the group of TPO-receptor mimetic agents. ROMIPLOSTIM is a fusion protein between a human IgG1-Fc domain and a TPO-receptor binding domain (“peptibody”), whereas ELTROMBOPAG is a non-peptidic small molecule targeting the TPO receptor. Both agents act as agonists and are approved for the treatment of severe thrombocytopenias [69]. Filgrastim (human recombinant G-CSF with an additional methionine, generated from bacteria) was the first CSF approved for the treatment of granulocytopenias. Similar to lenograstim (human recombinant G-CSF from eukaryotic cells), it promptly and selectively increases up to 100-fold the number of functionally active neutrophils, for instance, in patients with cytotoxic drug-induced neutropenias [68]. Treatment with G-CSF markedly reduced the incidence and severity of infections leading to hospital admissions in patients who have received chemotherapy because of malignant tumors. In tumor patients, however, the therapy has not led to an increase in life expectancy. All other CSFs have been evaluated in clinical trials, and some are approved in countries outside Europe.

Therapeutically administered CSFs are intended to substitute for the loss of a patient’s own differentiation factors. To increase their half-lives in vivo, CSFs can be pegylated, too, sometimes additionally via a carbohydrate linker. Despite of the close relationship between these “biologicals” and the endogenous CSFs, these drugs can cause side effects, too. For the CSFs the most prominent side effect is bone and muscle pain, since the administered CFS dose largely exceeds the endogenous concentration and thus induces massive proliferative bone marrow cell activity. Other side effects include dysuria, sometimes elevation of liver enzymes and uric acid, and rarely, a drop in blood pressure, eosinophilia, or allergic reactions [70].

7 Other Drugs Targeting Cytokine Signaling

Suppression of immune and inflammatory reactions can be achieved by inhibition of CYTOKINES in several ways: (1) the inhibition of CYTOKINE synthesis, (2) the decrease of CYTOKINES in free active form, (3) the blocking of the interaction with their receptor, or (4) the inhibition of CYTOKINE-dependent signaling (Fig. 6.4). For each mechanism, at least one clinically relevant example exists.

Fig. 6.4

Inhibition of cytokines. : cytokine, : cytokine antagonist, : functionally active cytokine receptors, : extracellular (“soluble”) domains of receptors

The by far most predominant target cell for immunosuppressants is the T helper lymphocyte. Cytostatic drugs such as AZATHIOPRINE decrease the number of (T) lymphocytes and thereby also of cytokine-producing cells. Monoclonal antibodies directed against T-cell epitopes in part also work by this mechanism; antibodies against CD3 or the INTERLEUKIN-2 receptor mainly prevent activation or proliferation, respectively. They are effective in preventing transplant rejection and also in the treatment of autoimmune diseases (an example being lupus erythematosus). The immunosuppressants CYCLOSPORINE A and TACROLIMUS very selectively block the synthesis of T-lymphocyte cytokines, predominantly of their growth factor IL-2 [71]. Thus, without being cytotoxic, these drugs interfere with T-cell receptor signaling and are approved for prophylaxis and treatment of transplant rejection. GLUCOCORTICOIDS (e.g., prednisone) are similarly immunosuppressive by interfering with the gene expression of IL-2 and other cytokines synthesized by T lymphocytes [72]. They also represent the most efficient anti-inflammatory drugs available at present. Although they affect multiple pro-inflammatory mechanisms, their efficacy largely relies on the capacity to block gene expression of most pro-inflammatory cytokines including IL-1 to IL-8, TNF, or IFNγ.

In the last decades, the signal-transduction pathways engaged by cytokine receptors have been investigated extensively. A core finding was that there exist several phosphorylation cascades in which key protein kinases offer targets for pharmacological interventions. Although quite a number of selective protein kinase inhibitors have been found, only very few of them so far have been approved. The inhibitors SIROLIMUS and EVEROLIMUS, both blocking the serine/threonine protein kinase mTOR (mechanistic target of rapamycin, a synonym for sirolimus), are approved for the prevention of transplant rejection. They prevent mTOR from being activated by IL-2 signaling, normally leading to proliferation of activated T lymphocytes [73].

Regarding the treatment of autoimmune or inflammatory diseases, components of the JAK/STAT signaling pathway seem to be a promising drug target, since over 50 different cytokines exert their effect by activating this common pathway. Janus kinases (JAK) selectively bind to cytokine receptors and, upon activation, transmit the signal by phosphorylating either themselves or other signal proteins, such as the transcription factor STAT (signal transducer and activator of transcription). The JAK1/JAK2 inhibitor RUXOLITINIB is approved for the therapy of myeloproliferative disorders (primary myelofibrosis and polycythemia vera), with a gain-of-function mutation of JAK2 being the most common cause of the disease. RUXOLITINIB is currently under evaluation for the treatment of RA and psoriasis. The US Food and Drug Administration (FDA) has granted approval for the JAK1/JAK3 inhibitor TOFACITINIB for the treatment of RA, but the European Medicines Agency (EMA) refused approval of the drug because of unresolved drug safety concerns. TOFACITINIB also showed beneficial effects in preclinical model of psoriasis and ulcerative colitis and is now being evaluated in clinical trials for its efficacy in these diseases [74].

8 Summary and Outlook

It was not long after their discovery and subsequent molecular characterization that cytokines were tested for their therapeutic potential. This was only made possible by gene technology, which allowed sufficient amounts to be produced in good quality. Some of them—interferons or the colony-stimulating factors were subsequently established as drugs with great medical and even economic importance (Tables 6.6 and 6.7; see Appendices 1 and 2). Not all high-flying hopes, however, have been fulfilled, especially with regard to the treatment of malignant tumors. Thus, after a period of drawbacks, new strategies have begun to evolve, which allow high local concentrations to be selectively generated, the most sophisticated approach involving the use of genetically altered cells.

Table 6.6 Therapeutically relevant cytokines

Table 6.7 Therapeutically relevant cytokine inhibitors

On the other hand, cytokines are now known to be crucial participants in the pathogenesis of many diseases. The realization that long-known and valuable drugs, such as the glucocorticosteroids, act predominantly by suppressing the synthesis of certain cytokines has prompted a search for mechanisms by which the synthesis or function of individual cytokines can be blocked more selectively. Even though cytokines or their inhibitors such as cytokine-specific antibodies have developed into indispensable drugs in important indications, it is certain that this is only the beginning. This assumption is based on the growing evidence that these molecules contribute to many more diseases than those anticipated originally; important examples are atherosclerosis, congestive heart failure, or neurodegenerative diseases.