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.     

The role of soluble receptors in cytokine biology: the agonistic properties of the sIL-6R/IL-6 complex

Cytokines perform ever-increasing roles in both, the regulation of general homeostasis and in orchestrating the immune response during disease. To ensure that control of the cytokine network is tightly regulated, nature has developed a series of systems designed for this purpose. In this respect, researchers have placed considerable emphasis on identifying and characterising the regulatory properties of soluble cytokine receptors. These proteins bind their ligands with similar affinities to those of their cognate transmembrane receptors and are effective at prolonging the circulating half-life of cytokines they bind. However, it is the individual capacity of these soluble receptors to act as either antagonists or agonists which has been the principal focus of most research studies. This review provides an overview of the activities of soluble cytokine receptors, but primarily concentrates on those that possess agonistic properties.     

Soluble cytokine receptors: their role in immunoregulation

A number of cytokine receptors exist in soluble form in the biological fluids of both animals and humans, a phenomenon that might have immunoregulatory implications in vivo. Although these soluble receptors specifically inhibit binding and activity of their respective cytokines in vitro, their actual function in vivo as cytokine inhibitors or as carrier proteins is unclear. Abnormalities in the production of these substances might contribute to the pathophysiology of immune and neoplastic diseases. Besides their role in regulating cytokine activity in vivo, soluble cytokine receptors hold significant potential for therapeutic use as very specific anticytokine agents and as indicators in diagnosis and assessment of immune parameters, prognosis, disease progression, response to treatment, etc., in a variety of autoimmune and malignant diseases.     

Soluble receptors for IL-1 and IL-4: biological activity and therapeutic potential.

Cytokine research has yielded a range of products which have now reached the stage of clinical trials and a plethora of novel therapeutics may be expected. Recently, cytokine receptors have also become an area of intensive research. Preliminary results indicate that recombinant soluble receptors can interfere with the biological functions of cytokines and thus may be appropriate for the treatment of certain pathological conditions where cytokine activity needs to be modulated.     

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.     

ADAM17 orchestrates Interleukin-6, TNFα and EGF-R signaling in inflammation and cancer

It was realized in the 1990s that some membrane proteins such as TNFα, both TNF receptors, ligands of the EGF-R and the Interleukin-6 receptor are proteolytically cleaved and are shed from the cell membrane as soluble proteins. The major responsible protease is a metalloprotease named ADAM17. So far, close to 100 substrates, including cytokines, cytokine receptors, chemokines and adhesion molecules of ADAM17 are known. Therefore, ADAM17 orchestrates many different signaling pathways and is a central signaling hub in inflammation and carcinogenesis. ADAM17 plays an important role in the biology of Interleukin-6 (IL-6) since the generation of the soluble Interleukin-6 receptor (sIL-6R) is needed for trans-signaling, which has been identified as the pro-inflammatory activity of this cytokine. In contrast, Interleukin-6 signaling via the membrane-bound Interleukin-6 receptor is mostly regenerative and protective. Probably due to its broad substrate spectrum, ADAM17 is essential for life and most of the few human individuals identified with ADAM17 gene defects died at young age. Although the potential of ADAM17 as a therapeutic target has been recognized, specific blockade of ADAM17 is not trivial since the metalloprotease domain of ADAM17 shares high structural homology with other proteases, in particular matrix metalloproteases. Here, the critical functions of ADAM17 in IL-6, TNFα and EGF-R pathways and strategies of therapeutic interventions are discussed.     

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).     

Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor.

Accumulating evidence suggests that in serum and other biological fluids, cytokine binding is a property associated with soluble proteins, including a high-affinity soluble version of the IL-4 receptor (sIL-4R). While it is tempting to speculate that sIL-4R might act as a serum carrier protein or serve to inhibit or modulate IL-4 action, specific biological roles for sIL-4R remain to be established. To further assess the immunoregulatory and therapeutic potential of sIL-4R and other soluble receptors, we have created transgenic mice which constitutively express elevated levels of biologically active sIL-4R. Phenotypic characterization of lymphoid organs in sIL-4R transgenic mice revealed normal numbers of B and T cells and normal surface marker expression. Splenic lymphocytes displayed normal in vitro activities as measured by the PFC response and generation of cytotoxic T cells. In addition, antigen-specific IgE and IgG1 in vivo responses were similar in control and transgenic mice. Despite the apparent developmental normality of the sIL-4R transgenic mice, these animals were markedly deficient in the ability to reject cardiac allografts, suggesting that IL-4 is critical for the generation of alloreactivity. The results further suggest that the ability of sIL-4R to regulate IL-4 activities may be under the control of complex interactions that remain to be elucidated.     

Effects of an intravenous catheter on the local production of cytokines and soluble cytokine receptors in healthy men

Like many aspects of physiology, functions of the immune system show considerable diurnal variation. Studies investigating diurnal variations in the circulating amounts of cytokines, in general, used blood samples obtained from an intravenous catheter. The results of such studies may be confounded by an effect of the catheter on local cytokine production. We measured the levels of IL-6, tumour necrosis factor alpha (TNF-alpha) and soluble TNF receptors (sTNF-R) p55 and p75 in 20 healthy men between 09:00 and 19:00 h in plasma samples obtained from an intravenous catheter and in one additional sample obtained by a simple needle stick in the contralateral arm 10 h after baseline. In plasma from the catheter the levels of IL-6 increased significantly over time, TNF-alpha levels slightly decreased and the time courses of TNF receptor levels showed significant trends of a higher order. Control levels of IL-6, TNF-alpha and sTNF-R p75 measured in plasma obtained by needle stick after 10 h did not differ from baseline, and those of sTNF-R p55 were even higher. We conclude that local alterations in the production of cytokines and soluble cytokine receptors induced by an intravenous catheter represent an important confounding factor for studies investigating diurnal variations in immune functions.     

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.     

Soluble cytokine receptors (sIL-2Rα, sIL-2Rβ) induce subunit-specific behavioral responses and accumulate in the cerebral cortex and basal forebrain

Soluble cytokine receptors are normal constituents of body fluids that regulate peripheral cytokine and lymphoid activity. Levels of soluble IL-2 receptors (sIL-2R) are elevated in psychiatric disorders linked with autoimmune processes, including ones in which repetitive stereotypic behaviors and motor disturbances are present. However, there is no evidence that sIL-2Rs (or any peripheral soluble receptor) induce such behavioral changes, or that they localize in relevant brain regions. Here, we determined in male Balb/c mice the effects of single peripheral injections of sIL-2Rα or sIL-2Rβ (0-2 μg/male Balb/c mouse; s.c.) on novelty-induced ambulatory activity and stereotypic motor behaviors. We discovered that sIL-2Rα increased the incidence of in-place stereotypic motor behaviors, including head up head bobbing, rearing/sniffing, turning, and grooming behavior. A wider spectrum of behavioral changes was evident in sIL-2Rβ-treated mice, including increases in vertical and horizontal ambulatory activity and stereotypic motor movements. To our knowledge, this is the first demonstration that soluble receptors induce such behavioral disturbances. In contrast, soluble IL-1 Type-1 receptors (0-4 μg, s.c.) didn't appreciably affect these behaviors. We further demonstrated that sIL-2Rα and sIL-2Rβ induced marked increases in c-Fos in caudate-putamen, nucleus accumbens and prefrontal cortex. Anatomical specificity was supported by the presence of increased activity in lateral caudate in sIL-2Rα treated mice, while sIL-2Rβ treated mice induced greater c-Fos activity in prepyriform cortex. Moreover, injected sIL-2Rs were widely distributed in regions that showed increased c-Fos expression. Thus, sIL-2Rα and sIL-2Rβ induce marked subunit- and soluble cytokine receptor-specific behavioral disturbances, which included increases in the expression of ambulatory activity and stereotypic motor behaviors, while inducing increased neuronal activity localized to cortex and striatum. These findings suggest that sIL-2Rs act as novel immune-to- brain messengers and raise the possibility that they contribute to the disease process in psychiatric disorders in which marked increases in these receptors have been reported.     

Circulating TNF-alpha and its soluble receptors during experimental acute pancreatitis

Clinical and experimental studies have shown increased concentrations of TNF-alpha and its soluble receptors in serum of patients with acute pancreatitis. In this work, we have investigated the time-course of TNF-alpha and its soluble receptors during taurocholate-induced acute pancreatitis. In addition, since TNF-alpha itself could mediate the shedding of its receptors, we have assessed the effect of inhibiting TNF-alpha production on the release of soluble TNF-alpha receptors in experimental acute pancreatitis. Our results indicate that soluble receptors are released in the early stages of the disease and this increase is concomitant with the release of TNF-alpha, which is mainly bound to specific proteins. The increased concentrations of its receptors strongly suggest that they could be these binding proteins. Inhibition of TNF-alpha generation with pentoxifylline abrogated the shedding of sTNF-alphaR1, but had no effect on sTNF-alphaR2. This finding suggests that the shedding of sTNF-alphaR1 is induced by TNF-alpha itself, but in the case of sTNF-alphaR2, the shedding appears to be induced by another mechanism.     

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.     

Bioassay for tumor necrosis factors-α and -β

Tumor necrosis factor (TNF) is a central cytokine in the pathogenesis of septic shock and other inflammatory states. Assay by immunoassay is convenient, but, because of circulating soluble receptors, does not accurately reflect biological activity of the cytokine. This article describes how to perform a bioassay for TNF, using its cytopathic effect on the murine cell line L929. By suitable manipluation, the assay can determine the two different forms of TNF, alpha and beta.     

TNF-alpha, soluble TNF receptor and interleukin-6 plasma levels in the general population

The cytokines tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptors p55 and p75, and interleukin 6 (IL-6) are involved in host defense against several microbiological agents, in the process of inflammation and also in body weight regulation. In the present study, we sought to assess the influence of age, gender, smoking, and body mass index on plasma levels of TNF-alpha, TNF receptors, and IL-6 in more than 550 adult subjects randomly selected from the Bavarian population. None of the cytokine parameters had a normal distribution and all distributions were significantly skewed. The cytokine plasma levels investigated increased significantly with age, while gender had a relatively weaker influence on the plasma levels. Plasma levels of TNF-alpha, TNF receptors, and IL-6 correlated significantly with the BMI. The study provides insights into factors influencing the cytokine levels investigated in a randomly chosen study sample.     

Bcl10 plays a divergent role in NK cell-mediated cytotoxicity and cytokine generation

Activating receptors such as NKG2D and Ly49D mediate a multitude of effector functions including cytotoxicity and cytokine generation in NK cells. However, specific signaling events that are responsible for the divergence of distinct effector functions have yet to be determined. In this study, we show that lack of caspase recruitment domain-containing protein Bcl10 significantly affected receptor-mediated cytokine and chemokine generation, but not cytotoxicity against tumor cells representing "missing-self" or "induced-self." Lack of Bcl10 completely abrogated the generation of GM-CSF and chemokines and it significantly reduced the generation of IFN-gamma (>75%) in NK cells. Commitment, development, and terminal maturation of NK cells were largely unaffected in the absence of Bcl10. Although IL-2-activated NK cells could mediate cytotoxicity to the full extent, the ability of the freshly isolated NK cells to mediate cytotoxicity was somewhat reduced. Therefore, we conclude that the Carma1-Bcl10-Malt1 signaling axis is critical for cytokine and chemokine generation, although it is dispensable for cytotoxic granule release depending on the activation state of NK cells. These results indicate that Bcl10 represents an exclusive "molecular switch" that links the upstream receptor-mediated signaling to cytokine and chemokine generations.     

Dynamics of diurnal changes in serum concentration of TNF-alpha soluble receptors in gastrointestinal cancer patients

The objective of the study was to investigate the dynamic changes in soluble TNFalpha p-55 and p-75 receptors in serum of advanced cancer patients during 24 hours. The group examined consisted of 42 patients suffering from advanced gastrointestinal neoplasms (colorectal, gastric and pancreatic cancer). Serum levels of the cytokine and both receptors in cancer patients were measured using ELISA type kits 6 times a day (8.00 a.m., 2.00 p.m., 6.00 p.m., 10.00 p.m., 2.00 a.m. and again 8.00 a.m.) as well as in healthy controls. The levels of TNFalpha and its soluble receptors were substantially increased in the examined group and displayed statistically significant circadian fluctuations. The presence of circadian rhythm of the cytokine was proved (acrophase - 00.36 a.m.), however no diurnal rhythm of soluble TNF receptors was observed. The concentration of p-55 receptor was distinctly lower then p-75. The peak p-55 value appeared at 10.00 p.m. while the p-75 reached its minimum level at the same time. Although there was no statistical correlation between the receptor concentrations the shapes of both curves remained inversely proportional. The present results may suggest the presence of complex self-regulation mechanisms in advanced gastrointestinal cancer patients.