Christoph Garbers

ADAM17: a molecular switch to control inflammation and tissue regeneration

A disintegrin and metalloproteinase 17 (ADAM17), also known as tumor necrosis factor-α converting enzyme (TACE), is a membrane-bound enzyme that cleaves cell surface proteins, such as cytokines (e.g. TNFα), cytokine receptors (e.g. IL-6R and TNF-R), ligands of ErbB (e.g. TGFα and amphiregulin) and adhesion proteins (e.g. L-selectin and ICAM-1). Here we examine how ectodomain shedding of these molecules can alter their biology and impact on immune and inflammatory responses and cancer development. Gene targeting of Adam17 is embryonic lethal, highlighting the importance of ectodomain shedding during development. Tissue-specific deletion, or hypomorphic knock-in, of Adam17 demonstrates an in vivo role for ADAM17 in controlling inflammation and tissue regeneration. The potential of ADAM17 as therapeutic target is also discussed.

Interleukin-6 and its receptors: A highly regulated and dynamic system

Interleukin-6 (IL-6) is a multifunctional cytokine with well-defined pro- and anti-inflammatory properties. Although only small amounts in the picogram range can be detected in healthy humans, IL-6 expression is highly and transiently up-regulated in nearly all pathophysiological states. IL-6 induces intracellular signaling pathways after binding to its membrane-bound receptor (IL-6R), which is only expressed on hepatocytes and certain subpopulations of leukocytes (classic signaling). Transduction of the signal is mediated by the membrane-bound β-receptor glycoprotein 130 (gp130). In a second pathway, named trans-signaling, IL-6 binds to soluble forms of the IL-6R (sIL-6R), and this agonistic IL-6/sIL-6R complexes can in principle activate all cells due to the uniform expression of gp130. Importantly, several soluble forms of gp130 (sgp130) are found in the human blood, which are considered to be the natural inhibitors of IL-6 trans-signaling. Most pro-inflammatory roles of IL-6 have been attributed to the trans-signaling pathway, whereas anti-inflammatory and regenerative signaling, including the anti-bacterial acute phase response of the liver, is mediated by IL-6 classic signaling. In this simplistic view, only a minority of cell types expresses the IL-6R and is therefore responsive for IL-6 classic signaling, whereas gp130 is ubiquitously expressed throughout the human body. However, several reports point towards a much more complex situation. A plethora of factors, including proteases, cytokines, chemical drugs, and intracellular signaling pathways, are able to modulate the cellular expression of the membrane-bound and soluble forms of IL-6R and gp130. In this review, we summarize current knowledge of regulatory mechanisms that control and regulate the dynamic expression of IL-6 and its two receptors.

Plasticity and cross-talk of Interleukin 6-type cytokines

Interleukin (IL)-6-type cytokines are critically involved in health and disease. The duration and strength of IL-6-type cytokine-mediated signaling is tightly regulated to avoid overshooting activities. Here, molecular mechanisms of inter-familiar cytokine cross-talk are reviewed which regulate dynamics and strength of IL-6 signal transduction. Both plasticity and cytokine cross-talk are significantly involved in pro- and anti-inflammatory/regenerative properties of IL-6-type cytokines. Furthermore, we focus on IL-6-type cytokine/cytokine receptor plasticity and cross-talk exemplified by the recently identified composite cytokines IL-30/IL-6R and IL-35, the first inter-familiar IL-6/IL-12 family member. The complete understanding of the intra- and extracellular cytokine networks will aid to develop novel tailor-made therapeutic strategies with reduced side effects.

The soluble Interleukin 6 receptor: Generation and role in inflammation and cancer

Soluble cytokine receptors are frequently found in human serum, most of them possessing antagonistic properties. The Interleukin 6 receptor (IL-6R) is found as a transmembrane protein on hepatocytes and subsets of leukocytes, but soluble isoforms of the IL-6R (sIL-6R) are generated by alternative splicing or by limited proteolysis of the ADisintegrin And Metalloproteinases (ADAM) gene family members ADAM10 and ADAM17. Importantly, the sIL-6R in complex with its ligand Interleukin 6 (IL-6) has agonistic functions and requires cells expressing the signal transducing ß-receptor gp130 but not the membrane-bound IL-6R. We have called this process IL-6 trans-signaling. Naturally occurring isoforms of soluble gp130 (sgp130), which are generated by alternative splicing, are natural inhibitors of IL-6 trans-signaling, leaving IL-6 classic signaling via the membrane-bound IL-6R unaffected. We used recombinant sgp130Fc protein and recently generated transgenic mice expressing high levels of sgp130Fc to discriminate between classic and trans-signaling in vivo, and demonstrated that IL-6 trans-signaling is critically involved in generation and maintenance of several inflammatory and autoimmune diseases including chronic inflammatory bowel disease, rheumatoid arthritis, peritonitis and asthma, as well as inflammation-induced colon cancer.

Interleukin-6: From basic biology to selective blockade of pro-inflammatory activities

Cytokines receptors exist in membrane bound and soluble form. A soluble form of the human IL-6R is generated by limited proteolysis and alternative splicing. The complex of IL-6 and soluble IL-6R stimulates target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. We have named this process trans-signaling. Soluble gp130 is the natural inhibitor of IL-6/soluble IL-6R complex responses. Recombinant soluble gp130 protein is a molecular tool to discriminate between gp130 responses via membrane bound and soluble IL-6R responses. Neutralizing monoclonal antibodies for global blockade of IL-6 signaling and the sgp130Fc protein for selective blockade of IL-6 trans-signaling have been used in several animal models of human diseases. Using the sgp130Fc protein or sgp130Fc transgenic mice we demonstrate in models of inflammatory bowel disease, peritonitis, rheumatoid arthritis, atherosclerosis pancreatitis, colon cancer, ovarian cancer and pancreatic cancer, that IL-6 trans-signaling via the soluble IL-6R is the crucial step in the development and the progression of the disease. Therefore, sgp130Fc is a novel therapeutic agent for the treatment of chronic inflammatory diseases and cancer and it undergoes phase I clinical trials as an anti-inflammatory drug since June 2013.

Species Specificity of ADAM10 and ADAM17 Proteins in Interleukin-6 (IL-6) Trans-signaling and Novel Role of ADAM10 in Inducible IL-6 Receptor Shedding

Hypomorphic ADAM17ex/ex mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17ex/ex mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.

Inhibition of Classic Signaling Is a Novel Function of Soluble Glycoprotein 130 (sgp130), Which Is Controlled by the Ratio of Interleukin 6 and Soluble Interleukin 6 Receptor

Background: IL-6 trans-signaling plays a critical role in chronic inflammation and cancer. Results: The trans-signaling inhibitor sgp130(Fc) also inhibits classic signaling depending on IL-6/sIL-6R ratios. Conclusion: The additional function of sgp130(Fc) suggests that in vivo only low therapeutic concentrations guarantee blockade of trans-signaling but not classic signaling. Significance: The demonstration that the trans-signaling inhibitor can also inhibit classic signaling is central for the field of IL-6 biology. IL-6 trans-signaling via the soluble IL-6 receptor (sIL-6R) plays a critical role in chronic inflammation and cancer. Soluble gp130 (sgp130) specifically inhibits IL-6 trans-signaling but was described to not interfere with classic signaling via the membrane-bound IL-6R. Physiological and most pathophysiological conditions are characterized by a molar excess of serum sIL-6R over IL-6 characterized by free IL-6 and IL-6 found in IL-6·sIL-6R complexes allowing both classic and trans-signaling. Surprisingly, under these conditions, sgp130 was able to trap all free IL-6 molecules in IL-6·sIL-6R·sgp130 complexes, resulting in inhibition of classic signaling. Because a significant fraction of IL-6 molecules did not form complexes with sIL-6R, our results demonstrate that compared with the anti-IL-6R antibody tocilizumab or the anti-trans-signaling monoclonal antibody 25F10, much lower concentrations of the dimeric sgp130Fc were sufficient to block trans-signaling. In vivo, sgp130Fc blocked IL-6 signaling in the colon but not in liver and lung, indicating that the colon is a prominent target of IL-6 trans-signaling. Our results point to a so far unanticipated role of sgp130 in the blockade of classic signaling and indicate that in vivo only low therapeutic concentrations of sgp130Fc guarantee blockade of IL-6 trans-signaling without affecting IL-6 classic signaling.

Interleukin-6 and interleukin-11: same same but different

Abstract The pleiotropic physiological functions of interleukin (IL-)6 type cytokines range from embryonic development and tissue homoeostasis to neuronal development and T cell differentiation. In contrast, imbalance of the well-controlled cytokine signaling network leads to chronic inflammatory diseases and cancer. IL-6 and IL-11 both signal through a homodimer of the ubiquitously expressed β-receptor glycoprotein 130 (gp130). Specificity is gained through an individual IL-6/IL-11 α-receptor, which does not directly participate in signal transduction, although the initial cytokine binding event to the α-receptor leads to the final complex formation with the β-receptors. Both cytokines activate the same downstream signaling pathways, which are predominantly the mitogen-activated protein kinase (MAPK)-cascade and the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. However, recent studies have highlighted divergent roles of the two related cytokines. Here, we will discuss how the biochemical similarities are translated into unique and non-redundant functions of IL-6 and IL-11 in vivo and illustrate strategies for cytokine-specific therapeutic intervention.

An Interleukin-6 Receptor-dependent Molecular Switch Mediates Signal Transduction of the IL-27 Cytokine Subunit p28 (IL-30) via a gp130 Protein Receptor Homodimer

Background: Anti-inflammatory signaling of IL-27, p28, and EBI3 is mediated by gp130 and Wsx-1. Results: Signaling of p28 via IL-6R is mediated by a gp130 homodimer. Conclusion: Signaling of p28 via IL-6R is likely not anti-inflammatory. Significance: We identify the signal receptor complex of p28/IL-6R. IL-27 consists of the cytokine subunit p28 and the non-signaling α-receptor EBI3. p28 was shown to additionally act via the non-signaling membrane-bound IL-6 α-receptor (IL-6R) as an agonistic cytokine but also as a gp130 β-receptor antagonist, leading to inhibition of IL-6 signaling. Here, we developed a strategy for bacterial expression, purification, and refolding of murine p28. We show that p28 did not interfere with IL-6- or IL-27-induced signaling, indicating that p28 has no antagonistic properties. Moreover, we demonstrate that murine p28 acts as an agonistic cytokine via the murine and human IL-6R, indicating that p28 exhibits no species specificity. p28 was able to induce p28-trans-signaling via the soluble IL-6R (sIL-6R), a characteristic property that was initially described for trans-signaling of IL-6 via the sIL-6R. Of notice, p28/sIL-6R trans-signaling was inhibited by the IL-6 trans-signaling antagonist, soluble gp130. At higher concentrations, p28 but not IL-6 was able to induce signaling even in the absence of IL-6R or EBI3. Although IL-27 signals via a heterodimer of the β-receptor chains gp130 and Wsx-1, p28/IL-6R specifically recruits two gp130 β-receptor chains for signal transduction. The binding of p28 to a gp130/Wsx-1 heterodimer or a gp130 homodimer is highly selective and controlled by a novel molecular switch induced by EBI3 or IL-6R, respectively.

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.