Abstract
Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.
Highlights
A soluble form of IL-6 receptor mediates pathogenic IL-6 trans-signaling
ELISA measurements revealed that soluble interleukin-6 receptor (sIL-6R) protein accumulated in the supernatant of THP-1 and U937 cells upon PMA stimulation, which could be blocked when metalloproteases were globally inhibited by Marimastat (Fig. 1, A and B)
To narrow down the metalloproteases involved in endogenous IL-6R shedding we took advantage of the specific inhibitor pair GI254023X/ GW280264X, which distinguishes between ADAM17- and ADAM10-mediated proteolysis
Summary
A soluble form of IL-6 receptor mediates pathogenic IL-6 trans-signaling. Results: ADAM10 and ADAM17 release IL-6 receptor from both human and murine monocytes/macrophages, whereas in the blood IL-6 receptor is present on microvesicles. The resulting binary cytokine complex exhibits agonistic properties and is able to activate cells that only express gp130 but lack membranebound IL-6R This alternative IL-6 signaling pathway has been termed “IL-6 trans-signaling” and greatly expands the spectrum of IL-6 responsive cells [3]. A soluble form of the IL-6R is present in human serum at relatively high levels and several reports have connected serum sIL-6R generation to alternative mRNA splicing [10, 11]. Hypomorphic ADAM17 mutant mice showing only residual proteolytic activity (5%) exhibit unaltered sIL-6R serum levels [17] This indicates that either an alternative protease, e.g. ADAM10, or even an entirely different mechanism is responsible for serum sIL-6R generation. We were able to locate membrane-associated IL-6R on circulating microvesicles, challenging the prevailing dogma that only metalloproteinases and, in humans, alternative splicing account for the release of sIL-6R into the circulation
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