Prof. Dr. Jürgen Scheller

The immunoregulatory cytokine Interleukin-6 (IL6) acts in a pro- and anti-inflammatory fashion. Synthesized by myeloid cells, fibroblasts and endothelial cells, IL6 on target cells, binds to the IL6 receptor (IL6R) and signals via complex formation with the ubiquitously expressed gp130 receptor. Paradoxically, most cells, which respond to IL6 during inflammatory states do not express the IL6R and are themselves not responsive to the cytokine. A naturally occurring soluble form of the IL6R renders all cells responsive to IL6. Interleukin 6 (IL6) trans-signaling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6 itself.

We show that intrinsic and extrinsic apoptotic stimulation by DNA damage, cytokine deprivation and Fas stimulation promotes shedding of sIL6R. Apoptosis induced shedding of the IL6R was caspase dependent but PKC independent, with inhibition of ADAM17 preventing IL-6R shedding. Such insight is relevant to the control of acute inflammation, where transition from the initial neutrophil infiltration to a more sustained population of mononuclear cells is essential for the resolution of the inflammatory process. This transitional event is governed by IL-6 trans-signaling. We demonstrate that IL6R is shed from apoptotic human neutrophils. In vivo studies in a murine inflammation model showed that neutrophil depletion resulted in reduced local sIL6R levels and a concomitant decrease in mononuclear cells, suggesting that apoptosis induced IL6R shedding from neutrophils promotes IL-6 trans-signaling and regulates the attraction of monocytic cells involved in the clearance of apoptotic neutrophils.

In murine models of Crohn''s disease, rheumatoid arthritis and colon cancer, interleukin (IL)-6 signalling via the soluble IL-6 receptor (sIL-6R, termed IL-6 trans-signalling) has been shown to promote the pathology associated with these conditions. These detrimental activities can however be selectively blocked by soluble forms of the gp130 receptor. Although soluble gp130 (sgp130) therefore represents a viable therapeutic modality for the treatment of these conditions, the mass manufacture of such biologics is often expensive. The advent of molecular farming has however provided an extremely cost effective strategy for the engineering of recombinant proteins. We described the expression and production of a biologically active sgp130 variant which is expressed in transgenic tobacco plants as an elastin-like pentapeptide (ELP) fusion protein (mini-gp130-ELP). Mini-gp130-ELP consists of the first three domains of gp130 (Ig-like domain and cytokine binding module) fused to 100 repeats of ELP. Expression of mini-gp130-ELP did not affect the growth rate or morphology of the transgenic plants, and purification was achieved using inverse transition cycling. This approach led to an overall yield of 141 µg of purified protein per gram of fresh leaf weight. The purified mini-gp130-ELP specifically inhibited sIL-6R-mediated trans-signalling as measured by binding to the IL-6/sIL-6R-complex and through its ability to block sIL-6R-mediated activation of STAT3 phosphorylation and proliferation in human hepatoma cells and murine pre-B-cells.

Consequently, this study validates the potential application of molecular farming in transgenic tobacco plants as a strategy for the expression and purification of therapeutically advantageous biologics such as sgp130.

Moreover, we developed a transgenic mouse strategy, which specifically blocks all IL6 responses mediated by the soluble IL6R but not by the membrane bound IL6R. The antagonist of IL6-Transsignaling is the soluble form of the transmembrane receptor sgp130, dimerized by an Fc-tag (sgp130Fc). The cDNA coding for sgp130Fc was placed under the liver specific promoter PEPCK and randomly inserted into the mouse genome. In these mice, acute inflammation is blocked to the same degree as in IL6 knock out mice strongly arguing for a major role of the soluble IL6R in vivo.

The mode of activation of gp130 and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. Upon expression in cells, L-gp130 stimulates ligand independent STAT3 and ERK1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the IL-3 dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Since gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells which completely blocked their differentiation in the absence of cytokine stimulation and consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases.