Martina Fischer

The designer cytokine hyper-interleukin-6 is a potent activator of STAT3-dependent gene transcription in vivo and in vitro.

Interleukin-6 (IL-6) triggers pivotal pathwaysin vivo. The designer protein hyper-IL-6 (H-IL-6) fuses the soluble IL-6 receptor (sIL-6R) through an intermediate linker with IL-6. The intracellular pathways that are triggered by H-IL-6 are not defined yet. Therefore, we studied the molecular mechanisms leading to H-IL-6-dependent gene activation. H-IL-6 stimulates haptoglobin mRNA expression in HepG2 cells, which is transcriptionally mediated as assessed by run-off experiments. The increase in haptoglobin gene transcription correlates with higher nuclear translocation of tyrosine-phosphorylated STAT3 and its DNA binding. As H-IL-6 stimulates STAT3-dependent gene transcription, we compared the molecular mechanism between IL-6 and H-IL-6. Transfection experiments were performed with a STAT3-dependent luciferase construct. The same amount of H-IL-6 stimulated luciferase activity faster, stronger, and for a longer period of time. Dose response experiments showed that a 10-fold lower dose of H-IL-6 stimulated STAT3-dependent gene transcription comparable with the higher amount of IL-6. Cotransfection with the gp80 and/or gp130 receptor revealed that the effect of H-IL-6 on STAT3-dependent gene transcription is restricted to the gp80/gp130 receptor ratio. High amounts of gp130 increased and high amounts of gp80 decreased the effect on H-IL-6-dependent gene transcription. To investigate the in vivo effect of H-IL-6 on gene transcription in the liver, H-IL-6 and IL-6 were injected into C3H mice. H-IL-6 was at least 10-fold more effective in stimulating the DNA binding and nuclear translocation of STAT3, which enhances haptoglobin mRNA and protein expression. Thus H-IL-6 stimulates STAT3-dependent gene transcription in liver cells in vitro and in vivo at least 10-fold more effectively than IL-6. Our results provide evidence that H-IL-6 is a promising designer protein for therapeutic intervention during different pathophysiological conditions also in humans.

The designer cytokine hyper-IL-6 mediates growth inhibition and GM-CSF-dependent rejection of B16 melanoma cells.

The low immunogenic B16 melanoma cell line was transfected with a mammalian expression vector containing the complementary DNA for a sIL-6R/IL-6 fusion protein, termed Hyper-IL-6 (H-IL-6), which was shown to have biological activities at 100–1000-fold lower concentrations than IL-6 in combination with sIL-6R. The secreted p84 glycoprotein was detected in the supernatant of transfected cells and was fully active on BAF3/gp130 cells, which respond to IL-6/sIL-6R but not to IL-6 alone. Administration of recombinant H-IL-6 to C57BL/6 mice resulted in a prolonged acute phase protein gene expression indicating long systemic persistence of the fusion protein. Transfected B16 cells (B16/H-IL6 cells) showed morphological alterations in combination with a dramatic growth inhibition in vitro. Subcutaneous injection in C57BL/6 mice resulted in an almost complete rejection of B16/H-IL6 cells. This effect was partially abolished in FVB/BL/6 mice transgenic for a GM–CSF receptor antagonist, indicating a GM–CSF-dependent rejection of H-IL-6 transfected B16 cells. These results demonstrate that the anti-tumor effect of cytokines like IL-6 which are secreted by transfected melanoma cells at least in part depends on GM–CSF activity.

The Membrane Proximal Cytokine Receptor Domain of the Human Interleukin-6 Receptor Is Sufficient for Ligand Binding but Not for gp130 Association

Interleukin-6 (IL-6) belongs to the family of the “four-helix bundle” cytokines. The extracellular parts of their receptors consist of several Ig- and fibronectin type III-like domains. Characteristic of these receptors is a cytokine-binding module consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif. On target cells, IL-6 binds to a specific IL-6 receptor (IL-6R), and the complex of IL-6·IL-6R associates with the signal transducing protein gp130. The IL-6R consists of three extracellular domains. The NH2-terminal Ig-like domain is not needed for ligand binding and signal initiation. Here we have investigated the properties and functional role of the third membrane proximal domain. The protein can be efficiently expressed in bacteria, and the refolded domain is shown to be sufficient for IL-6 binding. When complexed with IL-6, however, it fails to associate with the gp130 protein. Since the second and the third domain together with IL-6 can bind to gp130 and induce signaling, our data demonstrate the ligand binding function of the third domain and point to an important role of the second domain in complex formation with gp130 and signaling.

Alternative assay procedures for cytokines and soluble receptors of the IL-6 family

Human hepatoma cells (HepG2 cells) were transfected with expression vectors for human IL-6 (hIL-6) and rat IL-6R (rIL-6-R). The cell lines were used for testing the biological activity of different IL-6 species, soluble hIL-6R (shIL-6R) and some members of the IL-6 cytokine family by means of an ELISA procedure. The assay is based on induction of the gene expression of the acute phase protein haptoglobin in hepatoma cells and provides an alternative bioassay taking advantage of the hepatocyte stimulatory activity of IL-6 (as opposed to the B9 proliferative assay). A dose-response experiment with IL-6 showed that half-maximal stimulation was achieved with approx. 5 ng/ml of hIL-6 in HepG2 cells and with 5-10 ng/ml muIL-6 in HepG2-rIL-6R cells after 24 h. The same response was achieved with 10 ng/ml shIL-6R in HepG2-IL6 cells. In conclusion, the assay is fast and reliable and might be adopted for other cytokines and receptors with hepatocyte stimulating activity.