Designer Cytokines

Construction of chimeric proteins containing modules of different proteins belonging to the IL-6R family helped us to identify cytokine binding motives on receptor proteins (Kallen et al. 1999 2000; Aasland et al. 2002, 2003). On the basis of these experiments together with other structural information available on membrane bound and soluble cytokine receptors, we designed several cytokine-cytokine receptor molecules.

Covalent linkage of the domains of the IL-6 and sIL-6R that are necessary for biological activity via a flexible polypeptide linker resulted in a recombinant fusion protein, that was found to be 100-1,000 times more active than the native IL-6/sIL-6R complex. Because of this enhanced activity, we termed this cytokine Hyper-IL-6 (Fischer et al. 1997). Cells lacking the IL-6R, which as a consequence do not respond to IL-6 alone, such as embryonic stem cells, endothelial cells, hematopoietic progenitor cells, neuronal cells, and smooth muscle cells, show a remarkable biologic response to Hyper-IL-6. Adoption of this approach led to the construction of fusion proteins between IL-11 and the soluble IL-11R (Pflanz et al. 1999) Furthermore, proteins consisting of CNTF fused to the soluble CNTF-R have been constructed. These proteins show high neurotrophic activity on neural cells lacking a surface CNTF-R (Guillet et al. 2002; Sun et al. 2002).

By fusing the entire extracellular domain of gp130 protein to the Fc region of human IgG1, we constructed a soluble form of gp130 (sgp130Fc). Surprisingly, in HepG2 cells this protein inhibited only the production of the acute-phase protein antichymotrypsin (ACT) induced by Hyper-IL-6, whereas the response to IL-6 alone remained unaffected (Jostock et al. 2001). We concluded that sgp130Fc does not interfere with responses mediated by the membrane bound IL-6R but exclusively inhibits IL-6 trans-signaling via the soluble IL-6R (Fig. 1b).

Gestrichelter Kreis

Fig. 1a, b Classical and trans-signaling of IL-6 and the inhibitory mechanism of sgp130. a The two modes of IL-6 activation: classical IL-6 activation via the membrane-bound IL-6R and trans-signaling via a soluble form of the IL-6R. In both cases, signals are transmitted by membrane-bound gp130. b Sgp130 binds the IL-6/sIL-6R complex to antagonize IL-6 trans-signaling, whereas classical signaling remains unaffected

Fig. 1a, b Classical and trans-signaling of IL-6 and the inhibitory mechanism of sgp130. a The two modes of IL-6 activation: classical IL-6 activation via the membrane-bound IL-6R and trans-signaling via a soluble form of the IL-6R. In both cases, signals are transmitted by membrane-bound gp130. b Sgp130 binds the IL-6/sIL-6R complex to antagonize IL-6 trans-signaling, whereas classical signaling remains unaffected

Since gp130 is part of the receptors of the cytokines IL-6, IL-11, IL-27, CLC, CNTF, CT-1, LIF, OSM, and NPN, we asked whether the sgp130 inhibition was specific for the IL-6/sIL-6R complex or whether sgp130Fc also effected the biologic activity of the other IL-6-family cytokines (Jostock et al. 2001; Scheller et al. 2005). Our experiments showed that CNTF-mediated activities were unaffected by the sgp130Fc protein. To inhibit the proliferative activity of LIF and OSM on BAF/3 cells stably transfected with gp130 and LIF-R cDNAs, more than 100-fold higher sgp130Fc concentrations were needed than for the inhibition of Hyper-IL-6. These findings are in agreement with surface plasmon resonance experiments showing that Hyper-IL-6 and OSM bind to sgp130 with KD values of 6.9x10-9 M and 1.6x10-7 M, respectively (Richards et al. 2006). Because the recently found cytokine IL-27 also acts via a receptor complex consisting of gp130 and the related receptor protein WSX-1 (Pflanz et al. 2004), we examined whether sgp130Fc inhibited the biologic activity of IL-27 (Scheller et al. 2005). Our results clearly indicated that the sgp130Fc protein did not affect the IL-27-medi-ated STAT3 phosphorylation and proliferation of BAF/3 cells expressing gp130 and WSX-1. As a conclusion of these results, we postulated that the sgp130Fc protein is a specific inhibitor of the IL-6/sIL-6R trans-signaling complex.

Physiological Role of Soluble gp130

In experimental arthritis, colitis, and colon cancer, sgp130 has been shown to influence leukocyte trafficking and to reduce the severity of the diseases. Moreover, the phenotype of sgp130 transgenic mice resembles the phenotype of mice treated with sgp130 in vivo. Together these findings led us to the conclusion that sgp130 acts as the natural inhibitor only of IL-6/sIL-6R complexes. IL-6 does not directly bind sgp130, and as a consequence the classical IL-6 signal via the membrane forms of the IL-6R and gp130 remains unaffected. Figure 1b shows our concept of the molecular mechanism, by which soluble gp130 exerts selective inhibition of the IL-6/sIL-6R complex. The IL-6/sIL-6R complex shows equal affinity for the soluble and membrane-

bound variants of sgp130, and a molar excess of sgp130 inhibits IL-6 trans-signaling. The selective inhibition of IL-6 trans-signaling appears to be an important pathophysiological regulation mechanism in inflammatory diseases.

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