Jürgen Müllberg

The human cytomegalovirus glycoprotein, UL16, binds to two members of a novel family of molecules, the ULBPs, and to the MHC class I homolog, MICB. The ULBPs are GPI-linked glycoproteins belonging to the extended MHC class I family but are only distantly related to MICB. The ULBP and MICB molecules are ligands for the activating receptor, NKG2D/DAP10, and this interaction is blocked by a soluble form of UL16. The ULBPs stimulate cytokine and chemokine production from NK cells, and expression of ULBPs in NK cell-resistant target cells confers susceptibility to NK cell cytotoxicity. Masking of NK cell recognition of ULBP or MIC antigens by UL16 provides a potential mechanism by which human cytomegalovirus-infected cells might evade attack by the immune system.

Signal transduction in response to interleukin-6 (IL-6) requires binding of the cytokine to its receptor (IL-6R) and subsequent homodimerization of the signal transducer gp130. The complex of IL-6 and soluble IL-6R (sIL-6R) triggers dimerization of gp130 and induces responses on cells that do not express membrane bound IL-6R. Naturally occurring soluble gp130 (sgp130) can be found in a ternary complex with IL-6 and sIL-6R. We created recombinant sgp130 proteins that showed binding to IL-6 in complex with sIL-6R and inhibited IL-6/sIL-6R induced proliferation of BAF/3 cells expressing gp130. Surprisingly, sgp130 proteins did not affect IL-6 stimulated proliferation of BAF/3 cells expressing gp130 and membrane bound IL-6R, indicating that sgp130 did not interfere with IL-6 bound to IL-6R on the cell surface. Additionally, sgp130 partially inhibited proliferation induced by leukemia inhibitory factor (LIF) and oncostatin M (OSM) albeit at higher concentrations. Recombinant sgp130 protein could be used to block the anti-apoptotic effect of sIL-6R on lamina propria cells from Crohn disease patients. We conclude that sgp130 is the natural inhibitor of IL-6 responses dependent on sIL-6R. Furthermore, recombinant sgp130 is expected to be a valuable therapeutic tool to specifically block disease states in which sIL-6R transsignaling responses exist, e.g. in morbus Crohn disease.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise. The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) provides a comprehensive and forward-looking overview of industry’s experience with viral contamination of cell cultures used to produce recombinant proteins.

Interleukin 6 (IL-6) is considered an important mediator of acute inflammatory responses. Moreover, IL-6 functions as a differentiation and growth factor of hematopoietic precursor cells, B cells, T cells, keratinocytes, neuronal cells, osteoclasts, and endothelial cells. IL-6 exhibits its action via a receptor complex consisting of a specific IL-6 receptor (IL-6R) and a signal transducing subunit (gp130). Soluble forms of both receptor components are generated by shedding and are found in patients with various diseases such as acquired immune deficiency syndrome, rheumatoid arthritis, and others. The function of the soluble (s)IL-6R in vivo is unknown. Since human (h)IL-6 acts on human and murine target cells, but murine IL-6 on murine cells only, we constructed transgenic mice expressing the hsIL-6R. We report here that in the presence of hsIL-6R, mice are hypersensitized towards hIL-6, mounting an acute phase protein gene induction at significantly lower IL-6 dosages compared to control animals. Furthermore, in hsIL-6R transgenic mice, the detected acute phase response persists for a longer period of time. The IL-6/IL-6R complex prolongs markedly the Il-6 plasma half-life. Our results reinforce the role of the hsIL-6R as an agonistic protein, help to understand the function of the hsIL-6R in vivo, and highlight the significance of the receptor in the induction of the acute phase response.

Like many proteins with a single transmembrane domain the IL-6R exists in a membrane-associated and soluble form. The soluble IL-6R is generated by limited proteolysis of the membranous receptor. This process, also called shedding, is drastically enhanced by PMA, an activator of protein kinase C. The soluble receptor protein was purified to homogeneity from supernatants of COS-7 cells transfected with a cDNA coding for the transmembrane IL-6R. The COOH-terminus of the shed receptor protein was analyzed by carboxypeptidase treatment and subsequent amino acid analysis. The established cleavage site Gln357/Asp358 was extensively altered by point mutations and small deletions to define the structural requirements for cleavage. Although point mutations around the cleavage site reduced shedding of the IL-6R up to fivefold, deletions of 5 or 10 amino acids almost completely abolished shedding. Deletion of the cytoplasmic domain of the receptor had no influence on shedding of the protein. It turned out that a potential N-glycosylation site close to the proteolytic cleavage site of the IL-6R is used. However this N-glycosylation does not affect the efficiency of the shedding process. Furthermore, we demonstrate for the first time that the human IL-6R is constitutively phosphorylated and that this phosphorylation can be stimulated by PMA but is not correlated with shedding of the receptor protein. The knowledge of the mechanism by which the soluble IL-6R is generated will help to identify the processing enzyme involved and to analyze its regulation.

Many cytokines and soluble cytokine receptors are generated by limited proteolysis of membrane-bound precursors. We have examined the ability of the recently described metalloprotease inhibitor, TNF-alpha protease inhibitor (TAPI), and other protease inhibitors to modulate shedding. The membrane-bound forms of the ligands TNF-alpha and CSF-1, the p60 TNFR and the IL-6R, were expressed in COS-7 cells. As expected, TAPI blocked the spontaneous and PMA-induced release of TNF-alpha from transfected cells. Interestingly, TAPI also inhibited the release of soluble forms of p60 TNFR and IL-6R in COS-7 cells. However, the processing of CSF-1, which also requires proteolytic cleavage of a membrane protein, was not affected. The ability of TAPI to inhibit shedding was unique, since several other classes of protease inhibitors, including three other metalloprotease inhibitors, did not inhibit shedding of IL-6R. To determine whether TAPI would prevent shedding under more physiologic conditions, we demonstrated that TAPI was able to prevent unstimulated and PMA-induced release of the soluble forms of TNF-alpha, p60 TNFR, and IL-6R from the monocytic cell line, THP-1, and from human peripheral blood monocytes. In addition, TAPI was able to inhibit LPS-induced shedding of the p60 TNFR and TNF-alpha from monocytes. In summary, our results indicate that a metalloprotease or group of related metalloproteases is responsible for the proteolytic cleavage of several cell surface proteins.

ABSTRACT The Epstein-Barr virus (EBV) gH-gL complex includes a third glycoprotein, gp42. gp42 binds to HLA class II on the surfaces of B lymphocytes, and this interaction is essential for infection of the B cell. We report here that, in contrast, gp42 is dispensable for infection of epithelial cell line SVKCR2. A soluble form of gp42, gp42.Fc, can, however, inhibit infection of both cell types. Soluble gp42 can interact with EBV gH and gL and can rescue the ability of virus lacking gp42 to transform B cells, suggesting that a gH-gL-gp42.Fc complex can be formed by extrinsic addition of the soluble protein. Truncated forms of gp42.Fc that retain the ability to bind HLA class II but that cannot interact with gH and gL still inhibit B-cell infection by wild-type virus but cannot inhibit infection of SVKCR2 cells or rescue the ability of recombinant gp42-negative virus to transform B cells. An analysis of wild-type virions indicates the presence of more gH and gL than gp42. To explain these results, we describe a model in which wild-type EBV virions are proposed to contain two types of gH-gL complexes, one that includes gp42 and one that does not. We further propose that these two forms of the complex have mutually exclusive abilities to mediate the infection of B cells and epithelial cells. Conversion of one to the other concurrently alters the ability of virus to infect each cell type. The model also suggests that epithelial cells may express a molecule that serves the same cofactor function for this cell type as HLA class II does for B cells and that the gH-gL complex interacts directly with this putative epithelial cofactor.

A functionally and structurally diverse group of transmembrane proteins including transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteolytic activity responsible for pro-tumor necrosis factor alpha (proTNFalpha) processing has been identified and named TACE (TNFalpha converting enzyme). In experiments with TACE deficient (TACE-/-) fibroblasts we found that 4beta-phorbol 12-myristate 13-acetate (PMA)-induced shedding of the interleukin-6 receptor (IL-6R) is strongly reduced. A basal hydroxamate sensitive release of IL-6R, however, could still be detected. This result demonstrates that TACE plays a role in IL-6R processing and that additional metalloproteases might be involved. PMA-induced shedding of IL-6R in TACE deficient mouse fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL-6R and proTNFalpha we generated chimeric IL-6R and proTNFalpha proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNFalpha and IL-6R, respectively. Interestingly, proTNFalpha chimeric proteins showed only minimal shedding. In contrast, IL-6R chimeras containing the proTNFalpha CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNFalpha CS is similar to that of proTNFalpha itself. We conclude that the amino-acid sequence at the proteolytic CS contributes to the cleavage characteristics of a protein. However, this information alone is not sufficient to transfer cleavability as seen with proTNFalpha chimeras containing the IL-6R CS and which were resistant to shedding.

New members of the extended MHC class I-like family were identified based on their ability to bind human cytomegalovirus glycoprotein UL16 and/or their mutual homology. Soluble UL16 binding prteins (ULBP) competed with each other for binding to NK cells. Treatment of human and mouse NK cells with ULBP led to increased production of cytokines/chemokines, proliferation, cytotoxic activity and up-regulation of activation-associated surface molecules. The presence of ULBP during the stimulation phase of the CTL assay caused increased cytotoxic activity. Addition of soluble recombinant UL16 protein inhibited the biological activities mediated by ULBP, suggesting the existence of a novel mechanism utilized by CMV to evade elimination by the host immune system.

The Epstein-Barr virus BZLF2 gene encodes a glycoprotein that associates with gH and gL and facilitates the infection of B lymphocytes. In order to determine whether the BZLF2 protein recognizes a B-cell-specific surface antigen, a soluble protein containing the extracellular portion of the BZLF2 protein linked to the Fc portion of human immunoglobulin G1 (BZLF2.Fc) was expressed from mammalian cells. BZLF2.Fc was used in an expression cloning system and found to bind to a beta-chain allele of the HLA-DR locus of the class II major histocompatibility complex (MHC). Analysis of amino- and carboxy-terminal deletion mutants of the BZLF2.Fc protein indicated that the first 90 amino acids of BZLF2.Fc are not required for HLA-DR beta-chain recognition. Site-directed mutagenesis of an HLA-DR beta-chain cDNA and subsequent immunoprecipitation of expressed mutant beta-chain proteins using BZLF2.Fc indicated that the beta1 domain, which participates in the formation of peptide binding pockets, is required for BZLF2.Fc recognition. The addition of BZLF2.Fc to sensitized peripheral blood mononuclear cells in vitro abolished their proliferative response to antigen and inhibited cytokine-dependent cytotoxic T-cell generation in mixed lymphocyte cultures. Flow-cytometric analysis of Akata cells induced to express late Epstein-Barr virus antigens indicated that expression of BZLF2 did not result in reduced surface expression levels of MHC class II. The ability of BZLF2.Fc to bind to the HLA-DR beta chain suggests that the BZLF2 protein may interact with MHC class II on the surfaces of B cells.

cDNAs coding for the two receptor subunits of the interleukin-6 receptor have been stably expressed in Madine Darby canine kidney (MDCK) cells. The fate of the IL-6 binding protein (IL-6R) and of the signal transducing protein gp130 was studied independently. Both proteins were proteolytically cleaved from cells metabolically labeled with [35S]methionine/cysteine leading to the release of soluble receptor proteins of 55 kDa and 100 kDa, respectively. In contrast to the shedding of the IL-6R gp130 was inefficiently released from the cells and the process was not significantly stimulated by the phorbolester PMA. In addition we show that the soluble forms of the IL-6R and gp130 released by transfected cells can form a ternary complex with interleukin-6 indicating that such complexes also may occur in vivo.

An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-α-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.

As an approach to understanding the interaction of interleukin-6 (IL-6) and its 80-kDa receptor (gp80), we have constructed chimeric human/murine IL-6-molecules, which were expressed in Escherichia coli and analyzed for biological activity and receptor binding. This experimental strategy was based on the observation that human IL-6 acts on human and murine cells, whereas murine IL-6 stimulates only murine cells. The regions to be exchanged were chosen according to the four antiparallel helix model of the hematopoietic cytokine family. All 14 chimeras constructed showed biological activity on murine cells. From the differential biological activities on human cells we deduced that three out of four domains of IL-6 are involved in species specificity, whereas only two domains are necessary for specific recognition by the gp80 IL-6-receptor protein.

Abstract The genome of human herpes virus 8, which is associated with Kaposi’s sarcoma, encodes proteins with similarities to cytokines and chemokines including a homologue of IL-6. Although the function of these viral proteins is unclear, they might have the potential to modulate the immune system. For viral IL-6 (vIL-6), it has been demonstrated that it stimulates IL-6-dependent cells, indicating that the IL-6R system is used. IL-6 binds to IL-6R, and the IL-6/IL-6R complex associates with gp130 which dimerizes and initiates intracellular signaling. Cells that only express gp130 but no IL-6R cannot be stimulated by IL-6 unless a soluble form of the IL-6R is present. This type of signaling has been shown for hematopoietic progenitor cells, endothelial cells, and smooth muscle cells. In this paper we show that purified recombinant vIL-6 binds to gp130 and stimulates primary human smooth muscle cells. IL-6R fails to bind vIL-6 and is not involved in its signaling. A Fc fusion protein of gp130 turned out to be a potent inhibitor of vIL-6. Our data demonstrate that vIL-6 is the first cytokine which directly binds and activates gp130. This property points to a possible role of this viral cytokine in the pathophysiology of human herpes virus 8.

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4–helical bundle" cytokines of the IL–6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (<i>e.g.</i> IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.

Abstract The pleiotropic cytokine IL-6 has been predicted to be a protein with four antiparallel alpha-helices. Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of a set of chimeric human/murine IL-6 proteins has recently allowed us to define a new region (residues Lys41-Glu95) within the IL-6 molecule as being important for receptor binding and biologic activity. We subdivided and analyzed this region, which primarily corresponds to the loop between the first and second alpha-helix of IL-6 with respect to its role in the interaction with the ligand binding subunit of the IL-6 receptor complex and with the IL-6 signal-transducing protein gp130. By construction and analysis of human/murine chimeric IL-6 molecules with only 7 to 10 amino acid residues different from human IL-6 we show that two distinct parts of this region are responsible for receptor binding and signal transduction. On the basis of the recently published structure of granulocyte-CSF, we present a three-dimensional model for the tertiary structure of IL-6, which, together with the IL-6 receptor interaction data, allows for the rational design of human IL-6 receptor antagonists.

Interleukin-6 (IL-6) exerts its action via a cell surface receptor complex consisting of two subunits, the IL-6 receptor and the signal transducer gp130. We have studied the role of both transmembrane proteins for IL-6 internalization and ligand-induced down-regulation of cell surface receptors. Co-expression of wild-type and mutant forms of both the IL-6 receptor and gp130 in transiently transfected COS-7 cells revealed that gp130 is essential for efficient endocytosis and receptor down-regulation. Whereas the cytoplasmic domain of the IL-6 receptor is not significantly involved in the internalization process, deletion of the corresponding domain of gp130 resulted in an almost complete loss of the ability to endocytose IL-6. Mutants with different truncations within the intracellular domain of gp130 revealed that a 10-amino acid sequence TQPLLDSEER is crucial for efficient internalization. Since this sequence contains a putative di-leucine internalization motif, we suggest that a di-leucine motif directs the receptor mediated endocytosis of the IL-6 receptor complex.

C-terminally deleted analogs of human interleukin-6 (IL-6) have been constructed at the cDNA level, and after cell-free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL-6 resulted in 58% helix, 14% beta-structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL-6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% beta-structure, and 18% turn and coil.

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a β-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this β-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

We introduce a procedure for the rapid generation of fully human antibodies derived from “Fab-on-phage” display libraries. The technology is based on the compatibility of display vectors and IgG expression constructs, and allows reformatting of individual Fab clones to IgG, as well as reformatting of antibody repertoires. Examples of batch reformatting of an uncharacterized Fab repertoire and of a pool of Fabs, previously analyzed at the phage level, are presented. The average transient expression levels of the IgG constructs in HEK293T cells are above 10 μg/ml, allowing the use of conditioned media in functional assays without antibody purification. Furthermore, we describe a high-throughput purification method yielding IgG amounts sufficient for initial antibody characterization. Our technology allows the generation and production of antigen-specific complete human antibodies as fast or even faster than raising monoclonal antibodies by conventional hybridoma techniques.

Most transmembrane proteins are subjected to limited proteolysis by cellular proteases. The recent molecular cloning of the TNF-a converting enzyme (TACE) revealed that this shedding enzyme belongs to a family of metalloproteinases which contain a disintegrin domain (ADAM family). The activity of these proteases seems to be tightly regulated. Mice lacking functional TACE are not viable demonstrating the importance of this enzyme for body homeostasis. This review describes the current knowledge of shedding enzymes, the ADAM protein family, the mechanism of shedding as well as physiological consequences of shedding of cytokines and cytokine receptors for cytokine biology.

Pro-TNF alpha, Steel factor, type II IL-1R and IL-2R alpha were expressed in COS-7 cells and the generation of their soluble forms was examined. The release of all four proteins was strongly stimulated by the phorbol ester PMA and completely blocked by a hydroxamate-based inhibitor of metalloproteases. COS-7 cell membranes were found to cleave various synthetic pro-TNF alpha peptides with the same specificity as a partially purified TNF alpha converting enzyme purified from human monocytic cells, suggesting that the same enzyme may be responsible for at least some of the COS-7 cell shedding activity.

Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL-6 protein. vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly. Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL-6.

Degradation of extracellular matrix proteins is performed by metalloproteinases which are inhibited by tissue inhibitors of metalloproteinases (TIMP). We expressed the murine TIMP‐1 protein in E. coli and prepared a polyclonal antiserum against the recombinant protein. Using this antiserum we studied the biosynthesis and glycosylation of murine TIMP‐1 protein in COS‐7 cells transfected with a TIMP‐1 expression plasmid by metabolic labeling and indirect immunofluorescence studies. In primary rat hepatocytes we show for the first time that TIMP‐1 protein expression is up‐regulated upon stimulation with IL‐1β and IL‐6. Since TIMP‐1 is induced during the acute phase reaction it could possibly be involved in the pathogenesis of liver fibrosis.

Gp130 is the signal transducing subunit of the interleukin-6 receptor. Signaling is initiated by the complex formation of gp130 with IL-6 bound to the IL-6 receptor (IL-6R). We have subdivided the extracellular domain of gp130 in two parts and expressed the mutant proteins as soluble IgG fusion proteins in COS-7 cells. By studying the formation of the ternary complex we show that the membrane distal half of gp130 which contains a cytokine receptor domain is responsible for the interaction with the IL-6/IL-6R complex. Interestingly this is the same region which is believed to be involved in specific recognition of the related cytokines LIF, OM, and probably also of CNTF and IL-11.

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. 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. Interleukin-6-type cytokines (IL 1The abbreviations used are: ILinterleukinCT-1cardiotrophin-1CTNFciliary neurotrophic factorLIFleukemia inhibitory factorOSMoncostatin MCBMcytokine-binding modulePCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salinePODperoxidase. -6, IL-11, CT-1, CNTF, LIF, OSM) are cytokines with a characteristic helical fold (1Bazan J.F. Immunol. Today. 1990; 11: 350-354Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). All these cytokines act via receptor complexes, which contain at least one molecule of gp130, the common signal transducing protein of the IL-6 family of cytokines (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). IL-6 and IL-11 act via a homodimer of gp130, whereas CT-1, CNTF, LIF, and OSM require a heterodimer of gp130 and the related protein LIF-receptor (LIF-R) (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Recently it has been shown that in the human system OSM additionally binds and acts via a receptor complex consisting of gp130 and the OSM receptor, a protein related to gp130 and LIF-R (3Mosley B. De Imus C. Friend D. Boiani N. Thoma B. Park L.S. Cosman D. J. Biol. Chem. 1996; 271: 32635-32643Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). In the murine system, the gp130 and OSM receptor complex seems to be the only complex that is bound and activated by OSM (4Ichihara M. Hara T. Kim H. Murate T. Miyajima A. Blood. 1997; 90: 165-173Crossref PubMed Google Scholar). interleukin cardiotrophin-1 ciliary neurotrophic factor leukemia inhibitory factor oncostatin M cytokine-binding module polymerase chain reaction polyacrylamide gel electrophoresis enzyme-linked immunosorbent assay phosphate-buffered saline peroxidase. Interestingly, the cytokines IL-6, IL-11, CT-1, and CNTF first bind to their specific receptor proteins and induce intracellular signaling by subsequent association of this complex with a gp130/gp130 homodimer or a gp130/LIF-R heterodimer. In contrast, LIF and OSM bind directly to LIF-R and gp130, respectively, leading to the formation of heterodimeric gp130-LIF-R or gp130/OSM-R complexes (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Thus, LIF and OSM do not require specific cytokine receptor subunits as do IL-6, IL-11, CT-1, and CNTF. The three-dimensional structures of IL-6 (5Somers W. Stahl M. Seehra J.S. EMBO J. 1997; 16: 989-997Crossref PubMed Scopus (226) Google Scholar), CNTF (6McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (129) Google Scholar), and LIF (7Robinson R.C. Grey L.M. Staunton D. Vankelecom H. Vernallis A.B. Moreau J.F. Stuart D.I. Heath J.K. Jones E.Y. Cell. 1994; 77: 1101-1116Abstract Full Text PDF PubMed Scopus (194) Google Scholar) have been solved and have been shown to share a common “four-helix bundle” fold. The specific ligand binding receptors for IL-6, IL-11, and CNTF are membrane proteins, the extracellular parts of which consist of three domains. Cytokine binding occurs via the cytokine-binding module (CBM), which consists of the second (D2) and the third (D3) domains. The three-dimensional structure of the CBM domain of gp130 has recently been solved (8Bravo J. Staunton D. Heath J.K. Jones E.Y. EMBO J. 1998; 17: 1665-1674Crossref PubMed Scopus (119) Google Scholar). It is structurally highly related to the extracellular parts of the growth hormone receptor (9De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2028) Google Scholar), the prolactin receptor, and the erythropoietin receptor. The specific receptors for IL-6, IL-11, CT-1, and CNTF cytokines exist in a membrane bound and a soluble form (10Davis S. Aldrich T.H. Ip N.Y. Stahl N. Scherer S. Farruggella T. DiStefano P.S. Curtis R. Panayotatos N. Gascan H. Chevalier S. Yancopulos G.D. Science. 1993; 259: 1736-1739Crossref PubMed Scopus (329) Google Scholar, 11Müllberg J. Schooltink H. Stoyan T. Gunther M. Graeve L. Buse G. Mackiewicz A. Heinrich P.C. Rose-John S. Eur. J. Immunol. 1993; 23: 473-480Crossref PubMed Scopus (439) Google Scholar, 12Baumann H. Wang Y. Morella K.K. Lai C.F. Dams H. Hilton D.J. Hawley R.G. Mackiewicz A. J. Immunol. 1996; 157: 284-290PubMed Google Scholar, 13Pennica D. Arce V. Swanson T.A. Vejsada R. Pollock R.A. Armanini M. Dudley K. Phillips H.S. Rosenthal A. Kato A.C. Henderson C.E. Neuron. 1996; 17: 63-74Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In contrast to most soluble receptors for cytokines and growth factors, the soluble receptors of the IL-6 family complexed with their ligands can elicit a biological signal on cells that only express the signaling subunits gp130 and LIF-R. This process has recently been called transsignaling (14Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (692) Google Scholar). Whereas virtually all cells of the body express gp130, the proteins LIF-R, IL-6R, CNTF-R, and IL-11R are only expressed on some cell types (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Recently, it has been shown that cell types that respond exclusively to IL-6/sIL-6R, but not to IL-6 alone, include hematopoietic progenitor cells (15Peters M. Schirmacher P. Goldschmitt J. Odenthal M. Peschel C. Dienes H.P. Fattori E. Ciliberto G. Meyer zum Büschenfelde K.H. Rose-John S. J. Exp. Med. 1997; 185: 755-766Crossref PubMed Scopus (151) Google Scholar), endothelial cells (16Romano M. Sironi M. Toniatti C. Polentarutti N. Fruscella P. Ghezzi P. Faggioni R. Luini W. van Hinsbergh V. Sozzani S. Bussolino F. Poli V. Ciliberto G. Mantovani A. Immunity. 1997; 6: 315-325Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar), osteoclasts (17Udagawa N. Takahashi N. Katagiri T. Tamura T. Wada S. Findlay D.M. Martin T.J. Hirota H. Taga T. Kishimoto T. Suda T. J. Exp. Med. 1995; 182: 1461-1468Crossref PubMed Scopus (333) Google Scholar), and neuronal cells (18März P. Cheng J.-C. Gadient R.A. Patterson P. Stoyan T. Otten U. Rose-John S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3251-3256Crossref PubMed Scopus (286) Google Scholar). Structure-function analyses of the human IL-6R (19Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (180) Google Scholar, 20Vollmer P. Peters M. Ehlers M. Yagame H. Matsuba T. Kondo M. Yasukawa K. Büschenfelde K.H. Rose-John S. J. Immunol. Methods. 1996; 199: 47-54Crossref PubMed Scopus (23) Google Scholar) have shown that the NH2-terminal Ig-like domain of the receptor protein is not needed for ligand binding and biological activity. So far, the contribution of the single D2 and D3 domains to ligand binding and gp130 activation has not been analyzed. Here we show that the D3 domain alone is sufficient for IL-6 binding but cannot associate with gp130. Restriction enzymes were obtained from AGS (Heidelberg, Germany). Vent polymerase was from New England Biolabs (Beverly, MA). Isopropyl-β-d-thiogalactopyranoside was purchased from GERBU (Gaisheim, Germany). Guanidine hydrochloride was from Fluka (Buchs, Switzerland). The preparation of the polyclonal monospecific antisera against IL-6R (6.2) was described previously (21Stoyan T. Michaelis U. Schooltink H. Van Dam M. Rudolph R. Heinrich P.C. Rose-John S. Eur. J. Biochem. 1993; 216: 239-245Crossref PubMed Scopus (68) Google Scholar). Anti-rabbit IgG POD conjugate was purchased from Sigma (Deisenhofen, Germany). Precipitating and soluble BM blue substrates for detection of peroxidase activity were obtained from Boehringer Mannheim (Mannheim, Germany). Skimmed milk powder was from ICN (Meckenheim, Germany). The D3-encoding region of the IL-6R-cDNA was amplified by PCR using an IL-6R-cDNA (22Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar) as a template. NdeI and HindIII sites were introduced in the 5′- and 3′-primers, respectively, to enable cloning of the amplified DNA in the NdeI and HindIII sites of the procaryotic expression vector pRSET 5b (23Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar, 24van Dam M. Müllberg J. Schooltink H. Stoyan T. Brakenhoff J.P. Graeve L. Heinrich P.C. Rose-John S. J. Biol. Chem. 1993; 268: 15285-15290Abstract Full Text PDF PubMed Google Scholar) (sense-primer, 5′ CCG GCG CAT ATG GGA ATC TTG CAG CCT G 3′; antisense-primer, 5′ GGC CCA AGC TTA AGT AGT AAG TGC CTG C 3′). PCR was performed in a total volume of 50 μl of PCR buffer (10 mmKCl, 20 mm Tris-HCl, pH 8.8, 10 mm(NH4)2SO4, 2 mmMgSO4, 0.1% Triton X-100, 0.2 mm of each dNTP) containing 0.25 ng of template DNA, 0.5 unit of Vent DNA-polymerase, and 1 μm D3 sense and antisense primer pairs, respectively. Amplification of D3 DNA by PCR was performed with 35 cycles of denaturation (45 s, 94 °C), primer annealing (45 s, 60 °C), and elongation (45 s, 72 °C). PCR products were purified, subsequently digested with NdeI and HindIII, and ligated into the pRSET 5b vector opened with NdeI andHindIII. The D3 cDNA construct was verified by restriction analysis and automated DNA sequencing. E. coliBL21 (DE3) cells (23Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar, 24van Dam M. Müllberg J. Schooltink H. Stoyan T. Brakenhoff J.P. Graeve L. Heinrich P.C. Rose-John S. J. Biol. Chem. 1993; 268: 15285-15290Abstract Full Text PDF PubMed Google Scholar) were transformed with the D3 expression vector. 200 ml of minimal medium as has been described (25Neubauer P. Häggström L. Enfors S.-O. Biotechnol. Bioeng. 1995; 47: 139-146Crossref PubMed Scopus (108) Google Scholar) were supplemented with ampicillin (50 μg/ml), glucose (5 g/liter), and trace elements (25Neubauer P. Häggström L. Enfors S.-O. Biotechnol. Bioeng. 1995; 47: 139-146Crossref PubMed Scopus (108) Google Scholar), inoculated with a 2-ml culture of a single clone, and cultured overnight at 37 °C. A biofermentor (BIOFLO 3000, New Brunswick Scientific, Edison, NJ) was charged with 3 liters of minimal medium, inoculated with the total overnight culture, and incubated at 37 °C and a minimum oxygen saturation of 30%. Expression of the recombinant protein was induced by adding isopropyl-β-d-thiogalactopyranoside (final concentration: 0.4 mm) at an A600 of about 3 when a specific growth rate of μ = 0.7 was reached, and the fermentation process was maintained for a further 2 h. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mmTris-HCl, pH 7.5, 1 mm EDTA, 1% Tween 20, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride). Complete lysis of bacteria was achieved by three freeze/thaw steps followed by two cycles of sonification (3 min) and several cycles of washing and centrifugation resulting in a preparation of purified inclusion bodies. Supernatants and pellets were analyzed by SDS-PAGE and Western blotting. Inclusion bodies were solubilized in 6m guanidine HCl, 50 mm Tris-HCl, pH 8.0, 10 mm dithiothreitol. A Superdex200 (16/60) column (Amersham Pharmacia Biotech) was equilibrated with refolding buffer (50 mm sodium phosphate, pH 5, 250 mm NaCl, 1 mm dithiothreitol, 0.5 mm EDTA), loaded with 1.5 ml of solubilized inclusion bodies (200 μg of protein), and run with a constant flow rate of 1.5 ml/min. Fractions of 2 ml were collected, analyzed by SDS-PAGE, and significant fractions were pooled and concentrated. By this method 30 mg of purified protein were obtained from a single fermentation process (3 liters of bacterial culture). CD measurements were carried out on an Aviv (Aviv Associates, Lakewood, NJ) 62DS CD spectrometer equipped with a temperature control unit and a Jasco J-600 spectropolarimeter, both calibrated according to Chen and Yang (26Chen G.C. Yang J.T. Anal. Lett. 1977; 10: 1195-1207Crossref Scopus (397) Google Scholar). The spectral bandwidth was 1.5 nm. The time constant ranged between 1 and 4 s and the cell path length between 0.1 and 10 mm. For detection of D3, purified protein or inclusion bodies from different purification steps were separated on 12.5% SDS-polyacrylamide gels and electroblotted onto nylon filters (GeneScreen Plus, NEN Life Science Products). Filters were blocked and incubated with polyclonal rabbit antibody to IL-6R 1:2000 followed by anti-rabbit IgG POD conjugate 1:2000. Peroxidase activity was detected using precipitating BM blue POD substrate. Binding of D3 to IL-6 was analyzed by sandwich ELISA at 22 °C. Purified recombinant IL-6 was coated on ELISA plate wells (Microtest III, Falcon, Oxnard, CA) at 10 μg/ml. After blocking of unspecific binding with PBS containing 5% skimmed milk powder, D3 was applied at 10 μg/ml. After washing, bound protein was detected with polyclonal rabbit antibody to IL-6R 1:2000 followed by anti-rabbit IgG POD conjugate 1:2000. Peroxidase activity was detected with soluble BM blue POD. In every ELISA, recombinant soluble IL-6R (consisting of D2 and D3), expressed in Pichia pastoris and purified as described previously (20Vollmer P. Peters M. Ehlers M. Yagame H. Matsuba T. Kondo M. Yasukawa K. Büschenfelde K.H. Rose-John S. J. Immunol. Methods. 1996; 199: 47-54Crossref PubMed Scopus (23) Google Scholar), was applied in paralell at 10 μg/ml. For negative control the equivalent experiment was performed with the ELISA plate wells coated with PBS containing 5% skimmed milk. 100 ng of purified D3 protein or sIL-6R were incubated for 4 h at 4 °C with a 20-fold excess of human IL-6-Fc or a 10-fold excess of human gp130-Fc (alone or in combination with 300 ng of recombinant human IL-6) or a human IL6-sIL-6R fusion construct called H-IL-6-Fc (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). 2T. Jostock, S. Rose-John, and J. Müllberg, manuscript in preparation. All Fc proteins were obtained from supernatants of transiently transfected COS-7 cells and purified via protein A affinity chromatography. Concentrations were checked using standard protein assays. Immune complexes were precipitated with protein A-Sepharose, separated on 12.5% SDS-polyacrylamide gels, and blotted as described above. Both precipitated D3 protein and sIL-6R again were detected using polyclonal rabbit antibody to IL-6R. IL-6 was covalently immobilized to a carboxymethyl dextran matrix (Fisons, Loughborough, UK) at 2.25 μg/ml for 2 min in 10 mm sodium acetate buffer, pH 5.0, as recommended by the manufacturer (28Krebs B. Griffin H. Winter G. Rose-John S. J. Biol. Chem. 1998; 273: 2858-2865Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Binding experiments were performed at controlled temperature (25 °C) with 10 different concentrations of purified D3 protein using the IASYSTM(Fisons) optical biosensor. Association was monitored for at least 2 min, the sample was replaced by PBS/0.05% Tween 20 (PBST), and dissociation was monitored accordingly before the cuvette was regenerated with 5 mm HCl and equilibrated again in PBST. Association and dissociation affinograms were analyzed by nonlinear regression with the FASTfit (Fisons) software, which uses the Marquardt-Levenburg algorithm for iterative data fitting. For expression of D3 of IL-6R in E. coli, the respective cDNA fragment was amplified by PCR and cloned into the expression vector pRSET 5b (23Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar, 24van Dam M. Müllberg J. Schooltink H. Stoyan T. Brakenhoff J.P. Graeve L. Heinrich P.C. Rose-John S. J. Biol. Chem. 1993; 268: 15285-15290Abstract Full Text PDF PubMed Google Scholar). The expressed protein consisted of D3 together with the epitope recognized by the antibody 6.2 (Fig.1 A) (21Stoyan T. Michaelis U. Schooltink H. Van Dam M. Rudolph R. Heinrich P.C. Rose-John S. Eur. J. Biochem. 1993; 216: 239-245Crossref PubMed Scopus (68) Google Scholar). After expression in a biofermenter and lysis of cells, the protein was exclusively found in inclusion bodies (IB). No recombinant protein was found in the supernatant of the inclusion bodies (SN IB). The expressed protein was visualized as a 17-kDa band by SDS-PAGE (Fig.1 B) and Western blotting (Fig. 1 C). Refolding and purification of the highly enriched protein was achieved by solubilization of the inclusion bodies in guanidine hydrochloride and subsequent gel filtration on a Superdex200 column equilibrated with sodium phosphate buffer (29Müller-Newen G. Pflanz S. Hassiepen U. Stahl J. Wollmer A. Heinrich P.C. Grötzinger J. Eur. J. Biochem. 1997; 247: 425-431Crossref PubMed Scopus (8) Google Scholar). Upon rechromatography of the refolded protein, a single peak was observed indicating that no dimers or higher aggregates were formed during the recovery process of D3 (data not shown). The folding state of the protein was characterized by CD spectroscopy. Fig.2 A shows the far-UV CD spectrum of D3. The spectrum was indicative of a protein in a folded state. The prominent band at 232 nm is the positive lobe of a couplet, which can be attributed to interacting tryptophan side chains (30Grishina I.B. Woody R.W. Faraday Discuss. 1994; 1994: 245-262Crossref Google Scholar). It had also been observed in the far-UV CD spectrum of the corresponding domain of the granulocyte-colony-stimulating factor receptor (31Anaguchi H. Hiraoka O. Yamasaki K. Naito S. Ota Y. J. Biol. Chem. 1995; 270: 27845-27851Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and the third extracellular domain of gp130 (29Müller-Newen G. Pflanz S. Hassiepen U. Stahl J. Wollmer A. Heinrich P.C. Grötzinger J. Eur. J. Biochem. 1997; 247: 425-431Crossref PubMed Scopus (8) Google Scholar). This band seems to be characteristic of the WSXWS motif present in the COOH-terminal domain of the CBM in all class I cytokine receptors. Secondary structure analysis of the CD spectrum showed that the protein mainly consisted of β-sheet (D3: β-sheet = 56%; α-helix = 0%), confirming the secondary structure prediction (32Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1880) Google Scholar). The folded state of the protein was also evident from the near-UV CD spectrum (Fig. 3 B), which showed several distinct bands attributable to tyrosine and tryptophan side chains (30Grishina I.B. Woody R.W. Faraday Discuss. 1994; 1994: 245-262Crossref Google Scholar). These data demonstrated that purification and refolding of D3 from solubilized inclusion bodies could be achieved in a one-step procedure using size-exclusion chromatography.Figure 3Thermal unfolding of D3. Thermal unfolding of D3 was monitored by recording a series of far-UV CD spectra with increasing temperature. A, far-UV CD spectra of D3 at different temperatures (trace 1, 25 °C; trace 2, 28 °C; trace 3, 31 °C; trace 4, 34 °C; trace 5, 37 °C; trace 6, 40 °C; trace 7, 43 °C). B, normalized 232 nm ellipticities of D3 plotted versus temperature.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The thermal stability of the molecule at pH 6.8 was also studied by CD spectroscopy. Fig. 3 A shows a series of far-UV CD spectra recorded at different temperatures. With increasing temperature the prominent positive band at 232 nm decreases. The ellipticity at 232 nm was plotted as a function of temperature in Fig. 3 B. The transition midpoint for D3 at pH 6.8 is 35 °C, indicating a remarkable thermal instability of this domain. We next asked whether D3 was able to bind human IL-6. IL-6 was immobilized to a 96-well ELISA dish, and binding was assayed by indirect ELISA as described under “Materials and Methods.” D3 was recognized by the antibody 6.2 as efficiently as sIL-6R, as shown by Western blotting (Fig. 1 C). In Fig. 4, measurements of D3 and of sIL-6R binding to IL-6 are shown. Binding of IL-6 by D3 and by sIL-6R (consisting of domains D2 + D3) (20Vollmer P. Peters M. Ehlers M. Yagame H. Matsuba T. Kondo M. Yasukawa K. Büschenfelde K.H. Rose-John S. J. Immunol. Methods. 1996; 199: 47-54Crossref PubMed Scopus (23) Google Scholar) were comparable. When human IL-6 was immobilized to an IAsysTM cuvette (see “Materials and Methods”) real-time interaction between IL-6 and D3 could be analyzed. In Fig. 5 Aassociation curves recorded for 10 concentrations of D3 are shown. For clarity, dissociation, although measured for all concentrations, is shown only for the highest concentration. The obtained data were used to calculate an association rate, ka = 2.72 × 104 ± 1467.61 m−1s−1, and a dissociation rate, kd = 1.04 × 10−2 ± 0.0009 s−1, between IL-6 and D3. From these values an affinity of KD = 385 nm can be derived (Fig. 5 B). For comparison, reported affinities for the interaction of immobilized IL-6 and entire soluble IL-6R (33Weiergräber O. Hemmann U. Kuster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P. Hart M.H. Stabel S. Heinrich P. Eur. J. Biochem. 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar) or the immobilized entire sIL-6R and IL-6 (34Toniatti C. Cabibbo A. Sporena E. Salvati A.L. Cerretani M. Serafini S. Lahm A. Cortese R. Ciliberto G. EMBO J. 1996; 15: 2726-2737Crossref PubMed Scopus (51) Google Scholar), which also were mesured at controlled temperature (25 °C) (33Weiergräber O. Hemmann U. Kuster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P. Hart M.H. Stabel S. Heinrich P. Eur. J. Biochem. 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar, 34Toniatti C. Cabibbo A. Sporena E. Salvati A.L. Cerretani M. Serafini S. Lahm A. Cortese R. Ciliberto G. EMBO J. 1996; 15: 2726-2737Crossref PubMed Scopus (51) Google Scholar), were about 10-fold higher. We next asked whether binding of D3 to IL-6 could be detected in solution and whether a D3/IL-6-complex was able to associate with the signal transducing protein gp130. To answer these questions we incubated purified D3 protein with Fc fusion proteins of IL-6 and gp130 in the presence or absence of IL-6. We have recently constructed a fusion protein consisting of human IL-6 and human soluble IL-6R in which both proteins were covalently linked by a flexible polypeptide linker (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). This fusion protein was called Hyper-IL-6 (H-IL-6). This IL-6/sIL-6R fusion protein was expressed as an Fc fusion protein (H-IL-6-Fc). Since we have shown that in the H-IL-6 fusion protein, the IL-6R binding site of IL-6 is not accessible for free IL-6R (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar), this H-IL-6-Fc protein was used as a negative control. Bound protein was immunoprecipitated and detected by Western blotting. As shown in Fig.6 A, D3 could be precipitated with IL-6-Fc (left lane), but not with the combination of IL-6 and gp130-Fc. There was no binding of D3 to gp130-Fc alone or to H-IL-6-Fc. In Fig. 6 B the identical experiment is shown using the sIL-6R consisting of D2 and D3, which could be precipitated with both, IL-6-Fc, and the combination of IL-6 and gp130-Fc. Interestingly, the sIL-6R was almost quantitatively precipitated by IL-6-Fc, whereas only 5–10% of the D3 protein was captured by IL-6-Fc, possibly reflecting the lower affinity of D3 for IL-6 (see above). These results clearly show that D3 of sIL-6R, although being able to bind IL-6, could not mediate an association of the complex with gp130. Four conclusions can be drawn from our study: (i) the third domain of the human IL-6R can be expressed on its own and correctly refoldsin vitro. (ii) The protein is remarkably unstable to thermal perturbation. (iii) D3 on its own binds the ligand IL-6, albeit with a lower affinity than the complete human IL-6R. (iv) Although D3 is sufficient for IL-6 binding, the complex of D3 and IL-6 fails to associate with gp130. Receptors for the four-helix cytokines have been shown to be composed of Ig and fibronectin type III-like domains (35Sprang S.R. Bazan J.F. Curr. Opin. Struct. Biol. 1993; 3: 815-827Crossref Scopus (240) Google Scholar). Specific ligand recognition has been ascribed to the CBM, which consists of two of such fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif (32Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1880) Google Scholar). In the case of the growth hormone-growth hormone receptor complex, it has been demonstrated that both domains of the CBM are involved in ligand binding (9De Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2028) Google Scholar). The extracellular portion of human IL-6R consists altogether of three domains (22Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar). The NH2-terminal domain has been shown to be dispensable for ligand recognition and signal initiation (19Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (180) Google Scholar, 20Vollmer P. Peters M. Ehlers M. Yagame H. Matsuba T. Kondo M. Yasukawa K. Büschenfelde K.H. Rose-John S. J. Immunol. Methods. 1996; 199: 47-54Crossref PubMed Scopus (23) Google Scholar). Recent experiments might point to a role of the NH2-terminal domain of the IL-6R for intracellular processing and protein stability. 3P. Vollmer, B. Oppmann, N. Voltz, and S. Rose-John, submitted for publication. Ligand recognition by the CBM of human IL-6R is believed to occur via the loops which connect the β-strands within the two fibronectin type III-like domains (36Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). In analogy to the growth hormone-growth hormone receptor complex, the third domain of IL-6R is predicted to establish a contact with the respective domain of the CBM of gp130 (36Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). In fact, mutations in a membrane proximal loop region of the IL-6R, which was predicted to be involved in such a contact, turned out to prevent association of the IL-6·IL-6R complex with gp130 (37Salvati A.L. Lahm A. Paonessa G. Ciliberto G. Toniatti C. J. Biol. Chem. 1995; 270: 12242-12249Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Such IL-6R muteins turned out to be weak antagonists of IL-6 (37Salvati A.L. Lahm A. Paonessa G. Ciliberto G. Toniatti C. J. Biol. Chem. 1995; 270: 12242-12249Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Our results demonstrate that D3 of human IL-6R on its own is sufficient for ligand binding. The affinity we measured is about 1 order of magnitude lower than the one determined for the entire sIL-6R (33Weiergräber O. Hemmann U. Kuster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P. Hart M.H. Stabel S. Heinrich P. Eur. J. Biochem. 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar, 34Toniatti C. Cabibbo A. Sporena E. Salvati A.L. Cerretani M. Serafini S. Lahm A. Cortese R. Ciliberto G. EMBO J. 1996; 15: 2726-2737Crossref PubMed Scopus (51) Google Scholar). As suggested by our model of the IL-6·IL-6R complex (36Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar), there are two contact regions between IL-6 and IL-6R. The D2 portion of IL-6R is in contact with the 2c region (loop between the helix A and the helix B) (38Ehlers M. Grötzinger J. deHon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar). The D3 portion of the IL-6R touches the COOH-terminal part of helix D, which also has been implicated in receptor binding (39Krüttgen A. Rose-John S. Moller C. Wroblowski B. Wollmer A. Müllberg J. Hirano T. Kishimoto T. Heinrich P.C. FEBS Lett. 1990; 262: 323-326Crossref PubMed Scopus (71) Google Scholar, 40Leebeek F.W. Kariya K. Schwabe M. Fowlkes D.M. J. Biol. Chem. 1992; 267: 14832-14838Abstract Full Text PDF PubMed Google Scholar). A possible explanation for the low affinity between D3 and IL-6, which we observed in this study, is that loop residues of D2 contribute to ligand binding and thereby to the affinity of the IL-6R (36Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). As discussed above, D3 was shown to be essential for the contact between IL-6R and gp130. Our data demonstrate that this contact is not sufficient for association of the D3·IL-6 complex with gp130. This implies that D2 is needed for the formation of the complex between IL-6, IL-6R, and gp130. We have shown that D3 of the human IL-6R has a remarkably low thermal stability. Unfolding starts already at 28 °C. For comparison, the analogous domain of gp130 is stable up to 34 °C. One consequence of this instability of D3 is that this protein does not exhibit antagonistic activity on cells stimulated with human IL-6 (data not shown). Such assays have to be performed at 37 °C a temperature at which D3 is already unfolded. Since a recombinant version of sIL-6R consisting of D2 and D3 is stable at 37 °C (20Vollmer P. Peters M. Ehlers M. Yagame H. Matsuba T. Kondo M. Yasukawa K. Büschenfelde K.H. Rose-John S. J. Immunol. Methods. 1996; 199: 47-54Crossref PubMed Scopus (23) Google Scholar), it can be concluded that in this complex D2 and D3 must stabilize each other. It has been suggested recently that the complex of IL-6, IL-6R, and gp130 has a stochiometry of two molecules each of IL-6, IL-6R, and gp130 (41Ward L.D. Howlett G.J. Discolo G. Yasukawa K. Hammacher A. Moritz R.L. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Abstract Full Text PDF PubMed Google Scholar, 42Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A.L. Toniatti C. Ciliberto G. EMBO J. 1995; 14: 1942-1951Crossref PubMed Scopus (210) Google Scholar, 43Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (302) Google Scholar). Steric considerations led us to suggest a tetrameric model of the receptor complex containing one molecule each of IL-6 and IL-6R associated with two molecules of gp130 (36Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). Of note, our tetrameric model would predict a close contact of D2 of IL-6R with one gp130 protein (36Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins Struct. Funct. Genet. 1997; 27: 96-109Crossref PubMed Scopus (98) Google Scholar). In this respect it is worth mentioning that mutation of valine 190 in the human IL-6R resulted in loss of complex formation between IL-6·IL-6R and gp130 (19Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (180) Google Scholar), whereas IL-6 binding to IL-6R was unchanged (19Yawata H. Yasukawa K. Natsuka S. Murakami M. Yamasaki K. Hibi M. Taga T. Kishimoto T. EMBO J. 1993; 12: 1705-1712Crossref PubMed Scopus (180) Google Scholar). Valine 190 resides within D2 (22Yamasaki K. Taga T. Hirata Y. Yawata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (889) Google Scholar). A peptide corresponding to a part of D3 in the human IL-6R was shown to inhibit the biological activity of IL-6 without disturbing binding of IL-6 to IL-6R (44Grube B.J. Cochrane C.G. J. Biol. Chem. 1994; 269: 20791-20797Abstract Full Text PDF PubMed Google Scholar). From these data it can be concluded that both D2 and D3 seem to be involved in complex formation between IL-6·IL-6R and gp130. Our data add to the understanding of the molecular mechanism of the association of the components IL-6, IL-6R, and gp130 in the receptor complex. Futhermore no experimental structural information is available on human IL-6R, the reported strategy of bacterial expression and refolding of the IL-6R domain, which is important for ligand binding, might be the basis for experiments aiming at structure elucidation by NMR spectroscopy or x-ray crystallography.

Although regulated ectodomain shedding affects a large panel of structurally and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around cleavage sites, most proteins are shed in response to the stimulation of protein kinase C by phorbol esters. The signal-transducing receptor subunit gp130 is not a substrate of the regulated shedding machinery. We generated several chimaeric proteins of gp130 and the proteins tumour necrosis factor α (TNF-α), transforming growth factor α (TGF-α) and interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchanging small peptide sequences of gp130 for cleavage-site peptides of TNF-α, TGF-α and IL-6R we showed that these short sequences conferred susceptibility to spontaneous and phorbol-ester-induced shedding of gp130. Importantly, these chimaeric gp130 proteins were functional, as shown by the phosphorylation of gp130 and the activation of signal transduction and activators of transcription 3 (‘STAT3’) on stimulation with cytokine. To investigate minimal requirements for shedding, truncated cleavage-site peptides of IL-6R were inserted into gp130. The resulting chimaeras were susceptible to shedding and showed the same cleavage pattern as observed in the chimaeras containing the complete IL-6R cleavage site. Surprisingly, we could also generate cleavable chimaeras by exchanging the juxtamembrane sequence of gp130 for the corresponding region of leukaemia inhibitory factor (‘LIF’) receptor, a protein that like gp130 is not subject to regulated or spontaneous shedding. Thus it seems that there is no minimal consensus shedding sequence. We speculate that structural changes allow the access of the protease to a membrane-proximal region, leading to shedding of the protein.

Although regulated ectodomain shedding affects a large panel of structurally and functionally unrelated proteins, little is known about the mechanisms controlling this process. Despite a lack of sequence similarities around cleavage sites, most proteins are shed in response to the stimulation of protein kinase C by phorbol esters. The signal-transducing receptor subunit gp130 is not a substrate of the regulated shedding machinery. We generated several chimaeric proteins of gp130 and the proteins tumour necrosis factor alpha (TNF-alpha), transforming growth factor alpha (TGF-alpha) and interleukin 6 receptor (IL-6R), which are known to be subject to shedding. By exchanging small peptide sequences of gp130 for cleavage-site peptides of TNF-alpha, TGF-alpha and IL-6R we showed that these short sequences conferred susceptibility to spontaneous and phorbol-ester-induced shedding of gp130. Importantly, these chimaeric gp130 proteins were functional, as shown by the phosphorylation of gp130 and the activation of signal transduction and activators of transcription 3 ('STAT3') on stimulation with cytokine. To investigate minimal requirements for shedding, truncated cleavage-site peptides of IL-6R were inserted into gp130. The resulting chimaeras were susceptible to shedding and showed the same cleavage pattern as observed in the chimaeras containing the complete IL-6R cleavage site. Surprisingly, we could also generate cleavable chimaeras by exchanging the juxtamembrane sequence of gp130 for the corresponding region of leukaemia inhibitory factor ('LIF') receptor, a protein that like gp130 is not subject to regulated or spontaneous shedding. Thus it seems that there is no minimal consensus shedding sequence. We speculate that structural changes allow the access of the protease to a membrane-proximal region, leading to shedding of the protein.

Nat. Med. 6, 583–588 (2000); corrected after print 4 November 2010 In the version of this article initially published, the fourth image in Figure 4b was a duplication of the third image. The error has been corrected in the HTML and PDF versions of the article.

The interleukin-6-type family of cytokines bind to receptor complexes that share gp130 as a common signal-transducing subunit. So far, receptor antagonists for interleukin-6-type cytokines have been constructed that still bind to the specific ligand binding subunit of the receptor complex, but have lost the ability to stimulate gp130. Such receptor antagonists compete for a specific receptor of a member of the cytokine family. Interleukin-6 only binds to gp130 when complexed with the interleukin-6 receptor that exists as a membrane bound and soluble molecule. Here we have constructed fusion proteins that consist of the soluble form of the human interleukin-6 receptor covalently linked to interleukin-6 receptor antagonists. These fusion proteins directly bind to gp130. Moreover, at concentrations of 10–50 nm they completely neutralize not only the biological activity of interleukin-6 but also of other cytokines of the interleukin-6-type family that act via gp130 homodimers or gp130/LIF-R heterodimers. Therefore, these gp130 targeting cytokine antagonists might be useful therapeutic tools in disease states that are related to cytokines of the interleukin-6 family. The interleukin-6-type family of cytokines bind to receptor complexes that share gp130 as a common signal-transducing subunit. So far, receptor antagonists for interleukin-6-type cytokines have been constructed that still bind to the specific ligand binding subunit of the receptor complex, but have lost the ability to stimulate gp130. Such receptor antagonists compete for a specific receptor of a member of the cytokine family. Interleukin-6 only binds to gp130 when complexed with the interleukin-6 receptor that exists as a membrane bound and soluble molecule. Here we have constructed fusion proteins that consist of the soluble form of the human interleukin-6 receptor covalently linked to interleukin-6 receptor antagonists. These fusion proteins directly bind to gp130. Moreover, at concentrations of 10–50 nm they completely neutralize not only the biological activity of interleukin-6 but also of other cytokines of the interleukin-6-type family that act via gp130 homodimers or gp130/LIF-R heterodimers. Therefore, these gp130 targeting cytokine antagonists might be useful therapeutic tools in disease states that are related to cytokines of the interleukin-6 family. interleukin LIF receptor leukemia inhibitory factor ciliary neurotrophic factor cardiotrophin-1 oncostatin M enzyme-linked immunosorbent assay monoclonal antibody polymerase chain reaction. Interleukin-6-type cytokines (IL1-6, IL-11, CT-1, CNTF, LIF, OSM) share the same folding topology of a four-helix bundle (1Bazan J.F. Immunol. Today. 1990; 11: 350-354Abstract Full Text PDF PubMed Scopus (514) Google Scholar,2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). They all act via receptor complexes containing at least one molecule of gp130, the common signal-transducing protein of the IL-6-type family of cytokines (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Whereas IL-6 and IL-11 use a homodimer of gp130, CT-1, CNTF, LIF, and OSM require a heterodimer consisting of gp130 and the LIF-receptor (LIF-R) (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Recently it has been shown that, in the human system, OSM additionally binds and acts via a receptor complex consisting of gp130 and OSM-receptor, a protein related to gp130 and LIF-R (3Mosley B. De Imus C. Friend D. Boiani N. Thoma B. Park L.S. Cosman D. J. Biol. Chem. 1996; 271: 32635-32643Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). However, in mice, OSM only acts via the gp130 and OSM-receptor (4Ichihara M. Hara T. Kim H. Murate T. Miyajima A. Blood. 1997; 90: 165-173Crossref PubMed Google Scholar). Interestingly, IL-6, IL-11, CT-1, and CNTF bind first to specific receptor proteins and these complexes associate with the gp130 homodimer or the gp130/ILIF-R heterodimer. In contrast, LIF and OSM bind directly to LIF-R and gp130, respectively, leading to the formation of heterodimeric gp130·LIF-R or gp130·OSM-R complexes (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). An increased expression of IL-6 has been reported for several diseases like plasmacytoma/myeloma, Castleman's disease, mesanglial proliferative glomerulonephritis, osteoporosis, autoimmune diseases, and AIDS (5Akira S. Taga T. Kishimoto T. Adv. 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We have recently constructed a designer cytokine (Hyper-IL-6) consisting of the bioactive parts of IL-6 and sIL-6R fused by a flexible protein linker. This cytokine was 100–1,000 times more active than the separate proteins IL-6 and sIL-6R (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). Because the designer cytokine Hyper-IL-6 directly binds to and stimulates gp130, we speculated that the fusion of sIL-6R to IL-6 receptor antagonists via a flexible peptide linker would result in proteins that would bind to one gp130 protein without inducing its dimerization, thereby acting as an effective antagonist. Because gp130 is a constituent of all receptor complexes of the IL-6 family of cytokines, we analyzed whether these designer cytokine antagonists could inhibit receptor complexes consisting of gp130/gp130 homodimers or gp130/LIF-R heterodimers. Dulbecco's modified Eagle's medium with glutamax, minimum Eagle's medium, and penicillin/streptomycin were purchased from Life Technologies, Inc. (Eggenstein, Germany). Fetal calf serum was obtained from Seromed (Berlin, Germany). The human sIL-6R ELISA was purchased from CLB (Hiss Diagnostics, Freiburg, Germany). DEAE-dextran and Nonidet P-40 were obtained from Sigma (Taufkirchen, Germany). Protein A-Sepharose CL-4B was obtained from Amersham Pharmacia Biotech (Freiburg, Germany). Restriction enzymes, T4-DNA-ligase and Vent polymerase were from New England Biolabs (Schwalbach, Germany). Tran35S-label (44 TBq/mmol) was from ICN (Meckenheim, Germany) and [3H]thymidine was purchased from Amersham Pharmacia Biotech (Aylesbury, U.K.). X-ray films (X-OMAT-AR) were from Eastman Kodak Co. The following antibodies have been used: MT18, recognizing the NH2-terminal immunoglobulin domain of the human IL-6R (28Hirata Y. Taga T. Hibi M. Nakano N. Hirano T. Kishimoto T. J. 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Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar) as the common template. The PCR leading to the first fragment used the following oligonucleotides as primers 5′-CAGCATCACTGTGTCATCCAC-3′ (s1) sense and 5′-GAGGATATCCCGAATTTGTTTGTCAAT-3′ (as1) antisense for the second fragment the primers 5′-CGGGATATCCTCGACTTCATCTCAGCCCTGAGAAAG-3′ (s2) sense and 5′-TGTTCTCATCTGCACAGCTCTGGC-3′ (as2) antisense were used the third fragment was made with the primers 5′-AAAGACCTGATCCAGTTCCTGCAG-3′ (s3) sense and a pCDM8 reverse primer (as3). The mutated nucleotides are underlined. As the second step the fragments were ligated by digesting the first and the second fragment with EcoRV and ligating them. After phosphorylation, the ligation product was amplified by a second PCR round using the primers s1 and as2. The product of this PCR was ligated via blunt ends to the third PCR fragment of step one, and a final PCR was made using the primers s1 and as3 for amplification. As the third step the last PCR product was cloned into the pCDM8 expression vector. Thereafter, the PCR DNA and pCDM8 containing H-IL-6 were digested with the unique sites XhoI and NotI and then ligated. For the construction of a H-AIL-6 cDNA, aEcoNI/NotI fragment of the previously constructed IL-6 receptor antagonist cDNA IL-6–2a2/3C9 in the vector pRSET (15Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) was cloned into the pCDM8-H-IL-6 vector (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar) opened withEcoNI/NotI. COS-7 cells were transiently transfected using the DEAE-dextran (32Lopata M.A. Cleveland D.W. Sollner-Webb B. Nucleic Acids Res. 1984; 12: 5707-5717Crossref PubMed Scopus (517) Google Scholar). After 48 h, medium was replaced by Dulbecco's modified Eagle's medium with 0.5% fetal calf serum penicillin/streptomycin. After 72 h, the supernatant was collected and concentrated 10-fold by ultrafiltration. Transiently transfected COS-7 cells were metabolically labeled with 50 μCi/ml [35S]cysteine/methionine for 6 h in cysteine/methionine-free minimum Eagle's medium. The supernatant was harvested, supplemented with 0.5% Nonidet P-40, and pretreated with pansorbin (Calbiochem). After incubation with the appropriate antibody or fusion protein for 5 h at 4 °C, the immune complexes were precipitated with protein A-Sepharose preincubated with COS-7 supernatant of unlabeled cells, separated by SDS-polyacrylamide gel (7.5%) electrophoresis and visualized by fluorography. Proliferation assays were performed with stably transfected BAF/3 cells treated with increasing amounts of IL-6, LIF, OSM, CNTF, and H-IL-6 and antagonized with H-DFRD or H-AIL-6 at the concentrations indicated in the figures. Proliferation of the cells was measured after 72 h by pulse labeling with [3H]thymidine for 4.5 h. Radioactivity incorporated in the DNA was determined by scintillation counting. Supernatants of transiently transfected COS-7 cells were separated by SDS-polyacrylamide gel (7.5%) electrophoresis and blotted onto nitrocellulose membranes. The proteins were detected by a murine monoclonal IL-6 specific antibody (mAb-8) (29Brakenhoff J.P. Hart M. De Groot E.R. Di Padova F. Aarden L.A. J. Immunol. 1990; 145: 561-568PubMed Google Scholar) and visualized via peroxidase reaction by a secondary POD-linked rabbit IgG anti-mouse antibody. HepG2 cells were stimulated as described, and activation was measured as the secretion of the acute phase protein haptoglobin that was analyzed by ELISA (33Oppmann B. Stoyan T. Fischer M. Voltz N. März P. Rose-John S. J. Immunol. Methods. 1996; 195: 153-159Crossref PubMed Scopus (9) Google Scholar). IL-6 is a four-helix bundle cytokine that interacts with a receptor complex consisting of IL-6R and a homodimer of gp130. As shown in Fig. 1 A, the interaction sites of IL-6 with the receptor subunits have been named site I (contact site to IL-6R), site II (contact site to gp130) (14Savino R. Ciapponi L. Lahm A. Demartis A. Cabibbo A. Toniatti C. Delmastro P. Altamura S. Ciliberto G. EMBO J. 1994; 13: 5863-5870Crossref PubMed Scopus (129) Google Scholar), and site III (contact site to gp130) (11Ehlers M. Grötzinger J. de Hon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar, 15Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Ehlers M. Grötzinger J. Fischer M. Bos H.K. Brakenhoff J.P.J. Rose-John S. J. Interferon Cytokine Res. 1996; 16: 569-576Crossref PubMed Scopus (23) Google Scholar). A schematic representation of the IL-6 receptor antagonists (Fig. 1 B) and the fusion proteins (Fig. 1 C) used in this study is shown. H-IL-6 is a recently developed fusion protein with the sIL-6R covalently linked to human IL-6 by a 13-amino acid peptide linker (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). The sIL-6R is linked either to human IL-6 (H-IL-6) or to an IL-6 receptor antagonist with a defective site II (H-DFRD) (14Savino R. Ciapponi L. Lahm A. Demartis A. Cabibbo A. Toniatti C. Delmastro P. Altamura S. Ciliberto G. EMBO J. 1994; 13: 5863-5870Crossref PubMed Scopus (129) Google Scholar) or a defective site III (H-AIL-6) (11Ehlers M. Grötzinger J. de Hon F.D. Müllberg J. Brakenhoff J.P. Liu J. Wollmer A. Rose-John S. J. Immunol. 1994; 153: 1744-1753PubMed Google Scholar,15Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Ehlers M. Grötzinger J. Fischer M. Bos H.K. Brakenhoff J.P.J. Rose-John S. J. Interferon Cytokine Res. 1996; 16: 569-576Crossref PubMed Scopus (23) Google Scholar). The cDNAs coding for the fusion proteins were transfected into COS-7 cells, and the secreted proteins were quantified using a sIL-6R ELISA (Fig. 2 A). The supernatants were adjusted to equal fusion protein concentrations. As shown in Fig. 2 B, approximately equal amounts were detected on a Western blot using an anti-IL-6 monoclonal antibody. The somewhat weaker detection of the fusion protein H-AIL-6 might be because of reduced recognition of the mutated IL-6 within the fusion protein by the monoclonal antibody used for detection. The fusion protein H-IL-6 has been shown to directly stimulate gp130 expressed on target cells (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). The fusion proteins H-DFRD and H-AIL-6 each have one defective contact site to gp130, either site II or site III, respectively (14Savino R. Ciapponi L. Lahm A. Demartis A. Cabibbo A. Toniatti C. Delmastro P. Altamura S. Ciliberto G. EMBO J. 1994; 13: 5863-5870Crossref PubMed Scopus (129) Google Scholar, 15Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). They should, however, still be able to bind to gp130 via the second site. To test for the binding ability of the fusion proteins, we metabolically labeled COS-7 cells that had been transfected with H-IL-6, H-DFRD, and H-AIL-6 cDNAs. To confirm equal expression of the fusion proteins, the supernatants of the transfected COS-7 cells were immunoprecipitated with the mAb MT18, which recognized the NH2-terminal immunoglobulin domain of the human IL-6R (28Hirata Y. Taga T. Hibi M. Nakano N. Hirano T. Kishimoto T. J. Immunol. 1989; 143: 2900-2906PubMed Google Scholar). The precipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, and gels were subjected to fluorography. As shown in Fig. 3 A, this procedure led to the precipitation of proteins of an apparent molecular mass of 70 kDa. The approximately equal intensities indicate equal protein concentrations in the supernatant of transfected cells. In a parallel experiment, a fusion protein consisting of the extracellular portion of human gp130 fused to the constant part of human immunoglobulin (Fc) was added to the supernatants of transfected COS-7 cells. Supernatants were treated with protein A-Sepharose, and precipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and subjected to fluorography. As shown in Fig. 3 B, radiolabeled H-IL-6, H-DFRD, and H-AIL-6 were all precipitated by the gp130-Fc protein albeit with slightly different efficiencies. H-IL-6 and H-DFRD were precipitated at nearly equal amounts compared with H-AIL-6 that was precipitated with reduced efficiency. From this experiment it can be concluded that the three fusion proteins directly bind to the extracellular portion of gp130. We speculated that the fusion proteins H-DFRD and H-AIL-6, which both had only one intact contact site to gp130, would bind to and block gp130 by preventing homodimerization with gp130 or heterodimerization with LIF-R. We first tested whether the fusion proteins H-DFRD and H-AIL-6 exhibited biological activity on gp130-expressing cells. We used IL-3-dependent BAF/3 cells stably transfected with a human gp130 cDNA (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). As can be seen in Fig. 4, in the absence of IL-3 these cells proliferate when stimulated with 10 ng/ml H-IL-6. In contrast, stimulation of cells with H-DFRD and H-AIL-6 at concentrations up to 5 μg/ml led to no [3H]thymidine incorporation. This experiment clearly demonstrated that in contrast to H-IL-6, the fusion proteins H-DFRD and H-AIL-6 possessed no intrinsic biological activity. We next asked whether the proteins H-DFRD and H-AIL-6 were able to inhibit the growth of BAF/3 cells stably transfected with human gp130 and human IL-6R cDNAs. These cells can be stimulated by human IL-6 with half-maximal proliferation achieved at 0.5 ng/ml IL-6 (Fig. 5 A). To cells stimulated with 0.5 ng/ml IL-6 we added increasing amounts of the two fusion proteins H-DFRD and H-AIL-6. The H-DFRD and H-AIL-6 fusion proteins completely inhibited the proliferation of the cells with an IC50 of 300–500 ng/ml as shown in Fig. 5 B. An identical amount of COS-7 supernatants of mock transfected cells did not affect the IL-6-stimulated proliferation of gp130-transfected BAF/3 cells (data not shown). As an additional control we used the IL-6R specific IL-6 antagonists 2a2/3C9 (15Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and DFRD (14Savino R. Ciapponi L. Lahm A. Demartis A. Cabibbo A. Toniatti C. Delmastro P. Altamura S. Ciliberto G. EMBO J. 1994; 13: 5863-5870Crossref PubMed Scopus (129) Google Scholar) (Fig. 1 B). As described previously (14Savino R. Ciapponi L. Lahm A. Demartis A. Cabibbo A. Toniatti C. Delmastro P. Altamura S. Ciliberto G. EMBO J. 1994; 13: 5863-5870Crossref PubMed Scopus (129) Google Scholar, 15Ehlers M. de Hon F.D. Bos H.K. Horsten U. Kurapkat G. van De Leur H.S. Grötzinger J. Wollmer A. Brakenhoff J.P. Rose-John S. J. Biol. Chem. 1995; 270: 8158-8163Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) these proteins inhibited the IL-6-induced proliferation of BAF/3 cells transfected with gp130 and IL-6R cDNAs. The LIF, OSM, or CNTF-induced proliferation of BAF/3 cells stably transfected with gp130, LIF-R, and CNTF-R remained unaffected by the two IL-6 antagonists 2a2/3C9 and DFRD (data not shown). When BAF/3 cells stably transfected with human gp130 were used, proliferation can be stimulated with the fusion protein H-IL-6 (27Fischer M. Goldschmitt J. Peschel C. Kallen K.J. Brakenhoff J.P.J. Wollmer A. Grötzinger J. Rose-John S. Nat. Biotechnol. 1997; 15: 142-145Crossref PubMed Scopus (433) Google Scholar). Cells grown in the presence of 10 ng/ml H-IL-6 were treated with increasing doses of H-DFRD and H-AIL-6. As shown in Fig. 6, the two fusion antagonists inhibited growth of the gp130-transfected BAF/3 cells with an IC50 of about 1,000 ng/ml. Because the fusion antagonistic proteins were effective in blocking the activity of IL-6 and H-IL-6 due to the fact that the proteins directly bind to gp130, we tested whether these proteins might also block the biologic response of cytokines that induce heterodimerization of gp130 and LIF-R. We therefore tested the fusion proteins for their ability to inhibit proliferation of BAF/3 cells stably transfected with human gp130, human LIF-R and human CNTF-R cDNAs. These cells were stimulated with LIF, OSM, or CNTF. Fig. 7 A shows that BAF/3 cells stably transfected with these cDNAs proliferated in response to the cytokines LIF and CNTF with EC50 values of about 1 ng/ml. The EC50 for OSM was slightly higher, about 5 ng/ml. The proliferation induced by LIF was inhibited by both fusion proteins with an IC50 of around 100 ng/ml (Fig. 7 B). The inhibition of the biologic activity of OSM and CNTF, however, occurred with an IC50 dose of about 500 ng/ml (Fig. 7,C and D). On hepatoma cells, cytokines of the IL-6 family have been shown to induce the synthesis and secretion of acute phase proteins (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar, 35Gauldie J. Richards C. Harnish D. Lansdorp P. Baumann H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7251-7255Crossref PubMed Scopus (1376) Google Scholar). Therefore we tested if the constructed fusion proteins were able to block the biological activity on HepG2 cells of gp130/LIF-R requiring cytokines. HepG2 cells treated with increasing amounts of human LIF secreted the acute phase protein haptoglobin into the supernatant (36Mackiewicz A. Schooltink H. Heinrich P.C. Rose-John S. J. Immunol. 1992; 149: 2021-2027PubMed Google Scholar). Haptoglobin concentration can be measured by ELISA (33Oppmann B. Stoyan T. Fischer M. Voltz N. März P. Rose-John S. J. Immunol. Methods. 1996; 195: 153-159Crossref PubMed Scopus (9) Google Scholar). As shown in Fig. 8 A, HepG2 cells stimulated with LIF secreted haptoglobin into the medium with maximal stimulation at 25–50 ng/ml. When HepG2 cells stimulated with 25 ng/ml human LIF were treated with increasing amounts of the H-DFRD and H-AIL-6 fusion proteins, haptoglobin secretion was reduced to background levels. The inhibition of the biologic activity of LIF on HepG2 cells occurred with an IC50 dose of 200 ng/ml for H-AIL-6 and more than 1,000 ng/ml for H-DFRD. We have used the approach to covalently link IL-6 or IL-6 muteins with one defective contact site for gp130 (IL-6 receptor antagonists) to the soluble form of the IL-6R to create fusion proteins which directly bind to gp130. These fusion proteins directly target the extracellular portion of gp130 and therefore inhibit cytokines of the IL-6-type family, which interact with either a homodimer of gp130 (like IL-6) or with a heterodimer of gp130 and LIF-R (like CNTF, OSM and CNTF). The coprecipitation studies with the extracellular portion of gp130 linked to an Fc protein revealed that H-IL-6 and H-DFRD proteins bind equally well to gp130, whereas H-AIL-6 seemed to bind with somewhat reduced efficiency. Experiments with IL-6 receptor antagonists in the presence of soluble IL-6R and a soluble form of gp130 with a C-terminalmyc tag led to the conclusion that site II and site III act independently from each other as binding sites for gp130 (37Ciapponi L. Graziani R. Paonessa G. Lahm A. Ciliberto G. Savino R. J. Biol. Chem. 1995; 270: 31249-31254Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Although coprecipitation with a gp130-Fc fusion protein does not necessarily reflect the situation on the plasma membrane, these data might indicate that site III of IL-6 is more important for gp130 contact than site II. This notion is, however, not supported by the inhibition data presented in this mansucript. One would expect that H-DFRD being a better binder of gp130 would be a more effective antagonist of gp130. The fusion proteins H-AIL-6 and H-DFRD, however, are not significantly different in their inhibitory efficiency. Disregulation of the expression of cytokines of the IL-6-type family has been implicated in the pathophysiology of many diseases including Castleman's disease, multiple myeloma, osteoporosis, and the development of Kaposi's sarcoma (6Kallen K.-J. Meyer zum Büschenfelde K.H. Rose-John S. Exp. Opin. Invest. Drugs. 1997; 6: 237-266Crossref PubMed Scopus (22) Google Scholar). Efforts have been made to neutralize the activity of the IL-6-type cytokines by mAbs or cytokine receptor antagonists (7Klein B. Wijdenes J. Zhang X.G. Jourdan M. Boiron J.M. Brochier J. Liautard J. Merlin M. Clement C. Morel Fournier B. Liu Z.Y. Mannoni P. Sany J. Bataille R. Blood. 1991; 78: 1198-1204Crossref PubMed Google Scholar, 8Wendling D. Racadot E. Wijdenes J. J. 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Immunol. Today. 1994; 15: 455-458Abstract Full Text PDF PubMed Scopus (53) Google Scholar). Recently we reported on the development of a specific targeting strategy of cytokine-secreting cells by a bispecific diabody recognizing human IL-6 and CD3, which induced T-cell-mediated killing of these cells (40Krebs B. Griffin H. Winter G. Rose-John S. J. Biol. Chem. 1998; 273: 2858-2865Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). All these strategies, however, act to neutralize or block one given cytokine. IL-6 receptor antagonists or neutralizing mAbs do not neutralize the biologic activity of IL-11, LIF, OSM, CNTF, or CT-1. In the case of multiple myeloma cells, it has been shown that these cells not only proliferate in response to IL-6 but also in the presence of other if not all members of the IL-6 cytokine family (16Zhang X.G. Gu J.J. Lu Z.Y. Yasukawa K. Yancopoulos G.D. Turner K. Shoyab M. Taga T. Kishimoto T. Bataille R. Klein B. J. Exp. Med. 1994; 179: 1337-1342Crossref PubMed Scopus (222) Google Scholar). The gp130-interacting antagonist presented in this report has the great advantage of blocking gp130 directly and thereby blocking the biological activities all members of the IL-6-type family of cytokines. In addition, it has recently been demonstrated that IL-6 bound to soluble IL-6R has a longer plasma half-life in vivo than IL-6 alone (41Peters M. Jacobs S. Ehlers M. Vollmer P. Müllberg J. Wolf E. Brem G. Meyer zum Büschenfelde K.H. Rose-John S. J. Exp. Med. 1996; 183: 1399-1406Crossref PubMed Scopus (242) Google Scholar). Moreover, we have recently shown that the fusion protein H-IL-6 when injected into mice has a considerably longer bioavailability than IL-6 alone. 2M. Peters and S. Rose-John, manuscript in preparation. We therefore anticipate that our newly developed gp130-targeting fusion proteins will also show a more favorable pharmacokinetics than do IL-6 receptor antagonists (41Peters M. Jacobs S. Ehlers M. Vollmer P. Müllberg J. Wolf E. Brem G. Meyer zum Büschenfelde K.H. Rose-John S. J. Exp. Med. 1996; 183: 1399-1406Crossref PubMed Scopus (242) Google Scholar). On the other hand, one has to be aware that pleiotropic side effects upon injection of the fusion proteins might occur, because gp130 is present on all cells of the body and is involved in a wide spectrum of cellular activities (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). Recently, an inhibitor of LIF was constructed that exhibited a lowered affinity for gp130 because of introduced point mutations, whereas the affinity for LIF-R was unchanged (42Vernalis A.B. Hudson K.R. Heath J.K. J. Biol. Chem. 1997; 272: 26947-26952Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Consequently, this LIF-R antagonist was shown to possess antagonistic activity not only for LIF but also for the LIF-R binding cytokines CT-1, CNTF, and OSM (42Vernalis A.B. Hudson K.R. Heath J.K. J. Biol. Chem. 1997; 272: 26947-26952Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Taken together we have provided evidence that fusion proteins of IL-6 receptor antagonists and the soluble IL-6R can be used to directly target the receptor subunit gp130, which is a component of all receptor complexes of the IL-6 cytokine family. These fusion proteins when given at a molar excess are able to bind to the extracellular portion of gp130. Therefore we have for the first time constructed an antagonist with the potential to block all cytokines of the IL-6-type family. These novel inhibitors of IL-6-type cytokines might have therapeutic implications for diseases that have been connected with disregulation of the expression of these cytokines. We thank Dr. R. Palacios (Basel, Switzerland) for the gift of BAF/3 cells. The gift of BAF/3 cells stably transfected with gp130 and LIF-R cDNAs of recombinant LIF by B. Oppmann (Mainz, Germany) is gratefully acknowledged. We thank M. Fischer (Mainz, Germany) for help with expression and purification of recombinant proteins. We are most thankful for the gift of the IL-6 antagonistic protein 2a2/3C9 by Dr. M Ehlers (Mainz, Germany) and the gift of the IL-6R recognizing mAb MT18 by Dr. K. Yasukawa (Tokyo, Japan). We gratefully acknowledge the consent of the Dean of Medicine to publication of this work, which is part of the M.D. thesis of Christoph Renné.

Three forms of interleukin-6 (IL-6) have been constructed and stably transfected into human hepatoma cells (HepG2). Wild type IL-6 containing a signal peptide was rapidly secreted as a biologically active protein. IL-6 lacking the signal peptide accumulated within the cytoplasm of transfected cells. Surprisingly, IL-6 carrying a COOH-terminal extension of the amino acids Lys-Asp-Glu-Leu (KDEL) was not completely retained in the endoplasmic reticulum (ER). Complete retention in the ER was achieved when the 14 COOH-terminal amino acids of protein disulfide isomerase which include the KDEL signal were added to the COOH terminus of IL-6. This finding clearly demonstrates that the addition of the protein sorting signal KDEL alone is not sufficient for full retention of IL-6 in the ER. IL-6 accumulated in the cytoplasm and IL-6 retained in the ER failed to induce liver-specific acute-phase protein synthesis in the host cells, indicating that there is no intracellular role for IL-6 in signal transduction. Retention of IL-6 in the ER led to the prevention of surface expression of the IL-6 receptor protein gp80, making these cells unresponsive to IL-6. This phenomenon can be exploited in the future to generate transgenic animals which will become completely cytokine unresponsive in the tissues in which they express an ER retained cytokine.

Annals of the New York Academy of SciencesVolume 762, Issue 1 p. 207-221 The Soluble Interleukin-6 Receptora STEFAN ROSE-JOHN, Corresponding Author STEFAN ROSE-JOHN First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, GermanyAddress correspondence to Dr. Stefan Rose-John at the First Department of Internal Medicine, Section Pathophysiology, Obere Zahlbacher Strasse 63. Johannes Gutenberg Universitat, Mainz, 55101 Mainz, Germany. Tel: 49-6131-173363. E-Mail-ROSE@MZDMZA.ZDV.UNI-MAINZ.DE.Search for more papers by this authorMARC EHLERS, MARC EHLERS First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, Germany First Department of Internal Medicine, NMFZ, Langenbeckstrasse 63, Johannes Gutenberg Universitat Mainz, 55101, Mainz, Germany.Search for more papers by this authorJOACHIM GRÖTZINGER, JOACHIM GRÖTZINGER First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, GermanySearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, Germany Immunex Corp., 51 University Street, Seattle, WA 98101, USA.Search for more papers by this author STEFAN ROSE-JOHN, Corresponding Author STEFAN ROSE-JOHN First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, GermanyAddress correspondence to Dr. Stefan Rose-John at the First Department of Internal Medicine, Section Pathophysiology, Obere Zahlbacher Strasse 63. Johannes Gutenberg Universitat, Mainz, 55101 Mainz, Germany. Tel: 49-6131-173363. E-Mail-ROSE@MZDMZA.ZDV.UNI-MAINZ.DE.Search for more papers by this authorMARC EHLERS, MARC EHLERS First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, Germany First Department of Internal Medicine, NMFZ, Langenbeckstrasse 63, Johannes Gutenberg Universitat Mainz, 55101, Mainz, Germany.Search for more papers by this authorJOACHIM GRÖTZINGER, JOACHIM GRÖTZINGER First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, GermanySearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG First Department of Internal Medicine, Section Pathophysiology, University of Mainz, Obere Zahlbacher Str. 63, D-55101 Mainz, Germany Department of Biochemistry, RWTH Aachen, Pauwelsstraße 30, D-52057 Aachen, Germany Immunex Corp., 51 University Street, Seattle, WA 98101, USA.Search for more papers by this author First published: July 1995 https://doi.org/10.1111/j.1749-6632.1995.tb32327.xCitations: 18 a The experimental work described in this article has been supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume762, Issue1Interleukin-6-Type CytokinesJuly 1995Pages 207-221 RelatedInformation

We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.

Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in vivo of several neuronal cell types including sensory and motor neurons. The primary structure of CNTF suggests it to be a cytosolic protein with strong similarity to the alpha-helical cytokine family which is characterized by a bundle of four anti-parallel helices. CNTF exerts its activity via complexation with CNTF receptor (CNTF-R). This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction [gp130 and leukaemia inhibitory factor receptor (LIF-R)]. We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria. Biological activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably transfected with CNTF-R, gp130 and LIF-R cDNAs. CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active whereas the removal of 23 amino acids resulted in an inactive protein. In addition, 18 amino acid residues could be removed from the C-terminus of the CNTF protein without apparent loss of bioactivity, but further truncation at the C-terminus yielded biologically inactive proteins. The introduction of two point mutations into the CNTF protein at a site that presumably interacts with one of the two signal-transducing proteins resulted in a CNTF mutant with no measurable bioactivity. In addition, a model of the three-dimensional structure of human CNTF was constructed using the recently established structural co-ordinates of the related cytokine, granulocyte colony-stimulating factor. CD spectra of CNTF together with our mutational analysis and our three-dimensional model fully support the view that CNTF belongs to the family of alpha-helical cytokines. It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic properties.

Signal transduction in response to interleukin-6 (IL-6) results from homodimerization of gp130. This dimerization occurs after binding of IL-6 to its surface receptor (IL-6R) and can also be triggered by the complex of soluble IL-6R and IL-6. We fused IL-6 to the constant region of a human IgG1 heavy chain (Fc). IL-6Fc was expressed in COS-7 cells and purified via Protein A Sepharose. Using three different assays we found that the biological activity of this dimeric IL-6 protein is comparable with monomeric IL-6. Recently, we described the designer cytokine Hyper-IL-6 (H-IL-6) in which soluble IL-6R and IL-6 are connected via a flexible peptide linker. This molecule turned out to be 100-1000 times more effective than unlinked IL-6 and soluble IL-6R. Hyper-IL-6 acts on cells only expressing gp130 and is a potent stimulator of in vitro expansion of early hematopoietic precursors. Here we show that a Fc fusion protein of H-IL-6 (H-IL-6Fc) has the same biological activity on BAF/gp130 cells as H-IL-6. Furthermore, both H-IL-6 forms have a similar ability to induce the synthesis of acute phase proteins in human hepatoma cells HepG2 and in mice in vivo. The introduction of a thrombin cleavage site between H-IL-6 and the Fc portion of H-IL-6Fc made it possible to specifically recover biologically active monomeric H-IL-6 by limited proteolysis of the fusion protein. A more general use of cleavable immunoadhesins expressed in mammalian cells is discussed.

Conference Article| February 01 1999 Generation and function of the soluble interleukin-6 receptor J. Müllberg; J. Müllberg 1Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg Universität Mainz, Obere Zahlbacher Strasse 63, 55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar P. Vollmer; P. Vollmer 1Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg Universität Mainz, Obere Zahlbacher Strasse 63, 55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar K. Althoff; K. Althoff 1Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg Universität Mainz, Obere Zahlbacher Strasse 63, 55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar P. März; P. März 1Medizinische Klinik, Abteilung Pathophysiologie, Johannes Gutenberg Universität Mainz, Obere Zahlbacher Strasse 63, 55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar S. Rose-John S. Rose-John 1 1To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1999) 27 (2): 211–219. https://doi.org/10.1042/bst0270211 Article history Received: August 18 1998 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation J. Müllberg, P. Vollmer, K. Althoff, P. März, S. Rose-John; Generation and function of the soluble interleukin-6 receptor. Biochem Soc Trans 1 February 1999; 27 (2): 211–219. doi: https://doi.org/10.1042/bst0270211 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CHO, Chinese hamster ovary, CNTF, ciliary neurotrophic factor, CT-1, cardiotrophin 1, IL, interleukin, IL-6R, interleukin 6 receptor, LIF, leukaemia inhibitory factor, LIF-R, LIF receptor, OSM, oncostatin M, TGF-α, transforming growth factor α, TNF-α, tumour necrosis factor α This content is only available as a PDF. © 1999 Biochemical Society1999 Article PDF first page preview Close Modal You do not currently have access to this content.

PERB11 (MIC) is a gene family possessing multiple copies located within the MHC. Structurally, PERB11 is related to the MHC class I, neonatal IgG Fc receptor (FcRn) and Zn-alpha 2-glycoprotein molecules. The MHC class I family is complex in terms of its genomic arrangement, expression and function, and available evidence suggests that the PERB11 family may be similarly complex. We have adopted an approach to study the expression of such complex gene families by immunizing with multiple peptides and by screening the resulting antibodies against a large range of tissues. The amino acid sequences of PERB11.1 and PERB11.2 as well as those of other related molecules were analysed and compared. Peptides were chosen for immunization based upon (i) loop formation within the equivalent known structure of the MHC class I molecules; (ii) immunogenicity by computer analysis; and (iii) evolutionary relationships. Antibodies in serum from immunized rabbits bound to three out of six peptides used for immunization. ELISA and immunoprecipitation demonstrated binding both to the peptides and to the PERB11.2 recombinant protein. By immunofluorescent staining of various tissues of several species, the three antisera generated overlapping profiles of activity. These included reactions with kidney, small and large intestine, oesophagus, testis, ovary and human neutrophils. This is the first description of antibodies induced by the PERB11 peptides. The extreme complexity of these profiles requires further investigation, but may be explained in terms of antibodies against diverse products of the PERB11 gene family and/or related molecules.

Appropriate segregation within manufacturing facilities is required by regulators and utilized by manufacturers to ensure that the final product has not been contaminated with (a) adventitious viruses, (b) another pre-/postviral clearance fraction of the same product, or (c) another product processed in the same facility. However, there is no consensus on what constitutes appropriate facility segregation to minimize these risks. In part, this is due to the fact that a wide variety of manufacturing facilities and operational practices exist, including single-product and multiproduct manufacturing, using traditional segregation strategies with separate rooms for specific operations that may use stainless steel or disposable equipment to more modern ballroom-style operations that use mostly disposable equipment (i.e., pre- and postviral clearance manufacturing operations are not physically segregated by walls). Further, consensus is lacking around basic definitions and approaches related to facility segregation. For example, given that several unit operations provide assurance of virus clearance during downstream processing, how does one define pre- and postviral clearance and at which point(s) should a viral segregation barrier be introduced? What is a "functionally closed" system? How can interventions be conducted so that the system remains functionally closed? How can functionally closed systems be used to adequately isolate a product stream and ensure its safety? To address these issues, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a facility segregation project with the following goals: define "pre- and postviral clearance zones" and "pre- and postviral clearance materials"; define "functionally closed" manufacturing systems; and identify an array of facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products. This article reflects the current thinking from this collaborative endeavor.LAY ABSTRACT: Operations in biopharmaceutical manufacturing are segregated to ensure that the final product has not been contaminated with adventitious viruses, another fraction of the same product, or with another product from within the same facility. Yet there is no consensus understanding of what appropriate facility segregation looks like. There are a wide variety of manufacturing facilities and operational practices. There are existing facilities with separate rooms and more modern approaches that use disposable equipment in an open ballroom without walls. There is also no agreement on basic definitions and approaches related to facility segregation approaches. For example, many would like to claim that their manufacturing process is functionally closed, yet exactly how a functionally closed system may be defined is not clear. To address this, the member companies of the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have conducted a project with the goal of defining important manufacturing terms relevant to designing an appropriately segregated facility and identifying different facility segregation approaches that are used for the safe and effective production of recombinant biologics as well as plasma products.

Annals of the New York Academy of SciencesVolume 762, Issue 1 p. 462-464 TIMP-1 Protein Expression Is Stimulated by IL-1β and IL-6 in Primary Rat Hepatocytes ELKE ROEB, ELKE ROEB Medizinische Klinik IIISearch for more papers by this authorLUTZ GRAEVE, LUTZ GRAEVE Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorSIEGFRIED MATERN, SIEGFRIED MATERN Medizinische Klinik IIISearch for more papers by this authorSTEFAN ROSE-JOHN, STEFAN ROSE-JOHN Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this author ELKE ROEB, ELKE ROEB Medizinische Klinik IIISearch for more papers by this authorLUTZ GRAEVE, LUTZ GRAEVE Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorSIEGFRIED MATERN, SIEGFRIED MATERN Medizinische Klinik IIISearch for more papers by this authorSTEFAN ROSE-JOHN, STEFAN ROSE-JOHN Institut für Biochemie, RWTH Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this author First published: July 1995 https://doi.org/10.1111/j.1749-6632.1995.tb32368.xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume762, Issue1Interleukin-6-Type CytokinesJuly 1995Pages 462-464 RelatedInformation

Annals of the New York Academy of SciencesVolume 762, Issue 1 p. 400-402 Residues 77-95 of the Human Interleuken-6 Protein are Responsible for Receptor Binding and Residues 41-56 for Signal Transduction MARC EHLERS, MARC EHLERS Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJOACHIM GRÖTZINGER, JOACHIM GRÖTZINGER Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorFLORIS D. De HON, FLORIS D. De HON Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam 1066 CX, The NetherlandsSearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJUST P. J. BRAKENHOFF, JUST P. J. BRAKENHOFF Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam 1066 CX, The NetherlandsSearch for more papers by this authorAXEL WOLLMER, AXEL WOLLMER Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorSTEFAN ROSE-JOHN, STEFAN ROSE-JOHN Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this author MARC EHLERS, MARC EHLERS Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJOACHIM GRÖTZINGER, JOACHIM GRÖTZINGER Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorFLORIS D. De HON, FLORIS D. De HON Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam 1066 CX, The NetherlandsSearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJUST P. J. BRAKENHOFF, JUST P. J. BRAKENHOFF Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam 1066 CX, The NetherlandsSearch for more papers by this authorAXEL WOLLMER, AXEL WOLLMER Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorSTEFAN ROSE-JOHN, STEFAN ROSE-JOHN Department of Biochemistry, Rheinisch-Westfälische Technische Hochshule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this author First published: July 1995 https://doi.org/10.1111/j.1749-6632.1995.tb32347.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume762, Issue1Interleukin-6-Type CytokinesJuly 1995Pages 400-402 RelatedInformation

Annals of the New York Academy of SciencesVolume 762, Issue 1 p. 410-412 A Region within the Cytoplasmic Domain of the Interleukin-6 Signal Transducer gp130 Important for Ligand-induced Endocytosis of the IL-6 Receptor ELKE DITTRICH, ELKE DITTRICH Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorCLAUDIA GERHARTZ, CLAUDIA GERHARTZ Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorSTEFAN ROSE-JOHN, STEFAN ROSE-JOHN Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorTANJA STOYAN, TANJA STOYAN Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorPETER C. HEINRICH, PETER C. HEINRICH Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorLUTZ GRAEVE, Corresponding Author LUTZ GRAEVEAuthor to whom correspondence should be addressed: Institut fiir Biochemie, Rheinisch-Westralische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany. Tel: 0241-8088837; Fax: 0241-8888428.Search for more papers by this author ELKE DITTRICH, ELKE DITTRICH Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorCLAUDIA GERHARTZ, CLAUDIA GERHARTZ Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorSTEFAN ROSE-JOHN, STEFAN ROSE-JOHN Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorJÜRGEN MÜLLBERG, JÜRGEN MÜLLBERG Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorTANJA STOYAN, TANJA STOYAN Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorPETER C. HEINRICH, PETER C. HEINRICH Institut für Biochemie, Rheinisch-Westfálische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, GermanySearch for more papers by this authorLUTZ GRAEVE, Corresponding Author LUTZ GRAEVEAuthor to whom correspondence should be addressed: Institut fiir Biochemie, Rheinisch-Westralische Technische Hochschule Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany. Tel: 0241-8088837; Fax: 0241-8888428.Search for more papers by this author First published: July 1995 https://doi.org/10.1111/j.1749-6632.1995.tb32350.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume762, Issue1Interleukin-6-Type CytokinesJuly 1995Pages 410-412 RelatedInformation

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Conference Abstract| February 01 1999 SHEDDING OF THE INTERLEUKIN-6 RECEPTOR: MECHANISMS AND PHYSIOLOGICAL CONSEQUENCES Stefan Rose-John; Stefan Rose-John 1I. Medizinische Klinik-Abteilung Pathophysiology, Johnnes Gutenberg Universität Mainz, D-55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar Katja Althoff; Katja Althoff 1I. Medizinische Klinik-Abteilung Pathophysiology, Johnnes Gutenberg Universität Mainz, D-55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar Martina Fischer; Martina Fischer 1I. Medizinische Klinik-Abteilung Pathophysiology, Johnnes Gutenberg Universität Mainz, D-55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar Petra Vollmer; Petra Vollmer 1I. Medizinische Klinik-Abteilung Pathophysiology, Johnnes Gutenberg Universität Mainz, D-55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar Malte Peters; Malte Peters 1I. Medizinische Klinik-Abteilung Pathophysiology, Johnnes Gutenberg Universität Mainz, D-55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar Jürgen Müllberg Jürgen Müllberg 1I. Medizinische Klinik-Abteilung Pathophysiology, Johnnes Gutenberg Universität Mainz, D-55101 Mainz, Germany Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1999 Biochemical Society1999 Biochem Soc Trans (1999) 27 (1): A22. https://doi.org/10.1042/bst027a022c Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation Stefan Rose-John, Katja Althoff, Martina Fischer, Petra Vollmer, Malte Peters, Jürgen Müllberg; SHEDDING OF THE INTERLEUKIN-6 RECEPTOR: MECHANISMS AND PHYSIOLOGICAL CONSEQUENCES. Biochem Soc Trans 1 February 1999; 27 (1): A22. doi: https://doi.org/10.1042/bst027a022c Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1999 Biochemical Society1999 Article PDF first page preview Close Modal You do not currently have access to this content.