Petra Vollmer

IL-6 Receptor Independent Stimulation of Human gp130 by Viral IL-6

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.

Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells

The ability of tumour cells to resist apoptosis-inducing signals by cytotoxic T cells may decide the success or failure of tumour elimination. An important effector of apoptosis is the CD95/CD95 ligand system (APO-1/Fas) that mediates perforin-independent cytotoxic T-cell killing of tumour cells. We propose a new strategy by which tumour cells can resist CD95-induced apoptosis. We identified matrix metalloproteinase-7, MMP-7 (Martilysin), as the first physiologically relevant protease that can specifically cleave CD95. MMP-7 is of unique importance because it is produced by the tumour cells themselves at early stages of tumour development. Microsequencing of the positions in CD95 cleaved by MMP-7 revealed two sites in the N-terminal extracellular domain of CD95, important for preligand assembly of CD95. MMP-7 cleavage of CD95 results in reduced CD95 surface expression and decreased CD95-mediated apoptosis sensitivity of tumour cells. Treatment of MMP-7-positive HT-29 tumour cells with MMP-7-antisense oligonucleotides led to an increase in CD95-mediated apoptosis sensitivity. Finally, specific cytotoxic T-cell killing was reduced in the presence of MMP-7. Thus, MMP-7 expression in tumour cells may contribute to an apoptosis-resistant phenotype, which ultimately promotes immune escape. This activity may account for the well-established role of MMP-7 in early tumour development.

Receptor Recognition Sites of Cytokines Are Organized as Exchangeable Modules TRANSFER OF THE LEUKEMIA INHIBITORY FACTOR RECEPTOR-BINDING SITE FROM CILIARY NEUROTROPHIC FACTOR TO INTERLEUKIN-6

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 (e.g. 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.

Yeast expression of the cytokine receptor domain of the soluble interleukin-6 receptor.

The complex of the soluble interleukin-6 receptor (sIL-6R) and IL-6 (IL-6) is a potent agonist on cells expressing the signal transducing protein gp 130. In contrast, IL-6 alone only stimulates cells which express a membrane bound form of the IL-6R and gp 130. The natural occurring sIL-6R is generated by shedding of the membrane receptor and to a lesser extend by alternative splicing. We have inserted the coding sequence of the 323 amino acid residues of the human sIL-6R into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris. We obtained, however, no detectable expression and secretion of the recombinant protein. When we used only the coding sequence of the cytokine receptor domain of the sIL-6R for the construction of an expression plasmid, this truncated version of the sIL-6R accumulated in the supernatant to 1-5 mg/l. The protein was purified by a single affinity chromatography step using a monoclonal antibody directed against the human IL-6R. Following the same approach, we expressed a truncated splice variant of the sIL-6R. Both, the secreted truncated sIL-6R and the splice variant showed full agonistic biological activity on human hepatoma cells. The described expression strategy will be useful for large scale production of biologically active sIL-6R and might be adapted for the expression of other members of the hematopoietic cytokine receptor family.