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

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.

Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

“…After stable transfection of a cDNA coding for gp130, these cells (Ba/F3-gp130), in the absence of IL-3, gain the ability to proliferate in the presence of IL-6/sIL-6-R, hyper-IL-6 (a fusion protein of IL-6 and sIL-6-R) (21), or viral IL-6. Previous studies have shown that Ba/F3-gp130 cells proliferate in response to hyper-IL-6 at a concentration below 1 ng/ml, whereas 100 ng/ml of viral IL-6 was needed to induce Baf/3-gp130 cell proliferation (26,46).…”

“…COS-7 and HepG2 cells were bought from the ATCC (Rockville, MD) and were routinely grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). The vIL-6 DNA sequence was originally obtained from F. Neipel and B. Fleckenstein (University of Erlangen, Germany) (48) and has been used in previous studies (26,46). The PEL-derived BCBL-1 cells were a kind gift from M. Gramatzki's group at the University of Kiel (Germany) and were maintained in RPMI 1640 medium (BioWhittaker,Walkersville, MD) supplemented with 2 mmol/liter L-glutamine (Life Technologies, Grand Island, NY), 10% fetal calf serum (BioWhittaker), and 5 g/ml gentamicin (Life Technologies).…”

“…vIL-6 binds to the gp130 receptor, leading to JAK/STAT activation (46). Activated STAT3 was detected with an anti-phospho-STAT3 antibody.…”

“…There is still debate about the involvement of the IL-6-R in cellular responses to vIL-6. Whereas vIL-6 responses were shown to be blocked by neutralizing IL-6-R monoclonal antibodies (9), other groups have questioned the requirement of IL-6-R for vIL-6-induced activation of cells (33,44,46,78). Viral IL-6 is constitutively expressed in lytically infected Kaposi spindle cells and AIDS-related B-cell lymphoma cell lines (BCBL and PEL) (45) and in Castleman's disease (56) and induces proliferation, angiogenesis, and hematopoiesis in IL-6-dependent cell lineages (26,45).…”

“…The sgp130Fc protein is expected to be a valuable therapeutic tool to specifically block disease states in which soluble IL-6-R trans signaling responses exist (5,8,29,51) and might also be used to block vIL-6-induced signals (46).…”