Structural requirements for gp80 independence of human herpesvirus 8 interleukin-6 (vIL-6) and evidence for gp80 stabilization of gp130 signaling complexes induced by vIL-6

Human herpesvirus 8 interleukin-6 (vIL-6) displays 25% amino acid identity with human IL-6 (hIL-6) and shares an overall four-helix-bundle structure and gp130-mediated STAT/mitogen-activated protein kinase signaling with its cellular counterpart. However, vIL-6 is distinct in that it can signal through gp130 alone, in the absence of the nonsignaling gp80 alpha-subunit of the IL-6 receptor. To investigate the structural requirements for gp80 independence of vIL-6, a series of expression vectors encoding vIL-6/hIL-6 chimeric and site-mutated IL-6 proteins was generated. The replacement of hIL-6 residues with three vIL-6-specific tryptophans implicated in gp80 independence from crystallographic studies or the A and C helices containing these residues did not confer gp80 independence to hIL-6. The N- and C-terminal regions of vIL-6 could be substituted with hIL-6 sequences with the retention of gp80-independent signaling, but substitutions of other regions of vIL-6 (helix A, A/B loop, helix B, helix C, and proximal half of helix D) with equivalent sequences of hIL-6 abolished gp80 independence. Interestingly, the B helix of vIL-6 was absolutely required for gp80 independence, despite the fact that this region contains no receptor-binding residues. Point mutational analysis of helix C, which contains residues involved in physical and functional interactions with gp130 domains 2 and 3 (cytokine-binding homology region), identified a variant, VI120EE, that was able to signal and dimerize gp130 only in the presence of gp80. gp80 was also found to stabilize gp130:g130 dimers induced by a distal D helix variant of vIL-6 that was nonetheless able to signal independently of gp80. Together, our data reveal the crucial importance of overall vIL-6 structure and conformation for gp80-independent signaling and provide functional and physical evidence of the stabilization of vIL-6-induced gp130 signaling complexes by gp80.     

Detection of direct binding of human herpesvirus 8-encoded interleukin-6 (vIL-6) to both gp130 and IL-6 receptor (IL-6R) and identification of amino acid residues of vIL-6 important for IL-6R-dependent and -independent signaling

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6-receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130-IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.     

Identification of amino acid residues of gp130 signal transducer and gp80 alpha receptor subunit that are involved in ligand binding and signaling by human herpesvirus 8-encoded interleukin-6

Human herpesvirus 8-encoded interleukin-6 (vIL-6) signals through the gp130 signal transducer but is not dependent on the IL-6 receptor alpha subunit (IL-6R, gp80) that is required for signaling by endogenous IL-6 proteins; however, IL-6R can enhance vIL-6 activity and can enable signaling through a gp130 variant, gp130.PM5, that is itself unable to support vIL-6 signaling. These findings suggest that the vIL-6-gp130 interactions are qualitatively different from those of human IL-6 (hIL-6) and that vIL-6 signaling may be more promiscuous than that of hIL-6 but that IL-6R may play a role in vIL-6 signaling in vivo. To examine the receptor binding requirements of vIL-6, we have undertaken mutational analyses of regions of gp130 and IL-6R potentially involved in interactions with ligand or in functional complex formation and used these variants in functional, ligand-binding, and receptor dimerization assays. The data presented identify positions within two interstrand loops of the gp130 cytokine-receptor homology domain that are important for vIL-6 signaling and vIL-6-induced receptor dimerization and show that vIL-6, like hIL-6, can form complexes with IL-6R and gp130 but that the roles of putative cytokine-binding residues of IL-6R in ligand-induced functional complex formation are qualitatively different in the case of vIL-6 and hIL-6.     

The hepatic interleukin-6 receptor. Down-regulation of the interleukin-6 binding subunit (gp80) by its ligand.

Interleukin-6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6-binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp130). We studied the role of gp80 in binding, internalization and down-regulation of the hepatic IL6-receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp80 with parental cells indicate that gp80 is responsible for low affinity binding (Kd = 500 pM) of IL6. Furthermore, gp80 is rate-limiting in internalization and degradation of IL6. Internalization resulted in a rapid down-regulation (t1/2 approximately 15-30 min) of IL6-binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6-binding sites at the cell surface required greater than 8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down-regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.     

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.     

Shedding of the interleukin-6 (IL-6) receptor (gp80) determines the ability of IL-6 to induce gp130 phosphorylation in human osteoblasts

Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R.     

Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.     

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

Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.     

Human herpesvirus 8 viral interleukin-6 interacts with splice variant 2 of vitamin K epoxide reductase complex subunit 1

Viral interleukin-6 (vIL-6) specified by human herpesvirus 8 is, unlike its cellular counterpart, secreted very inefficiently and can signal via vIL-6(2):gp130(2) signaling complexes from the endoplasmic reticulum (ER) compartment. Intracellular, autocrine activities of vIL-6 are important for proproliferative and prosurvival activities of the viral cytokine in latently infected primary effusion lymphoma (PEL) cells. However, the molecular determinants of vIL-6 ER localization and function are unclear. Using yeast two-hybrid analysis, we identified the database-documented but uncharacterized splice variant of vitamin K epoxide reductase complex subunit 1 (VKORC1), termed VKORC1 variant 2 (VKORC1v2), as a potential interaction partner of vIL-6. In transfected cells, epitope-tagged VKORC1v2 was found to localize to the ER, to adopt a single-transmembrane (TM) topology placing the C tail in the ER lumen, and to bind vIL-6 via these sequences. Deletion mutagenesis and coprecipitation assays mapped the vIL-6-binding domain (vBD) of VKORC1v2 to TM-proximal residues 31 to 39. However, while sufficient to confer vIL-6 binding to a heterologous protein, vBD was unable to induce vIL-6 secretion when fused to (secreted) hIL-6, suggesting a VKORC1v2-independent mechanism of vIL-6 ER retention. In functional assays, overexpression of ER-directed vBD led to suppression of PEL cell proliferation and viability, effects also mediated by VKORC1v2 depletion and, as reported previously, by vIL-6 suppression. The growth-inhibitory and proapoptotic effects of VKORC1v2 depletion could be rescued by transduced wild-type VKORC1v2 but not by a vIL-6-refractory vBD-altered variant, indicating the functional relevance of the vIL-6-VKORC1v2 interaction. Notably, gp130 signaling was unaffected by VKORC1v2 or vBD overexpression or by VKORC1v2 depletion, suggesting an alternative pathway of vIL-6 activity via VKORC1v2. Combined, our data identify a novel and functionally significant interaction partner of vIL-6 that could potentially be targeted for therapeutic benefit.     

Insights into the viral G protein-coupled receptor encoded by human herpesvirus type 8 (HHV-8)

HHV-8-GPCR is a chemokine-like receptor encoded by KSHV, the etiologic agent of KS. HHV-8-GPCR is constitutively active. Although it is homologous to mammalian CXCR2, it binds CXC and CC chemokines. Structure-function analysis showed that chemokines bind primarily to the amino terminus whereas signaling occurs in the absence of: the amino terminus, which is, therefore, not a tethered agonist. In in vitro systems, HHV-8-GPCR signals via multiple transduction pathways including, activation of phospholipase C and PKC, inhibition of adenylyl cyclase, activation of nuclear factor-κB; activation PI 3-kinase, p42/44 MAPK and Akt/PKB, and activation of JNK/SAPK, p38 MAPK and RAFTK. HHV-8-GPCR is important in the HHV-8 life cycle because HHV-8-GPCR-deficient viruses do not replicate in response to chemokines and exhibit, less efficient reactivation from latency. Although the role of HHV-8-GPCR in the pathogenesis of KS has not been defined, expression of HHV-8-GPCR resulted in the development of angioproliferative, KS-like tumors in transgenic mice. As endothelial cells may be targets of HHV-8 infection, HHV-8-GPCR has been studied in endothelial cells in vitro in which it affects cell adhesion and migration, increases cell survival, and stimulates secretion of proinflammatory cytokines and proangiogenic factors. Based on these findings and the observation that HHV-8-GPCR is expressed in only a few endothelial- like "spindle cells" within KS lesions, we propose that HHV-8-GPCR is involved in KS pathogenesis by stimulating secretion of proinflammatory/proangiogenic factors that act in a paracrine fashion to cause tumorigenesis.     

Cerebrospinal fluid and serum protein levels of tumour necrosis factor-alpha (TNF-alpha) interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R gp80) in multiple sclerosis patients

The aim of this study was to evaluate soluble proteins of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-6 receptor subunit gp80 (sIL-6R gp80), as markers of multiple sclerosis (MS). Paired cerebrospinal fluid (CSF) and serum samples of 20 MS patients and 15 controls suffering from non-inflammatory neurological diseases have been assayed retrospectively using monoclonal antibodies-based ELISAs. While TNF-alpha could not be detected in CSF, it was measurable in 20% of total sera. Interleukin-6 was measurable in 5% of total CSF and in 10% of total sera only. However, soluble IL-6R gp80 protein subunit was readily measurable, showing sera concentration (pg/mL) about 34 times higher and specific content (pg/mg total protein) around five times lower than those in paired CSF, similarly for both group of patients. No significant difference of sIL-6R gp80 level, which could be disease-, gender- or age-related, and no correlation of CSF sIL-6R gp80 content with that of paired serum or with routine clinical data for CSF, have been observed. We have concluded that soluble proteins of TNF-alpha, IL-6 and sIL-6R gp80 assayed by monoclonal antibodies-based ELISAs could not serve as markers of the MS activity.     

Caspase-8 cleavage of the interleukin-21 (IL-21) receptor is a negative feedback regulator of IL-21 signaling

We screened a library of human single-transmembrane proteins (sTMPs), produced by a cell-free system, using a luminescent assay to identify those that can be cleaved by caspase-8 (CASP8). Of the 407 sTMPs screened, only the interleukin-21 receptor (IL21R), vezatin (VEZT), and carbonic anhydrase XIV were cleaved at Asp344, Asp655 and Asp53, respectively. We confirmed that IL21R and VEZT were also cleaved in apoptotic HeLa cells with the cleavage sites. Interestingly, IL21R was cleaved within 30 min after apoptosis induction. Furthermore the CASP8-cleaved form of IL21R did not induce phosphorylation at Tyr705 of STAT3. Our results suggest that the interleukin-21 signaling cascade is negatively regulated by CASP8.     

Molecular mechanisms for viral mimicry of a human cytokine: activation of gp130 by HHV-8 interleukin-6

Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8) encodes a pathogenic viral homologue of human interleukin-6 (IL-6). In contrast to human IL-6 (hIL-6), viral IL-6 (vIL-6) binds directly to, and activates, the shared human cytokine signaling receptor gp130 without the requirement for pre-complexation to a specific alpha-receptor. Here, we dissect the biochemical and functional basis of vIL-6 mimicry of hIL-6. We find that, in addition to the "alpha-receptor-independent" tetrameric vIL-6/gp130 complex, the viral cytokine can engage the human alpha-receptor (IL-6Ralpha) to form a hexameric vIL-6/IL-6Ralpha/gp130 complex with enhanced signaling potency. In contrast to the assembly sequence of the hIL-6 hexamer, the preformed vIL-6/gp130 tetramer can be decorated with IL-6Ralpha, post facto, in a "vIL-6-dependent" fashion. A detailed comparison of the viral and human cytokine/gp130 interfaces indicates that vIL-6 has evolved a unique molecular strategy to interact with gp130, as revealed by an almost entirely divergent structural makeup of its receptor binding sites. Viral IL-6 appears to utilize an elegant combination of both convergent, and unexpectedly divergent, molecular strategies to oligomerize gp130 and activate similar downstream signaling cascades as its human counterpart.     

Enhancement of IL-6 receptor beta chain (gp130) expression by IL-6, IL-1 and TNF in human epithelial cells.

Analysis of the IL-6 Receptor beta chain (gp130) mRNA expression on the two human epithelial cell lines UAC and Hep3B reveals that it is enhanced by IL-6, IL-1 and TNF treatment. In the case of UAC cells, TNF action might be mediated by IL-6. For Hep3B cells, TNF seems to exert a direct effect on gp130, as no IL-6 expression is detected after stimulation by this cytokine. On the same cells, increase of the binding of an anti-gp130 monoclonal antibody was observed after treatment by TNF, which denotes the effective appearance of new gp130 molecules on the cell surface. All this cytokines seem to act selectively on the beta chain of the IL-6 receptor. This probably reflects the importance for some cells to have gp130 represented on their membrane in inflammatory contexts.     

CD27-IgD- memory B cells are modulated by in vivo interleukin-6 receptor (IL-6R) blockade in rheumatoid arthritis

IntroductionEnhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ).MethodsDN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, V_H gene rearrangements and specific isotypes.ResultsPhenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (P <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05 to 2.06).ConclusionsIn RA, the heterogeneous DN B cell compartment is expanded and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0580-y) contains supplementary material, which is available to authorized users.