Research ArticleCytokines

Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses

See allHide authors and affiliations

Science Signaling  02 Oct 2018:
Vol. 11, Issue 550, eaar7388
DOI: 10.1126/scisignal.aar7388

Inside IL-6 inflammation

Members of the interleukin-6 (IL-6) family of proinflammatory cytokines, which includes IL-6 and IL-11, stimulate responses by binding to a complex of their cytokine-specific receptor and the gp130 signaling receptor. Both of these receptor subunits can be shed from the cell surface and regulate distinct mechanisms of gp130-mediated signaling. Lamertz et al. engineered a reductionist system to interrogate how cells respond to wild-type and synthetic IL-6 and IL-11 cytokines. The authors found that a soluble form of gp130 blocked responses to IL-6 and IL-11 that were presented in trans by a neighboring cell. In contrast, extracellular blockade of cell surface gp130 had no effect on the response to autocrine IL-6, suggesting that these responses could occur within the cell.

Abstract

Interleukin-6 (IL-6) is a proinflammatory cytokine of the IL-6 family, members of which signal through a complex of a cytokine-specific receptor and the signal-transducing subunit gp130. The interaction of IL-6 with the membrane-bound IL-6 receptor (IL-6R) and gp130 stimulates “classic signaling,” whereas the binding of IL-6 and a soluble version of the IL-6R to gp130 stimulates “trans-signaling.” Alternatively, “cluster signaling” occurs when membrane-bound IL-6:IL-6R complexes on transmitter cells activate gp130 receptors on neighboring receiver cells. The soluble form of gp130 (sgp130) is a selective trans-signaling inhibitor, but it does not affect classic signaling. We demonstrated that the interaction of soluble gp130 with natural and synthetic membrane-bound IL-6:IL-6R complexes inhibited IL-6 cluster signaling. Similarly, IL-11 cluster signaling through the IL-11R to gp130 was also inhibited by soluble gp130. However, autocrine classic and trans-signaling was not inhibited by extracellular inhibitors such as sgp130 or gp130 antibodies. Together, our results suggest that autocrine IL-6 signaling may occur intracellularly.

INTRODUCTION

The cytokine interleukin-6 (IL-6) critically has functions in health and disease (1). These include pro- and anti-inflammatory functions including acute-phase response, proliferation and differentiation of T and B cells, and homeostatic functions (2). To mediate its effects, IL-6 binds to the non–signal-transducing IL-6 receptor (IL-6R), which forms a complex with the signal-transducing glycoprotein 130 (gp130) co-receptor. IL-6 recognition stimulates homodimerization of gp130 and activates the Janus kinase/signal transducer and activator of transcription (JAK/STAT), the phosphatidylinositol 3-kinase (PI3K), and the mitogen-activated protein kinase (MAPK) downstream signaling pathways (3). Because IL-6 itself does not bind to the gp130 subunit alone, the IL-6R is mandatory for the interaction of IL-6 with gp130 (4). A soluble form of IL-6R is produced by proteolytic shedding and by alternative splicing (5).

Interaction of IL-6 with membrane-bound IL-6R and gp130 induces classic signaling, whereas binding of the complex of IL-6 and soluble IL-6R (sIL-6R) to gp130 induces trans-signaling. The IL-6R is mainly found on hepatocytes and immune cells; thus, these cell types are the only ones that can respond directly to IL-6 through classic signaling. This mode of signaling is necessary to induce the acute-phase response and exerts the homeostatic and anti-inflammatory functions of IL-6 (2, 6). In contrast, because gp130 is ubiquitously expressed, trans-signaling by the sIL-6R can activate virtually all cells of the body. IL-6 trans-signaling mainly regulates proinflammatory responses, and blocking of trans-signaling both by natural soluble gp130 (sgp130) and by sgp130Fc fusion proteins as artificial sgp130 dimers reduces disease burden in a variety of preclinical chronic and autoimmune disease models (7). Whereas a sgp130 specifically inhibits IL-6 trans-signaling, IL-6 classic signaling is largely unaffected by sgp130 (4, 8). Only under conditions with a large excess of sIL-6R over IL-6 can sgp130 also interfere with IL-6 classic signaling by trapping the few IL-6 molecules with a surplus of sIL-6R:sgp130 binding partners (8).

Cells can also respond to IL-6 through a process known as cluster signaling. In this receptor activation mode, membrane-bound IL-6:IL-6R complexes on transmitter cells triggered gp130 receptor activation on receiver cells. Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic T helper 17 (TH17) cells (9) and might also be involved in the development of cytotoxic T cells (10). These reports fail to show an inhibitory function of sgp130 on cluster signaling.

IL-11 is the only other member of the IL-6 family that signals through a homodimer of gp130 (11). Whereas IL-6 is more involved in controlling inflammatory reactions, IL-11 is required for regenerative processes such as gut and heart (11). Like IL-6, IL-11 also has no affinity toward gp130 in the absence of its specific, but non–signal-transducing IL-11R. The overall structural composition of the IL-11R is very similar to that of IL-6R. In analogy to IL-6 classic signaling, IL-11 induces signal transduction by binding to the IL-11R followed by binding recruitment of two gp130 molecules by the IL-11:sIL-11R complex (3). Soluble forms of IL-11R are found in human serum samples (12), and complexes of soluble IL-11R and IL-11 as well as designer cytokines composed of both proteins connected by a flexible linker sequence are biologically active and induce proliferation, hematopoiesis, and acute-phase response (12, 13). Similar to IL-6 trans-signaling, IL-11 trans-signaling can also be inhibited by sgp130 (12). However, the role of IL-11 cluster signaling remains unclear.

Here, we demonstrated that sgp130Fc interacted with membrane-bound IL-6:IL-6R complexes. In addition, we engineered a reductionist system of IL-6 cluster signaling and found that sgp130 inhibited IL-6 cluster signaling. Similarly, IL-11 was also able to induce cluster signaling in receiver cells, which was efficiently inhibited by sgp130Fc. To specifically mimic cluster signaling, we designed novel membrane-bound designer Hyper-cytokines, which behave like natural IL-6:IL-6R and IL-11:IL-11R complexes, albeit with a higher efficiency, and which were also bound and inhibited by sgp130. When expressed together with gp130 on the same cell, soluble and membrane-bound Hyper-cytokines facilitated constitutive gp130 receptor activation. Because this activity was not inhibited by extracellular blockade, these data suggest that gp130 may be activated intracellularly in this context.

RESULTS

sgp130Fc binds to membrane-bound IL-6:IL-6R complexes

To analyze whether sgp130 is able to interact with membrane-bound IL-6:IL-6R complexes (Fig. 1A), we used Ba/F3 cells that stably expressed human IL-6R but lacked gp130. We verified the presence of IL-6R on the cell surface of Ba/F3–IL-6R cells and that at least some IL-6 bound to membrane-bound IL-6R by flow cytometry (Fig. 1, B and C). When we incubated Ba/F3–IL-6R cells with IL-6 and sgp130Fc, we detected sgp130Fc on the cell surface by flow cytometry, which suggested that sgp130Fc bound to IL-6:IL-6R complexes on BaF/3–IL-6R (Fig. 1D) cells, but not to the IL-6R alone (Fig. 1E). sgp130Fc is a fusion protein consisting of the extracellular domains of gp130 linked to an Fc part of a human immunoglobulin G (IgG) antibody (4). Although the data suggested binding of sgp130Fc to IL-6:IL-6R complexes by flow cytometry, the shift detected was small, perhaps because of limited binding of IL-6 to IL-6R on the cell surface of Ba/F3–IL-6R cells (Fig. 1C). Therefore, we designed two membrane-bound Hyper-cytokines, in which IL-6 was connected to IL-6R by flexible peptide linkers.

Fig. 1 sgp130Fc binds to membrane-bound IL-6:IL-6R complexes.

(A) Simple model of how sgp130 (white) may bind to IL-6 (light gray):IL-6R (dark gray) complexes on the cell surface of Ba/F3 cells. (B) Flow cytometric analysis of cell surface IL-6R abundance on control (gray-filled) or IL-6R stably transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized mean fluorescence intensity (MFI) data are means ± SEM from all experiments. ** P = 0.01. (C) Flow cytometric analysis of recombinant IL-6 binding to control (gray-filled) or IL-6R transduced (line) or Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. ns, not significant. (D) Flow cytometric analysis of recombinant sgp130Fc binding to membrane-bound IL-6:IL-6R complexes on control (gray-filled) or IL-6R transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. (E) Flow cytometric analysis of recombinant sgp130Fc binding to membrane-bound IL-6R on control (gray-filled) or IL-6R transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments.

Membrane-bound Hyper–IL-6 (mbHIL-6) was derived from the original Hyper–IL-6 (HIL-6) designer cytokine (14), although the membrane anchorage sequence was fused to IL-6 and not to IL-6R (Fig. 2A). The second variant, named native-like mbHIL-6 (nmbHIL-6), consisted of IL-6 at the N terminus followed by the naturally occurring membrane-bound IL-6R at the C terminus (Fig. 2B). This native-like arrangement needed, however, a much longer linker peptide of about 10 nm to span the distance from the C terminus of IL-6 to the N terminus of IL-6R (Fig. 2B), when compared to the shorter distance (about 3 nm) from the C terminus of IL-6R to the N terminus of the IL-6R in the original HIL-6 (Fig. 2A). Moreover, domain 1 (D1) of the IL-6R was not included in nmbHIL-6 because only D2 and D3 of the human IL-6R are necessary for IL-6 binding and signal transduction by gp130 (14, 15). mbHIL-6 and nmbHIL-6 were efficiently expressed on the surface of Ba/F3–mbHIL-6 and Ba/F3–nmbHIL-6 cells, and both membrane-bound Hyper-cytokines facilitated binding of sgp130Fc (Fig. 2, C to H). We found that the binding of sgp130Fc to mbHIL-6 and nmbHIL-6 determined by flow cytometry was much stronger than that to the membrane-bound IL-6:IL-6R complex (Fig. 1D). This may be due to the increased IL-6:IL-6R complex stability within Hyper-cytokines compared to the individual proteins. Our results demonstrated that sgp130Fc can directly bind to membrane-bound IL-6:IL-6R complexes. Thus, the membrane-anchored Hyper-cytokines are suitable designer cytokines to study the effects of sgp130Fc on transactivation.

Fig. 2 sgp130Fc binds to mbHIL-6 complexes.

(A and B) Molecular model of mbHIL-6 (A) and nmbHIL-6 (B) that indicates IL-6 (yellow), D2 and D3 of the IL-6R (green), the linker between IL-6 and IL-6R (red), and the stalk region, transmembrane (TM), and intracellular domain (ICD) of IL-6R (gray). (C) Simple model of how sgp130 (white) may bind to mbHIL-6 (gray) on the cell surface of Ba/F3 cells. (D) Flow cytometric analysis of IL-6R abundance on control (gray-filled) and mbHIL-6 stably transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. *P ≤ 0.05. (E) Flow cytometric analysis of recombinant sgp130Fc binding to control (gray-filled) or mbHIL-6 transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. (F) Simple model of how sgp130 (white) binds to nmbHIL-6 (gray) on the cell surface of Ba/F3 cells. (G) Flow cytometric analysis of nmbHIL-6 abundance on control (gray-filled) and nmbHIL-6 transduced (line) Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. ***P = 0.001. (H) Flow cytometric analysis of recombinant sgp130Fc binding to nmbHIL-6 on Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SEM from all experiments. **P = 0.01.

sgp130Fc inhibits IL-6 cluster signaling by IL-6:IL-6R complexes

Cluster signaling by membrane-bound IL-6:IL-6R complexes on transmitter cells can activate gp130 receptor signaling in receiver cells. We set up a simple model system of IL-6 trans-presentation using two Ba/F3 cell lines: transmitter Ba/F3–IL-6R cells expressing IL-6R, whose proliferation was dependent on IL-3, and receiver Ba/F3-gp130 cells expressing gp130, whose proliferation was dependent on IL-6:sIL-6R or HIL-6 but not IL-3 (Fig. 3, A and B). Coculture of transmitter and receiver cells in the presence of IL-6 increased the proliferation of receiver cells, which was inhibited by the addition of sgp130Fc (Fig. 3B). The proliferation data were not normalized, meaning that in our Ba/F3-gp130 and Ba/F3–IL-6R coculture, proliferation based on trans-presentation and residual proliferation of Ba/F3-IL-6R cells induced by IL-3 were inseparable. IL-3 was needed to prevent apoptosis of transmitter cells, which would promote the shedding of IL-6R (16). Therefore, sgp130Fc-mediated inhibition of trans-presentation was reduced to the level of intrinsic proliferation induced by IL-3 (Fig. 3B). Trans-activation was significantly increased after the addition of VHH6, a recently described single-domain antibody that specifically stabilizes IL-6:sIL-6R complexes (Fig. 3B) (17). However, the VHH6-supported trans-presentation was efficiently inhibited by sgp130Fc.

Fig. 3 Transactivation by IL-6:IL-6R complexes is inhibited by sgp130Fc.

(A) Simple model of IL-6 transactivation, where IL-6 (light gray):IL-6R (dark gray) complexes on transmitter cells activate gp130 (white) on receiver cells. (B) CellTiter-Blue analysis of the proliferation of Ba/F3-gp130 cells, Ba/F3–IL-6R cells, or cocultures incubated with the indicated cytokines or recombinant proteins for 3 days. Data are means ± SD representative of three independent experiments. RFU, relative fluorescence unit. (C and D) Western blot analysis of pSTAT3 in lysates of Ba/F3–IL-6R or Ba/F3-gp130 cells (C) or cocultured Ba/F3–IL-6R and Ba/F3-gp130 cells stimulated with cytokines or recombinant proteins, as indicated. Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. (E) Western blot analysis of pSTAT3 in lysates of cocultured Ba/F3–IL-6R and Ba/F3-gp130 cells incubated with IL-6 and sgp130Fc, tocilizumab, or GW280264X (GW) as indicated. Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. **P = 0.01. (F) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells stimulated by 10% cellular supernatants collected after cocultivation experiments in (E). Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. *P ≤ 0.05 and ***P ≤ 0.001 by analysis of variance (ANOVA) test with Dunnett’s correction.

To determine whether cluster signaling augmented gp130-dependent signaling, we tested for downstream STAT3 activity by Western blotting. We found that IL-3 alone did not stimulate STAT3 phosphorylation (pSTAT3) in Ba/F3-gp130 and Ba/F3–IL-6R cells, consistent with its known role in mainly activating STAT5 (18). In contrast, stimulation of Ba/F3-gp130 cells with HIL-6 induced strong pSTAT3 (Fig. 3C). Coculture of transmitter and receiver cells for 30 min stimulated IL-6 dose-dependent pSTAT3, which was increased by the addition of VHH6 and inhibited by sgp130Fc (Fig. 3D). Tocilizumab, a humanized neutralizing IL-6R antibody approved for clinical therapy of rheumatoid arthritis and other diseases (7), also limited STAT3 activation stimulated by trans-presentation (Fig. 3E). Selective blockade of the IL-6R shedding proteases ADAM10 and ADAM17 (or A Disintegrin And Metalloprotease 10/17) by the inhibitor GW280264X did not inhibit pSTAT3 in the coculture system, suggesting that sIL-6R–mediated trans-signaling was not involved (Fig. 3E). To exclude that any IL-6:sIL-6R complexes were responsible for stimulating pSTAT3 through trans-signaling, we transferred supernatants obtained in the coculture experiments (Fig. 3E) to fresh Ba/F3-gp130 cells and probed for STAT3 activation (Fig. 3F). Conditioned supernatants did not stimulate pSTAT3, demonstrating that IL-6:sIL-6R complexes released from the cell surface were insufficient to stimulate Ba/F3-gp130 cells through trans-signaling.

sgp130Fc inhibits IL-6 cluster signaling by membrane-bound Hyper-cytokines

When we tested the novel membrane-bound HIL-6 designer cytokines, we found that mbHIL-6 and nmbHIL-6 increased signaling induced by IL-6:sIL-6R cluster signaling (Fig. 4A). Coculture of Ba/F3–mbHIL-6 or Ba/F3–nmbHIL-6 transmitter cells with Ba/F3-gp130 receiver cells increased the proliferation of receiver cells, without the need for external cytokine stimulation (Fig. 4B). Again, cluster signaling was significantly inhibited by sgp130Fc (Fig. 4B). Furthermore, treatment with HIL-6 stimulated in pSTAT3 Ba/F-gp130 receiver cells but not in Ba/F3–mbHIL-6 transmitter cells (Fig. 4C). Neither tocilizumab nor the ADAM10/17 inhibitor GW280264X prevented pSTAT3 activation induced by trans-presentation of mbHIL-6 and nmbHIL-6 (Fig. 4, D and E, and fig. S1, A and B). Similarly, tocilizumab does not inhibit HIL-6 (19). Although the affinities of IL-6 and tocilizumab for the IL-6R are within a comparable range (12, 13), it remains possible that tocilizumab may not bind the IL-6R in HIL-6 because the fusion of IL-6 and sIL-6R in HIL-6 locks IL-6 in the IL-6:sIL-6R complex and may obscure the IL-6R binding site. To exclude that shed, soluble mbHIL-6 or nmbHIL-6 were responsible for pSTAT3 through trans-signaling, we again used the supernatants obtained from coculture experiments to stimulate Ba/F3-gp130 cells (Fig. 4, D and E). As with earlier experiments (Fig. 3F), Ba/F3–mbHIL-6– or Ba/F3–nmbHIL-6–conditioned supernatants were not able to trigger pSTAT3 in Ba/F3-gp130 cells, demonstrating that any mbHIL-6 or nmbHIL-6 released was insufficient to stimulate these cells by trans-signaling (Fig. 4, F and G, and fig. S1, C and D). Together, our results supported that trans-presentation of IL-6 in complex with membrane-bound IL-6R activated cells that only expressed gp130 (9, 10). Moreover, we demonstrated that this trans-presentation can be inhibited by sgp130Fc.

Fig. 4 Transactivation by mbHIL-6 or nmbHIL-6 complexes is inhibited by sgp130Fc.

(A) Simple model of IL-6 transactivation, where IL-6 (light gray):IL-6R (dark gray) complexes encoded in mbHIL-6 and nmbHIL-6 on transmitter cells activate gp130 (white) on receiver cells. (B) CellTiter-Blue analysis of the proliferation by Ba/F3-gp130 cells, Ba/F3–IL-6R cells, or cocultures incubated with the indicated cytokines or recombinant sgp130Fc for 3 days. Data are means ± SD representative of three independent experiments. (C) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130, Ba/F3–mbHIL-6, or Ba/F3–nmbHIL-6 cells stimulated with HIL-6 or IL-3 for 30 min. Blots are representative of three independent experiments. Normalized band intensity data are means ± SEM from all experiments. (D and E) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells cocultured with Ba/F3–mbHIL-6 cells (D) or Ba/F3–nmbHIL-6 cells (E) and incubated with sgp130Fc or tocilizumab and GW280264X, as indicated. Blots are representative of three independent experiments. Normalized band intensity data are shown in fig. S1 (A and B). (F and G) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells stimulated with 10% supernatants collected from the coculture experiments in panel (D) (F) or panel (E) (G). Blots are representative of three independent experiments. Normalized band intensity data are shown in fig. S1 (C and D). *P ≤ 0.05 and ***P ≤ 0.001 by ANOVA test with Bonferroni correction (B) or ANOVA test with Dunnett’s correction (C).

sgp130Fc inhibits IL-11 cluster signaling

IL-11 is the only other member of the IL-6 cytokine family that induces signaling through gp130 homodimers (11). In analogy to IL-6, which must complex with the IL-6R or sIL6R to bind to gp130, IL-11 requires interaction with IL-11R or sIL-11R to bind gp130. IL-11R is homologous to IL-6R and also consists of three extracellular domains followed by stalk region, transmembrane domain, and intracellular domain (Fig. 5A) (11). When we generated Ba/F3–IL-11R cells and preincubated them with IL-11 and/or sgp130Fc, we found that sgp130Fc bound to membrane-bound IL-11:IL-11R complexes, but not to IL-11R alone (Fig. 5, B and C, and fig. S2, A and B). In the presence of IL-11, Ba/F3-gp130 responder cells proliferated and activated STAT3 when cocultured with Ba/F3–IL-11R transmitter cells (Fig. 5, D and E, and fig. S2, C and D). These effects of IL-11R trans-presentation were inhibited by sgp130Fc, but not influenced by the addition of the ADAM10/17 inhibitor GW280264X. These data confirm that IL-11 may be trans-presented.

Fig. 5 Transactivation by membrane-bound IL-11:IL-11R or mbHIL-11 complexes is inhibited by sgp130Fc.

(A) Simple model of IL-11 transactivation, where IL-11 (light gray): IL-11R (dark gray) or mbHIL-11 on transmitter cells activates gp130 (white) on receiver cells. (B) Flow cytometric analysis of human IL-11R cell surface abundance on control (gray-filled) and IL-11R, mbHIL-11, or mbHIL-11R355E stably transduced Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data from all experiments are shown in fig. S2A. (C) Flow cytometric analysis of recombinant sgp130Fc binding to membrane-bound IL-11:IL-11R complexes, mbHIL-11, or mbHIL-11R355E expressed on Ba/F3 cells. Histograms are representative of three independent experiments. Normalized MFI data from all experiments are shown in fig. S2C. (D) CellTiter-Blue analysis of cellular proliferation by Ba/F3–IL-11R, Ba/F3–mbHIL-11, or Ba/F3–mbHIL-11R355E cells co-incubated with Ba/F3-gp130 cells in the presence of the indicated cytokines and recombinant sgp130Fc for 3 days. Data are means ± SD representative of three independent experiments. (E) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 cells cocultured with Ba/F3–IL-11R, Ba/F3–mbHIL-11, or Ba/F3–mbHIL-11R355E cells and treated with IL-11, sgp130Fc, or GW as indicated. Blots are representative of two independent experiments. Normalized band intensity data from each experiment are shown in fig. S2D. (F) Western blot analysis of pSTAT3 in Ba/F3-gp130 cells stimulated with 10% supernatants collected from cocultivation in experiments in (E). Blots are representative of two independent experiments. Normalized band intensity data from each experiment are shown in fig. S2E. ***P ≤ 0.001 by ANOVA test with Bonferroni correction.

In analogy to mbHIL-6, we generated mbHIL-11 on the basis of the previously described Hyper-cytokine for IL-11 (HIL-11) (20). IL-6 and IL-11 are the only members of the IL-6 family of cytokines that signal by a gp130 homodimer (3). For both cytokines, the mode of signal transduction is very similar including binding of cytokine to the α receptor followed by recruitment of two gp130 molecules yielding signal transduction. This comparable mechanism is in line with the findings that sgp130 inhibits IL-6 and IL-11 trans-signaling. Thus, we were interested in whether IL-11 is also able to induce cluster signaling in a similar way to IL-6. Because IL-11R can be shed by ADAM proteases, leading to IL-11 trans-signaling (21), to reduce ADAM10-mediated shedding of mbHIL-11, we also generated the mbHIL-11R355E variant (Fig. 5A). This R355E variant is shed much less efficiently than wild-type IL-11R (21). The two variants mbHIL-11 and mbHIL-11R355E were expressed on the cell surface of Ba/F3 cells as shown by flow cytometry (Fig. 5B and fig. S2A). Binding of sgp130Fc to mbHIL-11 and mbHIL-11R355E was detectable by flow cytometry (Fig. 5C and fig. S2B). Coculture of mbHIL-11 and mbHIL-11R355E transmitter Ba/F3 cells with Ba/F3-gp130 receiver cells stimulated robust proliferation of receiver cells, which was inhibited by sgp130Fc (Fig. 5D). Coculture of the mbHIL-11 transmitter and gp130 receiver cells also activated pSTAT3, but not in the presence of sgp130Fc (Fig. 5E and fig. S2D). To excluded that shed, soluble IL-11:IL-11R, mbHIL-11, and mbHIL-11R355E were responsible for pSTAT3 through trans-signaling, we again used the supernatants obtained in coculture experiments (Fig. 5E and fig. S2E) to stimulate Ba/F3-gp130 cells. Neither Ba/F3-gp130 cocultured Ba/F3–IL-11:sIL-11R–, Ba/F3–mbHIL-11–, nor Ba/F3–mbHIL-11R355E–conditioned supernatants stimulated pSTAT3, again demonstrating that any released soluble IL-11:sIL-11R, smbHIL-11, and smbHIL-11R355E was not sufficient to stimulate Ba/F3-gp130 cells through trans-signaling (Fig. 5F). In conclusion, also IL-11 is able to stimulate gp130-mediated cluster signaling.

Autocrine IL-6 and IL-11 classic and trans-signaling is induced within the cell

We analyzed whether our novel Hyper-cytokines stimulated autocrine classic and trans-signaling using Ba/F3-gp130 cells coexpressing nmbHIL-6 or HIL-6 (Fig. 6, A to C, and fig. S3A). We found that Ba/F3–gp130–nmbHIL-6 and Ba/F3–gp130–HIL-6 cells proliferated without external cytokine stimulation. These data suggested that nmbHIL-6 and HIL-6 may activate classic or trans-signaling through autocrine or paracrine stimulation, respectively (Fig. 6D). Cellular proliferation induced by both endogenous HIL-6 and nmbHIL-6 was not inhibited by sgp130Fc, whereas sgp130Fc could inhibit cellular proliferation induced by exogenous HIL-6 (Fig. 6D). Thus, gp130-mediated proliferative signaling was initiated before the cell surface exposure (nmbHIL-6) or secretion (HIL-6) of the recombinant IL-6 Hyper-cytokines, which suggested an autocrine mechanism in a strict sense.

Fig. 6 Autocrine classic and trans-signaling by nmbHIL-6 or HIL-6 is not inhibited by extracellular inhibitors.

(A) Simple model of autocrine classic signaling through gp130 (white) by IL-6 (light gray):IL-6R (dark gray) encoded in nmbHIL-6 and trans-signaling by HIL-6. (B) Flow cytometric analysis of cell surface IL-6R abundance on control (gray-filled) and nmbHIL-6 stably transduced (line) Ba/F3-gp130 cells. Histograms are representative of three independent experiments. Normalized MFI data from all experiments are shown in fig. S3A. (C) Western blot (WB) analysis of HIL-6 abundance in cellular lysates (L) and cell culture supernatants (SN) of Ba/F3-gp130 control or HIL-6 stably transduced cells. Blots are representative of three independent experiments. (D) CellTiter-Blue analysis of cellular proliferation by Ba/F3-gp130, Ba/F3–gp130–nmbHIL-6, or Ba/F3–gp130–HIL-6 cells treated with HIL-6 or sgp130Fc, as indicated for 3 days. Data are means ± SD representative of three independent experiments. (E and F) Western blot analysis of pSTAT3 in lysates of Ba/F3-gp130 (E) or Ba/F3–gp130–nmbHIL-6, Ba/3–gp130–HIL-6, and Ba/F3–gp130–HIL-6R–IL-6 (F) cells treated with HIL-6, sgp130Fc, tocilizumab, P6, or B-R3, as indicated. Blots are representative of three independent experiments. Normalized band intensity data are means ± SD from all experiments (E) or are in fig. S3B (F). (G) Analysis of pSTAT3 in HEK 293 cells transfected with HIL-6Fc or sgp130Fc or treated with HIL-6Fc or sgp130Fc, as indicated. Blots are representative of three independent experiments. Normalized band intensity data from all experiments are shown in fig. S3C. *P ≤ 0.05 and ***P ≤ 0.001 by ANOVA test with Bonferroni correction (D) or ANOVA test with Dunnett’s correction (E).

To investigate whether such autocrine signaling was initiated from within the cell, we stimulated Ba/F3-gp130 cells with HIL-6 and analyzed pSTAT3 in the presence or absence of several inhibitors: sgp130Fc, tocilizumab, the pan-JAK inhibitor Pyridone 6 (P6) (22), and the neutralizing gp130 antibody B-R3 (23). As expected, sgp130Fc, B-R3, and P6 all reduced pSTAT3 in Ba/F3-gp130 cells stimulated by exogenous HIL-6, whereas tocilizumab did not (Fig. 6E). Ba/F3–gp130–IL-6R–IL-6 cells express gp130, IL-6R, and the cytokine IL-6, which activates gp130 signaling in an autocrine activation. All inhibitors except the intracellular JAK inhibitor P6 failed to inhibit pSTAT3 activation in Ba/F3–gp130–IL-6R–IL-6, Ba/F3–gp130–nmbHIL-6, and Ba/F3–gp130–HIL-6 cells, which suggested that they did not prevent autocrine classic and trans-signaling (Fig. 6F and fig. S3B). When we transiently coexpressed sgp130Fc in molar excess of HIL-6 in human embryonic kidney (HEK) 293 cells and analyzed IL-6 signaling, we found that only autocrine sgp130Fc but not sgp130 from the outside (paracrine stimulation) prevented activation of STAT3 (Fig. 6G and fig. S3C). These data support that gp130 signaling is initiated from an intracellular compartment.

We obtained similar results when we generated Ba/F3–gp130–IL-11R and Ba/F3–gp130–mbHIL-11 cells (Fig. 7, A and B). Ba/F3–gp130–mbHIL-11 cells also proliferated without external cytokine stimulation, and this process was not inhibited by sgp130Fc (Fig. 7C). We analyzed pSTAT3 in Ba/F3–gp130–IL-11R cells stimulated with IL-11 and found that P6 and B-R3 inhibited the response to exogenous IL-11, but the IL-6R–specific monoclonal antibody (mAb) tocilizumab and sgp130Fc did not (Fig. 7D). Similar to the results with IL-6 (Fig. 6F), the autocrine pSTAT3 induced in Ba/F3–gp130–mbHIL-11 cells was also not inhibited by sgp130Fc, tocilizumab, or B-R3, whereas P6 reduced pSTAT3 (Fig. 7E). Together, our results demonstrated that autocrine stimulation by HIL-6, nmbHIL-6, or mbHIL-11 sustained activation of gp130 that was unresponsive to extracellular inhibitors.

Fig. 7 Autocrine classic and trans-signaling by mbHIL-11 is not inhibited by extracellular inhibitors.

(A) Simple model of autocrine classic signaling through gp130 (white) by IL-11 (light gray):IL-11R (dark gray) complexes encoded in mbHIL-11. (B) Flow cytometric analysis of cell surface IL-11R abundance on control (gray-filled) and IL-11R or mbHIL-11 stably transduced (line) Ba/F3-gp130 cells. Histograms are representative of three independent experiments. Normalized MFI data are means ± SD from all experiments. (C) CellTiter-Blue analysis of cellular proliferation by Ba/F3-gp130 or Ba/F3–gp130–mbHIL-11 cells treated with HIL-6 and sgp130Fc, as indicated for 3 days. Data are means ± SD representative of three independent experiments. (D and E) Western blot analysis of pSTAT3 in lysates of Ba/F3–gp130–IL-11R (D) or Ba/F3–gp130–mbHIL-11 (E) cells treated with IL-11, sgp130Fc, tocilizumab, P6, or B-R3, as indicated. Blots are representative of at least two independent experiments. Normalized band intensity data are from each experiment (D) or are means ± SD from three experiments (E). *P ≤ 0.05 and ***P ≤ 0.001 by ANOVA test with Bonferroni correction (C) or ANOVA test with Dunnett’s correction (E).

DISCUSSION

In addition to the well-described classic and trans-signaling modes, IL-6 family cytokines can also activate cluster signaling, where transmitter cells present membrane-bound IL-6:IL-6R complexes to receiver cells carrying only gp130 (9, 10). Although endogenous sgp130 and the much more potent dimerized fusion protein sgp130Fc are specific trans-signaling inhibitors, these inhibitors leave classic IL-6 signaling and trans-presentation intact (4, 9, 10). However, it remains unclear why sgp130Fc does not interfere with trans-presentation in these studies. It is possible that steric hindrance of sgp130Fc might restrict binding to membrane-bound IL-6:IL-6R complexes. To directly address this question, we established a robust reductionist system of cluster signaling, enabled by novel membrane-bound Hyper-cytokines. Using this system, we demonstrated that sgp130Fc binds to membrane-bound IL-6:IL-6R and IL-11:IL-11R complexes. As a logical consequence, sgp130Fc was able to inhibit IL-6 and IL-11 cluster signaling (Fig. 8, A to D). A difference in our study was that IL-6 was loaded on the cells exogenously and not directly expressed by the cell that also expressed IL-6R. Although it is unlikely, coexpression of IL-6:sIL-6R and preassembly of the complex within the cell might also influence the inhibitory potential of sgp130, especially if the receiver cell is already in close contact with the transmitter cell.

Fig. 8 Schematic overview of sgp130 effects on IL-6 and IL-11 signaling modes.

(A) sgp130Fc did not inhibit IL-6/IL-11 classic signaling. (B) sgp130Fc binds IL-6:sIL-6R and IL-11:sIL-11R complexes and inhibits paracrine trans-signaling. (C) sgp130Fc did not inhibit autocrine IL-6 trans-signaling. PM, plasma membrane. (D) sgp130Fc binds IL-6:IL-6R and IL-11:IL-11R complexes on transmitter cells and inhibits transactivation on receiver cells. White, gp130; light gray, IL-6/IL-11; dark gray, IL-6R/IL-11R.

Our data indicated that autocrine IL-6 and IL-11 classic and trans-signaling in cells endogenously producing either membrane-bound or soluble Hyper-cytokines was not prevented by extracellular inhibitors such as sgp130Fc or the neutralizing gp130 antibody B-R3. These data highlight the mechanistic differences between autocrine and paracrine IL-6 and IL-11 signaling. Because autocrine HIL-6–induced signaling was inhibited by coexpression of sgp130Fc, our data suggest that autocrine signaling may be initiated within the cell. Viral IL-6 from human herpesvirus 8 signals intracellularly (24, 25). Moreover, intracellular signaling also occurs downstream of constitutively active gp130 variants (26, 27). In the case of intracellular complex formation of IL-6:IL-6R:gp130 and signal initiation from within the endoplasmic reticulum (ER)–Golgi network (Fig. 8C), we found that B-R3 and sgp130Fc simply cannot interfere with autocrine classic or trans-signaling, likely because they cannot reach these intracellular compartments. Although autocrine signaling occurs in cell culture, it remains to be seen whether this is also relevant in vivo.

We demonstrated that sgp130Fc was able to bind to membrane-bound IL-6:IL-6R complexes. However, sgp130Fc only inhibits paracrine classic signaling in the presence of extreme molar excesses of sIL-6R (8). This paradox might be explained by the unique receptor activation sequence of IL-6 and IL-11 in combination with a particular receptor arrangement on the plasma membrane. These factors might allow binding of natural sgp130 or sgp130Fc to IL-6:IL-6R complexes in the absence, but not in the presence, of gp130. gp130 exists as preformed dimers on the cell surface to enable rapid signal induction after IL-6 binding (28, 29). However, it remains unclear whether the IL-6R and IL-11R are also integrated into preformed complexes. Such preformed receptor complexes could hinder sgp130Fc accession. In addition, IL-6 does not bind gp130 in the absence of the (s)IL-6R, and the affinity of gp130 for IL-6:(s)IL-6R complexes is about 100 times stronger than the affinity of IL-6 for (s)IL-6R (13, 17, 30). Thus, low-affinity binding of IL-6 to IL-6R on the cell surface is rapidly followed by high-affinity binding of the IL-6 complex to membrane-bound gp130. Formation of IL-6:IL-6R:gp130 complexes would ultimately be accelerated by preformed complexes of IL-6R and gp130, which might also prevent significant interaction of sgp130Fc with IL-6:IL-6R complexes whenever gp130 is present on the same cell. However, IL-6:IL-6R:gp130 complexes might be additionally stabilized by other, thus far unknown factors. For example, it is unclear to what extent signaling by IL-6:IL-6R:gp130 complexes is initiated directly at the plasma membrane or whether signaling is specifically continued or even boosted after endocytosis. For other cytokines, such as tumor necrosis factor–α, signaling from endosomes plays an important role in signal transduction (31). At least to some extent, activation of STAT3 involves endosomal trafficking of the receptor complex (32).

Together, our data indicated that sgp130Fc binds to membrane-bound IL-6:IL-6R and IL-11:IL-11R complexes and thereby inhibits IL-6 and IL-11 trans-presentation but does not inhibit autocrine trans-signaling or paracrine and autocrine classic signaling. sgp130Fc is currently under clinical development for chronic inflammatory diseases (2), and it is of utmost importance to define the inhibitory profile of sgp130Fc in comparison to more general IL-6 and IL-6R blocking agents such as antibodies and JAK inhibitors. Finally, our finding reinforces the long-standing question why sgp130Fc does not interfere with classic signaling, which will be addressed in more detail in future studies.

MATERIALS AND METHODS

Cells and reagents

Ba/F3 cells (ACC-300) were purchased from the Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Murine Ba/F3-gp130 cells were obtained from Immunex (33). Murine Ba/F3–IL-6R cells were a gift from Garbers (University of Kiel, Germany). Ba/F3 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) high-glucose culture medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (Gibco, Life Technologies), penicillin (60 mg/liter), and streptomycin (100 mg/liter) (Genaxxon bioscience GmbH) at 37°C with 5% CO2. Proliferation was maintained in Ba/F3 cells by adding 0.2% conditioned medium of WEHI-3B cells [IL-3 (10 ng/ml)] (DSMZ ACC-26). Ba/F3-gp130 cells or variants thereof were maintained in the presence of HIL-6, a fusion protein of IL-6 and the sIL-6R, which mimics IL-6 trans-signaling (14). Either recombinant protein (10 ng/ml) or conditioned cell culture medium from a stable CHO-K1 clone secreting HIL-6 (10 ng/ml) was used. Expression and purification of human HIL-6 and human IL-6 were performed as described previously (14, 34). pSTAT3 (Tyr705) (D3A7), STAT3 (124H6), and myc (71D10) antibodies were obtained from Cell Signaling Technology. The anti–hIL-6R mAb 4 to 11 was described previously (16). Anti–IL-6 biotin mAb was obtained from ImmunoTools. Allophycocyanin (APC) anti-human IgG Fc (HP6017) was obtained from BioLegend. Rabbit anti-human IgG Fc–specific and peroxidase-conjugated secondary mAbs were obtained from Pierce (Thermo Fisher Scientific). APC-streptavidin was purchased from BD Biosciences. APC (115-136-146)–conjugated goat anti-mouse IgG polyclonal antibody was obtained from dianova. Alexa Fluor 488 goat anti-rabbit IgG was purchased from Life Technologies (A11070). Anti-CD130 (gp130) antibody B-R3 (ab34315) was purchased from Abcam. InSolution JAK Inhibitor I (pan-JAK inhibitor P6) was obtained from Merck. The metalloprotease inhibitor GW280264X (GW, selective for both ADAM10 and ADAM17) was a gift from GlaxoSmithKline (35). Inhibitor stock solutions were prepared in dimethyl sulfoxide. Recombinant sgp130Fc, IL-6, and VHH6 were produced and purified as described previously (14, 17). Rabbit anti-human IgG Fc HRP was obtained from Thermo Fisher Scientific.

Cloning of mbHIL-6, nmbHIL-6, nHIL-6, mbHIL-11, HIL-6-Fc, and gp130 variants

Corresponding expression vectors for mbHIL-6, mbHIL-11, and gp130 variants were cloned using standard cloning procedures. HIL-6Fc consists of amino acid residues 1 to 323 of the hIL-6R fused to hIL-6 (28 to 211) followed by an Fc fusion protein. The linker amino acid sequence connecting sIL-6R with IL-6 was N-GGGGSGGGGS-C. MbHIL-6 consists of amino acid residues 1 to 323 of the hIL-6R fused to hIL-6 (28 to 211) followed by the stalk region, transmembrane domain, and intracellular domain of the hIL-6R (315 to 468). The linker amino acid sequence connecting sIL-6R with IL-6 was N-GGGGSGGGGS-C. MbHIL-11 was generated in analogy to mbHIL-6. MbHIL-11 starts with the hIL-11R (1 to 315) fused to hIL-11 (31 to 199), followed by the stalk region, transmembrane domain, and intracellular domain of the hIL-11R (318 to 422). The linker sequence connecting IL-11R and IL-11 was 22 amino acid residues long (N-WTESRSPPARGGGGSGGGGSVEP-C). For the generation of mbHIL-11RR355E, a mutated DNA sequence encoding amino acids 331 to 422 of the human IL-11R was purchased from GeneArt (Thermo Fisher Scientific) and subcloned by the Not I and Eco RV restriction sites into pcDNA3.1–mbHIL-11. nmbHIL-6 starts with the amino acid residues of hIL-6 (1 to 212), followed by a linker of 29 amino acid residues (LEGGGGSEAAAAKPAPAPEAAAAKGGGGS) and the hIL-6R sequence without die D1 domain from amino acid residues 114 to 468. Complementary DNAs (cDNAs) encoding human receptors and designer cytokines were subcloned into the retroviral plasmids pMOWS-puro or pMOWS-hygro (36).

Transfection, transduction, and selection of cells

Ba/F3 or Ba/F3-gp130 cells were retrovirally transduced with constructs for mbHIL-6, IL-11R, mbHIL-11, and gp130 variants as described (37). Transduced cells were grown in standard DMEM supplemented with HIL-6 (10 ng/ml) or IL-3 (10 ng/ml). Selection of transduced Ba/F3 cells was performed with puromycin (1.5 μg/ml) or hygromycin B (1 mg/ml) (Carl Roth GmbH) or both. HEK 293 cells were grown in standard DMEM. Cells (4 × 105) were transiently transfected with Turbofect and 1 μg of plasmid DNA for sgp130Fc and 100 ng of plasmid DNA for HIL-6Fc. Six hours after transfection, the medium was exchanged to standard DMEM without transfection reagent. Cells were harvested 48 hours after stimulation for 30 min as indicated.

Cell surface detection of cytokine receptors

Ba/F3 or Ba/F3-gp130 cells expressing IL-6R, IL-11R, mbHIL-6, nmbHIL-6, or mbHIL-11 variants were washed with phosphate-buffered saline (PBS) plus 1% bovine serum albumin (BSA) (PBS-BSA) and incubated at 5 × 105 cells/100 μl of PBS-BSA supplemented with anti–HIL-6R mAbs 4 to 11 (mbHIL-6), anti–IL-6 biotin mAb (nmbHIL-6), or anti-myc mAb 71D10 for 2 hours on ice. After washing, cells were incubated in 100 μl of PBS-BSA containing a 1:100 dilution of APC (for HIL-6R) or FITC (fluorescein isothiocyanate) (for myc-tag) (dianova) for 1 hour at 4°C. Cells were washed, suspended in 200 μl of PBS-BSA, and analyzed by flow cytometry (BD FACSCanto II flow cytometer, BD Biosciences). Data were evaluated using the FCS Express software (De Novo Software).

Proliferation assays

Proliferation of Ba/F3 cell lines was determined 72 hours after cytokine stimulation using the CellTiter-Blue Cell Viability Assay (Promega). Cells were washed, and 5000 cells of each cell line were cultured for 3 days in a final volume of 100 μl with or without cytokines or inhibitors. The CellTiter-Blue Reagent was used to estimate the number of viable cells by recording the fluorescence (excitation, 560 nm; emission, 590 nm) using an Infinite M200 PRO plate reader (Tecan) immediately after adding 20 μl of reagent per well (time point 0) and up to 120 min after incubation. The fluorescent signal from the CellTiter-Blue Reagent is proportional to the number of viable cells. All of the values were measured in triplicates per experiment. Fluorescence values were normalized by the subtraction of time point 0 values. All experiments were performed at least three times, and one representative experiment was selected.

Transactivation assays

Ba/F3-gp130 cells were washed and starved for at least 3 hours in serum-free medium. Ba/F3–IL-6R–, Ba/F3–mbHIL-6–, Ba/F3–nmbHIL-6–, or Ba/F3–mbHIL-11–expressing cells were washed and cultured with IL-3 with or without inhibitors for 3 hours. Afterward, cells that were cultured with GW280264X (GW, selective for both ADAM10 and ADAM17; 3 μM) for inhibition of shedding were washed again three times and then incubated with GW and inhibitors. Subsequently, Ba/F3-gp130 cells were mixed with Ba/F3–IL-6R, Ba/F3–mbHIL-6, Ba/F3–nmbHIL-6, or Ba/F3–mbHIL-11 and variants thereof and incubated for 30 min (in case of Ba/F3–IL-6R cells, IL-6 was added as indicated). Cells were then harvested by centrifugation at 4°C for 5 min at 1500g. Supernatants were used for cellular stimulation of Ba/F3-gp130 cells, and the cell pellets were lysed in Laemmli sample buffer and heated for 10 min at 95°C. Analysis of pSTAT3 was done by Western blotting.

Stimulation assays

Ba/F3–IL-6R (106) and Ba/F3-gp130 cells/ml and variants thereof were washed and starved in serum-free medium for 6 hours. Afterward, cells were stimulated with the indicated cytokines or inhibitors for 30 min [IL-3 (10 ng/ml), HIL-6 (10 ng/ml), sgp130Fc (10 μg/ml), tocilizumab (10 μg/ml), B-R3 (1 μg/ml), P6 (10 mM), and GW (3 μM)]. The cells were harvested by centrifugation at 4°C for 5 min at 1500g. Cells were suspended in Laemmli sample buffer, and pSTAT3 analysis was done by Western blotting as described.

Western blotting

Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF (polyvinylidene difluoride) membranes. Membranes were blocked and probed with the indicated primary antibodies. After washing, membranes were incubated with secondary peroxidase-conjugated antibodies. The Immobilon Western Reagents (Millipore Corporation) and the ChemoCam Imager (Intas Science Imaging Instruments GmbH) were used for signal detection. Control STAT3 blots were produced with the same samples on separate gels. Western blotting data were quantified using ImageJ software. Band intensities for pSTAT3 were normalized on the respective unphosphorylated form of the protein.

Statistical analysis

Data are presented as means ± SEM for flow cytometry or means ± SD for proliferation assays and Western blotting. Fluorescence-activated cell sorting data are analyzed by an unpaired Student’s t test. For multiple comparisons, one-way ANOVA, followed by Bonferroni or Dunnett’s correction, was used (GraphPad Prism 6.0, GraphPad Software Inc.). Statistical significance was set at the level of P < 0.05.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/550/eaar7388/DC1

Fig. S1. Statistical analysis of membrane-bound HIL-6–mediated IL-6 cluster signaling.

Fig. S2. Statistical analysis of membrane-bound HIL-11–mediated IL-11 cluster signaling.

Fig. S3. Autocrine classic and trans-signaling by nmbHIL-6 or HIL-6 is not inhibited by extracellular inhibitors.

REFERENCES AND NOTES

Acknowledgments: We thank C. Garbers (Christian-Albrechts-University of Kiel, Germany) for supplying a sample of Ba/F3–IL-6R cells. R. Dvorsky supported modelling of the linker sequences for native membrane-bound Hyper–IL-6. Funding: This work was funded by grants from the Bundesministerium für Bildung und Forschung (to J.S.; project InTraSig) and Deutsche Forschungsgemeinschaft (SFB974). Author contributions: L.L. conducted most of the experiments and analyzed the data. F.R. and J.M.M. performed experiments with mbHIL-11. G.H.W., S.H., P.B., and D.M.F. supported cloning, recombinant protein expression, and cell culture experiments. All authors helped in writing the paper. J.M.M., D.M.F., and R.P. analyzed the data and performed statistical analysis. J.S. designed the study, analyzed the data, and wrote the paper. Competing interests: G.H.W. is employed by CONARIS Research Institute AG (Kiel, Germany), which is commercially developing sgp130Fc proteins as therapeutics for inflammatory diseases. In addition, he is an inventor on the following intellectual property owned by CONARIS pertaining to the sgp130Fc used in the present study: Australian Patent No. 2007263939; Brazilian Patent Application No. PI0713063-5; Canadian Patent No. 2,575,800 and Application No. 2,656,440; Chinese Patent No. ZL200780024879.1; Eurasian Patent No. 015620; European Patent Nos. 1873166 and 1630232; Indian Patent No. 265303; Japanese Patent Nos. 4615016 and 5417171; South Korean Patent No. 10-1474817; Ukrainian Patent No. 95636; and U.S. Patent Nos. 8,206,948, 8,895,012, 9,034,817, and 9,573,989. The other authors declare that they have no competing or financial interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
View Abstract

Stay Connected to Science Signaling

Navigate This Article