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Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5
Biochemical and Biophysical Research Communications xxx (2017) 1e5
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Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5 Samadhi Aparicio-Siegmund, Malte Deseke, Annett Lickert, Christoph Garbers* Institute of Biochemistry, Kiel University, 24118 Kiel, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2017 Accepted 28 January 2017 Available online xxx
IL-6 exerts its pleiotropic activities on its target cells via the IL-6 alpha-receptor (IL-6R), which is expressed on a limited number of cell types. IL-6 can further signal via soluble forms of its receptor (sIL6R), a process that has been termed trans-signaling. Recently, CD5 was described as an alternative alphareceptor for IL-6 on B cells leading to the phosphorylation of the transcription factor STAT3 via the signaltransducing b-receptor gp130 in a Jak2-dependent manner. In this study, we sought to investigate whether IL-6 was also able to signal via soluble CD5 (sCD5) analogous to IL-6 trans-signaling. We show that IL-6 indeed binds to sCD5, but that this does not lead to the activation of signal transduction or cell proliferation. Furthermore, sCD5 did also not interfere with IL-6 classic signaling, suggesting that the affinity between the two proteins was too weak to provoke a biological effect. Thus, trans-signaling of IL6 can only occur via sIL-6R, but not sCD5. © 2017 Elsevier Inc. All rights reserved.
Keywords: Interleukin-6 CD5 STAT3 Trans-signaling
1. Introduction Signaling via the ubiquitously expressed glycoprotein 130 (gp130) is a hallmark of all cytokines of the interleukin-6 (IL-6) family [1]. Specificity is gained through cell-dependent expression of other signaling and non-signaling receptors. Binding of IL-6 to its non-signaling IL-6 a-receptor (IL-6R), so-called classic signaling, leads to gp130 homodimerization and the activation of intracellular signaling cascades, including the Jak/STAT pathway [2]. Enhanced activities and increased serum levels of IL-6 are associated with inflammatory human diseases [3,4]. Most soluble cytokine receptors function as antagonistic decoy receptors, but the soluble IL-6R (sIL-6R) is a rare example of an agonist [5]. IL-6 binds to sIL-6R with the same affinity as to the membrane-bound IL-6R, and sIL-6R/IL-6 complexes can activate all cells of the human body via a gp130 homodimer. This mode of IL-6 signaling has been termed trans-signaling and represents an attractive therapeutic target [4,6]. The majority of the sIL-6R is generated by proteolytic cleavage of the membrane-bound precursor, and the two metalloproteases ADAM10 and ADAM17 have been described as efficient sheddases of the IL-6R [7e13]. Recently,
* Corresponding author. Institute of Biochemistry, Kiel University, Olshausenstrasse 40, 24118 Kiel, Germany. E-mail address: [email protected] (C. Garbers).
a third modality of IL-6 signaling has been described, in which IL-6 couples to the IL-6R intracellularly and is then transported to the cell surface, where it stimulates gp130 on a neighboring cell. This pathway has been termed IL-6 cluster signaling/trans-presentation [14]. CD5 is a type I transmembrane protein that belongs to the scavenger receptor cysteine-rich (SRCR) superfamily [15,16]. It consists of three extracellular domains and was initially used as a cell surface marker for T cells; however, it is also expressed on B cells [16]. CD5 has an important role in T cell receptor signaling [17] and can be activated by a variety of different ligands, including zymosan, gp150, gp77-80, gp35-70 and CD5 itself [16,18]. Different forms of CD5 are generated by alternative processing of its mRNA [19], and naturally occurring soluble forms of CD5 (sCD5) appear to be generated by proteolytic cleavage of membrane-anchored CD5 [20]. Recently, IL-6 was described as another ligand for CD5 on human B cells, which leads to the activation of the transcription factor STAT3 via gp130 and Jak2, thereby promoting tumor growth [21]. In the present study, we analyzed a possible role for sCD5 in IL-6 trans-signaling. We show that although IL-6 is able to bind to sCD5, this interaction is much weaker compared to IL-6/sIL-6R interaction and therefore sCD5/IL-6 complexes are not able to provoke a biological response.
http://dx.doi.org/10.1016/j.bbrc.2017.01.174 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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2. Materials and Methods Cells lines e Ba/F3-gp130 and Ba/F3-gp130-hIL-6R cells have been described previously [22]. HepG2 cells were obtained from DSMZ GmbH (Braunschweig, Germany). The cell lines were grown in DMEM high glucose culture medium (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum, penicillin (60 mg/l) and streptomycin (100 mg/l). Cells were cultured in a standard incubator with a water-saturated atmosphere at 37 C and 5% CO2. Ba/F3-gp130 cells were additionally supplemented with 10 ng/ml Hyper-IL-6, which is an IL-6/sIL-6R fusion protein. Ba/F3-gp130-hIL-6R cells were additionally supplemented with 10 ng/ml human IL-6 instead of Hyper-IL-6. Antibodies and proteins e Monoclonal antibodies against signal transducer and activator of transcription-1 (STAT1), STAT3, pSTAT1 and pSTAT3 were purchased from New England Biolabs (Frankfurt am Main, Germany). The b-actin antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). Secondary antibodies IRDye 800CW Goat anti-Mouse and IRDye 680RD Donkey anti-Rabbit were from LICOR Biosciences (Lincoln, NE, USA). Recombinant human soluble CD5 was from R&D Systems (Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany). Hyper-IL-6 was expressed and purified according to [23,24]. Expression and purification of human IL-6 and sIL-6R was described previously [22]. Stimulation and lysis of cells e For cytokine stimulation the individual Ba/F3 cell lines were washed twice in PBS and serumstarved for 2 h at 37 C in DMEM. Subsequently the cells were counted and 2 106 Ba/F3-gp130 or Ba/F3-gp130-hIL-6R cells per well were seeded on a 12-well-plate in 400 ml DMEM. Indicated amounts of IL-6 and sCD5 for each well were mixed in 100 ml DMEM and after preincubation at 37 C for 30 min were added to the cells for 15 min at 37 C. The cell pellet was resuspended in 60 ml Laemmli buffer and boiled. 20 ml of the resulting cell lysates were loaded on a SDS-gel. HepG2 cells were seeded for stimulation at a density of 1 106 cells per well in 6-well plates and after 16 h washed with PBS and serum-starved for 4 h at 37 C in 900 ml DMEM. IL-6 and sCD5 were preincubated and stimulation was done as described for Ba/F3 cells. Cells were scraped on ice and the pellet was lysed in 100 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1% IGEPAL [NP-40], 1% Triton X-100 and complete protease inhibitor cocktail tablets (Roche, Grenzach, Germany)). Western blotting e The protein concentration of HepG2 lysates was determined using BCA protein assay kit (Thermo Fisher scientific, Waltham, MA, USA). 30 mg of total protein were separated on a 10% SDS gel and afterwards transferred onto a nitrocellulose membrane by semi-dry blotting. Membranes were blocked with Odyssey Blocking Buffer (LICOR Biosciences, Lincoln, NE, USA) washed in TBS and incubated overnight at 4 C with antibodies against pSTAT1, STAT1, pSTAT3, STAT3 or b-actin diluted in TBS-T which contained 5% BSA. After three washing steps with TBS-T secondary antibodies IRDye 800CW Goat anti-Mouse and IRDye 680RD Donkey anti-Rabbit were diluted in TBS-T containing 5% BSA and incubated on the membranes for 1 h at room temperature. Following three washing steps fluorescence was detected on an Odyssey Fc Imaging System (LICOR Biosciences, Lincoln, NE, USA). Proliferation assays e Ba/F3-gp130 cells were washed three times with sterile PBS. Afterwards, cells were resuspended in DMEM containing 10% FCS and 1% Penicillin/Streptomycin. After counting, 5 103 cells per well were cultured in a final volume of 100 ml in a 96 well plate with IL-6, sIL-6R and sCD5 as indicated. Cells were incubated for 48 h at 37 C and cell viability was measured using the CellTiter-Blue Cell Viability Assay kit (Promega, Mannheim, Germany) following the manufacturer's instructions.
Values were determined in triplicates per experiment, and relative light units (RLU) obtained after 60 min were normalized by subtraction of control values obtained after 0 min. ELISA e 96 well ELISA plates were coated with either sIL-6R (2 mg/ml), sCD5 (2 mg/ml) or 5% BSA in PBS overnight. Wells were blocked for 1 h with 2% BSA/PBS at room temperature. After addition of IL-6 (0e2000 ng/ml) for 2 h, bound cytokine was determined with a biotinylated anti-IL-6 antibody (ImmunoTools, Friesoythe, Germany). Afterwards, streptavidin-coupled horseradish peroxidase (R&D Systems) was added, and the enzymatic reaction performed with BM blue POD (Roche Applied Science). Absorbance at 450 nm was determined on a Tecan infinite M200 PRO reader (Tecan, Maennedorf, Switzerland). Presentations of experimental data e Data are expressed as mean values ± SD unless indicated otherwise. For Western blot experiments, one experiment from at least three with similar outcome is shown. 3. Results 3.1. IL-6 binds to sCD5 IL-6 binds with high affinity to the IL-6R. Recently, Zhang et al. identified CD5 as a second IL-6-binding receptor, which activates intracellular signaling on B cells in the absence of the IL-6R and constitutes a feed-forward loop to promote tumor growth [21]. In order to confirm this finding, we first coated ELISA plates with recombinant sIL-6R and added increasing amounts of IL-6 (0e2000 ng/ml) to the plates. We used an anti-IL-6 antibody to determine binding of IL-6 to its receptor in a concentrationdependent manner by this approach (Fig. 1A). In order to compare binding of IL-6 to sIL-6R and sCD5, we next coated ELISA plates with recombinant sCD5 and added the same amounts of IL-6 as before. Although IL-6 clearly bound to sCD5 in a concentrationdependent manner, we observed less and weaker binding compared to sIL-6R (Fig. 1B). Thus, we could reproduce binding of IL-6 to CD5 described before [21], but the affinity appeared to be significantly lower compared to IL-6/IL-6R binding. 3.2. IL-6/sCD5 does not induce trans-signaling on Ba/F3-gp130 cells Having confirmed binding of sCD5/IL-6, we sought to investigate whether this complex would be able to activate cells via gp130 like sIL-6R/IL-6 complexes can do. Incubation of Ba/F3-gp130 cells with 10 ng/ml IL-6 and increasing amounts of sIL-6R (0e200 ng/ml) led to a dose-dependent proliferation of the cell line, as shown previously (Fig. 2A, [22,25]). When we incubated Ba/F3-gp130 cells with 10 ng/ml IL-6 and sCD5 (0e200 ng/ml), we did not observe any proliferation of the cell line, indicating that sCD5/IL-6 was not able to induce signaling (Fig. 2B). This was further confirmed by shortterm treatment of Ba/F3-gp130 cells and subsequent analysis of phosphorylation of the downstream transcription factors STAT1 and STAT3. Whereas 10 ng/ml Hyper-IL-6, a designer cytokine where IL-6 is fused to the sIL-6R that mimics IL-6 trans-signaling, led to the phosphorylation of both STAT1 and STAT3, even very high amounts of sCD5 up to 1 mg/ml in combination with 100 ng/ml IL-6 did not induce any STAT phosphorylation (Fig. 2C, D). 3.3. sCD5 does not interfere with IL-6 classic signaling As these first results indicate that sCD5 binds IL-6, but is unable to activate signal transduction in cells expressing gp130, we wanted to analyze whether sCD5 could act as an antagonistic decoy receptor. In IL-6 classic signaling, IL-6 binds to the membrane-bound IL-6R to initiate signaling, and the presence of sCD5 could thus
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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Fig. 1. IL-6 binds to sCD5. (A) ELISA plates were coated with either human sIL-6R or BSA as negative control. Binding of increasing amounts of human IL-6 (0e2000 ng/ml) was determined as described in Materials and Methods. (B) The experiment was performed as described under panel (A), but plates were coated with human sCD5 instead of sIL-6R. One representative experiment out of two performed (n ¼ 3, mean ± SD) is shown.
Fig. 2. sCD5 does not induce agonistic trans-signaling in combination with IL-6. (A) Equal numbers of Ba/F3-gp130 cells were incubated with 10 ng/ml IL-6 and increasing amounts of sIL-6R (0e200 ng/ml) as indicated. Proliferation of the cells was determined 48 h later. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (B) The experiment was performed as described for panel (A), but cells were incubated with increasing amounts of sCD5 instead of sIL-6R as indicated. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (C) Equal amounts of Ba/F3-gp130 cells were serum-starved for two hours, and afterwards stimulated for 15 min with IL-6/sCD5 at the indicated concentrations. Stimulation with 10 ng/ml Hyper-IL-6 was used as positive control, and phosphorylation of STAT1 was analyzed by Western blot. bactin was used to ensure equal protein loading. One representative experiment out of three performed is shown. (D) The experiment was performed as described for panel (C), but phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 and b-actin were used to ensure equal protein loading. One representative experiment out of three performed is shown.
compete for ligand binding and thereby reduce cellular proliferation. As a control, we added high amounts (10e100 ng/ml) of the agonistic sIL-6R to Ba/F3-gp130-hIL-6R cells stimulated with 10 ng/ ml, and as expected could not detect any influence of the sIL-6R (Fig. 3A). Interestingly, addition of sCD5 up to 1 mg/ml did not reduce IL-6 classic signaling on Ba/F3-gp130-hIL-6R (Fig. 3B), and we could also not detect any inhibitory influence of sCD5 on the phosphorylation of STAT3 when we stimulated Ba/F3-gp130-hIL-6R cells with IL-6 (Fig. 3C). In summary, our results indicate that sCD5 does not act as a decoy receptor that sequesters IL-6 classic signaling. 3.4. sCD5 does not significantly modulate IL-6 signaling on cells expressing endogenous IL-6R The previous experiments have been performed with rather artificial cell lines that overexpress gp130 either alone or in
combination with the human IL-6R. To exclude that this overexpression has an influence on our results, we performed similar experiments with the human hepatoma cell line HepG2, which expresses both gp130 and IL-6R endogenously at lower levels compared to the Ba/F3-gp130-IL-6R cells. Although we could detect a small increase in STAT1 phosphorylation when cells were stimulated with 10 ng/ml IL-6 in combination with sCD5 compared to cells stimulated with IL-6 alone (Fig. 4A), this subtle difference was not detectable for STAT3 phosphorylation (Fig. 4B). Furthermore, when we stimulated HepG2 cells with higher IL-6 concentrations of 20 ng/ml with or without the addition of sCD5, we could detect neither an influence on STAT1 nor of STAT3 phosphorylation (Fig. 4C, D). This indicates that sCD5 also does not act as an agonistic or antagonistic modulator of IL-6 signaling when cells express endogenous level of IL-6. In conclusion, our results indicate that despite an interaction between IL-6 and sCD5, the affinity between the two proteins is too low to trigger a biological response.
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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Fig. 3. sCD5 does not block IL-6 classic signaling. (A) Equal amounts of Ba/F3-gp130-hIL-6R cells were incubated with 10 ng/ml IL-6 and different amounts of sIL-6R (10e1000 ng/ ml) as indicated. Proliferation of the cells was determined 48 h later. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (B) The experiment was performed as described for panel (A), but cells were incubated with increasing amounts of sCD5 instead of sIL-6R as indicated. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (C) Equal amounts of Ba/F3-gp130-hIL-6R cells were serum-starved for two hours, and afterwards stimulated for 15 min with IL-6/sCD5 at the indicated concentrations. Phosphorylation of STAT3 was analyzed by Western blot. b-actin and total STAT3 were used to ensure equal protein loading. One representative experiment out of three performed is shown.
4. Discussion
Fig. 4. sCD5 does not modulate IL-6 signaling on HepG2 cells. (AeD) Equal amounts of HepG2 cells were serum-starved for four hours, and afterwards stimulated for 15 min with IL-6/sCD5 at the indicated concentrations. Phosphorylation of STAT1 or STAT3 was analyzed by Western blot. b-actin and total STAT1 or total STAT3 were used to ensure equal protein loading. One representative experiment out of three performed is shown.
The biological activities of cytokines like IL-6 have to be tightly controlled, because overshooting IL-6 signaling contributes critically to inflammation and cancer development in a STAT3dependent manner [26]. This confinement is achieved by cellspecific and restricted expression of the IL-6R, which limits the actions of IL-6 to certain target cells. Interestingly, a recent report described the scavenger receptor CD5 as a second receptor for IL-6 on B cells, which can perform IL-6 dependent signaling in the absence of the IL-6R [21]. Like the IL-6R, binding of IL-6 to CD5 induced homodimerization of gp130, which results in phosphorylation of the transcription factor STAT3 and subsequent cell proliferation thereby promoting cancer development [21]. Because the IL-6R is not expressed on all B cell subtypes [27], IL-6 signaling via CD5 significantly widens the spectrum of B cells that can be activated by IL-6. The biology of IL-6 is further complicated by the existence of sIL6R forms and the fact that IL-6/sIL-6R complexes act agonistically due to activation of gp130 on cells irrespective whether these express the IL-6R themselves. Because this trans-signaling pathway is believed to be causative for the pro-inflammatory activities of IL-6 [5,6], the existence of sCD5 in human serum opens up the possibility that sCD5 could act in the same manner as sIL-6R. In human sepsis patients, sCD5 levels are associated with higher mortality [28]. Furthermore, sCD5 appears to contribute to the pathogenesis of atopic dermatitis [29]. However, our results show that the affinity of IL-6 towards sCD5 is much lower compared to sIL-6R, and the resulting IL-6/sCD5 complexes are not able to induce STAT3 activation via gp130 on cells. This resembles our previous study where we could show that IL-30, the cytokine subunit of IL-27, was able to signal via membrane-bound IL-6R, but the affinity was too low to perform trans-signaling via the sIL-6R [30]. We found,
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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however, that a fusion protein of sIL-6R and IL-30, where both proteins are connected via a flexible peptide linker, induced cell proliferation and STAT3 phosphorylation, which might also be the case for sCD5/IL-6. Soluble cytokine receptors often act as antagonistic decoy receptors, and soluble receptors of tumor necrosis factor are prominent examples with therapeutic implications [31]. Therefore, we tested whether binding of IL-6 to sCD5 could potentially affect IL-6 classic signaling. However, we again could not detect any significant influence on cell proliferation and STAT3 activation. In summary, our results show that IL-6 trans-signaling is exclusively mediated by sIL-6R, but not by sCD5, and that sCD5 in vivo is unlikely to play an important regulatory role in IL-6 classic or transsignaling. Acknowledgments This work was funded by grants from the Bundesministerium für Bildung und Forschung (BMBF grant “InTraSig”, project B to C.G.), and by the Cluster of Excellence ‘Inflammation at Interfaces’ of the Deutsche Forschungsgemeinschaft (DFG). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.01.174. References €tzinger, S. Rose[1] C. Garbers, H. Hermanns, F. Schaper, G. Müller-Newen, J. Gro John, J. Scheller, Plasticity and cross-talk of Interleukin 6-type cytokines, Cytokine Growth Factor Rev. 23 (2012) 85e97. [2] J. Lokau, C. Garbers, Signal transduction of interleukin-11 and interleukin-6 areceptors, Recept. Clin. Investig. 3 (2016). [3] J. Wolf, S. Rose-John, C. Garbers, Interleukin-6 and its receptors: a highly regulated and dynamic system, Cytokine 70 (2014) 11e20. [4] C. Garbers, S. Aparicio-Siegmund, S. Rose-John, The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition, Curr. Opin. Immunol. 34 (2015) 75e82. [5] A. Chalaris, C. Garbers, B. Rabe, S. Rose-John, J. Scheller, The soluble Interleukin 6 receptor: generation and role in inflammation and cancer, Eur. J. Cell. Biol. 90 (2011) 484e494. [6] S.A. Jones, J. Scheller, S. Rose-John, Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling, J. Clin. Investig. 121 (2011) 3375e3383. [7] V. Matthews, B. Schuster, S. Schutze, I. Bussmeyer, A. Ludwig, C. Hundhausen, T. Sadowski, P. Saftig, D. Hartmann, K.J. Kallen, S. Rose-John, Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE), J. Biol. Chem. 278 (2003) 38829e38839. €nner, A. Chalaris, M.L. Moss, D.M. Floss, D. Meyer, F. Koch[8] C. Garbers, N. Ja Nolte, S. Rose-John, J. Scheller, Species specificity of ADAM10 and ADAM17 proteins in interleukin-6 (IL-6) trans-signaling and novel role of ADAM10 in inducible IL-6 receptor shedding, J. Biol. Chem. 286 (2011) 14804e14811. [9] J. Müllberg, H. Schooltink, T. Stoyan, M. Günther, L. Graeve, G. Buse, A. Mackiewicz, P. Heinrich, S. Rose-John, The soluble interleukin-6 receptor is generated by shedding, Eur. J. Immunol. 23 (1993) 473e480. [10] J. Lokau, R. Nitz, M. Agthe, N. Monhasery, S. Aparicio-Siegmund, € ller-Hackbarth, G.H. Waetzig, J. Gro € tzinger, N. Schumacher, J. Wolf, K. Mo G. Müller-Newen, S. Rose-John, J. Scheller, C. Garbers, Proteolytic cleavage governs interleukin-11 trans-signaling, Cell. Rep. 14 (2016) 1761e1773. €ft, K. Knittler, G. Dombrowsky, [11] S. Riethmueller, J.C. Ehlers, J. Lokau, S. Düsterho € tzinger, B. Rabe, S. Rose-John, C. Garbers, Cleavage site localization J. Gro differentially controls interleukin-6 receptor proteolysis by ADAM10 and ADAM17, Sci. Rep. 6 (2016) 25550.
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Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5 Samadhi Aparicio-Siegmund, Malte Deseke, Annett Lickert, Christoph Garbers* Institute of Biochemistry, Kiel University, 24118 Kiel, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2017 Accepted 28 January 2017 Available online xxx
IL-6 exerts its pleiotropic activities on its target cells via the IL-6 alpha-receptor (IL-6R), which is expressed on a limited number of cell types. IL-6 can further signal via soluble forms of its receptor (sIL6R), a process that has been termed trans-signaling. Recently, CD5 was described as an alternative alphareceptor for IL-6 on B cells leading to the phosphorylation of the transcription factor STAT3 via the signaltransducing b-receptor gp130 in a Jak2-dependent manner. In this study, we sought to investigate whether IL-6 was also able to signal via soluble CD5 (sCD5) analogous to IL-6 trans-signaling. We show that IL-6 indeed binds to sCD5, but that this does not lead to the activation of signal transduction or cell proliferation. Furthermore, sCD5 did also not interfere with IL-6 classic signaling, suggesting that the affinity between the two proteins was too weak to provoke a biological effect. Thus, trans-signaling of IL6 can only occur via sIL-6R, but not sCD5. © 2017 Elsevier Inc. All rights reserved.
Keywords: Interleukin-6 CD5 STAT3 Trans-signaling
1. Introduction Signaling via the ubiquitously expressed glycoprotein 130 (gp130) is a hallmark of all cytokines of the interleukin-6 (IL-6) family [1]. Specificity is gained through cell-dependent expression of other signaling and non-signaling receptors. Binding of IL-6 to its non-signaling IL-6 a-receptor (IL-6R), so-called classic signaling, leads to gp130 homodimerization and the activation of intracellular signaling cascades, including the Jak/STAT pathway [2]. Enhanced activities and increased serum levels of IL-6 are associated with inflammatory human diseases [3,4]. Most soluble cytokine receptors function as antagonistic decoy receptors, but the soluble IL-6R (sIL-6R) is a rare example of an agonist [5]. IL-6 binds to sIL-6R with the same affinity as to the membrane-bound IL-6R, and sIL-6R/IL-6 complexes can activate all cells of the human body via a gp130 homodimer. This mode of IL-6 signaling has been termed trans-signaling and represents an attractive therapeutic target [4,6]. The majority of the sIL-6R is generated by proteolytic cleavage of the membrane-bound precursor, and the two metalloproteases ADAM10 and ADAM17 have been described as efficient sheddases of the IL-6R [7e13]. Recently,
* Corresponding author. Institute of Biochemistry, Kiel University, Olshausenstrasse 40, 24118 Kiel, Germany. E-mail address: [email protected] (C. Garbers).
a third modality of IL-6 signaling has been described, in which IL-6 couples to the IL-6R intracellularly and is then transported to the cell surface, where it stimulates gp130 on a neighboring cell. This pathway has been termed IL-6 cluster signaling/trans-presentation [14]. CD5 is a type I transmembrane protein that belongs to the scavenger receptor cysteine-rich (SRCR) superfamily [15,16]. It consists of three extracellular domains and was initially used as a cell surface marker for T cells; however, it is also expressed on B cells [16]. CD5 has an important role in T cell receptor signaling [17] and can be activated by a variety of different ligands, including zymosan, gp150, gp77-80, gp35-70 and CD5 itself [16,18]. Different forms of CD5 are generated by alternative processing of its mRNA [19], and naturally occurring soluble forms of CD5 (sCD5) appear to be generated by proteolytic cleavage of membrane-anchored CD5 [20]. Recently, IL-6 was described as another ligand for CD5 on human B cells, which leads to the activation of the transcription factor STAT3 via gp130 and Jak2, thereby promoting tumor growth [21]. In the present study, we analyzed a possible role for sCD5 in IL-6 trans-signaling. We show that although IL-6 is able to bind to sCD5, this interaction is much weaker compared to IL-6/sIL-6R interaction and therefore sCD5/IL-6 complexes are not able to provoke a biological response.
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Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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2. Materials and Methods Cells lines e Ba/F3-gp130 and Ba/F3-gp130-hIL-6R cells have been described previously [22]. HepG2 cells were obtained from DSMZ GmbH (Braunschweig, Germany). The cell lines were grown in DMEM high glucose culture medium (Gibco/Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum, penicillin (60 mg/l) and streptomycin (100 mg/l). Cells were cultured in a standard incubator with a water-saturated atmosphere at 37 C and 5% CO2. Ba/F3-gp130 cells were additionally supplemented with 10 ng/ml Hyper-IL-6, which is an IL-6/sIL-6R fusion protein. Ba/F3-gp130-hIL-6R cells were additionally supplemented with 10 ng/ml human IL-6 instead of Hyper-IL-6. Antibodies and proteins e Monoclonal antibodies against signal transducer and activator of transcription-1 (STAT1), STAT3, pSTAT1 and pSTAT3 were purchased from New England Biolabs (Frankfurt am Main, Germany). The b-actin antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). Secondary antibodies IRDye 800CW Goat anti-Mouse and IRDye 680RD Donkey anti-Rabbit were from LICOR Biosciences (Lincoln, NE, USA). Recombinant human soluble CD5 was from R&D Systems (Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany). Hyper-IL-6 was expressed and purified according to [23,24]. Expression and purification of human IL-6 and sIL-6R was described previously [22]. Stimulation and lysis of cells e For cytokine stimulation the individual Ba/F3 cell lines were washed twice in PBS and serumstarved for 2 h at 37 C in DMEM. Subsequently the cells were counted and 2 106 Ba/F3-gp130 or Ba/F3-gp130-hIL-6R cells per well were seeded on a 12-well-plate in 400 ml DMEM. Indicated amounts of IL-6 and sCD5 for each well were mixed in 100 ml DMEM and after preincubation at 37 C for 30 min were added to the cells for 15 min at 37 C. The cell pellet was resuspended in 60 ml Laemmli buffer and boiled. 20 ml of the resulting cell lysates were loaded on a SDS-gel. HepG2 cells were seeded for stimulation at a density of 1 106 cells per well in 6-well plates and after 16 h washed with PBS and serum-starved for 4 h at 37 C in 900 ml DMEM. IL-6 and sCD5 were preincubated and stimulation was done as described for Ba/F3 cells. Cells were scraped on ice and the pellet was lysed in 100 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1% IGEPAL [NP-40], 1% Triton X-100 and complete protease inhibitor cocktail tablets (Roche, Grenzach, Germany)). Western blotting e The protein concentration of HepG2 lysates was determined using BCA protein assay kit (Thermo Fisher scientific, Waltham, MA, USA). 30 mg of total protein were separated on a 10% SDS gel and afterwards transferred onto a nitrocellulose membrane by semi-dry blotting. Membranes were blocked with Odyssey Blocking Buffer (LICOR Biosciences, Lincoln, NE, USA) washed in TBS and incubated overnight at 4 C with antibodies against pSTAT1, STAT1, pSTAT3, STAT3 or b-actin diluted in TBS-T which contained 5% BSA. After three washing steps with TBS-T secondary antibodies IRDye 800CW Goat anti-Mouse and IRDye 680RD Donkey anti-Rabbit were diluted in TBS-T containing 5% BSA and incubated on the membranes for 1 h at room temperature. Following three washing steps fluorescence was detected on an Odyssey Fc Imaging System (LICOR Biosciences, Lincoln, NE, USA). Proliferation assays e Ba/F3-gp130 cells were washed three times with sterile PBS. Afterwards, cells were resuspended in DMEM containing 10% FCS and 1% Penicillin/Streptomycin. After counting, 5 103 cells per well were cultured in a final volume of 100 ml in a 96 well plate with IL-6, sIL-6R and sCD5 as indicated. Cells were incubated for 48 h at 37 C and cell viability was measured using the CellTiter-Blue Cell Viability Assay kit (Promega, Mannheim, Germany) following the manufacturer's instructions.
Values were determined in triplicates per experiment, and relative light units (RLU) obtained after 60 min were normalized by subtraction of control values obtained after 0 min. ELISA e 96 well ELISA plates were coated with either sIL-6R (2 mg/ml), sCD5 (2 mg/ml) or 5% BSA in PBS overnight. Wells were blocked for 1 h with 2% BSA/PBS at room temperature. After addition of IL-6 (0e2000 ng/ml) for 2 h, bound cytokine was determined with a biotinylated anti-IL-6 antibody (ImmunoTools, Friesoythe, Germany). Afterwards, streptavidin-coupled horseradish peroxidase (R&D Systems) was added, and the enzymatic reaction performed with BM blue POD (Roche Applied Science). Absorbance at 450 nm was determined on a Tecan infinite M200 PRO reader (Tecan, Maennedorf, Switzerland). Presentations of experimental data e Data are expressed as mean values ± SD unless indicated otherwise. For Western blot experiments, one experiment from at least three with similar outcome is shown. 3. Results 3.1. IL-6 binds to sCD5 IL-6 binds with high affinity to the IL-6R. Recently, Zhang et al. identified CD5 as a second IL-6-binding receptor, which activates intracellular signaling on B cells in the absence of the IL-6R and constitutes a feed-forward loop to promote tumor growth [21]. In order to confirm this finding, we first coated ELISA plates with recombinant sIL-6R and added increasing amounts of IL-6 (0e2000 ng/ml) to the plates. We used an anti-IL-6 antibody to determine binding of IL-6 to its receptor in a concentrationdependent manner by this approach (Fig. 1A). In order to compare binding of IL-6 to sIL-6R and sCD5, we next coated ELISA plates with recombinant sCD5 and added the same amounts of IL-6 as before. Although IL-6 clearly bound to sCD5 in a concentrationdependent manner, we observed less and weaker binding compared to sIL-6R (Fig. 1B). Thus, we could reproduce binding of IL-6 to CD5 described before [21], but the affinity appeared to be significantly lower compared to IL-6/IL-6R binding. 3.2. IL-6/sCD5 does not induce trans-signaling on Ba/F3-gp130 cells Having confirmed binding of sCD5/IL-6, we sought to investigate whether this complex would be able to activate cells via gp130 like sIL-6R/IL-6 complexes can do. Incubation of Ba/F3-gp130 cells with 10 ng/ml IL-6 and increasing amounts of sIL-6R (0e200 ng/ml) led to a dose-dependent proliferation of the cell line, as shown previously (Fig. 2A, [22,25]). When we incubated Ba/F3-gp130 cells with 10 ng/ml IL-6 and sCD5 (0e200 ng/ml), we did not observe any proliferation of the cell line, indicating that sCD5/IL-6 was not able to induce signaling (Fig. 2B). This was further confirmed by shortterm treatment of Ba/F3-gp130 cells and subsequent analysis of phosphorylation of the downstream transcription factors STAT1 and STAT3. Whereas 10 ng/ml Hyper-IL-6, a designer cytokine where IL-6 is fused to the sIL-6R that mimics IL-6 trans-signaling, led to the phosphorylation of both STAT1 and STAT3, even very high amounts of sCD5 up to 1 mg/ml in combination with 100 ng/ml IL-6 did not induce any STAT phosphorylation (Fig. 2C, D). 3.3. sCD5 does not interfere with IL-6 classic signaling As these first results indicate that sCD5 binds IL-6, but is unable to activate signal transduction in cells expressing gp130, we wanted to analyze whether sCD5 could act as an antagonistic decoy receptor. In IL-6 classic signaling, IL-6 binds to the membrane-bound IL-6R to initiate signaling, and the presence of sCD5 could thus
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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Fig. 1. IL-6 binds to sCD5. (A) ELISA plates were coated with either human sIL-6R or BSA as negative control. Binding of increasing amounts of human IL-6 (0e2000 ng/ml) was determined as described in Materials and Methods. (B) The experiment was performed as described under panel (A), but plates were coated with human sCD5 instead of sIL-6R. One representative experiment out of two performed (n ¼ 3, mean ± SD) is shown.
Fig. 2. sCD5 does not induce agonistic trans-signaling in combination with IL-6. (A) Equal numbers of Ba/F3-gp130 cells were incubated with 10 ng/ml IL-6 and increasing amounts of sIL-6R (0e200 ng/ml) as indicated. Proliferation of the cells was determined 48 h later. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (B) The experiment was performed as described for panel (A), but cells were incubated with increasing amounts of sCD5 instead of sIL-6R as indicated. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (C) Equal amounts of Ba/F3-gp130 cells were serum-starved for two hours, and afterwards stimulated for 15 min with IL-6/sCD5 at the indicated concentrations. Stimulation with 10 ng/ml Hyper-IL-6 was used as positive control, and phosphorylation of STAT1 was analyzed by Western blot. bactin was used to ensure equal protein loading. One representative experiment out of three performed is shown. (D) The experiment was performed as described for panel (C), but phosphorylation of STAT3 was analyzed by Western blot. Total STAT3 and b-actin were used to ensure equal protein loading. One representative experiment out of three performed is shown.
compete for ligand binding and thereby reduce cellular proliferation. As a control, we added high amounts (10e100 ng/ml) of the agonistic sIL-6R to Ba/F3-gp130-hIL-6R cells stimulated with 10 ng/ ml, and as expected could not detect any influence of the sIL-6R (Fig. 3A). Interestingly, addition of sCD5 up to 1 mg/ml did not reduce IL-6 classic signaling on Ba/F3-gp130-hIL-6R (Fig. 3B), and we could also not detect any inhibitory influence of sCD5 on the phosphorylation of STAT3 when we stimulated Ba/F3-gp130-hIL-6R cells with IL-6 (Fig. 3C). In summary, our results indicate that sCD5 does not act as a decoy receptor that sequesters IL-6 classic signaling. 3.4. sCD5 does not significantly modulate IL-6 signaling on cells expressing endogenous IL-6R The previous experiments have been performed with rather artificial cell lines that overexpress gp130 either alone or in
combination with the human IL-6R. To exclude that this overexpression has an influence on our results, we performed similar experiments with the human hepatoma cell line HepG2, which expresses both gp130 and IL-6R endogenously at lower levels compared to the Ba/F3-gp130-IL-6R cells. Although we could detect a small increase in STAT1 phosphorylation when cells were stimulated with 10 ng/ml IL-6 in combination with sCD5 compared to cells stimulated with IL-6 alone (Fig. 4A), this subtle difference was not detectable for STAT3 phosphorylation (Fig. 4B). Furthermore, when we stimulated HepG2 cells with higher IL-6 concentrations of 20 ng/ml with or without the addition of sCD5, we could detect neither an influence on STAT1 nor of STAT3 phosphorylation (Fig. 4C, D). This indicates that sCD5 also does not act as an agonistic or antagonistic modulator of IL-6 signaling when cells express endogenous level of IL-6. In conclusion, our results indicate that despite an interaction between IL-6 and sCD5, the affinity between the two proteins is too low to trigger a biological response.
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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Fig. 3. sCD5 does not block IL-6 classic signaling. (A) Equal amounts of Ba/F3-gp130-hIL-6R cells were incubated with 10 ng/ml IL-6 and different amounts of sIL-6R (10e1000 ng/ ml) as indicated. Proliferation of the cells was determined 48 h later. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (B) The experiment was performed as described for panel (A), but cells were incubated with increasing amounts of sCD5 instead of sIL-6R as indicated. One representative experiment out of three performed (n ¼ 3, mean ± SD) is shown. (C) Equal amounts of Ba/F3-gp130-hIL-6R cells were serum-starved for two hours, and afterwards stimulated for 15 min with IL-6/sCD5 at the indicated concentrations. Phosphorylation of STAT3 was analyzed by Western blot. b-actin and total STAT3 were used to ensure equal protein loading. One representative experiment out of three performed is shown.
4. Discussion
Fig. 4. sCD5 does not modulate IL-6 signaling on HepG2 cells. (AeD) Equal amounts of HepG2 cells were serum-starved for four hours, and afterwards stimulated for 15 min with IL-6/sCD5 at the indicated concentrations. Phosphorylation of STAT1 or STAT3 was analyzed by Western blot. b-actin and total STAT1 or total STAT3 were used to ensure equal protein loading. One representative experiment out of three performed is shown.
The biological activities of cytokines like IL-6 have to be tightly controlled, because overshooting IL-6 signaling contributes critically to inflammation and cancer development in a STAT3dependent manner [26]. This confinement is achieved by cellspecific and restricted expression of the IL-6R, which limits the actions of IL-6 to certain target cells. Interestingly, a recent report described the scavenger receptor CD5 as a second receptor for IL-6 on B cells, which can perform IL-6 dependent signaling in the absence of the IL-6R [21]. Like the IL-6R, binding of IL-6 to CD5 induced homodimerization of gp130, which results in phosphorylation of the transcription factor STAT3 and subsequent cell proliferation thereby promoting cancer development [21]. Because the IL-6R is not expressed on all B cell subtypes [27], IL-6 signaling via CD5 significantly widens the spectrum of B cells that can be activated by IL-6. The biology of IL-6 is further complicated by the existence of sIL6R forms and the fact that IL-6/sIL-6R complexes act agonistically due to activation of gp130 on cells irrespective whether these express the IL-6R themselves. Because this trans-signaling pathway is believed to be causative for the pro-inflammatory activities of IL-6 [5,6], the existence of sCD5 in human serum opens up the possibility that sCD5 could act in the same manner as sIL-6R. In human sepsis patients, sCD5 levels are associated with higher mortality [28]. Furthermore, sCD5 appears to contribute to the pathogenesis of atopic dermatitis [29]. However, our results show that the affinity of IL-6 towards sCD5 is much lower compared to sIL-6R, and the resulting IL-6/sCD5 complexes are not able to induce STAT3 activation via gp130 on cells. This resembles our previous study where we could show that IL-30, the cytokine subunit of IL-27, was able to signal via membrane-bound IL-6R, but the affinity was too low to perform trans-signaling via the sIL-6R [30]. We found,
Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174
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however, that a fusion protein of sIL-6R and IL-30, where both proteins are connected via a flexible peptide linker, induced cell proliferation and STAT3 phosphorylation, which might also be the case for sCD5/IL-6. Soluble cytokine receptors often act as antagonistic decoy receptors, and soluble receptors of tumor necrosis factor are prominent examples with therapeutic implications [31]. Therefore, we tested whether binding of IL-6 to sCD5 could potentially affect IL-6 classic signaling. However, we again could not detect any significant influence on cell proliferation and STAT3 activation. In summary, our results show that IL-6 trans-signaling is exclusively mediated by sIL-6R, but not by sCD5, and that sCD5 in vivo is unlikely to play an important regulatory role in IL-6 classic or transsignaling. Acknowledgments This work was funded by grants from the Bundesministerium für Bildung und Forschung (BMBF grant “InTraSig”, project B to C.G.), and by the Cluster of Excellence ‘Inflammation at Interfaces’ of the Deutsche Forschungsgemeinschaft (DFG). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.01.174. References €tzinger, S. Rose[1] C. Garbers, H. Hermanns, F. Schaper, G. Müller-Newen, J. Gro John, J. Scheller, Plasticity and cross-talk of Interleukin 6-type cytokines, Cytokine Growth Factor Rev. 23 (2012) 85e97. [2] J. Lokau, C. Garbers, Signal transduction of interleukin-11 and interleukin-6 areceptors, Recept. Clin. Investig. 3 (2016). [3] J. Wolf, S. Rose-John, C. Garbers, Interleukin-6 and its receptors: a highly regulated and dynamic system, Cytokine 70 (2014) 11e20. [4] C. Garbers, S. Aparicio-Siegmund, S. Rose-John, The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition, Curr. Opin. Immunol. 34 (2015) 75e82. [5] A. Chalaris, C. Garbers, B. Rabe, S. Rose-John, J. Scheller, The soluble Interleukin 6 receptor: generation and role in inflammation and cancer, Eur. J. Cell. Biol. 90 (2011) 484e494. [6] S.A. Jones, J. Scheller, S. Rose-John, Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling, J. Clin. Investig. 121 (2011) 3375e3383. [7] V. Matthews, B. Schuster, S. Schutze, I. Bussmeyer, A. Ludwig, C. Hundhausen, T. Sadowski, P. Saftig, D. Hartmann, K.J. Kallen, S. Rose-John, Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE), J. Biol. Chem. 278 (2003) 38829e38839. €nner, A. Chalaris, M.L. Moss, D.M. Floss, D. Meyer, F. Koch[8] C. Garbers, N. Ja Nolte, S. Rose-John, J. Scheller, Species specificity of ADAM10 and ADAM17 proteins in interleukin-6 (IL-6) trans-signaling and novel role of ADAM10 in inducible IL-6 receptor shedding, J. Biol. Chem. 286 (2011) 14804e14811. [9] J. Müllberg, H. Schooltink, T. Stoyan, M. Günther, L. Graeve, G. Buse, A. Mackiewicz, P. Heinrich, S. Rose-John, The soluble interleukin-6 receptor is generated by shedding, Eur. J. Immunol. 23 (1993) 473e480. [10] J. Lokau, R. Nitz, M. Agthe, N. Monhasery, S. Aparicio-Siegmund, € ller-Hackbarth, G.H. Waetzig, J. Gro € tzinger, N. Schumacher, J. Wolf, K. Mo G. Müller-Newen, S. Rose-John, J. Scheller, C. Garbers, Proteolytic cleavage governs interleukin-11 trans-signaling, Cell. Rep. 14 (2016) 1761e1773. €ft, K. Knittler, G. Dombrowsky, [11] S. Riethmueller, J.C. Ehlers, J. Lokau, S. Düsterho € tzinger, B. Rabe, S. Rose-John, C. Garbers, Cleavage site localization J. Gro differentially controls interleukin-6 receptor proteolysis by ADAM10 and ADAM17, Sci. Rep. 6 (2016) 25550.
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Please cite this article in press as: S. Aparicio-Siegmund, et al., Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.01.174