Granulocytes Are Unresponsive to IL-6 Due to an Absence of Gp130

Published April 6, 2018, doi:10.4049/jimmunol.1701191 The Journal of Immunology

Granulocytes Are Unresponsive to IL-6 Due to an Absence of gp130

Andrew N. Wilkinson,*,† Kate H. Gartlan,*,† Greg Kelly,* Luke D. Samson,* Stuart D. Olver,* Judy Avery,*,‡ Nienke Zomerdijk,*,‡ Siok-Keen Tey,*,‡ Jason S. Lee,* Slavica Vuckovic,*,†,1 and Geoffrey R. Hill*,†,‡,1

IL-6 mediates broad physiological and pathological effects through its receptor signal transducing unit gp130. Due to the reportedly wide cellular expression of gp130, IL-6 is thought to signal ubiquitously via gp130 complex formation with membrane-bound IL-6Ra or soluble IL-6Ra. gp130 signaling primarily induces p-STAT3 and p-STAT1. In contrast to the previous dogma, we show in this article that circulating mouse and human granulocytes are unable to induce p-STAT3 or p-STAT1 after stimulation with IL-6 or an IL-6/soluble IL-6R complex. Furthermore, we demonstrate that this is due to a lack of gp130 expression on mouse and human granulocytes, despite their expression of membrane-bound IL-6R. Importantly, the absence of gp130 is not only a feature of mature granulocytes in healthy individuals, it is also observed after allogeneic stem cell transplantation. Moreover, granulocyte gp130 expression is lost during maturation, because granulocyte-monocyte progenitor cells express gp130 and respond to IL-6. Given that granulocytes constitute 50–70% of circulating leukocytes, this indicates a signiﬁcantly smaller scope of IL-6 signaling than previously anticipated and has important implications for therapeutic IL-6 inhibition and the mechanisms of action thereof. The Journal of Immunology, 2018, 200: 000–000.

nterleukin-6 is a pleiotropic cytokine that is capable of driving signaling pathways, that use a receptor system composed of the immunity to infection, hematopoiesis, and inflammation (1). ligand-specific IL-6Ra and the common gp130 signal transducer I Dysregulation of IL-6 is linked to many chronic inflamma- (1, 8, 9). tory and autoimmune diseases, which has led to its targeted in- IL-6 classical signaling requires IL-6Ra and gp130 expression hibition in the clinic (2). Inhibition of IL-6 responses with by the target cell and is driven by extracellular IL-6 binding to tocilizumab (TCZ), a humanized anti–IL-6Ra Ab, has been suc- membrane-bound IL-6Ra (mIL-6Ra), inducing gp130 recruit- cessfully used to treat patients with rheumatoid arthritis (RA), ment and homodimerization. Alternatively, IL-6 trans and cluster juvenile idiopathic arthritis, Castleman disease, and cytokine re- signaling requires only gp130 expression by the target cell, and lease syndrome (2–6). It also appears to be a promising approach IL-6/IL-6Ra complexes are formed externally. During trans sig- to prevent acute graft-versus-host disease (7). IL-6 mediates its naling, IL-6 binds to soluble (s)IL-6Ra that interacts with gp130 broad biological activities through versatile signaling mecha- expressed by the target cell. Cluster signaling has been proposed nisms, including classical, trans, and the recently described cluster in dendritic cell (DC)–T cell interactions, such that IL-6/IL-6Ra complexes are formed intracellularly by DCs prior to trafficking to *Immunology Department, QIMR Berghofer Medical Research Institute, Brisbane, the cell surface and subsequent interaction with gp130 expressed † Queensland 4006, Australia; School of Medicine, University of Queensland, by target T cells within the immunological synapse (8, 9). Herston, Queensland 4006, Australia; and ‡Royal Brisbane and Women’s Hospital, Brisbane, Queensland 4029, Australia All IL-6 signaling pathways result in gp130 homodimerization 1S.V. and G.R.H. contributed equally to this work. and phosphorylation of STAT3 and STAT1 proteins, which translocate to the nucleus to induce transcriptional modification of ORCIDs: 0000-0001-5008-6429 (N.Z.); 0000-0001-9567-382X (S.-K.T.); 0000- 0003-0879-934X (J.S.L.). target genes (10). Because gp130 is reported to be ubiquitously Received for publication August 18, 2017. Accepted for publication March 9, 2018. expressed (11–13), IL-6 trans signaling has been proposed to This work was supported by research grants from the National Health and Medical widen the spectrum of IL-6 to virtually all cells in the body, and a Research Council (Australia). G.R.H. is a National Health and Medical Research few studies have confirmed the direct ability of human leukocyte Council Senior Principal Research Fellow. populations to respond to IL-6 (14). Moreover, the effects of IL-6 A.N.W. and K.H.G. designed and performed experiments and wrote the manuscript; on neutrophil function has been unclear, with conflicting reports G.K., L.D.S., and S.D.O. performed experiments; J.A., N.Z., and S.-K.T. coordinated and collected the patient samples and data; J.S.L. designed experiments and provided regarding its influence on neutrophil apoptosis and downstream essential reagents; S.V. designed and performed experiments and helped to write the signaling (15–20). In this article, we demonstrate that gran- manuscript; and G.R.H. designed experiments and helped to write the manuscript. ulocytes (CD66b+Gr; neutrophils and eosinophils) are unrespon- Address correspondence and reprint requests to Prof. Geoffrey R. Hill, Bone Marrow sive to IL-6 classical and trans signaling due to their absence of Transplantation Laboratory, QIMR Berghofer Medical Research Institute, Level 9 CBCRC, 300 Herston Road, Herston, QLD 4006, Australia. E-mail address: Geoff. gp130 expression. Given that granulocytes constitute 50–70% of [email protected] circulating leukocytes, this demonstrates that the targets of IL-6 Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; allo-SCT, alloge- signaling are somewhat more restricted than previously thought. neic stem cell transplantation; BM, bone marrow; CMP, common myeloid progenitor; DC, dendritic cell; GMFI, geometric mean fluorescent intensity; H-IL-6, hyper–IL-6; HSPC, hematopoietic stem progenitor cell; Lin, lineage; LSK, Lin2Sca-1+c-kit+; Materials and Methods MEP, megakaryocyte-erythroid progenitor; mIL-6Ra, membrane-bound IL-6Ra; Samples qPCR, quantitative PCR; RA, rheumatoid arthritis; s, soluble; TCZ, tocilizumab. Peripheral blood was collected from healthy adult donors (n = 10) or clinical Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 trial patients (day 60 and 90 after allogeneic stem cell transplantation

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701191 2 GRANULOCYTES LACK gp130

[allo-SCT], ACTRN12614000266662, n = 28). Apheresis products were Biosciences) by magnetic separation (CD66abce MicroBeads Kit human; collected from G-CSF–mobilized stem cell donors for allo-SCT (G-CSF Miltenyi Biotec) and further purified by FACS (BD FACS Aria, .99% at 10 mg/kg daily for 4 or 5 d prior to collection). All mouse studies used granulocyte purity). Monocytes, CD4+ T cells, and CD8+ T cells were 8–12-wk-old female C57BL/6 mice that were purchased from the Animal collected by Ficoll-Paque density gradient of peripheral blood and sort Resources Center (Perth, WA, Australia). All studies were approved by the purified (BD FACSAria). In all sort protocols, cells were maintained at institutional ethics committees, and all patients signed informed consent. 4˚C, and dead cells were excluded by 7-aminoactinomycin D (7AAD) staining. mAbs Abs used in flow cytometry and high-throughput imaging were purchased Granulocyte (neutrophil) purification from BioLegend (mouse IgG1 [MOPC-21], mouse IgG2a [MOPC-173], rat Neutrophils were purified from the peripheral blood of healthy adults by IgG2a [RTK2758], rat IgG2b [RTK4530], anti-human CD45RA [HI100], Ficoll-Paque density gradient separation (.97% granulocyte purity), fol- CD38 [HIT2], CD123 [6H6], CD126 [IL-6Ra, UV4], CD8 [SK1], CD3 lowed by negative immunomagnetic isolation enrichment (EasySep Direct [HIT3a], CD4 [RPA-T4], CD14 [HCD14], CD10 [HI10a], anti-mouse Human Neutrophil Isolation Kit; STEMCELL Technologies, .99.8% CD11b [M1/70], Ly6C [HK1.4], Ly6G [1A8], CD4 [GK1.5], CD3ε granulocyte purity). Five milliliters of peripheral whole blood was diluted [145-2C11], c-kit [CD117, 2B8], and FcgII/IIIR [CD16/32, 93]), BD in 20 ml of saline, overlaid onto 20 ml of Ficoll-Paque Plus (GE Health- Biosciences (mouse IgG2a [MOPC-173], and anti-human p-STAT3 care), and centrifuged (515 3 g, 20 min, room temperature, no brake). [pY705, 4/P-STAT3], p-STAT1 [pY701, 4/A-STAT1], CD34 [581], CD33 After centrifugation, the top layers of plasma, the PBMC interface, and [WM53], gp130 [CD130, AM64], CD66b [G10F5], CD45 [HI30], anti- Ficoll-Paque were removed, leaving an RBC and granulocyte pellet. The mouse CD8a [53-6.7], and Sca-1 [D7]), eBioscience (anti-mouse IL-6Ra pellet was resuspended to 5 ml with saline, and neutrophils were enriched [CD126, D7715A7] and CD34 [Ram34]), and R&D Systems (anti-human by removing the RBCs and remaining contaminating cell types by negative gp130 [CD130, 28126] and anti-mouse gp130 [CD130, 125623]). For immunomagnetic isolation using an EasySep Direct Human Neutrophil murine stem cell and progenitor analysis, the following lineage (Lin) Ab Isolation Kit (STEMCELL Technologies), as per the manufacturer’s mixture was used (BioLegend): anti-mouse CD3ε (145-2C11), CD5 (53-7.3), protocol. Ter-119 (TER-119), Gr-1 (RB6-8C5), Mac-1 (M1/70), and B220 (30-F11). Apoptosis assay Cell preparation and analysis Neutrophils were purified from human peripheral blood by Ficoll-Paque Cells in whole unmanipulated human and mouse peripheral blood (100 ml) density separation (followed by negative immunomagnetic isolation), were surface stained and stimulated (15 min at room temperature) with plated at 1 3 106 cells per milliliter in IMDM supplemented with 10% recombinant human G-CSF (100 ng/ml; Amgen), human IL-6 (5–50 ng/ml; FCS, and stimulated with IL-6 (20 ng/ml) or G-CSF (20 ng/ml) at 37˚C, Life Technologies), mouse IL-6 (10–30 ng/ml; BioLegend), or hyper–IL-6 5% CO2 for 24 h or were left unstimulated. Neutrophil apoptosis was (H-IL-6; human or mouse, 15–150 ng/ml; R&D Systems) or were left determined using an Annexin V–Apoptosis Detection Kit (BD Biosci- unstimulated. A similar molar ratio of free IL-6:IL-6 within the H-IL-6 ences), as per the manufacturer’s protocol. construct (free IL-6 molecular mass ∼ 20 kDa versus H-IL-6 construct molecular mass ∼ 59 kDa IL-6/sIL-6R/linker protein complex) was used. Gene expression analysis For time-course experiments, granulocytes were enriched by Ficoll-Paque density separation and stimulated with recombinant human G-CSF For qPCR analysis, sort-purified CD66b+Gr, monocytes (CD14+Mo), (100 ng/ml) or human IL-6 (50 ng/ml) for the indicated time or were left CD4+ T cells, and CD8+ T cells were transferred to Buffer RLT (QIA- unstimulated. Erythrocytes were lysed, and cells were fixed with BD GEN) and stored at 280˚C. Total RNA was extracted using an RNeasy Phosflow Lyse/Fix Buffer (BD Biosciences). Cells undergoing intracellular Micro Kit (QIAGEN), and cDNAwas prepared with a Maxima H Minus staining were permeabilized with chilled Perm Buffer III (BD Biosciences) First Strand cDNA Synthesis Kit (Thermo Fisher). Gene expression was for 30 min on ice. Permeabilized cells were stained for p-STAT3 and determined by qPCR using TaqMan GE assays (Applied Biosystems) for p-STAT1 or IgG2a isotype controls for 1 h. Samples were washed and human IL6ST (Hs00174360_m1) and were run in parallel with the analyzed using flow cytometry (BD LSR Fortessa) and FlowJo software housekeeping gene B2M (Hs00187842_m1). Reactions were run and (v9.9). Unimodal p-STAT3 and p-STAT1 expression in myeloid cells was analyzed on a ViiA 7 Real-Time PCR System (Thermo Fisher). IL6ST displayed as geometric mean fluorescent intensity (GMFI) values minus gene expression was determined using the comparative Ct method isotype controls, and bimodal p-STAT3 and p-STAT1 expression in T cells (22DDCt) normalized relative to B2M, and one sample within the dataset was expressed as the percentage of positive cells after gating on isotype was used as a reference. controls. For murine studies, bone marrow (BM) was flushed, splenocytes were isolated by mechanical disruption, and both samples were treated with Western blot lysis buffer, to remove contaminating erythrocytes, and surface stained. For gp130 protein immunoblotting, protein cell lysates were prepared For high-throughput imaging analysis, cells were also stained with from sort-purified CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ Tcells Hoechst 33342 nuclear dye (0.3 mg/ml), and fluorescent images were vi- using RIPA lysis buffer (20 mmol/l Tris [pH 8], 150 mmol/l NaCl, 10% sualized and acquired via an ImageStream X Imaging Flow Cytometer glycerol, 1% Nonidet P-40) containing 13 cOmplete Protease Inhibitor (Amnis; EMD Millipore). Cocktail (Roche). For STAT3/p-STAT3 immunoblotting, granulocytes To assess gp130 expression on granulocyte progenitor cells, naive mouse were purified using a Ficoll-Paque density gradient, followed by negative BM was isolated, treated with lysis buffer to remove contaminating 2 immunomagnetic bead enrichment, and were washed and stimulated erythrocytes, and stained for Lin and progenitor cell markers (Lin Sca-1+c- 2 2 with IL-6 (50 ng/ml) or G-CSF (100 ng/ml) for 15 min or were left kit+ [LSK] progenitor cells and within the Lin Sca-1 c-kit+ gate, 2 2 unstimulated. After stimulation, cells were washed twice with PBS, and megakaryocyte-erythroid progenitor [MEP] cells [CD34 FcgII/IIIR ], protein cell lysates were prepared using RIPA lysis buffer containing 23 + g lo common myeloid progenitor [CMP] cells [CD34 Fc II/IIIR ], and cOmplete Protease Inhibitor Cocktail and 1 3 PhosSTOP Phosphatase + g hi granulocyte-monocyte progenitor cells [CD34 Fc II/IIIR ]) (21). To as- Inhibitor (Roche). Using a Bio-Rad Protein Assay Kit (Bio-Rad, Her- sess gp130 expression on human progenitor cells, CD34+ cells were + cules, CA) and the Bradford method, 20 mg of denatured proteins was enriched by magnetic depletion of Lin cells (BD IMag Human Lineage separated using 10% SDS-PAGE and transferred to polyvinylidene Cell Depletion Set) and stained for progenitor markers (human hemato- 2 2 difluoride membranes. The membranes were blocked for 1 h in 5% skim poietic stem progenitor cells [HSPCs; Lin CD34+CD38 ] and within the 2 2 2 milk or BSA in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween- Lin CD34+CD38+CD33+CD10 gate, CMP cells [CD123+CD45RA ] + + 20). Immunoblotting was performed with primary Abs anti-gp130 and granulocyte–monocyte progenitor cells [CD123 CD45RA ]) (22). The (1:500; Santa Cruz Biotechnology), anti-Erk1/2 (1:1,000), total STAT3 responsiveness of mouse and human granulocyte progenitor cells to IL-6 (1:2000, clone D3Z2G), or p-STAT3 (Tyr705, clone D3A7, 1:1,000) (Cell m was assessed as above for peripheral blood samples (100 l). Signaling Technology) and detected with HRP-conjugated anti-rabbit Cell sorting IgG (1:10,000; Cell Signaling Technology). After applying ECL detection reagents (GE Healthcare or Thermo Fisher Scientific), protein bands Peripheral blood was collected from healthy adults, and granulocytes (SSChi were visualized using x-ray film (Fujifilm). The intensity of p-STAT3 CD66b+CD142), monocytes (CD66b2CD14+), CD4+ T cells (CD3+CD4+ and STAT3 bands was scanned from films, and the level of STAT3 CD82), and CD8+ T cells (CD3+CD42CD8+) were purified for quantita- activation was calculated using the following formula: percentage of tive PCR (qPCR) and immunoblotting analysis. Granulocytes were STAT3 activation = intensity of p-STAT3/(intensity of STAT3 + intensity enriched from lysed whole blood samples (BD Pharm Lyse buffer; BD of p-STAT3) 3 100%. The Journal of Immunology 3

FIGURE 1. IL-6 classical and trans stimulation fails to upregulate p-STAT3 or p-STAT1 in human granulocytes. (A–D) Peripheral blood cells (in whole blood) were stimulated with G-CSF (100 ng/ml) or increasing concentrations of IL-6 (classical) or H-IL-6 (trans), as indicated. H-IL-6 is an IL-6/sIL-6R/ linker protein complex that mimics the natural IL-6/sIL-6Ra complex to induce IL-6 trans signaling. p-STAT3 and p-STAT1 expression was assessed in gated CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells by ﬂow cytometry. Representative line graphs (A and C) and percentage of cells upregulating p-STAT3 and p-STAT1 (B and D) relative to unstimulated cells (NIL) in response to G-CSF, IL-6, or H-IL-6 (n = 7 healthy adults). Each line in (B) and (D) represents an individual healthy donor.

Statistical analysis Unlike CD14+Mo and CD4+ T cells, CD8+ T cells showed a Stimulation data sets using IL-6, H-IL-6, and G-CSF were compared with heterogeneous response to IL-6 and H-IL-6 stimulation, with their unstimulated values using one-way ANOVA with the Dunnett ∼40% of cells upregulating p-STAT3, demonstrating diversity in multiple-comparisons test. Surface receptor expression was compared in- IL-6 signaling within this population (Figs. 1, 2C, 2D). Critically, dividually between cell types using repeated-measures one-way ANOVA, however, CD66b+Gr were unable to upregulate p-STAT3 or with the Tukey multiple-comparisons test. The Kruskal–Wallis test with the Dunnett multiple-comparisons test was used to evaluate differences in p-STAT1 in response to multiple concentrations of IL-6 or H-IL-6 gp130 protein expression and IL6ST and IL6R gene expression across (Figs. 1, 2A, 2B) or to upregulate p-STAT3 over multiple time multiple groups. Data are presented as mean 6 SEM, and a p value , 0.05 points (Fig. 2E). This inability to respond to IL-6 or H-IL-6 could was considered statistically significant. Statistical analyses were performed not be attributed to impaired cell function, because G-CSF stim- using Prism 7.01 (GraphPad). ulation of CD66b+Gr induced p-STAT3 expression as expected (Figs. 1A, 1C, 2A). Results We next confirmed this unresponsiveness of granulocytes to IL-6 classical and trans signaling does not induce STAT3 or IL-6 stimulation using immunoblotting. Because low-level impu- STAT1 phosphorylation in human granulocytes rities in granulocyte preparations can potentially alter experimental IL-6 signaling was investigated in circulating CD66b+Gr, CD14+ outcomes (23), we isolated granulocytes using Ficoll-Paque den- Mo, CD4+ T cells, and CD8+ T cells in healthy adult peripheral sity separation, followed by negative-immunomagnetic separation, blood without prior manipulation. Cells were stimulated with IL-6 which resulted in granulocyte populations with high purity (classical signaling) or rIL-6/IL-6Ra protein complex H-IL-6 (.99.8%, Fig. 2F). Minimal p-STAT3 was detected in unstimu- (trans signaling), and p-STAT3 and p-STAT1 levels were deter- lated granulocytes and, consistent with flow cytometry data, there mined (Figs. 1, 2). In response to IL-6 (Figs. 1A, 1B, 2A–D) and was no response to IL-6 stimulation (Fig. 2G, 2H). In contrast, H-IL-6 (Figs. 1C, 1D, 2A–D) stimulation, we observed a signifi- G-CSF stimulation elicited a strong p-STAT3 response. Further- cant and dose-dependent upregulation of p-STAT3 and, to a lesser more, culturing purified neutrophils in vitro with G-CSF, but not extent, p-STAT1 in CD14+Mo, CD4+ T cells, and CD8+ T cells. IL-6, protected granulocytes from apoptosis, consistent with the 4 GRANULOCYTES LACK gp130

FIGURE 2. IL-6 fails to upregulate p-STAT3 or protect human granulocytes from apoptosis. Peripheral blood cells (in whole blood as per Fig. 1) were left unstimulated or were stimulated with IL-6 (30 ng/ml), H-IL-6 (100 ng/ml), or G-CSF (100 ng/ml), and GMFI of p-STAT3 (A) and p-STAT1 (B) levels was determined in myeloid populations. Percentage of positive p-STAT3 (C) and p-STAT1 (D) T cells (mean 6 SEM, IL-6 and H-IL-6). *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVA with the Dunnett multiple-comparisons test. n.s., p . 0.05. (E) Ficoll-Paque–enriched granulocytes (.97%) were stimulated with IL-6 (50 ng/ml) or G-CSF (100 ng/ml) or were left unstimulated in a time-course analysis for the indicated times (minutes), and upregulation of p-STAT3 relative to unstimulated (Nil) samples was assessed by flow cytometry (n = 4 healthy adults). (F–J) Circulating granulocytes were isolated by Ficoll-Paque density separation of whole blood, followed by negative immunomagnetic isolation enrichment. (F) Representative flow cytometry plot of CD66b+Gr purity (.99.8%). Immunoblot of total STAT3 and p-STAT3 (Tyr705)(G) and percentage of STAT3 activation in purified granulocytes (H) in unstimulated conditions (Nil) or after a 15-minute stimulation with IL-6 (50 ng/ml) or G-CSF (100 ng/ml) (n = 5 healthy adults). **p , 0.01, one-way ANOVA with the Dunnett multiple-comparisons test. n.s., p . 0.05. Representative dot plots (I) and proportions (J) of purified live (annexin V27AAD2), apoptotic (annexin V+7AAD2), and necrotic (annexin V+7AAD+) granulocytes after a 24-h culture in unstimulated conditions (Nil) or with IL-6 (20 ng/ml) or G-CSF (20 ng/ml) (n = 4 healthy adults). latter’s inability to signal (Fig. 2I, 2J). These data suggest that, (Fig. 3A–C, 3E, 3F), consistent with their inability to respond to despite the pleiotropic effects of IL-6, circulating human CD66b+ IL-6 or H-IL-6 stimulation. Importantly, this absence of surface Gr are not targets of IL-6 signaling, through classical or trans gp130 protein was not limited to healthy adult CD66b+Gr; it was + signaling pathways. also observed in CD66b Gr 60 and 90 d after allo-SCT (Fig. 3D), a setting in which IL-6 dysregulation is profound, and IL-6 inhi- The absence of the gp130 signal transducer prevents IL-6 bition results in very low rates of acute graft-versus-host disease classical and trans signaling in human granulocytes (7). To exclude possible gp130 internalization or preferential ex- To understand the apparent differential responses to IL-6 stimu- pression of gp130 splice variants in CD66b+Gr, we confirmed the lation, we examined the key components of the IL-6R system absence of gp130 protein in purified (.99%, Fig. 3G) CD66b+Gr required by classical (gp130 and mIL-6Ra) and trans (gp130) by Western blot (Fig. 3H, 3I) and low IL6ST gene expression by signaling pathways in CD66b+Gr, CD14+Mo, CD4+ T cells, and qPCR (Fig. 3J). Moreover, IL6ST gene expression in granulocytes CD8+ T cells. Using flow cytometry (Fig. 3A–C) and high- was significantly lower than in CD14+Mo, CD4+ T cells, and throughput imaging analysis (Fig. 3E, 3F), we observed gp130 CD8+ T cells in the HemaExplorer public online database expression on CD14+Mo, CD4+ T cells, and CD8+ T cells. Ap- (Fig. 3K) (24). Finally, we confirmed the absence of gp130 proximately 40% of CD8+ T cells did not express gp130 (Fig. 3A, staining in neutrophils with a second mAb (Fig. 3L). We consis- 3B), reflecting their heterogeneous response to IL-6 stimulation tently observed mIL-6Ra expression on CD66b+Gr, despite the (Figs. 1, 2C, 2D). Surface gp130 was coexpressed with mIL-6Ra fact that mIL-6Ra is unable to signal in the absence of gp130 on ∼70% of CD14+Mo and ∼40% of CD4+ T cells, but on a (Fig. 3A–C). Therefore, granulocytes are refractory to IL-6 due to smaller fraction of CD8+ T cells (Fig. 3C). In contrast, healthy the absence or very low protein expression of the signal transducer adult CD66b+Gr exhibited a striking absence of gp130 expression gp130. The Journal of Immunology 5

FIGURE 3. Absence of the signal transducer gp130 on human granulocytes prevents IL-6 signaling. (A–C) Peripheral blood CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells were assessed for surface expression of gp130 and mIL-6Ra by flow cytometry. (A) Representative dot plots show gp130 (clone AM64) and mIL-6Ra expression relative to their isotype controls (mouse IgG1). Proportion of gp130+ cells and mIL-6Ra+ cells (B) and distribution of gp1302mIL-6Ra2, gp1302mIL-6Ra+, gp130+mIL-6Ra2, and gp130+mIL-6Ra+ subsets (C) within CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells (n = 10 healthy adults). *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVA. (D) Proportion of gp130+ cells within CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells in whole peripheral blood samples on days 60 and 90 post allo-SCT (D + 60, n = 28; D + 90, n = 11). **p , 0.01, ***p , 0.001, one-way ANOVA. Representative images (original magnification 360; red: CD66b, orange: CD14, magenta: CD3, green: gp130, blue: Hoechst 33342) (E) and line graph of collective cell fluorescence (.700 cells) of gp130 expression (F) in CD66b+Gr, CD14+Mo, and CD3+ T cells assessed by high-throughput imaging cytometry. (G–J) Healthy adult peripheral blood monocytes, CD4+ T cells, CD8+ T cells, and enriched granulocytes were sort purified for immunoblot and qPCR analysis. (G) Representative purity plot (.97%). Immunoblot (H) and normalized protein expression (I) of gp130 in CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells, as assessed by Western blot with Erk1/2 used as a loading control (n = 3 healthy adults). *p , 0.05, Kruskal-Wallis test with the Dunnett multiple-comparisons test. (J) Relative expression of gp130 signal transducer gene (IL6ST) in sorted CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells (n = 3 or 4 healthy adults). **p , 0.01, Kruskal–Wallis test with the Dunnett multiple-comparisons test. (K)In silico analysis of IL6ST and IL6R expression in polymorphonuclear neutrophils (PMN; derived from peripheral blood and BM), CD14+Mo, CD4+ T cells, and CD8+ T cells (http://servers.binf.ku.dk/bloodspot/) (24). *p , 0.05, **p , 0.01, ***p , 0.001, Kruskal-Wallis test with the Dunnett multiple- comparisons test. (L) Peripheral blood CD66b+Gr, CD14+Mo, CD4+ T cells, and CD8+ T cells (in whole blood) were stained for gp130 with an alternative clone (28126) and compared with isotype control by flow cytometry.

Murine neutrophils do not express gp130 and do not respond to absence was not restricted to circulating neutrophil populations, IL-6 classical or trans signaling because Ly6G+ derived from primary lymphoid tissue (spleen) and Given the broad use of murine models to understand IL-6 responses BM also lacked gp130 expression (Fig. 4G). Together, these data and delineate IL-6 signaling pathways (1, 8, 16, 25, 26), we further demonstrate that, consistent with human circulating granulocytes, investigated IL-6 responses in murine granulocytes. As observed murine neutrophils are refractory to IL-6 stimulation through IL-6 in humans, we identified a striking inability of circulating murine classical and trans signaling pathways due to the absence of neutrophils (Ly6G+) and eosinophils (Siglec-F+SSChi, data not gp130. shown) to upregulate p-STAT3 or p-STAT1 in response to IL-6 classical (IL-6) or trans (H-IL-6) signaling (Fig. 4A–E). This was Granulocytes lose gp130 and their capacity to respond to IL-6 in contrast to murine monocytes (Ly6ChiMo) and T cell subsets, during development for which significant upregulation of p-STAT3 was observed after Because previous studies have reported a significant role for IL-6 IL-6 and H-IL-6 stimulation. Importantly, granulocyte function signaling in granulopoiesis (27, 28), we next investigated the was not inherently defective in these assays, because parallel G- ability of murine and human granulocyte progenitor cells to re- CSF stimulation elicited a strong p-STAT3 response in Ly6G+ spond to IL-6. In this study, we detected gp130 expression in LSK (Fig. 4A, 4D). Despite gp130 expression in circulating murine cells, CMP cells, and granulocyte-monocyte progenitor cells in monocytes and T cells, the gp130 IL-6 signal transducer was not murine BM (Fig. 5A–C), as well as p-STAT3 upregulation when detected in murine neutrophils by flow cytometry (Fig. 4F). This stimulated with IL-6 (Fig. 5D, 5E). In contrast, MEP cells did not 6 GRANULOCYTES LACK gp130

FIGURE 4. Murine neutrophils also lack gp130 and are unresponsive to IL-6 stimulation. (A–F) Peripheral murine blood cells (in whole blood) were stimulated with human G-CSF (100 ng/ml) or increasing concentrations of murine IL-6 (classical) or H-IL-6 (trans), as indicated. p-STAT3 and p-STAT1 expression was assessed in gated Ly6G+, Ly6ChiMo, CD4+ T cells, and CD8+ T cells by flow cytometry. (A) Representative line graphs of p-STA expression in unstimulated cells (Nil) or in cells stimulated with IL-6 (30 ng/ml), H-IL-6 (100 ng/ml), or G-CSF (100 ng/ml). Dose- dependent upregulation of p-STAT3 and p-STAT1 relative to unstimulated cells (Nil) in response to IL-6 (B) or H-IL-6 (C)(n = 4 mice). GMFI of p-STAT3 (D) and percentage of p-STAT3 expression (E) by flow cytometry in unstimulated cells and cells stimulated with IL-6, H-IL-6, and G-CSF (n = 5). *p , 0.05, **p , 0.01, ***p , 0.001, one-way ANOVA with the Dunnett multiple-comparisons test. (F) Representative flow cytometric analysis of surface gp130 expression relative to isotype control (rat IgG2a) in murine peripheral blood gated on Ly6G+, Ly6ChiMo, CD4+ T cells, and CD8+ T cells. (G) Proportion of gp130+ cells within Ly6G+, Ly6ChiMo, CD4+ T cells, and CD8+ T cells derived from peripheral blood, spleen, and BM (n = 5 mice). ***p , 0.001, one-way ANOVA. n.s., p . 0.05. express gp130 and, consequently, did not respond with p-STAT3 current dogma, IL-6 does not directly influence granulocyte func- upregulation when stimulated with IL-6. Next, we investigated tion. The role of IL-6 in granulocytes has been unclear, with mul- whether human HSPCs, CMP cells, and granulocyte-monocyte tiple conflicting studies (15–20). For instance, IL-6 has been progenitor cells were also responsive to IL-6. In G-CSF–mobi- reported to promote STAT3 phosphorylation in granulocytes, but lized leukapheresis products, all three progenitor populations not to alter function or apoptosis (16), whereas Dienz et al. (15) expressed gp130, and IL-6 stimulation resulted in significant up- suggested that IL-6 promoted the expression of antiapoptotic genes regulation of p-STAT3 (Fig. 5F–J). Therefore, although mature and neutrophil survival but not STAT3 phosphorylation. Other granulocytes are not responsive to IL-6, IL-6 may instead drive studies have suggested that IL-6 can increase (17) or decrease (18– granulopoiesis via effects on their progenitors. 20) neutrophil apoptosis in vitro. Recent reports have shown that contaminating cell populations in common neutrophil-isolation Discussion procedures can affect experimental outcomes, including cytokine IL-6 is a pleotropic cytokine that modulates multiple components of expression in cultured neutrophils (23), although impurities have the adaptive and innate immune systems to promote and maintain been noted to have little impact on transcription profiles (30). Thus, inflammation (reviewed in Ref. 1). The cells mediating the down- these conflicting data sets likely reflect the difficulties in interpret- stream inflammatory cascades include T and B lymphocytes, in ing the effects of cytokines on mixed myeloid populations that addition to diverse myeloid lineages. Granulocytes represent the exhibit poor survival in vitro and the potential for indirect effects largest circulating innate population in humans and are well- of cytokine stimulation on signaling cascades. In support of this, described mediators of inflammation in a range of responses to adoptive transfer of labeled neutrophils into patients receiving infectious pathogens and autoimmune Ags, in which IL-6 is known IL-6R inhibition has recently demonstrated a definitive effect of to drive pathogenicity via the secretion of cytokines, chemokines, IL-6 on neutrophil migration (with retention in liver and spleen) in and proteases (29). In this article, we demonstrate that, contrary to RA patients, but it excluded any effect on apoptosis or function The Journal of Immunology 7

FIGURE 5. Murine and human granulocytes lose their capacity to respond to IL-6 stimulation during development. (A–E) Mouse BM progenitor cells were assessed for gp130 expression and their ability to respond to IL-6. (A) Representative dot plots show gating strategy used to deﬁne LSK progenitor cell, MEP cell (Lin2Sca-12c-kit+CD342FcgII/IIIR2), CMP cell (Lin2Sca-12c-kit+CD34+FcgII/IIIRlo), and granulocyte-monocyte progenitor cell (Lin2 Sca-12c-kit+CD34+FcgII/IIIRhi) populations. Representative line graphs of gp130 (B) and gp130 expression relative to isotype controls (C) on LSK progenitor cells, MEP cells, CMP cells, and granulocyte-monocyte progenitor cells (n = 4). Upregulation of p-STAT3 relative to unstimulated cells (Nil) in response to IL-6 (30 ng/ml) (D) and representative line graphs of p-STAT3 expression (E) in LSK progenitor cells, MEP cells, CMP cells, and granulocyte- monocyte progenitor cells (n=5). (F–J) Human progenitor cells enriched in G-CSF–mobilized leukapheresis products were assessed for gp130 expression and their responsiveness to IL-6 stimulation. (F) Representative dot plots show gating strategy used to deﬁne HSPCs (Lin2CD34+CD382), CMP cells (Lin2 CD33+CD102CD34+CD38+CD123+CD45RA2), and granulocyte-monocyte progenitor cells (Lin2CD33+CD102CD34+CD38+CD123+CD45RA+). Rep- resentative line graphs of gp130 (G) and gp130 expression relative to isotype controls (H) on HSPCs, CMP cells, and granulocyte-monocyte progenitor cells (n = 3). Upregulation of p-STAT3 relative to unstimulated cells in response to IL-6 (30 ng/ml) (I) and representative line graphs of p-STAT3 expression (J) in HSPCs and combined CMP cells and granulocyte-monocyte progenitor cells (CMP/GMP) (n = 3). ***p , 0.001, one-way ANOVA. n.s., p . 0.05.

(31). Our data clearly demonstrate that IL-6 does not induce have been shown to release sIL-6Ra upon apoptosis (34). More- p-STAT3 or p-STAT1 activation in granulocytes. Furthermore, we over, neutrophils have been proposed to play a key role in pro- conclude that this is due to a lack of gp130, which is required for moting IL-6 trans signaling, driving the transition from neutrophilic signal transduction of all known forms of IL-6 signaling; therefore, to mononuclear infiltration and resolving inflammation (25, 35). IL-6 does not directly affect granulocyte function or apoptosis. Thus, these data suggest that mIL-6Ra expression by granulocytes Thus, the clear effects of IL-6R inhibition on neutrophil migration may act as an important source of sIL-6Ra in vivo. Alternatively, in are likely to be indirect effects that are mediated by IL-6 signaling concert with IL-6 secretion, mIL-6Ra in mature granulocytes could in other cell types, such as endothelial cells or monocytes. Con- potentially provide cluster signaling to other gp130-expressing sistent with this, although (usually mild to moderate) neutropenia is target cells, such as that proposed during DC–T cell interactions observed in a significant fraction (,20%) of RA patients receiving (8, 9). However, there is no evidence that cluster signaling can be TCZ treatment, there has been no reported temporal link between a performed by non-APCs. decrease in circulating granulocytes and an increase in infection Although human peripheral T cells expressed gp130 and (32, 33). responded to IL-6, we did observe differential mIL-6Ra and gp130 Interestingly, we consistently observed mIL-6Ra expression on expression and the capacity to respond to IL-6. Furthermore, granulocytes, despite the absence of its signal transducer, gp130. gp130 expression on CD4+ and CD8+ T cells was much lower in sIL-6Ra, which is required for IL-6 trans signaling, is primarily patients after allo-SCT than in healthy adults, suggesting that generated by proteolytic cleavage of mIL-6Ra, and neutrophils gp130 is downregulated during inflammation. Previous studies 8 GRANULOCYTES LACK gp130 have reported that mouse T cells downregulate mIL-6Ra and interleukin-6 inhibition with tocilizumab to standard graft-versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial. Lancet gp130 upon TCR or IL-6 stimulation (36, 37). Moreover, this Oncol. 15: 1451–1459. downregulation of gp130 was reported to be associated with 8. Heink, S., N. Yogev, C. Garbers, M. Herwerth, L. Aly, C. Gasperi, V. Husterer, acquisition of memory markers, such as CD44, rendering cells A. L. Croxford, K. Mo¨ller-Hackbarth, H. S. Bartsch, et al. 2017. Trans- presentation of IL-6 by dendritic cells is required for the priming of patho- unresponsive to IL-6 (36, 38). Therefore, reductions in gp130 genic TH17 cells. [Published erratum appears in 2017 Nat. Immunol. 18: 474.] expression and responsiveness to IL-6 in human peripheral blood Nat. Immunol. 18: 74–85. T cells after allo-SCT are likely a reflection of activation in 9. Quintana, F. J. 2016. Old dog, new tricks: IL-6 cluster signaling promotes pathogenic TH17 cell differentiation. Nat. Immunol. 18: 8–10. response to alloantigen or pathogens within a lympho-deplete 10. Heinrich, P. C., I. Behrmann, S. Haan, H. M. Hermanns, G. Mu¨ller-Newen, and environment; however, this does not account for the lower ex- F. Schaper. 2003. Principles of interleukin (IL)-6-type cytokine signalling and its pression of gp130 in CD8+ T cells relative to CD4+ T cells in regulation. Biochem. J. 374: 1–20. 11. Calabrese, L. H., and S. Rose-John. 2014. IL-6 biology: implications for clinical peripheral blood of healthy donors. This is in contrast to murine targeting in rheumatic disease. Nat. Rev. Rheumatol. 10: 720–727. CD8+ T cells from naive mice that expressed high levels of gp130. 12. Hibi, M., M. Murakami, M. Saito, T. Hirano, T. Taga, and T. Kishimoto. 1990. Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell 63: This disparity is likely to reflect the relatively high abundance of 1149–1157. memory T cells in humans and the presence of significant numbers 13. Saito, M., K. Yoshida, M. Hibi, T. Taga, and T. Kishimoto. 1992. Molecular of additional CD8+ T cells, such as mucosal associated invariant cloning of a murine IL-6 receptor-associated signal transducer, gp130, and its regulated expression in vivo. J. Immunol. 148: 4066–4071. T cells, which did not express gp130 (data not shown). It is also 14. Oberg, H. H., D. Wesch, S. Gru¨ssel, S. Rose-John, and D. Kabelitz. 2006. important to note that, although mIL-6Ra expression on T cells Differential expression of CD126 and CD130 mediates different STAT-3 phos- was low, as long as these cells expressed gp130, they responded to phorylation in CD4+CD252 and CD25high regulatory T cells. Int. Immunol. 18: 555–563. IL-6. Moreover, this responsiveness of T cells could not be at- 15. Dienz, O., J. G. Rud, S. M. Eaton, P. A. Lanthier, E. Burg, A. Drew, J. Bunn, tributed to the presence of sIL-6Ra (and trans signaling) in our B. T. Suratt, L. Haynes, and M. Rincon. 2012. Essential role of IL-6 in protection against H1N1 influenza virus by promoting neutrophil survival in the lung. assay, because addition of the IL-6 trans signaling inhibitor Mucosal Immunol. 5: 258–266. sgp130:Fc failed to inhibit IL-6–induced STAT3 phosphorylation 16. Wright, H. L., A. L. Cross, S. W. Edwards, and R. J. Moots. 2014. Effects of IL-6 (data not shown). Thus, the expression of IL-6Ra is likely to be and IL-6 blockade on neutrophil function in vitro and in vivo. Rheumatology (Oxford) 53: 1321–1331. underreported by current flow cytometry approaches. 17. Afford, S. C., J. Pongracz, R. A. Stockley, J. Crocker, and D. Burnett. 1992. The Finally, although mature granulocytes are refractory to IL-6 due induction by human interleukin-6 of apoptosis in the promonocytic cell line to the absence of gp130 expression, their progenitors do express U937 and human neutrophils. J. Biol. Chem. 267: 21612–21616. 18. Biffl, W. L., E. E. Moore, F. A. Moore, and C. C. Barnett, Jr. 1995. Interleukin-6 gp130, confirming that this receptor subunit is lost during cellular suppression of neutrophil apoptosis is neutrophil concentration dependent. J. maturation. Thus, IL-6 can still influence granulopoiesis at an early Leukoc. Biol. 58: 582–584. stage of development, which may account for the neutropenia that 19. McNamee, J. P., P. V. Bellier, B. C. Kutzner, and R. C. Wilkins. 2005. Effect of pro-inflammatory cytokines on spontaneous apoptosis in leukocyte sub-sets is sometimes observed in RA patients after TCZ treatment (3, 32, within a whole blood culture. Cytokine 31: 161–167. 33, 39, 40). Alternatively, TCZ has been suggested to induce 20. Ottonello, L., G. Frumento, N. Arduino, M. Bertolotto, P. Dapino, M. Mancini, and F. Dallegri. 2002. Differential regulation of spontaneous and immune neutropenia by acting on mature neutrophil migration and mar- complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of gination (31); however, by virtue of signaling incompetence oxidants, Bax and caspase-3. J. Leukoc. Biol. 72: 125–132. within these cells, this is likely due to indirect effects. Importantly, 21. Mullally, A., C. Bruedigam, L. Poveromo, F. H. Heidel, A. Purdon, T. Vu, R. Austin, D. Heckl, L. J. Breyfogle, C. P. Kuhn, et al. 2013. 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