The Journal of Immunology

A Soluble Form of IL-27Ra Is a Natural IL-27 Antagonist

Ce´line Dietrich,*,† Sophie Candon,†,‡ Frank M. Ruemmele,x and Odile Devergne*,†

IL-27 is a of the IL-12 family that plays a key role in the regulation of inflammatory and responses. Its receptor is composed of IL-27Ra and gp130 and activates the STAT pathway. We show in this study, using an ELISA that we developed, that a naturally occurring soluble form of IL-27Ra (sIL-27Ra) is produced by human activated CD4+ and CD8+ T cells, B cells, myeloid cells, and various cell lines. sIL-27Ra is present at a mean concentration of 10,344 6 1,274 pg/ml in the sera from healthy individuals. Biochemical studies showed that sIL-27Ra is released as two N-glycosylated variants of ∼90 and ∼70 kDa. In IL- 27Ra–transfected COS7 cells, primary cells, and cell lines, production of sIL-27Ra is inhibited by the metalloprotease inhibitors GM6001 and TAPI-0. Importantly, natural sIL-27Ra binds rIL-27, inhibits IL-27 binding to its cell surface receptor, and is a potent inhibitor of IL-27 signaling, as shown by its ability to specifically block IL-27–mediated STAT activation, at low molar excess over IL-27. Also, we found that serum levels of sIL-27Ra were elevated in patients with Crohn’s disease, a Th1-mediated disease. These findings suggest that sIL-27Ra may play important immunoregulatory functions under normal and pathological conditions. The Journal of Immunology, 2014, 192: 5382–5389.

nterleukin-27 is a heterodimeric cytokine of the IL-12 family suggested that mouse p28 does not antagonize IL-27 activity, but composed of two subunits, EBV-induced gene 3 (EBI3) and p28 plays an agonistic role through the IL-6R (10). I (1, 2). In humans, it is expressed at high levels by monocytes/ Cytokine receptors can exist in both membrane and soluble and dendritic cells upon activation and by placental forms (11). These latter can bind to and modulate their trophoblasts (2–4). IL-27 displays broad immunological functions activity in different ways. They can either play an antagonistic and plays a key role in the regulation of inflammatory and T cell role by preventing cytokine association with the membrane form responses. Although it was initially described as a factor promoting of the receptor, or alternatively play a positive role by extending the initiation of Th1 responses, it was later found to play a major the half-life of the cytokine and potentiating its activity as de- T cell–suppressive function by limiting Th1 responses, inhibiting scribed for IL-15Ra or IL-7Ra (12, 13). Gp130 naturally exists as Th2 and Th17 cell differentiation, and regulating the development of a soluble form that inhibits IL-6/IL-6R complex signaling (14) but Tr1 and other T regulatory cell populations. In addition to its role as does not inhibit IL-27 signaling (15). Mouse neuronal cells ex- an immunoregulator, IL-27 also regulates angiogenesis, hemato- press an alternatively spliced IL-27Ra isoform of 33 kDa, lacking poiesis, and osteoclastogenesis (reviewed in Refs. 5, 6). exons 7–14, that encodes part of the extracellular domain (16).

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. IL-27 signals through a heterodimeric receptor composed of This truncated IL-27Ra isoform was reported, not to act as two chains, IL-27Ra (formerly called TCCR or WSX-1) and gp130, a dominant negative, but to associate with CNTF-R and gp130 and activates the STAT pathway, predominantly STAT1 and similarly to full-length membrane IL-27Ra, to form a tripartite STAT3 (7). Cytokine signaling and activity can be modulated by functional receptor for humanin, a neurotrophic peptide (16, 17). natural agonists or antagonists. In the case of IL-27, two studies In this study, we investigated whether human IL-27Ra can be have shown that, in mice, the p28 subunit of IL-27 can be secreted detected as a soluble form that could modulate IL-27 activity. We alone, independently of EBI3, and constitutes an IL-27 antagonist found that soluble forms of IL-27Ra (sIL-27Ra) are spontane- (8, 9). However, human p28 is not secreted in the absence of EBI3 ously released from cells as N-glysosylated proteins of 70/90 kDa, in transfected cells (2), and it is presently unknown whether in through proteolytic cleavage by metalloproteases. sIL-27Ra can

http://classic.jimmunol.org humans p28 is released naturally free of EBI3 and could act as bind rIL-27 in vitro, is complexed with IL-27 in vivo, and inhibits an IL-27 antagonist. In addition, controversially, a recent report IL-27 signaling. It was present at detectable levels in the sera from healthy individuals and was upregulated in the sera of patients with Crohn’s disease (CD). *Centre National de la Recherche Scientifique Unite´ Mixte de Recherche 8147, Uni- versite´ Paris Descartes, Sorbonne Paris Cite´, 75 015 Paris, France; †Institut Necker Enfants Malades, INSERM U1151, Centre National de la Recherche Scientifique Unite´ Materials and Methods Mixte de Recherche 8253, 75 015 Paris, France; ‡INSERM U1013, Universite´ Paris Downloaded from Descartes, Sorbonne Paris Cite´, 75 015 Paris, France; and xService de Gastroente´rologie Human cells and sera Pe´diatrique, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Necker-Enfants Malades, COS7, Hodgkin (KMH2), and Burkitt (BL2) B lymphoma cell lines were 75 015 Paris, France grown in DMEM (COS7) or RPMI 1640 medium (KMH2 and BL2) Received for publication December 23, 2013. Accepted for publication March 31, supplemented with 10–15% FBS, 1% glutamine, and 1% antibiotics (Life 2014. Technologies) (complete medium). COS7 cells were transfected by elec- Address correspondence and reprint requests to Dr. Odile Devergne, Institut Necker troporation (Bio-Rad) with pcDNA3 vector, empty or encoding full-length Enfants Malades, INSERM U1151, Centre National de la Recherche Scientifique C-terminal V5-6His–tagged IL-27Ra (gift of H. Gascan, Angers, France), Unite´ Mixte de Recherche 8253, Hoˆpital Necker, Baˆtiment Jean Hamburger, 161 and GFP expression vector (Clontech) and incubated for various times in rue de Se`vres, 75 015 Paris, France. E-mail address: [email protected] complete DMEM or Optimem medium. The online version of this article contains supplemental material. Human CD4+ T cells (purity .97%) or CD8+ T cells (purity .95%) Abbreviations used in this article: ADAM, a disintegrin and metalloproteinase; CD, were purified by negative magnetic cell isolation (Miltenyi Biotec) from Crohn’s disease; EBI3, EBV-induced gene 3; MMP, matrix metalloprotease; sIL- PBMC of adult healthy donors (Etablissement Franc¸ais du Sang, Paris, 27Ra, soluble form of IL-27Ra. France), as previously described (18). Purified T cells were cultured (1 3 106/ml) for 3–7 d in complete RPMI 1640 medium in the presence of IL-2 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 (20 U/ml; Roche), and either PHA (4 mg/ml; Sigma-Aldrich) or beads

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303435 The Journal of Immunology 5383

(ratio 1 bead per 2 cells) coated with CD2/CD3/CD28 Abs (T cell NaCl, 3% glycerol, 1.5 mM EDTA), 10- to 30-fold concentrated KMH2 activation/expansion kit; Miltenyi Biotec). Human tonsillar B cells (pu- culture supernatant, or normal human sera. All samples were supplemented rity .99%) were purified by magnetic depletion using CD2 microbeads with protease inhibitors (1 mM PMSF, 1 mg/ml pepstatin, and 1 mg/ml (Miltenyi Biotec) and cultured (2 3 106/ml) for 2–4 d in complete RPMI leupeptin) and precleared with protein G–Sepharose beads (GE Health- 1640 medium in the presence of CD40 Ab (0.5 mg/ml; R&D Systems), as care) before incubation with Abs. sIL-27Ra was immunoprecipitated using described (19). Monocytes (purity .97%) were purified from PBMC by either mouse mAb or goat polyclonal anti–IL-27Ra Ab (R&D Systems), in positive selection using CD14 microbeads (Miltenyi Biotec) and cultured parallel to control Abs (mouse MOPC141 mAb or purified goat IgG; (3 3 106/ml) for 3 d in the presence of LPS (100 ng/ml; Invivogen). Sigma Aldrich), followed by incubation with protein G–Sepharose beads, Monocyte-derived mature dendritic cells were generated from human or mouse anti-human IL-27Ra mAb covalently linked to agarose beads monocytes cultured for 6 d in the presence of GM-CSF, followed by using AminoLink Plus immobilization kit (Thermo Scientific). After a 2-d maturation with TNF-a (20). Two cell-permeable inhibitors of metal- washes in lysis buffer, immunoprecipitates were eluted, separated by SDS- loproteases, GM6001 or TAPI-0 (Merck Chemicals), were used at concen- PAGE, and transferred to nitrocellulose membranes. Primary Abs for im- trations that were not toxic to cells. Because inhibitors were reconstituted in munoblotting included goat biotinylated polyclonal anti-human IL-27Ra DMSO, DMSO was added at a similar dilution in the control cultures. Ab, mouse V5 mAb (Invitrogen), and 2G4H6 anti-EBI3 mAb (3). Binding Normal human sera were obtained from healthy, nonpregnant volunteers of primary Abs was detected using HRP-conjugated secondary reagents (n = 28). Sera (n = 43) from six women with normal pregnancies, collected and chemiluminescence reagents (Pierce). When indicated, immunopre- at various times during pregnancy (six to eight sera per individual) for cipitates were submitted to N-glycanase treatment with PGNase F set serological diagnosis (Be´cle`re Hospital), were used in this study. Sera from (Sigma-Aldrich) before analysis by SDS-PAGE and immunoblotting. pediatric patients (n = 52) with active CD were collected for diagnosis purpose (Necker Hospital) before initiation of treatment with infliximab. Purification of natural sIL-27Ra Studies were conducted in accordance with the Declaration of Helsinki and were approved by the hospital ethics committee. Natural sIL-27Ra was purified from the culture supernatant (450 ml) of KMH2 cells (106 per ml) incubated for 48 h in X-Vivo 15 media (Lonza). ELISA The culture supernatant was concentrated by 45-fold using Amicon Ultra centrifugal filter units and supplemented with protease inhibitors before To detect sIL-27Ra, a sandwich ELISA was developed by using mouse overnight incubation with anti–IL-27Ra beads. Beads were washed ex- anti-human IL-27Ra mAb (191106; R&D Systems) as coating Ab and tensively, and bound proteins were recovered by acidic elution. After goat biotinylated polyclonal anti-human IL-27Ra Ab (R&D Systems) as neutralization and addition of BSA as a carrier, the eluate was dialyzed detection Ab. Both Abs were raised against the extracellular domain of IL- against PBS and concentrated (Microcon centrifugal filter device; Milli- 27Ra. Binding of the detection Ab was detected using streptavidin HRP pore). Concentration of purified sIL-27Ra was determined by ELISA. conjugate (GE Healthcare) and tetramethylbenzidine (R&D Systems) as a substrate. Human rIL-27Ra-Fc fusion protein (R&D Systems) was used STAT activation assay as a standard. Because the m.w. of IL-27Ra-Fc is higher than that of sIL- After overnight starvation in serum-free media, BL2 cells were incubated in 27Ra, values for sIL-27Ra, expressed in pg/ml, were corrected based on 48-well plate (106 cells per 100 ml) in RPMI 1640 medium supplemented the molarity of each protein. The limit of sensitivity was 30 pg/ml sIL- with 0.5% BSA (RPMI/BSA media). Prior to addition to cells, rIL-27 (6His- 27Ra. This ELISA detects sIL-27Ra, bound or not to IL-27. In some tagged covalently linked EBI3-p28 fusion protein; R&D Systems) was in- cases, culture supernatants were concentrated using Amicon Ultra cen- cubated for 15 min at 37˚C in 100 ml RPMI/BSA media containing various trifugal filter device (Millipore) before ELISA. To investigate whether sIL- concentrations of rIL-27Ra-Fc chimera, recombinant gp130-Fc chimera 27Ra could form complexes with IL-27 in the sera, we developed an (R&D Systems), or purified natural sIL-27Ra. The mixture was then added ELISA by using mouse anti-human IL-27Ra mAb or a control isotype to BL2 cells for 15 min at 37˚C and 5% CO2. The reaction was stopped, and mAb as coating Abs, and goat biotinylated polyclonal anti-human IL-27 cells were lysed in ice-cold 1% Nonidet P-40 lysis buffer supplemented with Ab (R&D Systems) as detection Ab. Binding of the detection Ab was protease and phosphatase inhibitors. Phosphorylated and total STAT1 were by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. detected as indicated above. Serum levels of IL-27 were detected using a detected by immunoblot using rabbit polyclonal Abs from Cell Signaling commercial kit (R&D Systems) with some modifications of the procedure. Technology. Quantification of STAT1 inhibition was determined by densi- tometric analysis of autoradiography bands using ImageJ software. Immunoprecipitation, Western blotting, and N-glycanase treatment FACS analysis sIL-27Ra was immunoprecipitated from either the lysate of transfected Cells were first saturated in PBS containing 20% normal human serum. Cell COS7 cells (lysis buffer: 1% Nonidet P-40, 50 mM Tris [pH 7.4], 150 mM surface IL-27Ra was detected using PE-conjugated anti–IL-27Ra mAb http://classic.jimmunol.org

FIGURE 1. sIL-27Ra is released from differ- ent primary human cell types. (A) Purified CD4+ or CD8+ T cells were cultured in the presence of IL-2 and either PHA or CD2/CD3/CD28-coated beads. After the indicated time, cell culture

Downloaded from supernatants were collected and tested by sIL- 27Ra ELISA. Cell culture supernatants from CD40-stimulated purified B cells (B) or LPS- stimulated purified monocytes (C) were tested by sIL-27Ra ELISA before concentration (13)or after a 6- or 8-fold concentration (63 and 83). Data shown in (A)–(C) represent the mean value 6 SEM of three to four experiments performed with different donors. (D) sIL-27Ra levels were measured by ELISA in the culture supernatant from TNF-a–matured dendritic cells (DC) de- rived from nine different donors. ND, not detected. 5384 CHARACTERIZATION OF sIL-27Ra

(R&D Systems), in parallel to an IgG2b-PE control mAb. Cells were an- alyzed on FACSCalibur, and data were analyzed using FlowJo software. For binding experiments, BL2 cells were saturated in PBS containing 5% veinoglobulins and incubated for 40 min on ice with 6His-tagged rIL-27 that had been preincubated or not with natural purified sIL-27Ra for 15 min at 37˚C. Binding of IL-27 was detected with an anti-hexahistidine mAb (ThermoScientific), followed by PE-conjugated F(ab9)2 anti-mouse IgG (eBioscience). Statistical analysis Statistical significance was determined using Student’s paired t test (*p , 0.05, **p , 0.01, ***p , 0.001) or unpaired t test (p values indicated on the graphs). Pearson’s test was used for correlation. Pearson correlation coefficients (r2 values) and p values are indicated on the graphs. Results A soluble form of IL-27Ra is detected in the cell culture supernatant from various primary human cell types To investigate whether IL-27Ra exists as a soluble form, we first developed a sandwich ELISA, as described in Materials and Methods, and screened supernatants from various human primary cell types. Because activated human CD4+ T cells express high levels of IL-27Ra mRNA and protein (2, 18), we first tested these cells for sIL-27Ra production. Purified CD4+ T cells were stim- ulated for 3–7 d with PHA or beads coated with CD2/CD3/CD28 Abs in the presence of IL-2. In both conditions of stimulation, detectable levels of sIL-27Ra were measured in the culture su- pernatant after 3 d of stimulation, and these levels increased over the activation period (up to 339 pg/ml on average at day 7) (Fig. 1A, left). Comparable levels of sIL-27Ra were detected in the culture supernatant from CD8+ T cells activated with CD2/ CD3/CD28 beads (mean 401 6 13 pg/ml at day 7; Fig. 1A, right). Human tonsillar B cells stimulated with CD40 Ab for 2–4 d or FIGURE 2. ELISA analysis of sIL-27Ra in the sera of healthy indi- A CD14+ monocytes stimulated with LPS for 3 d also released sIL- viduals. ( ) Levels of sIL-27Ra were measured by ELISA in the sera from healthy individuals. (B) The correlation between sIL-27Ra and IL-27 se- 27Ra, albeit at lower levels (Fig. 1B, 1C). Mature monocyte- rum levels is shown. Although a logarithmic scale is used, linear regression derived dendritic cells that constitutively express high amounts was calculated using the actual values. (C) Sera from six pregnant women, by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. of IL-27Ra mRNA (7) produced variable amounts of sIL-27Ra, collected at various times during pregnancy (6–10 sera per time point), were constitutively (Fig. 1D). tested by sIL-27Ra ELISA. The mean values (6SEM) are represented. (D) Screening of supernatants from human cell lines revealed that Sera from four nonpregnant healthy individuals (Da–d) and four pregnant various cell lines, including B and T cell lines and melanoma cell women (De–h) were tested in a sandwich ELISA using the indicated Abs as lines, released sIL-27Ra. Of these, the KMH2 lymphoma cell line capture Abs and biotinylated anti-human IL-27 Ab as detection Ab. contained among the highest levels of sIL-27Ra (.250 pg/ml). (four nonpregnant and four pregnant individuals) were tested in Detection of sIL-27Ra in sera from healthy human subjects this ELISA. As shown in Fig. 2D, increased IL-27 binding over Next, we tested sera from healthy individuals (n = 28) for the the control was observed when anti–IL-27Ra mAb was used as http://classic.jimmunol.org presence of sIL-27Ra. As shown in Fig. 2A, sIL-27Ra was a capture Ab, indicating that a fraction of sIL-27Ra is associated detected in all samples, at concentrations ranging from 3,108 to with IL-27 in the serum. 31,757 pg/ml (mean 10,344 6 1,274 pg/ml). Serum levels of sIL- a 27Ra were compared with those of IL-27, as measured by IL-27 Biochemical analysis of sIL-27R ELISA. No significant correlation was observed between serum We further characterized the soluble form of IL-27Ra by using levels of IL-27 and sIL-27Ra (Fig. 2B). immunoprecipitation and Western blot. Mature human full-length

Downloaded from In previous studies (3, 4), we had shown that levels of EBI3, IL-27Ra contains a 482-aa extracellular domain comprising seven released by placental trophoblast cells, are strongly upregulated in potential N-glycosylation sites, a 26-aa transmembrane domain, the sera from pregnant women and gradually increase with ges- and a 96-aa intracellular domain, resulting in a transmembrane tational age. Determination of sIL-27Ra levels in sera from six protein with a predicted molecular mass of 66 kDa (22). pregnant women collected at various times during their pregnancy Western blot analysis of the cell lysate of COS7 cells transfected (43 sera, from 9 to 40 wk of pregnancy) showed that serum levels with a plasmid encoding a C-terminal V5-6His–tagged full-length of sIL-27Ra did not increase during pregnancy (Fig. 2C), in IL-27Ra, showed that cell-associated IL-27Ra is detected as contrast to the situation previously observed for EBI3. a doublet of ∼115 and ∼95 kDa (Fig. 3A). These isoforms cor- Soluble forms of cytokine receptors can be complexed with responded to two different N-glycosylated variants, as immuno- the cytokine in the serum (21). To investigate whether sIL-27Ra precipitation of IL-27RaV5His and removal of N-linked sugars by forms complexes with IL-27 in the serum, we developed an ELISA treatment of the immunoprecipitate with N-glycanase resulted in by using an anti–IL-27Ra mAb as a capture Ab and a biotinylated a single band of ∼80 kDa (Fig. 3B). This apparent molecular mass anti–IL-27 Ab as a detection Ab. As positive and negative con- is higher than the predicted one (68 kDa) and suggests that the trols, an anti–IL-27 Ab or a control isotype mAb was used, re- specific amino acid composition of human IL-27Ra results in spectively, as capture Ab. Sera from eight healthy individuals aberrant migration on SDS-PAGE. The Journal of Immunology 5385

FIGURE 3. Biochemical characterization of sIL- 27Ra. Lysates from COS7 cells transiently transfected with vector control or vector expressing IL-27RaV5His (A), and anti–IL-27Ra immunoprecipitates from the lysate of IL-27RaV5His–transfected COS7 cells before or after N-glycanase treatment (B), were analyzed by immunoblot using anti–IL-27Ra Ab. (C) Concentrated culture supernatant from KMH2 cells (left) or human sera (right) was submitted to immunoprecipitation with goat or mouse anti–IL-27Ra Abs, followed by protein G beads, or with anti–IL-27Ra beads, as indicated, and analyzed by anti–IL-27Ra immunoblot. (D) Treatment of anti–IL-27Ra immunoprecipitate from KMH2 cul- ture supernatant with N-glycanase causes a shift in the apparent molecular mass of sIL-27Ra from 90/70 to 60 kDa. Cross-reacting IgH is indicated by an asterisk. Kilodaltons of molecular size markers are reported on the left.

Immunoprecipitation of sIL-27Ra with goat polyclonal or However, in activated CD4+ T cells, a small but significant in- mouse anti–IL-27Ra mAbs from the concentrated supernatant of crease of surface IL-27Ra was observed in cells treated with KMH2 cells (Fig. 3C, left) or from normal human serum (Fig. 3C, GM6001 or TAPI-0 (Fig. 4G, 4H). right) resulted in each case in detection of two diffuse bands of sIL-27Ra binds rIL-27 and antagonizes IL-27 signaling ∼90 and ∼70 kDa, as shown by anti–IL-27Ra immunoblot. Treatment of anti–IL-27Ra immunoprecipitate with N-glycanase Former coimmunoprecipitation studies performed with a re- reduced the apparent molecular mass of sIL-27Ra to a single band combinant soluble extracellular form of IL-27Ra have shown of ∼60 kDa (Fig. 3D), slightly above the predicted size of the that it could bind EBI3/p28 heterodimer, but not individual sub- extracellular domain of IL-27Ra (52.2 kDa). units (2). In addition, a recombinant soluble dimeric IL-27R-Fc fusion protein was reported to act as an inhibitor of IL-27 activity, a Role of metalloproteases in sIL-27R production whereas recombinant soluble gp130-Fc fusion protein had no ef- by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. Soluble forms of cytokine receptors can be generated by alter- fect (15, 25). Therefore, we verified that natural and presumably native splicing or by proteolytic cleavage and shedding of the monomeric sIL-27Ra behaves similarly to the recombinant pro- cell surface cytokine receptor. Because former Northern blot teins and could bind IL-27 and inhibit its biological activity. analyses of human IL-27Ra (22, 23) did not evidence alternative Immunoprecipitation of natural sIL-27Ra from concentrated transcripts that could code for the entire extracellular domain of supernatant of KMH2 cells in the presence of rIL-27 (100 ng per IL-27Ra andnoalternativesplicingwaspredictedinASPic immunoprecipitate) resulted in coprecipitation of rIL-27, but not database, we investigated a potential role of metalloproteases in of free EBI3 naturally present at high levels in KMH2 supernatant the generation of sIL-27Ra. (500 ng per immunoprecipitate) (Fig. 5A), consistent with the Transient transfection of COS7 cells with full-length IL- findings described above. http://classic.jimmunol.org 27RaV5His resulted in cell surface expression of the protein, as To investigate a potential inhibiting role of natural sIL-27Ra,we expected (Fig. 4A), but also in the production of sIL-27Ra that took advantage of the BL2 cell line that responds to low con- accumulated over time in the culture medium, as detected by centrations of IL-27. Titration experiments revealed that 2 ng/ml Western blot (Fig. 4B) or ELISA (Fig. 4C). Production of this (33 pM) rIL-27 induced significant activation of STAT1, but not soluble form was strongly reduced in the presence of two chem- other STATs, in these cells. In this assay, preincubation of rIL-27 ical inhibitors of metalloproteases, GM6001 or TAPI-0 (respec- with IL-27R-Fc prior to cell stimulation resulted in a marked re- Downloaded from tively .80 and 90% inhibition on average, at 48 h) (Fig. 4C). This duction of STAT1 activation in a dose-dependent manner, whereas decrease was specific to the soluble form given that no significant preincubation with gp130-Fc had no effect, even at high doses, as decrease of full-length IL-27RaV5His expression was detected by expected (Fig. 5B). When used at higher concentrations (5 and 10 immunoblot analysis of the cell lysate (Fig. 4D). ng/ml), rIL-27 also activated STAT3 in BL2 cells. Interestingly, Similarly, incubation of KMH2 cells (Fig. 4E) or CD2/CD3/ not only STAT1, but also STAT3 activation was inhibited when CD28-activated CD4+ T cells (Fig. 4F) with GM6001 or TAPI-0 IL-27 was preincubated with either low (10 ng/ml) or high (200 significantly decreased sIL-27Ra production, in a dose-dependent ng/ml) doses of sIL-27R-Fc (Supplemental Fig. 1). This latter find- manner (∼90% inhibition in KMH2 cells and .60% inhibition ing indicates that IL-27 complexed with its soluble receptor does in CD4+ T cells, at the highest concentrations tested), without not activate the gp130/STAT3 axis by trans-signaling, as has been affecting cell proliferation. In transfected COS7 cells or in KMH2 described for IL-6/IL-6Ra complex (14). cells, inhibition of IL-27Ra cleavage did not substantially in- Importantly, preincubation of rIL-27 with almost equimolar crease its cell surface level (data not shown), indicating that only doses of purified natural sIL-27Ra (2 ng/ml, 22–29 pM) induced a small proportion of IL-27Ra was spontaneously shed from the a 50% inhibition of STAT1 activation, whereas a complete inhi- cell surface, as commonly observed for cytokine receptors (24). bition was observed at 10 ng/ml (3- to 4-fold molar excess) 5386 CHARACTERIZATION OF sIL-27Ra by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. FIGURE 4. Role of metalloproteases in the production of sIL-27Ra.(A) COS7 cells were cotransfected with GFP vector and pcDNA3 control vector or pcDNA3 encoding IL-27RaV5His. Two days after transfection, cells were stained with isotype control (filled gray histogram) or anti–IL-27Ra (bold line) Abs and analyzed by FACS. Staining observed in GFP-positive cells is shown. (B) Cell culture supernatants from transfected COS7 cells were tested by Western blot for detection of sIL-27R. Only Abs recognizing the extracellular domain of IL-27Ra, but not its intracellular C terminus (V5 mAb), detected sIL-27Ra.(C)Cell culture supernatants from IL-27RaV5His–transfected COS7 cells, incubated for the indicated times with GM6001, TAPI-0 (both at 50 mM), or DMSO control, were tested by sIL-27Ra ELISA. No sIL-27Ra was detected in the culture supernatant from vector control-transfected COS7 cells. (D) Cell lysates from COS7 cells collected 48 h after transfection were analyzed by immunoblot with anti-V5 Ab to monitor IL-27RaV5His expression levels. On a longer exposure, a smaller band that was weaker in the presence of metalloprotease inhibitors and might correspond to the transmembrane and intracellular domains was observed (data not shown). One representative experiment of two to three is shown in (A)–(D). (E) KMH2 cells were incubated for 3 d with various con-

http://classic.jimmunol.org centrations of GM6001, TAPI-0, or DMSO. Levels of sIL-27Ra measured by ELISA in the culture supernatants and cell numbers determined by trypan blue exclusion are shown as the mean 6 SEM of relative levels of three independent experiments. (F) Purified CD4+ T cells were stimulated for 5 d with CD2/CD3/ CD28 beads and IL-2 and various concentrations of GM6001, TAPI-0, or DMSO. Relative levels of sIL-27Ra and cell numbers observed in four independent experiments performed with different donors are shown. Inhibitor doses .25 mM were not used in CD4+ T cells because they resulted in cell toxicity. (G)Cell membrane IL-27Ra expression (mIL-27Ra) was analyzed by FACS in activated CD4+ T cells cultured for 5 d in the presence of GM6001, TAPI-0 (both at 25 mM), or DMSO. The mean fluorescence intensity of mIL-27Ra staining is indicated. (H) The mean fluorescence intensity of mIL-27Ra staining observed in GM6001 or TAPI-0–treated CD4+ T cells relative to that observed in the DMSO control is shown as mean 6 SEM of four independent experiments. *p , 0.05,

Downloaded from **p , 0.01, ***p , 0.001.

(Fig. 5C). This inhibition was specific to IL-27, because preincubation CD, a Th1-associated granulomatous inflammatory bowel disease of another STAT1-inducing cytokine, IFN-g, with recombinant or characterized by the accumulation of activated myeloid and CD4+ natural sIL-27Ra did not inhibit STAT activation (Fig. 5D). T cells and formation of epithelioid granulomas at the sites of Inhibition of IL-27 signaling by sIL-27Ra was most likely due to disease. We previously showed that IL-27 is produced in situ by a competion effect and inhibition of IL-27 binding to its cell surface epithelioid granulomatous cells of CD patients (26). In addition, receptor. Flow cytometry experiments confirmed that preincubation genetic studies identified IL-27 p28 as a candidate gene for CD of rIL-27 with natural sIL-27Ra inhibited IL-27 binding to BL2 susceptibility and suggested that dysregulated IL-27 expression cells (Fig. 5E). Taken together, these data indicate that natural sIL- could contribute to the pathology (27–29). 27Ra is a potent and specific antagonist of IL-27. Analysis of sera from CD patients with active disease (n = 52) showed that serum levels of sIL-27Ra were significantly higher a Serum levels of sIL-27R are elevated in CD patients than those of healthy controls (Fig. 6A). In certain patients, values Next, we investigated whether altered serum levels of sIL-27Ra were up to 23-fold higher than the average value observed in could be observed under pathological conditions. We focused on controls. Similarly, serum IL-27 levels were higher (by 10-fold The Journal of Immunology 5387

FIGURE 5. Natural sIL-27Ra antagonizes IL-27 signaling. (A) Concentrated cell culture supernatant of KMH2 cells was submitted to immuno- by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. precipitation with control or anti–IL-27Ra Abs, in the absence or presence of 100 ng rIL-27. Immunoprecipitates (lanes 1–4)andafractionofsu- pernatant before immunoprecipitation (lane 5) were analyzed by anti–IL-27Ra and anti-EBI3 immunoblots. (B and C) rIL-27 was preincubated for 15 min at 37˚C with various concentrations of rIL-27R-Fc or gp130-Fc fusion proteins, or purified natural sIL-27Ra, before addition to BL2 cells for 15 min. Cell lysates were analyzed for STAT1 activation with anti–phospho-STAT1 Ab and subsequently with STAT1 Ab to monitor STAT1 levels. (D) Similar experiment was conducted using IFN-g in place of IL-27. (E) Inhibition of IL-27 binding to BL2 cells. rIL-27 (2 ng) was preincubated or not with purified natural sIL-27Ra (2 ng) before incubation with BL2 cells. IL-27 binding was measured by FACS. One representative experiment of two to three is shown.

http://classic.jimmunol.org on average) in CD patients than in controls (Fig. 6B). Overall, from differential N-glycosylation. We demonstrated in in vitro a positive correlation was observed between serum levels of experiments that natural sIL-27Ra can bind IL-27 and inhibits sIL-27Ra and IL-27 in CD patients (Fig. 6C). However, not all its signaling. Because preparations of purified sIL-27Ra contained patients had a concomitant increase of IL-27 and sIL-27Ra levels, both isoforms of sIL-27Ra, we do not know whether both isoforms and the molar ratio between serum levels of IL-27 and sL-27Ra are equally efficient at inhibiting IL-27 signaling. Of note, only was highly variable among patients, ranging from ,0.01 to 20.6 a few fold molar excess of sIL-27Ra over IL-27 was sufficient to

Downloaded from (Fig. 6D). block IL-27 signaling. It should be noted that all in vitro experi- ments were performed using recombinant covalently linked EBI3- Discussion p28 heterodimer. Although we cannot exclude that the affinity of IL-27 is a potent immunoregulator that has drawn interest as rIL-27 for sIL-27Ra might be different from that of natural IL-27, a tool or a target for immunotherapy. Therefore, understanding the our results suggest that sIL-27Ra, at concentrations found in the mechanisms by which IL-27 activity can be modulated is impor- sera of healthy individuals, could inhibit IL-27 activity. tant. In this study, to our knowledge, we report the first evidence IL-27 and its receptor are produced by different cell types. that the IL-27Ra component of the IL-27R complex exists natu- Whereas in normal conditions IL-27 is predominantly expressed by rally as a soluble form that antagonizes IL-27 activity. activated cells of the myeloid lineage, IL-27Ra is constitutively We show that sIL-27Ra is spontaneously released from different expressed by numerous immune on nonimmune cells. This non- human lymphoid and myeloid primary cell types and from cell overlapping spectrum of production may account for the non- lines and is present in the serum of healthy individuals. Immu- correlated levels of IL-27Ra and IL-27 detected in sera from noblot analysis of sIL-27Ra immunoprecipitates from culture healthy individuals. In pregnant women, release of EBI3 and IL- supernatants or from human sera indicated that sIL-27Ra is pro- 27 by placental cells into maternal blood was not associated with duced as glycosylated proteins of ∼90 and ∼70 kDa that result increased levels of sIL-27Ra in the serum, suggesting that pla- 5388 CHARACTERIZATION OF sIL-27Ra

FIGURE 6. Analysis of sIL-27Ra and IL-27 levels in the sera of CD patients. Serum levels of sIL-27Ra (A) and IL-27 (B) were determined by ELISA in 52 CD patients and 28 healthy individuals. The correlation between sIL-27Ra and IL-27 serum levels (C) and the molar ratio between IL-27 and sIL-27Ra serum values (D) are shown. Although a logarithmic scale is used in (C), linear regression was calculated using the actual values.

cental cells are not a major source of sIL-27Ra production. Also, specific sheddase(s), ADAM-17 or others, responsible for IL- we showed by ELISA that part of IL-27 is complexed with sIL- 27Ra cleavage. 27Ra in human sera. Of note, the Abs used to detect IL-27 in this IL-27Ra shedding leads to release of an IL-27 antagonist and may ELISA are polyclonal Abs recognizing both EBI3 and p28, and also, by decreasing levels of cell surface IL-27Ra, lower subsequent therefore able to detect not only IL-27 heterodimer, but also free IL-27 response in producing cells. Production of sIL-27Ra by acti- subunits. However, because IL-27Ra does not bind individual vated cells can therefore constitute a negative feedback mechanism to subunits, the association we detected in serum between IL-27 and dampen IL-27 effects in vivo. sIL-27Ra mayplayabeneficialrolein sIL-27Ra is unlikely to be due to direct interaction between sIL- pathological settings characterized by excessive IL-27 production 27Ra and free EBI3 or p28. Nevertheless, it remains unknown and response linked to the severity of the pathology such as psoriasis whether this association results from direct interaction between (30, 31). Conversely, in pathologies associated with defective IL-27

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. IL-27 and sIL-27Ra or from an indirect association of IL-27 and production/response such as asthma (32, 33), production of sIL-27Ra sIL-27Ra with other serum proteins. by activated T cells may worsen the pathology. Two different mechanisms have been described for the gener- Increased production of IL-27 has been observed in sarcoidosis, ation of soluble cytokine receptors, as follows: synthesis of an tuberculosis, CD, visceral leishmaniasis, psoriasis, rheumatoid ar- alternatively spliced mRNA or proteolytic cleavage of the mem- thritis, and cancer (26, 34–37). The exact role of IL-27 in CD remains brane-anchored receptor. Several lines of evidence support the to be elucidated. IL-27 can both promote and/or limit excessive Th1 involvement of the second mechanism in the generation of sIL- response and inhibit Th17 cell differentiation, and, in murine studies 27Ra. First, there is to date no evidence for a spliced isoform of of colitis, IL-27 has been associated with either promotion or atten- human IL-27Ra coding the entire extracellular domain. Second, uation of the disease, depending on the model (38–41). Interestingly, http://classic.jimmunol.org all cell lines and primary cell types (T, B, and myeloid cells) that human genetics studies identified polymorphisms and epigenetic produce sIL-27Ra in the culture supernatant also exhibit mem- modifications in p28 locus, suggesting that dysregulated p28/IL-27 brane expression of IL-27Ra. Third, transfection of COS7, which production may contribute to CD pathology (27–29). The elevated does not express endogenous IL-27Ra, with full-length IL-27Ra levels of IL-27 that we observed in the sera of CD patients most cDNA resulted in sIL-27Ra production in the culture supernatant. likely result from high levels of IL-27 production by epithelioid cells Last, in transfected COS7, KMH2 cells, and activated CD4+ of granuloma. With respect to sIL-27Ra, both activated T cells and

Downloaded from T cells, production of sIL-27Ra was blocked by the addition of epithelioid cells could contribute to its production. Further studies two hydroxamate-based metalloprotease inhibitors, GM6001 and would be necessary to determine whether variable ratios between TAPI-0. The fact that metalloproteases are involved in sIL-27Ra serum IL-27 and sIL-27Ra levels may be indicative of different production does not exclude, however, that sIL-27Ra could be clinical features and outcome of disease. generated by alternative splicing in other cell types and/or under different conditions. Acknowledgments The specific metalloproteases involved in the cleavage of We thank Hugues Gascan (Angers, France) for the gift of IL-27Ra-V5-6His surface IL-27Ra remain to be identified. Previous studies have plasmid. shown that a given receptor is often cleaved by more than one metalloprotease (11). GM6001 is a broad spectrum inhibitor of Disclosures matrix metalloproteases (MMP) that inhibits numerous MMPs and The authors have no financial conflicts of interest. members of the ADAM (a disintegrin and metalloproteinase) family. Similarly, TAPI-0, a potent inhibitor of ADAM-17, References inhibits other ADAM family members and several MMPs. 1. Devergne, O., M. Hummel, H. Koeppen, M. M. Le Beau, E. C. Nathanson, Therefore, further investigation will be required to uncover the E. Kieff, and M. Birkenbach. 1996. A novel -12 p40-related protein The Journal of Immunology 5389

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