IL-17RA/IL-17RC Receptor Complex Cytokine Signals Through the The

The Human IL-17F/IL-17A Heterodimeric Cytokine Signals through the IL-17RA/IL-17RC Receptor Complex

This information is current as Jill F. Wright, Frann Bennett, Bilian Li, Jonathan Brooks, of October 1, 2021. Deborah P. Luxenberg, Matthew J. Whitters, Kathleen N. Tomkinson, Lori J. Fitz, Neil M. Wolfman, Mary Collins, Kyri Dunussi-Joannopoulos, Moitreyee Chatterjee-Kishore and Beatriz M. Carreno J Immunol 2008; 181:2799-2805; ; doi: 10.4049/jimmunol.181.4.2799 Downloaded from http://www.jimmunol.org/content/181/4/2799

References This article cites 32 articles, 14 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/181/4/2799.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Human IL-17F/IL-17A Heterodimeric Cytokine Signals through the IL-17RA/IL-17RC Receptor Complex

Jill F. Wright,1* Frann Bennett,2* Bilian Li,2† Jonathan Brooks,* Deborah P. Luxenberg,* Matthew J. Whitters,* Kathleen N. Tomkinson,3* Lori J. Fitz,* Neil M. Wolfman,* Mary Collins,* Kyri Dunussi-Joannopoulos,* Moitreyee Chatterjee-Kishore,4† and Beatriz M. Carreno5*

החתום conditions. We recently reported that activated human CD4؉ T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface Downloaded from plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be http://www.jimmunol.org/ exploited to differentially affect the activity of these cytokines. The Journal of Immunology, 2008, 181: 2799–2805.

nterleukin 17A (IL-17A) is the founding member of the likely that IL-17A and IL-17F adopt a similar structure. Recently, proinﬂammatory IL-17 cytokine family. This cytokine family we have shown that activated human CD4ϩ T cells not only ex- I consists of six members (A–F), with IL-17A and IL-17F press IL-17A and IL-17F homodimers, but also an IL-17F/IL-17A sharing the closest amino acid sequence identity of ϳ50%. IL-17A heterodimer, and that the conserved cysteines used in the knot and IL-17F are homodimeric cytokines produced by the Th17 T formation of IL-17A and IL-17F homodimers are the cysteines cell lineage and share similar biological activities, including in-

involved in the disulfide linkage of the IL-17F/IL-17A heterodimer by guest on October 1, 2021 duction of cytokines and chemokines involved in inflammatory (20). The murine IL-17F/IL-17A heterodimer has also been shown responses (1–7). IL-17A and IL-17F have been implicated in a to be expressed by differentiated Th17 cells (21, 22). variety of autoimmune diseases, such as rheumatoid arthritis, mul- The IL-17 receptor family consists of five members: RA, RB, tiple sclerosis, inflammatory bowel disease, asthma, and psoriasis RC, RD, and RE. IL-17A has been shown to bind IL-17RA (also (5, 6, 8–16). referred to as IL-17R) with high affinity, and IL-17RA is required The crystal structure of IL-17F has been reported (17) and for the biological activity of IL-17A (17, 23, 24). Binding of IL- shows that IL-17F forms a disulfide-linked dimer that contains a 17F to IL-17RA was not detected by surface plasmon resonance; cysteine knot motif similar to that reported for members of the however, weak binding of IL-17F to cells expressing IL-17RA was nerve growth factor and TGF-␤ superfamilies (18, 19). Given the detected, suggesting that both IL-17A and IL-17F utilize IL-17RA high degree of amino acid homology between IL-17A and IL-17F as part of their receptor complex (17). The IL-17RA receptor is a and the conservation of the four cysteines that form the knot, it is preformed multimeric complex and is thought to undergo a conformational change upon binding to IL-17A or IL-17F (25). *Department of Inflammation and †Biological Technologies, Wyeth Research, Cam- It has been shown that IL-17A cannot bind to T cells, B cells, bridge, MA, 02140 and myeloid cells that are deficient in IL-17RA (24). Toy et al. Received for publication August 23, 2007. Accepted for publication June 2, 2008. showed that human IL-17A or IL-17F could not elicit a response Ϫ/Ϫ The costs of publication of this article were defrayed in part by the payment of page on mouse IL-17RA cells and the activity could only be re- charges. This article must therefore be hereby marked advertisement in accordance gained by the cotransfection of human IL-17RA and IL-17RC, with 18 U.S.C. Section 1734 solely to indicate this fact. demonstrating that both IL-17RA and IL-17RC are necessary for 1 Address correspondence and reprint requests to Dr. Jill F. Wright, Department of Inflammation, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140. the biological activity of IL-17A and IL-17F (26). More recently, E-mail address: [email protected] it has been shown that a soluble form of IL-17RC could neutralize 2 F.B. and B.L. contributed equally to this work. the activity of IL-17A and IL-17F on human bronchial epithelial 3 Current address: Acceleron Pharma, 24 Emily Street, Cambridge, MA 02139. cells stimulated with IL-17 cytokine plus TNF-␣ (27). 4 Current address: Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL In this report, we investigate the roles of IL-17RA and IL-17RC 60064. receptors in mediating the biological activity of the IL-17F/IL-17A 5 Current address: Washington University School of Medicine, Division of Oncology, heterodimer. We find that the heterodimer, along with IL-17A and 660 South Euclid Avenue, Campus Box 8007, St. Louis, MO 63110. IL-17F homodimers, requires IL-17RA and IL-17RC receptors for Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 signaling. Additionally, our results strongly suggest that different www.jimmunol.org 2800 IL-17F/IL-17A SIGNALS THROUGH IL-17RA/IL-17RC COMPLEX

Table I. siRNAs used for human IL-17RA and IL-17RC knockdown

siRNA Cat. No. Sequence

Hs_IL17R_1_HP S100104979 CAG CGG TCT GGT TAT CGT CTA Hs_IL17R_2_HP S100104986 CGG CAC CTA CGT AGT CTG CTA Hs_IL17R_3_HP S100104993 CAG GAA GGT CTG GAT CAT CTA Hs_IL17R_4_HP S100105000 CAG GTT TGA GTT TCT GTC CAA Hs_IL17RC_1_HP S100144165 ACC GCA GAT CAT TAC CTT GAA Hs_IL17RC_2_HP S100144172 CAG GTA CGA GAA GGA ACT CAA Hs_IL17RC_3_HP S100144179 CGG GAC TTA AAT AAA GGC AGA Hs_IL17RC_4_HP S100144186 CCG CGC GGC TCT GCT CCT CTA

biological activities among homodimers and heterodimer of IL- ng/ml) was added to plates and incubated for 3 h, followed by serial di- 17A and IL-17F cytokines may be attributed to afﬁnity differences lutions of biotinylated IL-17A, IL-17F, and IL-17F/IL-17A for2hatroom among these cytokines for IL-17RA and IL-17RC receptors. temperature. ELISA was developed with poly-HRP strepavidin (Pierce Biotechnology) and tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories). Materials and Methods Reagents siRNA

Human IL-17F, IL-17A, and IL-17F/IL-17A were puriﬁed as previously Twenty-four hours before transfection, BJ cells were seeded at 104 cells/ Downloaded from described (20) and used in all experiments except small interfering RNA well in 96-well plates. Cells were transfected with siRNA (20 nM) using (siRNA)6 experiments, where IL-17A from R&D Systems was used. IL- DharmaFECT 1 transfection reagent (Dharmacon) according to the man- 17F, IL-17A, and IL-17F/IL-17A were biotinylated according to the manufacturer’s instructions. Each transfection contained one of eight siRNAs ufacturer’s protocol using FluoReporter Minibiotin-XX protein labeling kit listed in Table I. siRNAs were obtained from Qiagen. At 24 h, transfection (Molecular Probes). Anti-IL-17RA and anti-IL-17RC polyclonal Abs used medium was removed and replaced with fresh medium or culture medium in functional assays were purchased from R&D systems. Human IL- containing IL-17A, IL-17F, or IL-17F/IL-17A at the indicated concentrations. After 16 h, cells were harvested for mRNA preparation and super-

17RA.Fc and IL-17RC.Fc were puriﬁed as described below. http://www.jimmunol.org/ natants were collected and analyzed for GRO-␣ production by ELISA Cloning of the human IL-17 receptors (R&D Systems).

Full-length human IL-17RA and IL-17RC were PCR amplified from un- TaqMan stimulated MG63 cDNA and sequence confirmed. The extracellular portions of human IL-17RA (residues 1–317) and human IL-17RC (residues The TaqMan gene expression assay probe-primer sets for IL-17R 1–452) were fused in frame with a GSGSGSG linker and the human IgG1 (Hs00234888_m1, 20ϫ) and IL-17RC (Hs00262062_m1, 20ϫ) were ob- Fc portion (28). PCR-derived fusion receptors were subcloned into a CMV tained from Applied Biosystems. BJ cell mRNA was isolated using promoter-driven mammalian expression construct and sequence verified. TurboCapture mRNA kit (Qiagen) according to the manufacturer’s instructions; mRNA was eluted in 50 ␮l of elution buffer. Ten microliters of Expression and purification of the human IL-17RA.Fc and ϫ mRNA per sample and 1 of probe-primer mixture per samples were used by guest on October 1, 2021 IL-17RC.Fc receptors in 25 ␮l TaqMan PCR reactions (30 min at 48°C, 10 min at 95°C, then 40 cycles each of 15 s at 95°C and 1 min at 60°C on MicroAmp optical HEK 293 cells were transiently transfected with IL-17RA.Fc or IL- 96-well plates/caps) performed on an ABI Prism 7700 DNA sequence de- 17RC.Fc expressing vectors using TransIT-LT1 (Mirus Bio). Twenty-four tector (Applied Biosystems). Each plate contained triplicates of the test hours post transfection, media was removed and replaced with serum-free mRNA templates and no-template controls for each reaction mix. The ex- media. Conditioned media was harvested at 48 h and protein production ␤ pression for each gene was normalized to human 2-microglobulin gene evaluated by Western blot analysis. Media containing IL-17RA.Fc or IL- expression. 17RC.Fc was flowed over a protein A column (Amersham Biosciences), washed with PBS, and protein eluted with 20 mM citric acid, 200 mM Western blot analysis NaCl (pH 3). Protein aggregates were removed by size exclusion chromatography (PBS (pH 7.2)) and the resulting preparations dialyzed (PBS (pH HEK 293 cells (1.2 ϫ 105 cells/ml) stably transfected with human IL- 7.2)) and characterized by SDS-PAGE, Western blot analysis, and analyt- 17RA or IL-17RC plasmid were seeded in 96-well plates. After 16–24 h ical size-exclusion chromatography. the cells were transfected with 20 nM of siRNA and DharmaFECT transfection reagent (Dharmacon) according to the manufacturer’s protocol. At Cell-based assay 48 h, cells were washed once with PBS and lysed on ice using M-PER, mammalian protein extraction reagent (Pierce Biotechnology). Extracted BJ human foreskin fibroblast cells (American Type Culture Collection) protein was then loaded on an SDS-PAGE gel, transferred to a nylon mem- were maintained in DME ϩ 10% FCS, 2 mM glutamine, 1 mM sodium brane, and the membrane blocked with 5% nonfat dried milk in PBS con- pyruvate, 0.1 mM MEM nonessential amino acids, and 100 U/ml penicillin ϩ taining 0.1% Tween 20. IL-17RA or IL-17RC Ab was added at 1/4000 and 100 ␮g/ml streptomycin. BJ cells were seeded at 5 ϫ 103 cells/well into incubated overnight at 4oC. Membranes were washed three times (PBS 96-well flat-bottom microtiter plates in which cytokines had been predi- with 0.1% Tween 20) and incubated with donkey anti-goat IgG-HRP (1/ luted in culture medium with or without soluble receptors. In treatments 2000 dilution, Santa Cruz Biotechnology). Proteins were visualized using where Abs to cell-surface receptors were used, Abs to the receptors were Western Lightning Western Blot Chemiluminescence Reagent Plus added (20 ␮g/ml) to the cells before the addition of cytokine at the indi- (PerkinElmer). cated concentration. Cells were incubated at 37°C for 16–24 h, and supernatants were collected and analyzed for growth-related oncogene (GRO)-␣ production by ELISA (R&D Systems). Binding kinetics of the IL-17 cytokines with receptors Kinetic analysis was performed using a Biacore 2000 (Biacore). All ex- ELISA periments were performed at 22°C. IL-17RA.Fc or IL-17RC.Fc was im- Binding of human IL-17A, IL-17F, and IL-17F/IL-17A to human IL- mobilized between 1000 and 2000 resonance units (RU) using amine 17RA.Fc and IL-17RC.Fc was determined by indirect sandwich ELISA. coupling as directed by the manufacturer. Human IL-17F, IL-17A, and ELISA plates (Costar) were coated with goat anti-human IgG-Fc (10 ␮g/ IL-17F/IL-17A heterodimer were each diluted into HBST buffer (10 mM ml, Bethyl Laboratories). Human IL-17RA.Fc (6 ng/ml) or IL-17RC.Fc (30 HEPES with 0.15 M NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) at concentrations of 400, 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 nM. Each sample was injected at 50 ␮l/min for 3 min followed by 10-min dissoci- 6 Abbreviations used in this paper: siRNA, small interfering RNA; GRO-␣, growth- ation. The surface was regenerated by a 30-s injection of a solution of related oncogene-alpha. 0.549 M MgCl2, 0.138 M potassium thiocyanate, 0.276 M urea, and 0.549 The Journal of Immunology 2801 Downloaded from http://www.jimmunol.org/

FIGURE 2. IL-17F/IL-17A heterodimer can bind independently to IL- 17RA and IL-17RC. Goat anti-human IgG (10 ng/ml) was used to capture FIGURE 1. Functional activity of IL-17 cytokines. A, Human primary

IL-17RA.Fc (6 ng/ml) or IL-17RC.Fc (30 ng/ml), and biotinylated IL-17A, by guest on October 1, 2021 ϫ 3 foreskin fibroblast (BJ) cells were seeded at 5 10 cells/well into 96-well IL-17F, or IL-17F/IL-17A was added at the indicated concentrations. ELISA plates containing IL-17A (1 ng/ml), IL-17F/IL-17A heterodimer (5 ng/ml), was developed as described in Materials and Methods. A, IL-17A, IL-17F, and or IL-17F (50 ng/ml) and incubated at 37°C for 16–24 h. Supernatants IL-17F/IL-17A binding to IL-17RC.Fc. B, IL-17A, IL-17F, and IL-17F/IL- ␣ were collected and analyzed in triplicate for GRO- , IL-6, and IL-8 pro- 17A binding to IL-17RA.Fc. Data are representative of two experiments. duction by ELISA. Stimulation index represents the data normalized to those obtained with media control. Data represent three experiments. B, Human primary foreskin fibroblast (BJ) cells were seeded at 5 ϫ 103 cells/ well into 96-well plates containing serial dilutions of purified IL-17A, IL- the absence of cytokine stimulation and the increased signal-to- 17F/IL-17A, or IL-17F and incubated at 37°C for 16–24 h. Supernatants background ratio (8- to 12-fold) upon cytokine stimulation. IL-17F were collected and analyzed in triplicate for GRO-␣ production by ELISA. was less potent than IL-17A, and the IL-17F/IL-17A heterodimer Data are representative of seven experiments. was found to be more potent than IL-17F but less potent than IL-17A (Fig. 1B). The potency differences are in agreement with data reported previously on human bronchial epithelial cells (20) M guanidine-HCl followed by two consecutive 60-s HBST buffer injec- and on mouse embryonic fibroblast cells (21). tions. Data were double referenced (29) using Scrubber2 software (Bio- Logic Software), then fit to a 1:1 binding model using Biacore evaluation IL-17F, IL-17A, and IL-17F/IL-17A bind to the IL-17RA/IL-17RC software version 3.2. receptor complex with different kinetics Indirect sandwich ELISAs were done to evaluate the binding of Results IL-17F, IL-17A, and IL-17F/IL-17A to IL-17RC.Fc and IL- Functional activity of IL-17A, IL-17F, and IL-17F/IL-17A on 17RA.Fc (Fig. 2). We confirmed, as previously reported (30), that BJ cells IL-17A and IL-17F homodimers can bind independently to IL- We have previously reported that IL-17F, IL-17A, and IL-17F/IL- 17RA or IL-17RC. IL-17F/IL-17A heterodimer can also bind in- 17A induce the secretion of GRO-␣ from the BEAS-2B human dependently to either IL-17RC or IL-17RA (Fig. 2). All three of bronchial epithelial cell line (20). We have extended this observa- the IL-17 cytokines bind to IL-17RC.Fc with approximately the tion to BJ, a primary human foreskin fibroblast cell. ELISA anal- same EC50 of 12–17 ng/ml (Fig. 2A). However, the EC50 for the ysis of conditioned medium obtained from BJ cells cultured with IL-17 cytokines are quite different for binding to IL-17RA.Fc. purified IL-17F, IL-17A, or the IL-17F/IL-17A heterodimer The lowest EC50 occurs between IL-17A and IL-17RA.Fc with a showed that all three cytokines can induce GRO-␣, IL-6, and IL-8 value of 19 ng/ml, whereas IL-17F binds weakly to IL-17RA.Fc Ͼ secretion, albeit with different potencies (Fig. 1A). We have chosen with an EC50 2000 ng/ml. The IL-17F/IL-17A heterodimer has ␣ to evaluate functional activity of IL-17 cytokines using GRO- an EC50 of 329 ng/ml, which is 10-fold higher than the binding of production as a readout due to low levels of GRO-␣ secretion in IL-17A but ϳ10-fold lower than the IL-17F, as shown in Fig. 2B. 2802 IL-17F/IL-17A SIGNALS THROUGH IL-17RA/IL-17RC COMPLEX

Table II. Kinetic rate constants of IL-17 cytokines binding to IL-17RA.Fc and IL-17RC.Fc as determined by Biacorea

Ϫ1 Injected Analyte Immobilized Ligand kon (M/s) koff (s ) Kd (M) IL-17A IL-17RA-Fc 1.39 Ϯ 0.15 ϫ 105 2.94 Ϯ 0.70 ϫ 10Ϫ4 2.15 Ϯ 0.73 ϫ 10Ϫ9 IL-17F IL-17RA-Fc 9.43 Ϯ 0.38 ϫ 103 1.64 Ϯ 0.10 ϫ 10Ϫ3 1.74 Ϯ 0.07 ϫ 10Ϫ7 IL-17F/IL-17A IL-17RA-Fc 4.28 Ϯ 1.46 ϫ 104 1.03 Ϯ 0.01 ϫ 10Ϫ3 2.55 Ϯ 0.83 ϫ 10Ϫ8 IL-17A IL-17RC-Fc 8.92 Ϯ 0.39 ϫ 104 1.79 Ϯ 0.08 ϫ 10Ϫ3 2.01 Ϯ 0.18 ϫ 10Ϫ8 IL-17F IL-17RC-Fc 1.28 Ϯ 0.07 ϫ 105 2.12 Ϯ 0.20 ϫ 10Ϫ3 1.66 Ϯ 0.06 ϫ 10Ϫ8 IL-17F/IL-17A IL-17RC-Fc 1.44 Ϯ 0.15 ϫ 105 1.51 Ϯ 0.16 ϫ 10Ϫ3 1.06 Ϯ 0.22 ϫ 10Ϫ8

a Shown are the means and SDs from three independent experiments.

To better characterize the binding kinetics of human IL-17F, the Kd of the IL-17F/IL-17A heterodimer (26 nM) is intermediate IL-17A, and IL-17F/IL-17A to each receptor component, the as- between IL-17A and IL-17F. In contrast to their different binding sociation and dissociation rate constants were measured by surface kinetics for IL-17RA.Fc, IL-17F, IL-17A, and IL-17F/IL-17A, plasmon resonance. The calculated dissociation constants are each bind IL-17RC.Fc with similar on and off rates and hence also shown in Table II. Each IL-17 cytokine tested binds IL-17RA.Fc have similar Kd values of 10–20 nM. Altogether, these results with a different Kd, which is reflected by differences in the on and indicate that IL-17A binds better to IL-17RA than IL-17RC, IL- 5 off rates. IL-17A binds with the fastest on rate (1.4 ϫ 10 M/s) and 17F binds to IL-17RC with ϳ10-fold higher affinity than to IL- Downloaded from ϫ Ϫ4 slowest off rate (2.9 10 /s), which result in the lowest Kd of 2.2 17RA, while IL-17F/IL-17A binds with similar affinity to both nM. IL-17F has about a 10-fold slower on rate (9.4 ϫ 103 M/s) and receptors. 10-fold slower off rate (1.6 ϫ 10Ϫ3/s), which results in about a IL-17RA and IL-17RC siRNA decrease IL-17F, IL-17A, and 100-fold higher Kd for IL-17RA.Fc (170 nM) compared with IL- 17A. Interestingly, the IL-17F/IL-17A heterodimer has an on rate IL-17F/IL-17A activity ϫ 4 (4.3 10 M/s) that is intermediate between IL-17A and IL-17F We used siRNA to knock down the expression of the IL-17RA and http://www.jimmunol.org/ Ϫ whereas the off rate (1.0 ϫ 10 3/s) is the same as IL-17F. Overall, IL-17RC receptors to investigate the requirement of these two receptors for the functional activity of IL-17F, IL-17A, and IL-17F/ by guest on October 1, 2021

FIGURE 3. Effect of siRNA on the expression of IL-17RA and IL- 17RC. BJ cells were transfected with either IL-17RA or IL-17RC siRNA as described in the Materials and Methods. A, The amount of knockdown FIGURE 4. Effect of IL-17RA and IL-17RC siRNA on IL-17 cytokine for IL-17RA and IL-17RC in the transfected BJ cells was measured by response. BJ cells were transfected with IL-17RA or IL-17RC siRNAs quantitative PCR and plotted as percentage response to a siRNA control identiﬁed in Fig. 3. The transfection medium was removed and replaced (NTC1). B, HEK 293 cells expressing IL-17RA or IL-17RC were trans- with culture medium containing IL-17A (A), IL-17F (B), or IL-17F/IL-17A fected with either IL-17RA or IL-17RC siRNA. At 48 h, cells were col- (C) at indicated concentrations. After 16 h, supernatants were collected and lected, protein was extracted, and receptor expression was analyzed by analyzed in triplicate for GRO-␣ secretion by ELISA. Data are represen- Western blot. tative of three experiments. The Journal of Immunology 2803

to IL-17RA and IL-17RC can block the activity of IL-17F and IL-17A (30). BJ cells were stimulated with suboptimal concentrations of cytokines in the presence or absence of Abs to IL-17RA and IL-17RC. As shown in Fig. 5A, IL-17A-, IL-17F/IL-17A-, and IL-17F-dependent induction of GRO-␣ was signiﬁcantly decreased when the cells were treated with anti-human IL-17RA or anti-human IL-17RC Abs. However, anti-IL-17RC Ab had a more profound effect on the activity of IL-17F than on the activities of IL-17A and IL-17F/IL-17A heterodimer. These results, in con- junction with the siRNA knockdown experiments, strongly sup- port the notion of a common receptor complex for IL-17A, IL-17F, and IL-17F/IL-17A.

Preferential inhibition of homodimers and heterodimer of IL-17A and IL-17F cytokines by soluble forms of IL-17RA and IL-17RC Our surface plasmon resonance results demonstrated different binding affinities between IL-17 cytokines and IL-17RA and IL- 17RC receptor components (Table II), so we investigated whether the soluble form of the receptors would affect differentially the Downloaded from functional activity of IL-17A, IL-17F, and IL-17F/IL-17A. Soluble receptor forms were generated by in-frame fusing of the extracellular portions of IL-17RA or IL-17RC to human IgG1 Fc portions, and protein production was done in HEK 293 cells. The ability of FIGURE 5. Effect of Abs to IL-17RA or IL-17RC or soluble forms of the soluble receptors to inhibit cytokine activity was determined at the receptor on IL-17 cytokine activity. A, BJ cells were seeded at 5 ϫ 103 a 50-fold molar excess of receptor to cytokine. As shown in Fig. http://www.jimmunol.org/ cells/well into plates containing either anti-IL-17RA (20 ␮g/ml) or anti- 5B, IL-17RA.Fc and IL-17RC.Fc significantly neutralize the ac- IL-17RC (20 ␮g/ml) Abs. IL-17A (1 ng/ml), IL-17F/IL-17A (5 ng/ml), or tivity of IL-17A or IL-17F, respectively, at Ͼ50% inhibition. Nei- IL-17F (50 ng/ml) was then added and cells incubated at 37°C for 16–24 ther IL-17RA.Fc nor IL-17RC.Fc alone could significantly inhibit ␣ h, followed by analysis of the supernatants for GRO- secretion by ELISA. the activity of the IL-17F/IL-17A heterodimer. Interestingly, a GRO-␣ production for the various Ab/cytokine conditions was normalized combination of soluble IL-17RA and IL-17RC neutralized ϳ50% to that obtained with cells culture in cytokine alone. Data represent an average of three experiments. B, IL-17A (1 ng/ml), IL-17F/IL-17A (5 ng/ of the activity of IL-17F/IL-17A heterodimer. These findings sug- ml), or IL-17F (50 ng/ml) was preincubated with soluble receptors at a gest that a combination of both receptors is required to neutralize the activity of the IL-17F/IL-17A heterodimer whereas only the 50-fold molar excess, isotype control, or media followed by addition of BJ by guest on October 1, 2021 cells. Supernatants were harvested between 16 and 20 h and analyzed for higher binding affinity receptor, either IL-17RA or IL-17RC, is GRO-␣ secretion by ELISA. GRO-␣ production for the various Ab/cyto- required for neutralizing the activity of IL-17A or IL-17F ho- kine conditions was normalized to that obtained with cells cultured in cy- modimers, respectively. tokine alone. Data represent an average of three experiments. Discussion In this study, we show that human IL-17F/IL-17A heterodimer, the IL-17A. BJ cells were transfected with various IL-17RA (RA-1 to recently identified member of the IL-17 cytokine family, utilizes RA-4) or IL-17RC (RC-1 to RC-4) siRNAs, and the level of IL- the same receptor complex as the IL-17F and IL-17A cytokines. 17RA and IL-17RC transcript expression was determined by quan- Using various experimental approaches, including surface plasmon titative PCR (Fig. 3A). Additionally, the effect of siRNA on IL- resonance and siRNA gene knockdown, we characterized the 17RA and IL-17RC protein expression in HEK 293 cells physical and functional interactions of IL-17F/IL-17A, IL-17F, overexpressing IL-17RA or IL-17RC was determined by Western and IL-17A with the IL-17RA and IL-17RC receptors and propose blot analysis (Fig. 3B). Based on the specific inhibition of both that all three cytokines require both receptors for their biological mRNA transcripts and protein, two siRNAs per receptor were se- activity. lected to evaluate IL-17RA (RA-3 and RA-4) and IL-17RC (RC-2 Toy et al. demonstrated that human IL-17A or IL-17F could not and RC-4) activity in functional assays. IL-17F, IL-17A, and IL- induce CXCL1 expression in IL-17RAϪ/Ϫ fibroblast cells and that 17F/IL-17A were added at three different concentrations to BJ cells transfection of human IL-17RA did not rescue the expression of transfected with IL-17RA, IL-17RC, or control (NTC1) siRNAs CXCL1 as was seen in wild-type cells (26). When the cells were (Fig. 4). IL-17RA and IL-17RC siRNAs decreased GRO-␣ pro- cotransfected with both human IL-17RA and IL-17RC and treated duction in response to IL-17A, IL-17F, or IL-17F/IL-17A at all with either human IL-17A or IL-17F, production of CXCL1 was three concentrations tested relative to the siRNA control. These restored, suggesting that a heterodimeric IL-17RA/IL-17RC recep- results suggest that the IL-17F/IL-17A heterodimer activity de- tor was required for signaling (26). Recently, it has been reported pends on both IL-17RA and IL-17RC and thus, this cytokine that IL-17RA is also required for murine IL-17F/IL-17A signaling shares a common receptor complex with IL-17A and IL-17F. (21). We confirm and extend these findings by assessing the function of human IL-17F/IL-17A heterodimer in more physiological Anti-IL-17RA and anti-IL-17RC decrease the functional activity primary human foreskin fibroblast cells. We find that Ab blockade of IL-17F, IL-17A, and IL-17F/IL-17A or siRNA knockdown of either receptor component leads to loss of To further delineate the role of IL-17RA and IL-17RC, we eval- both homodimeric and heterodimeric cytokine activity. These data uated whether Abs to IL-17RA and IL-17RC could block IL-17F/ are consistent with a model in which both receptor chains are re- IL-17A functional activity. It has been previously shown that Abs quired to form a functional receptor complex. Although IL-17A 2804 IL-17F/IL-17A SIGNALS THROUGH IL-17RA/IL-17RC COMPLEX and IL-17F homodimers can bind independently to IL-17RA and has been shown that IL-17RA is a preformed multimeric complex IL-17RC (Table II), both chains are required for IL-17A and IL- and that receptor association is reduced in the presence of IL-17A 17F biological function. or IL-17F, suggesting a conformational change of the receptor due In our study, direct binding measurements with purified IL-17 to ligand binding (25). Furthermore, it has been proposed that li- cytokines and receptors indicate that while all three IL-17 cyto- gand binding alters the conformation of IL-17RA to facilitate a kines bind with similar affinities to IL-17RC, they bind very dif- functional, heterotypic interaction with IL-17RC (26). It is tempt- ferently to IL-17RA. The order of binding to the receptors and the ing to speculate that one of the receptors binds to the large cavity binding affinities for receptor chains may determine the molecular present in the homodimer or heterodimer interface, leading to a mechanisms that differentiate the biological functions of these cy- conformational change and recruitment of the second receptor. tokines. Based on the binding kinetics, we propose that IL-17F is The recent emergence of the effector Th17 T cell lineage has likely to associate with IL-17RC and not IL-17RA, unless IL- reshaped the Th1/Th2 landscape and presents a new arm of adap- 17RA is in great abundance. On the other hand, IL-17A would be tive immunity that provides explanations to some aspects of im- predicted to preferentially bind IL-17RA complexes. The IL-17F/ mune regulation and immune pathogenesis. While considerable IL-17A heterodimer is likely to interact equally with either recep- data related to the expression of IL-17A and IL-17F cytokines tor chain complex. The pattern of binding affinities that we dem- have been generated recently, detailed functional studies of the onstrate for IL-17A and IL-17F is the same as in previous reports IL-17 receptor family have not yet been reported. Currently, it is (17, 27), but the dissociation constants differ. The difference in not known if IL-17F, IL-17A, and IL-17F/IL-17A have redundant relation to IL-17RA may be attributed to the monomeric form of roles in certain disease settings. Along these lines, studies have the IL-17RA protein used in previous experiments (17) vs the been shown that IL-17A-deficient mice are resistant to the devel- dimeric IL-17RA receptor Ig Fc fusion construct used in the opment of collagen-induced arthritis, suggesting that IL-17F can- Downloaded from present study. Consistent with the higher affinity of IL-17A com- not compensate for IL-17A in the pathogenesis of collagen-in- pared with IL-17F for IL-17RA, we show that the IL-17RA.Fc duced arthritis (15). By contrast, intranasal administration of either fusion protein inhibits cellular responses mediated by IL-17A bet- IL-17A or IL-17F adenovirus leads to pulmonary neutrophilia and ter than those mediated by either IL-17F or IL-17F/IL-17A het- inflammatory gene expression in the lungs (32). erodimer (Fig. 5B and Ref. 30). Both soluble receptors, IL- Based on our finding of a shared use of receptors by IL-17A, 17RA.Fc and IL-17RC.Fc, which have approximately the same IL-17F, and IL-17F/IL-17A, an interesting question arises from http://www.jimmunol.org/ binding affinity for the IL-17F/IL-17A heterodimer, are required to our studies as to whether IL-17F/IL-17A contributes partially to inhibit the activity of IL-17F/IL-17A. The affinities we report for the activity observed for IL-17A in vitro and in vivo. Tissue ex- IL-17A and IL-17F binding to IL-17RC are weaker than a previous pression comparing IL-17F/IL-17A heterodimer, IL-17F, and IL- report even though both studies used the same spliced form of 17A might define the role of the heterodimer. In conclusion, we IL-17RC and both used a receptor Ig Fc reagent (27). However, have shown that IL-17F/IL-17A, IL-17F, and IL-17A share a com- these studies differed in how the receptor was immobilized on the mon receptor complex. However, due to binding affinity differ- sensor chip surface, which, in turn, may affect binding affinity of ences between the IL-17 cytokines and soluble receptors, each the ligand (31). receptor component may be exploited to target either IL-17A, IL- by guest on October 1, 2021 Biological activity of the IL-17 family may differ among cell 17F, or IL-17F/IL-17A cytokine. Elucidating the complexity re- types as a result of IL-17 receptor composition (homodimeric and lated to regulation of the expression of various IL-17A–F family heterodimeric forms). For instance, we observed that soluble IL- members and the biologic significance of their interactions with 17RC.Fc, when used at 50-fold molar excess, can neutralize the their cognate receptor(s) will enhance their potential as therapeutic biological activity of IL-17F but not IL-17A on primary human targets in inflammation and autoimmunity. foreskin fibroblast cells even though they have a similar binding affinity for the IL-17RC (Fig. 5B). On human bronchial epithelial Disclosures cells, Kuestner et al. (27) have reported neutralization of IL-17A The authors have no financial conflicts of interest. activity (in combination with TNF-␣) by IL-17RC used at a 217- fold molar excess. These disparate results may be partially attrib- References uted to differences in receptor composition among cell lines tested 1. Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, and A. L. Gurney. 2003. 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