Clinical and Experimental Immunology
OR I GI NA L ART IC LE
doi:10.1111/cei.12751
Interleukin-21 induces migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis
R. Xing,* Y. Jin,* L. Sun,* L. Yang,* C. Li,* Z. Li,† X. Liu* and J. Zhao* *Department of Rheumatology and †
Immunology, and Department of Anesthesiology, Peking University Third Hospital, Beijing, PR China
Accepted for publication 1 December 2015 Correspondence: X. Liu and J. Zhao, Department of Rheumatology and Immunology, Peking University Third Hospital, No. 49, North Garden Road, Beijing 100191, China. E-mail: [email protected]; [email protected]
Summary Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast-like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)-21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL-21 on the migration, invasion and matrix metalloproteinase (MMP) expression in RA-FLS. We found that IL-21 promoted the migration, invasion and MMP (MMP-2, MMP-3, MMP-9, MMP-13) production in RA-FLS. Moreover, IL-21 induced activation of the phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription-3 (STAT-3) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, and blockage of these pathways [PI3K/protein kinase B (AKT) inhibitor LY294002, STAT-3 inhibitor STA-21 and ERK1/2 inhibitor PD98059] attenuated IL-21-induced migration and secretion of MMP-3 and MMP-9. In conclusion, our results suggest that IL-21 promotes migration and invasion of RA-FLS. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA. Keywords: fibroblast-like synoviocytes, interleukin-21, invasion, matrix metalloproteinases, migration
Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joints characterized by synovial inflammation, hyperplasia of synovial tissues, synovial pannus and cartilage and bone destruction [1]. Among the various pathological events in the synovium, fibroblast-like synoviocytes (FLS) are reported to play a pivotal role. The FLS from RA patients (RA-FLS) are stimulated to migrate from affected synovium to healthy synovium [2], contributing to the spread of arthritis and destruction of distant joints [3]. RA-FLS invade the extracellular matrix (ECM) directly and secrete matrix metalloproteinases (MMPs) into synovial fluid, which destroys cartilage and bone and exacerbates joint damage [4]. Cytokines play a critical role in the onset and progression of RA. Several proinflammatory cytokines, such as tumour necrosis factor-(TNF)-a and interleukin (IL)-6, are essential to RA development owing to their role in synoviocyte activation [5]. Regulating these proinflammatory cytokines may prevent disease progression, and are candidate RA therapies.
IL-21 is a member of the IL-2 family of cytokines, and binds the IL-21 receptor (IL-21R), a heterodimer composed of IL-21R and the common g-chain that is shared with receptors specific for IL-2, IL-4, IL-7, IL-9 and IL-15 [6]. IL-21 is expressed primarily by CD41 T cells, including T helper type 1 (Th1), Th2, Th17 and natural killer (NK) T cells [7–9]. Notably, IL-21R is expressed highly by RA-FLS in rheumatoid synovium tissue [10]. Increased IL-21 plasma levels are associated with enhanced disease activity and radiographic status in RA patients [11]. Inhibition of IL-21 with the IL-21 receptor Fc fusion protein (IL-21R.Fc) reduces inflammatory cytokine production and attenuates the progression of arthritis in collagen-induced arthritis (CIA) animal models [12]. In contrast, IL-21R deficiency protects against severe inflammation and joint destruction in streptococcal cell wall arthritis [13]. A previous study has demonstrated that IL-21 promotes MMP-1, MMP-2, MMP-3 and MMP-9 expression in intestinal fibroblasts [14]. MMPs play important roles in the degradation of basement membranes and ECM, and are
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crucial for the migration and invasion of many types of cells [15,16]. However, we still know little about the effect of IL-21 on RA-FLS migration and invasion, which prompted us to investigate the potential effect of IL-21. Further study of IL-21 may help to elucidate the precise role of IL-21 in disease, and therapies targeting IL-21 may represent novel alternative treatments for diverse human immunological conditions. In the present study, we explored the effects of IL-21 on migration and invasion of RA-FLS and observed the effect of IL-21 on MMPs and tissue inhibitor of metalloproteinases (TIMPs) production to determine the potential mechanisms underlying the action of IL-21 in RA-FLS.
Materials and methods Isolation of synovial cells and cell culture FLS were prepared from synovectomized tissues of RA and osteoarthritis (OA) patients undergoing joint replacement surgery. Synovial tissues from five RA patients [female, aged 32, 31, 51, 49 and 60 years, disease-modifying antirheumatic drugs (DMARDs) application irregularity] and two OA patients (female, aged 67 and 69 years) were minced mechanically, washed in cold sterile phosphatebuffered saline (PBS) and digested with 150 mg/ml of Dispase II (Roche, Reinach, Switzerland) for 4 h at 378C, with gentle agitation. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; GIBCO, Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin (GIBCO) and 100 mg/ml streptomycin (GIBCO) in a 378C incubator containing a 5% CO2-enriched atmosphere. Cells from passages 4–7 were used in all experiments. Cells at passages 4–7 were identified by flow cytometric analysis. Ethical approval was obtained from the Ethics Committee of Peking University Third Hospital.
CD41 T cells and NK cells purification from human donors Whole blood was obtained from healthy human donors from Peking University Third Hospital. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density-gradient centrifugation (GE Healthcare Life Sciences, Fairfield, CT, USA), according to the manufacturers’ protocol. CD41 T cells were purified by negative selection using the EasySepTM human CD41 T cell isolation kit (Stemcell Technologies Inc., British Columbia, Canada), as recommended by the manufacturer. NK cells were purified by negative selection using the EasySepTM human NK cell selection kit (Stemcell). The CD41 T cells and NK cells were cultured in 24-well plates in 1 ml medium (RPMI-1640 supplemented with 10% heatinactivated FBS) with 50 ng/ml phorbol myristate acetate 148
(PMA)/ionomycin (Selleck Chemicals, Houston, TX, USA) for 18 h, then the supernatants were cultured with RA-FLS or OA-FLS in the lower chambers of Transwell plates.
Reverse transcription–polymerase chain reaction (RT–PCR) Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, with cDNA synthesis and RT–PCR analysis performed as described previously [17]. The following primers were used for each molecule: MMP-1: 50 -ACTCT GGAGTAATGTCACACC-30 (sense) and 50 -GTTGGTCCACC TTTCATCTTCA-30 (anti-sense); MMP-2: 50 -CCGTCGCCC ATCATCAAGTT-30 (sense) and 50 -CTGTCTGGGGCAGT CCAAAG-30 (anti-sense); MMP-3: 50 -AGTCTTCCAATCC TACTGTTGCT-30 (sense) and 50 -TCCCCGTCACCTCCAAT CC-30 (anti-sense); MMP-9: 50 -GGGACGCAGACATCGTCA TC-30 (sense) and 50 -TCGTCATCGTCGAAATGGGC-30 (anti-sense); MMP-13: 50 -GCTGCCTTCCTCTTCTTGA-30 (sense) and 50 -TGCTGCATTCTCCTTCAGGA-30 (anti-sense); TIMP-1: 50 -CCTTCTGCAATTCCGACCTCGTC-30 (sense) and 50 -CGGGCAGGATTCAGGCTATCTGG-30 (anti-sense); TIMP2: 50 -GATTAGGGCCAAAGCGGTCAGTG-30 (sense) and 50 GCCTGATGCCCGTTGATGCTC-30 (anti-sense); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 50 -GAAGGT CGGAGTCAACGG-30 (sense) and 50 -GGAAGATGGTGAT GGGATT-30 (anti-sense).
Enzyme-linked immunosorbent assay (ELISA) Cell culture supernatants were stored at 2808C until assayed. Supernatant cytokine concentrations were measured using ELISA, according to the manufacturer’s protocol. MMP-3 and MMP-9 ELISA kits were obtained from eBioscience (San Diego, CA, USA).
Immunoblotting After cell samples were lysed in lysis buffer, protein concentration was estimated using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Proteins were loaded onto a 10% sodium dodecyl sulphatepolyacrylamide gel and transferred electrophoretically to polyvinylidene fluoride membranes (PVDF; Merck Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 2 h, membranes were incubated with primary antibodies according to the supplier’s protocol, followed by incubation with a horseradish peroxidaseconjugated secondary antibody at room temperature for 1 h. Blots were developed using an enhanced chemiluminescence detection kit (Pierce). Antibodies used were as follows: anti-p-protein kinase B (AKT) (Ser473), anti-AKT, anti-phosphoinositide 3-kinase (PI3K)p110a, anti-p-signal transducer and activator of transcription-3 (STAT-3) (Ser727), anti-p-STAT-3 (Tyr705), anti-STAT-3, anti-p-
C 2016 British Society for Immunology, Clinical and Experimental Immunology, 184: 147–158 V
IL-21 induces invasion of RA-FLS
extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) (T202/Y204), anti-ERK1/2, anti-p-mitogenactivated protein kinase kinase (MEK1/2) (Ser217/221), anti-MEK1/2, anti-TIMP-1 and anti-TIMP-2 antibody from Cell Signaling Technology (Danvers, MA, USA), and anti-MMP-1, anti-MMP-2, anti-MMP-3, anti-MMP-9 and anti-MMP-13 from Abcam (Cambridge, UK).
Cell migration and invasion assays Cell migration was determined using 65 mm Transwell chambers with 8 lm pores (Corning, NY, USA). Briefly, a total of 5 3 104 cells in 100 ll DMEM were added in triplicate to the top chambers of Transwell plates, and the lower chambers were filled with DMEM containing TNF-a (20 ng/ml) or IL-21 (1, 10, 50, 100 ng/ml) with 10% FBS. For kinase pathways blocking experiments, PD98059 (20 lM), an ERK inhibitor, LY294002 (10 lM), an AKT inhibitor or STA21 (50 lM), a STAT-3 inhibitor (Cell Signaling Technology) and AG490 (Selleck Chemicals, Houston, TX, USA) were used. After culturing at 378C for 24 h, cells were fixed with 100% methanol for 30 min at room temperature. After removal of methanol and subsequent washing with PBS, the cells that invaded the matrix were stained with 01% crystal violet (Merck Millipore) solution for 30 min at room temperature. The nonmigrating cells were removed from the upper surface by cotton swabs. The number of cells that migrated through the membrane to the lower surface was counted in five representative microscopic fields (3100 magnification). The number of migrated cells was averaged from three 310 field-of-view images and normalized to control. Cell invasion ability was determined using Matrigel invasion chambers (BD Biosciences, Tokyo, Japan), according to the manufacturer’s instructions. The upper chambers were freshly coated with Matrigel, and medium was added to the lower chamber as described above. For blocking experiments, FLS were incubated with IL-21 (50 ng=ml) plus IL-21R.Fc (001, 01, 1, 10 lg/ml) or immunoglobulin (Ig)G.Fc (10 lg/ml) for 48 h. Soluble recombinant human IL-21R chimera (IL-21R.Fc) (991-R2) and recombinant human IgG1 chimera chimera (IgG. Fc) (110-HG) were purchased from R&D Systems (Abingdon, UK). Cell invasion was allowed to occur for 48 h and the gel and cells on the top membrane surface were removed with cotton swabs. Cells that had penetrated to the bottom were counted.
Flow cytometric analysis Following the required time in culture, 5 3 105 FLS were harvested in DMEM, cells were washed with PBS and incubated with saturating antibody concentrations for 20 min at room temperature. Flow cytometry was performed using a fluorescence activated cell sorter (FACS)Aria II flow cytometer (Becton Dickinson, San Jose, CA, USA).
Cells were gated using forward- versus side-scatter to remove any dead cells and cellular debris and thus provide a uniform population of FLS. For each sample, 10 000 cells were analysed. Results were expressed as the corrected mean fluorescence intensity (MFI) following subtraction of non-specific IgG control fluorescence. Antibodies used were as follows: vimentin-Alexa Fluor 488 and Alexa Fluor 488 isotype were from R&D Systems; and extracellular surface markers with fluorescently conjugated antibodies specific for CD4 (BD Biosciences), CD14, CD68, CD56, intercellular adhesion molecule 1 (ICAM-1) (eBioscience), vascular cell adhesion molecule 1 (VCAM-1) and cadherin-11 (R&D Biosystems). Flow cytometry data were analysed using FlowJo 274 software (TreeStar Inc., San Carlos, CA, USA).
Statistical analysis Data are expressed as the mean 6 standard error of the mean (s.e.m.). Data were compared using one-way analysis of variance (ANOVA), followed by Turkey’s method for multiple comparison (GraphPad Prism version 60; GraphPad Software Inc., La Jolla, CA, USA). A P-value less than 005 was considered significant in all statistical analyses.
Results Migration induced by IL-21 from CD41 and NK cells For CD41 T and NK cell enrichment from healthy human donors, cellular purity was detected with a flow cytometer before and after sorting using the immunomagnetic isolation method. The purity of CD41 T and NK cells was 98 and 88.9%, respectively (Fig. 1a). The IL-21 concentration in the supernatant of CD41 T and NK cells were up to 55 ng/ml and 119 ng/ml (Fig. 1b). When cultured with the CD41 T and NK cell supernatant or recombinant human IL-21 (50 ng/ml), the RA-FLS migration was increased significantly (Fig. 1c). However, when OA-FLS were cultured with CD41 T and NK cell supernatant, the CD41 T cell supernatant-induced OA-FLS migration was increased significantly, while NK cells and the IL-21 group were not changed (Fig. 1d).
IL-21 promotes migration and invasion of RA-FLS in vitro FLS at passages 4–7 were identified by flow cytometric analysis as a homogeneous population with the phenotype of
OR I GI NA L ART IC LE
doi:10.1111/cei.12751
Interleukin-21 induces migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis
R. Xing,* Y. Jin,* L. Sun,* L. Yang,* C. Li,* Z. Li,† X. Liu* and J. Zhao* *Department of Rheumatology and †
Immunology, and Department of Anesthesiology, Peking University Third Hospital, Beijing, PR China
Accepted for publication 1 December 2015 Correspondence: X. Liu and J. Zhao, Department of Rheumatology and Immunology, Peking University Third Hospital, No. 49, North Garden Road, Beijing 100191, China. E-mail: [email protected]; [email protected]
Summary Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast-like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)-21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL-21 on the migration, invasion and matrix metalloproteinase (MMP) expression in RA-FLS. We found that IL-21 promoted the migration, invasion and MMP (MMP-2, MMP-3, MMP-9, MMP-13) production in RA-FLS. Moreover, IL-21 induced activation of the phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription-3 (STAT-3) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, and blockage of these pathways [PI3K/protein kinase B (AKT) inhibitor LY294002, STAT-3 inhibitor STA-21 and ERK1/2 inhibitor PD98059] attenuated IL-21-induced migration and secretion of MMP-3 and MMP-9. In conclusion, our results suggest that IL-21 promotes migration and invasion of RA-FLS. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA. Keywords: fibroblast-like synoviocytes, interleukin-21, invasion, matrix metalloproteinases, migration
Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joints characterized by synovial inflammation, hyperplasia of synovial tissues, synovial pannus and cartilage and bone destruction [1]. Among the various pathological events in the synovium, fibroblast-like synoviocytes (FLS) are reported to play a pivotal role. The FLS from RA patients (RA-FLS) are stimulated to migrate from affected synovium to healthy synovium [2], contributing to the spread of arthritis and destruction of distant joints [3]. RA-FLS invade the extracellular matrix (ECM) directly and secrete matrix metalloproteinases (MMPs) into synovial fluid, which destroys cartilage and bone and exacerbates joint damage [4]. Cytokines play a critical role in the onset and progression of RA. Several proinflammatory cytokines, such as tumour necrosis factor-(TNF)-a and interleukin (IL)-6, are essential to RA development owing to their role in synoviocyte activation [5]. Regulating these proinflammatory cytokines may prevent disease progression, and are candidate RA therapies.
IL-21 is a member of the IL-2 family of cytokines, and binds the IL-21 receptor (IL-21R), a heterodimer composed of IL-21R and the common g-chain that is shared with receptors specific for IL-2, IL-4, IL-7, IL-9 and IL-15 [6]. IL-21 is expressed primarily by CD41 T cells, including T helper type 1 (Th1), Th2, Th17 and natural killer (NK) T cells [7–9]. Notably, IL-21R is expressed highly by RA-FLS in rheumatoid synovium tissue [10]. Increased IL-21 plasma levels are associated with enhanced disease activity and radiographic status in RA patients [11]. Inhibition of IL-21 with the IL-21 receptor Fc fusion protein (IL-21R.Fc) reduces inflammatory cytokine production and attenuates the progression of arthritis in collagen-induced arthritis (CIA) animal models [12]. In contrast, IL-21R deficiency protects against severe inflammation and joint destruction in streptococcal cell wall arthritis [13]. A previous study has demonstrated that IL-21 promotes MMP-1, MMP-2, MMP-3 and MMP-9 expression in intestinal fibroblasts [14]. MMPs play important roles in the degradation of basement membranes and ECM, and are
C 2016 British Society for Immunology, Clinical and Experimental Immunology, 184: 147–158 V
147
R. Xing et al.
crucial for the migration and invasion of many types of cells [15,16]. However, we still know little about the effect of IL-21 on RA-FLS migration and invasion, which prompted us to investigate the potential effect of IL-21. Further study of IL-21 may help to elucidate the precise role of IL-21 in disease, and therapies targeting IL-21 may represent novel alternative treatments for diverse human immunological conditions. In the present study, we explored the effects of IL-21 on migration and invasion of RA-FLS and observed the effect of IL-21 on MMPs and tissue inhibitor of metalloproteinases (TIMPs) production to determine the potential mechanisms underlying the action of IL-21 in RA-FLS.
Materials and methods Isolation of synovial cells and cell culture FLS were prepared from synovectomized tissues of RA and osteoarthritis (OA) patients undergoing joint replacement surgery. Synovial tissues from five RA patients [female, aged 32, 31, 51, 49 and 60 years, disease-modifying antirheumatic drugs (DMARDs) application irregularity] and two OA patients (female, aged 67 and 69 years) were minced mechanically, washed in cold sterile phosphatebuffered saline (PBS) and digested with 150 mg/ml of Dispase II (Roche, Reinach, Switzerland) for 4 h at 378C, with gentle agitation. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; GIBCO, Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin (GIBCO) and 100 mg/ml streptomycin (GIBCO) in a 378C incubator containing a 5% CO2-enriched atmosphere. Cells from passages 4–7 were used in all experiments. Cells at passages 4–7 were identified by flow cytometric analysis. Ethical approval was obtained from the Ethics Committee of Peking University Third Hospital.
CD41 T cells and NK cells purification from human donors Whole blood was obtained from healthy human donors from Peking University Third Hospital. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density-gradient centrifugation (GE Healthcare Life Sciences, Fairfield, CT, USA), according to the manufacturers’ protocol. CD41 T cells were purified by negative selection using the EasySepTM human CD41 T cell isolation kit (Stemcell Technologies Inc., British Columbia, Canada), as recommended by the manufacturer. NK cells were purified by negative selection using the EasySepTM human NK cell selection kit (Stemcell). The CD41 T cells and NK cells were cultured in 24-well plates in 1 ml medium (RPMI-1640 supplemented with 10% heatinactivated FBS) with 50 ng/ml phorbol myristate acetate 148
(PMA)/ionomycin (Selleck Chemicals, Houston, TX, USA) for 18 h, then the supernatants were cultured with RA-FLS or OA-FLS in the lower chambers of Transwell plates.
Reverse transcription–polymerase chain reaction (RT–PCR) Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, with cDNA synthesis and RT–PCR analysis performed as described previously [17]. The following primers were used for each molecule: MMP-1: 50 -ACTCT GGAGTAATGTCACACC-30 (sense) and 50 -GTTGGTCCACC TTTCATCTTCA-30 (anti-sense); MMP-2: 50 -CCGTCGCCC ATCATCAAGTT-30 (sense) and 50 -CTGTCTGGGGCAGT CCAAAG-30 (anti-sense); MMP-3: 50 -AGTCTTCCAATCC TACTGTTGCT-30 (sense) and 50 -TCCCCGTCACCTCCAAT CC-30 (anti-sense); MMP-9: 50 -GGGACGCAGACATCGTCA TC-30 (sense) and 50 -TCGTCATCGTCGAAATGGGC-30 (anti-sense); MMP-13: 50 -GCTGCCTTCCTCTTCTTGA-30 (sense) and 50 -TGCTGCATTCTCCTTCAGGA-30 (anti-sense); TIMP-1: 50 -CCTTCTGCAATTCCGACCTCGTC-30 (sense) and 50 -CGGGCAGGATTCAGGCTATCTGG-30 (anti-sense); TIMP2: 50 -GATTAGGGCCAAAGCGGTCAGTG-30 (sense) and 50 GCCTGATGCCCGTTGATGCTC-30 (anti-sense); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 50 -GAAGGT CGGAGTCAACGG-30 (sense) and 50 -GGAAGATGGTGAT GGGATT-30 (anti-sense).
Enzyme-linked immunosorbent assay (ELISA) Cell culture supernatants were stored at 2808C until assayed. Supernatant cytokine concentrations were measured using ELISA, according to the manufacturer’s protocol. MMP-3 and MMP-9 ELISA kits were obtained from eBioscience (San Diego, CA, USA).
Immunoblotting After cell samples were lysed in lysis buffer, protein concentration was estimated using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Proteins were loaded onto a 10% sodium dodecyl sulphatepolyacrylamide gel and transferred electrophoretically to polyvinylidene fluoride membranes (PVDF; Merck Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA) for 2 h, membranes were incubated with primary antibodies according to the supplier’s protocol, followed by incubation with a horseradish peroxidaseconjugated secondary antibody at room temperature for 1 h. Blots were developed using an enhanced chemiluminescence detection kit (Pierce). Antibodies used were as follows: anti-p-protein kinase B (AKT) (Ser473), anti-AKT, anti-phosphoinositide 3-kinase (PI3K)p110a, anti-p-signal transducer and activator of transcription-3 (STAT-3) (Ser727), anti-p-STAT-3 (Tyr705), anti-STAT-3, anti-p-
C 2016 British Society for Immunology, Clinical and Experimental Immunology, 184: 147–158 V
IL-21 induces invasion of RA-FLS
extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) (T202/Y204), anti-ERK1/2, anti-p-mitogenactivated protein kinase kinase (MEK1/2) (Ser217/221), anti-MEK1/2, anti-TIMP-1 and anti-TIMP-2 antibody from Cell Signaling Technology (Danvers, MA, USA), and anti-MMP-1, anti-MMP-2, anti-MMP-3, anti-MMP-9 and anti-MMP-13 from Abcam (Cambridge, UK).
Cell migration and invasion assays Cell migration was determined using 65 mm Transwell chambers with 8 lm pores (Corning, NY, USA). Briefly, a total of 5 3 104 cells in 100 ll DMEM were added in triplicate to the top chambers of Transwell plates, and the lower chambers were filled with DMEM containing TNF-a (20 ng/ml) or IL-21 (1, 10, 50, 100 ng/ml) with 10% FBS. For kinase pathways blocking experiments, PD98059 (20 lM), an ERK inhibitor, LY294002 (10 lM), an AKT inhibitor or STA21 (50 lM), a STAT-3 inhibitor (Cell Signaling Technology) and AG490 (Selleck Chemicals, Houston, TX, USA) were used. After culturing at 378C for 24 h, cells were fixed with 100% methanol for 30 min at room temperature. After removal of methanol and subsequent washing with PBS, the cells that invaded the matrix were stained with 01% crystal violet (Merck Millipore) solution for 30 min at room temperature. The nonmigrating cells were removed from the upper surface by cotton swabs. The number of cells that migrated through the membrane to the lower surface was counted in five representative microscopic fields (3100 magnification). The number of migrated cells was averaged from three 310 field-of-view images and normalized to control. Cell invasion ability was determined using Matrigel invasion chambers (BD Biosciences, Tokyo, Japan), according to the manufacturer’s instructions. The upper chambers were freshly coated with Matrigel, and medium was added to the lower chamber as described above. For blocking experiments, FLS were incubated with IL-21 (50 ng=ml) plus IL-21R.Fc (001, 01, 1, 10 lg/ml) or immunoglobulin (Ig)G.Fc (10 lg/ml) for 48 h. Soluble recombinant human IL-21R chimera (IL-21R.Fc) (991-R2) and recombinant human IgG1 chimera chimera (IgG. Fc) (110-HG) were purchased from R&D Systems (Abingdon, UK). Cell invasion was allowed to occur for 48 h and the gel and cells on the top membrane surface were removed with cotton swabs. Cells that had penetrated to the bottom were counted.
Flow cytometric analysis Following the required time in culture, 5 3 105 FLS were harvested in DMEM, cells were washed with PBS and incubated with saturating antibody concentrations for 20 min at room temperature. Flow cytometry was performed using a fluorescence activated cell sorter (FACS)Aria II flow cytometer (Becton Dickinson, San Jose, CA, USA).
Cells were gated using forward- versus side-scatter to remove any dead cells and cellular debris and thus provide a uniform population of FLS. For each sample, 10 000 cells were analysed. Results were expressed as the corrected mean fluorescence intensity (MFI) following subtraction of non-specific IgG control fluorescence. Antibodies used were as follows: vimentin-Alexa Fluor 488 and Alexa Fluor 488 isotype were from R&D Systems; and extracellular surface markers with fluorescently conjugated antibodies specific for CD4 (BD Biosciences), CD14, CD68, CD56, intercellular adhesion molecule 1 (ICAM-1) (eBioscience), vascular cell adhesion molecule 1 (VCAM-1) and cadherin-11 (R&D Biosystems). Flow cytometry data were analysed using FlowJo 274 software (TreeStar Inc., San Carlos, CA, USA).
Statistical analysis Data are expressed as the mean 6 standard error of the mean (s.e.m.). Data were compared using one-way analysis of variance (ANOVA), followed by Turkey’s method for multiple comparison (GraphPad Prism version 60; GraphPad Software Inc., La Jolla, CA, USA). A P-value less than 005 was considered significant in all statistical analyses.
Results Migration induced by IL-21 from CD41 and NK cells For CD41 T and NK cell enrichment from healthy human donors, cellular purity was detected with a flow cytometer before and after sorting using the immunomagnetic isolation method. The purity of CD41 T and NK cells was 98 and 88.9%, respectively (Fig. 1a). The IL-21 concentration in the supernatant of CD41 T and NK cells were up to 55 ng/ml and 119 ng/ml (Fig. 1b). When cultured with the CD41 T and NK cell supernatant or recombinant human IL-21 (50 ng/ml), the RA-FLS migration was increased significantly (Fig. 1c). However, when OA-FLS were cultured with CD41 T and NK cell supernatant, the CD41 T cell supernatant-induced OA-FLS migration was increased significantly, while NK cells and the IL-21 group were not changed (Fig. 1d).
IL-21 promotes migration and invasion of RA-FLS in vitro FLS at passages 4–7 were identified by flow cytometric analysis as a homogeneous population with the phenotype of
