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Keywords:

  • interleukin-2 immune complex;
  • regulatory T cell;
  • rheumatoid arthritis;
  • signal transducer and activator of transcription 5;
  • T helper type 17

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In this study, we investigated the effects of administration of interleukin-2 (IL-2)/JES6-1 (anti-IL-2 monoclonal antibody) immune complexes on the expansion and activation of regulatory T (Treg) cells, the down-regulation of T helper type 17 (Th17) cells, and the control of the severity of collagen-induced arthritis (CIA). Wild-type and CIA-induced wild-type mice were injected intraperitoneally (i.p.) with IL-2 or IL-2/JES6-1 complex three times at 2-day intervals. Treg cell surface markers were analysed by flow cytometry. After injecting IL-2 or IL-2/JES6-1, the time kinetics of IL-2 signalling molecules was examined by FACS and Western blotting. Concentrations of IL-17 and IL-10 were measured by ELISA. Injection of IL-2/JES6-1 increased the proportion of Foxp3+ Treg cells among splenic CD4+ T cells, which reached the highest level on day 4 after injection. Up-regulation of CTLA4, GITR and glycoprotein-A repetitions predominant (GARP) was observed. Activation of p-signal transducer and activator of transcription 5 (STAT5) was apparent within 3 hr after injection of IL-2/JES6-1 complexes. Expression of IL-2 signalling molecules, including p-AKT and p-p38/mitogen-activated protein kinase, was also higher in splenocytes treated with IL-2/JES6-1 complexes. Injection of IL-2/JES6-1 complexes suppressed the induction of CIA and the production of IL-17 and inflammatory responses while increasing the level of IL-10 in the spleen. The expansion of Treg cells (via STAT5) and the concomitant increase in IL-2 signalling pathways by IL-2/JES6-1 complexes suggests their potential use as a novel therapeutic agent for the treatment of autoimmune arthritis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Regulatory T (Treg) cells prevent the onset of overt autoimmunity and immunopathology by suppressing excessive immune responses against self and foreign antigens. Treg cells are believed to use one or more suppressive mediators available to them, including cytotoxic T lymphocyte antigen 4 (CTLA4), glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR), lymphocyte activation gene-3, CD25, transforming growth factor-β (TGF-β), interleukin-10 (IL-10) and IL-35, to provide a customized inhibitory effect according to the pathological condition at hand.[1-5]

Treg cells can suppress both the initial activation and subsequent effector functions of pathogenic T cells and can inhibit antigen-specific immune responses.[6, 7] Increasing Treg cell function is used widely as a viable immunosuppressive method in many pathological conditions, including autoimmune diseases, such as rheumatoid arthritis.

The most widely studied population of Treg cells is that of naturally occurring CD25+ CD4+ cells.[8, 9] Expression of CD25, the α-subunit of the IL-2 receptor (IL-2Rα, CD25), requires the action of the transcription factor Foxp3.[10] In addition to displaying IL-2Rα, Treg cells are characterized by the presence of IL-2Rβ (CD122) and the common γ-chain (CD132) on the cell surface, which are displayed as the high-affinity trimeric IL-2R. Signalling through the IL-2R is transmitted through the Janus kinase (JAK) 1/3 and signal transducer and activator of transcription 5 (STAT5) pathways,[11] and is required for the generation, differentiation, growth, survival and function of Treg cells.[12, 13] Stimulation of naive CD4+ T cells with IL-2 and TGF-β facilitates the binding of STAT5 to the Foxp3 promoter, leading to the generation of induced Treg cells in the periphery.[11, 14]

Recent reports have shown that immune complexes of IL-2 and anti-IL-2 monoclonal antibodies (mAbs) can exhibit two distinct activities depending on the specificity of the bound mAb. They can either elicit expansion of mostly memory CD8+ and natural killer cells or induce the selective expansion of primarily Treg cells.[14, 15] In mouse T cells, IL-2/mAb complexes generated with the anti-IL-2 mAb clone S4B6 target primarily memory CD8+ and natural killer cells, whereas complexes produced with the anti-IL-2 mAb clone JES6-1 induce the expansion of Treg cells. The antibody S4B6 binds to the region of IL-2 that interacts with the CD25 binding site, whereas 5JES6-1 covers the region of IL-2 that makes contact with CD122 receptors. Hence, complexes of IL-2 with the S4B6 mAb lead to vigorous stimulation of CD122hi cells, whereas IL-2 bound to the JES6-1 mAb selectively stimulates CD25+ cells.[16] Because IL-2/S4B6 and IL-2/JES6-1 complexes appear to mediate their subset-specific functions through selective interaction with the low-affinity dimeric IL-2R (CD122/CD132) and the high-affinity trimeric IL-2R (CD25/CD122/CD132), respectively, they are also called IL-2/mAbCD122 (the low-affinity dimer) and IL-2/mAbCD25 (the high-affinity trimer) complexes.[17] Interestingly, IL-2/JES6-1 complexes can suppress experimental autoimmune encephalomyelitis by effectively expanding Treg cells.[18] This result suggests a possible application of IL-2/JES6-1 complexes in the modulation of the immune response in a variety of autoimmune disorders.

In this study, we examined the ability of IL-2/JES6-1 complex-induced Treg cells to ameliorate collagen-induced autoimmune arthritis (CIA) in normal DBA1/J mice. We found that IL-2/JES6-1 induced the expansion of CD25+ Foxp3+ Treg cells and sustained the increased expression of CD25, phospho-STAT5 (p-STAT5), and p-p38/mitogen-activated protein kinase (MAPK) signalling. Administration of IL-2/JES6-1 complexes effectively suppressed joint inflammation in the CIA mouse model. Our results provide additional support for the potential use of IL-2/JES6-1 complexes as a novel therapeutic immunomodulator in the treatment of human autoimmune disease.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Animals

Six-week-old male DBA1/J mice (SLC, Inc., Shizuoka, Japan) were maintained in groups of five in polycarbonate cages in a specific pathogen-free environment and were fed standard mouse chow (Ralston Purina, Gray Summit, MO) and water ad libitum. All experimental procedures were examined and approved by the Animal Research Ethics Committee at the Catholic University of Korea.

Induction of arthritis and injection of IL-2/JES6-1 complexes

To evaluate the effects of administration of IL-2/JES6-1 complexes, DBA1/J mice were divided into three groups, PBS, IL-2 (1·5 μg; eBioscience, San Diego, CA) and IL-2/JES6-1 complex (1·5 μg/7·5 μg; eBioscience) and were injected intraperitoneally (i.p.) on days 0, 2 and 4. To examine the effect of IL-2/JES6-1 complexes on CIA, IL-2/JES6-1 complexes were injected i.p. three times at 2-day intervals before CIA induction. To induce CIA, 100-μg bovine type II collagen (CII) and complete Freund's adjuvant CFA (Chondrex, Inc.,Redmond, WA) (Arthrogen-CIA) were injected intradermally into the base of the tail. Starting on the next day, three independent observers examined the severity of arthritis three times per week. The severity of arthritis was recorded using the mean arthritis index on a scale of 0–4, as reported previously.[19] The final value represented the average index from all four legs recorded by the three independent observers.

Flow cytometric analyses of p-STAT5 and Treg cell surface markers

Cell pellets were prepared from the spleens of DBA1/J mice injected with IL-2/JES6-1 complexes. To examine the population of Treg cells, cells were stained with anti-CD4-peridin chlorophyll protein (PerCP) mAb (eBioscience) and anti-CD25-allophycocyanin (APC; eBioscience). Cells were permeabilized and fixed with CytoFix/CytoPerm (BD Pharmingen, San Diego, CA) according to the manufacturer's protocol, stained further with anti-Foxp3-FITC (eBioscience) and/or anti-p-STAT5-phycoerythrin (PE; eBioscience). To examine the expression of surface markers, cells were stained with anti-CD4-PerCP, anti CD25-APC, anti-Foxp3-PE, anti-GITR-FITC, anti-GARP-PE, anti-ICAM-FITC, anti-CD103-PE, anti-PD1-FITC and anti-CD103-PE (all from eBioscience). For intracellular staining, the cells were stained with anti-CTLA4-PE (eBioscience).

Regulatory T-cell isolation

Regulatory T cells were isolated from spleens of mice injected with PBS, IL-2 or IL-2/JES6-1. The spleen cells were stained with CD4 microbeads (Miltenyi Biotec, Auburn, CA). Anti-CD4 microbeads were used essentially as recommended by the manufacturer. Anti-CD4 microbeads (10 μl) were added and incubated for 10 min at 4°. The CD4+ cells were isolated by MACS separation column (Miltenyi Biotec) and the isolated cells were stained with anti-CD4-PerCP and anti-CD25-APC for 30 min at 4°. CD4+ CD25+ Treg cells were sorted by FACSAria III sorter (BD Bioscience, San Jose, CA).

Real-time polymerase chain reaction

Relative expression of specific mRNAs was quantified by real-time PCR using SYBR Green I (Roche Diagnostics, Mannheim, Germany). The following sense and antisense primers were used: for Foxp3, 5′-GGC CCT TCT CCA GGA CAG A-3′ and 5′-GCT GAT CAT GGC TGG GTT GT-3′, for IL-10, 5′-AAG TGA TGC CCC AGG CA-3′ and 5′-TCT CAC CCA GGG AAT TCA AA-3′, for suppressor of cytokine signalling 1 (SOCS1), 5′-CTT AAC CCG GTA CTC CGT GA-3′ and 5′-GAG GTC TCC AGC CAG AAG TG-3′, for SOCS3, 5′-CGC CTC AAG ACC TTC AGC TC-3′ and 5′-CTG ATC CAG GAA CTC CCG AA-3′, for CD25, 5′-AGA ACA CCA CCG ATT TCT GG-3′ and 5′-AGC TGG CCA CTG CTA CCT TA-3′, and for β-actin, 5′-GAA ATC GTG CGT GAC ATC AAA G-3′ and 5′-TGT AGT TTC ATG GAT GCC ACA G-3′.

Measurement of IL-17 concentrations

Concentrations of IL-17 in serum were measured by sandwich ELISA. Anti-mouse IL-17 mAb (R&D Systems, Minneapolis, MN) was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°. The wells were treated with blocking solution (PBS containing 1% BSA and 0·05% Tween-20), samples and the standard recombinant IL-17 (R&D Systems) were added to the 96-well plate, and the plate was incubated. Biotinylated IL-17 polyclonal antibody (R&D Systems) was added, and the reactions were allowed to proceed. The plates were washed, 2000-fold diluted ExtrAvidin-alkaline phosphate (Sigma Aldrich, St Louis, MO) was added, and the reactions were allowed to proceed. The plates were washed, and p-nitrophenyl phosphate disodium salt (50 μl; Pierce Chemical Company, Rockford, IL), diluted in diethanolamine buffer, was applied.

Measurement of immunoglobulin concentrations and collagen-specific IgG2a titres

The serum concentrations of IgG, IgG1 and IgG2a were measured by ELISA, using mouse IgG, IgG1 and IgG2a ELISA quantification kits (Bethyl Laboratories, Montgomery, TX). For collagen-specific IgG2a analysis, serum was collected at 40 days after the first immunization. Bovine type II collagen (40 μg/ml in PBS) was used to coat 96-well flat-bottomed plates (Nunc) at 4° overnight. Serially diluted serum samples were applied and incubated at room temperature for 1 hr. Then, the wells were washed with washing buffer (PBS containing 50 mm Tris–HCl, 0·14 m NaCl and 0·05% Tween-20), followed by addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2a (Bethyl Laboratories). The HRP activity was measured using tetramethyl benzidine solution (eBioscience). Results were analysed by measuring the absorbance at 450 nm.

Western blotting for p-STAT5, p-AKT and p-p38/MAP

Spleen tissues were collected from mice at 3, 6, 24, 48 and 72 hr after injection of PBS, IL-2 or IL-2/JES6-1. Protein samples were separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were stained with primary antibodies to p-STAT5, p-AKT, p-p38/MAPK, STAT5, AKT and p38/MAPK (all from Cell Signaling, Danvers, MA) and β-actin. The HRP-conjugated secondary antibody was then added.

Staining for confocal microscopy

Spleen tissues were obtained 7 weeks after primary immunization. The tissues were stained using anti-Foxp3-FITC (eBioscience), anti-IL-17 (eBioscience), anti-CD25-APC (BioLegend, San Diego, CA), anti-p-STAT3-PE (eBioscience), anti-p-STAT5-PE (eBioscience), anti-CD4-biotin (BD Biosciences, San Jose, CA), and streptavidin Cy3 in PBS. Stained sections were analysed using a confocal microscopy system (LSM 510 Meta; Carl Zeiss, Thornwood, NY).

Immunohistochemistry

Mouse joint tissues were obtained 12 and 26 days after the first injection and fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier, and embedded in paraffin. Joint tissues were sectioned at 7-μm thickness, dewaxed using xylene, rehydrated through a gradient of alcohols, and then stained with haematoxylin and eosin (H&E) and Safranin O to detect proteoglycans. Endogenous peroxidase activity was quenched with methanol–3% H2O2. Immunohistochemistry was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Tissues were incubated with the first primary anti-mouse Foxp3 mAb and anti-mouse IL-17 mAb overnight at 4°. The primary antibodies were detected with a biotinylated secondary linking antibody, followed by incubation with streptavidin–peroxidase complex for 1 hr. The final colour product was developed using DAB chromogen (Dako, Carpinteria, CA). Positive cells were counted, and results are expressed as means ± SD.

Histological evolution

Mouse joint tissues were obtained 40 days after the first injection. Tissues were stained with H&E and Safranin O to detect proteoglycans. Scoring was as follows: inflammation score 0 = no inflammation, score 1 = slight thickening of lining layer or some infiltrating cells in sub-lining layer, score 2 = slight thickening of lining layer plus some infiltrating cells in sub-lining layer, score 3 = thickening of lining layer, influx of cells in sub-lining layer and presence of cells in the synovial space, and score 4 = synovium highly infiltrated with many inflammatory cells. For cartilage damage, the scoring was 0 = no destruction, 1 = minimal erosion, limited to single spots, 2 = slight-to-moderate erosion in a limited area, 3 = more extensive erosion, and 4 = general destruction.[20]

JAK/STAT PCR array

RNA was extracted from the spleens of mice stimulated with PBS, IL-2 or IL-2/JES6-1 complexes 6 h after injection. Complementary DNA was synthesized from the isolated RNA and analysed using the RT2 Profiler JAK/STAT PCR array kit (SABiosciences, Santa Clarita, CA). The data were evaluated using the ABI suite (Applied Biosystems, Carlsbad, CA).

Statistical analyses

All data are expressed as means ± SD. Statistical analyses were performed using the spss software (ver. 10.0 for Windows; SPSS, Chicago, IL). Differences between groups were analysed using an unpaired Student's t-test, assuming equal variances. P-values < 0·05 were considered to indicate statistical significance.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In vivo expansion of Treg cells in DBA1/J mice injected with IL-2/JES6-1 complexes

Before determining whether expansion of endogenous Foxp3+ Treg cells could ameliorate the severity of CIA induced in DBA1/J mice, we first analysed the ability of IL-2/JES6-1 complexes to induce expansion of Treg cells in normal DBA1/J mice. As observed in other mouse strains,[18] injection of IL-2/JES6-1 complexes at 2-day intervals more effectively elicited expansion of Treg cells in the spleen than did injection of IL-2 or PBS (Fig. 1). A rapid increase in the number of Treg cells in mice injected with IL-2/mAb complexes versus IL-2 was observed as early as day 2. This effect was most pronounced on day 4 and persisted until days 11–12 (Fig. 1a). Treg cell expansion in spleen was clearly visible by confocal microscopy of spleen tissue (Fig. 1b). Expansion of Treg cells was also evident in peripheral and mesenteric lymph nodes,[21] albeit to a lesser degree than in the spleen, whereas none was observed in the thymus (Fig. 1c).

image

Figure 1. In vivo expansion of regulatory T (Treg) cells after treatment with interleukin-2 (IL-2)/JES6-1 complexes. PBS (= 5), IL-2 (= 5), or IL-2/JES6-1 complexes (= 5) were injected intraperitoneally into DBA1/J mice on days 0 (first injection), 2 (second injection), and 4 (third injection). (a,b) Spleen cells from mice were stained for CD4, CD25 and Foxp3 at the times indicated. (a) Frequency (left) and total cell number (right) of Foxp3+ Treg cells. (b) Confocal images of spleen tissues stained for CD4 (red), CD25 (blue) and Foxp3 (green). The graph on the right represents the average numbers of Foxp3+ cells in five typical peri-arterial lymphoid sheath areas. (c) On day 4 after injection, cells from the spleen, peripheral (p) lymph nodes (LN), mesenteric (m) LN, and thymus were stained for CD4, CD25 and Foxp3 and analysed by flow cytometry. The frequency of Foxp3+ Treg cells is indicated. (d) The surface expression of CD25, GITR, GARP, ICAM, CD103 and PD1, and the intracellular expression of Foxp3 and CTLA4 were determined in splenic CD4+ CD25+ Foxp3+ Treg cells. (e) Messenger RNA levels of Foxp3, IL-10 and suppressor of cytokine signalling 3 (SOCS3) in CD4+ CD25high Treg cells were analysed by real-time PCR on day 4. (f) Joint tissues were stained for Foxp3, IL-10, and SOCS3 on day 4. The data represent the means ± SD of two or three independent experiments (*< 0·05, ** < 0·005, *** < 0·0005).

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As observed previously, expanded Treg cells in DBA1/J mice induced with IL-2/JES6-1 complexes expressed higher levels of Foxp3 than Treg cells from IL-2-injected mice. Treg cells induced by IL-2/JES6-1 injection also exhibited higher levels of suppressive molecules, such as CD25, CTLA4, GITR and GARP[22-24] (Fig. 1d). Elevated expression of the adhesion molecules ICAM-1, CD103, PD1, CD44 and inducible costimulator was also observed (Fig. 1d and other data not shown). Up-regulation of Foxp3 in IL-2/JES6-1 complex-induced Treg cells was detected at the mRNA level, as measured by real-time PCR analysis (Fig. 1e). Treatment with IL-2/JES6-1 complexes, but not with IL-2, increased the transcription of the suppressive cytokine IL-10 and SOCS3, which is induced by activation of STAT5[25, 26] (Fig. 1e). Immunohistochemical analysis of the joint tissues showed that the Foxp3, IL-10 and SOCS3 proteins were highly expressed in mice injected with IL-2/JES6-1 complexes compared with those injected with IL-2 or PBS (Fig. 1f and other data not shown).

Prominent induction of p-STAT5 in Treg cells after treatment with IL-2/JES6-1 complexes

Upon binding to its receptor on T cells, IL-2 activates the JAK/STAT signalling pathway, leading to phosphorylation of the STAT5 transcription factor, which is pivotal in inducing T-cell activation and Treg cell differentiation.[27] To compare the extent of STAT5 activation induced by IL-2/JES6-1 complexes, intracellular staining and Western blotting were used to compare the levels of p-STAT5 in Treg cells from DBA1/J mice after injection of IL-2/JES6-1 complexes and in those injected with IL-2. Injection of IL-2/JES6-1 complexes rapidly and potently induced a high level of p-STAT5, which was detectable within 3 hr, followed by a gradual dissipation of p-STAT5 staining over the next 12–24 hr (Fig. 2a–c). In contrast, injection of IL-2 alone induced a much lower level of p-STAT5, although the duration of induction was similar to that with IL-2/JES6-1 complexes (Fig. 2a–c).

image

Figure 2. Induction of p-signal transducer and activator of transcription 5 (STAT5) level in expanded regulatory T (Treg) cells after treatment with interleukin-2 (IL-2)/JES6-1 complexes. PBS, IL-2, or IL-2/JES6-1 complexes were injected intraperitoneally into DBA1/J mice on days 0, 2 and 4. Spleen tissues were collected from mice at the times indicated (3–48 hr). (a) p-STAT5 expression on CD4+ CD25+ Foxp3+ Treg cells was determined and compared among the three groups by flow cytometry. (b) The results are presented as mean fluorescence intensities. (c) Expression levels of p-STAT5, p-AKT, and p-p38/MAPK were determined by Western blotting at the times indicated. (d) Messenger RNA levels of CD25 and suppressor of cytokine signalling 1 (SOCS1) in spleen cells were determined by real-time PCR 6 hr after injection. The data are presented as the means ± SD of two or three independent experiments (*< 0·05, ** < 0·005, *** < 0·0005).

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The potent induction of p-STAT5 was translated into strong activation of downstream signalling pathways and the synthesis of other molecules. In particular, prompt induction of p-p38/MAPK in Treg cells was observed within 3 hr after injection of IL-2/JES6-1 complexes, and was sustained for the next 24 hr (Fig. 2c). In contrast, injection of IL-2 led to a much weaker induction of the corresponding pathways (Fig. 2c). In terms of other IL-2-signalling molecules, real-time PCR analyses showed that the synthesis of CD25 and SOCS1 was induced much more strongly in Treg cells by injection of IL-2/JES6-1 complexes than with IL-2 (Fig. 2d). Expression of these genes is known to be induced by activation of STAT5.[28] In contrast, the expression of genes induced by STAT3, such as Mmp3, Crp and A2m, was significantly reduced in the same group (data not shown). These results suggest that IL-2/JES6-1 complexes triggered the activation of STAT5, leading to increased expression of its downstream genes. It is possible that IL-2/JES6-1 complexes also affected the regulation of STAT3 in a way that decreased its activation and so also the expression of associated genes. However, the mechanism responsible for this negative regulation remains to be determined.

Suppression of collagen-induced arthritis development in IL-2/JES6-1-treated DBA1/J mice

To assess whether inducing the expansion of Treg cells before immunization with bovine CII could prevent the onset of CIA, DBA1/J mice were given three injections of IL-2/JES6-1 complexes and then immunized with CII. Control mice received IL-2 or PBS before CII immunization. As expected, symptoms of CIA appeared in all control mice (pre-injected with PBS) starting ~ 5 weeks after CII immunization. These mice exhibited severe disease symptoms over the next 2 weeks (Fig. 3a). A similar result was observed among mice pre-injected with IL-2 alone (Fig. 3a), indicating that this pre-treatment conferred minimal or no protection. In contrast, mice pre-treated with IL-2/JES6-1 complexes were highly resistant to CIA induction, and ~ 25% of these mice were affected by only very mild disease during the observation period (Fig. 3a).

image

Figure 3. Suppression of arthritis after treatment with interleukin-2 (IL-2)/JES6-1 complexes. Mice were injected intraperitoneally three times at 2-day intervals (days 0, 2, 4) with PBS, IL-2 or IL–2/JES6-1 complexes. Arthritis was induced on day 5 by injecting 100 μg of collagen type II (CII) and complete Freunds' adjuvant into the base of the tail. Three independent observers examined the severity of arthritis three times per week for up to 47 days. (a) Arthritis severity was recorded as the mean arthritis index score and incidence score. (b) Ankle joint tissues were obtained from mice with collagen-induced arthritis (CIA) that were previously treated with PBS, IL-2 or IL-2/JES6-1 complexes on day 40 after start of the experiment and stained with toluidine blue, Safranin O, and haematoxylin & eosin. The inflammation and cartilage scores are shown in bar graphs (right). (c) Total IgG, IgG1, IgG2a, and CII-specific IgG2a levels were measured in serum from each group on day 40. Data are presented as the means ± SD of two or three independent experiments (*P < 0·05, ** P < 0·005, *** < 0·0005).

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Consistent with the arthritis score, histological analysis showed severe inflammation and cartilage destruction in PBS-treated and IL-2-treated mice, whereas minimal signs of inflammation and tissue destruction were observed in mice injected with IL-2/JES6-1 complexes (Fig. 3b).

One possible mechanism involved in the suppression of the onset of CIA is blocking the production of anti-CII antibodies.[29, 30] Analysis of serum from the three groups of mice 40 days after CII immunization showed that mice injected with IL-2/JES6-1 displayed lower concentrations of anti-CII IgG2a antibodies than did mice injected with IL-2 or PBS (Fig. 3c, far right). Mice injected with IL-2/JES6-1 complexes also exhibited less IgG, and the IgG1 level was considerably lower than that of IgG2a, compared with control mice (Fig. 3c).

Prolonged suppression of Th17 cells in CIA mice after treatment with IL-2/JES6-1 complexes

The pro-inflammatory cytokine IL-17, produced by Th17 cells, is a major mediator of joint inflammation in arthritis.[29, 31, 32] To determine whether CIA amelioration by treatment with IL-2/JES6-1 complexes involved suppression of IL-17 production, serum levels of IL-17 were measured in mice pre-treated with IL-2/JES6-1 complexes or with PBS. Interleukin-17 concentrations were measured at various times before the onset of disease on days 7–26. In control mice, a spike in the IL-17 concentration was clearly detectable from day 12, and this concentration was sustained for the next 2 weeks (Fig. 4a). In contrast, IL-2/JES6-1-injected mice displayed a much smaller spike in IL-17 concentration on day 12, and the level returned quickly to basal levels, within 1 week (Fig. 4a).

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Figure 4. Prolonged suppression of interleukin-17 (IL-17) levels in mice with collagen-induced arthritis (CIA) treated with IL-2/JES6-1. Mice were injected intraperitoneally three times at 2-day intervals (days 0, 2, and 4) with PBS or IL-2/JES6-1 complexes, and arthritis was induced on day 5. (a) Serum levels of IL-17 were measured in each group by ELISA on days 7, 9, 12, 19 and 26. (b) On day 12 after injection, the mRNA levels of Foxp3, IL-10 and suppressor of cytokine signalling 3 (SOCS3) in the splenocytes of untreated and IL-2/JES6-1-treated CIA mice were determined by real-time PCR. (c) Spleen tissue was obtained from untreated and IL-2/JES6-1-treated CIA mice on days 12 and 26 after the injection and stained for CD4 (white) and IL-17 (red). To analyse CD4+ CD25+ Foxp3+ regulatory T cells, spleen tissues were stained for CD4 (red), CD25 (blue) and Foxp3 (green). Cell counts are shown in the form of bar graphs (right). (d) Confocal image of p-signal transducer and activator of transcription 3 (STAT3)- and p-STAT5-positive cells in the spleen tissue on days 12 and 26. Cell counts are shown in the form of bar graphs (right). The data are presented as the means ± SD of two or three independent experiments (*< 0·05, **P < 0·005, *** < 0·0005).

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The differences in serum levels of IL-17 in PBS-injected versus IL-2/JES6-1-injected mice correlated with suppressive conditions in the spleen. On day 12, the mRNA levels of Foxp3, IL-10 and SOCS3, a negative regulator of STAT3 that activates Th17,[25, 33] were significantly higher in the spleens of IL-2/JES6-1-injected mice than in PBS-treated CIA control mice (Fig. 4b). Differences in the Th17 level and Treg cell numbers were also detected by immunohistochemistry. On days 12 and 26, the expression of IL-17 was significantly lower and the presence of Foxp3+ Treg cells was significantly higher in the spleen of IL-2/JES6-1-injected mice compared with PBS-treated CIA mice (Fig. 4c).

Next, we investigated whether treatment with IL-2/JES6-1 complexes was associated with activation of STAT3 and STAT5, which are the key transcription factors for Th17 and Treg cell differentiation, respectively.[34] We examined levels of p-STAT3 in spleen tissue on days 12 and 26; the level of p-STAT3 was decreased whereas that of p-STAT5 was increased in IL-2/JES6-1-injected mice (Fig. 4d). These results suggest that injection of IL-2/JES6-1 complexes decreased the serum IL-17 level by increasing the number of Foxp3+ Treg cells via inducing phosphorylation of STAT5.

Increased Treg cell frequency in the joint tissue of IL-2/JES6-1-treated CIA mice

To investigate whether the suppressive mechanisms evident in the spleen induced by IL-2/JES6-1 complexes were also elicited in the joints, histological analyses of ankle joints from the mice described above were performed on days 12 and 26. As observed in the spleen, the frequency of Foxp3+ cells was significantly higher in tissues from mice treated with IL-2/JES6-1 complexes than in those from control mice on both days 12 and 26 (Fig. 5a,b). Moreover, the expression of IL-17 was significantly lower in the tissues from IL-2/JES6-1-injected mice than in control mice (Fig. 5).

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Figure 5. Increased presence of regulatory T (Treg) cells in the joints of mice with collagen-induced arthritis (CIA) treated with interleukin-2 (IL-2)/JES6-1. (a) Ankle joint tissues were obtained from each group of mice used in Fig. 4 on days 12 and 26 after the start of the experiment and were stained with haematoxylin & eosin, anti-mouse Foxp3, and anti-mouse IL-17. (b) The results are represented as graph of positive cell number (*< 0·05, **< 0·005, ***< 0·0005).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Treg cells can regulate excessive antigen-specific immune responses and so are a potent target for the customized treatment of autoimmune diseases, such as rheumatoid arthritis. However, the mechanism responsible for Treg cell differentiation is not fully understood, and little is known about effective methods for increasing Treg cell activity in vivo. In the current study, we found that the expansion of Treg cells induced by treatment with IL-2/JES6-1 immune complexes effectively suppressed joint inflammation in a mouse model of autoimmune arthritis. Our results demonstrate that the autoimmune response can be controlled by inducing the expansion of host Treg cells. In this CIA model, IL-2/JES6-1-induced expansion of Treg cells significantly ameliorated the severity of arthritis by suppressing the production of CII-specific antibodies and by inhibiting Th17 cells and IL-17.

Binding of IL-2 to its surface receptor triggers the JAK/STAT signalling pathway, leading to phosphorylation of STAT5.[27] In this study, we examined the ability of the IL-2/JES6-1 immune complexes to stimulate IL-2R-mediated signalling by measuring the level of p-STAT5, which is key to the expansion of Foxp3+ Treg cells. Consistent with their potent biological activity, IL-2/JES6-1 immune complexes induced a much stronger and extended STAT5 activation than did IL-2 alone. Létourneau et al.[13] reported recently that binding of the JES6-1 mAb endows IL-2 with an increased half-life by fortifying the interaction between the mAb and the neonatal Fc receptor. We propose that the protracted half-life of IL-2/JES6-1 complexes caused a robust and sustained activation of p-STAT5, leading to the expansion of the Treg cell population. Consistent with this idea, Treg cells from mice injected with IL-2/JES6-1 immune complexes displayed increased expression of CD25, CTLA4 and GITR on the surface, up-regulated SOCS1, SOCS3 and oncostatin M (Osm), which are known to be induced by STAT5, and produced more IL-10 than did Treg cells from mice injected with IL-2. A higher expression level of CD25 (IL-2Rα), in particular, is likely to contribute significantly to inducing strong expansion and activation of Treg cells by further strengthening the binding of IL-2. The lack of this positive feedback mechanism for Treg cells could be one of the main reasons why injection of IL-2 tends to augment, rather than reduce, joint inflammation,[35] presumably by stimulating recently activated T cells, rather than Treg cells. Hence, the IL-2/JES6-1 immune complex could be suppressing joint inflammation because it causes a faster and stronger induction of CD25 on Treg cells than pathogenic cells, translating into selective expansion and activation of Treg cells.

In the current study, JES6-1-expanded Treg cells delayed and ameliorated the onset of CIA by suppressing both the humoral and cell-mediated immune responses against collagen. Formation of germinal centre and activation of follicular helper CD4+ T cells have been shown to trigger various autoimmune diseases by mediating disease pathology. Hence, in CIA, the levels of CII-specific antibodies are elevated in the serum and can directly induce arthritic inflammation.[36] We have found that production of CII-specific antibodies was suppressed in IL-2/JES6-1-treated CIA mice, indicating that Treg cells suppressed production of CII-specific antibodies. It would be interesting to determine whether Treg cells induced by IL-2/JES6-1 are capable of directly affecting germinal centre formation and plasma/memory B-cell activation. In this regard, while Treg cells were initially described to suppress pathogenic T-cell subsets, such as Th1 and Th17 cells, more recent work has suggested the presence of Treg cells among follicular helper T cells that can suppress antibody production.[21, 37, 38] These follicular regulatory Treg cells were reported to display features we found in the IL-2/JES6-1-expanded Treg cells, including high expression CTLA4, GITR and IL-10.[21, 37, 38] Foxp3+ Treg cells suppressed the expansion of auto-reactive B cells and isotype switching of IgG1 in a diabetes animal model.[39] This result suggests that Foxp3+ Treg cells may regulate autoantibody production of B cells directly. However, further investigation is needed to clarify whether increased Treg cells are a direct cause of the decreased autoantibody production as a result of IL-2/JES6-1.

In terms of cell-mediated immunity, IL-2/JES6-1-induced Treg cells seemed to modulate the cytokine production profile in CIA. In the synovium of autoimmune arthritis patients, a complex interaction of accumulated immune cells contributes to the destruction of joint tissue.[29, 31, 32, 40] Furthermore, the pathogenic role of IL-17 is particularly pronounced.[41-43] Our results showed that the expression of IL-17 in the inflamed joint was significantly reduced after IL-2/JES6-1 complex injection. This change was accompanied by an increased proportion of Foxp3+ Treg cells in the joint, demonstrating that IL-2/JES6-1 complexes could directly control pathology in the inflamed tissue. Nonetheless, it is unclear whether these Treg cells were expanded in situ, or had been systemically expanded first, then transported to the inflamed joint. Regardless of the cellular mechanism, the suppressive effect was sustained for more than 3 weeks by repeated injection of IL-2/JES6-1.

In addition to suppression of Th17 cells, indirectly mediated by Treg cells, a direct inhibition of Th17 cells by IL-2 was also a likely mechanism for the reduced level of IL-17. The ability of IL-2 to suppress the generation of Th17 cells was described recently[44, 45] and is thought to be one reason why IL-2-deficient mice exhibit increased Th17 and IL-17 production and develop autoimmune disorders. Consistent with this, we observed that injection of IL-2/JES6-1 complexes before immunization of CII induced an increased level of p-STAT5 and a lower level of p-STAT3 than in control mice. Moreover, IL-2/JES6-1-treated mice had increased expression of IL-10 and SOCS3, compared with controls. The significance of these findings is that STAT3 transmits IL-6-induced signals required for Th17 cell development and STAT3 is inhibited by SOCS3. Hence, such disparate regulation of STAT5 and STAT3 functionally translates into expansion of Foxp3+ Treg cells and concomitant contraction of IL-17-producing Th17 cells. The opposing effects of IL-2 and STAT5 activation on Th17 cells were also observed previously with activation of STAT3/RORγt in pSTAT5-deleted T cells.[44, 45]

Studies on the role of IL-2 in T-cell activation have a long and circuitous history. In short, the key concept is the pleiotropic nature of IL-2 in the overall immune response. This is because IL-2 can be recognized by multiple cell types with at least two forms of receptors (IL-2Rαβγ and IL-2Rβγ) and can mediate opposing effects, depending on the state of activation and differentiation of responding cells. In this sense, IL-2 can either ameliorate or aggravate arthritis depending on the timing of administration,[35] illustrating the importance of targeting IL-2 to specific subsets of T cells at the right time in the immune response. Use of IL-2/mAb complexes is a good step towards targeting IL-2 to the relevant T cells and further investigations on IL-2/mAb complexes with and without other immune modulators may lead to even better regimens to induce maximum suppression of autoimmune arthritis.

In summary, injection of IL-2/JES6-1 complexes was sufficient to suppress the inflammatory response and the development of pathogenic T cells in a CIA animal model. This effect may have been achieved by the robust induction of p-STAT5 and by activation of other downstream mediators in the IL-2R signalling pathway, together leading to the expansion of Treg cells. The management of autoimmune arthritis calls for concomitant manipulation of Treg cells and Th17 cells. Further clinical application of IL-2/JES6-1 complexes might involve the prospective use of these complexes as a therapeutic agent for treatment of human autoimmune diseases.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

This research was supported by the Public Welfare & Safety Research Programme through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2011-0020951) and by the World Class University (WCU) Programme, NRF, Ministry of Education, Science and Technology (MEST), Korea (R31–10105).

Disclosure

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors have no conflict of interest to disclose.

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/AOF3Lu.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References