Interleukin-15 receptor α expression in inflammatory bowel disease patients before and after normalization of inflammation with infliximab

Authors

  • Clémentine Perrier,

    Corresponding author
    1. Laboratory of Clinical Immunology, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium
    • Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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    • Both authors contributed equally to this work.
  • Ingrid Arijs,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
    2. Gene Expression Unit, Department of Molecular Cell Biology, KU Leuven, Leuven, Belgium
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    • Both authors contributed equally to this work.
  • Dominiek Staelens,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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  • Christine Breynaert,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
    2. Laboratory of Clinical Immunology, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium
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  • Isabelle Cleynen,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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  • Kris Covens,

    1. PharmAbs, The KU Leuven Antibody Centre, Leuven, Belgium
    2. Centre for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium
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  • Marc Ferrante,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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  • Gert Van Assche,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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  • Séverine Vermeire,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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  • Gert de Hertogh,

    1. Department of Morphology and Molecular Pathology, University Hospitals, Leuven, Belgium
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  • Frans Schuit,

    1. Gene Expression Unit, Department of Molecular Cell Biology, KU Leuven, Leuven, Belgium
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  • Paul Rutgeerts,

    1. Department of Gastroenterology, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium
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  • Jan L. Ceuppens

    1. Laboratory of Clinical Immunology, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium
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Correspondence: C. Perrier, Translational Research Centre for Gastrointestinal Disorders (TARGID), KU Leuven, Herestraat 49, box 811, 3000 Leuven, Belgium. Email: Clementine.Perrier@med.kuleuven.be

Senior author: Jan L. Ceuppens, email: jan.ceuppens@uzleuven.be

Summary

Interleukin-15 (IL-15) is a pro-inflammatory cytokine thought to contribute to the inflammation in inflammatory bowel diseases (IBD). The specific receptor chain IL-15Rα can be expressed as a transmembranous signalling receptor, or can be cleaved by a disintegrin and metalloprotease domain 17 (ADAM17) into a neutralizing, soluble receptor (sIL-15Rα). The aim of this study is to evaluate the expression of IL-15Rα in ulcerative colitis (UC) and Crohn's disease (CD) patients before and after infliximab (IFX) therapy. Gene expression of IL-15Rα, IL-15 and ADAM17 was measured at the mRNA level by quantitative reverse transcription-PCR in mucosal biopsies harvested before and after first IFX therapy. Concentrations of sIL-15Rα were measured in sera of patients by ELISA and IL-15Rα protein was localized in the gut by immunohistochemistry and immunofluorescence. Mucosal expression of IL-15Rα is increased in UC and CD patients compared with controls and it remains elevated after IFX therapy in both responder and non-responder patients. The concentration of sIL-15Rα in serum is also increased in UC patients when compared with controls and does not differ between responders and non-responders either before or after IFX. CD patients have levels of sIL-15Rα comparable to healthy controls before and after therapy. In mucosal tissues, IL-15Rα+ cells closely resemble activated memory B cells with a pre-plasmablastic phenotype. To conclude, IBD patients have an increased expression of IL-15Rα mRNA in the mucosa. Expression is localized in B cells, suggesting that IL-15 regulates B-cell functions during bowel inflammation. No change in release of sIL-15Rα is observed in patients treated with IFX.

Introduction

Interleukin-15 (IL-15) is a pro-inflammatory cytokine produced mainly by activated monocytes, macrophages and dendritic cells, and it promotes the survival, proliferation and functional activity of immune cells such as memory T cells, natural killer cells, B cells, mast cells, monocytes, dendritic cells and macrophages. The IL-15 signal is transmitted through a receptor complex including IL-2/15Rβ, the common γ (γC) chain and the specific, high-affinity IL-15 receptor α (IL-15Rα) chain.[1] The IL-15Rα chain is expressed by many cell types including monocytes, dendritic cells, natural killer cells, T cells and fibroblasts. Several isoforms of IL-15Rα exist and are generated either by alternative splicing[2] or by proteolytic cleavage.[3] Hence, IL-15Rα can be found as a membrane receptor or as a soluble receptor (sIL-15Rα); and as most isoforms of the receptor contain a sushi domain allowing very-high-affinity binding of IL-15,[4] signalling or regulatory functions can be attributed to the receptor. Competition between soluble and membrane receptors can result in reduced biological activity of IL-15,[5] though a superagonist effect of the soluble form was also observed.[6] The signalling of IL-15 appears more complicated than for other cytokines because IL-15 primarily exists as a complex bound to IL-15Rα.[7] When IL-15/IL-15Rα complexes are shuttled to the cell surface, they can stimulate opposing cells through the β/γC receptor complex (trans-presentation), or alternatively may allow an autocrine stimulation (cis-presentation).[7] Currently, the regulation of such mechanisms remains largely unknown.

Interleukin-15 plays an important protective role in infectious disease,[8] and is also thought to be involved in many autoimmune diseases such as multiple sclerosis, psoriasis and rheumatoid arthritis.[1] Inflammatory bowel disease (IBD) patients also have increased expression of IL-15 and IL-15Rα in mucosal tissues,[9] and lamina propria mononuclear cells of IBD patients produce more IL-15 upon stimulation than cells from controls.[10] These studies suggest that this cytokine might contribute to the pathological inflammation in the bowel. A recent study also suggested that neutralization of IL-15 through sIL-15Rα could explain in part the success of infliximab (IFX) therapy in patients with Crohn's disease (CD).[11]

We intended to investigate the potential role of IL-15Rα in ulcerative colitis (UC) and CD patients during inflammation and after control of inflammation with IFX therapy. As the receptor can transmit a signal via the membrane receptor or act as an antagonist when secreted as a soluble receptor, we compared the concentration of sIL-15Rα in serum and the gene expression of all isoforms of the protein in mucosal tissues in patients with IBD versus controls. Using immunohistochemistry, we also determined the localization of the receptor in mucosal tissues.

Materials and methods

Patients

Quantification of sIL-15Rα was performed in the sera of 101 patients with active IBD (36 with UC and 65 with CD) before and 4–6 weeks after IFX therapy, and in the sera of 14 healthy controls. The baseline characteristics of patients tested for the presence of sIL-15Rα in serum are summarized in Tables 1 and 2. To focus on unambiguous responders and non-responders, clinical response and non-response were defined as follows. Clinical response for UC patients was assessed at week 10 and defined as absence of blood in stools and normalization of stool frequency with complete mucosal healing. Clinical non-response was defined as no amelioration in clinical disease activity and no mucosal healing. Response in CD patients was defined as a drop bigger than 150 points of the Crohn's disease activity index (CDAI) and to CDAI < 150 within the 10 weeks after first administration of IFX. Non-response was defined as no improvement and an increase of CDAI score within 10 weeks following administration.

Table 1. Baseline characteristics of patients with ulcerative colitis
 All (n = 36)Responders (n = 25)Non-responders (n = 11) P
  1. IFX, infliximab; IQR, interquartile range; UC, ulcerative colitis.

  2. a

    P < 0.05.

Male/Female (%)20/16 (56)13/11 (52)7/4 (64)0·718
Median (IQR) age at diagnosis (years)25·6 (19·9–40·9)24·1 (18·9–34·7)40·2 (30·0–54·5)0·016a
Median (IQR) age at first IFX (years)37·7 (27·3–50·3)30·2 (24·5–42·1)49·9 (37·8–62·1)0·00
Median (IQR) duration of disease before first IFX (year)5·7 (2·6–13·0)4·5 (2·4–11·6)7·6 (2·8–13·9)0·247
Median C-reactive protein at first IFX (mg/dl)8·9 (4·0–23·7)11·0 (3·8–24·8)4·0 (4·0–20·0)0·477
Median (IQR) endoscopic Mayo score at first IFX3 (2–3)2 (2–3)3 (3–3)0·031a
Active smoking at first IFX3 (5·5)2 (8·0)1 (9·1)1·000
Extent of disease
Left-sided ulcerative colitis (%)14 (39)10 (40)4 (36)1·000
Pancolitis (%)22 (61)15 (60)7 (64)1·000
Concomitant medication at first IFX
5-Aminosalicylates21/3615/256/111·000
Corticosteroids13/369/254/111·000
Azathioprine/6-mercaptopurine20/3616/254/110·159
Methotrexate0/360/250/111·000
Corticosteroids + immunosuppressants8/367/251/110·388
Table 2. Baseline characteristics of patients with Crohn's disease
 All (n = 65)Responders (n = 52)Non-responders (n = 13) P
  1. CDAI, Crohn's disease activity index; IFX, infliximab; IQR, interquartile range.

  2. a

    P < 0.05.

Male/Female (%)23/42 (35·4)17/35 (32·7)6/7 (46·2)0·364
Median (IQR) age at diagnosis (years)22·8 (18·8–27·225·8 (16·6–31·4)21·2 (18·8–26·8)0·409
Median (IQR) age at first IFX (years)30·1 (25·6–44·3)29·7 (25·3–42·03)41·2 (24·7–47·3)0·333
Median (IQR) duration of disease before first IFX (year)9·2 (4·0–14·9)7·1 (3·8–14·8)14 (8·1–18·5)0·081
Median C-reactive protein at first IFX (mg/dl)16·3 (4·9–30·9)20·0 (10·7–33·1)3·5 (1·5–4·6)0·0001a
Median (IQR) CDAI score at first IFX278·0 (226·5–361·5)303·5 (257·3–368·3)199·0 (56·5–248·5)0·0001a
Active smoking at first IFX10 (15·4)9 (17·3)1 (7·7)0·672
Extent of disease
Ileum (%)30 (46)24 (46)6 (46)0·096
Colon (%)47 (72)40 (77)7 (54)1·000
Concomitant medication at first IFX
5-Aminosalicylates16/6514/522/130·492
Corticosteroids21/6515/526/130·233
Azathioprine/6-mercaptopurine46/6539/527/130·134
Methotrexate3/652/521/130·494
Corticosteroids + immunosuppressants15/6512/523/131·000

For mucosal expression studies, biopsy specimen and patient characteristics and definition of response to IFX therapy are reported in previous publications from our group.[12, 13]

Quantitative reverse transcription-PCR

Quantitative reverse transcription (qRT-) PCR was performed to measure the mucosal mRNA expression levels of IL-15Rα, IL-15, a disintegrin and metalloprotease domain 17 (ADAM17) and β-actin. Total RNA was isolated from the biopsy specimens using the RNeasy Mini Kit (Qiagen, Benelux B.V., Venlo, the Netherlands). Total RNA integrity and quantity were determined with a 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE), respectively. Primers and dual-labelled probes were designed using oligoanalyzer software (www.idtdna.com/analyzer/Applications/OligoAnalyzer/) and synthesized by Sigma-Genosys (Haverhill, UK). Primer and probe sequences are shown in supplementarymaterial, Table S1S. Complementary DNA was synthesized from 0·5 μg total RNA using a reverse transcriptase kit (RevertAid H Minus First Strand cDNA synthesis kit; Fermentas, St Leon-Rot, Germany). Multiplex real-time PCR was performed in a final reaction volume of 25 μl on a Rotor-Gene 3000 instrument (Corbett Research, Mortlake, Australia), using QuantiTect Multiplex PCR NoROX Kit (Qiagen). All samples were amplified in duplicate reactions. Cycle threshold values were determined by rotor-gene 6.0.16 software. The relative expression of target mRNA levels was calculated as a ratio relative to the β-actin reference mRNA.[14]

ELISA for sIL-15Rα and IL-15

Antibodies and standard IL-15Rα Fc Chimera were purchased from R&D Systems (Minneapolis, MN). Briefly, NUNC maxisorp ELISA plates were coated overnight with 4 μg/ml monoclonal anti-hIL-15Rα (MAB1471) in carbonate–bicarbonate buffer. Wells were blocked with 5% BSA for at least 2 hr at 37° and sera diluted 1 : 2 and 1 : 4 in 0·5% BSA PBS were incubated for 1 hr at room temperature. The standard curve was set up in a pool of sera from healthy volunteers, which were previously tested to be negative for IL-15Rα. The IL-15Rα Fc chimera (147-IR) was diluted in 50% serum : 50% 0·5% BSA PBS from 1000 pg/ml to 5 pg/ml to generate a standard curve. After extensive washing, detection was performed with polyclonal goat anti-human IL-15Rα biotinylated antibody (BAF847) and traditional colorimetric substrate reactive to horseradish peroxidase (HRP). The assay allowed the detection of 10–20 pg/ml protein diluted in 50% serum : 50% 0·5% BSA PBS.

ELISA to quantify IL-15 was purchased from R&D Systems (DuoSet DY247) and performed according to the manufacturer's instructions.

Immunohistochemistry

Immunohistochemistry was performed on 5-μm-thick sections from paraffin blocks of formalin-fixed endoscopically derived mucosal biopsies and resection specimens from IBD patients and controls. After drying, deparaffinization and rehydration, epitope retrieval was performed at high pH (Dako PT Link machine, Dako Belgium NV, Heverlee, Belgium). Sections were then washed for 5 min three times (Envision Flex wash buffer, Dako) and Envision Flex Peroxidase-Blocking Reagent (Dako) was applied for 10 min at room temperature. After a second washing step, sections were incubated with an anti-human IL-15Rα rabbit polyclonal antibody (Proteintech Group, IL, USA, dilution 1 : 75) for 30 min at room temperature. Following a third washing step, bound primary antibody was visualized by incubating the slides for 30 min with Envision Flex/HRP (Dako) and application of the Envision AB+ Chromogen (Dako) for 10 min at room temperature. After rinsing, the slides were counterstained with haematoxylin, dehydrated, cleared and mounted. The primary antibody was omitted in the negative controls. The stains were evaluated by an experienced gastrointestinal pathologist (GdH). Pictures were acquired using an Olympus BX41 microscope mounted with an Olympus SC30 camera (Olympus, Tokyo, Japan) at a magnification of 100 ×.

Immunofluorescence

Cellular localization was performed with double staining for IL-15Rα and CD19 or CD38. Sections were deparaffinized and brought to water. Antigen retrieval was performed in 0·01 m citrate buffer at pH 6 for 20 min in a boiling water bath, slides were then allowed to cool down for 20 min in the hot retrieval solution. Endogenous peroxidase was inhibited by exposure to 0·1% H2O2 in methanol, 20 min at room temperature. Sections were then blocked for 30 min with TNB blocking solution (Perkin Elmer, Groningen, the Netherlands) and incubated with primary antibody (anti-IL-15RA, Proteintech 16744-1-AP, 1/100; and either anti-CD38 or anti-CD19, Abcam ab93941 or ab31947, respectively, 1/100), for 1 hr at room temperature. Slides were washed and incubated with secondary antibody (HRP donkey anti-rabbit, 1/500; Alexa Fluor®488 donkey anti-mouse, 1/400, Jackson ImmunoResearch), 30 min at room temperature. IL-15RA signal was then amplified using the tyramide signal amplification (TSA) method (Perkin Elmer, NEL704A Cy3 kit) following the manufacturer's instructions. Nuclei were stained with DAPI. Finally, slides were mounted with FluorSave (Calbiochem, San Diego, CA) and stored at 4°. Pictures were acquired with a Zeiss AxioVert 100M microscope (Zeiss, Oberkochen, Germany).

Statistics

Wilcoxon signed-rank test was used for paired samples and Mann–Whitney U-test for unpaired samples, using spss software (SPSS, Chicago, IL).

Results

Gene expression of IL-15, IL-15Rα and ADAM17 in mucosal tissues

We first measured the gene expression of IL-15 and IL-15Rα in endoscopically derived mucosal biopsies of IBD patients with active disease collected before and after first the IFX therapy. Gene expression was measured in 12 controls (six colon and six ileum), 24 active UC, 18 active Crohn's colitis (CDc) and 17 active Crohn's ileitis (CDi) patients before and 4–6 weeks after first infusion of IFX by qRT-PCR.[12] The expression of IL-15 mRNA was not significantly increased in patients with active UC and CD before therapy when compared with healthy controls and the expression did not change with anti-tumour necrosis factor-α treatment, either in responders or in non-responders (Fig. 1a,b). Of note, expression of IL-15 was more pronounced in the ileum than in the colon. The expression of IL-15 in CDi patients before therapy, both in responders and non-responders, tended to be higher than in control ileums, although the difference did not reach significance (P = 0·069).

Figure 1.

Expression of mRNA of interleukin-15 (IL-15) (a and b), IL-15 receptor α (IL-15Rα) (c and d) and a disintegrin and metalloprotease domain 17 (ADAM17) (e and f) was measured by quantitative reverse-transcription-PCR in endoscopic biopsies of control colons (n = 6), control ileums (n = 6), ulcerative colitis (UC) patients (R: n = 6 and NR: n = 16), Crohn's colitis (CDc) patients (R: n = 10 and NR: n = 7) and Crohn's ileitis (CDi) patients (R: n = 7 and NR: n = 10). A line between two points represents the change in expression before and after therapy for one inflammatory bowel disease patient.

The expression of IL-15Rα mRNA in mucosal biopsies was increased in UC, CDc and CDi patients before therapy compared with control tissues (Fig. 1c,d; P < 0·001, P = 0·001 and P < 0·001, respectively). These results confirm a previous report on the expression of IL-15Rα in UC and CD patients.[9] The expression of the receptor remains elevated after IFX treatment in responder patients while inflammation is controlled and did not further increase after IFX therapy in either responders or in non-responders. Because sIL-15Rα can be released in patients responding to IFX through the action of the protease ADAM17,[11] we quantified the expression of ADAM17 in mucosal biopsies before and after IFX therapy and found no differences. ADAM17 was not up-regulated in inflamed tissues when compared with healthy controls, and no increase of ADAM17 expression after therapy was observed for both responders and non-responders (Fig. 1e,f).

sIL-15Rα concentrations in serum remain unchanged before and after IFX therapy in UC and CD patients

Expression of ADAM17 mRNA might not reflect the activity of the metalloprotease, so we further quantified sIL-15Rα at the protein level in sera of patients using a sandwich ELISA. This ELISA detects free sIL-15Rα, as well as complexes of IL-15/sIL-15Rα with the same sensitivity. To avoid the fact that intermediate responders blur the results, we focused on patients with a very good response to IFX and patients that did not respond at all to the therapy (see Materials and methods for response criteria). We compared these data with the concentration of sIL-15Rα in healthy volunteers (n = 14).

Sera of 36 patients with UC were collected before and 4–6 weeks after the first IFX infusion; among them, 25 were responders and 11 were non-responders. Baseline characteristics of UC patients before the start of therapy are summarized in Table 1. Non-responder patients were older at diagnosis and at start of therapy and had a higher endoscopic Mayo score. Duration of disease, C-reactive protein before therapy, active smoking, localization of the disease and concomitant medication were similar between responders and non-responders.

We observed that the concentration of sIL-15Rα was slightly increased in UC patients before therapy when compared with healthy volunteers (6·1 pmol/l; 4·0–8·2 versus 2·9 pmol/l 0·8–5·9; P = 0·0069; median and interquartile range). No difference between responders and non-responders before therapy was observed (6·3; 3·8–8·3 versus 6·1; 5·0–6·3; P = 0·85). Concentration of sIL-15Rα did not increase after treatment in either group (Fig. 2a).

Figure 2.

Concentrations of soluble interleukin-15 receptor-α (sIL-15Rα) were measured by ELISA in serum of 14 healthy volunteers, 36 ulcerative colitis (UC) patients (R: n = 25 and NR: n = 11) (a) and 73 Crohn's disease (CD) patients (R: n = 58 and NR: n = 15) (b) before and after infliximab (IFX) therapy.

Using the same ELISA, we analysed the sera of CD patients undergoing their first treatment with IFX, including 52 responders and 13 non-responders. Baseline characteristics of these patients are summarized in Table 2. No differences were observed between responders and non-responders for the age of diagnosis, the age at start of therapy, the duration of the disease, smoking, location of the disease and concomitant medications. We observed a lower C-reactive protein concentration and CDAI score before the start of the therapy in non-responder patients in comparison with responders, suggesting a lower load of inflammation.

The concentration of sIL-15Rα in patients with active CD before therapy was not different from healthy controls (2·4 pmol/l; 1·7–3·8 versus 2·9 pmol/l; 0·8–5·9; P = 0·98). There was no difference between responder and non-responder patients before and after therapy (Fig. 2b). Furthermore, no increase of sIL-15Rα concentration after therapy was detected in responders (2·4 pmol/l; 1·8–3·9 versus 2·4 pmol/l; 1·7–3·4; P = 0·544), nor in non-responders (1·8 pmol/l; 0·9–3·6 versus 1·6 pmol/l (1·2–2·1); P = 0·397). Some outlier patients (4/51) in the responder group had an elevated concentration of sIL-15Rα after therapy, but except for one of them, this was not a substantial increase linked to the therapy.

As IL-15–IL-15Rα complexes can also be detected by this ELISA, we aimed to determine if sIL-15Rα was loaded with bioactive IL-15. Using a commercial kit for IL-15, which detects free IL-15 and IL-15Rα–IL-15 complexes, we quantified IL-15 in samples with high levels of sIL-15Rα (n = 11) and in 14 additional patients with clear clinical inflammation. Interleukin-15 was detected in low amounts in only three samples out of 11 samples with high concentrations of sIL-15Rα, suggesting that most of the soluble receptors are not loaded with IL-15. In addition, in all other patients tested (n = 14) IL-15 remained below the detection limit (< 20 pg/ml).

Overall, this shows that sIL-15Rα is not released in serum of IBD patients upon treatment with IFX, and suggests that IL-15Rα is mainly expressed as a membrane receptor. To speculate about the role of IL-15Rα, we aimed to localize the expression of the protein.

IL-15Rα expression is restricted to B cells in the lamina propria

The localization of IL-15Rα was first evaluated by immunohistochemistry in endoscopically derived mucosal biopsies of UC patients and controls, and on resection specimens of CD patients. Based on morphological characteristics, the expression of IL-15Rα was found mainly in cells with a phenotype of antibody-producing cells in UC patients (Fig. 3a,b). No expression was found in epithelial cells or intraepithelial lymphocytes. Furthermore, regions with T cells and non-stimulated B cells located in germinal centres were also negative for the staining. The number of immune cells and cells positive for IL-15Rα was decreased in UC patients in remission (Fig. 3c) and in patients treated successfully with IFX (Fig. 3d).

Figure 3.

Mucosal tissues stained by immunohistochemistry for interleukin-15 receptor-α (IL-15Rα). Mucosal biopsies of patients with active ulcerative colitis patients (a and b), ulcerative colitis patients in remission (c) and ulcerative colitis patient after infliximab therapy (d) were stained with a polyclonal antibody targeting IL-15Rα. Resection specimens of Crohn's disease patients (e and f) were stained according to the same procedure. Black bars represent 100 μm.

Resection specimens of CD patients showed a similar distribution pattern to UC patients (Fig. 3e,f). Cells with an antibody-producing phenotype located in the lamina propria express the receptor. Epithelial cells, intraepithelial lymphocytes, T cells and B cells located in germinal centres were negative for IL-15Rα staining. Deeper layers of the tissues were also negative for IL-15Rα (data not shown). To better define cells expressing the receptor, double staining for IL-15Rα and CD19 and CD38 was performed using immunofluorescence (Fig. 4). All IL-15Rα-positive cells were CD19-positive, demonstrating a B-cell lineage. Furthermore, CD38, a marker of T-cell and B-cell activation, was not present or was weakly expressed in IL-15Rα-positive cells, suggesting that IL-15Rα-positive cells are memory B cells or pre-plasmablast, yet with an antibody-producing phenotype.

Figure 4.

Immunofluorescence staining of mucosal tissues. Staining of interleukin-15 receptor-α (IL-15Rα) (red, a) together with CD19 (green, b) show clear co-localization with yellow staining (c), whereas staining of IL-15Rα (red, d) together with CD38 (green, e) show distinct pattern of expression with almost no co-localization (f).

These data suggest that the receptor might play a role in B-cell activation and differentiation, and are in line with a previous report showing positive staining for IL-15Rα in CD20+ B cells in mucosal tissues.[9]

Altogether, these data show that in both diseases, IL-15Rα is expressed by B cells producing antibodies. We could not find any expression of the protein on epithelial cells, nor on T cells.

Discussion

In this study, we aimed to evaluate the role of IL-15 signalling in IBD patients before and after first IFX therapy. Using qRT-PCR we could not detect a significant increase of IL-15 expression in mucosal biopsies, as previously observed in UC patients,[9] although ileal expression of IL-15 in CD patients was higher than in controls. In a former publication, we observed an increased secretion of IL-15 by lamina propria mononuclear cells of IBD patients when these latter were stimulated by lipopolysaccharide or interferon-γ.[10] It was further identified by immunohistochemistry on mucosal biopsies that macrophages were the main producers of IL-15 in IBD patients.[10] It is possible that the sensitivity of the assay used for this study was not high enough to detect increased IL-15 mRNA expression in a specific subset of cells (most likely macrophages) present in the mucosal biopsies without any additional stimulation. Alternatively, it has been shown that IL-15 can also be regulated in a post-transcriptional manner,[15] which would explain the absence of a difference at the mRNA level. Serum level of IL-15 remained below the detection limit before and after therapy in the majority of patients tested, despite clear inflammation. Because IL-15Rα has a very high affinity for IL-15,[4] free IL-15 might have immediately been scavenged by IL-15Rα to form a complex. However, IL-15 was detected in very low amounts in only three patients with high sIL-15Rα levels. This demonstrates that the release of IL-15 in the serum of an IBD patient before or after IFX therapy is limited, and suggests that IL-15 production remains local.

In contrast, we observed a significantly increased expression of IL-15Rα mRNA in UC and CD patients before therapy when compared with control tissues, suggesting a potential role of the receptor in bowel inflammation. It is interesting to note that the expression of IL-15RA mRNA is not normalized in patients after successful IFX therapy; so even when inflammation is reduced, the receptor remains elevated, which might point towards a primary pathogenic role of the receptor in bowel inflammation. Supporting these observations, it was shown that IL-15 knockout mice are less susceptible to dextran sodium sulphate acute and chronic colitis than wild-type animals, suggesting a potential pathogenic role for IL-15 signalling.[16]

To determine whether the receptor was released as neutralizing sIL-15Rα, we first checked the expression of ADAM17 mRNA, the enzyme responsible for cleavage of membrane IL-15Rα into sIL-15Rα. Gene expression was similar in IBD patients to that in healthy controls and did not change after IFX therapy. It is interesting to note that all patients had normal levels of ADAM17 mRNA expression, because it was recently shown that a loss of function mutation in ADAM17 might be the cause of a particular form of IBD.[17] The activity of the enzyme might not be reflected by the mRNA expression level, therefore, we quantified sIL-15Rα in serum of UC and CD patients before and after IFX therapy. We first observed a slight increase of sIL-15Rα in the serum of UC patients compared with healthy volunteers, suggesting that sIL-15Rα is implicated in UC inflammation. This increase was not observed in CD patients, who display the same concentration of sIL-15Rα as healthy volunteers. As far as IFX therapy is concerned, the concentration of sIL-15Rα remained stable before and after therapy in both diseases. Hence, we do not observe an association between normalization of inflammation and neutralization of IL-15 via sIL-15Rα in CD or UC patients, denying that release of the decoy receptor sIL-15Rα could be a major mechanism of IFX therapy. The difference between our study and the one from Bouchaud et al.[11] is unexpected. Although the time of investigation is slightly different between the two studies (4–6 weeks after first IFX infusion versus after third IFX infusion), it cannot explain the difference, because serum levels of sIL-15Rα after the third IFX therapy were not increased (data not shown). Importantly, in the study of Bouchaud et al.,[11] only a limited number of responder patients displayed an increased sIL-15Rα (eight out of 32), which demonstrates that increase of sIL-15Rα is obviously not a major process for the success of infliximab therapy.

We identified cells with a phenotype of antibody-producing cells as the main cell type expressing IL-15Rα in mucosal tissues of IBD patients. With immunofluorescence we further characterized IL-15Rα+ cells as CD19+ confirming the B-cell lineage. The limited expression of CD38 staining of IL-15Rα+ cells suggests that the receptor is expressed in newly differentiated plasmablast. These data are in accordance with the observation from Nishiwaki showing expression of IL-15Rα on CD20+ B cells in the mucosa of IBD patients,[9] which further supports a role for IL-15Rα in B cells in IBD. Furthermore, Neither study shows any expression of IL-15Rα in epithelial cells and this using two different commercially available antibodies. This is a second discrepancy with the study from Bouchaud et al.[11] and it is possible that the specificity of antibody used explains the differences between these two studies.

Mucosal biopsies of UC patients in remission or after successful IFX therapy display a reduction of antibody-producing cells, and concordantly of IL-15Rα-positive cells; however, IL-15Rα mRNA expression is not decreased after IFX therapy. It has been shown that IL-15Rα is recycled and remains functional for a long period,[18] hence it is possible that the protein accumulates during inflammation, despite equal mRNA expression. This could explain why the mRNA levels remain unchanged before and after therapy.

At the cellular level, the expression of IL-15Rα is localized at the surface of cells and intracellularly, probably in the endoplasmic reticulum, as has been observed previously in other cell types.[2] No apparent staining was observed in the nucleus of CD19+ cells. Four main isoforms of IL-15Rα have been described (the full length IL-15Rα, the δ2 IL-15Rα which lacks the IL-15-binding domain, the δ3 IL-15Rα, which lacks a linkerhinge region and the δ23 IL-15Rα2) and appear to localize differently within cells. Isoforms lacking exon 2 are localized in the endoplasmic reticulum, Golgi and cytoplasmic vesicles, whereas the full length protein is associated with the nuclear membrane.[2] Based on the intracellular localization of IL-15Rα expressed in the bowel of IBD patients, it is possible that the ‘regulatory’ isoform lacking an IL-15 binding domain is preferentially expressed. This, however, does not exclude the possibility that the full-length receptor is not expressed and further investigations are needed to clarify this.

Although IL-15 has been mainly studied for its role on T cells and NK cells, B cells are also responsive to IL-15; IL-15 together with CpG stimulates the proliferation of memory B cells.[19] In addition, IL-15 together with B-cell-activating factor and lipopolysaccharide induce the production of IgA and IgG from memory B cells.[20] Direct interaction between B cells and monocytes/macrophages leads to proliferation of B cells,[21] and these may then become further activated by CD40–CD40 ligand interaction between B cells and T cells. Furthermore, in allergic individuals, IL-15 decreases the production of allergen-specific IgE and favours the production of IgG,[22] showing an important role for IL-15 in the functions of antibody-producing cells. We can hypothesize that IL-15 produced by macrophages[10] contributes to the inflammation in the bowel by stimulating B cells to proliferate and produce antibodies. The absence or weak staining of CD38 in many IL-15Rα-positive cells suggests that the cells are memory B cells differentiating into pre-plasmablasts and plasmablasts.[23] In vitro studies showed that IL-15 allows activation of memory B cells and their differentiation toward plasmablast.[24] This suggests that IL-15Rα might be involved in the differentiation of plasmablast in IBD patients. It remains unclear whether IL-15Rα+ cells are the producers of anti-neutrophil cytoplasm antibodies (pANCA) and anti-Saccaromyces cerevisiae antibodies (ASCA), typically observed in IBD patients, which might participate in the deleterious response observed in these diseases.

Alternatively, it is not excluded that B cells also express IL-15, because this has been shown in patients with multiple sclerosis,[25] and might therefore act as stimulating cells presenting the complex IL-15–IL-15Rα on the cell surface. The presence of IL-15, however, needs to be demonstrated for B cells, as so far, only macrophages[10] and possibly stromal cells were shown to produce IL-15.[26]

Altogether, these data suggest that IL-15 signalling contributes to the inflammation in IBD patients, by stimulating B cells in the lamina propria. It is tempting to speculate that blocking IL-15 would be an interesting target for future treatment of IBD. However, it is not clear yet what are the implications of B-cell stimulation, and if its prevention would help to resolve the inflammation. In our studies, clinical response to IFX is not associated with an increase of sIL-15Rα in the serum of IBD patients. We therefore believe that an increase of sIL-15Rα after IFX infusion is not a major mechanism that contributes to the success of the therapy.

Acknowledgements

This work was supported by a grant from the Swiss Science Research Foundation (PBLAP3-129427/1) to CP, a grant from the Fund for Scientific Research-Flanders (FWO-Flanders) Belgium (FWO project nr.G.0440·06 and nr.G.0479·10) to IA, a grant from the Agency for Innovation by Science and Technology in Flanders (IWT) to CB, and an SBO grant from IWT to KC. IA, IC, MF are postdoctoral fellow of FWO-Flanders. SV is a clinical researcher of FWO-Flanders.

Disclosures

PR discloses consulting roles for Centocor, Schering-Plough, UCB, Abbott, Genentech, Pfizer, Hoffmann-La Roche, Bristol Myers Squibb, Millenium Pharmaceuticals, Tillotts, GSK, ChemoCentryx, and has received lectures and research support from Centocor, Schering Plough, UCB and Abbott. SV discloses speaker or consultancy roles for Pfizer, Ferring, MSD, Abbott and UCB and grant support from Abbott and UCB. GvA discloses speaker or consultancy roles to the University of Leuven from Pfizer, Ferring, Novartis, MSD, Abbott and UCB and grant support from Abbott and MSD. The other authors have no competing interests.

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