Increased IL-15 production of muscle cells in polymyositis and dermatomyositis
Tomoko Sugiura1,
Masayoshi Harigai1,
Yasushi Kawaguchi1,
Kae Takagi1,
Chikako Fukasawa1,
Satomi Ohsako-Higami1,
Shuji Ohta1,
Michi Tanaka1,
Masako Hara1 and
Naoyuki Kamatani1
1 Institute of Rheumatology, Tokyo Womens Medical University, 10-22 Kawada-cho, Shinjuku-ku, Tokyo 162-0054, Japan
Correspondence to: T. Sugiura; E-mail: tsugiura{at}ior.twmu.ac.jp
Transmitting editor: T. Watanabe
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Abstract
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In polymyositis (PM)/dermatomyositis (DM), various cytokines, especially macrophage-derived cytokines such as IL-1
, IL-1ß and tumor necrosis factor (TNF)-
, are expressed in the inflammatory foci. We previously reported that IL-15, a novel cytokine with a biological activity similar to that of IL-2, is expressed in muscle cells in PM/DM. In the present study, we set out to investigate the regulation of IL-15 in cultured myoblasts. Myoblasts constitutively produced a low level of IL-15 and the production was augmented by stimulation with IFN-
, IL-1
, IL-1ß, TNF-
or lipopolysaccharide (LPS) in a dose-dependent manner. These stimuli also enhanced the expression of IL-15 mRNA. About 3040% of IL-15 was detected intracellularly, while the rest was released into the culture supernatant. Immunohistochemical staining revealed that intracellular IL-15 was localized in the perinuclear area of the cytoplasm in the myoblasts. Despite the considerable amounts of intracellular IL-15, the myoblasts predominantly expressed authentic IL-15 mRNA isoform. This isoform generates IL-15 with long signal peptide preprotein, which is all to be secreted. The biological activity of IL-15 secreted from the myoblasts was examined using an IL-15-dependent murine T cell line, CTLL-2. Culture supernatants of the myoblasts induced a proliferative response of CTLL-2 and this was specifically inhibited by anti-IL-15 antibody. These results suggest that inflammatory stimuli induce the production of IL-15 in the muscle cells in PM/DM, and IL-15 may contribute to the immunopathogenesis by augmenting recruitment and activation of the infiltrating T cells. Blocking of IL-15 production might be of therapeutic value in PM/DM.
Keywords: dermatomyositis, IL-15, muscle cells, polymyositis
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Introduction
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The expression of various cytokines has been reported in the muscle tissue of idiopathic inflammatory myopathy (IIM) (14). A common finding of these studies is the predominant expression of monokines such as IL-1
, IL-1ß and tumor necrosis factor (TNF)-
in the muscle tissue, and this cytokine profile is common among patients with the different subsets of IIM including polymyositis (PM), dermatomyositis (DM) and inclusion body myositis. Among these cytokines, the expression of IL-1
is most frequently reported. IL-1
is expressed in the endothelial cells, infiltrating mononuclear cells (MNC) and, to a lesser extent, in the muscle cells themselves in IIM. This aberrantly expressed IL-1
can induce endothelial cells to express cell adhesion molecules such as intercellular adhesion molecule-1 (1,5), thus promoting extravasation of the inflammatory cells to the affected muscle tissue. TNF-
possesses similar functions to IL-1 in activating the endothelial cells (6). Because these cytokines are not expressed or only sparsely expressed in normal muscle tissue, they are expected to be involved in the development of inflammatory processes.
In contrast to the predominant expression of monokines, lymphokines such as IL-2 or IFN-
are less commonly expressed in the muscle tissue of IIM (14). Nevertheless, infiltrating T cells are in their activated state because they express HLA-DR antigen (7) and leukocyte function-associated antigen-1 (8), and have a memory phenotype (9). The discordance in the scarce expression of T cell-derived cytokines, despite a large number of activated T cells in the inflammatory site, has been explained by the difference in the phase of the inflammatory process. It is possible that IFN-
is produced at the initiation of inflammation and down-regulated at the point of muscle biopsy, although the expression of IFN-
might be dependent on the severity of the inflammation. We previously reported that IFN-
is expressed on infiltrating MNC in eight of 10 PM/DM patients and these IFN-
-expressing muscle tissues showed severe inflammation (10). These results are consistent with those of other investigators (11). Thus, IFN-
could be a potent T cell activator in IIM. Nevertheless, are there any cytokines that are commonly expressed and can activate T cells in IIM? In the present study, we propose that IL-15 produced by the muscle cells could act as a T cell activator in IIM.
IL-15 is a member of the 4
-helix bundle cytokine family and is a novel cytokine with IL-2-like activity, which includes stimulation of the proliferation and activation of T cells and NK cells, locomotion and chemotaxis of T cells, induction of cytotoxic effector cells, and Ig production by B cells (1214). In contrast to IL-2, IL-15 mRNA is expressed in various non-lymphoid tissues and cells, such as the placenta, skeletal muscle, kidney, lung, heart, fibroblasts, keratinocytes and monocytes, but not in resting or activated T cells (12). IL-15 meditates its function through the ß and
chain of the IL-2 receptor and its own unique
chain (15). We first reported in a previous study that IL-15 is expressed in the muscle cells in PM/DM (10). In the present study, we focused on the regulation of IL-15 production in cultured myoblasts and we also examined whether it possesses any biological activity.
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Methods
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Patients
Muscle specimens were obtained from three patients with PM and two patients with DM. All the subjects fulfilled the diagnostic criteria of Bohan and Peter (16). None were receiving any immunosuppressive treatment at the time of muscle biopsy. As normal controls, we chose three individuals who were suspected to have muscle disorders, but whose biopsied muscle tissues showed normal histological findings.
Immunohistochemistry
Serial 4-µm thick sections were air-dried and fixed in 2% paraformaldehyde for 30 min at 4°C. Immunohistochemical staining was performed using a commercial kit (Vectastain Universal Quick Kit; Vector, Burlingame, CA). Briefly, all the sections were incubated with 2.5% normal horse serum for 30 min, followed by incubation with anti-IL-15 mAb (10 µg/ml; R & D Systems, Minneapolis, MN), anti-CD68 mAb (10 µg/ml; Dako, Glostrup, Denmark) or mouse IgG1 (Dako) for 30 min. After being washed with PBS, the sections were incubated for 10 min with horse biotinylated secondary antibody. After an extensive wash with PBS, all the sections were exposed to a streptavidin/peroxidase preformed complex for 5 min and then covered with diaminobenzidine tetrahydrochloride for 2 min. All the sections were counter-stained with hematoxylin.
Myoblast culture
Myoblasts of human skeletal muscle were obtained from biopsied muscle tissues by enzymatic digestion and purified by magnetic cell separation using anti-CD56 mAb (10). Briefly, the muscle tissue was minced and digested by 0.05% trypsin. The resulting cell suspension was incubated for 30 min to make the fibroblasts adhere. Non-adhesive cells were removed and incubated in a new gelatin-coated dish in DMEM containing 10% FCS. After becoming confluent, the myoblasts were purified by magnetic cell separation using anti-CD56 mAb (Becton Dickinson, San Jose, CA) and goat anti-mouse IgG-coated magnetic microbeads (Miltenyl Biotec, Bergisch Gladbach, Germany). These purified human myoblasts easily formed myotubes, and they were CD56+ and expressed acetylcholine receptor (data not shown).
Immunofluorescent staining
The myoblasts were cultured in chamber slides, and fixed in 50% methanol and 50% acetone for 10 min at 4°C. After being blocked with 2.5% horse serum, the slides were incubated with anti-IL-15 mAb for 30 min followed by incubation with phycoerythrin-labeled goat anti-mouse IgG (Becton Dickinson) for 30 min. Confocal microscopy analysis and scanning were performed using a CSU10 (Yokogawa Electric, Tokyo, Japan).
RT-PCR
Total cellular RNA was isolated from the myoblasts with TRIzol (Gibco, Frederick, MD) according to the manufacturers protocol. Total RNA was reverse transcribed to cDNA using Superscript II reverse transcriptase (Gibco). For PCR, 2 µl of reverse transcriptase product was used in a total volume of 50 µl containing the following reagents: 1.5 mM MgCl2, PCR buffer (1 x 50 mM KCl and 10 mM TrisHCl, pH 8.3), 0.2 mM each of dNTPs, 1 U of Ampli Taq polymerase (Boehringer Mannheim, Mannheim, Germany), and forward and reverse primers (20 µM each). The sequences of the primers were as follows: IL-15 forward(a), 5'-GGATTTACCGTGGCTTTGAGT AATGAG-3'; IL-15 forward(b), 5'-GCCTTCATGGTATTGGG AAC-3'; IL-15 reverse, 5'-GAATCAATTGCAATCAAGAAGTG-3'; ß-actin forward, 5'-AAGAGAGGCATCCTCACCCT-3'; and ß-actin reverse, 5'-TACATGGCTGGGGTGTTGAA-3'. The thermocycle conditions were 35 cycles at 95°C for 1 min (denaturing), 60°C for 30 s (annealing) and 72°C for 1 min (extension). The PCR products were electrophoresed in a 2% agarose gel and visualized with ethidium bromide.
Quantitative analysis of mRNA expression
To assess whether various stimulations increase the expression of IL-15 mRNA, we performed quantitative analysis of the mRNA expression using a Pre-developed TaqMan Assay Reagents System (Perkin-Elmer, Foster City, CA) according to the manufacturers protocol. Briefly, at the end of culture total RNA was isolated from the myoblasts and reverse transcribed as described above. cDNA was amplified using a pre-developed mixture containing primers for IL-15 and fluorescent-labeled probe (FAM) or primers for endogenous GAPDH and fluorescent-labeled probe (VIC). The PCR reaction and quantitative analysis of mRNA were performed by ABI Prism 7700 Sequence Detection System (Perkin-Elmer). The quantity of mRNAs for IL-15 and GAPDH was tentatively calculated for each sample using a standard sample of a known quantity. Finally, the quantity of IL-15 mRNA was divided by that of GAPDH mRNA of the same sample and expressed as relative quantity of IL-15 mRNA.
Preparation of cell extracts
Myoblasts were detached by trypsinization, washed 3 times in PBS, and lysed in 100 µl of 50 mM TrisHCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 1% NP-40, containing 1 mM PMSF (Sigma, St Louis, MO) and 0.2 U/ml aprotinin (Sigma). The extracts were centrifuged at 5000 g and the supernatants were used for ELISA.
ELISA
To determine the concentrations of IL-15 in the culture supernatants or cell extracts, a specific ELISA was developed. Briefly, ELISA plates (Costar, Cambridge, MA) were preabsorbed with capture antibodies for IL-15 (2 µg/ml; R & D Systems). After an overnight incubation at 4°C, the plates were washed with PBS containing 0.05% Tween 20 (PBST) and blocked with PBS containing 10% FCS for 2 h at room temperature. After washing with PBST, serial dilutions of recombinant IL-15 (R & D Systems) or samples were applied to the plates in duplicate. After an overnight incubation at 4°C, the plates were washed 3 times with PBST and then incubated with biotin-conjugated anti-IL-15 antibody (200 ng/ml; R & D Systems) for 45 min at room temperature. After washing with PBST, all the plates were incubated with avidinperoxidase (10 mg/ml; Sigma) for 30 min at room temperature. After washing, an ABTSperoxidase substrate mixture (Kirkegaard & Perry, Gaithersburg, MD) was added to each well and the absorbance was measured using an ELISA plate reader (Immuno Mini NJ-2300; Nippon Intermed, Tokyo, Japan) with a test wavelength of 414 nm and a reference wavelength of 490 nm. The sensitivity of the assay was 7.81 pg/ml.
Proliferation assay
A murine IL-15-dependent T cell line, CTLL-2, was kindly provided by Dr H. Kato (Department of Immunology and Microbiology, Tokyo Womens Medical University) and maintained in 10% FCS/RPMI 1640 supplemented with 5 x 105 M 2-mercaptoethanol and 100 pg/ml of recombinant human IL-15 (Genzyme/Techne, Cambridge, MA). CTLL-2 was rested for 6 h in IL-15-free medium and plated in 96-well microtiter plates at 5 x 104 cells/well. Supernatants of the myoblasts were prepared by incubating the cells with 10 ng/ml of IL-1
for 120 h. CTLL-2 was stimulated with fresh medium containing 50% supernatants of the myoblasts along with either anti-IL-15 mAb (R & D Systems), anti-IL-2 mAb (R & D Systems) or control mouse IgG1 (Cosmo Bio., Tokyo, Japan) for 48 h. During the last 4 h, the cell proliferation was analyzed by a Premix WST-1 Cell Proliferation Assay System (Takara, Tokyo, Japan), which is similar to a MTT assay, according to the manufacturers protocol. The absorbance was measured with a test wavelength of 450 nm and a reference wavelength of 630 nm.
Statistical analysis
Comparison of data was performed using the paired Students t-test in Fig. 2 and unpaired Students t-test in Fig. 3. P < 0.05 was considered significantly different.

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Fig. 2. Effect of cytokines and LPS on the IL-15 production of the myoblasts. The myoblasts obtained from three PM/DM patients were stimulated with various concentrations of cytokines and LPS for 72 h to collect the culture supernatants and cell extracts. The concentrations of IL-15 were determined by ELISA. The results are expressed as mean ± SE (n = 3). Closed bars: intracellular IL-15; open bars: secreted IL-15. *P < 0.05; **P < 0.01.
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Fig. 3. Quantitative analysis of IL-15 mRNA expression. Six hours after stimulation with various cytokines or LPS, total RNA was isolated from the myoblasts of PM/DM, and IL-15 and GAPDH mRNAs were quantified by an ABI Prism 7700 sequence detection system. The quantity of IL-15 mRNA was divided by that of GAPDH mRNA of the same sample and expressed as relative quantity of IL-15 mRNA. The myoblasts constitutively expressed small amounts of IL-15mRNA (oval white full). Stimulation with IFN- (oval right diagonal, 250 U/ml), IL-1 (oval dot, 5 ng/ml), IL-1ß (oval dot full, 5 ng/ml), TNF- (oval left diagonal, 10 ng/ml) or LPS (oval black full, 50 ng/ml) significantly increased the expression of IL-15 mRNA.
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Results
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Muscle cells in PM/DM strongly expressed IL-15 compared to normal controls
The cytoplasm of the muscle cells was strongly stained with anti-IL-15 mAb in the five patients including both PM and DM, although that of the normal controls was only marginally stained (Fig. 1B and D). In contrast to its predominant expression in the muscle cells, IL-15 was sparsely expressed in the infiltrating MNC, even in the macrophages (Fig. 1A and B).


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Fig. 1. IL-15 was expressed predominantly in muscle cells in PM/DM. The cytoplasm of the muscle cells was strongly stained with anti-IL-15 mAb in a representative PM patient (B), although that of the normal control was marginally stained (D). In contrast to the muscle cells, only occasional infiltrating MNC were stained with IL-15 (B). The staining with CD68 in the PM patient is shown (A). Most of CD68+ cells were negative for IL-15 (A and B). Arrows indicate CD68+ cells. Similar results were observed in other PM/DM patients. The staining with the control mouse IgG1 in the PM patient (C) and a normal control (E) is shown.
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Stimulation with cytokines or LPS enhanced the production of IL-15 by myoblasts
To investigate the role of cytokines on IL-15 production in the cultured myoblasts, semi-confluent myoblasts were stimulated with various concentrations of IFN-
(PharMingen), IL-1
(R & D Systems), IL-1ß (R & D Systems), TNF-
(PharMingen) or LPS (Sigma) for 96 h. At the end of the culture, the culture supernatants and cell extracts were collected, and supplied for ELISA. Without stimulation, the myoblasts constitutively produced low levels of IL-15 (Fig. 2). There was no significant difference in the basal levels of IL-15 production between the myoblasts obtained from three different patients. Stimulation with IFN-
, IL-1
, IL-1ß, TNF-
or LPS enhanced the IL-15 production in a dose-dependent manner (Fig. 2). About 3040% of IL-15 was detected in the cell extracts of the stimulated myoblasts.
Stimulation with cytokines and LPS enhanced the IL-15 mRNA expression
We next analyzed whether the stimulation with cytokines or LPS increases the expression of IL-15 mRNA. Six hours after stimulation, the expression of IL-15 mRNA was quantitatively analyzed as described in Methods. The myoblasts constitutively expressed small amounts of IL-15 mRNA. Stimulation with IFN-
, IL-1
, IL-1ß, TNF-
or LPS significantly increased the expression of IL-15 mRNA (Fig. 3).
Immunocytochemically staining of IL-15 in unstimulated myoblasts
Because 3040% of IL-15 was detected in the cell extracts, we examined the intracellular distribution of IL-15 in the cultured myoblasts by immunocytochemistry. Using conventional immunocytochemistry, intracellular IL-15 was found to localize predominantly in the perinuclear cytoplasm (Fig. 4A and B). Confocal microscopic analysis of the immunofluorescent staining confirmed the distribution of intracellular IL-15 in the unstimulated myoblasts (Fig. 4C). We also investigated the staining of myoblasts those were stimulated by 250 U/ml of IFN
for 48 h. The staining pattern and localization of IL-15 was similar to that of unstimulated myoblasts (not shown).

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Fig. 4. Cellular staining for IL-15. The unstimulated myoblasts cultured in chamber slides were incubated with control msIgG1 (A) or anti-IL-15 mAb (B and C). Immunocytochemical (A and B) and immunofluorescent (C) staining revealed that intracellular IL-15 located particularly in the perinuclear area of the myoblasts. Immunofluorescent staining was analyzed by confocal microscope (C). N = nucleus
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Expression of IL-15 mRNA isoform in myoblasts
There are two different isoforms in human IL-15 mRNA. The authentic IL-15 mRNA encodes a preprotein with a 48-amino-acid signal peptide [long signal peptide (LSP)]. An alternative form of IL-15 mRNA, generated by retention of exon A, has a shorter open reading frame due to stop codons within exon A and is followed by a new AUG codon, encoding an IL-15 precursor protein with a 21-amino-acid signal peptide [short signal peptide (SSP)] (17,18). In some cancer cell lines such as small cell lung cancer, this alternative form of IL-15 mRNA is predominantly expressed. To clarify whether myoblasts express this alternative isoform, PCR was performed using isoform-specific primers. The primers were set up to distinguish two IL-15 mRNA isoforms according to a previous report (17). As shown in Fig. 5(A), primer a was set at exon 1, upstream of the alternative exon A, while the lower common primer was set at exon 6. The expected size for authentic IL-15 mRNA is 524 bp, while the alternative product is 643 bp due to the additional exon. Primer b was set within exon A. If the alternative form of IL-15 mRNA exists, the PCR product with a size of 430 bp is generated. As shown in Fig. 5(B), the myoblasts strongly expressed the authentic IL-15 mRNA, but only weakly expressed the alternative form of IL-15 mRNA. The peripheral blood mononuclear cells expressed both forms of IL-15 mRNA.


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Fig. 5. IL-15 mRNA isoform expression in the myoblasts. (A) RT-PCR was performed using upper primer a or b and a common lower primer to detect the existence of an alternative form. The expected size of primer a is 524 bp, while that of alternative product is 643 bp due to additional exon A. However, if alternative IL-15 mRNA exists, the PCR product with a size of 430 bp could be generated from primer b as shown schematically. SP = signal peptide; aa = amino acid. (B) IL-15 mRNA isoform expression in the unstimulated myoblasts, IFN- -stimulated myoblasts and PBMC. Lanes 13, upper primer a; lanes 46, upper primer b; lanes 1 and 4, PBMC; lanes 2 and 5, unstimulated myoblasts; lanes 3 and 6, IFN- -stimulated myoblasts. The IFN- -stimulated myoblasts as well as the unstimulated myoblasts only weakly expressed alternative IL-15 mRNA compared to PBMC. (C) ß-Actin mRNA expression. Lane 1, PBMC; lane 2, unstimulated myoblasts; lane 3; IFN- -stimulated myoblasts. Product size for the ß-actin gene is 218 bp.
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The supernatants of myoblasts contain biologically active IL-15
CTLL-2 was stimulated with the supernatants of the IL-1
-stimulated myoblasts along with anti-IL-15 mAb, anti-IL-2 mAb or control mouse IgG1 for 48 h and cell proliferation was analyzed by a WST-1 assay as described in Methods. As shown in Fig. 6, CTLL-2 proliferated in response to the supernatants of the myoblasts and this was almost completely inhibited by anti-IL-15 mAb. In contrast, neither control mouse IgG1 nor anti-IL-2 mAb blocked this proliferative response. Anti-IL-2 mAb at 2 µg/ml completely blocked the IL-2-driven CTLL-2 proliferation (data not shown). Additionally, CTLL-2 did not respond to IL-1
(data not shown).

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Fig. 6. The supernatants of the myoblasts contain biologically active IL-15. Supernatants of the myoblasts were prepared by stimulation with 10 ng/ml of IL-1 for 120 h. CTLL-2 was rested in IL-15-free medium for 6 h and then incubated with fresh medium containing 50% culture supernatants of the myoblasts along with various concentrations of anti-IL-15 mAb, 2000 ng/ml of anti-IL-2 mAb or control IgG1 for 48 h. During the last 6 h of incubation, the cells were pulsed with 10 µl of Premix WST-1 solution and the absorbance at 450 nm was measured. Anti-IL-15 mAb inhibited CTLL-2 proliferation by the myoblasts culture supernatants in a dose-dependent manner. Anti-IL-15 mAb at 3.2 ng/ml completely inhibited the effect of the culture supernatants of myoblasts on the CTLL-2 proliferation. Neither anti-IL-2 mAb nor isotype-matched mouse control IgG1 showed inhibitory effects in this system. The results are expressed as the mean ± SE of a triplicate assay. *P < 0.05; **P < 0.01. myoblast sup# = myoblast supernatant
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Discussion
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In the present study, we showed that cultured myoblasts constitutively produced low levels of IL-15 and the production was enhanced by the stimulation with inflammatory cytokines. In vivo, the muscle cells in PM/DM strongly expressed IL-15 compared with those of the normal controls. Considering its biological functions, overproduced IL-15 from the muscle cells could be involved in the development of immune responses in PM/DM.
There have been several reports about the expression of IL-15 in chronic inflammatory diseases such as pulmonary sarcoidosis, inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) (1924). In IBD, the expression of IL-15 mRNA is significantly higher than that of normal controls (21). Liu et al. (22) showed that mucosal macrophages expressed IL-15 in IBD. They also reported that the lamina propria (LP) T cells from IBD patients strongly expressed CD40 ligand (CD40L), which induced TNF and IL-12 production from monocytes via CD40CD40L interaction. Recombinant IL-15 strongly enhances this capacity of LP T cells by inducing the expression of CD40L. In RA, the synovial lining cells produce IL-15 and the synovial fluids contain high concentrations of IL-15. Synovial fluid T cells obtained from RA patients proliferate in response to recombinant IL-15, although synovial fluid has chemotactic activity for T cells and this activity is partially inhibited by anti-IL-15 antibody (23). Furthermore, IL-15 induces monocytes to produce TNF-
, which plays a crucial role in the development of synovitis of RA (24). These observations suggest the involvement of IL-15 in the pathogenesis of chronic inflammatory diseases, especially in respect of T cell recruitment and activation. In these diseases, the main source of IL-15 is proposed to be infiltrating macrophages. In contrast to these previous reports, a unique point of the present study is that the predominant source of IL-15 in PM/DM is the muscle cells themselves. This indicates that the muscle cells are not only the target of autoimmune reaction in PM/DM, but can also be a potent enhancer of inflammation by producing IL-15.
In the mesenchymal cells, IL-15 would exert other biological functions than in the lymphoid cells in physiological conditions. Cultured myoblasts increase their mass upon incubation with recombinant IL-15 (25,26) and this cytokine prevents muscle protein wasting in tumor-bearing rats (27). These data indicate that constitutively produced IL-15 might have some anabolic effects in myoblasts. The effect of IL-15 on myoblast per se in PM/DM should be investigated in future studies.
As for the inducer of IL-15 production in the muscle cells of PM/DM in vivo, inflammatory cytokines are most reasonable candidates. In the present data, cultured myoblasts produced IL-15 in response to the stimulation of cytokines including IL-1
, IL-1ß, TNF-
and IFN-
. By immunohistochemical staining, the expression of these cytokines has been reported in the muscle tissues of PM/DM. Among them, IL-1
and TNF-
were commonly reported to be expressed (14), while the expression of IFN-
was rather weak (10). There is another candidate for an IL-15-inducer. In our previous study (10), we reported that the ligation of CD40 molecule expressed on the cultured myoblasts using a trimeric form of recombinant CD40L increased IL-15 production from the myoblasts. Immunohistochemistry revealed that muscle cells and infiltrating MNC in PM/DM expressed CD40 and CD40L respectively, which were not expressed in normal controls. Hence, we suggest that CD40 ligation is the second candidate. These inflammatory cytokines and cell surface molecules could be relevant to the increased expression of IL-15 in the muscle cells of PM/DM.
The expression of IL-15 is regulated at the levels of transcription, translation, and intracellular trafficking and translocation, while IL-2 is predominantly controlled at the level of transcription and mRNA stabilization. Indeed, IL-15 mRNA is constitutively expressed in a broad range of cells, but it is difficult to demonstrate IL-15 protein in the supernatants of such cells (12,28). In the myoblasts, the increase in IL-15 mRNA reflected the production of the IL-15 protein, both intracellularly and extracellularly (Fig. 2). As described above, IL-15 has two isoforms with different signal peptides. IL-15 with LSP is all secreted, whereas IL-15 with alternative SSP is not secreted but stored intracellularly, localizing in nuclear and cytoplasmic components. The predominance of either isoform of IL-15 mRNA depends on the cell types. Our data indicate that the myoblasts predominantly express IL-15 with LSP which is to be secreted. However, 3040% of the IL-15 protein was detected in the cell extracts of the myoblasts (Fig. 2). Hence, it is possible that most of IL-15 detected intracellularly was the secretory form of IL-15 protein in transport vesicles from the endoplastic reticulum/Golgi apparatus.
In conclusion, IL-15 is overproduced from the muscle cells with inflammatory stimuli in PM/DM. The overexpressed IL-15 may augment recruitment and/or activation of autoreactive T cells, but this hypothesis is yet to be examined. Treatment of an animal model with autoimmune-mediated myositis using anti-IL-15 antibody will give an answer to this question. If this is the case, the blocking of IL-15 activity could be a therapeutic candidate for PM/DM.
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Acknowledgements
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We thank Dr H. Kato for providing us with CTLL-2 and Dr Y. Simoda (Medical Research Institute) for technical assistance with confocal microscopy. This study was supported by a grant from the Autoimmune Diseases Research Committee organized by the Ministry of Health and Welfare of Japan
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Abbreviations
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CD40LCD40 ligand
DMdermatomyositis
IBDinflammatory bowel disease
IIMidiopathic inflammatory myopathies
LPlamina propria
LPSlipopolysaccharide
LSPlong signal peptide
MNCmononuclear cell
PMpolymyositis
RArheumatoid arthritis
SSPshort signal peptide
TNFtumor necrosis factor
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