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The Journal of Immunology, 2004, 173: 5180-5188.
Copyright © 2004 by The American Association of Immunologists

Endogenous IL-15 Sustains Recruitment of IL-2R{beta} and Common {gamma} and IL-2-Mediated Chemokine Production in Normal and Inflamed Human Gingival Fibroblasts1

Akiko Ozawa*,{dagger},{ddagger}, Hiroyuki Tada{dagger}, Yumiko Sugawara§, Akiko Uehara*,{dagger}, Takashi Sasano§, Hidetoshi Shimauchi{ddagger}, Haruhiko Takada{dagger} and Shunji Sugawara2,*

* Division of Oral Immunology, {dagger} Division of Oral Microbiology, and {ddagger} Division of Periodontology and Endodontology, Department of Oral Biology, and § Division of Oral Diagnosis, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently reported that anti-IL-15 neutralizing mAb has been shown to inhibit production of MCP-1 in response to IL-2 from normal human gingival fibroblasts (HGF), the major constituent of gingival tissue. In the present study, we examined the expression of IL-2R and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2R{beta} and common {gamma}-chain ({gamma}c) but not IL-2R{alpha} or IL-15R{alpha}, whereas inflamed HGF expressed IL-2R{alpha}, IL-15R{alpha}, IL-2R{beta}, and {gamma}c, as assessed by RT-PCR and flow cytometry. Exogenous IL-2 and IL-15 induced production of MCP-1 but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed the 48 aa-IL-15 isoform, and the isoform was not actively secreted but rather existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb for 24 h completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Jak 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2R{beta} and {gamma}c expression at mRNA and protein levels. Furthermore, excess amounts of IL-2 restored the inhibitory effect of anti-IL-15, inhibition of NF-{kappa}B abrogated the expression of IL-2R{beta} and {gamma}c, and IL-2-induced-nuclear translocation of NF-{kappa}B was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2R{beta} and {gamma}c through activation of NF-{kappa}B in HGF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fibroblasts and their extracellular matrix products play pivotal roles in maintaining the structural integrity of connective tissues, in healing processes, and in pathological alterations (1, 2). Fibroblasts are not a homogeneous population in different anatomical regions or even within a single tissue, and are considered to actively define the structure of microenvironments and modulate immune cell behavior by conditioning the local and cellular microenvironment (1, 2). Human gingival fibroblasts (HGF),3 the major constituent of gingival connective tissue, have been shown to express immunologic receptors, such as a bacterial pattern recognition receptor CD14 (3, 4), TLRs (5), and costimulatory molecule CD40 (1, 6) and to produce various cytokines, such as IL-1, IL-6, IL-8, and MCP-1, by interaction with their ligands (3, 4, 5, 7). These observations indicate that HGF also actively participate in immune responses and inflammatory processes.

IL-2 is a principal growth factor that induces the proliferation and differentiation of T and NK cells (8) and shares many biological activities and the use of the IL-2R{beta}-chain and common {gamma}-chain ({gamma}c) with IL-15 (8, 9). IL-2R and IL-15R have their own {alpha}-chain and the heterotrimers form fully functional high affinity IL-2R and IL-15R complexes. These subunits can be expressed individually or in various combinations, resulting in distinct receptor complexes that bind IL-2 and IL-15 with different affinities (8, 9). Activation through IL-2R{beta} and {gamma}c is directly associated with phosphorylation of Jak1 and Jak3, resulting in STAT3 and STAT5 phosphorylation, respectively (10). It has been shown that human monocytes also bear functional IL-2R{beta} and {gamma}c (11), human fibroblasts from adult bone marrow, embryonic skin, and lung express IL-2R{alpha} and IL-2R{beta} (12, 13, 14), and fibroblast-like synoviocytes express functional IL-2R{beta} and {gamma}c (15). In addition, we have recently shown that normal HGF express functional IL-2R{beta} and {gamma}c and that IL-2 stimulation induced production of MCP-1 and expression of ICAM-1 by HGF, resulting in the augmentation of ICAM-1-mediated neutrophil adhesion, and that the IL-2-induced MCP-1 production was significantly inhibited by pretreatment with anti-IL-15 neutralizing mAb (16). These observations suggest that IL-2 has a wide variety of biological activities in cells other than the lymphoid cell population, and that IL-15 expressed by HGF sustains IL-2-mediated signaling in HGF.

There are two isoforms of IL-15, one with a 48 aa-leading peptide and one with a 21 aa leader peptide (17, 18). The 48 aa-IL-15 is targeted to the secretory pathway with low secretion potential (19, 20), whereas the 21 aa-IL-15 appears to be restricted to the cytoplasm and nucleus (19, 21). Recent reports showed that IL-15 is constitutively expressed on fibroblasts from human spleen and skin and also on human monocytes and leukemic progenitors without any IL-15 secretion (22, 23, 24, 25, 26). However, it is still unclear which of the isoforms are expressed on the cell surface.

Periodontal disease is clinically identified as an inflammation of periodontal tissues caused by Gram-negative organisms, and is characterized by the infiltration of T and B cells as well as neutrophils into inflamed gingival tissues, resulting in direct and indirect destruction of periodontal tissue (27). The T cell features in diseased periodontal tissues can be compared with those in rheumatoid arthritis. These observations led us to investigate 1) whether HGF from normal and inflamed regions differentially express IL-2R and IL-15R subunits and respond to IL-2 and IL-15 and 2) the role of endogenous IL-15 in IL-2-mediated signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human rIL-2 was kindly provided by Shionogi Pharmaceutical (Osaka, Japan). Anti-human IL-2R{alpha} no. 22722.2 (mouse IgG1), anti-human IL-2R{beta} no. 27302.1 (mouse IgG1), anti-human {gamma}c no. 38024.11 (mouse IgG1), neutralizing anti-human IL-15 no. 34505.11 (mouse IgG), and human rIL-15 were obtained from Genzyme/Techne (Minneapolis, MN). Anti-human IL-15 polyclonal Ab H-114 (rabbit IgG) and anti-NF-{kappa}B p65 polyclonal Ab (rabbit IgG) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PE-labeled anti-human IL-2R{beta} Mik-{beta}3 (mouse IgG1) and PE-labeled {gamma}c AG184 (mouse IgG1) were obtained from BD Biosciences (Mountain View, CA). Anti-human IL-15R{alpha} polyclonal Ab (goat IgG) was obtained from R&D Systems (Minneapolis, MN). A NF-{kappa}B inhibitor, pyrrolidine dithiocarbamate (PDTC), was obtained from Calbiochem-Novabiochem (La Jolla, CA). All isotype control mAbs were obtained from Beckman Coulter (Miami, FL) and dialyzed against PBS. LPS of Escherichia coli O55:B5 and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.

Cells and cell culture

HGF were prepared from the explants of normal and inflamed gingival tissues of patients with adult periodontitis undergoing periodontal surgery under informed consent and cultured in {alpha}-MEM supplemented with 10% FCS (Life Technologies, Grand Island, NY) in 100-mm diameter tissue culture dishes (Falcon; BD Labware, Lincoln Park, NJ), as previously described (4). Cells were detached from the culture dishes by treatment with nonenzymatic cell dissociation solution (CDS) (Sigma-Aldrich). Cells were used as confluent monolayers at subculture levels 3–10. The Ethical Review Board of Tohoku University Graduate School of Dentistry (Sendai, Japan) approved the experimental procedures.

PBMCs from heparinized (10 U/ml) peripheral venous blood were isolated by Lympholyte-H (Cedarlane Laboratories, Hornby, Ontario, Canada) gradient centrifugation at 800 x g for 20 min at room temperature (28). The isolated PBMCs were washed three times with PBS at 4°C. The viability of the cells was greater than 98%, as judged by trypan blue dye exclusion.

RT-PCR

Total RNA was isolated from HGF (2 x 106 cells) using a Total RNA Isolation kit (Isogen; Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. PBMCs were used as positive controls. PCR for IL-2R{alpha}, IL-2R{beta}, {gamma}c, and human GAPDH was performed as described (16). The primers used for IL-15R{alpha}, IL-15, and 21 aa-IL-15 were as follows: IL-15R{alpha}, forward 5'-GCCAGCGCCACCCTCCACAGTAA-3' and reverse 5'-GCCAGCGGGGGAGTTTGCCTGAC-3' (29); IL-15, forward 5'-GGCTTTGAGTAATGAGAATTTCGA-3' and reverse 5'-ATCAGTTGCAATCAAGAAGTGTTG-3' (17); and 21 aa-IL-15, forward 5'-GCCTTCATGGTATTGGGAAC-3' and reverse 5'-GAATCAATTGCAATCAAGAAGTG-3' (20). The primers for IL-15R{alpha}, IL-15, and 21 aa-IL-15 were constructed to generate fragments of 402, 513/632, and 427 bp, respectively. Amplification was performed in a model MP TP3000 PCRthermal cycler (Takara, Tokyo, Japan) as follows: for IL-15R{alpha}, 35 cycles ofdenaturation at 94°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 2 min; for IL-15, 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, and followed by a final extension at 72°C for 7 min; and for 21 aa-IL-15, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s, and followed by a final extension at 72°C for 7 min. Amplified samples were visualized by electrophoresis on 2% agarose gels and staining with ethidium bromide and photographed under UV light.

Flow cytometry

Flow cytometric analyses were performed with a FACScan (BD Biosciences) (28). For immunofluorescence staining, confluent HGF were collected by nonenzymatic CDS and washed in PBS. HGF were stained with PE-labeled anti-IL-2R{beta}, anti-{gamma}c, or isotype control mAb at 4°C for 30 min. HGF were also stained with anti-IL-2R{alpha} mAb, anti-IL-2R{beta} mAb, anti-{gamma}c mAb, polyclonal anti-IL-15, polyclonal anti-IL-15R{alpha}, or control Abs at 4°C for 30 min, and then incubated with FITC-conjugated goat anti-rabbit IgG, goat anti-mouse IgG, or rabbit anti-goat IgG (BioSource International, Camarillo, CA) at 4°C for a further 30 min. The arithmetic mean was used in the computation of the mean fluorescence intensity. Percent expression of surface molecules was calculated as described previously (28).

Detection of cytokines by ELISA

Confluent HGF were collected by trypsinization and washed in PBS three times. The cells (104 cells/200 µl) were seeded in {alpha}-MEM with 10% FCS in wells of 96-well flat-bottom plates (Falcon). After incubation for 4 days at 37°C in a 5% CO2 atmosphere, confluent monolayers of HGF were washed with {alpha}-MEM three times, and a test stimulant was added in 200 µl of {alpha}-MEM with 1% FCS for 24 h. For the inhibition experiments, HGF in 96-well plates were preincubated with antagonists for 30 min-1 h at 37°C and were then stimulated with agonists for 24 h at 37°C. After the incubation, the supernatants were collected and the levels of MCP-1, IL-8, and IL-15 produced by HGF in the supernatants were determined with OptEIA human MCP-1, IL-8, and IL-15 ELISA kits (BD Pharmingen, San Diego, CA). The concentrations of the cytokines in the culture supernatants were determined with the Softmax data analysis program (Molecular Devices, Menlo Park, CA). Each sample was assayed in triplicate.

Detection of Jak1 and Jak3 phosphorylation

Cells were lysed with a lysing buffer for 30 min at 4°C with mixing as described (16). After centrifugation, the supernatants were precleared by incubation with protein G-agarose (Santa Cruz Biotechnology) for 1 h at 4°C. The samples were then immunoprecipitated with rabbit anti-human Jak1 or Jak3 polyclonal Ab (Santa Cruz Biotechnology) and protein G-agarose for 2 h at 4°C. The immunoprecipitates were separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Atto Instruments, Tokyo, Japan). To detect phosphotyrosine content, the membrane was sequentially bound with an anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) at a 1/1000 dilution and peroxidase-conjugated goat anti-mouse Ig (Santa Cruz Biotechnology) at a 1/3000 dilution. The membrane was reprobed with anti-Jak1 or -Jak3 polyclonal Ab to determine the amount of Jak1 or Jak3 in each lane.

RNA interference

Transfection for targeting endogenous IL-15 was conducted using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) and small interfering RNA (siRNA) (final concentration, 200 nM), according to manufacturer’s instruction. The sequence of target for IL-15 mRNA used in this study is 5'-AACTTCTCTCAGACTTACTTT-3'.

Assay for NF-{kappa}B activity

Activated NF-{kappa}B was measured with an NF-{kappa}B assay kit specific for the p65 subunit according to the manufacturer’s instructions (Active Motif, Carlsbad, CA). Briefly, samples of whole cell extracts (1–10 µg of protein/well) were added to 96-well plates coated with an oligonucleotide containing the NF-{kappa}B consensus site (5'-GGGACTTTCC-3'), and incubated for 1 h at room temperature with mild agitation. After washing three times, NF-{kappa}B p65 Ab was added and incubated for 1 h without agitation followed by addition of HRP-conjugated anti-mouse IgG1. Colorimetric reactions were developed, stopped, and measured at 450 nm. The specificity of binding was also examined using an oligonucleotide containing a wild-type or mutated NF-{kappa}B consensus binding site.

Immunostaining

HGF were grown on eight-chamber glass slides (Falcon) and treated with or without IL-2 for 30 min, fixed with 4% paraformaldehyde for 15 min at room temperature, treated with 0.5% Triton X-100 for 15 min, and washed with PBS. Cells were then incubated with rabbit anti-human NF-{kappa}B p65 polyclonal Ab (1/100; Santa Cruz Biotechnology) for 2 h at room temperature, followed by HRP-labeled goat anti-rabbit Envision plus system (DAKO, Kyoto, Japan) for 1 h. The chromogen used was 3,3'-diaminobenzidine tetrahydrochloride (DAKO).

Statistical analysis

All of the experiments in this study were performed at least three times to confirm the reproducibility of the results. Experimental values are given as means ± SD of triplicate assays. The statistical significance of differences between two means was evaluated by a one-way ANOVA using the Bonferroni or Dunnett method, and values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-2R and IL-15R subunits by normal and inflamed HGF

We first examined the expression of IL-2R and IL-15R subunits in HGF prepared from normal and inflamed regions. Normal HGF expressed IL-2R{beta} and {gamma}c mRNAs, but the expression of IL-2R{alpha} and IL-15R{alpha} mRNAs could not be detected by RT-PCR (Fig. 1A). In contrast, inflamed HGF expressed IL-2R{alpha} and IL-15R{alpha} mRNAs in addition to IL-2R{beta} and {gamma}c mRNAs. Corresponding expression patterns at the protein level were confirmed by flow cytometric analyses. Fig. 1B shows representative FACS profiles of IL-2R and IL-15R subunit expression on normal and inflamed HGF.



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  		FIGURE 1. Expression of IL-2R and IL-15R by HGF. A, Total RNA was extracted from confluent normal (left) and inflamed (right) HGF (lanes 2, 4, 6, and 8). PBMCs (lanes 1, 3, 5, and 7) were used as a positive control. cDNA was prepared and analyzed for the mRNA expression of IL-2R{alpha} (lanes 1 and 2), IL-2R{beta} (lanes 3 and 4), {gamma}c (lanes 5 and 6), IL-15R{alpha} (lanes 7 and 8) (upper panels), and GAPDH (lower panels) by RT-PCR. M, m.w. marker. B, Confluent normal and inflamed HGF were collected by CDS and stained with anti-IL-2R{alpha}, anti-IL-15R{alpha}, anti-IL-2R{beta}, and anti-{gamma}c (solid lines), or isotype control Abs (dotted lines), and analyzed by flow cytometry. The results presented are representative of four different experiments with similar results.

 
Production of chemokines by normal and inflamed HGF in response to IL-2 and IL-15

We next examined the responsiveness of normal and inflamed HGF to exogenous IL-2 and IL-15. LPS was used as a positive control (4). In normal HGF, IL-2 and IL-15 significantly induced production of MCP-1 but were not effective in inducing IL-8 production (Fig. 2). In inflamed HGF, IL-2 and IL-15 induced IL-8 production that was comparable to that induced by LPS, and induced more than 2-fold higher MCP-1 production as compared with that in normal HGF (Fig. 2). The production of MCP-1 from inflamed HGF in response to LPS was extremely high (37 ± 1.0, 76 ± 1.1, and 95 ± 1.4 ng/ml at 1, 10, and 100 ng/ml LPS, respectively).



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  		FIGURE 2. Production of chemokines by HGF upon stimulation with IL-2 and IL-15. Confluent normal and inflamed HGF were stimulated with the indicated concentrations of IL-2, IL-15, or LPS for 24 h. The concentrations of IL-8 and MCP-1 in the culture supernatants were determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. *, p < 0.01 compared with unstimulated control (medium alone). The results presented are representative of three differentexperiments showing similar results.

 
Expression of IL-15 in HGF as a membrane-bound form

There are two isoforms of IL-15, one with a 48 aa and one with a 21 aa leader peptide (17, 18). As it has been shown that pretreatment with anti-IL-15 neutralizing mAb for 1 h significantly inhibited the IL-2-induced MCP-1 production in HGF (16), we next examined whether HGF express IL-15 mRNA and produce IL-15 isoforms. PBMCs were used as a positive control. RT-PCR analysis revealed that PBMCs mainly expressed IL-15 mRNA with 513 bp, and weakly expressed IL-15 mRNA with 632 bp (Fig. 3A), the transcripts encoding 48 aa-IL-15 and 21 aa-IL-15, respectively (17). In contrast, both normal and inflamed HGF strongly expressed only IL-15 mRNA with 513 bp, the transcript encoding 48 aa-IL-15. The results were confirmed by using the 21 aa-IL-15-specific primer (Fig. 3B). Both types of HGF spontaneously produced a small amount (~100 pg/ml) of IL-15 for 24 h, but the production was not further augmented by stimulation with IL-2 or LPS (Fig. 3C). Instead, flow cytometric analyses showed that IL-15 is expressed on the cell surface of HGF and the expression by inflamed HGF was higher than that of normal HGF and that excess rIL-15 reduced the binding of anti-IL-15 (Fig. 3D), which excludes the possibility that IL-15 surface expression is not result from nonspecific binding of anti-IL-15 Ab. Furthermore, the expression was not influenced by treatment with IL-2 or LPS (data not shown). These results indicate that HGF mainly transcribe the 48 aa-IL-15 isoform, and that the isoform is not actively secreted but rather is constitutively expressed as a membrane-bound form on the cell surface.



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  		FIGURE 3. Expression of IL-15 by HGF. A and B, Total RNA was extracted from normal (lane 2) and inflamed HGF (lane 3), and cDNA was prepared and analyzed for the mRNA expression of IL-15 and GAPDH by RT-PCR. PBMCs (lane 1) were used as a positive control. M, m.w. marker. Primers allowing the detection of both IL-15 mRNA isoforms (A) and 21 aa-IL-15 isoform (B) were used. C, Confluent normal and inflamed HGF were stimulated with the indicated concentrations of IL-2 or LPS for 24 h. The concentrations of IL-15 in the culture supernatants were determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. D, Confluent normal and inflamed HGF were collected by CDS, stained with anti-IL-15 polyclonal or control Abs in the presence or absence of IL-15 at indicated concentrations, and analyzed by flow cytometry. The results presented are representative of three different experiments showing similar results.

 
Effect of anti-IL-15 on production of chemokines and signaling induced by exogenous IL-2

Both normal and inflamed HGF were pretreated with anti-IL-15 neutralizing mAb for 24 h, and the production of MCP-1 induced by IL-2, IL-15, and LPS was examined. The results showed that the pretreatment completely inhibited the production of MCP-1 induced by IL-2 and IL-15 to the control level in both HGF (Fig. 4). However, pretreatment with anti-IL-15 mAb did not alter the responsiveness to LPS.



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  		FIGURE 4. Effect of anti-IL-15 mAb on IL-2-induced MCP-1 production by HGF. Confluent normal (A) and inflamed HGF (B) were pretreated with or without anti-IL-15 neutralizing mAb or isotype control mAb (10 µg/ml each) for 24 h, and then stimulated with the indicated concentrations of IL-2, IL-15, or LPS for 24 h. The concentration of MCP-1 in the culture supernatants was determined by ELISA. The results are expressed as the mean ± SD for triplicate cultures. *, p < 0.01 compared with respective control (stimulant alone). The results presented are representative of three different experiments showing similar results.

 
As activation through IL-2R{beta} and {gamma}c is directly associated with activation of Jak1 and Jak3, respectively (10), we next examined the tyrosine phosphorylation of Jak1 and Jak3 induced by IL-2 in HGF. Phosphorylation of Jak1 and Jak3 was not detected in unstimulated normal HGF, and addition of IL-2 to the culture induced the phosphorylation of Jak1 and Jak3, which peaked at 30 min and declined thereafter (Fig. 5). The results clearly demonstrated that HGF are activated through IL-2R{beta} and {gamma}c followed by signal transduction via Jak1 and Jak3, respectively. Furthermore, the phosphorylation of Jak1 and Jak3 in normal HGF induced by IL-2 was totally abolished by the pretreatment with anti-IL-15 mAb. Inflamed HGF showed the same results (data not shown). These results suggest that anti-IL-15 mAb affects the signaling and expression of IL-2R and IL-15R subunits but not LPS receptor.



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  		FIGURE 5. Involvement of IL-15 in signaling of IL-2 in HGF. Confluent normal HGF were left untreated (upper panels) or pretreated with anti-IL-15 neutralizing mAb (10 µg/ml) (lower panels) for 24 h. Cells were then stimulated with IL-2 (10 ng/ml) for the time indicated. The cell lysates were immunoprecipitated with anti-Jak1 or -Jak3 polyclonal Ab, subjected to SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The blot was probed with anti-phosphotyrosine mAb 4G10. The blot was reprobed with the anti-Jak1 or -Jak3 polyclonal Ab to determine the amount of Jak1 or Jak3 in each lane. The results presented are representative of three different experiments showing similar results.

 
Involvement of endogenous IL-15 in the expression of IL-2R{beta} and {gamma}c in HGF

Therefore, we examined the expression of the IL-2R and IL-15R subunits after treatment with anti-IL-15 neutralizing mAb. The expression of IL-2R{alpha} and IL-15R{alpha} mRNAs was unchanged in inflamed HGF after treatment with anti-IL-15 mAb for 24 h, whereas the expression of IL-2R{beta} and {gamma}c mRNAs in normal and inflamed HGF disappeared as a result of the treatment (Fig. 6A). Corresponding expression patterns at the protein level were confirmed by flow cytometric analyses (Fig. 6B). However, the expression of IL-15 mRNA was unchanged by the treatment with anti-IL-15 mAb for 24 h in both types of HGF (data not shown), indicating that IL-15 is constitutively transcribed in HGF.



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  		FIGURE 6. Effect of anti-IL-15 mAb on the expression of IL-2R and IL-15R. A, Confluent normal (lanes 2 and 3) and inflamed (lanes 4 and 5) HGF were left untreated (lanes 2 and 4) or treated with anti-IL-15 neutralizing mAb (lanes 3 and 5) (10 µg/ml each) for 24 h. Total RNA was then extracted from the cells, and cDNA was prepared and analyzed for the mRNA expression of IL-15R{alpha}, IL-2R{alpha}, IL-2R{beta}, {gamma}c (upper panels), and GAPDH (lower panels) by RT-PCR. PBMCs were used as a positive control (lane 1). M, m.w. marker. B, Confluent normal and inflamed HGF were treated with or without anti-IL-15 neutralizing mAb (10 µg/ml) for 24 h, collected by CDS, and stained with anti-IL-2R{beta}, and anti-{gamma}c (solid lines), or control Abs (dotted lines), and analyzed by flow cytometry. The results presented are representative of four different experiments with similar results.

 
To further confirm the involvement of endogenous IL-15 in the expression of IL-2R{beta} and {gamma}c, we used siRNA to block the expression of IL-15 in HGF. Transfection of normal HGF with IL-15-specific siRNA resulted in inhibition of IL-15 mRNA but not GAPDH mRNA from 24 to 72 h of culture (Fig. 7A). Inflamed HGF showed the same result (data not shown). In transfected normal and inflamed HGF for 72 h, the expression of IL-2R{beta} and {gamma}c was reduced to control level (Fig. 7B), and the transfection in the presence of exogenous IL-15 prevented the receptor down-regulation as assessed by flow cytometry (Fig. 7C). However, the expression of IL-15R{alpha} in inflamed HGF was not affected by the transfection. These results suggest that an autocrine regulatory loop of endogenous IL-15 is critical for the expression of IL-2R{beta} and {gamma}c in HGF.



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  		FIGURE 7. Inhibition of IL-2R{beta} and {gamma}c expression in HGF by IL-15-specific siRNA. A, Confluent normal HGF were transfected with IL-15-specific siRNA for the time indicated. Total RNA was then extracted from the cells, and cDNA was prepared and the mRNA expression of IL-15 and GAPDH was analyzed by RT-PCR. M, m.w. marker. B, Normal and inflamed HGF were transfected with or without IL-15-specific siRNA for 72 h, collected by CDS and stained with anti-IL-15R{alpha}, anti-IL-2R{beta}, and anti-{gamma}c (solid lines), or control Abs (dotted lines), and analyzed by flow cytometry. C, Normal and inflamed HGF were transfected with IL-15-specific siRNA in the presence of exogenous IL-15 (100 and 1000 ng/ml) for 72 h, collected by CDS and stained with anti-IL-2R{beta} and anti-{gamma}c (solid lines), or control Abs (dotted lines), and analyzed by flow cytometry. The results presented are representative of three different experiments with similar results.

 
Involvement of NF-{kappa}B in the expression of IL-2R{beta} and {gamma}c in HGF

The above finding also indicates that signals through IL-2R{beta} and {gamma}c elicited by endogenous IL-15 sustain the expression of IL-2R{beta} and {gamma}c, and raises the question of whether exogenous IL-2 could substitute for endogenous IL-15 because IL-2 and IL-15 share IL-2R{beta} and {gamma}c. To examine this possibility, normal and inflamed HGF were treated with anti-IL-15 in the presence of excess IL-2 for 24 h, and we analyzed the expression of IL-2R{beta} and {gamma}c by RT-PCR and flow cytometry. The result showed that exogenous IL-2 at 1 µg/ml restored the expression of IL-2R{beta} and {gamma}c in anti-IL-15-treated normal and inflamed HGF (Fig. 8).



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  		FIGURE 8. Effect of exogenous IL-2 on anti-IL-15-induced suppression of IL-2R{beta} and {gamma}c. Confluent normal and inflamed HGF were treated with or without anti-IL-15 neutralizing mAb (10 µg/ml) in the presence or absence of IL-2 at the indicated concentration for 24 h. A, Total RNA was then extracted from the cells, and cDNA was prepared and analyzed for the mRNA expression of IL-2R{beta}, {gamma}c and GAPDH by RT-PCR. PBMCs (P) were used as a positive control. M, m.w. marker. B, Cells were collected by CDS, stained with anti-IL-2R{beta} and anti-{gamma}c (solid lines), or control Abs (dotted lines), and analyzed by flow cytometry. The results presented are representative of three different experiments with similar results.

 
As it was reported that anti-IL-15 neutralizing mAb inhibited nuclear localization of NF-{kappa}B (30), we measured NF-{kappa}B activity in HGF. The basal level of NF-{kappa}B activity in normal HGF was gradually decreased to 3 h after treatment with anti-IL-15 neutralizing mAb and was restored thereafter (Fig. 9A). Transfection of normal HGF with IL-15-specific siRNA for 72 h also reduced NF-{kappa}B activity in the cells (Fig. 9B). Furthermore, inhibition of NF-{kappa}B with PDTC for 24 h completely inhibited IL-2R{beta} and {gamma}c mRNAs in normal HGF (Fig. 9C). Inflamed HGF showed the same results (data not shown). Consistent with the mRNA expression, flow cytometric analyses showed that treatment of normal and inflamed HGF with PDTC inhibited the expression of IL-2R{beta} and {gamma}c on the cell surface (Fig. 9D). Microscopic examination showed that stimulation of normal HGF with exogenous IL-2 (10 ng/ml) for 1 h clearly induced NF-{kappa}B translocation to nuclei (Fig. 10, A and B) and the translocation was completely inhibited by transfection with IL-15-specific siRNA for 72 h (Fig. 10, C and D). Inflamed HGF showed the same results (data not shown). These results indicate that constitutive activation of NF-{kappa}B by endogenous IL-15 is essential for the expression of IL-2R{beta} and {gamma}c in HGF.



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  		FIGURE 9. Involvement of NF-{kappa}B in the expression of IL-2R{beta} and {gamma}c in HGF. A, Confluent normal HGF were treated with or without anti-IL-15 neutralizing mAb (10 µg/ml) for the time indicated, and active NF-{kappa}B was measured. B, Confluent normal HGF were transfected with IL-15-specific siRNA for 72 h, and active NF-{kappa}B was measured. The results are expressed as the mean ± SD for triplicate cultures. *, p < 0.01 and **, p < 0.05 compared with untreated control (medium alone). C, Confluent normal HGF (lanes 2, 3, 5, and 6) were incubated with (lanes 3 and 6) or without PDTC (100 µM) (lanes 2 and 5) for 24 h. PBMCs were used as a positive control (lanes 1 and 4). Total RNA was then extracted from the cells, cDNA was prepared and analyzed for the mRNA expression of IL-2R{beta} (lanes 1-3), {gamma}c (lanes 4-6) (upper panel), and GAPDH (lower panel) by RT-PCR. M, m.w. marker. D, Confluent normal and inflamed HGF were incubated with or without PDTC (100 µM) for 24 h, collected by CDS, stained with anti-IL-2R{alpha}, anti-IL-15R{alpha}, anti-IL-2R{beta} and anti-{gamma}c, or control Abs, and analyzed by flow cytometry. The results presented are representative of three different experiments with similar results.

 


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  		FIGURE 10. Microscopic examination of NF-{kappa}B nuclear translocation. Normal HGF were left untreated (A and B) or transfected with IL-15-specific siRNA for 72 h (C and D) and incubated in the absence (A and C) or presence of IL-2 (10 ng/ml) (B and D) for 1 h. After fixation, cells were stained with anti-human NF-{kappa}B p65 polyclonal Ab. The results presented are representative of three different experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that normal HGF express IL-2R{beta} and {gamma}c but not IL-2R{alpha} and IL-15R{alpha}, whereas inflamed HGF expressed IL-2R{alpha}, IL-15R{alpha}, IL-2R{beta}, and {gamma}c; that exogenous IL-2 and IL-15 induced production of MCP-1 but not IL-8 in normal HGF and induced the production of both chemokines in inflamed HGF; and that endogenous IL-15 plays an important role in the expression of IL-2R{beta} and {gamma}c through activation of NF-{kappa}B in the cells.

There are two isoforms of IL-15, one with a 48 aa and one with a 21 aa leader peptide (17, 18). The 48 aa-IL-15 is targeted to the secretory pathway with a low secretion potential (19, 20) and expression of IL-15 is controlled mainly posttranscriptionally at the levels of translation and secretion (9), while the 21 aa-IL-15 is not secreted but rather appears to be restricted to the cytoplasm and nucleus (19, 21). The present study showed that normal and inflamed HGF expressed the 48 aa-IL-15 mRNA (Fig. 3, A and B) and spontaneously produced only a small amount (~100 pg/ml) of IL-15 in the supernatants during 24 h (Fig. 3C). The production was not further augmented by stimulation with IL-2 and LPS; rather, IL-15 existed as a membrane-bound form on the cell surface of HGF (Fig. 3, C and D). This observation suggests that translation or intracellular trafficking of IL-15 in HGF is regulated, which is in agreement with recent reports showing that IL-15 is constitutively expressed on fibroblasts from human spleen and skin and also on human monocytes and leukemic progenitors without any IL-15 secretion (22, 23, 24, 25, 26). Considering these findings, it is conceivable that HGF express endogenous IL-15 as the membrane-bound IL-15, which is the transcript encoding 48 aa-IL-15.

Activation through IL-2R{beta} and {gamma}c is directly associated with the activation of Jak1 and Jak3, respectively (10), and addition of IL-2 induced the phosphorylation of Jak1 and Jak3 in HGF (Fig. 5). These results clearly indicate that HGF are activated through IL-2R{beta} and {gamma}c followed by signal transduction via Jak1 and Jak3, respectively. Although the phosphorylation of Jak1 and Jak3 in inflamed HGF induced by IL-2 at 10 ng/ml showed the same result as normal HGF, it is possible that phosphorylation in inflamed HGF is induced at low concentrations of IL-2, as indicated in Fig. 2, because of the IL-2R{alpha} expression by the cells. Pretreatment with anti-IL-15 neutralizing mAb for 24 h totally abrogated the induction of MCP-1 production by IL-2 and IL-15 but not by LPS (Fig. 4) and also abolished phosphorylation of Jak1 and Jak3 in HGF (Fig. 5), as a result of complete inhibition of IL-2R{beta} and {gamma}c expression at mRNA and protein levels, without affecting IL-2R{alpha} and IL-15R{alpha} expression (Fig. 6). Furthermore, the observation was confirmed by blocking of endogenous IL-15 with IL-15-specific siRNA (Fig. 7). Another neutralizing anti-IL-15 mAb showed the same results and cross-linking of IL-15/IL-2R{beta}/{gamma}c or IL-2R{beta}/{gamma}c by addition of mAbs to the molecules followed by anti-mouse IgG did not alter the expression of IL-2R{beta} and {gamma}c (data not shown), indicating that neutralizing anti-IL-15 mAb inhibited binding of IL-15 to IL-2R{beta} and {gamma}c on the cell surface, and excludes the possibility that the anti-IL-15 induced inhibitory effect by cross-linking of IL-15/IL-2R{beta}/{gamma}c. Recent reports showed that neutralizing the anti-IL-15 mAb inhibited {gamma}c expression in CD34+ hemopoietic progenitors (26, 30), but had no clear effect on IL-2R{beta}. Another report showed that the formation of the IL-15/IL-15R{alpha} complex on lymphoid cell surfaces induced a trans-endosomal recycling of IL-15 leading to the persistence of surface-bound IL-15 (31). Taking into account these observations, it is conceivable that the anti-IL-15 mAb acts as an inhibitor of the autocrine loop of endogenous IL-15, that the autocrine loop of endogenous IL-15 is critical for sustaining the transcription and expression of IL-2R{beta} and {gamma}c, and that IL-15R{alpha} does not participate in the autocrine loop, because the anti-IL-15 mAb and IL-15-specific siRNA showed equivalent effects on IL-15R{alpha}-deficient normal and IL-15R{alpha}-expressing inflamed HGF without affecting IL-15R{alpha} expression (Figs. 6 and 7).

To delineate the precise mechanisms, HGF were treated with anti-IL-15 neutralizing mAb in the presence of an excess amount of exogenous IL-2, and the result showed that excess IL-2 restored the inhibitory effect of the anti-IL-15 mAb (Fig. 8). Furthermore, pretreatment with anti-IL-15 mAb and transfection with IL-15-specific siRNA inhibited NF-{kappa}B activity (Fig. 9, A and B), inhibition of NF-{kappa}B with PDTC totally abrogated expression of IL-2R{beta} and {gamma}c mRNA (Fig. 9C) and protein (Fig. 9D), and transfection with IL-15-specific siRNA inhibited nuclear translocation of NF-{kappa}B induced by IL-2 (Fig. 10). In agreement with this observation, a recent study showed that anti-IL-15 neutralizing mAb inhibited nuclear translocation of NF-{kappa}B (30). Therefore, the present study suggests that endogenous IL-15 plays a role in sustaining NF-{kappa}B activity, which leads to the constitutive expression of IL-2R{beta} and {gamma}c at mRNA and protein levels. The present study also indicated that endogenous IL-15 does not participate in the LPS-mediated signaling cascade, as anti-IL-15 mAb had no effect on LPS-induced chemokine production (Fig. 4).

It has been reported that lung myofibroblasts represent the differentiated counterpart of normal lung fibroblasts and express {alpha}-smooth muscle actin (32), and that lung myofibroblasts, in contrast to normal lung fibroblasts, constitutively express {gamma}c (33). However, we found that HGF prepared from normal and inflamed regions expressed only a low level of {alpha}-smooth muscle actin, and that no differences in the expression were detected by immunohistochemistry (data not shown). This observation suggests that HGF do not differentiate into myofibroblasts in the course of the inflammatory process and expression of the IL-2R and IL-15R subunits in HGF is characteristic of periodontal tissue. Inflamed HGF expressed IL-2R{alpha} and IL-15R{alpha} in addition to IL-2R{beta} and {gamma}c (Fig. 1) and produced IL-8 and about two times more MCP-1 compared with normal HGF in response to treatment with IL-2 or IL-15 (Fig. 2), which may partly be due to formation of trimeric high affinity IL-2R and IL-15R complexes. These observations also suggest that HGF in inflamed regions are actively involved in the inflammatory process by producing high concentrations of chemokines in response to exogenous IL-2/IL-15 as well as LPS. It has previously been shown that normal HGF primed with IFN-{gamma} produce increased levels of chemokines in response to LPS through up-regulation of CD14 and MyD88 mRNA expression (5). IFN-{gamma} treatment (1000 IU/ml for 3 days) induced the expression of IL-15R{alpha} mRNA, but had no effect on the expression of IL-2R{alpha} mRNA and surface expression of IL-15 (Ref.16 and data not shown). In the present study, HGF were used as confluent monolayers at subculture levels 3–10, and inflamed HGF at longer subculture levels up to 15 did not alter the phenotype of IL-2R and IL-15R or the level of surface IL-15 expression (data not shown), indicating that inflammatory conditions prevalent in the tissue environment are not involved in the expressions of IL-2R{alpha} and IL-15R{alpha}. We do not know the reason why inflamed HGF expressed IL-2R{alpha} and IL-15R{alpha} at this point, but studies using IFN-{gamma} and related cytokines may provide clues to the mechanism. Constitutive activation of member(s) of the IFN regulatory factor family may occur in inflamed HGF.

Periodontal disease is caused by infection with anaerobic Gram-negative organisms such as Porphyromonas gingivalis, and T and B cells as well as neutrophils are found in the dense inflammatory infiltrate in this disease (16, 17). In addition, Th1- or Th2-type T cells and their associated cytokines contribute to the onset and healing of this disease (27). IL-2 is secreted from activated T cells and is a key cytokine in both Th1 and Th2 responses (34). The concentration of IL-2 in the secretory vesicles of T cells is estimated to be in the millimolar range, and >90% of the secreted IL-2 would be expected to remain in the vicinity via the formation of cytokine reservoirs on the cell surface (35), at concentrations considerably higher than those used in this study (1–1000 ng/ml, i.e., 0.065–65 nM). A high concentration of IL-2 produced by infiltrating T cells may also occur in inflamed periodontal tissue, and IL-2-mediated activation of HGF is thus likely to occur in vivo.

The present study showed that IL-2 activates HGF to produce MCP-1 and IL-8 (Figs. 2 and 4). MCP-1 plays a critical role in the activation and migration of monocytes, T cells, and NK cells, and is an important factor in the development of Th1 and Th2 responses (36, 37). MCP-1 is also produced by cytokine-primed and -differentiated neutrophils, and has been implicated in the regulation of the transition from innate to adaptive immunity (38). IL-8 is a major chemokine responsible for the activation of neutrophils and the migration of neutrophils and T cells to inflammatory sites (39). Therefore, the activation of HGF by IL-2, which is released by activated T cells, may play an important role in innate immunity against periodontal pathogens by controlling Th1 and Th2 responses at the periodontitis site.

In addition to the biological actions of IL-15 originally described (9), the membrane-bound IL-15 expressed on fibroblasts from human spleen and skin also regulates NK cell differentiation from blood CD34+ progenitors and the proliferation of activated T cells, respectively (22, 23). Thus, the endogenous membrane-bound IL-15 on HGF may participate in the development, survival, and activation of NK and T cells in the oral mucosa. Finally, the observation that endogenous IL-15 sustains recruitment of IL-2R{beta} and {gamma}c and IL-2-mediated chemokine production in normal and inflamed HGF may also apply to IL-15-expressing nonimmunological cells such as other fibroblasts and epithelial cells (9), and the interaction of IL-2, IL-15, and their receptors in lymphoid cells should be examined.


   Acknowledgments
 
We thank E. Nemoto (Tohoku University Graduate School of Dentistry) for helpful discussions, T. Yamaguchi and K. Watanabe (Tohoku University Graduate School of Agricultural Science, Sendai, Japan) for excellent suggestions, and D. Mrozek (Medical English Service, Kyoto, Japan) for reviewing the paper.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Grant-in-Aid (15390551) for Scientific Research from Japan Society for the Promotion of Science and the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Back

2 Address correspondence and reprint requests to Dr. Shunji Sugawara, Division of Oral Immunology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 Japan. E-mail address: sugawars{at}mail.tains.tohoku.ac.jp Back

3 Abbreviations used in this paper: HGF, human gingival fibroblast; {gamma}c, common {gamma}-chain; PDTC, pyrrolidine dithiocarbamate; CDS, cell dissociation solution; siRNA, small interfering RNA. Back

Received for publication March 1, 2004. Accepted for publication August 3, 2004.


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