Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0103044 on June 16, 2003

Published online before print June 16, 2003
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(Journal of Leukocyte Biology. 2003;74:352-359.)
© 2003 by Society for Leukocyte Biology

Expression of IL-2 receptor ß and {gamma} chains by human gingival fibroblasts and up-regulation of adhesion to neutrophils in response to IL-2

Akiko Ozawa*,{dagger}, Hiroyuki Tada*,{dagger}, Riyoko Tamai*, Akiko Uehara*, Kouichi Watanabe{ddagger}, Takahiro Yamaguchi{ddagger}, Hidetoshi Shimauchi{dagger}, Haruhiko Takada* and Shunji Sugawara*,1

Departments of
* Microbiology and Immunology and
{dagger} Periodontics and Endodontics, Tohoku University Graduate School of Dentistry, and
{ddagger} Department of Animal Production Science, Tohoku University Graduate School of Agricultural Science, Sendai, Japan

1Correspondence: Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: sugawars{at}mail.cc.tohoku.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the role of human gingival fibroblasts (HGF), the major constituents of gingival tissue in periodontal inflammatory disease, the expression of interleukin-2 receptor (IL-2R) {alpha}, ß, and {gamma} chains was examined. Immunohistochemistry showed a pronounced accumulation of CD8+ T cells in the inflamed lamina propria of gingival tissue from patients with adult periodontitis. HGF express IL-2Rß and IL-2R{gamma} at mRNA and protein levels, but the expression of IL-2R{alpha} could not be detected, as assessed by reverse transcriptase-polymerase chain reaction and flow cytometry. IL-2Rß, and -{gamma} expressed on HGF were functionally active, as addition of neutralizing anti-IL-2Rß and -{gamma} antibodies caused inhibition of the IL-2-induced production of monocyte chemoattractant protein-1 (MCP-1), and addition of IL-2 induced phosphorylation of Janus tyrosine kinase 3, which is critical in signaling through IL-2R{gamma} in HGF. The IL-2-induced MCP-1 production was significantly inhibited by pretreatment with neutralizing antibody to IL-15. Addition of IL-2 also induced a marked up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of HGF, which in turn, significantly augmented the adhesion of human neutrophils, which were inhibited by an anti-ICAM-1 antibody. These results suggest that HGF express functional IL-2Rß{gamma}, respond to IL-2 from infiltrated T cells, and actively participate in the inflammatory process in the periodontal region and that IL-15 produced by HGF sustains IL-2-mediated signaling in HGF.

Key Words: gingival tissue • cytokine receptor • chemokine • adhesion molecule • periodontitis


   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 among 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), the major constituent of gingival connective tissue, have been shown to express immunologic receptors, such as a bacterial pattern recognition receptor CD14 [3 , 4 ], Toll-like receptors (TLRs) [5 ], and a costimulatory molecule CD40 [1 , 6 ] on the cell surface and to produce various cytokines, such as interleukin (IL)-1, IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1), by interaction with their ligands [3 4 5 , 7 ], indicating that HGF also actively participate in inflammatory processes and immune responses.

IL-2 is a 15.5-kDa glycoprotein produced principally by activated T cells following the specific interaction of the antigen–major histocompatibility complex with the T cell antigen receptor complex, and its biologic targets have been presumed to be limited largely to the lymphocyte population, including T cells, B cells, and natural killer (NK) cells [8 ]. A fully functional, high-affinity IL-2 receptor (IL-2R) complex consists of three molecularly distinct membrane proteins: p55 (IL-2R{alpha} chain, CD25), p75 (IL-2Rß chain, CD122), and p64 (IL-2R{gamma} chain, CD132). These subunits can be expressed individually or in various combinations, resulting in distinct IL-2R complexes that bind IL-2 with different affinities; heterodimers of {alpha}ß and ß{gamma} chains show intermediate affinity for IL-2 [8 ]. In addition, human monocytes also bear functional IL-2Rß and -{gamma} chains [9 ], human fibroblasts from adult bone marrow (BM); embryonic skin and lung express IL-2R{alpha} and -ß [10 11 12 ]; and fibroblast-like synoviocytes express functional IL-2Rß and -{gamma} chains [13 ]. These observations indicate that IL-2 has a wide variety of biological activities in cells other than the lymphocyte population.

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 destruction of direct and indirect periodontal tissue [14 ]. The T cell features in diseased periodontal tissues can be compared with those in rheumatoid arthritis. These observations suggest that HGF and infiltrated T cells interact in the lesions and led us to investigate whether HGF respond to a T cell-derived major cytokine, IL-2, and express IL-2Rs. In this study, we showed that HGF express a functional IL-2Rß and -{gamma} chain but not -{alpha} chain; IL-2 stimulation induced production of MCP-1 and expression of intercellular adhesion molecule-1 (ICAM-1, CD54), one of the major adhesion molecules by HGF, resulting in the augmentation of ICAM-1-mediated neutrophil adhesion; and the underlying mechanisms were discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Lipopolysaccharide (LPS) of Escherichia coli O55:B5 was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human (rh)IL-2 was kindly provided by Shionogi Pharmaceutical Co. (Osaka, Japan). The Hayashibara Biomedical Laboratories (Okayama, Japan) kindly provided human natural interferon-{gamma} (IFN-{gamma}; antiviral activity, 8.0x106 IU/mg protein). Anti-human CD25 H31 [mouse immunoglobulin G (IgG)1; ref. 15 ] and anti-human CD122 TU27 (mouse IgG1) [16 ] monoclonal antibodies (mAb) were a gift from Kazuo Sugamura (Tohoku University Graduate School of Medicine, Sendai, Japan). Anti-human CD132 #38024.11 (mouse IgG1), neutralizing anti-human IL-15 #34505.11 (mouse IgG), and rhIL-15 were obtained from Genzyme/Techne (Minneapolis, MN). Neutralizing anti-human CD54 (ICAM-1) 84H10 (mouse IgG1), and all isotype-control mAb were obtained from Beckman Coulter (Miami, FL) and dialyzed against phosphate-buffered saline (PBS). All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.

Immunohistochemistry
Human gingival tissues were obtained from patients with adult periodontitis undergoing periodontal surgery with informed consent. Immunohistochemistry was performed using anti-CD8 (BD PharMingen, San Diego, CA) and anti-IL-2 (Santa Cruz Biotechnology, Santa Cruz, CA), as described previously [17 ]. The sections were also stained with periodic acid-Schiff (PAS). The immunoreactivity was observed using a confocal laser microscope (Bio-Rad Laboratories, Hercules, CA). Normal and inflamed regions of the specimens were pathologically confirmed. The Ethical Review Board of Tohoku University Graduate School of Dentistry (Sendai, Japan) approved the experimental procedures.

Cells and cell culture
HGF were prepared from the explants of normal gingival tissues of patients under informed consent and cultured in {alpha}-minimum essential medium ({alpha}-MEM) supplemented with 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY) in 100 mm-diameter tissue-culture dishes (Falcon, BD Labware, Lincoln Park, NJ), as described previously [4 ]. Cells were used as confluent monolayers at subculture levels 3–10.

Peripheral blood mononuclear cells (PBMCs) from heparinized (10 U/ml) peripheral venous blood were isolated by Lympholyte-H (Cederlane Laboratories, Hornby, Ontario, Canada) gradient centrifugation at 800 g for 20 min at room temperature [18 ]. 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.

Neutrophils from heparinized (10 U/ml) peripheral venous blood were isolated by density-gradient centrifugation on Mono-Poly resolving medium (ICN Biomedical, Costa Mesa, CA) at 300 g for 30 min at room temperature. The fraction containing neutrophils was harvested and washed twice with PBS at 4°C. The purity of neutrophils was above 95% morphologically.

Flow cytometry
Flow cytometric analyses were performed with a fluorescence-activated cell sorter (FACScan, BD Biosciences, Mountain View, CA) [18 ]. For immunofluorescence staining, confluent HGF were collected by trypsinization and washed in PBS. HGF were stained with anti-CD25, anti-CD122, anti-CD132, anti-CD54, or isotype-control mAb at 4°C for 30 min and were then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse 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 (MFI).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from HGF (2x106 cells) using a total RNA isolation kit (Isogen, Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. PBMCs were used as positive controls. The primers used for PCR were as follows; CD25, forward 5'-ACAACCAATGTCAATGCACAAGCT-3' and reverse 5'-TCTGTTCCCGGCTTCTTACCAAGA-3' [19 ]; CD122, forward 5'-CCGTGGCTCGGCCACCTC-3' and reverse 5'-TAGGGGTCGTAAGTAAAGTACACC-3' [13 ]; CD132, forward 5'-CCAGGACCCACGGGAACCCA-3' and reverse 5'-GGTGGGAATTCGGGGCATCG-3' [11 ]; and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and reverse 5'-CATGTGGGCCAATGAGGTCCACCAC-3' [4 ]. The primers for CD25, CD122, CD132, and GAPDH were constructed to generate fragments of 634/419, 437, 493, and 983 bp, respectively. Amplification was performed in a model MP TP3000 PCR thermal cycler (Takara, Tokyo, Japan) as follows: with CD25, 30 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 2 min; with CD122, 40 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 45 s; with CD132, 40 cycles of denaturation at 94°C for 1 min, annealing at 63°C for 1 min, and extension at 72°C for 45 s; and with human GAPDH, 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. Amplified samples were visualized on 2% agarose gels stained with ethidium bromide and photographed under UV light.

Detection of cytokine by enzyme-linked immunosorbent assay (ELISA)
Confluent HGF were collected by trypsinization and washed in PBS three times. The cells (105 cells/200 µl) were seeded in {alpha}-MEM with 10% FCS in wells of 96-well flat-bottom plates (Falcon, BD Labware). 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 {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 and IL-8 produced by HGF in the supernatants were determined with OptEIATM human MCP-1 and IL-8 ELISA kits (BD PharMingen). 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 Janus tyrosine kinase (Jak)3 phosphorylation
Cells were lysed with a buffer containing 1% Nonidet-P40, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 100 µg/ml aprotin, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM Na2MoO4, and 10 mM NaF for 30 min at 4°C with mixing [20 ]. 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 Jak3 polyclonal antibody (Santa Cruz Biotechnology) and protein G-Agarose for 2 h at 4°C. The immunoprecipitates were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride (PVDF) membrane (Atto Instruments, Tokyo, Japan). To detect phosphotyrosine content, the membrane was sequentially bound with an antiphosphotyrosine 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-Jak3 polyclonal Ab to determine the amount of Jak3 in each lane.

Adhesion assay
Confluent HGF in wells of a 96-well plate coated with collagen I (Falcon, BD Labware) were treated with anti-CD54 84H10 or isotype-control IgG (10 µg/ml each) for 30 min at 37°C and were then washed with warmed medium three times. Neutrophils (5x106 cells/ml) were labeled with 5 µM calcein acetoxymethyl (Molecular Probes, Eugene, OR) in RPMI-1640 medium for 30 min at 37°C. The labeled neutrophils (5x105 cells/well) were then added to cell monolayers and incubated for 30 min at 37°C. At the end of the incubation, the cells were washed with warmed PBS three times, and adherent neutrophils were evaluated using a cuvette fluorometer (Versa Fluor, Bio-Rad Laboratories) at 494 nm excitation and 510 nm emission. Serial dilutions of calcein-labeled neutrophils were used as a standard to calculate the number of adherent neutrophils.

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


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Infiltration of T cells in inflamed gingiva
As several lines of evidence suggest that periodontal diseases result from or cause imbalances in the regulation of the local immune response after an infection of periodontopathic bacteria [14 ], we first examined whether immune cells are infiltrated in inflamed regions of gingival tissues. The results of immunohistochemistry showed that many cells had infiltrated the lamina propria in the inflamed region of cryosections of gingival tissue from adult periodontitis patients, as compared with normal regions of the sections (Fig. 1A and 1C ). Furthermore, numerous CD8+ T cells, an effector subset of T cells, infiltrated the lamina propria in the inflamed region (Fig. 1B and 1D) . Infiltration of CD4+ T cells was also observed in the region, and infiltrating cells in the section of inflamed gingival tissue expressed IL-2, as assessed by immunohistochemistry (data not shown). The observation indicates that the interaction of T cells with HGF, the major resident cells in the lamina propria, could occur in the inflamed gingiva.



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  		Figure 1. Infiltration of T cells in inflamed gingiva. Cryosections of gingival tissue from a patient with adult periodontitis were stained with PAS (A, C) or the anti-CD8 mAb in green (B, D). Cell nuclei were counterstained with propidium iodide in red. Normal (A, B) and inflamed (C, D) regions of the section. GE, Gingival epithelium; LP, lamina propria. Original bars = 50 µm. The results presented are representative of three different experiments demonstrating similar results.

 
Expression of IL-2Rß (CD122) and -{gamma} (CD132) by HGF
Activated T cells principally produce IL-2 [8 ]. We, therefore, examined the expression of IL-2R subunits in HGF. PBMCs were used as a positive control. In flow cytometric analyses, unstimulated HGF substantially expressed IL-2Rß (CD122; MFI, 28.8 for CD122 vs. 4.8 for control) and IL-2R{gamma} (CD132; MFI, 43.3 for CD132 vs. 4.8 for control), but the expression of IL-2R{alpha} (CD25) was below the detectable limit (Fig. 2A ). Consistent with this, unstimulated HGF express IL-2Rß and IL-2R{gamma} mRNA (Fig. 2B , lanes 4 and 7) and did not express IL-2R{alpha} mRNA (Fig. 2B , lane 1), as assessed by RT-PCR. Pretreatment of HGF with IFN-{gamma} (100 IU/ml) for 24 h had no effect on the expression of IL-2Rß and IL-2R{gamma} or IL-2R{alpha} at the mRNA (Fig. 2B , lanes 2, 5, and 8) and protein levels (data not shown). In addition, the expression of IL-2Rß and -{gamma} in HGF was confirmed by using HGF from several donors.



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  		Figure 2. Expression of IL-2R subunits by HGF. (A) HGF were collected by trypsinization and stained with anti-CD25, anti-CD122, and anti-CD132 (solid lines) or isotype-control mAb (dotted lines) and were analyzed by flow cytometry. (B) Confluent HGF were treated with medium alone (lanes 1, 4, and 7) or with 100 IU/ml IFN-{gamma} for 24 h (lanes 2, 5, and 8). PBMCs (lanes 3, 6, and 9) were used as a positive control. Total RNA was extracted from the cells, and cDNA was prepared and analyzed for the mRNA expression of CD25 (lanes 1–3), CD122 (lanes 4–6), CD132 (lanes 7–9; upper panel), and GAPDH (lower panel) by RT-PCR. M, Molecular weight marker. The results presented are representative of four different experiments demonstrating similar results.

 
Production of MCP-1 by HGF in response to IL-2
To examine whether the IL-2Rß and -{gamma} expressed on HGF are functionally active, exogenous IL-2 was added to HGF at the indicated concentrations (1–1000 ng/ml) for 24 h. LPS (1 and 10 ng/ml) was used as a positive control. HGF produced IL-8 upon stimulation with LPS, as described previously [4 ], but IL-2 did not induce the production of IL-8 (Fig. 3A ). HGF spontaneously produced MCP-1, and the production was significantly increased in response to LPS (Fig. 3B) . Furthermore, when IL-2 was added to HGF, an increase in MCP-1 production was also seen over a range of 1–1000 ng/ml, with a peak at 300 ng/ml.



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  		Figure 3. Production of MCP-1 by HGF upon stimulation with IL-2. Confluent HGF were stimulated with the indicated concentrations of IL-2 or LPS for 24 h. ELISA determined concentrations of IL-8 and MCP-1 in the culture supernatants. 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 different experiments showing similar results.

 
Blocking CD122 and CD132 with a neutralizing Ab inhibits MCP-1 production by HGF
To investigate whether the direct ligation of IL-2R by IL-2 resulted in production of MCP-1, neutralizing Ab to CD25, CD122, and CD132 (5, 10, 20, and 40 µg/ml each) was added to the HGF cultures 45 min before IL-2. Addition of IL-2 significantly up-regulated MCP-1 production, which was significantly inhibited by the prior administration of anti-CD122 and anti-CD132 mAb but not by anti-CD25 mAb (Fig. 4 ), indicating that IL-2Rß and -{gamma} chains expressed on HGF were functionally active, and the activation of HGF by IL-2 was mediated through IL-2Rß and -{gamma}.



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  		Figure 4. Inhibition of MCP-1 production caused by blocking CD122 and CD132. HGF were cultured with (+) or without (–) neutralizing mAb to CD25 and CD132 (5, 10, 20, 40 µg/ml each) in {alpha}-MEM alone or with IL-2 (10 ng/ml). Neutralizing mAb were added 45 min before addition of IL-2, and the supernatants were collected after 24 h. The results are expressed as the mean ± SD for triplicate cultures. *, P < 0.01, compared with IL-2 alone. The results presented are representative of three different experiments demonstrating similar results.

 
Activation of Jak3 in HGF by IL-2
To confirm that the production of MCP-1 by HGF is a result of direct activation with IL-2, we next examined tyrosine phosphorylation of Jak3 induced by IL-2 in HGF, as activation through IL-2R{gamma} is directly associated with activation of Jak3 [21 ]. Phosphorylation of Jak3 was not detected in unstimulated HGF, and addition of IL-2 to the culture induced the phosphorylation of Jak3, which peaked at 30 min and declined thereafter (Fig. 5 ). The results clearly demonstrate that HGF are activated through IL-2R{gamma} followed by signal transduction via Jak3.



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  		Figure 5. IL-2-induced Jak3 phosphorylation in HGF, which were stimulated with IL-2 for the time indicated. The cell lysates were immunoprecipitated with anti-Jak3 polyclonal Ab, subjected to SDS-PAGE, and transferred to a PVDF membrane. The blot was probed with antiphosphotyrosine mAb 4G10. The blot was reprobed with the anti-Jak3 polyclonal Ab to determine the amount of Jak3 in each lane. The results presented are representative of four different experiments showing similar results.

 
Involvement of IL-15 in signaling of IL-2 in HGF
As IL-15R consists of three chains, {alpha}, ß and {gamma}, and the ß and {gamma} chains are shared between IL-2 and IL-15 [22 ], we next investigated the involvement of IL-15 in signaling IL-2 in HGF. Although HGF did not express IL-15R{alpha} (data not shown), HGF produced MCP-1 but not IL-8 in response to nanogram concentrations of exogenous IL-15 in a dose-dependent manner (Fig. 6A ), indicating that IL-15 also had the same properties on HGF. Additional IL-15 at 100 ng/ml showed only a marginal additive effect on the production of MCP-1 caused by IL-2 at 1 and 10 ng/ml. However, pretreatment with neutralizing anti-IL-15 mAb significantly inhibited the IL-2-induced MCP-1 production (Fig. 6B) , suggesting that a small amount of IL-15 produced by HGF sustained the IL-2-induced activation of HGF.



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  		Figure 6. Involvement of IL-15 in signaling of IL-2 in HGF. (A) Confluent HGF were stimulated with the indicated concentrations of IL-2 and IL-15 for 24 h. (B) Confluent HGF were pretreated with or without neutralizing anti-IL-15 or isotype-control IgG (10 µg/ml each) for 1 h and were then stimulated with the indicated concentrations of IL-2 for 24 h. 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 (IL-2 alone). The results presented are representative of three different experiments showing similar results.

 
Increase in ICAM-1-mediated neutrophil adhesion to HGF by IL-2
A previous report [23 ] showed that in immunohistological analysis, the expression of ICAM-1 on fibroblasts was greater in periodontal tissues than in normal gingival tissues and that cell-surface ICAM-1 expression was up-regulated by stimulation with inflammatory cytokines such as IL-1, tumor necrosis factor {alpha} (TNF-{alpha}), IFN-{gamma}, and LPS. Therefore, we then examined whether IL-2 induces ICAM-1 expression on the surface of HGF. Figure 7 showed that unstimulated HGF only slightly express ICAM-1 on the cell surface, IL-2 at 1 ng/ml induced a fivefold increase in the expression, and higher concentrations of IL-2 induced a slightly greater increase in the expression. IFN-{gamma} (100 IU/ml), a positive control, had a more pronounced effect on the ICAM-1 induction on the cell surface. As the infiltration of neutrophils into gingival tissues is an early event in gingival inflammation [24 ], the observation leads to the question whether IL-2 promotes neutrophil adhesion to HGF. Pretreatment of HGF with IL-2 significantly augmented the adhesion of neutrophils, which was inhibited by the anti-ICAM-1 mAb (Fig. 8 ). IFN-{gamma} was used as a positive control, and the same pattern was obtained as with IL-2, although the number of adherent neutrophils was more than that induced by IL-2. These results indicate that IL-2 induced ICAM-1 expression on HGF and ICAM-1-mediated neutrophil adhesion to HGF.



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  		Figure 7. Up-regulation of ICAM-1 expression on HGF after treatment with IL-2. HGF were stimulated with IL-2 at the concentration indicated or with IFN-{gamma} (100 IU/ml) for 24 h. After the incubation, cells were collected by trypsinization, HGF were stained with anti-ICAM-1 or isotype-matched mAb, and the expression of ICAM-1 on HGF was analyzed by flow cytometry. The number indicates the degree of increase in ICAM-1 expression after the treatments evaluated on the basis of the value obtained with untreated cells. The results presented are representative of three different experiments demonstrating similar results.

 


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  		Figure 8. Increased ICAM-1-mediated adhesion of neutrophils to HGF in response to IL-2. HGF were stimulated with medium alone or medium containing IL-2 (10 ng/ml) or IFN-{gamma} (100 IU/ml) as a positive control for 24 h. HGF washed with PBS were then treated with or without anti-ICAM-1 mAb 84H10 or isotype-control IgG (10 µg/ml each) for 30 min, and calcein-labeled neutrophils (5x105 cells/well) were added. After 30 min, nonadherent cells were removed, and the number of adherent cells was quantified using a spectrophotometer. The results are expressed as the mean ± SD for triplicate cultures. *, P < 0.01, compared with respective control. Results are expressed as the number of adherent cells. The results presented are representative of three different experiments demonstrating similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study showed that HGF express functional IL-2Rß and -{gamma} chains, and exogenous IL-2 induced the production of MCP-1 and the expression of ICAM-1 on the cell surface, which in turn, caused the augmentation of ICAM-1-mediated neutrophil adhesion to HGF. RT-PCR and flow cytometry confirmed the lack of IL-2R{alpha} in HGF (Fig. 2) , and neutralizing anti-IL-2R{alpha} mAb did not inhibit IL-2-induced MCP-1 production by HGF (Fig. 4) . IL-2Rß{gamma} formed an IL-2R of intermediate affinity [8 ], and IL-2R{gamma} is an essential component for a functional IL-2R complex [25 , 26 ]. We showed that IL-2Rß{gamma} expressed on HGF was functionally active, as addition of neutralizing anti-IL-2Rß and -{gamma} mAb caused inhibition of the IL-2-induced MCP-1 production (Fig. 4) , and IL-2 induced the phosphorylation of Jak3, which is critical in signaling through IL-2R{gamma} [21 ] in HGF (Fig. 5) . The phosphorylation of Jak3 in HGF peaked at 30 min stimulation with IL-2, whereas the phosphorylation in a human T cell leukemia virus-I-transformed human T cell line peaked at 10–15 min after IL-2 stimulation [20 , 27 ], indicating that signal transduction in HGF is slower than that in lymphocytes.

It has been reported that human monocytes also bear functional IL-2Rß and -{gamma} chains [9 ], that human fibroblasts from adult BM, embryonic skin, and lung express IL-2R{alpha} and -ß [10 11 12 ], and that fibroblast-like synoviocytes express functional IL-2Rß and -{gamma} but not -{alpha} chains [13 ]. As it is reported that fibroblasts in different anatomical regions and even in the same tissues consist of heterogeneous populations [1 , 2 , 4 ], we consider that expression of IL-2Rß and -{gamma} in HGF is characteristic in periodontal tissues. A recent report showed that lung myofibroblasts, contrary to normal lung fibroblasts, constitutively express the {gamma} chain [28 ]. However, HGF prepared form normal and inflamed regions express only a low level of {alpha}-smooth muscle actin, and there were no differences in the expression by immunohistochemistry (data not shown). This observation suggests that HGF do not differentiate into myofibroblasts in the evolution of the inflammatory process. However, HGF from inflamed gingival tissues produce about two times more MCP-1 compared with normal ones, and experiments are under way to clarify this point. Although IL-2R{gamma} expression in human monocytes was reported to be up-regulated by IL-2 and IFN-{gamma} [9 ], we could not observe the up-regulation of IL-2Rß and -{gamma} and induction of IL-2R{alpha} by IFN-{gamma} (Fig. 2) or IL-2 (data not shown), which was consistent with observations in fibroblast-like synoviocytes [13 ]. The previous reports showed that unstimulated fibroblasts from human embryonic lung in confluent culture substantially expressed ICAM-1, and the expression was down-regulated by exogenous IL-2 [10 ], but in sparse culture of the cells where ICAM-1 expression was low, IL-2 induces up-regulation of the expression [12 ]. The present study showed that unstimulated HGF in confluent culture express ICAM-1 at a low level, which was up-regulated by addition of IL-2 as well as IFN-{gamma} (Fig. 7) . This discrepancy also suggests that the fibroblasts from different anatomical regions are not the same in their characteristics. Furthermore, even at a concentration of 1000 ng/ml, IL-2 was not so effective in the induction of MCP-1 production (Fig. 3) and ICAM-1 expression (Fig. 7) , which may be explained by the expression of IL-2R with intermediate affinity (ß{gamma}) in HGF or also by characteristics of fibroblasts in periodontal tissues.

The present study showed that LPS induced production of IL-8 and MCP-1, whereas IL-2 induced the production of MCP-1 but not IL-8 (Fig. 3) . Activation of mammalian cells by LPS is mediated by a complex of TLR4 and MD-2; is followed by the sequential activation of myeloid differentiation factor 88, IL-1R-associated protein kinase, and TNFR-activated factor 6; and finally, induces the translocation of nuclear factor-{kappa}B into the nucleus [29 ]. The IL-2R{alpha} increases the affinity of IL-2 binding but lacks a signaling domain, and the IL-2Rß and -{gamma} were reported to associate with protein tyrosine kinases Jak1 and Jak3, respectively, followed mainly by activation of signal transducer and activator of transcription-3 and -5 [21 ]. Therefore, the difference in the induction of cytokines between LPS and IL-2 in HGF may account for the different signal-transduction pathway.

Periodontal disease is caused by an infection of 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 [14 ]. In addition, T helper cell type 1 (Th1) or Th2 and their associated cytokines could contribute to the onset of healing of this disease [14 ]. The immunohistochemistry of gingival tissues actually showed that numerous CD8+ T cells are accumulated in the lamina propria of inflamed regions (Fig. 1) . IL-2 is secreted from activated T cells and a key cytokine in Th1 and Th2 responses [30 ]. The concentration of IL-2 in the secretory vesicles of T cells is estimated to be in the millimolar range, and more than 90% of the IL-2 after secretion would be expected to remain in the vicinity by the formation of cytokine reservoirs on the cell surface [31 ] in 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 also could occur in inflamed periodontal tissue, and IL-2-mediated activation of HGF is likely to occur in vivo. The present study showed that IL-2 activates HGF to produce MCP-1 and up-regulate ICAM-1 expression via IL-2Rß{gamma} on the cells, indicating that T cell-derived IL-2 has an ability to activate not only T cells and monocytes but also resident fibroblasts in the lesions of periodontitis.

IL-2Rß and -{gamma} chains are also IL-15R with intermediate affinity [22 ]. HGF did not express IL-15R{alpha} (data not shown), and exogenous IL-15 actually activated HGF to produce MCP-1 but not IL-8 (Fig. 6A) , which indicates that IL-15 also had the same properties on HGF. Intriguingly, pretreatment with neutralizing anti-IL-15 mAb significantly inhibited the IL-2-induced MCP-1 production (Fig. 6B) . A recent report showed that IL-15 is present as a membrane-bound cytokine not only on inflammatory infiltrating cells such as macrophages but also on target cells that may recycle this cytokine on their membrane in a functional form [32 ]. Moreover, membrane-bound IL-15 is expressed on fibroblasts from human spleen and skin and regulates differentiation of progenitors and proliferation of T cells, respectively [33 , 34 ]. Thus, the present study suggests that a small amount of IL-15 produced by HGF sustained the IL-2-induced activation of HGF in an autocrine loop, although HGF did not express high-affinity IL-15R{alpha}.

ICAM-1 is one of the major adhesion molecules interacting with the ß2-integrin family including lymphocyte-function associated antigen-1 (LFA-1) and Mac-1, present on neutrophils, monocytes, and T cells [35 ], and is a key molecule in the adhesion and migration of neutrophils and T cells in the inflamed region in periodontitis [36 , 37 ]. A previous report showed that in immunohistological analysis, the expression of ICAM-1 on fibroblasts was greater in periodontal tissues than in normal gingival tissues and that cell-surface ICAM-1 expression on HGF was up-regulated by stimulation with inflammatory cytokines such as IL-1, TNF-{alpha}, IFN-{gamma}, and LPS [23 ]. In agreement with these reports, Figure 8 showed that the up-regulation of ICAM-1 expression on HGF in response to IL-2 as well as IFN-{gamma} contributes extensively to the interaction with neutrophils. It is also reported that fibroblasts can be stimulated by the direct surface interaction of ICAM-1 with its ligands on inflammatory cells [38 ], indicating that direct ICAM-1-mediated interaction of HGF with neutrophils may cause further activation of HGF. 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 [39 , 40 ]. MCP-1 is also produced by cytokine-primed and -differentiated neutrophils and implicated the importance in the regulation of the transition from innate to adaptive immunity [41 ]. A recent report indicates that ICAM-1/LFA-1 ligation favors human Th1 development [42 ]. Therefore, the activation of HGF by IL-2, which are released by activated T cells, may play an important role in innate immunity against periodontal pathogens and in adaptive immunity in controlling Th1 and Th2 responses at the periodontitis site.

As IL-2R{gamma} has been revealed to be an indispensable subunit of the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors to date and is now referred to as the common {gamma}-chain [27 , 43 ], there is a possibility that HGF express common {gamma}-chain-associated cytokine receptors. An investigation is under way to clarify this point.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Grants-in-Aid for Scientific Research (12470380, 13671894, and 14370576) from the Japan Society for the Promotion of Science. We thank K. Sugamura (Tohoku University Graduate School of Medicine) for providing anti-IL-2R mAb and E. Nemoto (Tohoku University School of Dentistry) for helpful discussions.

Received January 27, 2003; revised May 2, 2003; accepted May 3, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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