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Keywords:

  • bone resorption;
  • T-lymphocyte;
  • potassium channel;
  • RANKL;
  • osteoprotegerin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The effects of the potassium channel (Kv1.3) blocker kaliotoxin on T-cell-mediated periodontal bone resorption were examined in rats. Systemic administration of kaliotoxin abrogated the bone resorption in conjunction with decreased RANKL mRNA expression by T-cells in gingival tissue. This study suggests a plausible therapeutic approach for inflammatory bone resorption by targeting Kv1.3.

Introduction: Kv1.3 is a critical potassium channel to counterbalance calcium influx at T-cell receptor activation. It is not known if Kv1.3 also regulates RANKL expression by antigen-activated T-cells, and consequently affects in vivo bone resorption mediated by activated T-cells.

Materials and Methods:Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein-specific Th1-clone cells were used to evaluate the expression of Kv1.3 (using reverse transcriptase-polymerase chain reaction [RT-PCR] and Western blot analyses) and the effects of the potassium channel blocker kaliotoxin (0–100 nM) on T-cell activation parameters ([3H]thymidine incorporation assays and ELISA) and expression of RANKL and osteoprotegerin (OPG; flow cytometry, Western blot, and RT-PCR analyses). A rat periodontal disease model based on the adoptive transfer of activated 29-kDa outer membrane protein-specific Th1 clone cells was used to analyze the effects of kaliotoxin in T-cell-mediated alveolar bone resorption and RANKL and OPG mRNA expression by gingival T-cells. Stimulated 29-kDa outer membrane protein-specific Th1 clone cells were transferred intravenously on day 0 to all animals used in the study (n = 7 animals per group). Ten micrograms of kaliotoxin were injected subcutaneously twice per day on days 0, 1, 2, and 3, after adoptive transfer of the T-cells. The control group of rats was injected with saline as placebo on the same days as injections for the kaliotoxin-treated group. The MOCP-5 osteoclast precursor cell line was used in co-culture studies with fixed 29-kDa outer membrane protein-specific Th1-clone cells to measure T-cell-derived RANKL-mediated effects on osteoclastogenesis and resorption pit formation assays in vitro. Statistical significance was evaluated by Student's t-test.

Results: Kaliotoxin decreased T-cell activation parameters of 29-kDa outer membrane protein-specific Th1 clone cells in vitro and in vivo. Most importantly, kaliotoxin administration resulted in an 84% decrease of the bone resorption induced in the saline-treated control group. T-cells recovered from the gingival tissue of kaliotoxin-treated rats displayed lower ratios of RANKL and OPG mRNA expression than those recovered from the control group. The ratio of RANKL and osteoprotegerin protein expression and induction of RANKL-dependent osteoclastogenesis by the activated T-cells were also markedly decreased after kaliotoxin treatments in vitro.

Conclusion: The use of kaliotoxin or other means to block Kv1.3 may constitute a potential intervention therapy to prevent alveolar bone loss in periodontal disease.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Normal bone remodeling requires precise control of the rates of bone formation by osteoblasts and degradation by osteoclasts.(1) RANKL; its cellular receptor, RANK; and the decoy receptor, osteoprotegerin (OPG) have been identified as the key molecular regulation system for bone remodeling.(2–5) RANKL is the main stimulatory factor for the formation of mature osteoclasts and is essential for their survival.(3,4) The effects of RANKL are counteracted by OPG that is secreted by various tissues and prevents the binding of RANKL to its receptor RANK on osteoclasts and thereby suppresses osteoclastogenesis.(5)

Activated T-cells have been associated with increased osteoclast formation and accelerated bone resorption under inflammatory conditions in vivo(6–7) and in vitro.(8) In fact, the alveolar bone destruction observed in periodontal infections has been shown to be mediated in part by microorganism-triggered induction of RANKL expression on T-cells and the subsequent activation of osteoclasts.(9–11) Hence, development of therapeutic approaches to target RANK-RANKL interactions between activated T-cells and osteoclast precursor cells or to regulate RANKL expression by activated T-cells constitute promising areas for intervention in periodontal disease pathogenesis and other inflammatory bone resorptive diseases such as rheumatoid arthritis.

Optimal T-cell activation requires several hours of calcium entry from the external milieu through Ca2+ release-activated Ca2+ channels.(12) To maintain Ca2+ entry over the time scale required for gene transcription, a balancing efflux of K+ ions should also occur through potassium channels, including the voltage-gated potassium channel Kv1.3.(12) Blocking of T-lymphocyte Kv1.3 with high affinity blockers has been shown to attenuate the Ca2+ signaling pathway leading to decreased proliferation and cytokine production by activated T-cells in vitro.(12) Furthermore, selective blockade of Kv1.3 has been reported to inhibit T-cell activation in vivo in a mini pig model of delayed-type hypersensitivity(13,14) and in a rat model for experimental autoimmune encephalomyelitis.(15,16) However, the effect of blocking Kv1.3 in T-cell-mediated inflammatory bone resorption in vivo has not yet been described.

An experimental rat model for the study of T-cell-mediated inflammatory bone resorption has been previously reported.(17) In this model, local gingival stimulation of Th1-type memory T-cells specific for A. actinomycetemcomitans Omp29 that involve major histocompatibility complex (MHC) class II+/B7+ antigen-presenting cells (APC) could induce alveolar bone resorption in 10 days.(17)

Because RANKL is expressed by T-cells after T-cell receptor (TCR) stimulation(9,18) and its transcriptional upregulation is calcium-dependent,(18,19) we hypothesized that blocking Kv1.3 with kaliotoxin, a peptide from scorpion venom, should decrease the expression of RANKL by gingival T-cell infiltrates and reduce Th1-mediated bone resorption in this rat periodontal disease model.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animals

Female Rowett rats, 8–12 weeks of age (inbred at The Forsyth Institute for more than 20 generations in plastic isolators) were maintained under pathogen-free conditions in laminar flow cabinets.(17,20) Experiments using these animals were performed under the approval of The Forsyth Institute's Internal Animal Care and Use Committee.

Bacteria, bacterial antigens, and lipopolysaccharide

Actinobacillus actinomycetemcomitans, American Type Culture Collection 43718 (strain Y4; Manassas, VA, USA) was grown in pleuro-pneumonia-like organism broth (Difco Laboratories, Detroit, MI, USA) with glucose (3 g/liter) and sodium bicarbonate (1 g/liter) for 72 h at 37°C under increased CO2 (candle jar). The cultured bacteria at log-growth phase were killed with formalin (5%), washed five times in PBS, and resuspended in Roswell Park Memorial Institute (RPMI) medium to serve as T-cell antigen. The A. actinomycetemcomitans Omp29 and A. actinomycetemcomitans lipopolysaccharide (LPS) were prepared as previously described.(21,22) Purified Omp29 protein was kindly provided by Dr Hitoshi Komatsuzawa (Hiroshima University, Japan).

Adoptive transfer of Th1-type clone cells and treatments with kaliotoxin or OPG-Fc in vivo

Rowett strain female rats received three palatal gingival injections (1 μl/site) on the mesial of the first molar and in the papillae between first and second and third molars on the right and left sides of the maxilla. Gingival injections consisted of 500 μg/ml A. actinomycetemcomitans Omp29 plus 500 μg/ml A. actinomycetemcomitans Omp29 LPS (left, experimental side) and PBS as a control (right, control side) on day 0.

G23 Th1-type clone cells specific for A. actinomycetemcomitans Omp29(20) were stimulated with sterile formalin-killed A. actinomycetemcomitans and irradiated (3300 rad) syngeneic rat spleen cells (APC) for 3 days on 24-well plates. Stimulated G23 T-cells (4 × 106) were isolated by gradient centrifugation and transferred intravenously on day 0 as previously described.(17,20) Ten micrograms of kaliotoxin (MW: 4021.8; Alomone Labs, Jerusalem, Israel) were injected subcutaneously in the back of the neck twice per day on days 0, 1, 2, and 3 after adoptive transfer of the G23 T-cells. The control group of rats was injected with saline as placebo on the same days as the kaliotoxin-treated group.

One hundred micrograms of OPG-Fc (a fusion protein of human OPG and human IgG-Fc kindly provided by Dr Colin Dunstan from Amgen Inc., Thousand Oaks, CA, USA) or 100 μg of the irrelevant peptide L6-Fc (control group) were injected intraperitoneally on days 0, 2, and 4 after adoptive transfer of the G23 T-cells.

Proliferation assays

T-cells recovered from the spleens of rats receiving gingival challenge of Omp29/LPS and adoptive transfer of T clone cells showed proliferative responses to antigen presentation by APC up to 10 days after adoptive transfer.(17) Therefore, T-cells (>95% CD3+ T-cells) were recovered from rat spleens 10 days after adoptive transfer as described(17,20) and cultured in 96-well plates (5 × 103 cells/well) in the presence of irradiated normal splenic APC (5 × 105 cells/well) with or without killed A. actinomycetemcomitans bacteria (107/well) as antigen. [3H]Thymidine (0.5 μCi/well) was applied for the last 16 h of a total of 3 days in culture. Samples were harvested onto glass fiber filters, and radioactivity (cpm) was measured in a β-scintillation spectrometer (Beckman Coulter, Fullerton, CA, USA). Antigen-specific proliferation of the splenic T-cells was calculated and expressed as the antigen-specific stimulation index = (cpm of cells cultured in the presence of antigen/cpm of cells cultured in the absence of antigen).

In vitro proliferation assays with Omp29-specific Th1 clone cells (G21 or G23)(20,23) were performed as previously described in the presence of irradiated APC with or without killed A. actinomycetemcomitans bacteria.(20) Kaliotoxin (25–100 nM) was added to the T clone cell cultures 15 minutes before the antigen, and the APCs were added.

ELISA determinations

ELISA using horseradish peroxidase conjugated anti-rat IgG2a (sheep, Binding Site, San Diego, CA, USA) and o-phenylenendiamine (Sigma) as substrate was used to detect IgG2a antibody levels to Omp29 in rat serum at day 10 as previously described.(17) Hyperimmune serum from rats immunized subcutaneously with Omp29 was used as a reference standard. The OD at 490 nm of this serum diluted at 1:8500 was chosen as 100 ELISA units, and all rat serum IgG2a reactions to Omp29 were evaluated based on a reference curve provided by dilution of this hyperimmune serum.

The concentrations of interferon-γ (IFN-γ) in the culture supernatants of Omp29-specific Th1 clone cells were determined by DuoSet ELISA kits from R&D Systems Inc. (Minneapolis, MN, USA) following the manufacturer's recommendations.

Measurement of bone resorption in vivo

Ten days after adoptive transfer, rats were killed, and the jaws were defleshed. Periodontal bone resorption was measured on the palatal surface of the maxillary molars essentially as previously described.(17) The distances from the cemento-enamel junction (CEJ) to the alveolar ledge (AL) of experimental-injected sites (upper left palatal side injected with LPS and Omp29) and saline-injected control sites (upper right palatal side) were measured using a reticule eyepiece at 25× magnification. A total of five measurements were evaluated in each quadrant, including two roots of the first molar, both roots of the second molar, and one root of the third molar. The evaluation of bone resorption was calculated and expressed as percentage bone resorption = [(total CEJ-AL distance of five points on the left experimental side) − (total CEJ-AL distance of five points on the right control side)]/(total CEJ-AL distance on the five points of right control side) × 100.

Isolation of T-lymphocytes from rat gingival tissue

In the experimental periodontal disease model used herein, mononuclear cells isolated from gingival tissue of the experimental side demonstrated the highest Omp29-specific T-cell proliferation on the second and third days after the T-cell adoptive transfer.(17) However, bone resorption could only begin to be detected after 5 days,(17) with the maximum bone resorption observed 10 days after the T-cell transfer.(17) Therefore, gingival tissues were collected on days 0, 2, and 10 for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. CD3+ T-cells infiltrating into the gingival tissue were isolated as previously described.(17) To obtain an average of 105 CD3+ T-cells for RT-PCR analyses, gingival samples were pooled from the experimental sides of two rats on day 2 and four rats on day 10. Recovery of CD3+ T-cells was negligible from the experimental side on day 0 (n = 2 gingival samples, data not shown) or the control side on days 0, 2, or 10 (n = 2 gingival samples, data not shown). Briefly, gingival tissue was cut into small pieces and digested in RPMI/10% fetal calf serum (FCS) containing 2 mg/ml collagenase (Worthington Biochemical Corp., Freehold, NJ, USA) for 1 h at 37°C. Cell debris was excluded by passing cells through a steel mesh. Mononuclear cells were isolated by density gradient centrifugation using Histopaque (density 1.077 ± 0.001; Sigma Diagnostics, Inc., St Louis, MO, USA). The lymphocyte-containing interface layer was collected and washed twice with RPMI/10% FCS. CD3+ T-cells were enriched by positive selection with mouse monoclonal anti-rat αβ TCR antibody (R73) followed by Dynabeads (Dynal Biotech, NY, USA) coated with anti-mouse IgG as described.(17) The αβ TCR antibody R73 was kindly provided by Dr Thomas Hünig (University of Wurzberg, Germany).

RT-PCR analysis

Total RNA was extracted by using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) from approximately 105 T-cells when isolated from gingival tissue or 106 cultured G23 T-cells. Freshly isolated RNA was reverse transcribed with a SuperScript first-strand synthesis system (Invitrogen) following the manufacturer's recommendations to increased sensitivity. The resulting cDNA was amplified by PCR with HotStartTaq DNA polymerase (Qiagen) as recommended by the manufacturer. The sequences of the primers used in the amplification of rat RANKL were as follows: 5′-TCAGGTGGTCTGCAGCATCGCTCTG-3′ and 5′-AGTACGTCGCATCTTGATCCGGATC-3′ (PCR product size : 0.7 kb). The sequences of the primers used in the amplification of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: 5′-CATAGACAAGATGGTGAAGGTCGGTGTC-3′ and 5′-CTTACTCCTTGGAGG CCATGTAGGCCA-3′ (PCR product size: 0.9 kb). The sequences of the primers used in the amplification of rat OPG were as follows: 5′-TGAACAAGTGGCTGTGCTGTGCACT-3′ and 5′-AGTTTGCTCTTGCGAGCTGTGTCTC-3′ (PCR product size: 0.7 kb). The sequences of the primers used in the amplification of rat Kv1.3 were as follows: 5′-TGCCAGTTCCCTGAGACGCTGCTAGGCGAC-3′ and 5′-TGGCAATCGTATCATCTATTAGACATCAGT-3′ (PCR product size: 1.4 kb). All primers used in this study were purchased from Integrated DNA Technologies (Coralville, IA, USA) at the 25-nM scale. Linearity of the PCR conditions (in terms of cDNA amount and number of cycles) was determined for each primer pair in preliminary experiments (data not shown). To include a similar quantity of mRNA in each PCR reaction, preliminary GAPDH amplifications were also performed with cDNA derived from cells isolated from the experimental gingival tissue and compared with known amounts of cDNA and RNA obtained from cultured G23 T-cells used as standard (data not shown). PCR amplifications were performed with 200 pg of RNA derived from cultured G23 T clone cells and an estimated 150 pg of RNA for samples derived from gingival tissues (either total tissue or CD3+ T-cells infiltrating into the tissue). Total RNA was isolated from whole gingival tissue of the experimental side (injected with LPS and antigen) on day 0 and from the control side (injected with PBS) on days 0, 2, and 10, because recovery of CD3+ T-cells was negligible. Images of the amplified products in 1% agarose gels were processed with Molecular Analyst Software (BioRad, Hercules, CA, USA) and analyzed by Adobe Photoshop 5.0 (Adobe Systems Inc., San Jose, CA, USA) and Scion Image 1.62 (Scion Image, Frederick, MD, USA).

Preparation of protein lysates and Western blot analysis

Total cell lysates from cultured Omp29-specific T-cell clones (1–2 × 106) were prepared with the Mammalian Cell-PE LB buffer system supplemented with the Protease-Arrest protease inhibitor cocktail from GenoTechnology Inc. (St Louis, MO, USA). Protein extracts were resolved on 4–12% Novagen SDS-PAGE gels (Invitrogen) and transferred to Invitrolon polyvinylidene fluoride (PVDF) membranes (Invitrogen). Blots were incubated in blocking solution (2.5% nonfat milk in PBS) for 1 h at room temperature. Primary and secondary antibodies required in the detection of RANKL, OPG, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except the antibody to Kv1.3, which was obtained from Alomone Labs. Primary antibodies (1:400 dilution) were incubated in blocking solution containing 0.02% sodium azide at 4°C overnight. Secondary horseradish peroxidase linked anti-IgG antibodies were incubated in 1% nonfat milk in PBS for 1 h at room temperature. Blots were visualized using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) as recommended by the manufacturer.

Flow cytometry

RANKL surface expression was detected using OPG-Fc fusion protein essentially as described by others.(18) Briefly, single T-cell suspensions were incubated with OPG-Fc (10 μg/ml) or L6-Fc irrelevant fusion protein (10 μg/ml) and incubated with FITC anti-human IgG secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) as recommended by the manufacturer. Flow cytometry analyses was carried out to monitor FITC fluorescence intensity by using an EPICS ALTRA Flow cytometer (Beckman Coulter). Logarithmic amplification of fluorescence intensity was collected from at least 10,000 T-cells as determined by forward light scatter intensity.

Assessment of TRACP+ multinucleated cells and bone resorption pit formation

G23 T-cells were fixed with a cold solution of 50% acetone and 35% ethanol for 5 minutes. Fixed T-cells (105 cells/well) were cocultured with mouse MOCP-5 osteoclast precursor cells(24) (5 × 102 cells/well) in α-MEM with 15% fetal bovine serum (FBS) on 96-well plates for 7 days. The MOCP-5 osteoclast precursor cell line was kindly provided by Dr Yi-Ping Li (The Forsyth Institute, Boston, MA, USA). Cocultures were performed both in the presence and absence of human recombinant OPG (500 ng/ml; Peprotech Inc.) to confirm that the observed effects on the osteoclastogenesis of MOCP-5 were mediated through RANKL-RANK interactions. Differentiated osteoclasts were identified as red-stained cells with three or more nuclei by cytochemical staining for TRACP as described previously.(24,25) To evaluate bone resorption pit formation, cocultures of fixed T-cells and MOCP-5 were incubated on BD BioCoat Osteologic discs (BD Biosciences, Ontario, Canada) for 7 days. To measure the areas of resorption lacunae, cells were removed with bleach as recommended by the manufacturer, and digital images of the osteologic discs were taken under dark field microscopy. Resorbed areas on the digital images were measured with the Scion Image 1.62 software (Scion Corp.) and expressed as percentage of resorbed well area.

Statistical analysis

Results obtained from animal studies were expressed as mean ± SD. Data compiled from proliferation assays with Omp29-specific T clone cells in vitro, IFN-γ measurements, TRACP staining, and bone resorption assays in vitro were expressed as mean ± SE. Student's t-test was used to evaluate significance using the software package Origin 6.1 (OriginLab, Northampton, MA, USA). Values of p < 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Kaliotoxin inhibits antigen-specific proliferation of activated Omp29-specific T-cell clones in vitro

To determine whether the voltage-gated potassium channel Kv1.3 could potentially play a role in the antigen-specific stimulation of G23 T clone cells(20) in vitro, we first monitored the Kv1.3 mRNA expression pattern in G23 T clone cells stimulated by APC in the presence or absence of specific antigen (Fig. 1A). RT-PCR experiments followed by gel image analyses showed that G23 T-cells stimulated by APC for 24 or 72 h expressed an average of 4-fold higher Kv1.3 mRNA levels in the presence than in the absence of their specific antigen (Fig. 1A). Western blot analysis indicated that G23 cells stimulated in the presence of APC and antigen for 72 h expressed approximately 3-fold higher Kv1.3 protein content than cells cultured in the absence of the antigen (Fig. 1B). Similar results were found when a different Omp29-specific Th1 clone (G21)(23) was stimulated under the same experimental conditions (Fig. 1C). The significant upregulation of Kv1.3 in activated Omp29-specific T-cell clones prompted us to test whether antigen-dependent proliferation of the G23 rat T-cell clone could be inhibited by the Kv1.3 selective blocker kaliotoxin. As shown in Fig. 2, antigen-driven proliferation of G23 T clone cells was decreased in a dose-dependent fashion by kaliotoxin. Treatment with 25 nM kaliotoxin resulted in a decrease of approximately 50% proliferation. At higher doses of kaliotoxin (50–100 nM), the [3H]-thymidine incorporation of activated G23 T-cells was reduced to approximately correspond with that of cells cultured in the absence of the antigen (Fig. 2). Kv1.3 blockade was not toxic to rat G23 T-cells, as evidenced by trypan blue dye exclusion (data not shown). Similar results were found when G21 T-cells were treated with kaliotoxin under the same cell culture conditions as for the G23 T-cell clone (data not shown).

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Figure FIG. 1.. Antigen-specific stimulation of Kv1.3 in activated Omp29-specific T clone cells in vitro. (A) RT-PCR analysis of Kv1.3 mRNA levels in G23 T clone cells. G23 T-cells were cultured in 24-well plates in the absence or presence of APC and in the absence or presence of sterile killed A. actinomycetemcomitans bacteria for 24 or 72 h. Kv1.3 amplification was performed by two-step RT-PCR with 200 pg of RNA per reaction and 35 cycles. The constant amount of mRNA was demonstrated by amplification of GAPDH with 200 pg RNA per reaction and 25 cycles. Normalized Kv1.3 mRNA levels were expressed as the ratio between the intensity of the Kv1.3 and GAPDH bands at the bottom of the figure. (B) Immunoblot analysis of Kv1.3 protein levels in G23 clone T-cells activated for 72 h. SDS-PAGE and Western blot analysis were performed as detailed in the Materials and Methods section. Normalized Kv1.3 protein levels were expressed as the ratio between the intensity of the Kv1.3 and GAPDH protein bands (shown at the bottom of the figure). (C) Immunoblot analysis of Kv1.3 protein levels in G21 clone T-cells activated for 72 h. Normalization of Kv1.3 protein levels was performed as in B.

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Figure FIG. 2.. Kaliotoxin decreased the [3H]thymidine incorporation of activated G23 T-cells in vitro. G23 T-cells were cultured on 96-well plates (5 × 103 cells/well) in the presence of splenic APC (5 × 105 cells/well) with or without killed A. actinomycetemcomitans bacteria (107/well) as antigen. Kaliotoxin (25–100 nM) was added to the G23 cell cultures 15 minutes before the antigen and the APC. [3H]Thymidine (0.5 μCi/well) was applied overnight for the last 16 h of 3 days in culture. The data are mean ± SE cpm values of one representative experiment performed in triplicate. G23 cells grown with APC in the absence of antigen had a [3H]thymidine incorporation of 956 ± 121 cpm.

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Culture supernatants were taken at 48 h from Omp29-specific Th1 clone cells to monitor the effects of kaliotoxin on the production of IFN-γ. ELISA measurements indicated that in the absence of antigen, G21 T-cells produced very low levels of IFN-γ (45 ± 44 ng/ml). When stimulated in the presence of APC and antigen, G21 T-cells produced an average of 492 ± 126 ng/ml of IFN-γ. Doses of kaliotoxin ranging from 25 to 100 nM decreased the production of IFN-γ by an average of 40% in the Omp29-specific T clone cells (p < 0.05, n = 3). Specifically, G21 T-cells activated in the presence of 25 and 100 nM kaliotoxin produced 261 ± 22 and 301 ± 10 ng/ml of IFN-γ, respectively.

Kaliotoxin decreases alveolar bone resorption in experimental periodontal disease

Based on the ability of kaliotoxin to inhibit proliferation of two Omp29-specific T-cell clones, we sought to analyze the immunological influence of blocking Kv1.3 on adoptively transferred G23 clone T-cells in an experimental model for periodontal disease previously described.(17) In this model, local gingival stimulation of G23 clone T-cells by MHC class II+/B7+ APC leads to increased migration of G23 clone T-cells to gingival tissue and cervical lymph nodes. Some G23 T-cells migrate to the spleen and maintain responsiveness to Omp29 plus APC in vitro up to 10 days after adoptive T-cell transfer.(17) Furthermore, both the increase in the serum IgG2a antibody levels to A. actinomycetemcomitans Omp29 and the antigen-specific periodontal bone resorption can be detected 10 days after T-cell transfer.(17)

To block Kv1.3, 10 μg of kaliotoxin was injected subcutaneously twice per day for 4 days (days 0–3), whereas the control group of animals was injected with saline as placebo on the same days. Then, 10 days after adoptive transfer, T-cells were recovered from the spleens of kaliotoxin- and saline-treated rats and tested for their ability to proliferate in the presence of specific antigen and irradiated APC in vitro. Our data showed that the antigen-specific-stimulation index of splenocytes from kaliotoxin-treated animals was 65% lower than that of the splenocytes from the saline-treated group (Fig. 3A). It is noteworthy that treatment with kaliotoxin did not affect the size of the spleen or other organs, including lung, heart, kidney, stomach, or intestine in this rat experimental system (data not shown).

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Figure FIG. 3.. Kaliotoxin decreased the antigen-specific effects of G23 T-cells in experimental periodontal disease. Ten days after adoptive transfer of G23 T-cells and gingival injection of Omp29/LPS, rats in each group that had received kaliotoxin (n = 7) or saline as placebo (n = 7) were killed. Then, spleens, serum samples, and heads were collected for further analysis. (A) Splenic cells were cocultured with irradiated APC with or without Omp29 as an antigen for 3 days. The proliferation of antigen-specific T-cells was measured by [3H]thymidine incorporation (cpm). The antigen-specific-stimulation index was expressed as a ratio of the cpm obtained with cells cultured in the presence and absence of antigen (mean ± SD).aSignificantly different from the saline-treated group (Student's t-test, p < 0.001). (B) Serum samples were used to measure specific IgG2a response to the purified A. actinomycetemcomitans Omp29 antigen by ELISA. ELISA units (EU) were calculated based on rat hyperimmune serum to the Omp29.aSignificantly different from the saline-treated group (Student's t-test, p < 0.001). (C) Rat heads were processed to measure periodontal bone resorption as previously described.(17) Total CEJ-AL measurements of left maxilla − total CEJ-AL measurements of right maxilla were 0.4 ± 0.2 and 0.01 ± 0.1 mm for saline and kaliotoxin-treated groups, respectively. Bone resorption was expressed as percentage bone loss = [(total CEJ-AL measurements of left maxilla − total CEJ-AL measurements of right maxilla)/total CEJ-AL measurements of right maxilla] × 100.aSignificantly reduced compared with the saline-treated group (Student's t-test, p < 0.001).

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To further examine the in vivo effects of kaliotoxin on the immunological influence of G23 T-cells in recipient animals, 10 days after transfer of the Th1-clone cells, the serum IgG2a antibody levels to Omp29 were determined by ELISA. Our results showed that the sera from the kaliotoxin-treated rats exhibited 74% lower levels of IgG2a antibody to Omp29 than the saline-treated group (Fig. 3B). Next we tested whether the inflammatory alveolar bone resorption induced by adoptive transfer of activated G23 Th1 clone cells could be decreased by systemic administration of kaliotoxin compared with the saline-injected control group. Bone resorption measurements demonstrated that kaliotoxin administration decreased alveolar bone resorption by 84% with respect to that induced in the saline-treated group (Fig. 3C).

Kaliotoxin decreases the ratio of RANKL to OPG mRNA levels in activated G23 T-cells in vitro and in vivo

RT-PCR analyses were later performed to determine whether the observed reduction of Th1-mediated bone resorption by kaliotoxin could be attributed to this potassium channel blocker partly decreasing RANKL expression by activated G23 T-cells. To that end, CD3+ cells were isolated from gingival tissue of the experimental sides (injected with purified antigen and LPS) of the saline- or kaliotoxin-treated group of animals on days 2 and 10 after adoptive transfer. Whole gingival tissue isolated from the experimental sides on day 0 (Fig. 4A) or from the control sides on days 0, 2, or 10 (injected with PBS, data not shown) did not express detectable levels of RANKL mRNA. However, CD3+ T-cells isolated from the experimental sides of saline-treated animals expressed significant levels of RANKL mRNA on days 2 and 10 (Fig. 4A). Consistent with the ability of kaliotoxin to inhibit T-cell activation and therefore the antigen-specific upregulation of RANKL expression, CD3+ T-cells isolated from the experimental sides of kaliotoxin-treated animals showed almost undetectable RANKL mRNA levels (Fig. 4A).

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Figure FIG. 4.. Kaliotoxin downregulated the ratio of RANKL to OPG expression levels in activated Omp29-specific T clones cells. (A) Two-step RT-PCR with 150–200 pg of RNA per reaction was used to monitor the expression levels of RANKL and OPG mRNA by CD3+ T-lymphocytes or total gingival tissue of rats with experimental periodontal disease (n = 2–4 rats/time point). The mRNA expression levels of RANKL and OPG were tested in control animals injected with PBS and killed on days 0, 2, and 10 after the adoptive transfer or injected with kaliotoxin and killed on days 0, 2, and 10. GAPDH amplification was performed for normalization of mRNA quantity. The intensity of the amplified bands was quantified by Scion Image and expressed in arbitrary units as the ratio of RANKL/OPG expression normalized with GAPDH expression (data are shown at the bottom of the figure). (B) RT-PCR analyses were performed essentially as described in A, but with G23 T-cells cultured with APC alone, with APC and antigen, or with APC, antigen, and 50 nM of kaliotoxin for 24 or 72 h. Analysis and normalization of the data were performed as in A. (C) G23 T-cells activated in the presence and absence of 50 nM kaliotoxin for 72 h were used to analyze their RANKL and OPG protein levels by 4–12% SDS-PAGE and Western blot analysis. The protein expression of GAPDH was monitored for normalization purposes. Data corresponding to the normalization of RANKL to OPG protein levels were the average of two representative experiments. (D) G21 T-cells activated in the presence and absence of 50 nM kaliotoxin for 72 h were used to analyze their RANKL expression by flow cytometry. Logarithmic fluorescence intensity of OPG-Fc-FITC indicated the expression of surface RANKL expression and that of L6-Fc-FITC (negative control) indicated the background fluorescence.

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Because the decoy receptor OPG has been described to be a crucial negative regulator of RANK-RANKL interactions,(5) we analyzed its expression by RT-PCR and calculated the ratio of RANKL to OPG mRNA expression. Our results indicated that expression of OPG was also downregulated by kaliotoxin treatments, although to a lesser extent than that of RANKL (Fig. 4A). Overall, the ratio of RANKL to OPG mRNA expression was more than 90% lower on the experimental gingival sides of kaliotoxin-treated animals than in control animals on days 2 and 10 after the G23 T-cell transfer (Fig. 4A). Consistent with the results obtained with the isolated gingival CD3+ T-cells from our rat experimental model, G23 T-cells activated in vitro in the presence of 50 nM kaliotoxin also exhibited a clear downregulation of their ratio of RANKL to OPG mRNA expression levels (Fig. 4B). Furthermore the RANKL protein levels were significantly decreased by the kaliotoxin treatments as detected by both Western blot (Fig. 4C) and fluorescence-activated cell sorter (FACS) analyses (Fig. 4D) of G23 and G21 T-cell clones, respectively. OPG protein levels were also decreased by kaliotoxin but to a lesser extent than that of RANKL (Fig. 4C).

Kaliotoxin decreases the ability of G23 T-cells to interact with RANK

The kaliotoxin-mediated downregulation of RANKL expression by the G23 T-cells might be expected to decrease the ability of G23 T-cells to induce osteoclastogenesis. To test this possibility, we established a coculture system with fixed G23 T-cells (which were activated in the presence and absence of kaliotoxin) with the committed osteoclast precursor cell line MOCP-5.(24,25) The influence of T-cell-derived RANKL in inducing osteoclastogenesis and bone resorption in vitro was tested by TRACP staining and by monitoring the formation of bone resorption pits by the MOCP-5 cells. G23 T-cells cultured in the absence of the specific antigen did not induce the formation of multinucleated TRACP+ cells (Fig. 5A) or form resorption areas in osteologic discs (Fig. 5B). However, G23 T-cells activated in the presence of their specific antigen were able to induce the formation of TRACP+ cells with three or more nuclei (Fig. 5A) and bone resorption pits by MOCP-5 cells in vitro (Fig. 5B). These effects occurred in a RANKL-dependent fashion, as demonstrated by their abolition in the presence of 500 ng/ml of human recombinant OPG (Figs. 5A and 5B). However, cocultures of MOCP-5 cells with G23 T-cells that were activated in the presence of kaliotoxin exhibited the formation of significantly fewer multinucleated TRACP+ cells (Fig. 5A) and produced smaller resorption areas (Fig. 5B) compared with G23 T-cells activated in the absence of kaliotoxin.

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Figure FIG. 5.. Kaliotoxin decreased the ability of activated G23 T-cells to induce osteoclastogenesis and bone resorption in vitro. 5 × 105 G23 T-cells were cultured with APC and antigen in the presence and absence of 50 nM kaliotoxin on 24-well plates for 72 h. Then, 5 × 102 MOCP-5 cells/well were cultured on 96-well plates with 105 fixed G23 T-cells/well in α-MEM with 15% FBS for 7 days. Some cocultures were performed in the presence of recombinant human OPG (500 ng/ml) to test for RANKL-mediated osteoclastogenesis. (A) Differentiated MOCP-5 cells were identified by cytochemical staining and quantified as TRACP+ cells with three or more nuclei. Each point in the graph was expressed as mean ± SE of the number of multinucleated TRACP+ cells (n = 6 wells).a,bSignificantly different with respect to control coculture of MOCP-5 and activated G23 T-cells (with APC and antigen) as determined by Student's t-test (ap < 0.001,bp < 0.01). (B) To evaluate the ability of activated G23 T-cells to induce the formation of bone resorption pits after the treatments with kaliotoxin, fixed G23 T-cells were obtained as described above and cocultured with MOCP-5 cells on BD BioCoat Osteologic discs for 7 days. The resorption areas on each disc were quantified by Scion Image 1.62 software and expressed as percentage Resorbed area = Resorbed area per disc/total disc area × 100. Each point in the graph was expressed as mean ± SE (n = 4 osteologic discs).a,bSignificantly different with respect to control coculture of MOCP-5 and activated G23 cells (with APC and antigen) as determined by Student's t-test (ap < 0.001,bp < 0.01).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We found that Kv1.3 expression was upregulated at the mRNA and protein levels in an antigen-dependent fashion in activated Omp29-specific Th1 clone cells. Furthermore, the Kv1.3 blocker kaliotoxin inhibited their antigen-specific proliferation and production of IFN-γ in vitro. Our results are in agreement with previous reports showing that rat memory T-cell clones repeatedly stimulated with their specific antigens exhibited a high Kv1.3 expression, and their proliferation and production of Th1-like cytokines could be inhibited by kaliotoxin or other Kv1.3 potassium channel blockers in in vitro experiments.(15,16)

Our study further demonstrated that kaliotoxin downregulated several T-cell activation parameters in vivo, including antigen-specific proliferation, antibody production to Omp29, and most importantly, reduced Th1-mediated bone resorption in a rat experimental model for periodontal disease. Our findings suggest that kaliotoxin or its analogs may be used to interfere with periodontal disease pathology mediated by Th1 cells. The potential for therapeutic use of kaliotoxin is enhanced because of its apparent safety. In fact, because of the restricted expression of Kv1.3 to cells of the hematopoietic lineage,(12) blockade of Kv1.3 channels by systemic administration of specific potassium channel blockers has been previously reported to lack severe toxicity or side effects in animal trials.(13–16) Furthermore, the use of Kv1.3 blockers in vivo has been proven to lack effects on the function of normal peripheral rat lymphocytes, whose resting membrane potential is not set by Kv1.3.(26,27) In particular, kaliotoxin has been shown to inhibit a delayed-type hypersensitivity response to tuberculin in mini swine(13,14) and improve experimental autoimmune encephalomyelitis in rats, further emphasizing its therapeutic potential.(15,16)

In the experimental model for periodontal disease used herein, systemic administration of OPG-Fc resulted in a severe inhibition of alveolar bone resorption (T Kawai and MA Taubman, unpublished data, 2002). Specifically, intraperitoneal administration of 100 μg of OPG-Fc on days 0, 2, and 4 after the adoptive transfer inhibited alveolar bone resorption by 90% (p < 0.05) with respect to that induced in the control group injected with the unrelated fusion protein L6-Fc (L6-Fc: percentage bone resorption = 24.4 ± 10.7, n = 6; OPG-Fc: percentage bone resorption = 2.4 ± 7.3, n = 5). These results demonstrated that Omp29-specific Th1 clone cells induced bone resorption in a RANKL-dependent fashion. Unlike kaliotoxin, OPG-Fc administration did not affect the antigen-specific stimulation index of rat splenocytes or the serum IgG2a antibody levels to Omp29 (T Kawai and MA Tauban, unpublished data, 2002). T-cell-derived RANKL has also been shown to contribute to alveolar bone resorption and tooth loss in a mouse model of periodontal disease.(10) In that model, the alveolar bone resorption around the teeth could be inhibited by systemic injection of OPG-Fc without affecting periodontal inflammation, implying that even abundant local proinflammatory cytokines do not contribute to the destruction of bone in the absence of a functional RANK-RANKL system.(10) These findings suggested the importance of controlling the local ratio of RANKL to OPG expression as a potential mechanism to treat or prevent T-cell-mediated alveolar bone resorption.

RT-PCR analysis performed with CD3+ T-cells recovered from gingival tissue of saline-treated and kaliotoxin-treated rats indicated that both RANKL and OPG were upregulated in an antigen-dependent fashion and that kaliotoxin decreased the ratio of RANKL to OPG mRNA expression levels. Western blot and/or flow cytometry analyses further demonstrated that kaliotoxin determined a clear downregulation in RANKL and OPG protein levels by Omp29-specific Th1-type clone cells activated in the presence of kaliotoxin in vitro. Most importantly, kaliotoxin decreased the ratio of RANKL to OPG at the protein and mRNA levels. These results were consistent with the decreased ability of gingival T-cells to induce bone resorption in kaliotoxin-treated rats compared with saline-treated rats. The activation-induced expression of RANKL in T-cells has been previously shown to be dependent on the Ca2+/calcineurin signaling pathway and to be blocked by cyclosporin,(18,19) as well as the calcium mobilization inhibitor TMB-8.(19) However, our study is the first showing the ability of a potassium channel blocker to decrease the ratio of RANKL to OPG in T-cells. Furthermore, our coculture experiments with fixed G23 T-cells and MOCP-5 osteoclast precursor cells indirectly confirmed the ability of kaliotoxin to decrease the expression of RANKL by activated G23 T-cells, consequently resulting in diminishing their ability to interact with RANK. Unlike Kv1.3, the phosphatase calcineurin is expressed in most cells, which may limit the potential therapeutic use of immunosuppressant drugs such as cyclosporine A or FK-506 to ameliorate T-cell-mediated bone resorption. In fact, these immunosuppressants have been implicated in the pathogenesis of post-transplant osteoporosis,(28,29) which is consistent with their ability to induce osteoclastic activity and bone resorption in animal studies.(30,31) These effects might be partly mediated through the upregulation of the ratio of RANKL to OPG expression in osteoblasts, as recently described for osteoblast cell cultures treated with cyclosporine A or FK-506 in vitro.(32)

Neither the migration nor the retention of the adoptively transferred G23 T-cells to the gingival tissue seemed to be severely compromised by kaliotoxin. In fact, gingival CD3+ T-cells from saline- or kaliotoxin-treated animals expressed comparable levels of GAPDH mRNA, suggesting that a similar number of CD3+ T-cells migrated to the gingival tissue in either group. Kv1.3 has been suggested to play a role in the adhesion and migration of T-cells in certain mammalian species.(33) Blocking of Kv1.3 with kaliotoxin or other potassium channel blockers has been shown to inhibit β1 integrin-mediated adhesion and migration of human peripheral blood T-cells in vitro.(33) Furthermore, a decreased infiltration of T-cells caused by Kv1.3 blocking was reported in the mini swine in vivo model.(13) However, neither the levels of adhesion molecules nor the extent of migration of T-cells seemed to change because of kaliotoxin treatments in a rat model for experimental autoimmune encephalomyelitis.(15)

The primary mechanism responsible for acidification of the osteoclast-bone interface is vacuolar H+-adenosine triphosphatase (ATPase) coupled with chloride conductance localized to the ruffled membrane.(34,35) Other osteoclastic ion channels, including Kv1.3, might provide pathways to compensate for charge accumulation arising from the electrogenic transport of protons across the ruffled membrane of the actively bone-resorbing osteoclasts.(34) Considering that Kv1.3 might play a relevant role in the rat osteoclasts of our experimental model, our results do not exclude the hypothesis that kaliotoxin treatments in vivo partly block Kv1.3 channels in actively bone-resorbing osteoclasts and therefore contribute to the overall reduction of Th1-mediated bone resorption. This plausible mode of action of kaliotoxin will be the focus of our future studies.

In conclusion, our data support the notion that kaliotoxin acts as a potent inhibitor of T-cell-mediated inflammatory bone resorption in experimental periodontal disease by partly decreasing the ratio of RANKL to OPG expression by activated Th1 clone cells. Therefore, the use of selective potassium channel blockers may constitute a potential intervention therapy to prevent bone loss in some inflammatory bone resorptive disorders such as periodontal disease or rheumatoid arthritis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by The John W. Hein Fellowship from The Forsyth Institute and National Institute of Dental and Craniofacial Research Grants DE-03420 and DE-14551.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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