Etidronate inhibits human osteoblast apoptosis by inhibition of pro-apoptotic factor(s) produced by activated T cells

Etidronate inhibits human osteoblast apoptosis by inhibition of pro-apoptotic factor(s) produced by activated T cells YASUYO ABE, ATSUSHI KAWAKAMI, TOMOKI NAKASHIMA, ERI EJIMA, KAORU FUJIYAMA, TAKESHI KIRIYAMA, AKANE IDE, NOBUKO SERA, TOSHIRO USA, TAN TOMINAGA, KIYOTO ASHIZAWA, NAOKATA YOKOYAMA, and KATSUMI EGUCHI NAGASAKI, JAPAN

Humoral factors produced by activated T cells are thought to be important in the development of bone loss in patients with rheumatoid arthritis (RA). We investigated the inhibitory effect of etidronate disodium (EHDP) on apoptosis of human osteoblasts induced by supernatants from in vitro activated T cell cultures. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells were used in the present study as human osteoblasts. T cells were incubated with interleukin-2 and further activated with 12-o-tetradecanoyl-phorbol 13-acetate and ionomycin, either in the presence or absence of EHDP. After we carried out the cultivation, we examined the cytotoxicity of cultured T cell supernatants toward MG63 cells and human primary osteoblast-like cells. Supernatants from activated but not resting T cell cultures efficiently induced apoptosis of MG63 cells and primary osteoblast-like cells. Supernatants from activated T cell cultures, incubated with EHDP, exhibited significantly less cytotoxicity than did supernatants incubated in the absence of EHDP. In contrast, the cytotoxicity of activated T cell culture supernatants was not affected by direct treatment of human osteoblasts with EHDP. The concentration of soluble Fas ligand in activated T cell culture supernatants was actually increased by EHDP. However, EHDP did not influence soluble Fas and tumor necrosis factor-α concentrations in the supernatant. Furthermore, treatment of human osteoblasts with EHDP did not alter their expression of Bcl-2/Bcl-xL or their sensitivity to anti-Fas immunoglobulin M–induced apoptosis. Our results suggest that EHDP inhibits the production of soluble factor that induces apoptosis of human osteoblasts and thus exhibits a protective action toward human osteoblast apoptosis induced by activated T cell culture supernatants. Although the exact EHDPregulated molecule that induces apoptosis of human osteoblasts is unknown at present, our study may explain part of the therapeutic action of bisphosphonates in RA complicated by bone loss. (J Lab Clin Med 2000;136:344-54) Abbreviations: EHDP = etidronate disodium; ELISA = enzyme-linked immunosorbent assay; FBS = fetal bovine serum; HBSS = Hanks’ balanced salt solution; IgG = immunoglobulin G; IL-2 = interleukin-2; mAb = monoclonal antibody; PBS = phosphate-buffered saline solution; PBS-T = PBS containing 0.1% Tween 20; PMA = 12-o-tetradecanoyl-phorbol 13-acetate; RA = rheumatoid arthritis; sFas = soluble Fas; sFasL = soluble Fas ligand; TNF-α = tumor necrosis factor-α

From the The First Department of Internal Medicine, The First Department of Physiology, and the Department of Hospital Pharmacy, Nagasaki University School of Medicine. Submitted for publication October 18, 1999; revision submitted May 23, 2000; accepted June 30, 2000. Reprint requests: Katsumi Eguchi, MD, Professor and Chairman, The First Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Copyright © 2000 by Mosby, Inc. 0022-2143/2000 $12.00 + 0 5/1/109757 doi:10.1067/mlc.2000.109757 344

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isphosphonates are currently the treatment of choice in disorders characterized by increased bone resorption, including osteoporotic complications in patients with RA.1-5 Their precise mode of action remains to be elucidated. However, the main focus has centered on their effects on osteoclasts, with the demonstration that these drugs suppress bone resorption by inhibiting osteoclast functions.6,7 In vivo and in vitro studies have shown that bisphosphonates inhibit the recruitment and activity of osteoclasts, and

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this is thought to play a role in their therapeutic effect.8-10 Furthermore, recent studies have indicated that one of therapeutic actions of bisphosphonates is thought to be attributed to the induction of apoptosis in osteoclasts.6,7,11,12 Osteoblasts are among the major cellular constituents of bone architecture. They produce bone matrix and enhance new bone formation.13 Mouse models of glucocorticoid-induced bone loss have suggested that bone loss in these mice is partly attributed to an increase in osteoblast apoptosis.14 Furthermore, osteoblast apoptosis has been found in areas of periarticular osteoporosis in patients with RA.15 Thus it is suggested that osteoblast apoptosis is another mechanism that regulates bone metabolism.16 Humoral factors, produced by activated T cells in the peripheral blood and synovial tissues of patients with RA, are thought to be important in the development of bone loss in RA.17-20 This is supported by the fact that findings associated with RA, such as increased C-reactive protein and erythrocyte sedimentation rate correlate well with decreases in appendicular and axial bone mineral density.21,22 In relation to this, we recently reported that sFasL derived from activated T cells induces apoptosis of cultured human osteoblastic cell line MG63 cells and primary osteoblast-like cells.16 Furthermore, cultivation of activated T cells with dexamethasone suppresses both the production of sFasL and the cytotoxicity of cultured T cell supernatants toward MG63 cells and primary osteoblast-like cells.23 Previous reports have indicated that bisphosphonates exert an inhibitory action on cells of the mononuclear lineage.24-26 In the present study we examined whether EHDP, the first bisphosphonate developed for clinical use, inhibits apoptosis induced in human osteoblastic cell line MG63 cells and human primary osteoblast-like cells by activated cultured T cell supernatants. METHODS Cell culture. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells were used as human osteoblasts in the present study. Human primary osteoblastlike cells, obtained from normal bone of subjects who underwent corrective surgery for accidental injury, were used in the present study, as described previously by Takahashi et al27 with some modifications. In brief, bone fragments obtained at surgery were washed extensively in PBS and were minced. The bone fragments were incubated with collagenase (100 µg/mL; Sigma Chemical Co, St Louis, MO) and dispase (3.33 mg/mL; Godo Shuse Chemical Co, Tokyo, Japan) in HBSS at 37°C for 30 minutes. Enzyme-HBSS solution was exchanged three times. The cells isolated in fractions were cultured in RPMI 1640 supplemented with 10% FBS. The alkaline phosphatase activity of these cells was assayed by the method of Lowry et al,28 showing that the alkaline phos-

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phatase activity of the cells was clearly augmented by the addition of 1,25-(OH)2D3 (data not shown). In addition, almost all cells were positively stained when the cells were incubated with 2-amino-2-methyl-1,3-propandiol buffer (Wako Pure Chemical Industries, Osaka, Japan) containing naphthol AS-MX phosphatase and fast blue RR salt (data not shown; Sigma). None of the patients had any known metabolic bone disease or endocrine disorder. A signed consent was obtained from each patient. Peripheral blood T cells were isolated from consenting healthy donors. In brief, mononuclear cells were isolated from heparinized peripheral blood by Ficoll-Conray gradient centrifugation (Daiichi Pharmaceutical Co, Tokyo, Japan). The supernatants were depleted of adherent cells by incubation in Petri dishes (Falcon 3003; Becton Dickinson, Oxnard, CA) for 2 hours. Nonadherent cells were collected and incubated overnight with sheep red blood cells (Nippon Bio-Test Laboratories Inc, Tokyo, Japan) in RPMI 1640 supplemented with 10% FBS. T cells forming rosettes were isolated by Ficoll-Conray gradient centrifugation, and the sheep red blood cells were lysed with 0.8% ammonium chloride. Isolated T cell populations were more than 90% reactive with anti-CD3 mAb. The experiments described in this study were performed with both resting and activated T cells. T cells were activated by culturing for 7 days in RPMI 1640 supplemented with 10% FBS containing 500 IU/mL of recombinant human IL-2 (Takeda Pharmaceutical Co, Osaka, Japan), were washed, and were further activated with PMA (10 ng/mL; Sigma) and ionomycin (500 ng/mL; Sigma) for 24 hours in the presence or absence of various concentrations of EHDP (Sumitomo Pharmaceutical Co, Osaka, Japan). After incubation, cultured supernatants were collected and filtered through a 0.45-µm membrane (Millipore Corp, Bedford, MA) and stored at –20°C until use. Induction of apoptosis of MG63 cells and primary osteoblast-like cells by supernatants obtained from activated T cell cultures. Chromium 51–labeled (Amersham

International) MG63 cells and primary osteoblast-like cells (5 × 103/well) were incubated with cultured supernatants from T cells in 96-well round-bottomed microtiter plates (Falcon 3799; Becton Dickinson) for 8 hours in a total volume of 200 µL. After incubation, the plates were centrifuged, and 100 µL aliquots of the supernatants were assayed for radioactivity with a gamma counter. The spontaneous release of 51Cr was determined by incubating the target cells with medium only, while the maximum release was determined by adding Triton X-100 to a final concentration of 1%. The percentage of specific lysis was determined as follows: Lysis (%) =

Experimental 51Cr release – spontaneous 51Cr release Maximum

51Cr

release – spontaneous

51Cr

× 100

release

Apoptosis of human osteoblasts induced by supernatants from cultured T cells was confirmed by Hoechst 33258 dye staining and detection of the cells with hypodiploid DNA, as previously described.16,23 In brief, MG63 cells and primary osteoblast-like cells, incubated with cultured supernatants from T cells, were fixed with 2% glutaraldehyde solution for 10 minutes and stained with 0.2 mmol/L Hoechst 33258 dye for visual localization of DNA. The cells were examined

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under a fluorescence microscope to determine fragmentation of nuclei or condensation of chromatin (AHB-LB; Olympus, Tokyo, Japan). The cells with hypodiploid DNA were determined by flow cytometric analysis. In brief, the cells were fixed with 70% ethanol and treated with ribonuclease (100 µg/mL; Sigma) and then stained with propidium iodide (100 µg/mL; Sigma) for 30 minutes on ice. After incubation, the stained cells were analyzed with a flow cytometer to detect the presence of hypodiploid DNA+ cells (Epics XL; Beckman Coulter, Hialeah, FL). In some experiments, the inhibitor of caspase-3, Ac-Asp-Glu-Val-Asp-aldehyde (300 µmol/L; DEVD-CHO, Peptide Institute, Osaka, Japan), was added to the culture, and the above assays were also performed. Expression of Fas, Bcl-2, and Bcl-xL in MG63 cells and primary osteoblast-like cells. Fas expression in MG63 cells

and primary osteoblast-like cells was examined as described previously.16,23 In brief, cells were cultured in RPMI 1640 supplemented with 2% FBS in the absence or presence of various concentrations of EHDP. After cultivation, the cells were detached from the plate by adding 0.265 mmol/L EDTA; then the cells were washed twice with PBS containing 1% FBS. Fas expression was detected by an indirect immunofluorescence method with anti-human Fas mAb (IgG1; MBL, Nagoya, Japan) followed by phycoerythrin-conjugated goat anti-mouse IgG (MBL). In brief, cells were incubated with saturating amounts of anti-Fas mAb for 30 minutes at 4°C, washed three times with PBS, and resuspended in phycoerythrin-conjugated anti-mouse IgG. After incubation for 30 minutes at 4°C, the cells were washed, and Fas expression was determined by using a flow cytometer (Epics-Profile II; Coulter Immunology, Hialeah, FL). Bcl-2 and Bcl-xL expression in human osteoblasts was examined by Western blot analysis. For this purpose, MG63 cells and primary osteoblast-like cells were cultured with or without EHDP in RPMI 1640 supplemented with 2% FBS for 24 hours. They then underwent lysis by the addition of lysis buffer (50 mmol/L Tris buffer [pH 8], 150 mmol/L NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 100 µg/mL phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1% NP-40, and 0.5% sodium deoxycholate) for 20 minutes at 4°C. Insoluble material was removed by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was collected and the protein concentration determined with the BioRad protein assay kit (Bio-Rad, Melville, NY). An identical amount of protein from each lysate preparation (20 µg/well) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose filter that was subsequently blocked for 1 hour with 5% dried nonfat milk in PBS-T. The filter was then washed with PBST and incubated at room temperature for 2 hours in a 1:100 dilution of anti-human Bcl-2 mAb (DAKO Japan, Kyoto, Japan) or 1:500 dilution of anti-human Bcl-xL mAb (Trevigen Inc, Gaithersburg, MD). The filter was washed with PBS-T and incubated with a 1:1000 dilution of anti-mouse IgG (Amersham) coupled to horseradish peroxidase. The enhanced chemiluminescence system (Amersham) was used for detection. Induction of apoptosis in MG63 cells and primary osteoblast-like cells by anti-Fas IgM. Cultured MG63 cells

and primary osteoblast-like cells were examined for anti-Fas

IgM-induced apoptosis with a 51Cr release assay. 51Cr-labeled MG63 cells and primary osteoblast-like cells (5 × 103/well) cultured in the presence or absence of EHDP for 24 hours were incubated for an additional 8 hours with anti-Fas IgM (1000 ng/mL; MBL) in a total volume of 200 µL RPMI 1640 supplemented with 2% FBS in 96-well flat-bottomed microtiter plates (Falcon 3072; Becton Dickinson). After incubation, the plates were centrifuged and the percentage of specific 51Cr release was calculated as described before. Detection of sFasL, sFas, and TNF-α in cultured T cell supernatants. Concentrations of sFasL in cultured T cell

supernatants were determined by a sandwich ELISA kit with two anti-human FasL mAbs (MBL). Concentrations of sFas in cultured supernatants from T cells were also determined by a sandwich ELISA kit with two anti-Fas mAbs (MBL). The optical density of each well was measured at 450 nm with a microplate reader (ImmunoReader NJ-2001; InterMed, Tokyo, Japan), and the concentrations of sFasL and sFas were calculated from a dose-response curve derived from reference standards. TNF-α in cultured supernatants was also measured by ELISA (Otsuka Pharmaceutical Co, Tokushima, Japan). In brief, plates precoated with a mAb to TNF-α were incubated with the samples, further incubated with polyclonal rabbit anti-TNF-α antibody, and then reacted with goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase. Absorbance of the chromogenic compound produced by addition of the enzyme substrate was measured at 490 nm. The sensitivity of detection was 20 pg/mL. Effect of EHDP on proliferation of T cells. The proliferative response of resting or activated T cells cultured with or without EHDP was examined by a tritiated thymidine incorporation assay. In brief, isolated resting T cells (1 × 105/well) were plated in 96-well round-bottomed microtiter plates in the presence of variable concentrations of EHDP for 24 hours with 0.5 µCi of tritiated thymidine (New England Nuclear, Boston, MA) and then harvested on a glass filter with a semiautomatic cell harvester (Labo Mash; Labo Science, Tokyo, Japan). The radioactivity of each sample was determined in a liquid scintillation counter (LSC-5100; Aloka, Tokyo, Japan). The effect of EHDP on the proliferation of activated T cells was also examined. In brief, T cells were cultured for 7 days in the presence of 500 IU/mL of recombinant IL-2 as described above, were washed, and were further incubated with PMA (10 ng/mL) and ionomycin (500 ng/mL) for 24 hours in the presence of 0.5 µCi of tritiated thymidine. After cultivation, the radioactivity was determined as described. Statistical analysis. Values were expressed as mean ± SD. Differences between groups were tested for statistical significance by analysis of variance. Values of P < .05 were considered significant. RESULTS Induction of apoptosis of human osteoblasts by activated T cell culture supernatants. We have recently

reported that cultured supernatants from activated T cells are cytotoxic to human osteoblasts and induce apoptosis in these cells.16,23 Therefore we initially examined the effect of EHDP on this cytotoxic activity

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Fig 1. Inhibitory effect of EHDP on the cytotoxicity of activated T cell supernatants toward MG63 cells, as determined by 51Cr release assay. T cells were cultured with IL-2 (500 IU/mL) for 7 days, washed, and further activated by PMA (10 ng/mL) and ionomycin (500 ng/mL) for 24 hours in the absence or presence of various concentrations of EHDP. After incubation, the cytotoxicity of cultured supernatants toward MG63 cells was determined as described in Methods. Minus sign indicates addition of supernatants from resting T cells. Plus sign indicates addition of supernatants from activated T cells. *P < .01 versus activated T cells incubated without EHDP. Values are expressed as mean ± SD of four experiments for each EHDP concentration.

A

B

C

Fig 2. MG63 cell apoptosis induced by activated T cell supernatants as determined by Hoechst 33258 dye staining. MG63 cells were incubated with activated T cell culture supernatant with or without EHDP (as in Fig 1), and apoptotic cells were examined by Hoechst 33258 dye staining as described in Methods. A, MG63 cells incubated with supernatants from resting T cells. B, MG63 cells incubated with supernatants from activated T cells without EHDP. C, MG63 incubated with supernatants from activated T cells with 1 µmol/L EHDP. Note that MG63 cells with nuclear consolidation or fragmentation were observed in B, and this was suppressed proportionally in the presence of EHDP (C). The results shown are representative of six experiments (original magnification ×400).

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A

A

B

B

C

C

Fig 3. A, Determination of MG63 cell apoptosis induced by activated T cell supernatants by the presence of cells with hypodiploid DNA. MG63 cells were incubated with (a) resting T cell culture supernatant, (b) activated T cell culture supernatant, or (c) activated T cell culture supernatant in the presence of 300 µmol/L DEVDCHO. After incubation, the apoptosis of MG63 cells was examined by the presence of cells with hypodiploid DNA. Note that a clear induction of apoptotic cell death in MG63 cells by activated T cell culture supernatant was seen (b); however, it was almost suppressed in the presence of DEVD-CHO (c). Numbers in parentheses represent the percentage of cells with hypodiploid DNA. The results shown are representative of five experiments. B, Inhibition of MG63 cell apoptosis induced by activated T cell supernatants by cultivation of T cells with EHDP, which was determined by the presence of cells with hypodiploid DNA. MG63 cells were cultured with activated T cell supernatants incubated with or without EHDP (1 µmol/L); then the apoptotic cell death of MG63 cells was examined. Note that the induction of MG63 cell apoptosis by activated T cell supernatants was clearly suppressed by the cultivation of activated T cells with EHDP. Values are expressed as mean ± SD of four experiments. *P < .01 versus activated T cells incubated without EHDP.

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A

Fig 4. Inhibition of cytotoxicity in primary osteoblast-like cells induced by activated T cell supernatants by cultivation of T cells with EHDP, which was determined by 51Cr release assay. Primary osteoblast-like cells were cultured with activated T cell supernatants incubated with different concentrations of EHDP; then the cytotoxicity toward primary osteoblast-like cells was examined. Note that the cytotoxicity by activated T cell supernatants was clearly suppressed by the cultivation of activated T cells with EHDP. 51Cr release from primary osteoblast-like cells not treated with EHDP was calculated as 100%. Values are expressed as mean ± SD of four experiments for each EHDP concentration.

by using MG63 cells and primary osteoblast-like cells. For this purpose, T cells were stimulated with IL-2, followed by PMA and ionomycin in the presence or absence of EHDP, and the resulting cytotoxicity to human osteoblasts was examined. As shown in Fig 1, cultured supernatants from activated T cells efficiently killed MG63 cells, but those from resting T cells did not. MG63 cells incubated with activated T cell culture supernatants showed characteristic features of apoptosis such as nuclear consolidation and fragmentation (Fig 2, B), and flow cytometric analysis showed the increase in cells with hypodiploid DNA in MG63 cells incubated with activated T cell culture supernatants (Fig 3). In addition, apoptotic cell death of MG63 cells induced by cultured supernatants from activated T cells was almost completely inhibited by the caspase-3 inhibitor DEVD-CHO (Fig 3). Cytotoxicity of activated T cell culture supernatants toward primary osteoblast-like cells was also determined (Fig 4), and their cytotoxicity toward MG63 cells and primary osteoblast-like cells was inhibited by cultivation of T cells with EHDP (Figs 1, 3, and 4); however, proliferation of activated T cells was not affected by EHDP (Fig 5). These data indicate that soluble factor derived from activated T cells induced apoptosis of human

B

Fig 5. Proliferation of T cells was not affected by EHDP. Resting or activated T cells were incubated with variable concentrations of EHDP for 24 hours, and the proliferative response of the cells was determined by tritiated thymidine incorporation assay as described in the text. A, Resting T cells. B, Activated T cells. Note that the proliferative response of resting and activated T cells was not affected by EHDP. Values are expressed as mean ± SD of four experiments for each EHDP concentration. NS, No significant difference.

osteoblasts in a caspase-3–dependent fashion, and the production of caspase-3 was significantly suppressed after incubation with EHDP (Figs 1 through 4). EHDP did not act on human osteoblasts to suppress apoptosis of the cells induced by supernatants from activated T cells. In contrast with the results shown in Figs

1 through 4, direct EHDP treatment with MG63 cells and primary osteoblast-like cells themselves did not affect the cytotoxicity of activated T cell supernatants (Figs 6 and 7). We previously reported that MG63 cells

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Fig 6. EHDP treatment of MG63 cells did not reduce the cytotoxicity of activated T cell culture supernatants as determined by 51Cr release assay. MG63 cells were cultured with various concentrations of EHDP for 24 hours. After cultivation, the cells were labeled with 51Cr and further incubated with activated T cell supernatants. The cytotoxicity of activated T cell supernatants toward MG63 cells was determined as described in Methods. Minus sign indicates addition of supernatants of resting T cells; plus sign indicates addition of supernatants from activated T cells. Values are expressed as mean ± SD of four experiments for each EHDP concentration. NS, No significant difference.

A

B

C

D

Fig 7. EHDP treatment of MG63 cells did not alter apoptosis induced by activated T cell culture supernatants, as determined by Hoechst 33258 dye staining. MG63 cells were cultured with or without EHDP (1 µmol/L) for 24 hours. After incubation, MG63 cells were further incubated with activated T cell supernatants, and apoptotic cells were examined by Hoechst 33258 staining as described in Methods. A, MG63 cells cultured in the absence of EHDP (medium only). B, MG63 cells cultured in the absence of EHDP, followed by supernatants from resting T cells. C, MG63 cells cultured in the absence of EHDP, followed by supernatants from activated T cells. D, MG63 cells cultured in the presence of EHDP (1 µmol/L), followed by supernatants from activated T cells. Note that there was no significant difference between C and D in the relative proportion of apoptotic MG63 cells. The results are representative of six experiments (original magnification ×400).

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a

b

A

B

Fig 8. EHDP did not affect either Fas expression or anti-Fas IgM-induced apoptosis in MG63 cells. A, MG63 cells were cultured with or without EHDP (1 µmol/L) for 24 hours. After incubation, Fas expression was determined by using a flow cytometer as described in Methods. a, MG63 cells incubated without EHDP. b, MG63 incubated with 1 µmol/L EHDP. Light shading, Negative control. Deep shading, Anti-Fas staining. The percentages of positive cells are given in parentheses. Note that Fas expression in MG63 cells was not altered by EHDP treatment. Results are representative of four experiments. B, MG63 cells were cultured with or without EHDP (1 µmol/L) for 24 hours. After cultivation the cells were labeled with 51Cr and incubated further with anti-Fas IgM. The cytotoxicity was determined by using the 51Cr release assay as described in Methods. The 51Cr release from MG63 cells not treated with EHDP was calculated as 100%. Note that anti-Fas IgM-induced cytotoxicity toward MG63 cells was not reduced by direct EHDP treatment of MG63 cells. Values are expressed as mean ± SD of four experiments. NS, No significant difference.

and primary osteoblast-like cells express high levels of Fas and are efficiently committed to anti-Fas IgMinduced apoptosis.16 We therefore examined the effect of EHDP on Fas expression and anti-Fas IgM-induced apoptosis in human osteoblasts. As shown in Fig 8, A, Fas was highly expressed in MG63 cells. However, neither Fas expression nor anti-Fas IgM-induced apoptosis of MG63 cells were changed by treatment of these cells with EHDP (Fig 8, A and B). Similar results regarding Fas-mediated cytotoxicity were obtained by the use of primary osteoblast-like cells (primary osteoblast-like cells without treatment of EHDP, 15.8% ± 1.2%; primary osteoblast-like cells with treatment of 1 µmol/L EHDP, 16.4% ± 1.1%; determined by 51Cr release assay from four experiments). Both Bcl-2 and Bcl-xL are proto-oncogene products that inhibit several apoptotic processes including Fas-mediated apoptosis.29,30 We thus investigated the effect of EHDP on the expression of Bcl-2 and Bcl-xL in human osteoblasts. As shown in Fig 9, Bcl-2 expression in MG63 cells was not changed by treatment of these cells with EHDP. Bcl-xL was not expressed in MG63 cells and was not

induced by EHDP treatment (data not shown). Similar results were obtained by the use of primary osteoblastlike cells (data not shown). Detection of sFasL, sFas, and TNF-α in activated T cell culture supernatants. We previously reported that cul-

tured supernatants from activated T cells contain sFasL and induce apoptosis of human osteoblasts.16 Therefore we examined whether the presence of EHDP in activated T cell culture supernatants affects the production of sFasL and sFas. Although sFasL was not detected in the supernatants from resting T cells (data not shown), it was found in supernatants from activated T cells. However, its concentration was actually increased by EHDP (Fig 10, A). sFas was also detected in cultured supernatants from activated T cells, but its concentration was unaltered by EHDP (Fig 10, B). TNF-α is known to induce bone loss, and we thus examined concentrations of this cytokine in cultured supernatants from T cells. TNF-α was not detected in cultured supernatants from resting T cells (data not shown). However, TNF-α was clearly detected in activated T cell supernatants (Table I), but its concentration was not influenced by EHDP.

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Fig 9. EHDP did not change Bcl-2 expression in MG63 cells. MG63 cells were cultured with or without various concentrations of EHDP for 24 hours. After incubation, Bcl-2 expression in MG63 cells was determined by Western blot analysis. Note that Bcl-2 expression in these cells was not changed by EHDP. The results are representative of six experiments.

B

A

Fig 10. Treatment of activated T cells with EHDP increased sFasL production but did not change sFas production in activated T cell culture supernatants. T cells were cultured with IL-2 (500 IU/mL) for 7 days, washed, and further activated for 24 hours by incubation with PMA (10 ng/mL) and ionomycin (500 ng/mL) in the presence or absence of various concentrations of EHDP. After incubation, sFasL (A) and sFas (B) concentrations in the supernatants were measured by ELISA, as described in Methods. sFasL concentrations were increased in the supernatants by the addition of EHDP. However, sFas concentrations were not changed by EHDP. *P < .01 versus activated T cells incubated in the absence of EHDP. Values are expressed as mean ± SD of four experiments. NS, No significant difference.

DISCUSSION

Generalized and periarticular osteoporosis is recognized as a common complication of RA.31 A reduction in both appendicular and axial skeletal bone mass has been found by several investigators.32,33 However, recent reports have shown an increase in the bone mass of patients with RA who were treated with bisphosphonates.5,34 Humoral factors, including cytokines and prostaglandins, derived from activated T cells are major contributors to the induction of bone loss in patients with RA.17-20,35 Previous in vivo and in vitro studies with mononuclear lineage cells have speculated on the anti-inflammatory properties of bisphosphonates.24-26

We recently reported that cultured supernatants from in vitro activated T cells induce apoptosis in cells of the human osteoblastic cell line MG63 as well as in primary osteoblast-like cells. Major therapeutic action of bisphosphonates is suggested to be mediated through inhibition of osteoclast function; however, the protective function of bisphosphonates toward osteoblasts may also be a mechanism that suppresses bone loss.36 Therefore in the present study we examined whether EHDP modulates apoptosis triggered in human osteoblasts by activated T cell culture supernatants. We demonstrated that EHDP does indeed have an inhibitory effect on apoptosis of MG63 cells and pri-

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mary osteoblast-like cells. However, direct treatment of these cells with EHDP did not affect apoptosis induced by activated T cell supernatants. Its inhibitory effect was evident only when EHDP was added to activated T cell culture. We showed that T cell proliferation was not changed by EHDP. In addition, the apoptosis of T cells (determined by the presence of cells with hypodiploid DNA), either resting or activated T cells, was not affected by EHDP (data not shown). These data indicate that EHDP inhibits the production of soluble factor from activated T cells to induce apoptotic cell death of human osteoblasts. We recently reported that the triggering of Fas in MG63 cells and primary osteoblast-like cells by anti-Fas IgM or sFasL, derived from activated T cells, induces apoptosis of the cells.16 Therefore we also examined the effect of EHDP on this process. EHDP did not affect anti-Fas IgMinduced apoptosis in human osteoblasts, because EHDP treatment of MG63 cells and primary osteoblast-like cells directly did not have an influence on the apoptotic process. The concentration of sFasL in activated T cell supernatants was reduced by incubating the cells with dexamethasone, and this correlates well with its decreased cytotoxicity to cultured human osteoblasts.23 However, the concentration of sFasL in the cultured supernatants was actually increased by incubating activated T cells with EHDP. The concentration of sFas, which is considered a competitive inhibitor of sFasL,37 was not changed by EHDP. These findings suggest that humoral factors other than the sFas/sFasL system appear to be responsible for mediating the inhibitory effect of EHDP. TNF-α has been shown to induce bone loss.38,39 TNF receptor is preferentially expressed on osteoblasts, and increases in osteoblast apoptosis that include a Fasmediated one are found by treating these cells with TNF-α.40-42 A high concentration of TNF-α was detected in activated T cell culture supernatants but was not affected by EHDP. Furthermore, the increased Fasmediated apoptosis in human osteoblasts by treatment of the cells with TNF-α42 was not reduced by adding EHDP to the MG63 cell or primary osteoblast-like cell culture (data not shown). This suggests that EHDP affects neither the production of TNF-α nor the action of TNF-α to suppress an apoptosis of human osteoblasts induced by activated T cell supernatants. In conclusion, we have demonstrated in the present study the protective effect of EHDP against the apoptosis of MG63 cells and primary osteoblast-like cells induced by activated T cell supernatants. However, the molecules responsible for this effect remain to be determined. The cytotoxicity of supernatants from activated T cell cultures toward human osteoblasts was not

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altered by treating the cells with EHDP. In addition, the expression of the anti-apoptotic molecules Bcl-2 and Bcl-xL did not change in MG63 cells and primary osteoblast-like cells subjected to EHDP treatment. Therefore our results indicate that other soluble apoptotic or anti-apoptotic molecules are involved. These could include the soluble form of TNF-related apoptosisinducing ligand or CD40 ligand produced by activated T cells, the production of which might be affected by EHDP. A recent report has found that treatment of a murine osteocytic cell line with bisphosphonates inhibits the apoptosis of the cells induced by several apoptotic stimuli through activation of extracellular signal–regulated kinase, which have contributed in part to the efficacy of bisphosphonates for bone loss.36 We showed in the present study that EHDP appears to inhibit the production of soluble factor from activated T cells that induce apoptosis in human osteoblasts; however, EHDP treatment with osteoblasts did not suppress an apoptotic process of the cells induced by activated T cell culture supernatants, suggesting that activation of extracellular signal–regulated kinase may not be induced by EHDP in human osteoblasts, or that extracellular signal–regulated kinase in human osteoblasts activated by EHDP may not be involved in activated T cell supernatant-induced apoptosis in human osteoblasts. Although further studies are necessary to elucidate molecular mechanisms of EHDP that produce anti-apoptotic effects toward human osteoblasts, our study may support the therapeutic efficacy of EHDP in the treatment of bone loss in patients with RA. REFERENCES

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