Pro-inflammatory effects of the aminobisphosphonate ibandronate in vitro and in vivo

P. J. Richards, N. Amos, A. S. Williams and B. D. Williams

Rheumatology Research Laboratory, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK

Correspondence to: P. J. Richards, Rheumatology Research Laboratory, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To investigate the effects of the aminobisphosphonate, ibandronate, on the course of joint inflammation in rat antigen-induced arthritis (AIA) and the release of pro-inflammatory cytokines in partially purified human peripheral blood mononuclear cells (PBMC).

Methods. Rats with AIA received a single intra-articular injection of ibandronate (1 mg) 7 days post-arthritis induction and knee swelling was measured for 7 days thereafter. The effects of ibandronate (300 µg/ml) on PBMC cytokine production and activation marker expression were determined using polymerase chain reaction (PCR)/ELISA and FACS analysis, respectively.

Results. Joint swelling, associated with AIA, was sustained in ibandronate-treated rats compared with saline-treated control rats. Ibandronate stimulated the production of interferon gamma (IFN-{gamma}) in adherent PBMC, and increased the surface expression of Fc{gamma}RI and HLA DP, DQ, DR on the adherent monocyte population. Activation by lipopolysaccharide (LPS) of PBMC previously incubated with ibandronate led to enhanced levels of tumour necrosis factor alpha (TNF-{alpha}) secretion, and this could be partially inhibited by neutralizing antibodies to IFN-{gamma}.

Conclusions. The enhanced production of TNF-{alpha} by ibandronate-treated PBMC in vitro involves stimulation of adherent monocytes by IFN-{gamma} prior to LPS-induced activation. Similar cellular interactions may be involved in the pro-inflammatory effects of ibandronate in vivo.

KEY WORDS: Ibandronate, Antigen-induced arthritis, Tumour necrosis factor {alpha}, Interferon {gamma}, Mononuclear cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bisphosphonates are a diverse group of stable pyrophosphate analogues, which share a common phosphate–carbon–phosphate backbone structure [1]. They are extensively used in the treatment of osteoporosis, Paget's disease and hypercalcaemia of malignancy [2]. Phagocytosis of liposomally encapsulated clodronate facilitates entry into cells, where it initiates apoptosis [37] and inhibits the production of pro-inflammatory monokines interleukin (IL)-1ß, IL-6 and tumour necrosis factor alpha (TNF-{alpha}) [8, 9]. The depletion of macrophages by liposomal clodronate has been utilized in the treatment of experimental models of chronic inflammatory arthritis, where the elimination of macrophages from the synovium or the reticuloendothelial system (RES) resulted in reduced inflammation and joint destruction [1014].

In rheumatoid arthritis (RA), continued recruitment and activation of monocytes/macrophages can result in tissue destruction and related pathology [15]. In the inflamed synovium of patients with RA, activated macrophages are found in abundance [16] and are present in strategic sites related to the distribution of destructive pannus [17]. Their secretory products (monokines) dominate the cytokine profile of synovial tissue and fluid [1823]. The importance of monokines in the pathogenesis of both experimental models of arthritis and RA has been demonstrated using neutralizing monoclonal antibodies. The pre-treatment of murine antigen-induced arthritis (AIA) with anti-IL-1{alpha}/ß polyclonal antibodies totally prevented suppression of cartilage proteoglycan synthesis [24]. In type II collagen-induced arthritis (CIA), a single i.p. injection of either anti-TNF-{alpha} or anti-IL-1{alpha}/ß suppressed both inflammation and cartilage damage in arthritic joints [25]. The administration of anti-TNF-{alpha} antibodies to RA patients caused a decrease in markers of disease activity, including swollen joint counts and C-reactive protein (CRP) levels [2628].

The aminobisphosphonate, ibandronate [1-hydroxy-3-(methylpentyl-amino) propylidene-bisphosphonate], is 500 times more potent than clodronate at inhibiting bone resorption in the rat [29] and may prove to be an effective agent for the treatment of chronic inflammatory joint disease. However, ibandronate induces TNF-{alpha} production in human whole blood in vitro [30] and its infusion in patients with cancer-associated hypercalcaemia often leads to fever [31].

In the present study, we evaluate the use of ibandronate as a potential treatment for chronic inflammation by examining its effect on joint swelling in an experimental model of arthritis and on cytokine production in vitro using partially purified human PBMC.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Ibandronate was donated by Proctor and Gamble Pharmaceuticals (Ohio, USA) and contained <0.005 EU/ml of endotoxin as determined by the kinetic chromographic Limulus assay (KQCL) from Biowhittaker. RPMI, Hank's Balanced Salt Solution (HBSS), fetal calf serum (FCS), L-glutamine, penicillin and streptomycin were obtained from Life Technologies, and contained <1 EU/ml of endotoxin. MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue), propidium iodide (PI) and lipopolysaccharide (LPS; Escherichia coli ) were obtained from Sigma.

Animals
Male inbred Lewis rats (150 g) were obtained from Bantin and Kingman (The Field Station, Grimston, Hull, UK). The animals were housed in cages of five, allowed food and water ad libitum, and kept in the Biomedical Services Department for 1 week prior to their first immunization. The animals were housed in light/dark cycles of 12 h.

Induction and treatment of arthritis
Arthritis was induced in the right knee joints of immunized rats using 100 µl of methylated bovine serum albumin (mBSA) (1 mg) (Sigma) as described previously [32]. The development of arthritis was monitored at regular intervals by measuring knee diameters (mean of three readings), with the joint flexed at an angle of 90°, using a digital micrometer. Rats with established arthritis were treated with a single intra-articular injection of either sterile saline (0.9% w/v) or ibandronate (1 mg) 7 days after arthritis induction. Animals were assigned to either control or treatment groups so that they were matched for knee swelling on day 7. To assess the effect of the respective treatments, joint swelling was expressed as the difference in diameters between right and left knees.

Preparation of adherent peripheral blood mononuclear cells (PBMC)
Human mononuclear cells were isolated from peripheral blood of normal volunteers using Ficoll-Hypaque (Sigma) as previously described [33]. Adherent cells were incubated in RPMI 1640 supplemented with 10% heat inactivated FCS, L-glutamine (0.3 mg/ml), penicillin (50 IU/ml) and streptomycin (50 µg/ml), and consisted of 73% monocytes, 15% T cells and 12% B cells as determined by FACS analysis using monoclonal antibodies against CD14, CD3 and CD19, respectively.

Cell viability
The cell viability after each treatment was assessed using the MTT assay as previously described [34]. The percentage of viable cells remaining after ibandronate treatment was compared to untreated cells (control).

Effect of ibandronate on monocyte cell surface marker expression
Following incubation with ibandronate (300 µg/ml) for 24 h, cells were washed in HBSS and adjusted to 1x107 cells/ml in phosphate-buffered saline (PBS) containing 1% BSA, 0.1% NaN3 . To 100 µl of cell suspension were added 10 µl of normal rabbit serum (Sigma), followed by incubation for 15 min on ice. After this time, fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 (5 µg/ml) (Sigma), CD16 (10 µg/ml) (Dako), CD64 (20 µg/ml) (Serotec) or HLA-DP, DQ, DR (5 µg/ml) (Dako) were added and left on ice for 30 min in the dark. The cells were then washed and PI added at 1 µg/ml in PBS, followed by analysis on a FACS (Becton Dickinson), and the data converted into frequency histograms using the LysysII system. FACS analysis was restricted to a region previously shown to contain CD14+ monocytes only. All experiments were performed in triplicate.

Quantification of secreted cytokines
TNF-{alpha} was measured using a standard sandwich ELISA as previously described [33]. Monoclonal mouse anti-human TNF-{alpha} (Biogenesis) was used as the capture antibody and polyclonal rabbit anti-human TNF-{alpha} (Genzyme) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) were used as the detecting antibodies (all at a 1:500 final dilution factor). Recombinant TNF-{alpha} was used as a standard (donated by Dr N. Toppley, Cardiff Royal Infirmary, UK). Interferon gamma (IFN-{gamma}) was measured using the Human IFN{gamma} DuosetTM ELISA development system from Genzyme.

Effect of ibandronate on cytokine secretion
Cells were incubated with ibandronate (300 µg/ml) for a maximum of 24 h, followed by a further 24 h incubation with LPS (10 µg/ml). Supernatants were centrifuged and stored at -70°C prior to quantification of TNF-{alpha} and IFN-{gamma}. For the measurement of IFN-{gamma} and TNF-{alpha} specific mRNA by reverse transcription-polymerase chain reaction (RT-PCR), cells were lysed in RNAzol prior to the extraction of mRNA as described below.

RNA isolation and reverse transcription
Cells were lysed in RNAzol for 1 h on ice and RNA extracted according to the manufacturer's instructions (Biogenesis). cDNA was synthesized from pd(N)6-primed RNA RT with M-MLV superscript reverse transcriptase. The total RNA mixture was incubated with 400 U M-MLV reverse transcriptase, 0.1 M dithiothreitol, 6 µl RT buffer (all from Gibco BRL), 0.02 U pd(N)6, 5 mM dNTPs (Pharmacia Biotech), 60 U Rnasin (Promega), 100 µg/ml whole RNA and 6 µl of H2O in a total volume of 30.5 µl for 2 h at 37°C.

Inhibition of ibandronate LPS-induced TNF-{alpha} production
Adherent human PBMC were incubated with ibandronate (300 µg/ml) and 20 µg/ml of either polyclonal goat IgG, monoclonal goat anti-human IFN-{gamma} or polyclonal goat anti-human IFN-{gamma} (all from R&D Systems). Control cultures consisted of isolated human PBMC incubated with each antibody in medium without ibandronate. After 24 h of incubation, LPS (10 µg/ml) was added and the cells incubated for a further 24 h. After this time, the supernatants were centrifuged and stored at -70°C prior to TNF-{alpha} quantification.

Polymerase chain reaction
Reaction mixtures for PCR consisted of 5 µl of sample cDNA, 1 pmol of each primer and pre-mixed Ready-To-GoTM PCR beads (Pharmacia Biotech). PCR was performed on a thermal cycler (Sanyo electric Co. Ltd, Japan). Human mRNA specific for the housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH), was measured and used as an internal control. Reaction times were optimized for GAPDH, TNF-{alpha} and IFN-{gamma}, and were 94°C for an initial 4 min, followed by five cycles of 94°C for 30 s, 60°C for 45 s, 72°C for 1 min and either 25 cycles for IFN-{gamma}/GAPDH or 15 cycles for TNF-{alpha}/GAPDH of 94°C for 10 s, 60°C for 30 s, 72°C for 45 s. The reaction was completed with a 15 min extension at 72°C followed by 30°C for 1 min.

The primers for GAPDH (EMBL accession no. X02231) [35] and TNF-{alpha} (EMBL accession no. D00475) [36] were prepared on site (University of Wales College of Medicine, Cardiff), and those for IFN-{gamma} (EMBL access no. X01992) [37] were obtained from Life Technologies. Primer sequences: GAPDH (552 bp) 5'-GAA CGG GAA GCT TGT CAT CA-3' for the upstream primer and 5'-TGA CCT TGC CCA CAG CCT TG-3' for the downstream primer; IFN-{gamma} (356 bp) 5'-TCG TTT TGG GTT CTC TTG GC-3' for the upstream primer and 5'-TGC TTT GCG TTG GAC ATT CA-3' for the downstream primer; TNF-{alpha} (289 bp) 5'-GCC AAY GCC CTC CTG GCC AAT G-3' for the upstream primer and 5'-CCC TTC TCC AGC TGG AAG AC-3' for the downstream primer. Ethidium bromide (1%)-stained agarose gels (3%) were photographed using Gel 1000 station (Biorad) and the amount of cDNA quantified by densitometry (Molecular Analyst, Biorad). The optical density value (proportional to the amount of cDNA) for each RT-PCR product was then divided by the optical density value of the GAPDH cDNA product from the same sample run in parallel. Results were expressed as abundance of cDNA relative to abundance of control GAPDH cDNA.

Statistical analysis
The statistical significance between groups was evaluated using two-tailed Student's t-test, where a P value of <0.05 was considered statistically significant. Values are expressed as the mean±S.E.M.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of a single intra-articular injection of either ibandronate (1 mg) or saline (control) (0.9% w/v) on joint swelling was investigated in rats 7 days after arthritis induction (Fig. 1Go). By day 7, the majority of cells within the arthritic joint are monocyte/macrophages, and it was therefore considered to be the most appropriate time to administer bisphosphonate. On day 7, there was no significant difference in knee swelling between the treatment groups, where baseline knee swelling mean±S.E.M. in saline- and ibandronate-treated rats was 3.73±0.15 and 3.4±0.28 mm, respectively. Knee swelling in ibandronate-treated rats remained high for 4 days after treatment and during the course of the experiment was significantly greater than that in saline-treated control rats.



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FIG. 1.  Effect of a single intra-articular injection of saline (control) (0.9% w/v) or ibandronate (1 mg) on knee joint swelling in rat AIA. *P<0.01, **P<0.001 as compared to control rats (number of rats per treatment group=5).

 
The effects of ibandronate on pro-inflammatory cytokine release was investigated in vitro using adherent human PBMC. The viability of cells incubated with ibandronate (300 µg/ml) was >85% for each experiment. This concentration of ibandronate was equivalent to those used for other bisphosphonates in similar studies [8, 9] and was the maximum concentration tolerated by adherent human monocytes.

The effect of ibandronate (300 µg/ml) on the spontaneous release of TNF-{alpha} and IFN-{gamma} by adherent PBMC was investigated. Significant levels of secreted IFN-{gamma} were detected at 16 h (P<0.05) and 24 h (P<0.01) following incubation with ibandronate, and this was associated with increased expression of IFN-{gamma}-specific mRNA (Fig. 2Go). No significant increases in the level of TNF-{alpha}-specific mRNA or secreted TNF-{alpha} protein were observed in ibandronate-treated cells.



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FIG. 2.  Time-dependent production of IFN-{gamma} by adherent PBMC incubated with ibandronate (300 µg/ml). PCR-assisted amplification of IFN-{gamma}-specific mRNA was identified using semi-quantitative densitometric analysis, and was expressed as a ratio of the internal control GAPDH. The secretion of IFN-{gamma} protein was identified by ELISA. *P<0.05, **P<0.01 as compared to cells incubated in the absence of ibandronate. IFN-{gamma} levels in the absence of ibandronate were below detectable limits.

 
The adherent monocyte population incubated with ibandronate for 24 h showed increased expression of the activation markers Fc{gamma}RI (CD64) and HLA DP, DQ, DR (MHC Class II) (Fig. 3Go). The expression of CD14 was slightly reduced and Fc{gamma}RIII (CD16) remained unaffected.



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FIG. 3.  FACS analysis of adherent human monocyte cell surface antigens. Cells were incubated for 24 h in medium (control) (—) or medium supplemented with ibandronate (300 µg/ml) (- - - -) and labelled with FITC-conjugated clones UHCM-1 (CD14), DJ130c (CD16), CR3/43 (HLA-DP, DQ, DR) or 10.1 (CD64). The histograms are representative of three separate experiments.

 
LPS stimulation of PBMC previously incubated with ibandronate resulted in enhanced production of TNF-{alpha} (Fig. 4Go). TNF-{alpha} secretion was significantly greater than controls by 2 h, increased up to 8 h and was associated with a sustained expression of TNF-{alpha}-specific mRNA.



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FIG. 4.  Time-dependent production of TNF-{alpha} by LPS-stimulated adherent human PBMC. Cells were incubated for 24 h in medium (control) or medium supplemented with ibandronate (300 µg/ml). LPS (10 µg/ml) was added and TNF-{alpha}-specific mRNA and secreted protein measured over a 24 h period. PCR-assisted amplification of TNF-{alpha}-specific mRNA was identified using semi-quantitative densitometric analysis, and was expressed as a ratio of the internal control GAPDH. The secretion of TNF-{alpha} protein was identified by ELISA. *P<0.05, **P<0.0001 as compared to control.

 
The enhanced production of TNF-{alpha} by ibandronate-treated LPS-stimulated cells was significantly inhibited by monoclonal (P<0.05) and polyclonal (P<0.01) anti-IFN-{gamma} antibodies when compared with an irrelevant IgG polyclonal antibody (Fig. 5AGo). The production of TNF-{alpha} by LPS-stimulated cells in the absence of ibandronate was not significantly affected by any of the antibodies tested (Fig. 5BGo).



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FIG. 5.  Effects of IFN-{gamma} neutralizing antibodies on TNF-{alpha} production by adherent human PBMC. Cells were incubated for 24 h in (A) medium supplemented with ibandronate (300 µg/ml) or (B) medium alone (control) and 20 µg/ml of either polyclonal goat IgG (IgG), monoclonal goat anti-human IFN-{gamma} (mAb) or polyclonal goat anti-human IFN-{gamma} (pAb) for 24 h. Following a further 24 h incubation in the presence of LPS (10 µg/ml), supernatants were removed and tested for the presence of TNF-{alpha} by ELISA. *P<0.05, **P<0.01 as compared to the IgG. TNF-{alpha} production was calculated as a percentage of TNF-{alpha} produced by LPS-stimulated cells incubated with either (A) ibandronate or (B) medium alone in the absence of antibody.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is increasing interest in the possible use of bisphosphonates in the treatment of human RA due to their effectiveness in reducing the severity of the inflammation and degree of synovitis in experimental models of arthritis.

Early studies with free etidronate, clodronate and pamidronate demonstrated a marked inhibition of pannus formation, inflammatory erosion of cartilage and the pathological bone formation associated with rat adjuvant arthritis (AA) [3840]. The anti-inflammatory effects of free clodronate have also been reported in murine AIA [41] and in rat CIA [42]. Upon liposomal encapsulation, the anti-inflammatory effects of clodronate are greatly enhanced [1014].

The anti-inflammatory effects of free bisphosphonates have also been reported in RA patients. Patients treated with pamidronate displayed a reduction in joint swelling, and reported a decrease in morning stiffness and pain and an increased range of joint movement [43, 44]. An anti-inflammatory effect was also observed in patients administered free clodronate, as demonstrated by a significant improvement in the acute-phase response (APR) [45].

In the present study, we report the effects of the aminobisphosphonate, ibandronate, on joint inflammation in rat AIA. Ibandronate has been shown to be a potent inhibitor of osteoclast-induced bone resorption in the rat, being 50 times more potent than pamidronate and 500 times more potent than clodronate [29]. Ibandronate was therefore considered to be a potentially more effective bisphosphonate in the treatment of chronic inflammation.

However, when ibandronate (1 mg) was administered to established arthritis of 7 days duration, it induced a significant pro-inflammatory effect and, furthermore, sustained knee swelling when compared with saline-treated arthritic controls. We have also demonstrated this pro-inflammatory effect of ibandronate (1 mg) in normal rats, where a significant knee swelling was induced within 24 h (data not shown). This knee swelling was of short duration, with no significant knee swelling recorded 72 h post-ibandronate injection.

The aminobisphosphonate, pamidronate, has been shown to induce an APR when administered i.v. to patients with malignancy, and this was associated with increased plasma levels of IL-6 and TNF-{alpha} [30]. Both pamidronate and ibandronate induced TNF-{alpha} production in human whole blood in vitro. We therefore carried out in vitro studies to establish whether ibandronate could induce TNF-{alpha} production and thus offer an explanation for the effects observed in vivo.

Owing to the limited availability of rat immunological reagents at the time these studies were conducted, human PBMC were used. Ibandronate enhanced TNF-{alpha} secretion in LPS-stimulated adherent human PBMC and this secretion was associated with an increase in the duration of TNF-{alpha}-specific mRNA expression. In the absence of LPS, ibandronate was unable to induce TNF-{alpha} production. However, it did induce the production of small quantities of IFN-{gamma}. The induction of IFN-{gamma} mRNA expression demonstrates that ibandronate actively stimulates IFN-{gamma} production rather than release from intracellular stores. The induction of IFN-{gamma} was associated with the increased expression of activation markers, HLA-DP, DQ, DR and CD64. Enhanced TNF-{alpha} production at the level of both mRNA and protein regulation, and increased expression of activation markers, are hallmarks of IFN-{gamma} [4651]. Indeed, our study demonstrates that the enhanced production of TNF-{alpha} in ibandronate-treated PBMC is partly dependent on the presence of IFN-{gamma} prior to the addition of LPS.

The source of the IFN-{gamma} is not known, although the secretion of IFN-{gamma} in human blood is believed to be limited to two normal cell types: T cells and the large granular lymphocytes (LGL) [5256]. CD3+ T cells (15%) were identified as a contaminant in the adherent PBMC population used in our in vitro experiments. To date, there has been no report of a direct effect of ibandronate on T cell or LGL function. A recent finding where ibandronate failed to alter TNF-{alpha} production in a pure population of LPS-stimulated RAW 264 macrophages [57] supports the necessary involvement of other cell mediators.

An alternative explanation for the pro-inflammatory effects of ibandronate in vivo may involve induction of the histamine-forming enzyme, histidine decarboxylase (HDC). Nakamura et al. [58] demonstrated that two aminobisphosphonates, cycloheptyl-aminomethylene bisphosphonate (CHAMP) and alendronate, exacerbated the arthritis induced in mice by co-injection of type II collagen and Freund's adjuvant. This was associated with an elevation of HDC activity in spleen and bone marrow. Alendronate also increased HDC activity in normal mice, resulting in increased levels of histamine in bone, liver and spleen [59].

We conclude that attention must be paid to the pro-inflammatory actions of ibandronate when considering its use in the treatment of human RA. Although a potent anti-resorptive agent, ibandronate is capable of inducing IFN-{gamma} and enhancing TNF-{alpha} production in vitro. The production of such cytokines may partly be responsible for the pro-inflammatory effects of ibandronate in rat AIA.


    Acknowledgments
 
The authors would like to thank Dr Philippe Gasque (Department of Medical Biochemistry, University of Wales College of Medicine, UK) for his assistance in the molecular biology techniques used and Dr Simon Jackson (Department of Medical Microbiology, University of Wales College of Medicine, UK) who performed the Limulus assay.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 15 December 1998; revised version accepted 28 April 1999.



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