Liposomal clodronate eliminates synovial macrophages, reduces inflammation and ameliorates joint destruction in antigen-induced arthritis

P. J. Richards, A. S. Williams, R. M. Goodfellow and B. D. Williams

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

Correspondence to: P. J. Richards.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To investigate the efficacy of a single i.v. dose of clodronate encapsulated within small unilamellar vesicles in suppressing joint inflammation and the histological progression of rat antigen-induced arthritis (AIA).

Methods. Rats with AIA received a single i.v. injection of 20 mg of clodronate encapsulated within small unilamellar vesicles (SUVc) or larger multilamellar vesicles (MLVc) 7 days post-arthritis induction. Free clodronate or saline were used as negative controls.

Results. SUVc was shown to be more effective than MLVc, sustaining a significant reduction in knee swelling for up to 7 days after the initial systemic administration. Knee swelling in free clodronate-treated animals was not significantly affected. The increased efficacy of SUVc in reducing inflammation and joint destruction was associated with a significant depletion of resident ED1+, ED2+ and ED3+ macrophages from the synovial membrane (SM).

Conclusions. SUVc is more efficient than MLVc in reducing the severity of inflammation and joint destruction in rat AIA, and is associated with the specific elimination of macrophage subpopulations from the SM.

KEY WORDS: Clodronate, Small unilamellar vesicles, Synovial macrophages, Reticuloendothelial system.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In many chronic inflammatory diseases, continued recruitment and activation of the monocytes/macrophages can result in tissue destruction and related pathology [1]. In the inflamed synovium of patients with rheumatoid arthritis, activated macrophages are found in abundance [2] and are present in strategic sites related to the distribution of destructive pannus [3]. Their secretory products (monokines) dominate the cytokine profile of synovial tissue and fluid [49].

Experimental models of arthritis have shown that macrophages are present in the inflamed synovium and that different subpopulations can be identified using different monoclonal antibodies [1012]. Newly arrived immature macrophages/monocytes (identified with the monoclonal antibody ED1) outnumber the mature resident macrophages (ED2+). In chronically inflamed synovial tissue, ED3+ macrophages, normally only found in close association with T cells in lymphoid tissue, are also present [12]. Synovial macrophages are in the activated state, they express Class II antigens, and secrete tissue-damaging enzymes, the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-{alpha}), interleukin (IL)-1ß and IL-6, prostaglandins and several reactive oxygen species [13]. They also show enhanced phagocytic activity. Local production of TNF-{alpha} and IL-1ß by macrophages in the inflamed synovium occurs in both antigen-induced arthritis (AIA) and adjuvant arthritis (AA) models [1417]. Macrophages in synovial tissue are highly phagocytic and are capable, therefore, of ingesting liposomes which localize to the synovium.

Clodronate is a first-generation bisphosphonate which, when encapsulated within liposomes, is able to enter phagocytic cells and initiate apoptosis [1822]. Systemically administered clodronate encapsulated in large multilamellar vesicles (MLV), sterically stabilized with polyethylene glycol MS400 stearate (PEG-S), is capable of suppressing paw inflammation in rat AA [23]. These liposomes exerted their effect via a central immunoregulatory mechanism rather than by the removal of macrophages from synovial tissue.

It is known that liposome size influences their localization to synovial tissue [24]. Small unilamellar vesicles (SUV) have been shown to accumulate within inflamed paws of rats with AA to a much greater extent than large MLV. Systemically administered clodronate encapsulated in SUV can produce complete resolution of the clinical indices of inflammation, a normalization of the histology and a resolution of the bone changes seen in AA-treated rats [25]. This is associated with a significant reduction in ED1+ synovial macrophages in the inflamed paws.

In the present study, we compare the effects of a single i.v. injection of either unencapsulated clodronate, MLV encapsulating clodronate (MLVc) or SUV encapsulating clodronate (SUVc) on the severity of inflammation and degree of synovitis in rat AIA. We investigate the influence of SUVc treatment on macrophage subpopulations in organs of the reticuloendothelial system (RES) and also within the synovial membrane (SM).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Male inbred Lewis rats 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 of arthritis
Arthritis was induced in the right knee joint of each rat by following the method previously described [26]. Briefly, on two occasions 1 week apart, male Lewis rats (150 g) were injected s.c. with an emulsion of equal volumes of methylated bovine serum albumin (mBSA; 0.5 mg; Sigma Chemical Co.) and Freund's complete adjuvant (0.25 mg heat-killed Mycobacterium tuberculosis; Sigma Chemical Co.). Fourteen days after the second immunization, arthritis was induced with 100 µl of mBSA (1 mg) injected intra-articularly into the right knee. 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.

Liposome entrapment of clodronate
MLVs and SUVs encapsulating clodronate were prepared as described previously [27]. MLVs were composed of egg phosphatidylcholine and cholesterol (molar ratio 2:1). SUVs were produced by probe sonication (MSC Soniprep 150, 10 mm probe) of MLVc composed of egg phosphatidylcholine, cholesterol and dipalmitoyl phosphatidic acid (molar ratio 7:7:1); and a mean size of 100 nm was achieved after one 6-µm-amplitude burst for 5 min and two 10-µm-amplitude bursts for 10 min. The concentration of encapsulated clodronate within both MLV and SUV was determined by calculating the amount of tracer [99mTc]clodronate remaining using a Wallac 1261 multigamma counter (LKB).

Treatment of AIA
Seven days after arthritis induction, animals were divided into four matched groups (six rats per group). At this time (day 0), animals were injected i.v. with either 2 ml of sterile saline (0.9% w/v), free clodronate (20 mg), MLVc (20 mg) or SUVc (20 mg). To assess the effect of the respective treatments, joint swelling was expressed as the difference in diameters between right and left knees.

Histological grading of knee joint sections
Three rats from each treatment group were killed 3 days after the respective treatments (10 days after arthritis induction). The knee joints were removed, trimmed, and simultaneously fixed and decalcified in Decalcifier I® (Surgipath). Joints were embedded in paraffin wax, then sectioned in the sagittal plane at 5 µm and stained with haematoxylin and eosin (H&E). All sections were coded prior to assessment to eliminate observer bias and subsequently scored by an independent observer. The sections were graded subjectively using three parameters: degree of cartilage destruction and bone erosions (0–3), severity of synovial infiltration and inflammatory exudate (0–3), and degree of SM thickening (0–2).

Immunohistochemistry
Three rats from different treatment groups were killed 3 days after the respective treatments (10 days after arthritis induction). Cryostat sections (8 µm) of liver, spleen and undecalcified knee joints mounted on Superfrost/Plus slides (Scientific Lab. Supplies Ltd, Nottingham, UK) were used for immunohistochemical analysis. The following monoclonal antibodies (mAb) were used for the identification of macrophage subpopulations: ED1 for immature resident monocyte/macrophages; ED2 and ED3 for mature resident macrophages (all from Serotec, UK). All mAb were diluted in 1% BSA in phosphate-buffered saline (pH 7.4) (PBS/1% BSA) where indicated.

Prior to immunostaining, sections were fixed in acetone for 10 min at 4°C and allowed to air dry. Non-specific staining was blocked by incubation with normal rabbit serum (NRS; 1:10 in PBS/1% BSA) for 30 min in a humidity chamber at room temperature. The NRS was then removed and mAb ED1 (1:200), ED2 (1:400) or ED3 (1:500) added for 1 h at room temperature. After three, 1 min washes in PBS, sections were incubated with horseradish peroxidase-conjugated rat-absorbed rabbit anti-mouse IgG (1:50) for 1 h at room temperature. After washing, peroxidase was developed in 100 ml of PBS containing 50 mg of diaminobenzidine and 40 µl of 30% H2O2 . For controls, the same staining procedure was performed, but the specific mAb were replaced by an isotype-matched mouse mAb at identical concentrations.

Image analysis
Macrophage subpopulations were analysed quantitatively using a modification of the method previously described [28]. Briefly, slides were imaged using a Leica DMLB light microscope (Milton Keynes, UK) with a x10 objective and analysed with the computer-based image analysis system Improvision Density Slicing (OpenLab, Coventry, UK). Illumination voltage, camera set-up and calibration parameters were kept constant throughout all measurements. Minimum object boundaries were defined with 35 pixels and manual correction of selected fields was performed to achieve a complete match between the visual screen mask and the original microscope fields. In each slide, at least three consecutive representative fields were evaluated, and the mean area of brown immunoperoxidase staining determined. A total of three rats per treatment group and three sections per rat were analysed.

Statistical analysis
The two-tailed Student's t-test was used to determine whether knee swelling was significantly different between saline-, free clodronate-, MLVc- or SUVc-treated rats, where a P value of <0.05 was considered statistically significant. Values are expressed as the mean±S.E.M. This method of statistical analysis was also used to determine whether the area of positive staining in immunohistochemically stained frozen sections was significantly different between saline-, MLVc- or SUVc-treated rats.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effect of MLVc and SUVc on joint swelling
On day 0 (7 days post-arthritis induction), baseline knee swelling (mean±S.E.M.) in saline-, free clodronate-, MLVc and SUVc-treated rats was 3.43±0.2, 3.47±0.2, 3.31 ± 0.3 and 3.36±0.3 mm, respectively. There was no significant difference in knee swelling between the groups.

Free clodronate- and saline-treated rats were included as controls. When these two groups were compared, there was no significant difference in knee swelling from day 0 to day 14 (Fig. 1Go). Knee swelling in MLVc-treated rats was significantly less than in saline-treated rats at days 2 (P<0.05) and 3 (P<0.05) only. When compared to free clodronate-treated rats, no significant reduction in swelling was observed at any of the time points.



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FIG. 1.  Effect of single i.v. injection of clodronate (20 mg) on knee joint swelling in rat AIA. Arthritic rats were injected i.v. with either saline (control), free clodronate, MLVc or SUVc 7 days after arthritis induction (day 0). Knee swelling was determined for each animal by the difference in diameter between the arthritic right knee and normal left knee. *P< 0.05, **P<0.01 as compared to saline-treated AIA rats (number of rats per treatment group=6).

 
Knee swelling in rats treated with SUVc was significantly less than in saline-treated rats at days 2 (P<0.05), 3 (P<0.01), 4 (P<0.01), 5 (P<0.05) and 7 (P<0.01). There was also a significant reduction in swelling when compared to the free clodronate-treated animals at days 3 (P<0.05), 4 (P<0.01), 5 (P<0.05) and 7 (P<0.01). However, the decrease in swelling due to SUVc was not significantly different from that observed using MLVc at any of the time points.

Histopathological effects
The results of the histological examination of decalcified joint sections are summarized in Table 1Go and illustrated in Fig. 2A and BGo. SUVc-treated rats consistently displayed a total inflammatory score well below that of saline-, free clodronate- and MLVc-treated rats (Table 1Go). The reduced inflammatory score was associated with a large reduction in synovial infiltrate, as well as total abolition of invasive pannus, inflammatory exudate, cartilage destruction and bone erosion (Fig. 2BGo). Statistical analysis was not possible due to the limited number of animals available for experimental study.


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TABLE 1.  Histological grading of the knee joints isolated from rats with AIA 3 days after treatment with either saline, free clodronate, MLVc or SUVc
 


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FIG. 2.  H&E paraffin wax sections of knee joints isolated from rats, 3 days after treatment with either saline (control) or SUVc (10 days after arthritis induction). (A) Saline-treated rat knee joint with large inflammatory infiltrate, invasive pannus and severe cartilage destruction and bone erosion; histological grading=8. (B) SUVc-treated rat knee joint with minimal inflammatory infiltrate and well-preserved cartilage and bone structure; histological grading=2. (Original magnification x40.)

 
Elimination of macrophages from organs of the RES
Spleen.
Both MLVc and SUVc treatment resulted in a highly significant depletion of ED3+ macrophages (P<0.001) from the periarteriolar lymphatic sheath (PALS) and marginal zone as compared to saline-treated control rats (Fig. 3Go). MLVc treatment also resulted in a significant depletion of ED1+ macrophages (P<0.05). ED2+ macrophages were not significantly affected by either treatment. The differences in macrophage depletion between MLVc and SUVc treatments were not significant for any of the macrophage subpopulations. Both MLVc and SUVc significantly depleted ED1+ (P<0.05) and ED2+ (P<0.01) macrophages from the red pulp as compared to saline-treated control rats (Fig. 4Go). The differences in macrophage depletion between MLVc and SUVc treatment were not significant for any of the macrophage subpopulations.



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FIG. 3.  Combined analysis of macrophage subpopulations in the periarteriolar lymphatic sheath (PALS) and marginal zone of spleen in rats, 3 days after treatment with either saline (control), MLVc or SUVc (10 days after arthritis induction). Following a single i.v. injection of either MLVc or SUVc, a large significant reduction in ED3+ macrophages was observed. MLVc-treated rats also demonstrated a significant reduction in ED1+ macrophages. The ED2+ macrophage subpopulation was not significantly reduced by either treatment. All values are quantitative, with an average of at least three fields of analysis per section being performed. *P<0.05, ***P<0.001 as compared to saline-treated AIA rats (number of rats analysed per treatment group=3).

 


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FIG. 4.  Identification of macrophage subpopulations in the red pulp of spleen in rats, 3 days after treatment with either saline (control), MLVc or SUVc (10 days after arthritis induction). Following a single i.v. injection of either MLVc or SUVc, a large significant reduction in both ED1+ and ED2+ macrophages was observed within the red pulp. All values are quantitative, with an average of at least three fields of analysis per rat being performed. *P<0.05, **P<0.01 as compared to saline-treated AIA rats (number of rats analysed per treatment group=3).

 
Liver.
Both MLVc and SUVc significantly reduced ED1+ macrophages (P<0.05) and totally eliminated ED2+ macrophages (P<0.01) from the liver as compared to saline-treated control rats (Fig. 5Go). The differences in macrophage depletion between MLVc and SUVc treatment were not significant for any of the macrophage subpopulations.



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FIG. 5.  Identification of macrophage subpopulations in the liver of rats, 3 days after treatment with either saline (control), MLVc or SUVc (10 days after arthritis induction). Following a single i.v. injection of either MLVc or SUVc, a large significant reduction in ED1+ macrophages was observed. Unlike ED1+ macrophages, ED2+ macrophages were totally depleted from the liver. All values are quantitative, with an average of at least three fields of analysis per rat being performed. *P<0.05, **P<0.01 as compared to saline-treated AIA rats (number of rats analysed per treatment group=3).

 
Depletion of macrophages in the SM
A single i.v. injection of SUVc resulted in significant depletion of ED1+ (P<0.001), ED2+ (P<0.05) and ED3+ (P<0.05) macrophage subpopulations within the SM as compared to saline-treated control rats (Figs 6 and 7GoGo). SUVc treatment also resulted in a significantly larger depletion of ED1+ macrophages when compared to MLVc-treated rats (P<0.05). Although treatment with MLVc resulted in macrophage depletion, statistical significance was never attained.



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FIG. 6.  Identification of macrophage subpopulations in the SM of rats, 3 days after treatment with either saline (control), MLVc or SUVc (10 days after arthritis induction). Following a single i.v. injection of SUVc, a large significant reduction in ED1+ macrophages was observed. Both ED2+ and ED3+ subpopulations were also significantly depleted, although not to the same degree. Treatment with MLVc failed to result in significant depletion of any of the macrophage subpopulations. All values are quantitative, with an average of at least three fields of analysis per rat being performed. *P<0.05, ***P<0.001 as compared to saline-treated AIA rats (number of rats analysed per treatment group=3).

 


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FIG. 7.  Immunohistological identification of resident macrophages in cryostat sections of undecalcified knee joints isolated from rats, 3 days after treatment with either saline (control) or SUVc (10 days after arthritis induction). Resident macrophages present within the knee joints of rats treated with saline (A–C) or SUVc (D–F) were detected using the markers ED1 (A and D), ED2 (B and E) and ED3 (C and F). A large, significant depletion of all macrophage subpopulations was observed in SUVc-treated rat knee joints. Macrophage cells are identified by brown horseradish peroxidase staining (original magnification x80 for A, B, D and E, and x160 for C and F).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our study clearly demonstrates that a single i.v. dose of SUVc (20 mg) is more effective in increasing the rate of resolution of experimental arthritis than an equivalent dose of MLVc or free drug. Furthermore, this anti-inflammatory effect is associated with both local and systemic depletion of macrophage populations staining positively for ED1, ED2 and ED3. In previous studies using rat AIA, three consecutive i.v. doses of a sterically stabilized liposomal clodronate preparation (PEG-S MLV) resulted in only a moderate, short-lived reduction in knee swelling which did not alter the histological progression of the disease [29].

In the present report, a significant reduction in knee swelling was observed in SUVc-treated animals 2 days after treatment and was sustained for a further 5 days compared to the saline control. This was attributed to effective elimination of resident macrophages from the knee joint. MLVc produced a significant decrease in knee swelling on days 2 and 3 only. The limited effectiveness of MLVc was attributed to its inability to deplete macrophages from the SM significantly. MLVc significantly depleted macrophages from the liver and spleen to an equal or greater degree than SUVc.

MLVc has been utilized in rat AA to achieve a greater understanding of the role of macrophages in the pathogenesis of joint destruction. Intravenously injected liposomal clodronate was most effective in reducing synovitis and inflammation in chronic rat AA [23, 29]. In these studies, the reduction in clinical and histological signs of arthritis was attributed to the elimination of macrophages present in the liver, spleen and lymph nodes, and not in the inflamed SM.

In addition to MLVc, SUVc is also effective in systemically eliminating macrophages [30]. As with MLVc, SUVc significantly reduces inflammation in rat AA [25, 30]. A single systemic dose of SUVc (20 mg) induced a significant improvement in inflammatory parameters which was sustained for 2 weeks following treatment. The amelioration of arthritis was associated with a significant reduction in resident ED1+ macrophages in the SM. Systemically administered MLVc (20 mg) also produced a significant reduction in synovial macrophages, although not as marked as that observed with SUVc. These effects were specifically due to the actions of the encapsulated clodronate, as demonstrated by the inability of free clodronate (20 mg) to induce any significant improvement in inflammatory parameters when administered together with either empty SUV or MLV.

In our study, the increased efficacy of SUVc to deplete resident macrophages in the SM may be attributed to the ability of small liposomes to sustain a longer circulation time of entrapped clodronate within the blood. It is known that liposomes injected i.v. accumulate in the RES due to uptake by resident macrophages [31]. This can be disadvantageous in terms of diverting drug-bearing vesicles, such as liposomal clodronate, away from other sites of interaction (e.g. synovial macrophages). The clearance of liposomes from the circulation is determined by, amongst other things, vesicle size [32]. SUV exhibited a half-life of 7.5 h, as opposed to only 2 h by large unilamellar liposomes (LUV). Upon systemic administration, negatively charged SUV were shown to accumulate within the inflamed tissue of arthritic rats [24]. Up to 5.3% of the total quantity of SUV (95 nm) injected accumulated in all four inflamed paws of adjuvant arthritic rats, as compared to only 0.6% of MLV (1150 nm). A similar finding was demonstrated in human rheumatoid arthritis (RA) patients [33] where i.v. injected SUV accumulated in areas of active synovial proliferation. Such an increase in liposomal accumulation would, therefore, account for the increased depletion of resident macrophages upon SUVc treatment demonstrated in the present study.

In our study, the depletion of macrophages from the SM by SUVc also appeared to inhibit the histological progression of AIA. The importance of macrophages in the progression of experimental arthritis has been demonstrated using monoclonal antibodies directed against their secretory products [34, 35]. The pre-treatment of murine AIA with anti-IL-1{alpha}/ß polyclonal antibodies totally prevented suppression of cartilage proteoglycan synthesis [34]. 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 [35]. The intra-articular administration of MLVc, 7 days prior to CIA induction, resulted in total depletion of synovial lining macrophages [36]. Lining-depleted joints expressed significantly lower IL-1 mRNA in the synovium and were unresponsive to arthritis induction using IL-1 or TNF-{alpha}.

In conclusion, encapsulation of clodronate in SUV greatly enhances its anti-inflammatory effects in rat AIA. This is attributed to increases in its ability to deplete macrophages significantly at sites of chronic inflammation, and thus supports a central role for synovial macrophages in the progression of inflammation and joint destruction in experimental arthritis. With the optimization of liposomal accumulation of clodronate at specific sites of inflammation, the potential for treatment of lesions such as human RA is very promising.


    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 computer-based image analysis system Improvision Density Slicing.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 15 December 1998; revised version accepted 24 March 1999.