Rheumatology 1999;38:818–825
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
Abstract
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 postarthritis 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.
K : Clodronate, Small unilamellar vesicles, Synovial macrophages,
Reticuloendothelial system.
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 [4–9].
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 [10–12]. 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-a),
interleukin (IL)-1b and IL-6, prostaglandins and several
reactive oxygen species [13]. They also show enhanced
phagocytic activity. Local production of TNF-a and
IL-1b by macrophages in the inflamed synovium occurs
in both antigen-induced arthritis (AIA) and adjuvant
arthritis (AA) models [14–17]. 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 [18–22].
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
Submitted 15 December 1998; revised version accepted 24 March
1999.
Correspondence to: P. J. Richards.
818
© 1999 British Society for Rheumatology
Liposomal clodronate in antigen-induced arthritis
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
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 ml
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-mm-amplitude burst
for 5 min and two 10-mm-amplitude bursts for 10 min.
The concentration of encapsulated clodronate within
both MLV and SUV was determined by calculating the
819
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 IB
(Surgipath). Joints were embedded in paraffin wax, then
sectioned in the sagittal plane at 5 mm 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 mm) 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. Nonspecific 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 ratabsorbed 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 ml of 30% H O . For controls, the same staining
2 2
procedure was performed, but the specific mAb were
replaced by an isotype-matched mouse mAb at identical
concentrations.
820
P. J. Richards et al.
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
×10 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 ± ...
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 SUVctreated rats.
F. 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.
Results
Effect of MLVc and SUVc on joint swelling
On day 0 (7 days post-arthritis induction), baseline knee
swelling (mean ± ...) 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. 1). 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.
Histopathological effects
The results of the histological examination of decalcified
joint sections are summarized in Table 1 and illustrated
in Fig. 2A and B. SUVc-treated rats consistently displayed a total inflammatory score well below that of
saline-, free clodronate- and MLVc-treated rats
( Table 1). 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. 2B). Statistical analysis was not possible due to
T 1. Histological grading of the knee joints isolated from rats with AIA 3 days after treatment with either saline, free clodronate, MLVc
or SUVc
Parameter
Saline
Cartilage destruction/bone erosion (0–3)
Synovial infiltrate/inflammatory exudate (0–3)
SM thickening (0–2)
Total (maximum = 8)
3,
3,
2,
8,
Number of rats per treatment group = 3.
3,
3,
2,
8,
3
3
2
8
Free clodronate
2,
2,
2,
6,
2,
3,
2,
7,
3
3
2
8
MLVc
SUVc
1,
1,
2,
4,
0,
1,
1,
2,
0,
1,
1,
2,
1
1
2
4
0,
0,
1,
1,
0
0
1
1
Liposomal clodronate in antigen-induced arthritis
821
(A)
(B)
F. 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).
F. 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 ×40.)
the limited number of animals available for experimental study.
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 salinetreated control rats (Fig. 3). 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. 4). The differences in macrophage depletion
between MLVc and SUVc treatment were not significant
for any of the macrophage subpopulations.
F. 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).
822
P. J. Richards et al.
F. 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 salinetreated 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. 5). The differences in macrophage depletion between MLVc and
SUVc treatment were not significant for any of the
macrophage subpopulations.
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 7). 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.
Discussion
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 antiinflammatory 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
F. 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).
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.
Liposomal clodronate in antigen-induced arthritis
(A)
(B)
(C)
(D)
(E)
(F)
823
F. 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
×80 for A, B, D and E, and ×160 for C and F ).
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 sig-
nificant 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
824
P. J. Richards et al.
can be disadvantageous in terms of diverting drugbearing 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-1a/b 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-a or
anti-IL-1a/b 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-a.
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.
Acknowledgement
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.
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