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Long-term amelioration of rat adjuvant arthritis following systemic elimination of macrophages by clodronate-containing liposomes.

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ARTHRITIS & RHEUMATISM
Vol. 38, No. 12, December 1995, pp 1777-1790
0 1995, American College of Rheumatology
1777
LONG-TERM AMELIORATION OF RAT ADJUVANT ARTHRITIS
FOLLOWING SYSTEMIC ELIMINATION OF MACROPHAGES
BY CLODRONATE-CONTAINING LIPOSOMES
RAIMUND W. KINNE, CARSTEN B. SCHMIDT-WEBER, RALPH HOPPE, EBERHARD BUCHNER,
ERNESTA PALOMBO-KINNE, EBERHARD NURNBERG, and FRANK EMMRICH
Objective. To determine whether systemic elimination of macrophages by means of clodronatecontaining liposomes counteracts inflammation and
joint destruction in rats with established adjuvant arthritis (AA).
Methods. Rats with AA received a total of 2.7 mg
of clodronate encapsulated in liposomes in 3 intravenous
doses on days 10, 11, and 12 of arthritis. Phosphate
buffered saline (PBS), PBS-laden liposomes, or free
clodronate were used as negative controls. Clinical,
hematologic, and histopathologic signs of AA were monitored, and depletion of macrophages by clodronateliposomes was evaluated both in the synovial membrane
(SM) and in organs of the mononuclear phagocyte
system (MPS).
Results. Clodronate-laden liposomes led to significant, long-term amelioration of the clinical signs of AA,
a reduction in the erythrocyte sedimentation rate (ESR),
and counteraction of joint destruction, not only immediately after treatment, but also for 2 weeks thereafter.
Free clodronate induced moderate clinical improvement
and a significant decrease in the ESR, but only during
the late phase of AA. Drug-free vesicles even aggravated
the joint destruction. Clodronate-laden liposomes did
Presented in part at the Sixth Bertine Koperberg Conference, Nijmegen, The Netherlands, April 28-30, 1994.
Dr. Kinne’s work and Professor Emmrich’s work was supported by the German Ministry for Research and Technology (BMFT;
FKZ OlVM9311 and OlVM 8702). Mr. Schmidt’s work and Mr.
Buchner’s work was supported by the Graduiertenkolleg Erlangen.
Raimund W. Kinne, MD, Carsten B. Schmidt-Weber,
Eberhard Buchner, Ernesta Palombo-Kinne, MD, Eberhard Nurnberg, PhD: University of Erlangen-Nuremberg, Erlangen, Germany;
Ralph Hoppe, PhD: Goedecke AG, Berlin, Germany; Frank Emmrich, MD: University of Leipzig, Leipzig, Germany.
Address reprint requests to Raimund W. Kinne, MD,
Immunology Unit, Department of Medicine 111, University of
Erlangen-Nuremberg, Schwabachanlage 10, D - 91054 Erlangen,
Germany.
Submitted for publication May 5 , 1995; accepted in revised
form July 31, 1995.
not induce significant depletion of resident macrophages
in the SM, but rather, in the paracortical region of
popliteal lymph nodes, in the liver, and in the marginal
zone and periarteriolar lymphatic sheaths of the spleen.
Conclusion. Clodronate-laden liposomes induce
long-term amelioration of AA, even if administered
for a brief period during the florid phase of the disease.
The amelioration is paralleled by the elimination of
macrophages in immunocompetent areas of the spleen
and draining lymph nodes, but not locally in the SM.
This suggests an influence of the treatment on the
immunoregulatory rather than effector, functions of
macrophages.
As in rheumatoid arthritis (RA) in humans (1,2),
macrophages are abundantly present in the inflamed
synovial membrane (SM) of rats with adjuvant arthritis
(AA) (3-6). Macrophages are thought to play a major
role in arthritis, either as effector cells of tissue destruction, by secreting tissue-degrading enzymes or
pro-inflammatory cytokines (7-lo), or by virtue of
their immunoregulatory functions in the course of
antigen-driven responses (1 l), i.e., processing and
presentation of exogenous antigens to T helper cells in
the form of class I1 major histocompatibility complexantigenic determinant complexes (1 1). The latter functions may be particularly relevant to the pathogenesis
of RA (1,12), in that overrepresentation of certain
HLA-DR alleles on antigen-presenting cells (13-15)
may favor the presentation of select arthritogens to T
cells (12). In addition, macrophages may contribute to
the pathogenesis of arthritis by providing second signals that are necessary for full activation of (auto)reactive T cells (11,16).
A critical role of macrophages in the development of experimental AA is indicated by the fact that,
similar to the situation in human RA (17-19), macro-
1778
KINNE ET AL
phages in the synovium and in several body compartments of rats with AA are in a state of activation (6);
this activation has been shown to precede clear manifestations of arthritis (4,6), indicating that these cells
play a pivotal role in the development of this experimental condition.
Based on the critical roles of macrophages in
the course of autoimmune AA, we attempted the
systemic elimination of these cells to evaluate both
their contribution to the development of AA and the
feasibility of using antimacrophage pharmaceuticals as
disease-controlling antirheumatic agents (20).
Selective elimination of macrophages was pursued by systemic administration of vesicles laden with
cytotoxic drugs (21), a method described as “suicide
technique” (22). Liposomes containing clodronate, a
diphosphonate that becomes toxic when concentrated
intracellularly (22-24), were prepared according to a
formulation known to lead to radical depletion of
macrophages from the liver and spleen (25). Rats were
treated intravenously on days 10, 11, and 12, i.e., between the onset and the peak of clinical signs of arthritis.
A novel preparation of liposomes consisting of
polyethylene glycol MS400 stearate (PEG-S) instead
of phosphatidylcholine was used in the present study,
based on improved stability and biological performance both in vivo (26) and in vitro (SchmidtWeber et al: Unpublished observations). The effects of
clodronate-laden liposomes were compared with those
of the appropriate negative controls (phosphate buffered saline [PBS], free clodronate, or PBS-laden liposomes) and with those of oral dexamethasone as
conventional antiarthritis medication. To estimate
whether clinical responses were dose-related, 2 intraliposomal concentrations of clodronate-laden liposomes
were also tested. The effects of treatment were assessed
by monitoring clinical, hematologic, and histopathologic
parameters of AA. The question of the relationship
between the clinical efficacy of antimacrophage therapy
and localization of macrophage depletion was addressed
by performing a broad immunohistologic survey of
macrophage subpopulations in the SM and in organs of
the mononuclear phagocyte system (MPS) on 2 different days, 1 day after completion of treatment (day 13 of
AA) and 2 weeks thereafter (day 28 of AA).
MATERIALS AND METHODS
Preparation of liposomes. Multilamellar liposomes
were prepared according to the method described by Van
Rooijen and Claassen (25), by evaporating 10 ml of a
chloroform/methanol mixture containing 119 mg (0.18
mmoles) of PEG-S (Caesar and Loretz, Hilden, Germany),
13 mg (0.045 mmoles) of sodium dodecyl sulfate, and 68 mg
(0.18 mmoles) of cholesterol (both from Sigma, Deisenhofen,
Germany) (molar ratio 4:1:4) under vacuum at 60°C in a
rotation evaporator. Twenty milliliters of PBS (0.15M NaCI,
6.5 mM Na,HPO,, 1.5 mM KH,PO,, pH 7.4) or PBS
containing disodium clodronate (molecular weight 362.9; a
kind gift of Boehringer Mannheim, Mannheim, Germany)
was added to the lipid film; this led to the spontaneous
formation of PBS- or clodronate-liposome suspensions with
10 gm of lipid per liter.
Intraliposomal encapsulation of clodronate was obtained by adding PBS containing either 50 gmhiter (high
dose), or 10 gmfliter (low dose) of clodronate to the lipid film.
The suspensions were rotated without vacuum for 1 hour at
60°C. Free clodronate was removed by dialysis (up to 300 ml of
liposome suspension, 3 8-hour dialyses against 5 liters of PBS).
Before application, the suspension was centrifuged
for 10 minutes at 338g in a cell culture centrifuge (Hettich,
Tuttlingen, Germany) to remove excessively large liposomes
or particles. The intraliposomal concentration of clodronate
was determined as previously described (27); the final preparations contained a total of 2.7 mg (high dose) or 1.7 mg (low
dose) of clodronate 8.5 in ml of PBS.
As a prerequisite for the present study, the performance of the novel PEG-S liposome formulation was compared with that of conventional liposomes made of phosphatidylcholine and cholesterol. The relative in vivo uptake
of the synthetic PEG-S liposome formulation in arthritic
joints, assessed by radiolabeling techniques and external
gamma camera imaging, was consistently equal or superior
to that of phosphatidylcholine liposomes. In addition, monocytes in culture showed improved incorporation of PEG-S
liposomes, along with stronger cytotoxicity in response to
incubation with PEG-S clodronate-laden liposomes
(Schmidt-Weber et al: Unpublished observations).
Induction of AA. Female Lewis rats (weight 140-190
gm, ages 7-10 weeks; Charles River Laboratories, Sulzfeld,
Germany) were injected intradermally into the base of the
tail with 0.5 mg of heat-killed Mycobacterium tuberculosis
(R37 Ra; Difco, Detroit, MI) in 0.1 ml of paraffin oil (Riedel
de Haen AG, Seelze, Germany).
Treatments. On days 10, 1 1 , and 12 following induction of arthritis, rats were treated with 3 consecutive intravenous injections of PEG-S liposomes (3, 3, and 2.5 ml/day,
respectively) containing a total of 2.7 mg (high dose) or 1.7
mg (low dose) of clodronate encapsulated in the vesicles.
PBS alone, PBS-filled liposomes, or free clodronate in
matched volumes were used as negative controls.
Solutions of clodronate to be applied as free drug
were obtained by dissolving 15 mg of clodronate in 8.5 ml of
PBS. This dose was chosen on the basis of initial determinations of the total intraliposomal content of clodronate
using the murexide method (28); the intraliposomal content
of clodronate that was subsequently measured by highperformance liquid chromatography (27) was lower than the
initially measured content. A total of 15 mg of free clodronate was nevertheless used for treatment, to arbitrarily
compensate for the enhanced delivery of the drug through
encapsulation.
SYSTEMIC MACROPHAGE ELIMINATION AMELIORATES AA
Dexamethasone (Sigma) was suspended in 0.5% sodium carboxylmethyl cellulose (200 d m l ) , and used as a
positive treatment control-a standard medication with clear
antirheumatic e f f e c t o n d -0.75 ml(1 mgkg of body weight/
day) was given orally for 3 consecutive days.
The effects of the various treatments were monitored
by repeated measurement of the arthritis score, hindpaw
volume, body weight, erythrocyte sedimentation rate (ESR),
leukocyte count, and the differential cell count, as well as by
histopathologic evaluation of the degree of joint destruction.
One or 2 days before initiating treatment, the animals were
subdivided into groups with equal mean arthritis scores. The
mean body weight of the animals in all treatment groups was
normalized to the same value on day 9 (i.e., 1 day before
initiation of treatment) to allow meaningful statistical comparison.
Clinical parameters. Arthritis score. Inflammation in
each paw was graded in a blinded manner according to the
extent of erythema and edema of the periarticular tissues
(29), using a scale of 0-4, where 0 = no inflammation, 1 =
unequivocal inflammation of 1joint of the paw, 2 = unequivocal inflammation of at least 2 joints of the paw, or moderate
inflammation of 1joint; 3 = severe inflammation of 1 or more
joints; and 4 = maximum inflammation of 1 or more joints in
the paw. The scores for each paw were then added to get the
total arthritis score (maximum possible score 16 per animal).
Hindpaw volume. Swelling of the hindpaws was
measured using a plethysmometer chamber (H. Basile, Comerio, Italy). The hindpaw volume was expressed as the mean
of the volumes of both hindpaws.
Body weight. The loss of body weight typical of this
model of arthritis was monitored with a 1-gm precision
balance (Sartorius, Gottingen, Germany).
Hematologic parameters. ESR. This systemic parameter of AA was determined by mixing 45 pl of blood with
10 pl of sodium citrate (5% in distilled water), then drawing
the mixture into a glass capillary tube, allowing the erythrocytes to settle for 1 hour, and measuring the distance (in mm)
from the meniscus of the serum to the edge of the sedimented erythrocytes.
Leukocyte counts. Cells were counted in heparinized
peripheral blood using a Sysmex E-5000 M/CS cell counter
(Sysmex TOA Medical Electronics, Hamburg, Germany).
Differential cell counts were performed on blood smears that
had been stained with Pappenheim’s solution (30). The
relative percentages of lymphocytes, polyrnorphonuclear
neutrophils (PMN), and monocytes were evaluated by microscopy. A total of 100 cells in each blood smear were
counted.
Histologic analysis. On day 13 and day 28 during the
course of AA, the animals were killed, and the ankle and
tarsal joints were excised. The samples were frozen in
methylbutane cooled in liquid nitrogen, and embedded in 8%
gelatin. Ten undecalcified, 8 pn-thick, serial cryostat sections were prepared from each joint as previously described
(31) and stained with Giemsa solution.
To estimate the degree of cartilage and bone destruction, a semiquantitative score of 0-4 was used, where 0 = no
erosion of cartilage or bone, 1 = unequivocal erosion of
510% of cartilage and bone cross sections, 2 = erosion of
16
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1779
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Figure 1. Time course of changes in clinical parameters in normal
rats (n = 6) and in rats with adjuvant arthritis (AA) treated with
phosphate buffered saline (AAIPBS; n = 1l), free clodronate ( A N
Free - clo; n = 6), PBS-laden liposomes (AA/PBS - Lipos; n = 12),
or high-dose (2.7 mg of clodronate given in 3 intravenous doses
[arrows]) clodronate-laden liposomes (AAIClo - Lipos [HD] ;
n = 12). Values are the mean 2 SEM. Clodronate-liposomes caused
a major, long-lasting reduction in the arthritis score (A) and in
hindpaw swelling (B);the loss of body weight typical of AA was also
significantly counteracted (C). * = P 5 0.01 and ** = P 5 0.005
versus AAIPBS.
1780
d o % , 3 = erosion of 50-90%, and 4 = erosion of >90% of
cartilage and bone cross sections.
To estimate the degree of inflammatory infiltration, a
subjective semiquantitative score of 0-3 was used, where 0
= no infiltration, 1 = mild, 2 = moderate, and 3 = severe
infiltration. Sections were independently scored in a blinded
manner by 2 experienced observers (RWK, CBS-kV).
Jmmunohistochemical analysis. Cryostat sections of
liver, spleen, and popliteal lymph nodes or undecalcified,
unfixed cryostat sections of the joints 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;
ED7 for both PMN and macrophages (32,33) (all from
Serotec, Wiesbaden, Germany).
The MAb were used as ascites (diluted 1:lOO or
1 :1,000 in Tris buffered saline [TBS]/l% bovine serum albumin [BSA]) or as cell culture supernatants (undiluted or
diluted 1:lO). The antibodies were added and incubated for
30 minutes in a humid chamber at room temperature. After
washing, a peroxidase-coupled rabbit anti-mouse antibody
(diluted 1:30 in TBS/20% rat serum; Dakopatts, Hamburg,
Germany) was applied for 30 minutes. Afterwards, a
peroxidase-coupled swine anti-rabbit antibody (diluted 1:40;
Dakopatts) was added for 30 minutes. The peroxidase was
developed in a solution of 2.5 mg of diaminobenzidine
(Sigma) in 5 ml of PBS, pH 7.4. Shortly before use, the
solution was filtered, and 20 pl of 30% H,O, was added. For
controls, the same staining procedure was performed, but
the specific antibodies were replaced by an isotype-matched
mouse MAb at identical concentrations.
For the evaluation of macrophage depletion, the
density of cells stained with the different antibodies was
determined in SM, popliteal lymph node, and spleen, using a
semiquantitative approach, where a score of 0 = no positive
cells, 1 = a few positive cells, 2 = between 10% and 25%
positive cells, and 3 = between 25% and 50% positive cells.
In the liver, the absolute number of positive cells per defined
field of view was determined using a counting grid.
Fluorocytometry. Bone marrow was flushed from the
femurs and tibias of normal rats, or of arthritic rats subjected
to the above treatments, by using ice-cold RPMI medium
plus 10% fetal calf serum (FCS) (both from Gibco, Eggenstein, Germany). Peritoneal cells from the same groups of
animals were harvested by flushing the peritoneal cavity
twice with 20 ml of ice-cold PBS/l% FCS. Cells were stained
with antibodies at saturating concentrations. For immunofluorescence labeling, 2 X lo5 nucleated cells were suspended in 100 pl of PBS/O.5% BSA/O.O5% NaN,. Unlabeled
primary MAb was added, and detected with a secondary
fluorescein isothiocyanate-labeled antibody in PBS buffer as
above, plus 20% rat serum to avoid cross-reactions with rat
IgG contained in the cell preparation. Cells were washed
with PBS (as above) between every step.
Specificity of staining was confirmed using isotypematched control MAb for both staining procedures. Analyses were performed on an Epics XL flow cytometer (Coulter, Krefeld, Germany). Forward and side scatter gates were
set to include all viable nucleated cells.
Statistical analysis. The nonparametric MannWhitney U test was applied to analyze differences for all
KINNE ET AL
parameters examined. The effects of treatments were compared with those of PBS. For multiple comparisons (arthritis
score, hindpaw volume, body weight), significant differences
were accepted at P 5 0.01, otherwise (ESR, leukocyte
count, differential blood cell count, joint destruction score,
and depletion of macrophages), at P 5 0.05.
RESULTS
Clinical and hematologic effects of PBS and
PBS-laden liposomes. Arthritic rats treated with PBS
alone or with PBS-filled liposomes showed a fullblown arthritic syndrome throughout the course of the
experiment, with marked arthritis scores, augmented
hindpaw volume, and dramatic loss of body weight
(Figure 1).
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20
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Time (days)
Figure 2. Time course of changes in hematologic parameters in the
same groups of animals as in Figure 1. Values are the mean +. SEM.
Arrows show the dates of treatment. Clodronate-laden liposomes
significantly reduced the erythrocyte sedimentation rate during the
late phase of AA, an effect also observed with free clodronate (A).
Clodronate-laden liposomes induced transient leukocytosis (B). * =
P 5 0.05 and ** = P c: 0.01 versus AAIPBS.
SYSTEMIC MACROPHAGE ELIMINATION AMELIORATES AA
-m-
AAIPBS
--A-A-
AA I PBS Lip08
A A I Clo Lip08 (HD)
1781
Clinical and hematologic effects of free clodronate. The arthritis score, hindpaw volume, and body
weight were not significantly different from those of
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Figure 3. Time course of changes in the differential blood cell
count, expressed as the percentage of leukocytes, in the same
groups of animals as in Figure 1. Values are the mean f SEM.
Arrows show the dates of treatment. Clodronate-laden liposomes
induced a transient decrease in both the polymorphonuclear neutrophils (PMN) and monocytes at completion of treatment (A and C),
whereas lymphocytes were transiently increased upon treatment
(B). * = P 5 0.05 and ** = P 5 0.01 versus AA/PBS.
Diseased animals treated with PBS or PBSladen liposomes displayed high ESRs and increased
leukocyte counts. The 1ymphocyte:PMN ratio was
also inverted (Figures 2 and 3).
$50:
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0
5
10
4H
15
20
25
30
Time (days)
Figure 4. Comparison of the effects of 2 intrdiposomal concentrations of clodronate on clinical parameters of arthritis. Rats were
treated intravenously with PBS (n = 11) or with high-dose (HD) (2.7
mg of clodronate; n = 12) or low-dose (LD) (1.7 mg; n = 6)
clodronate-laden liposomes in 3 intravenous administrations (arrows). Values are the mean f SEM. The high-dose clodronate-laden
liposomes induced earlier and stronger amelioration of the arthritis
score (A). The low dose, in turn, was initially more effective in
counteracting the loss of body weight (C). * = P 5 0.01 and ** = P
I0.005 versus AA/PBS, and
= P 5 0.01 for comparison of the 2
doses. Six normal rats were studied. See Figure 1 for other definitions.
+
1782
the rats with AA that had been treated with PBS,
although there was an improvement in hindpaw volume and body weight that approached statistical significance (0.01 < P < 0.05) on day 20-28, i.e., during
'
the late phase of AA (Figure 1).
A significant decrease in the ESR compared
with PBS-treated rats was also noted in the late phase
of the experiment (day 20 and 28). The other parameters remained unaffected (Figures 2 and 3).
Clinical and hematologic effects of clodronateladen liposomes. The preparation containing the highdose clodronate (2.7 mg in 3 intravenous administrations) induced a marked, significant reduction in the
arthritis score from day 15 to day 21 compared with
PBS treatment. Concurrently, the swelling of the
hindpaws was significantly less severe compared with
PBS-treated animals from day 16 until day 28 (up to 2
weeks after the end of the 3-day treatment). The loss
of body weight that is typical of AA was also significantly counteracted from days 16 to 19 (Figure 1).
Treatment with high-dose clodronate-laden liposomes significantly reduced the ESR on day 28, a
late time point in the course of this experimental model
of arthritis. At the end of treatment (day 12), there was
a transient increase of the leukocyte count in clodronate liposome-treated animals compared with PBStreated animals (Figure 2). The relative percentages of
circulating PMN and monocytes were transiently decreased and those of lymphocytes were transiently
increased on days 12 and 14, respectively, during and
immediately after the end of treatment (Figure 3).
Clinical effects of low-dose clodronate-laden liposomes. The liposome preparation containing the lowdose clodronate (1.7 mg in 3 intravenous administrations) also significantly counteracted AA (Figure 4).
The onset of the amelioration, however, was slower
than in the case of high-dose clodronate, and the
duration of the effects was also significantly shorter;
both of these features suggest a dose-related response.
The counteracting of the loss of body weight was
significantly quicker with the low-dose preparation
(Figure 4).
Clinical and hematologic effects of dexamethasone. Oral treatment with the positive control dexamethasone on days 10, 11, and 12 of AA led to a
significant, but transient, reduction of the arthritis
score and hindpaw volume on days 12 and 13 (P 5
0.005) (data not shown). On day 14, there was already
a strong rebound of the clinical signs. In the case of the
arthritis score, the values in the dexamethasonetreated group exceeded those of the PBS-treated group
from day 26, with differences approaching statistical
significance (0.01 < P < 0.05). Treatment with dexa-
KINNE ET AL
day 13
4
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2
1
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CI
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Free-Clo PBS-Lipos Clo-Lipos
Figure 5. Changes in the joint destruction score, as assessed by
histopathologic analysis of ankle and tarsal joints, 13 and 28 days
following induction of adjuvant arthritis: 1 day and 2 weeks following treatment with either phosphate buffered saline (PBS) (n = 12
and n = 10, respectively), free clodronate (Free-Clo) (n = 6 and n =
12, respectively), PBS-laden liposomes (PBS-Lipos) (n = 12 and n =
12, respectively), or high-dose clodronate-laden liposomes (CloLipos) (n = 7 and n = 12, respectively). To correct for variations in
the destruction in different areas of the joint, 10 serial sections were
scored per joint. Values are the mean 2 SEM. Treatment with
clodronate-laden liposomes had a long-lasting effect on joint destruction, which decreased by -80% on day 13 and by -25% on day
28, compared with PBS-treated rats. In contrast, PBS-liposomes
aggravated the joint destruction. * = P s 0.05 and ** = P 5 0.01
versus AAIPBS.
methasone worsened the loss of body weight compared with PBS treatment on days 14, 26, and 28, but
the differences only approached statistical significance
(0.01 < P < 0.05) (data not shown).
There was an early, significant decrease in the
ESR on days 12 and 14 (P5 0.05) and a late decrease
in the peripheral blood leukocyte count on days 20 and
28 compared with PBS-treated rats. Significant
lymphopenia and granulocytosis were observed on
days 14,20, and 28 of AA (P 5 0.05) (data not shown).
day 13
day 28
a
BJ
w
Figure 6. Giemsa-stained undecalcified cryostat sections of ankle joints from rats with adjuvant arthritis, on day 13 (A, C, and E) and day 28
(B, D, and F): 1 day and 2 weeks following treatment with phosphate-buffered saline (PBS) (A and B), PBS-laden liposomes (C and D), or
high-dose clodronate-laden liposomes (E and F). Arrowhead in A, C, and E indicates different degrees of marginal cartilage and bone erosion;
arrow in B and D indicates remnants of cartilage and bone in almost completely destroyed joints; arrowheads in F show limited marginal erosion
on treatment with clodronate-laden liposomes. s = synovial membrane; b = bone; p = pannus; c = cartilage. (Original magnification x 93.)
1784
Histopathologic effects. Joint destruction. In
contrast to the lack of clinical effects, treatment with
PBS-laden liposomes induced significant aggravation
of joint destruction during the late phase of the disease
(day 28) compared with PBS treatment alone (Figures
5 and 6). Free clodronate did not influence the degree
of ankle joint destruction in either the early phase or
the late phase of AA (Figure 5 ) .
High-dose clodronate-laden liposomes significantly reduced the degree of ankle joint destruction
immediately after treatment (day 13) and for 2 weeks
thereafter (day 28), as compared with PBS treatment
(Figures 5 and 6). Oral dexamethasone also initially
reduced the degree of ankle joint destruction (day 13;
P I0.05); however, on day 28, the severity of the joint
destruction was indistinguishable from that in PBStreated rats (data not shown).
Inflammatory infiltration. None of the treatments, including conventional oral dexamethasone,
significantly counteracted the degree of joint infiltration by inflammatory cells, except for free clodronate
on day 13 (P I0.03 versus PBS-treated rats) (data not
shown). On day 28, clodronate-laden liposomes had
decreased the level of joint infiltration by- -20%, but
this difference did not reach statistical significance.
Depleting effects on macrophages in the synovial
membrane. Treatment of arthritic rats with clodronateladen liposomes did not induce significant depletion of
either immature or mature resident macrophages in the
SM. On day 13 of AA (1 day after completing treatment with clodronate-laden liposomes), there was
some decrease in the number of EDl- and ED3positive macrophage subpopulations, but in neither
case was statistical significance attained (Table 1)
(Figure 7).
Depleting effects on macrophagesin the mononuclear phagocyte system. Popliteal lymph node. Two
weeks following completion of treatment (day 28 of
AA), the paracortical region showed significant depletion of ED1-positive, immature resident macrophages.
A similar degree of depletion was already seen soon
after completion of treatment (day 13), but statistical
significance was not attained (Table 1).
Spleen. In the spleen, treatment with clodronate-laden liposomes induced focused depletion of
resident macrophages in the marginal zone and in the
periarteriolar lymphatic sheaths (PALS). The changes
did not occur simultaneously for all subpopulations;
the ED3-positive subpopulation was depleted in both
the marginal zone and the PALS on day 13 (1 day after
completing treatment); the depletion was also strikingly evident among ED3-positive marginal metallophilic macrophages (Figure 8). The ED1-positive sub-
KINNE ET AL
set was significantly depleted on day 13 in the PALS
and on day 28 in the marginal zone, whereas the
EDZpositive subset was depleted only in the PALS on
day 28 (Table 1).
Liver. Clodronate-liposome treatment induced
marked depletion of all subpopulations of liver macrophages on day 13 of AA. Repopulation was only
partially completed on day 28 (Table 1).
Bone marrow. Fluorocytometric analysis of
bone marrow cells revealed that clodronate-laden
liposomes induced no apparent changes in ED2positive macrophages or in CD4-positive cells (data
not shown).
DISCUSSION
Short-term systemic treatment with the antimacrophage pharmaceutical clodronate-laden liposomes during the florid phase of established AA in rats
led to long-lasting amelioration of clinical and histopathologic signs of the disease. Macrophages, therefore, appear to play a critical role in the course of AA,
and more generally in experimental arthritides, since
antigen-induced arthritis is also extremely sensitive to
such treatment (34,35). This concept extends to other
autoimmune disorders, since experimental encephalomyelitis and autoimmune neuritis are also prevented
following systemic elimination of macrophages (36,37).
The clinical benefits of clodronate-laden liposomes persisted for more than 2 weeks after only 3
days of treatment. This is interesting in view of the fact
that AA has a fulminant time course (3) and that the
chain of immunologic events leading to full-blown
arthritis was already quite advanced at the time of
treatment (38,39). In comparison to the strong efficacy
of this antimacrophage pharmaceutical, the benefits of
most of the immunotherapies investigated to date in
AA, e.g., anti-pan-T cell, anti-CD4, or anti-T cell
receptor d p MAb treatment (4042), are not as longlasting and are mostly dependent on continuous administration of these agents. In fact, such protocols
are more effective for preventing disease rather than
for treating established disease (41,42).
Clodronate-laden liposomes also improved systemic parameters of the disease, such as the ESR and
body weight. Thus, despite a very drastic treatment,
not only the local signs of arthritis, but also the general
health status of the treated animals improved (Figures
1 and 2). This finding is relatively surprising, since
treatment is based on a toxic principle. A broad survey
of the main organs (liver, spleen, lungs, and kidneys)
of arthritic rats treated with clodronate-laden liposomes confirmed the relative safety of this treatment in
Clo-lipos
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2.0 f 0.0 1.8 f 0.2
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ED1
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Clo-lipos
MacrophagesPMN,
ED7
Day 28
PBS
2
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Clo-lipos
MacrophagesPMN,
ED7
* In the synovial membrane, there were no significant changes in the macrophage subpopulations on either day. In the paracortical region of the popliteal lymph nodes, there was a significant
depletion of ED1-positive macrophages on day 28 (2 weeks after treatment). In the marginal zone and periarteriolar lymphatic sheath (PALS) of the spleen, macrophages were depleted on day
13 and day 28, although not simultaneously for all subpopulations (possibly related to different dynamics of repopulation and/or maturation of macrophage subsets). In the liver, all macrophage
subpopulations were significantly depleted on day 13, with still-incomplete repopulation on day 28. The lack of depletion of ED7-positive cells in all tissues examined, except the liver, indirectly
indicated a sparing of polymorphonuclear neutrophils (PMN). Macrophage depletion was assessed using a semiquantitative scoring system (0 = no positive cells, 1 = a few positive cells, 2
= 10-25% positive cells, and 3 = 25-50% positive cells), except in the liver, where the absolute number of positive cells per defined field of view was determined using a counting grid. Values
are the mean f SEM. Clo-lipos = clodronate-laden liposomes.
t P 5 0.05 versus phosphate buffered saline (PBS).
$ P 5 0.01 versus PBS.
Liver
Spleen
Red pulp
Marginal
zone
PALS
Popliteal lymph
nodes
2.0 f 0.4 1.0 f 0.0
Cortex
Paracortex
2.6 f 0.2 2.0 f 0.0
2.0 f 0.0 1.3 f 0.3
Synovial
membrane
PBS
ED1
Macrophages
Day 13
Table 1. Depletion of macrophages from the synovial membrane and organs of the mononuclear phagocyte system of rats with adjuvant arthritis following treatment with clodronate-laden
liposomes*
1786
KINNE ET AL
day 13
day 28
I -
' m
v)
Figure 7. Lack of depletion of macrophages in synovial membranes obtained from the ankle joints of rats with adjuvant arthritis.
Immunohistologic detection of resident macrophages (brown) (undecalcified cryostat sections, using the ED1 marker and the peroxidase
technique) on day 13 (A and C) and day 28 (B and D): I day and 2 weeks following treatment with phosphate buffered saline (A and B) or
high-dose clodronate-laden liposomes (C and D).s = synovial membrane; c = cartilage; b = bone; p = pannus. Arrowheads in B and D indicate
the bone-pannus or cartilage-pannus junction. (Original magnification x 186.)
rats (data not shown): no overt pathologic alterations
were found, other than granulomas typical of AA (43).
Such a degree of safety following treatment with
clodronate-laden liposomes may result from the extreme selectivity of this preparation for macrophages.
This is indicated by the sparing of PMN, a finding
consistent with results of other investigations that
have employed clodronate-laden liposomes in vivo
(44).Selectivity for macrophages rather than PMN or
other inflammatory cells has also been observed in vitro
(Schmidt-Weber et al: Unpublished observations).
Another factor that may add to the relative
safety of clodronate-laden liposomes is that, in vitro,
they cause cell death in macrophages via apoptosis
(45); because cellular debris resulting from macrophage cell death remains confined to apoptotic bodies,
secondary damage due to the release of enzymes
and/or mediators into the surrounding tissues may be
prevented, therefore limiting the side-effects of therapy. This hypothesis is supported by the lack of
inflammatory infiltrates in all tissues/organs subjected
to macrophage depletion (data not shown). Another
1787
SYSTEMIC MACROPHAGE ELIMINATION AMELIORATES AA
day 13
day 28
r
I
.
Figure 8. Depletion of macrophages from the spleen of rats with adjuvant-induced arthritis, following treatment with high-dose clodronateladen liposomes. Immunohistologic detection of resident macrophages (brown; performed as described in Figure 7, but using the ED3 marker)
on day 13 (A and C) and day 28 (B and D): 1 day or 2 weeks following treatment with phosphate buffered saline (A and B) or high-dose
clodronate-laden liposomes (C and D). Clodronate-laden liposomes led to virtually complete depletion of marginal-zone macrophages; depletion
was also striking among marginal metallophilic macrophages (arrowheads in A) at the border between the periarteriolar lymphatic sheaths (P)
and the marginal zone (M) on day 13 (C). The normal configuration of this subset of macrophages was basically restored on day 28 (D); on this
date, significant depletion of marginal zone and periarteriolar lymphatic sheath macrophages was still evident using the markers ED1 and ED2
(see Table 1). (Original magnification x 186.)
positive outcome of the present study was that massive elimination of macrophages in immunocompetent
areas did not increase the susceptibility of rats to
infectious diseases, even under standard conditions of
animal care.
In the present study, clodronate-laden liposomes proved at least as beneficial as standard oral
treatment with dexamethasone (46). This drug signifi-
cantly improved almost all parameters of AA, but only
in a transient manner, which was followed by a rebound even exceeding the severity of PBS-treated AA.
Such a rebound did not occur in rats treated with
clodronate-laden liposomes.
Control treatments were, for the most part,
ineffective. Free clodronate, however, significantly
reduced the clinical severity of AA and reduced the
1788
ESR, but only in the late phase of the disease, as has
been shown in previous studies (47,48). The limited
efficacy of clodronate in the free form was observed
despite administration of a dose in excess of the
encapsulated counterpart (15 mg versus 2.7 mg, respectively). This finding indicates that a major and
prompt effect is only seen when the drug is encapsulated and efficiently delivered in high concentrations to
macrophages. The antiarthritic properties of this drug
can therefore be highly amplified through selective
delivery to macrophages, complementing its anti-bone
resorption properties as a free substance (47-49).
PBS-laden liposomes, in turn, even aggravated
the degree of joint destruction on day 28 of AA,
suggesting that, to achieve antiarthritic effects, it is not
sufficient to merely interfere with the phagocytic function of macrophages. This result is consistent with
significant clinical aggravation in a different model of
arthritis, antigen-induced arthritis, using the same rat
strain and the same treatment protocols (ref. 34 and
Buchner et al: Unpublished observations). The uptake
of nontoxic liposomes may result in a potentiation of
the pro-inflammatory properties of macrophages,
which is consistent with the immunoadjuvant properties of liposomes (50) and/or with the stimulatory effects
exerted by drug-free liposomes on monocytes in vitro
(51 3 2 ) .
There was not sufficient support for one of the
main hypotheses of the present study-that amelioration of arthritis may result from the elimination of
resident macrophages from the SM, which conceivably act as effectors of tissue destruction. There was in
fact some reduction of the subpopulations carrying the
ED1 and the ED3 markers immediately after completion of treatment with clodronate-laden liposomes, but
these changes did not reach statistical significance.
Also, these populations were completely restored on
day 28, a time in which the antiarthritic effects of
clodronate-laden liposomes were still evident. The
lack of effect at a local level in the SM is confirmed by
the fact that local intraarticular injection of clodronateladen liposomes in the course of antigen-induced arthritis, a model with a stronger local character than
AA, does not ameliorate arthritis (34). Together, these
observations indicate that the efficacy of antimacrophage treatment in AA is not at the level of the SM,
but perhaps at a more systemic level. The prolonged
efficacy of the treatment long after it was stopped also
points to a counteracting of some of the basic features
of AA, rather than sole pharmacologic control of
KINNE ET AL
pro-inflammatory properties of macrophages at the
local level.
Treatment with clodronate-laden liposomes induced a significant decrease in the percentage of
circulating monocytes, but this was only transient, and
therefore not likely to cause diminished recruitment of
macrophages into the inflamed joint. The quick restoration of circulating monocytes also indicates that the
capacity of bone marrow monocyte progenitors to
replenish monocytes remained intact, an interpretation reinforced by the fact that mature bone marrow
resident macrophages were spared by clodronateladen liposomes.
The clinical efficacy of antimacrophage therapy
may be related to the systemic effects of clodronateladen liposomes in liver, popliteal lymph node, and
spleen (Table 1). This hypothesis is based on the
observations that, in these organs, macrophages remained depleted long after treatment was completed,
and paralleled the long-term efficacy of the treatment.
In addition, depletion was quite pronounced in areas
of the spleen that are critically involved in immunoregulatory functions (5334) as well as in liver
macrophages that produce pro-inflammatory mediators such as tumor necrosis factor a (44), both factors
probably being crucial to the development of AA (55).
Furthermore, preliminary data on the reactivity of
spleen T cells, derived from rats with AA treated with
clodronate-laden liposomes, indicate that systemic
depletion of macrophages significantly increases the T
cell response to concanavalin A compared with rats
with untreated arthritis (ref. 55 and Kinne et al:
Unpublished observations). This is compatible with
the hypothesis that clinical amelioration by clodronateladen liposomes may be associated with selection of
regulatory T cell subpopulations (55).
The pattern of depletion of spleen macrophages
observed in rats with AA appears different from that in
normal rats subjected to a similar treatment (25). In the
latter case, in fact, depletion affects all macrophage
subpopulations in all areas of the spleen (25), whereas
the effects seen in the present study in arthritic animals
were restricted to immunocompetent areas of the
spleen. One factor that may render these areas particularly sensitive to the effects of clodronate-laden liposomes is the activation status of local macrophages.
Such activation, in turn, may be determined by the
processing of arthritogens during the course of the
immunization phase in AA or by the reciprocal interaction between T cells and macrophages that follows
presentation of the antigen to T helper cells (11 , 5 3 3 ) .
SYSTEMIC MACROPHAGE ELIMINATION AMELIORATES AA
Indeed, activated monocytes are more sensitive to the
cytotoxic effects of clodronate-laden liposomes in
vitro than in resting monocytes (Schmidt-Weber et al:
unpublished observations); furthermore, macrophages
isolated from several body compartments of rats with
AA are activated (4,6,56), which includes a %fold
potentiation of their phagocytic properties (56). The
incorporation of clodronate-laden liposomes may
therefore be particularly enhanced in activated macrophages compared not only with resting macrophages,
but also with other phagocytic cells, such as PMN
(44). Moreover, activated macrophages may more
efficiently process clodronate-laden liposomes following their internalization (52), thus enhancing the selectivity of the cytotoxic effects. A further potentiation of
the affinity for activated macrophages may reside in
the surface characteristics of the liposome formulation
used in the present investigation (57), as also suggested by our unpublished in vitro findings.
Preferential elimination of activated macrophages from immunocompetent areas of lymph nodes
and spleen, together with the clear influence on spleen
T cell reactivity, suggest that the antiarthritic effects of
clodronate-laden liposomes in AA occur through interference with the immunoregulatory functions of
macrophages rather than through mere elimination of
effector macrophages from the inflamed SM. Investigations are, however, currently being performed to
exclude changes in functional properties of synovial
effector macrophages.
Since selective antimacrophage therapy exerts
a true disease-controlling effect on AA by objectively
counteracting structural joint destruction (20), specific
antimacrophage principles may also be applicable to
human RA to control the mutilating course of the
disease.
ACKNOWLEDGMENTS
The authors thank U. Vorderwiilbecke, B. Muller, J.
Harzendorf, and B. Niescher for expert technical assistance;
Dr. M. Meyer (Institute for Medical Statistics and Documentation, University of Erlangen-Nuremberg) for advice on
statistical analysis; Dr. P. Rower for performing the FACS
analysis of bone marrow cells; Dr. U. Feige for helpful
discussion; Prof. W. Mohr for advice on pathologic analyses
and critical review of the manuscript; and Dr. C. Dijkstra for
invaluable advice on immunohistochemical analyses.
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adjuvant, following, long, systemic, rat, ameliorative, elimination, clodronate, containing, terms, macrophage, arthritis, liposomes
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