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Efficient induction of collagen arthritis by the use of a synthetic muramyl dipeptide.

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993
EFFICIENT INDUCTION OF
COLLAGEN ARTHRITIS BY THE USE OF
A SYNTHETIC MURAMYL DIPEPTIDE
TOSHITAKA KOGA, SHIGERU SAKAMOTO, KAORU ONOUE, SHOZO KOTANI,
and AKINOBU SUMIYOSHI
We developed an efficient method for production
of experimental polyarthritis. Native collagen (type 11)
from human costal cartilage was emulsified with incomplete Freund’s adjuvant (IFA) containing the synthetic adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), and injected into one hind footpad of the
PVG/c rat. This method reproducibly induced severe
arthritis with high incidence, whereas the collagen alone
in IFA produced mild arthritis with low incidence. MDP
alone in IFA was shown to be minimally arthritogenic
under identical conditions.
Collagen-induced arthritis in the rat was described by Trentham et al (1) as a unique model of autoimmunity and rheumatoid arthritis. They have shown
that type I1 collagens of human, rat, and chick cartilage
(1) or bovine vitreous (2) emulsified with incomplete
Freund’s adjuvant (IFA) are able to produce an inflammatory arthritis in the rat. They suggested that this animal model is apparently distinct from other models
which usually require injection of complete Freund’s
From the Department of Biochemistry, School of Dentistry,
Kyushu University, Fukuoka 8 12, the Department of Microbiology,
Osaka University Dental School, Osaka 530, and the Department of
Pathology, Miyazaki Medical College, Kiyotake, Miyazaki 889- 16,
Japan.
Supported by grant in aid for scientific research from the
Ministry of Education, Science, and Culture, and the Ministry of
Health and Welfare of Japan.
Toshitaka Koga, MD: Professor of Biochemistry; Shigeru
Sakamoto, MD: Research Associate; Kaoru Onoue, MD: Professor of
Biochemistry; Shozo Kotani, MD: Professor of Microbiology; Akinobu Sumiyoshi, MD: Professor of Pathology.
Address reprint requests to Toshitaka Koga, MD, Department of Biochemistry, School of Denistry, Kyushu University, Fukuoka 812, Japan.
Submitted for publication September 29, 1979; accepted in
revised form April 15, 1980.
Arthritis and Rheumatism, Vol. 23, No. 9 (September 1980)
adjuvant (CFA) with tissue antigenic components, since
IFA was as effective as CFA for production of the collagen-induced arthritis.
The present study was undertaken to establish an
efficient method to develop collagen-induced arthritis.
The results reported here clearly show that production
of collagen-induced arthritis in the rat could be augmented by using a simple synthetic compound, N-acetylmuramyl-L-alanyl-D-isoglutamineor muramyl dipeptide (MDP). This compound has been found to be a
minimally effective structure for immunoadjuvant activity of the cell walls of mycobacteria and other bacteria (3-8). Furthermore, the MDP has been shown to
be nonarthritogenic (9- 1 1) or minimally arthritogenic,
at least under the present experimental conditions.
MATERIALS AND METHODS
Rats. Inbred PVG/c rats, 8-10 weeks old and
weighing 150-200 gm, were purchased from a local animal
supplier. Some of the rats of this strain were maintained and
bred in our laboratory. They were placed in plastic cages in
groups of six or less and fed ad libitum with standard laboratory chow and water.
Type I1 cartilage. Acetone powder of human costal
cartilage was treated twice with either 4M guanidinium chloride or 2M MgC1, to remove the majority of proteoglycan
(12,13). Collagen was then solubilized by pepsin digestion in
acid solution as described by Herbage et a1 (13). The pepsin
digests were clarified by centrifugation and collagen was precipitated 2-3 times from acid solution by the addition of sodium chloride to 0.9M or 1.OM and subsequent dialysis
against 0.02M disodium hydrogen phosphate, pH 9.4. The
precipitate was finally dissolved in 0.5M acetic acid, dialyzed
against 0.01M acetic acid, and lyophilized. This collagen
preparation before purification contained 86 residues of hydroxyproline per 1000 total amino acid residues of the collagen preparation as determined with an automatic amino acid
analyzer (Hitachi Model 835).
994
KOGA ET AL
Table 1. Production of inflammatory polyarthritis in PVG/c rats by cartilage collagen alone, cartilage
collagen + MDP, and MDP alone in incomplete Freund’s adjuvant
No. of rats with
polyarthritis scoring
Material (mg)
Cartilaget alone
1.6
0.8
0.4
Cartilage collagen -k M D O
0.8
0.4
0.2
0.08
MDP alone
-
MDP
(mg)
Incidence*
0
1-5
6-10
11-16
-
2/7
6/14
4/14
5
8
10
2
6
4
0
0
0
0
0
0
0.4
0.2
0.1
0.4
25/25
12/12
4/6
2/7
0
0
2
5
5
15
9
1
5
1
2
2
0
0
1.6
0.8
0.4
0.2
0/5
2/6
0/12
0/4
014
5
4
12
4
4
0
0
0
1
0
0
1
0
0
0
0
0
0
0
0
-
0.1
2
1
* Number positive/number tested.
t Cartilage collagen before purification.
Further purification of the cartilage type I1 collagen
was performed by DEAE-cellulose chromatography to remove residual proteoglycan and noncollagenous components
according to the method of Miller et a1 (14). The fractions
containing the collagen peak which were eluted with the initial buffer were combined and rechromatographed under
identical conditions. Additional purification by differential
salt precipitation was included to remove contaminating type
I collagen as described by Trelstad et a1 (15). Briefly, the collagen-containing fraction was dialyzed against 0.4 ionic
strength phosphate buffer, pH 7.6, and an equal volume of
cold 4.4M NaCl was added to precipitate type I collagen,
which was removed by centrifugation.
The collagen preparation thus purified contained 94
residues of hydroxyproline per 1000 total amino acid residues
of the collagen preparation, and no amino sugars as measured
with the automatic amino acid analyzer. Uronic acid assay
(16) revealed no amino sugars. Polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate (17) of the highly purified collagen preparation revealed a single band peculiar to
type I1 collagen as described by Herbage et a1 (13), whereas
cartilage collagen before purification revealed other bands
possibly due to contamination of type I collagen.
Muramyl dipeptide. The synthetic muramyl dipeptide
(MDP) was obtained through the courtesy of Dr. Atsuro Inoue (Daiichi Seiyaku Co., Tokyo). The synthesis of this compound was previously reported by Kusumoto et a1 (18). MDP
was dissolved in 0.15M phosphate buffered saline (PBS), pH
7.2, (l:l, v/v, mixture of 0.15M NaCl and 0.15M phosphate
buffer) at various concentrations before use.
Preparation and injection of the inocula. Collagen was
dissolved in 0.1M acetic acid at various concentrations and
dialyzed against PBS at 4°C for 24 hours. The concentration
of collagen was determined on the basis of hydroxyproline
content or the optical density at 235 nm of samples heat-denatured at 50°C for 30 minutes.
Equal volumes of collagen suspension and MDP solution in PBS were mixed immediately before preparation of the
inocula. Ten volumes of MDP solution, collagen suspension,
or a mixture of MDP and collagen were added drop by drop
to 8.5 volumes of liquid paraffin and 1.5 volumes of Arlacel A
(Sanko Junyaku Co Ltd, Tokyo) in a mortar to make a waterin-oil emulsion.
Rats were intracutaneously injected in the pad of one
hind leg with 0.2 ml of the above inocula. The volume of the
inocula (0.2 ml) was fairly large, but it was necessary to inject
a large amount of collagen since the final concentration of
collagen in the water-in-oil emulsion could not be made
higher than 8 mg/ml because of its high viscosity.
Evaluation of collagen arthritis. The rats were examined daily for 2 months to determine the day of onset and severity of arthritis. The severity of arthritis was graded from 0
to 4 for each of the extremities (except for the injected foot)
and the tail, according to the extent of the erythema, swelling,
and ankylosis of the periarticular tissues as described previously for evaluation of adjuvant arthritis (19). The maximum possible score was 16.
Histopathology. Some of the rats were killed, and involved paws were amputated 3 weeks after injection. After
immersion in 10% neutral formalin, the joints were decalcified, imbedded in paraffin, sectioned, and stained with hematoxylin and eosin.
RESULTS
Production of chronic inflammatory polyarthritis
in the PVG/c rat with cartilage collagen and MDP. Results in Table 1 show the arthritogenic ability of carti-
995
INDUCTION OF COLLAGEN ARTHRITIS
Table 2. Production of inflammatory polyarthritis in PVG/c rats by
MDP and purified Type I1 collagen
Type 11
collagen
(mg)
0.8
0.4
0.2
0.1
0.05
-
No. of rats with
polyarthritis scoring
MDP
(mg)
0.4
0.4
0.4
0.4
0.4
0.4
-
Incidence*
0
1-5
6-10
l1-16
10/10
9/10
9/10
4/7
I /7
0/8
0/8
0
1
3
2
0
4
3
3
1
3
0
0
0
* Number positive/number
1
3
6
8
8
I
0
0
4
8
1
0
0
0
0
tested.
lage collagen with and without MDP. Twelve of 35 rats
(34%) injected with 0.4-1.6 mg of cartilage collagen in
IFA developed polyarthritis (Table 1). Day of arthritis
onset ranged from day 17 to 43 in this group. The arthritis was efficiently induced when 0.2 to 0.4 mg MDP
was added to 0.4 and 0.8 mg collagen (Table 1). All 37
rats in this group developed polyarthritis, mostly with
severe arthritic signs, after a latent period of 10 to 18
days. Two of 7 rats injected with the smallest amount of
collagen (0.08 mg) with 0.4 mg of MDP developed mild
arthritis.
As shown in Table 1, rats injected with various
doses of MDP (0.1- 1.6 mg/rat) alone emulsified in IFA
failed to develop polyarthritis, except one group in
which 2 of 6 rats developed definite arthritic signs after
administration of 0.8 mg MDP. Additionally, a few rats
in other groups occasionally had flares of inflammation
on the injected paw 2-3 weeks after inoculation but no
sign of arthritis was observed on uninjected extremities
and tail.
Since a small amount of type I collagen was contained in the above collagen preparation, the type I1
collagen preparation highly purified from human costal
cartilage was tested for its arthritogenic capacity. As
shown in Table 2, a dose-related incidence of polyarthritis was observed in the groups of rats injected wtih
various doses of the collagen with a fixed amount of
MDP in IFA. Neither MDP without collagen in IFA
nor IFA alone produced arthritis.
Histopathologic features. Sections of the affected
joints revealed that injection of type I1 collagen with
MDP produced a chronic proliferative synovitis (Figure
1A) and, at a later stage, secondary destruction of articular cartilage and bone (Figure 1B).
Sections of other cartilaginous tissues including
costal cartilage, trachea, and ear showed no signs of
generalized cartilage inflammation.
DISCUSSION
In this study we confirmed the previous reports
(1,2) showing that type I1 collagen in IFA is able to produce polyarthritis in rats. Frequency of arthritis induction in our study approximated that of the report by
Trentham et a1 (1). However, we showed that the incorDoration of the synthetic MDP into the inocula containing type 11 collagen resulted in the efficient induction of
polyarthritis with regard to the frequency and severity
of the disease.
These findings raise two possibilities. First, MDP
enhanced the induction of polyarthritis by collagen
through its adjuvant effect. Using homogenate of guinea
pig spinal cord as an antigen, we previously demonstrated the adjuvant activity of MDP for induction of
experimental allergic encephalomyelitis (EAE) in the
PVG/c rat (11). Secondly, type I1 collagen may act as
an adjuvant for induction of arthritis by MDP. The synthetic MDP may be arthritogenic by itself, as indicated
by a recent report of Nagao and Tanaka (20). They observed that MDP, when injected in IFA of Difco Laboratories (Arlacel A:Bayol F = 1.5:8.5, v/v), but not of
Arlacel A and Drackeol 6 VR, induced arthritis in
about 50% (incidence in two experiments: 3 of 5 and 3
of 7, respectively) of WKA rats at a dose of 0.1 mg. In
the present study we used IFA with Arlacel A and liquid paraffin oil (1.5:8.5, v/v) and observed only 2 cases
of arthritis in 11 PVG/c rats receiving much higher
doses (0.8-1.6 mg) of MDP, and none in the group that
received 0.4 mg or less. Although the difference in the
oil component of the IFA used may affect the frequency
and seventy of the arthritis, further study is necessary to
clarify the reason underlying these discrepancies. It
should be noted that arthritogenicity of collagen itself
was clearly shown in the present experiment as well as
in the previous report (l), and the collagen-induced arthritis was definitely augmented by the use of MDP under the present conditions in which MDP alone revealed essentially no arthritogenicity.
Trentham et a1 (1) reported the equivalent frequency (average 50%) of collagen-induced arthritis in
outbred Wistar, SD, or inbred Wistar-Lewis strains. In
the preliminary study we found that the Lewis strain is
much less susceptible to collagen-induced arthritis.
Since the Lewis strain is known to be one of the most
susceptible to adjuvant arthritis (2 l), we compared the
susceptibility of the PVG/c and Lewis strains to adjuvant arthritis by using M tuberculosis cell wall. Both
strains developed severe arthritis (average score was 13
for each) in incidences of 10 of 10 and 13 of 13, respec-
996
KOGA ET AL
A
B
Figure 1. A, Photomicrograph 01 me bursae around the affected joint showing proliferation of synovial lining cells and chronic inflammatory infiltrate with fibrosis in the sublining stroma. B, Photomicrograph of the interphalangeal joint showing thickening of the joint capsule with a dense
infiltration of chronic inflammatory cells and fibrosis. Extension of inflammatory pannus containig fibrinoid exudate is also noted over the partly
destructed and damaged articular cartilage (Both figures hematoxylin and eosin; magnification X 70).
tively. This finding shows that collagen-induced arthritis does not correlate with adjuvant arthritis in the strain
susceptibility.
In agreement with the report by Trentham et a1
(1) on collagen-iaduced arthritis, one of the prominent
features of collagen and MDP-induced arthritis was the
complete absence of the inflammatory lesions of mucocutaneous tissues, and the granulomatous lesions of
liver, spleen, lung, and kidney (unpublished data),
which are known to be frequently ancillary to adjuvant
INDUCTION OF COLLAGEN ARTHRITIS
arthritis (22). We observed that rats developed severe
arthritis with cartilage collagen and MDP (Table 1) but
no ancillary lesions. By contrast, all of 10 PVG/c rats
injected with 0.4 mg of M tuberculosis cell wall developed not only arthritis but also definite signs of various
mucocutaneous lesions.
The efficient method developed in this study for
producing collagen-induced arthritis may open a promising approach not only in the study of the mechanism
of this particular model, but also in searches for possible
arthritogenic activity of collagen other than type I1 or
proteoglycan of the connective tissues. We are currently
undertaking studies along these lines.
ACKNOWLEDGMENTS
We wish to thank Dr. Atsuro Inoue (Research and
Development Division, Daiichi Pharmaceutical Co., Tokyo)
for the supply of a synthetic muramyl dipeptide. We also
thank Ms Kyoko Higuchi for helping us with the preparation
of this manuscript.
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