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Experimental allergic myositis in rats.

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Experimental Allergic Myositis in Rats
Graeme Morgan, J. B. Peter and Brian B. Newbould
Experimental allergic myositis (EAMI closely resembling human polymyositis was
produced in rats by a single injection into two inguinal lymph nodes with either
homogenates or purified subcellular fractions of homologous muscle in Freund's
complete adjuvant. EAM is not RocaMarily eccompanied by clinically evident
arthritis and is manifest by elevated serum enzymes, and widespread but focal
necrosis and phagocytosis of muscle fibers. EAM was transferred to normal
animals of the same strain by infusion of washed lymphocytes from affected
animals.
Experimental myopathy has been reported in rats, rabbits and guinea pigs after
injection of homogenates of skeletal muscle
together with Freund's complete adjuvant
(1-5). T h e muscle preparations used were
of homologous or heterologous origin and
various routes of injection were employed.
T h e resultant histopathology included
muscle fiber degeneration, with or without
accompanying inflammatory change.
Pearson (1) and Kakulas (4) described
myositis in rats after the injection of muscle homogenates with Freund's complete
adjuvant. They reported that every rat
which developed myositis also had adjuvant-induced arthritis and that myositis
was sometimes found in rats with arthritis
after the injection of adjuvant alone. It was
therefore uncertain whether the myositis
that developed after injection of muscle
homogenate and adjuvant was different
from that which accompanies arthritis due
to injection of adjuvant alone. Pearson (1)
and Kakulas (4) used adjuvants containing,
respectively, 2.5 and 3.0 mg of tubercle
bacilli per milliliter. I n studies reported
herein we used adjuvants with lower concentrations of tubercle bacilli (0.25 to 0.5
mg/ml) with progressively higher concentrations of muscle hornogenate. This
caused a widespread florid myositis with
elevation of serum enzymes but without
accompanying arthritis.
From the Department of Medicine, UCLA School
of Medicine, Los Angeles, Calif.
Supported by US Public Health Service grants NS
07587, HD 02584 and GM 15759.
GRAEME MORGAN, MB, BS, MRACP: Department Of
Pediatrics, Prince of Wales Hospital, Randwick,
2031, NSW, Australia. BRIAN B. NEWBOULD, PhD: Imperial Chemical Industries, Macclesfield, Cheshire,
England. J. P. PETER, MD, PhD: Department Of Medicine, UCLA School of Medicine, Los Angeles, Calif
T h e rats were of Wistar
or Lewis strains, weighing 200-250 g a t the start of
the experiments.
90024.
Address reprint requests to: Dr. J. B. Peter.
Submitted for publication Jan 29, 1971; accepted
Mar 8. 1971.
MATERIALS AND METHODS
Experimental animals.
Preparation of homoganata~of whola muscle
and of fractions of whole murclo homogonates.
Skeletal muscle from the hind lega of Wistar rats
was used for all preparations. The muscle was
trimmed and minced and then homogenized in a
Bronwill homogenizer, according to the technic of
Peter (6). When a homogenate of whole muscle
was to be used, the preparation was then further
homogenized in a mechanical tiaue grinder of
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
599
MORGAN E l A 1
Potter-Elvehjem type, passed through cheese cloth
and then through a fine wire gauze. Only preparations which passed through a needle of 0.3 mm bore
were used. When a Fmogenate of a subfraction of
whole muscle homogenate was to be used, fractionation by differential cenqifugation was employed.
The procedures described by. Peter (6) were used
to separate the fractions use&{n Experiment 1 and
described as mitochondrial fraction and sarcotubular vesicle fraction (fragmented sarcpplasmic reticulum) . Modifications of those procedures were used
to prepare the fraction described below as “270-40,000 X g,” which contained both mitochondria and
sarcotubular vesicles. In each instance the protein
Concentration of the final suspension in 0.9% saline
was estimated by the method of Lowry (7). This
concentration was 30 mg/ml in the mitochondrial
fraction and 15 mg/ml in the fraction containing
sarcotubular vesicles in Experiment 1, and was as
indicated below in the other experiments.
Preparatlon of the emulsion for injection. The
oil phase of the emulsion contained dead, dried
tubercle bacilli (strains PN, DT and C) , Arlacel A
(5% v/v) and Squibb heavy mineral oil. The bacilli
were carefully incorporated into the oil with the
aid of a glass pestle and mortar to yield a fine
suspension. The aqueous phase consisted of saline
homogenates of various muscle fractions which were
prepared as previously described. Equal volumes of
each phase were emulsified by repeated passage
through a fine cannula connecting two 1-ml syringes
(8). The resultant emulsion was considered satisfactory if it failed to spread when dropped on the
surface of ice-cold water.
Injections into the inguinal lymph nodes. One
node on each side was injected with about 0.005 ml
of material by the technic of Newbould (8) .
Serum enzyme studles. Blood for serum enzyme studies was drawn from the tail veins under
ether anesthesia. Creatine phosphokinase was estimated by the method of Rosalki (9). using a CPK
Stat-Pack.* Aldolase and glutamate-oxaloacetate
transaminase were determined by modifications of
the methods of Sibley and Lehninger (10) and
Sigma-Frankel (11) , using reagent kits.?
Muscle specimens for
histol6gic
examlnatlon.
Muscle specimens were taken immediately after
*Calbiochem, Los Angeles, Calif.
?Sigma Chemical Company, St. Louis, Mo.
600
death, fixed in 10% formalin, cut to 5
and stained with hematoxylin and eosin.
thickness
Preparation of lymph node cell suspenslon for
cell transfer studies. Donor animals were killed
with ether and the lower abdominal and internal
axillary nodes on both sides removed and placed in
a Petri dish containing Hank’s Balanced Salt Sohtion (HBSS). A second operator transferred the
nodes to another Petri dish containing a domeshaped piece of 100 mesh, stainless steel sieve
bathed in HBSS. The nodes on the stainless steel
sieve were gently teased with a scalpel blade to
produce a lymph node cell suspension. The debris
was placed on one side of the dish for subsequent
washing, and when all the nodes had been removed
and processed, the suspension and the washings
from the debris were passed through a 100-mesh
stainless steel sieve into a centrifuge tube and spun
at 1800 rpm X 10 minutes. The supernatant was
removed and discarded and an appropriate amount
of fresh HBSS added to the lightly compacted cells
to enable 2 ml of the final cell suspension to be
available for each recipient rat. The cells were
resuspended with the aid of a Pasteur pipette. Two
milliliters of the final suspension, containing 7.8 X
10” cells (85% viable by trypan blue and aqueous
eosin), were injected slowly into the tail vein of
recipients anesthetized with ether. All equipment
was sterilized before use, but no attempt was made
to maintain complete asepsis throughout the
procedure.
EXPERIMENTAL PROCEDURES
AND RESULTS
Experiment 1
Eighteen female Wistar rats were injected once into each inguinal lymph node
with an emulsion of a muscle preparation
with Freund’s adjuvant containing 0.5 mg
tubercle bacilli per milliliter. Three muscle
preparations were used. I n Group lA, 8
rats were injected with emulsion made
from a suspension of whole muscle homogenate. Group 1B consisted of 5 rats injected with emulsion made from a fraction
consisting mainly of mitochondria and in
Group IC, 5 rats were injected with emulsion made from a fraction consisting mainly of sarcotubular vesicles. One animal
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
ALLERGIC MYOSlTlS
from each group was killed 3 weeks after
injection, and the remainder were killed 7
weeks after injection. Sections for histologic examination were made from right and
left triceps, quadriceps, tibialis anterior
and medial and lateral heads of gastrocnemius. Blood was drawn for serum enzyme
studies before the injection was given and
again immediately before death. T h e rats
were weighed daily and examined daily for
signs of adjuvant disease.
Details are given in Table 1.
Results.
I n each group, the rat sacrificed at 3 weeks
then showed mild, focal myositis. At 7
weeks, focal myositis was found in 2 of 7, 2
of 4 and 2 of 4 rats injected with whole
muscle, mitochondria or vesicles, respectively. Myositis in the absence of arthritis
occurred in all three groups, but was not
correlated with weight changes or serum
enzyme elevation.
Experiment 2
Six male Wistar rats received one injection into the inguinal lymph nodes of an
emulsion made from a fraction containing
both mitochondria and sarcotubular vesicles (27040,000 x g) together with adjuvant containing 0.5 mg tubercle bacilli
per milliliter. Two rats were killed every 2
weeks for 6 weeks. Three male Wistar rats
were injected similarly with Freund’s adjuvant alone and one was killed every 2
weeks. Histologic examination, serum enzyme studies, weighing and examination
for ‘adjuvant disease were performed as in
the first experiment.
None of the rats showed eviResults.
dence of myositis. Two developed arthritis
(Table 1).
Experiment 3
Sixteen female Lewis rats received one
injection into the inguinal lymph nodes of
a n emulsion. Group 3A consisted of 6 rats
injected with a 27040,000 x g fraction
(protein concentration 25 mg/ml) emulsified with an equal volume of adjuvant
containing 0.25 mg tubercle bacilli per
milliliter. In Group 3B, 6 rats were injected with adjuvant containing 0.25 mg tubercle bacilli per milliliter emulsified with
saline. Group 3C consisted of 4 rats injected with a 27040,000 x g fraction of muscle
(protein concentration 25 mg/ml) emulsified with Freund’s incomplete adjuvant
(ie, no tubercle bacilli). T h e animals were
killed 2 weeks after injection. Serum enzyme studies, weighing and inspection for
adjuvant disease were performed as in the
first experiment. Histologic sections were
made from rectus abdominus, right and left
biceps brachii, triceps, rectus femoris, vastus lateralis, tibialis anterior and medial
and lateral heads of gastrocnemius. I n addition, 4 animals were injected with an oil
and saline emulsion as controls for Group
3C above, but were not examined histologically because of negative findings in Group
3c.
Results.
Myositis, more severe than
that observed i n Experiment 1, was detected in 4 of 6 rats injected with the mitochondria-vesicle fraction in complete
Freund’s adjuvant. One rat had very mild
arthritis of one joint. Injection of either
the muscle fraction without adjuvant or of
complete adjuvant without muscle caused
neither significant myositis nor arthritis in
10 rats. Serum enzymes did not rise in any
of the rats (Table 1).
Experiment 4
Nineteen female Lewis rats were injected. I n Group 4A, 7 rats were injected into
both inguinal lymph nodes with an emulsion made from 27040,000 x g fraction
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
601
MORGAN I3 A 1
Table 1. Experiments 1, 2, 3,
4t
Concentration of
~.
tubercle
Protein
bacilli i n
concentra- adjuvant
tion
(mg/ml
No. of
(mg/ml) dry weight) animals Arthritis
Muscle Preparation
~
Group Strain
Sex
Fraction
1A
1B
Wistar
Wistar
F
F
1C
Wistar
F
2
3A
Wistar
Lewis
M
F
Whote muscle
Mitechondrirl
fraction
Sarcotubular
vesicle fraction
270-40,000 X g
27040,000 X g
*
0.5
8
0.5
5
1
2
Foe4 myositis in 3
30
15
0.5
5
0
Focal myositis in 3
30
25
0.5
0.25
6
6
2
None
Myositis in 4, more
severe than in Experiment 1, lesions in
1-3 muscles each animal
None
Occasional necrotic
fiber with no inflammation in one animal
None at 8 days. A t 14
days severe focal myositis in 2 of 4 ani-.
mals, with lesions in
8 or 9 muscles, ser u m enzymes elevated (Table 2)
None
None
8
-
-
1
(very
mild)
0
3C
Lewis
Lewis
F
F
270-40,000 X g
25
0.25
0
6
4
0
4A
Lewis
F
270-40,000 X g
75
0.25
7
0
48
4C
Lewis
Lewis
F
F
270-40.000 X g
0.25
0.25
4
4
0
0
3B
-
Myos itis
75
Focal myositis in 3
* Starting concentration 18% (w/v) wet weight of muscle
t All injections were into both inguinal lymph nodes; group4C injections into hind footpads were
also
given
(protein concentration 75 mg/ml) and
Freund's adjuvant containing 0.25 mg tubercle bacilli per milliliter. Group 4B consisted of 4 rats injected into both inguinal
lymph nodes with an emulsion of saline
and Freund's adjuvant containing 0.25 mg
tubercle bacilli per milliliter. In Group 4C,
4 rats were injected into both inguinal
lymph nodes with the same preparation as
in Group 4A and simultaneously received
602
injections of 0.1 ml of the same preparation into each of the hind footpads. In
Group 4D, 4 rats received an emulsion of
saline with adjuvant containing 0.25 mg
tuber'cle bacilli per milliliter, injected in
the Bame way as for Group 4C above.
Three of the animals in Group 4A were
killed 8 days after injection. T h e other 4
rats in Group 4A and the animals in
Groups 4B, 4C and 4D were killed 14 days
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
ALLERGIC MYOSlTlS
after injection. Serum enzyme studies,
weighing and inspection foi adjuvant disease were performed as in the first experiment. Sections for histologic examination
were made from right and left rectus
femoris, vastus lateralis, tibialis anterior
and medial and lateral heads of gastrocnemius of all the animals except those of
Group 4D which were not examined because of negative results in Group 4C.
Results.
Immunization was similar to
that of Experiment 3 except that the
concentration of the muscle fraction injected was tripled (Table 2). None of the three
rats examined at 8 days had myositis but 2
of 4 rats at 14 days postinjection had a
much more severe and generalized myositis
than that which developed in Experiments
1 and 3. Lesions were focal and readily
detected in 8 and 9 of the muscles examined from each rat. Serum enzyme changes
were clearly correlated with myositis (Table 2). Arthritis did not develop in any of
the 15 rats and myositis was not found in
the rats injected with the adjuvant alone
(4B) or when lymph node injections were
combined with footpad injections (4C).
Experiment 5
Thirty-six female Lewis rats received
Table 2.
injections into both inguinal lymph nodes
of a n emulsion made from 27040,000 x g
fraction and Freund's adjuvant containing
0.25 mg tubercle bacilli per milliliter.
Eighteen of the donor animals were injected 7 days before the transfer and 18 eight
days before the transfer. All the rats were
killed on the same day, their lymph node
cells were pooled and 7.8 x 108 cells were
injected into 6 female Lewis rats, 3 of
which had been adrenalectomized one day
before the transfer. One of the adrenalectomized animals died 2 days after the transfer. T h e remainder of the recipients were
killed 10 days after the transfer and sections for histologic examination made from
10 muscles as in Experiment 4.
Results.
Neither of the adrenalectomized recipients of lymph node cells
showed evidence of myositis. Two of the
3 nonadrenalectomized recipients showed
myositic lesions in one of the muscles examined. T h e lesions were similar to those
seen in affected animals in previous experiments.
Histopathology of the Myositis
Lesions in the muscles were focal and
were clearly demarcated from normal tissue
(Fig 1, 2, 4 and 5). T h e principal changes
Serum Enzyme Studies, Group 4A
CPK
Animal
1
2
3
4
Myositis
+
+
0
0
Aldolase
GOT
A
B
A
B
A
B
122
93
122
110
261
220
133
139
46
28
37
37
140
238
42
40
110
88
94
137
230
286
48
1W
Results are expressed in mU/ml, Sibley-Lehninger U/ml and U/ml serum for CPK, aldolase and GOT
respectively
A = result on blood drawn before injection
B = result on blood drawn 2 weeks after injection
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
603
MORGAN ET A1
Fig 1. Right tibialis anterior of affected animal in Group 4A demonstrates focal nature of lesion surrounded by normal fibers. There is necrosis of fibers and perivascular infiltration with inflammatory cells
(X 75).
Fig 2. Left tibialis anterior (same animal as Figure 1) shows similar extensive lesion with necrotic fibers,
raplacement by inflammatory cells and giant cells with ringed arrangement of nuclei (X 75).
604
Arthritis and Rheumatism, Voi. 14, No. 5 (September-October 1971)
ALLERGIC MYOSlTlS
Fig 3. Higher magnification of Figure 2 shows normal vessel walls, necrotic fiber (no striations), inflammatory cells and e giant cell with a ring of nuclei (X 320).
Fig 4. Lateral head of gastrocnemius (Experiment 4A) shows extensive lesion. The narrow, oblong
structures are regenerating fibers (x 75).
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
605
MORGAN ET A 1
Fig 5. Higher magnification of Figure 4 shows regenerating fibers with vesicular nuclei and prominent
nucleoli. The normal striation of an adjacent fiber is apparent (x 500).
were necrosis and phagocytosis of muscle
fibers and infikration by inflammatory cells
in and between fibers (Fig 1-5). I n the
more extensive lesions, evidence of muscle
fiber regeneration was seen (Fig 4 and 5).
Lesions varied markedly in extent. Minimal lesions, consisting of isolated necrotic
fibers, were more often seen in the earlier
experiments, whereas in Experiment 4 (in
which more antigen was injected) the
lesions involved areas of 20 or more contiguous fibers and could be seen in stained
sections with the naked eye (Fig 2 and 4).
T h e inflammatory exudate consisted mostly
of mononuclear cells with smaller numbers
of polymorphonuclear cells and plasma
cells (Fig 3). Muscle fiber regeneration was
evidenced, in the larger lesions, by narrow
fibers which stained blue with hematoxylin
and eosin and which had vesicular nuclei
with prominent nucleoli. Giant cells were
also seen; these were usually circular, with
606
a ring of nuclei (Fig 3). Lesions were
usually perivascular and extensive lesions
were always so. T h e vessel walls appeared
normal (Fig 2 and 3).
Histopathology in the affected recipients
of the lymphocytes from injected animals
showed the same characteristics as i n actively immunized animals. Some foci consisted of necrosis and phagocytosis of single
muscle fibers (Fig 6); others were characterized by perivascular collections of inflammatory cells replacing muscle fibers (Fig 7
and 8). T h e lesions resembled in extent
those seen in the earlier experiments rather
than the very florid lesions observed in
Experiment 4.
DISCUSSION
Previous reports (1-5) have described the
production of experimental myositis in
rats, rabbits and guinea pigs after injections of muscle homogenate with Freunds
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
ALLERGIC MYOSlTlS
Fig 6. Single necrotic fiber with phagocytosis from the rectus femoris of non-adrenalectomized recipient
of lymph node cells (Experiment 5). The section contained four isolated fibers with this appearance which is
identical with that seen in actively induced myositis in Experiment 1 (x 500).
Fig 7.
Inflammatory infiltration in the same section as Figure 6
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
(x 75).
607
MORGAN ET A 1
Fig 8. Higher magnification of Figure 7
adjuvant. Evidence relating to its pathogenesis has been slight and inconclusive.
However, the technics used to induce it
have been like those used to induce other
experimental allergic diseases, suggesting
the provisional use of the term experimental allergic myositis (EAM).
In previous reports (1,4), myositis induced in rats has always been accompanied
by arthritis. It is therefore uncertain
whether the disease was independent of the
myositis (1, 4), which may accompany arthritis after injection of adjuvant alone. We
have produced myositis in rats by giving a
single injection into the inguinal lymph
nodes of emulsions made from muscle homogenates and Freunds adjuvant. Our
studies show ithat when the concentration
of muscle homogenate in the emulsion is
increased, and the concentration of tubercle bacilli in the adjuvant is decreased, an
increasingly florid myositis is produced
608
(x 500).
with a decreasing incidence of arthritis. We
conclude from this that experimental allergic myositis is independent of the myositis
which accompanies adjuvant-induced arthritis.
Serum enzyme changes have not been
referred to in previous reports of experimental allergic myositis. T h e myositis induced by high concentrations of muscle
(Experiment 4) is severe and is accompanied by elevation of serum creatine phosphokinase, serum glutamate-oxaloacetate
transaminase and serum aldolase. This
makes the experimental model a much
more useful one, for example in observing
the course of the disease and the influence
upon it of agents or procedures which
might either exacerbate or ameliorate it.
T h e results so far of our attempts to
induce experimental allergic myositis by
passive transfer of isogeneic lymph node
cells suggest that the disease is a manifesta-
Arthritis and Rheumatism, Vol. 14, No. 5 (September-October 1971)
ALLERGIC MYOSlTlS
tion of a cell-mediated immune response.
We are undertaking studies t o examine t h e
possibility that myositis i n the recipients of
t h e lymph node cells was d u e to transfer of
antigen rather t h a n of sensitized cells, b u t
this possibility seems remote. The relative
mildness of the passively induced disease
may be d u e to t h e very large mass of the
target organ compared, for example, with
t h e target organ i n experimental allergic
thyroiditis. O u r findings differ in this respect from those of Takayanagi ( 5 ) who
observed more severe myositis in recipient
guinea pigs after irradiation a n d transfer of
cells, t h a n in guinea pigs with actively
induced disease. Unfortunately Takayanagi’s report does n o t describe any studies i n
animals receiving irradiation -only and
hence the significance of his passive transfer data is dubious.
REFERENCES
1. Pearson CM: Development of arthritis,
periarthritis and periositis in rats given
adjuvants. Proc Soc Exp Biol Med 91:95101, 1956
2. T a l C, Liban E: Experimental productian
of muscular dystrophy-like lesions in rabbits and guinea pigs by an autoimmune
process. Brit J Exp Path 43:525-529, 1962
3. Dawkins RL: Experimental myositis associated with hypersensitivity to muscle.
J Path Bact 90:619-625, 1965
4. Kakulas BA: Destruction of differentiated
muscle cultures by sensitized lymphoid
cells. J Path Bact 91:495-503, 1966
5. Takayanagi T: Immunohistological studies
of experimental myositis in relation to
human polymyositis. Folia Psychiat Neurol
Jap 21:117-127, 1967
6. Peter JB: Studies of human skeletal muscle
mitochondria. Biochem Med 2:179-189,
1968
7. Lowry OH, Rosebrough NJ, Farr AL, et al:
Protein measurement with the Folin
Phenol Reagent. J Biol Chem 193:265-275,
1951
8. Newbould BB: Production of allergic encephalomyelitis in rats by injections of
spinal cord adjuvant into the inguinal
lymph nodes. Immunology 9:613-614, 1965
9. Rosalki SB: An improved method for
serum creatine phosphokinase determination. J Lab Clin Med 69:696-705, 1967
10. Sigma Technical Bulletin, No 750, Dec,
1965, Sigma Chemical Co, 3500 DeKalb
Street, St Louis, Missouri
11. Sigma Technical Bulletin, No 505, Sept,
1964. Sigma Chemical Co, 3500 DeKalb
Street, St Louis, Missouri
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