J Neuropathol Exp Neurol
Copyright Ó 2005 by the American Association of Neuropathologists, Inc.
Vol. 64, No. 9
September 2005
pp. 790–796
ORIGINAL ARTICLE
Laminin-Induced Autoimmune Myositis in Rats
Jiro Nakano, MSc, Toshiro Yoshimura, MD, Minoru Okita, PhD, Masakatsu Motomura, MD,
Shintaro Kamei, PhD, Hidenori Matsuo, MD, and Katsumi Eguchi, MD
Abstract
The present study aimed to examine if immunization with laminin
causes myositis in rats and whether the pathologic findings mirror
human polymyositis and dermatomyositis. Rats were immunized with
an emulsion of laminin and complete Freund’s adjuvant. As a result,
muscle fiber necrosis with infiltrating macrophages was frequently
observed and mononuclear cells were observed in the endomysium.
These mononuclear cells were composed of CD41 cells, CD81 T
cells, and macrophages. CD41 cells and CD81 T cells were mainly
located in the endomysium, whereas a large number of macrophages
were located in the endomysium and infiltrating muscle fibers. A
small number of B cells, detected by immunohistochemical staining,
were mainly located in the perimysium. The nonnecrotic muscle fiber
to which CD41 T cells, CD81 T cells, and perforin1 cells adhered
was negative for antimerosin and antidystrophin antibodies. Muscle
fiber necrosis in rats immunized with laminin may occur after denaturation of basement membrane proteins. In conclusion, the immunization with laminin induces moderate to severe myositis. We suggest
that laminin may be an important antigen for connective tissue diseases such as polymyositis and dermatomyositis.
Key Words: Connective tissue diseases, Laminin, Macrophage,
Myositis, T cell.
INTRODUCTION
Polymyositis and dermatomyositis (PM/DM) represent
autoimmune diseases in which T cells mediate destruction of
muscle cells (1–4); however, the precise trigger for this process
remains unknown. The autoantibody detected most frequently
in patients with PM/DM is anti-histidyl-tRNA synthetase antibody (Jo-1). The detection rate is, however, only 20% to 30%
(5), and immunization with histidyl-tRNA synthetase does not
induce myositis (6, 7). Serum antibodies that bind to other
From the School of Health Sciences (JN, TY), Nagasaki University, Nakasaki,
Japan; the Faculty of Care and Rehabilitation (MO), Division of Physical
Therapy, Seijoh University, Fukinodai, Tokai-Shi, Aichi, Japan; the First
Department of Internal Medicine (JN, MM, KE), Nagasaki University
School of Medicine, Nagasaki, Japan; the Blood Products Research
Department (SK), The Chemo-Sero-Therapeutic Research Institute
(Kaketsuken), Kumamoto, Japan; and Nagasaki Medical Center of
Neurology (HM), Nagasaki, Japan.
Send correspondence and reprints requests to: Katsumi Eguchi, MD, First
Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1, Sakamoto, Nagasaki 852-8501, Japan; E-mail: eguchi@net.
nagasaki-u.ac.jp
790
amino-acyl-tRNA synthetases have also been identified in the
serum of patients with PM/DM, although they are rare (5). To
clarify the etiology of PM/DM, experimental autoimmune
myositis is studied by immunization with xenogeneic muscle
homogenates or partially purified myosin (8–14). However, no
antimyosin antibodies have been reportedly detected in human
PM/DM. It is still unclear what antigen induces PM/DM.
Because PM/DM shows inflammation not only in muscle, but
also internal organs and dermis, especially in interstitial lung
disease (15, 16) and vasculitis (17), the pathogenic antigen
should also exist in tissues such as skin, muscle, blood vessels,
and lung. We proposed laminin as a candidate PM/DMassociated antigen because it is distributed in skin, muscle,
lungs, blood vessels, kidney, and other organs (18, 19). Additionally, PM/DM have considerable relevance to malignant
tumors in which laminin is also distributed (20).
In this study, we examined whether immunization with
laminin causes myositis in rats and to clarify if the pathologic
findings are similar to human PM/DM.
MATERIALS AND METHODS
Animals
Female Wistar rats were purchased from Charles Rivers
Laboratories, Japan, and bred in our animal facility. Twentythree 8-week-old rats were used in this study. The experimental
protocol was approved by the Ethics Review Committee for
Animal Experimentation at our institution.
Antibodies
The antibodies used in this study are listed in Table 1.
Purified peroxidase-conjugated goat antirabbit/mouse immunoglobulin was purchased from Vector Laboratories Inc.
(Burlingame, CA). Antibodies were used in immunohistochemical assay except for the antilaminin antibody, which was
used as a positive control in Western blot analysis as well.
Antigen and Immunization
Eighteen Wistar rats were immunized 3 times by intradermal injection every other week with laminin (lamininimmunized rat). Laminin (1 mg/mL) from murine EngelbrethHolm-Swarm tumors (Sigma, St. Louis, MO) was emulsified
with an equal amount of complete Freund’s adjuvant
(Mycoplasma tuberculosis, 5 mg/mL), and 0.3 mL of this
emulsion containing 150 mg of laminin was injected each time
into multiple sites of the back region. Additionally, rats received an injection of 2 mg pertussis toxin (List Biological
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
Laminin-Induced Autoimmune Myositis
TABLE 1. Antibodies Used in this Study
Antigen
Clone
Laminin
Merosin (laminin-a2 chain)
CD4
CD8
B cell
CD11b (macrophage)
Perforin 1
Dystrophin (N-terminus)
Dystrophin (C-terminus)
Dystrophin (rod domain)
–
5H2
W3/25
CBL1507
RLN-9D3
OX-42
–
Dy10/12B2
Dy8/6C5
Dy4/6D3
Dilution
Antibody Type
Immunogen Source
1:500
1:500
1:500
1:500
1:500
1:1000
1:500
1:500
1:500
1:500
Rabbit, poly
Mouse, mono
Mouse, mono
Mouse, mono
Mouse, mono
Mouse, mono
Rabbit, poly
Mouse, mono
Mouse, mono
Mouse, mono
Mouse, EHS-sarcoma
Human, merosin
Rat, thymocyte membrane
Mouse, ascites
Mouse, ascites
Mouse, myeloma
Human, perforin 1
Bacterial fusion protein
Bacterial fusion protein
Bacterial fusion protein
Labs, Campbell, CA) at each time of immunization as
described previously (13). As controls, 5 Wistar rats were
injected with an equal volume of 0.01 M phosphate-buffered
saline (PBS) with complete Freund’s adjuvant and pertussis
toxin using the same protocol (adjuvant-injected rat).
Blood and Tissue Sampling
Two weeks after the last immunization, rats were anesthetized with intraperitoneal sodium pentobarbital (40 mg/g).
Blood was obtained by venipuncture, allowed to clot at room
temperature for 1 to 3 hours, and centrifuged at 1500 g. The
sera were removed, divided into aliquots, and stored deepfrozen. Skeletal muscles of soleus, extensor digitorum longus,
tibialis anterior, and gastrocnemius were extracted from right
hind limbs under anesthesia. The muscles embedded in
tragacanth gum were then frozen in isopentane chilled by
liquid nitrogen and stored deep-frozen.
Western Immunoblot for
Antilaminin Autoantibody
Laminin was boiled for 5 minutes in a buffer containing
0.001% bromphenol blue, 0.01% brilliant green, 15% glycerol, 2% sodium dodecyl sulfate (SDS), and 62.5 mM TrisHCl (pH6.8) with 4% b-mercaptoethanol in some experiments. An agarose–polyacrylamide gel with low concentration
was used because laminin was a high molecular protein (21).
Ten micrograms of protein was then applied to a 5-cm-wide
lane of 2.5% SDS polyacrylamide gel containing 1% agarose
before electrophoresis. In each experiment, molecular mass
standards (Prestained SDS-PAGE standards broad range; BioRad Laboratories, Hercules, CA) were included. The gels were
run at 20 mA for 30 minutes in running buffer (25 mmol/L
Tris, 192 mmol/L glycine, and 0.1% SDS), then transferred to
Immobilon-P polyvinylidene diflouride (PVDF; Millipore
Corp., Bedford, MA) paper at 50 V for 2 hours in transfer
buffer (25 mmol/L Tris, 192 mmol/L glycine, and 20% methanol, pH 8.3). The strips containing protein were blocked with
4% skim milk in Tris-buffered saline (50 mmol/L Tris,
150 mmol/L NaCl and 0.1% Tween, 0.01% Briji-58, pH 7.5;
TNB), then incubated with diluted (1:500) normal rat serum or
antilaminin antibody at room temperature (RT) for 1 hour. Each
strip was washed with TNB and incubated with horseradish
peroxidase (HRP) conjugated antirat/rabbit IgG diluted (1:1000)
with TNB at RT for 30 minutes. Strips were again washed with
q 2005 American Association of Neuropathologists, Inc.
TNB and developed with a 4-chloro-1-naphthol (4CN) substrate kit for peroxidase (Vector Laboratories) for 10 minutes
according to the manufacturerÕs instructions. Finally, strips
were soaked in distilled water for 5 minutes.
Histologic Analysis
Serial frozen cross-sections of muscle, 7 mm in thickness, were stained with hematoxylin and eosin (H&E). For
analysis of histologic changes, the number of necrotic muscle
fibers was counted in the area of cross-section with approximately 500 muscle fibers included in H&E-stained sections.
Necrotic muscle fiber was chosen as the fiber undergoing
phagocytosis retaining original form. Additionally, the MannWhitney U test was used for statistical quantitative analysis
for histologic change in respective skeletal muscles. The
difference between laminin-immunized rats and adjuvantinjected rats was considered significant when the p value was
less than 0.05.
Immunohistochemical Analysis
To characterize the nature of inflammatory cells in
laminin-immunized rats, an immunohistochemical study was
performed using the following antibodies: CD4 (W3/25), CD8
(CBL1507), CD11b (OX42), anti-B cell (RLN-9D3), and
antiperforin 1(H-315).
To examine the condition of membrane of muscle fibers
in laminin-immunized rats, the sections were stained using
monoclonal antibodies: antimerosin (antilaminin a2 chain;
5H2) and 3 types of antidystrophin antibodies (N-terminus;
Dy10/12B2, C-terminus; Dy8/6C5, rod domain; Dy4/6D3).
The protocol of immunohistochemical staining was as follows:
serial 8-mm frozen cross-sections were air-dried and fixed in
ice-cold ether for 10 minutes. The sections were blocked with
10% bovine albumin in 0.1 mmol/L PBS (pH 7.4) for
20 minutes. The primary antibodies were applied to the
sections overnight at 4°C. The sections were rinsed in PBS for
15 minutes, after which biotinylated sheep antimouse/rabbit
IgG (1:100) was applied for 30 minutes at RT and rinsed in
PBS. To inhibit endogenous peroxidase, these sections were
then incubated with 0.3% H2O2 in methanol for 20 minutes at
RT. After rinsing in PBS, the sections were allowed to react
with avidin–biotin peroxidase complex (Vectastain Elite kit;
Vector Laboratories) for 30 minutes at RT. HRP-binding sites
791
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
Nakano et al
were visualized with 0.05% 3,3-diaminobenzidine, 0.01%
H2O2 in 0.5 mol/L Tris-HCl buffer at RT.
RESULTS
During experimentation, rats were observed and body
weight recorded daily. Approximately 10 days after the first
injection, both laminin-immunized rats and adjuvant-injected
rats showed mild to severe swelling in part of the ankle joint
indicating arthritis. Some laminin-immunized rats and adjuvantinjected rats trailed hind limbs and their body weights had
decreased. However, there is no significant difference in
clinical signs between laminin-immunized rats and adjuvantinjected rats (data not shown).
Antilaminin Autoantibody in Rats
Western immunoblot analysis using antilaminin antibody as positive control without 4% b-mercaptoethanol treatment showed a band at approximately 800 kDa, which
corresponded to laminin. Sera from all laminin-immunized
rats without 4% b-mercaptoethanol treatment produced a
similar band at approximately 800 kDa, whereas sera from all
adjuvant-injected rats without 4% b-mercaptoethanol treatment were negative (Fig. 1A). Using antilaminin antibody as
positive control and sera from all laminin-immunized rats with
4% b-mercaptoethanol treatment, reaction was separated into
2 bands at approximately 400 kDa and 200 kDa. However, sera
from all adjuvant-injected rats treated with 4% b-mercaptoethanol were negative (Fig. 1B). Thus, antilaminin autoantibody
was detected in laminin-immunized rats only.
Histologic Analysis
As shown in Figure 2, there was mononuclear cell
infiltration in the endomysium and muscle fiber necrosis
throughout the specimens from the laminin-immunized rats.
Necrosis was usually observed in single discrete fibers, but
also often in 2 or 3 adjacent fibers. Soleus muscles were affected more severely than extensor digitorum longus, tibialis
anterior, and gastrocnemius (Fig. 2A–C). In contrast, the
numbers of necrotic muscle fibers in all adjuvant-injected rats
were less than 5 in each skeletal muscle area examined (Fig. 2D).
The number of necrotic fibers in each skeletal muscle area is
summarized in Table 2. The number of necrotic fibers was
significantly increased in laminin-immunized rats compared
with adjuvant-injected rats in each skeletal muscle tested
(p , 0.05, Mann-Whitney U test).
Immunohistochemical Analysis
In the muscle of adjuvant-injected rats, a small number
of CD41 T cell (Fig. 3F) and OX421 macrophages were
observed in the perimysium and endomysium, but they did not
infiltrate muscle fibers. CD81 T cells and B cells were not
observed (data not shown). In the muscle of laminin-immunized
rats, as shown in Figure 3, we observed the following: 1) a
large number of CD41 and CD81 T cells were mainly located
in the endomysium; 2) a small number of CD81 T cells were
located in the internal muscle fiber; 3) a large number of
OX421 macrophages were located in the endomysium and
the internal muscle fiber; and 4) a few B cells were observed
only in the perimysium. The necrotic muscle fibers showing
phagocytosis by OX421 macrophage were negative for antimerosin and 3 types of antidystrophin antibodies (Fig. 4).
Furthermore, nonnecrotic muscle fibers with adhering CD41
T cells, CD81 T cells, and perforin1 cells were all negative
for antimerosin and antidystrophy antibodies (Fig. 5).
DISCUSSION
In this study, immunization with laminin induced inflammatory myopathy. Myosin protein such as purified myosin
(13, 14, 22), purified C-protein (14), and myosin B fraction
(12, 23, 24) are more commonly used as the antigen to induce
myositis. This is the first report that laminin, one of the protein
components in skeletal muscle other than myosin, induces
myositis; however, which protein acts as antigen to induce
myositis in human PM/DM is unknown. In our study, laminin
from murine Engelbreth-Holm-Swarm tumors was used as the
FIGURE 1. Western immunoblot
analysis for antilaminin autoantibody
using sera from laminin-immunized
rats and adjuvant-injected rats. (A)
Western immunoblot analysis without 4% b-mercaptoethanol treatment. Lane 1 was positive control
for antilaminin antibody. Sera from
laminin-immunized rats (lanes 2 and
3) detected a band (arrow), whereas
sera from all adjuvant-injected rats
were negative (lanes 4 and 5). (B)
Western immunoblot analysis with
4% b-mercaptoethanol treatment.
Lane 1 was positive control for
antilaminin antibody. In sera from
laminin-immunized rats (lanes 2 and
3), reaction was observed as 2
separate bands (arrows). Sera from
all adjuvant-injected rats was negative (lanes 4 and 5).
792
q 2005 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
Laminin-Induced Autoimmune Myositis
FIGURE 2. Histologic staining with
hematoxylin and eosin in the muscle
of laminin-immunized rats and adjuvant-injected rats. Inflammatory
lesions are observed in the muscle
of laminin-immunized rats (A–C).
(A) Endomysial inflammation without necrosis (arrowhead) and necrotic muscle fiber (arrow). (B)
Hyalinized muscle fiber with cellular
infiltration (arrow) and mononuclear
cells adhering to muscle fiber (arrowhead). (C, D) Image of inflammation with low magnification
(1003) of laminin-immunized rats
(C) and adjuvant-injected rats (D).
The muscles were soleus in all
photographs. Scale bars = (A, B)
50 mm; (C, D) 200 mm.
collagen vascular disease using an enzyme-linked immunosorbent assay (33).
In the experimental autoimmune myositis induced by
immunization with partially purified myosin, CD81 T cells
and OX421 macrophages infiltrate muscle fibers and CD41
T cells are mainly located in the endomysium (13, 14). It is
also frequently observed in human PM/DM muscle that
T-cytotoxic cells invade nonnecrotic muscle fibers, and that
T-helper cells exist mainly next to the nonnecrotic muscle fiber
(1, 2, 4). Additionally, CD81 T cells adhering to nonnecrotic
muscle fibers recognize an antigen on the muscle basement
membrane and/or plasma membrane and secrete perforin to
damage the muscle fibers (34). In this study, the immunohistochemical aspects of inflammatory cells were the same as that
described previously (1, 2, 4, 13, 14, 34). According to the
antigen and is composed of a1 (400 kDa), b1 (210 kDa), and
g1 (200 kDa) chains (laminin-1). Laminin in the basement
membrane of adult skeletal muscle fibers contains rich b1 and
g1 chains (25), and polyclonal antibodies against laminin-1
bind to the basement membrane of muscle fibers (26). Laminin
b1 and g1 chains are distributed among various organs and
tissues such as skin, lungs, blood vessels, nerve, and kidney in
addition to muscle (19, 27), and they have important roles for
various types of cell adhesion (28, 29).
Patients with streptococcal-related diseases, juvenile rheumatoid arthritis, American cutaneous leishmaniasis, Raynaud’s
phenomenon, and systematic sclerosis show elevated antilaminin autoantibody in their serum with low-high frequency
(30–32). Only 8 patients with DM showed antilaminin autoantibodies, but no elevation was seen among 109 patients with
TABLE 2. Numbers of Necrotic Muscle Fibers
Animals
Muscles*
n
0
1–5
6–10
11–20
. 21
Laminin-immunized
SOL
EDL
TA
Gastro
SOL
EDL
TA
Gastro
18
18
18
18
5
5
5
5
0
0
0
0
2
0
5
3
3
9
4
6
3
4
0
2
5
6
8
6
0
1
0
0
8
2
5
5
0
0
0
0
2
1
1
1
0
0
0
0
Adjuvant-injected
Mean 6 SD
10.1
6.4
10.1
8.1
2.0
0.4
6
6
6
6
6
6
0
0.6 6
7.3**
11.3**
8.6**
5.9**
2.1
0.9
0.9
*, The numbers of necrotic muscle fibers from 500 muscle fibers examined are given. **, significant difference compared with the adjuvant-injected rats in the same skeletal muscle
(p , 0.05).
SOL, soleus; EDL, extensor digitorum longus; TA, tibialis anterior; Gastro, gastrocnemius.
q 2005 American Association of Neuropathologists, Inc.
793
Nakano et al
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
FIGURE 3. Immunohistochemical
stainings showed inflammatory cells
in the muscle of laminin-immunized
rats and adjuvant-injected rats.
CD41 and CD81 T cells were
mainly located in the endomysial
region and endomysial connective
tissue ([A, B], arrows). Some CD81
T cells infiltrated into the muscle
fibers ([B], arrowhead). OX421
macrophages were located in various regions (C). In contrast, almost
all B cells were located in the
perimysium ([D], arrow). (E) Negative control for immunohistochemical staining. The observation of
CD41 T cell was shown on behalf
of the results of adjuvant-injected
rats (F). (A, F): CD4 (W3/24), (B):
CD8 (CBL1507), (C): CD11b
(OX42), (D): B cell (RLN-9D3) antibody reactivity. Scale bar = 100 mm.
character of the infiltrating T cells described here, in our
experimental autoimmune myositis, cellular immunity mainly
comprised the autoimmune mechanism. However, lamininimmunized rats showed serum antilaminin antibody and B
cells in the perimysium were observed. Humoral immunity
may comprise part of the immune response leading to our
experimental myositis.
The nonnecrotic muscle fibers showed CD41 and
CD81 T cell adherence, and perforin1 cells were negative for
antimerosin and antidystrophin antibodies. These results
showed that merosin (laminin-2) and dystrophin might have
degenerated in the early stage of muscle fiber necrosis in
laminin-immunized rats. Laminin in the basement membrane
of muscle fibers is known to play an important role to maintain
FIGURE 4. Immunohistochemical
stainings show macrophage (A),
merosin (B), and dystrophin (C) in
the muscle of laminin-immunized
rats. The arrows in each photograph
indicate the muscle fiber undergoing
necrosis/phagocytosis by an OX421
macrophage. All muscle fibers undergoing necrosis/phagocytosis were
negative for antimerosin and antidystrophin antibodies. (A): CD11b (OX-42), (B): merosin (laminin a2 chain; 5H2),
(C): dystrophin (C-terminus; Dy8/6C5) antibody reactivity. Scale bar = 100 mm.
794
q 2005 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
Laminin-Induced Autoimmune Myositis
FIGURE 5. Immunohistochemical
staining of muscle fiber with inflammatory response and without
phagocytosis in laminin-immunized
rats. The nonnecrotic muscle fibers
marked by the asterisk in respective
photographs were the same muscle
fiber. The nonnecrotic fiber with
adherent CD41 T cell ([B], arrowhead) and CD81 T cell ([C], arrowhead) also showed an adherent
perforin1 cell ([D], arrowhead). This
fiber was also negative for antimerosin (E) and each type of antidystrophin (F) antibodies. (A): Hematoxylin
& eosin, (B): CD4 (W3/24), (C): CD8
(CBL1507), (D): perforin, (E): merosin
(laminin a2 chain; 5H2), (F): dystrophin (C-terminus; Dy8/6C5) antibody
reactivity. Scale bar = 100 mm.
function of skeletal muscle fibers and binds to important
molecules on sarcolemma such as dystroglycan complex and
dystrophin or integrins (35–39). Defective expression of the
basement membrane protein merosin (laminin-2) results in the
muscle necrosis in congenital muscular dystrophies (40–44).
Merosin (laminin-2) is composed of a2, b1, and g1 chains
(25). Laminin-1 used for immunization in this study contains
b1 and g1 chains but not a2 chain. Thus, disruption of laminin
may play a key role in the degeneration of muscle fibers in
myositis, and we think that laminin might be one of the key
antigens for PM/DM.
In conclusion, immunization with laminin induced moderate to severe myositis in the rat. The character of infiltrating T cells was the same as in human PM/DM. Lamininimmunized myositis may be an experimental model of human
PM/DM and laminin might be one of the key antigens in
human PM/DM.
ACKNOWLEDGMENTS
The authors thank Rumiko Onitsuka (Blood Products
Research Department, The Chemo-Sero-Therapeutic Research
Institute, Kaketsuken) for expert technical assistance and
Yasuki Kikuchi (School of Health Sciences, Nagasaki University) for advice on statistical methods.
REFERENCES
1. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells
in myopathies. I: Quantitation of subsets according to diagnosis and sites
of accumulation and demonstration and counts of muscle fibers invaded
by T cells. Ann Neurol 1984;16:193–208
2. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells
in myopathies. IV: Cell-mediated cytotoxicity and muscle fiber necrosis.
Ann Neurol 1988;23:168–73
3. Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells
in myopathies. V: Identification and quantitation of T81 cytotoxic and
T81 suppressor cells. Ann Neurol 1988;23:493–9
4. Engel AG, Arahata K. Monoclonal antibody analysis of mononuclear cells
in myopathies. II: Phenotype of autoinvasive cells in polymyositis and
inclusion body myositis. Ann Neurol 1984;16:209–15
q 2005 American Association of Neuropathologists, Inc.
5. Mimori T. Structures targeted by the immune system in myositis. Curr
Opin Rheumatol 1996;8:521–7
6. Blechynden LM, Lawson MA, Tabarias H, et al. Myositis induced by
naked DNA immunization with the gene for histidyl-tRNA synthetase.
Hum Gene Ther 1997;8:1469–80
7. Miller FW, Waite KA, Biswas T, Plotz PH. The role of an autoantigen,
histidyl-tRNA synthetase in the induction and maintenance of autoimmunity. Proc Natl Acad Sci U S A 1990;87:9933–7
8. Dawkins RL. Experimental myositis associated with hypersensitivity to
muscle. J Pathol Bacteriol 1965;90:619–25
9. Currie S. Experimental myositis: The in-vivo and in-vitro activity of
lymph-node cells. J Pathol 1971;105:169–85
10. Morgan G, Peter JB, Newbould BB. Experimental allergic myositis in rats.
Arthritis Rheum 1971;14:599–60
11. Rosenberg NL, Ringel SP, Kotzin BL. Experimental autoimmune myositis
in SJL/J mice. Clin Exp Immunol 1987;68:117–29
12. Matsubara S, Okumura S. Experimental autoimmune myositis in SJL/J
mice produced by immunization with syngeneic myosin B fraction. Transfer
by both immunoglobulin G and T cells. J Neurol Sci 1996;144:171–5
13. Kojima T, Tanuma N, Aikawa Y, Shin T, Sasaki A, Matsumoto Y. Myosininduced autoimmune polymyositis in the rat. J Neurol Sci 1997;151:141–8
14. Kohyama K, Matsumoto Y. C-protein in the skeletal muscle induces
severe autoimmune polymyositis in Lewis rats. J Neuroimmunol 1999;
98:130–5
15. Jansen TL, Barrera P, van Engelen BG, Cox N, Laan RF, van de Putte LB.
Dermatomyositis with subclinical myositis and spontaneous pneumomediastinum with pneumothorax: Case report and review of literature.
Clin Exp Rheumatol 1998;16:733–5
16. Ito M, Kaise S, Suzuki S, et al. Clinico-laboratory characteristics of
patients with dermatomyositis accompanied by rapidly progressive
interstitial lung disease. Clin Rheumatol 1999;18:462–7
17. Koh ET, Seow A, Ong B, Ratnagopal P, Tjia H, Chng HH. Adult onset
polymyositis/dermatomyositis: Clinical and laboratory features and treatment response in 75 patients. Ann Rheum Dis 1993;52:857–61
18. Miner JH, Patton BL, Lentz SI, et al. The laminin alpha chains: Expression, developmental transitions, and chromosomal locations of alpha1-5,
identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3
isoform. J Cell Biol 1997;137:685–701
19. Miner JH, Sanes JR. Collagen IV alpha 3, alpha 4, and alpha 5 chains in
rodent basal laminae: Sequence, distribution, association with laminins,
and developmental switches. J Cell Biol 1994;127:879–91
20. Barnes BE, Mawr B. Dermatomyositis and malignancy. A review of the
literature. Ann Intern Med 1976;84:68–76
21. Elkon KB, Jankowshi PW, Chu JL. Blotting intact immunoglobulins and
other high-molecular-weight proteins after composite agarose-polyacrylamide gel electrophoresis. Anal Biochem 1984;140:208–13
795
Nakano et al
J Neuropathol Exp Neurol Volume 64, Number 9, September 2005
22. Nemoto H, Bhopale MK, Constantinescu CS, Schotland D, Rostami A.
Skeletal muscle myosin is the autoantigen for experimental autoimmune
myositis. Exp Mol Pathol 2003;74:238–43
23. Matsubara S, Kitaguchi T, Kawata A, Miyamoto K, Yagi H, Hirai S.
Experimental allergic myositis in SJL/J mouse. Reappraisal of immune
reaction based on changes after single immunization. J Neuroimmunol
2001;119:223–30
24. Wada J, Shintani N, Kikutani K, Nakae T, Yamauchi T, Takechi K.
Intravenous immunoglobulin prevents experimental autoimmune myositis
in SJL mice by reducing anti-myosin antibody and by blocking complement deposition. Clin Exp Immunol 2001;124:282–89
25. Patton BL, Miner JH, Chiu AY, Sanes JR. Distribution and function of
laminins in the neuromuscular system of developing, adult, and mutant
mice. J Cell Biol 1997;139:1507–21
26. Schuler F, Sorokin LM. Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo. J Cell Sci 1995;108:3795–80
27. Sasaki T, Mann K, Miner JH, Miosge N, Timpl R. Domain IV of mouse
laminin beta1 and beta2 chains. Eur J Biochem 2002;269:431–42
28. Nomizu M, Kuratomi Y, Ponce ML, et al. Cell adhesive sequences in
mouse laminin beta1 chain. Arch Biochem Biophys 2000;378:311–20
29. Kadoya Y, Salmivirta K, Talts JF, et al. Importance of nidogen binding to
laminin gamma1 for branching epithelial morphogenesis of the submandibular gland. Development 1997;124:683–91
30. Avila JL, Rojas M, Velázquez-Avila G, Rieber M. Antibodies to basement
membrane protein nitrogen and laminin in sera from streptococcal-related
diseases and juvenile rheumatoid arthritis patients. Clin Exp Immunol
1987;70:555–61
31. Avila JL, Rojas M, Rieber M. Antibodies to laminin in American
cutaneous leishmaniasis. Infect Immun 1984;43:402–6
32. Gabrielli A, Montroni M, Rupoli S, Caniglia ML. A retrospective study of
antibody against basement membrane antigens (type IV collagen and
laminin) in patients with primary and secondary Raynaud’s phenomenon.
Arthritis Rheum 1988;31:1432–36
796
33. Cohen DE, Kaufman LD, Varma AA, Seibold JR, Stiller M, Gruber BL.
Anti-laminin autoantibodies in Collagen vascular diseases: The use of
adequate control in studies of autoimmune response to laminin. Ann
Rheum Dis 1994;53:191–93
34. Goebels N, Michaelis D, Engelhardt M, et al. Differential expression of
perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis. J Clin Invest 1996;97:2905–10
35. Campbell KP. Three muscular dystrophies: Loss of cytoskeleton–extracellular matrix linkage. Cell 1995;80:675–79
36. Jannapureddy SR, Patel ND, Hwang W, Boriek AM. Merosin deficiency
leads to alteration in passive and active skeletal muscle mechanics. J Appl
Physiol 2003;94:2524–33
37. Vachon PH, Loechel F, Xu H, Wewer UM, Engvall E. Merosin and
laminin in myogenesis; specific requirement for merosin in myotube
stability and survival. J Cell Biol 1996;134:1483–97
38. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion.
Cell 1992;69:11–25
39. Ozawa E, Hagiwara Y, Yoshida M. Creatine kinase, cell membrane and
Duchenne muscular dystrophy. Mol Cell Biochem 1999;190:143–51
40. Di Blasi C, Mora M, Pareyson D, et al. Partial laminin alpha2 chain
deficiency in a patient with myopathy resembling inclusion body myositis.
Ann Neurol 2000;47:811–16
41. Ringelmann B, Röder C, Hallmann R, et al. Expression of laminin alpha1,
alpha2, alpha4, and alpha5 chains, fibronectin, and tenascin-C in skeletal
muscle of dystrophic 129ReJ dy/dy mice. Exp Cell Res 1999;246:165–82
42. Hayashi YK, Engvall E, Arikawa-Hirasawa E, et al. Abnormal localization of laminin subunits in muscular dystrophies. J Neurol Sci 1993;
119:53–64
43. Tomé FM, Evangelista T, Leclerc A, et al. Congenital muscular dystrophy
with merosin deficiency. C R Acad Sci III 1994;317:351–57
44. Kuang W, Xu H, Vachon PH, et al. Merosin-deficient congenital muscular
dystrophy. Partial genetic correction in two mouse models. J Clin Invest
1998;102:844–52
q 2005 American Association of Neuropathologists, Inc.