- Email: [email protected]
Skeletal muscle myosin is the autoantigen for experimental autoimmune myositis
Available online at www.sciencedirect.com R
Experimental and Molecular Pathology 74 (2003) 238 –243
www.elsevier.com/locate/yexmp
Skeletal muscle myosin is the autoantigen for experimental autoimmune myositis Hiroshi Nemoto,a Mahendra K. Bhopale,a Cris S. Constantinescu,b Donald Schotland,a and Abdolmohamad Rostamia,* a b
Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Division of Clinical Neurology, University Hospital, Queen’s Medical Centre, Nottingham NG7 2UH, UK Received 19 August 2002
Abstract Experimental autoimmune myositis (EAM) is a rodent model for human inflammatory muscle disease (IMD). It can be induced by immunization of rodents with skeletal muscle homogenate and adjuvant. The specific myositogenic autoantigen has not been clearly identified although some evidence points to skeletal muscle myosin. In this report we strengthen this evidence, showing that Lewis rats immunized with purified skeletal muscle myosin develop EAM with the same pattern and severity as EAM induced by whole rabbit skeletal muscle homogenate (WRM). Multiple inflammatory lesions are detected histopathologically in the biceps, quadriceps, and gastrocnemius muscles. Myosin-reactive T cells from animals immunized either with myosin or with WRM have similar patterns of antigen-induced proliferation. The results show that myosin, a component of skeletal muscle, is at least one autoantigen in EAM. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Experimental autoimmune myositis; Polymyositis; Dermatomyositis; Inflammatory muscle disease; Lewis rat; Complete Freund’s adjuvant; Pertussis toxin
Introduction Experimental autoimmune myositis (EAM) is an inflammatory autoimmune disease of skeletal muscle, which serves as a useful model for human inflammatory muscle disease (IMD), such as polymyositis and dermatomyositis, with which it shares similarities (Constantinescu et al., 1998). EAM can be induced in guinea pigs, mice, and rats by immunization with skeletal muscle homogenate (Dawkins, 1965; Kakulas, 1966; Kalden et al., 1973; Esiri and MacLennan, 1974, 1975; Rosenberg et al., 1987) or skeletal muscle protein (Kalden et al., 1973; Mogan et al., 1993). The resulting pathology consists of muscle fiber necrosis with vacuolation and infiltration with T cells (both
* Corresponding author. Department of Neurology, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, PA 19104, USA. Fax: ⫹1-215-573-2107. E-mail address: [email protected] (A. Rostami).
CD4⫹ and CD8⫹) and macrophages. There is up-regulation of statement of MHC class I and II molecules, adhesion molecules, immunoglobulins (Ig), and complement. All these features are similar to those of human IMD, therefore making EAM an attractive model system to investigate the pathogenesis of IMD. Further, it can be used for finding new therapies to suppress inflammation and muscle necrosis such as the successful treatment of EAM with intravenous gamma globulin (Wada et al., 2001). Cell-mediated and humoral immune mechanisms cooperate to produce pathology in IMD and EAM (Constantinescu et al., 1998; Dalakas, 1991). The identity of the inciting myositogenic autoantigen is not known with certainty. Myosin is an important protein component of skeletal muscle. Patients with IMD have anti-myosin autoantibodies, making myosin a strong candidate as an important autoantigen (Wada et al., 1983). We were the first to show that purified skeletal muscle myosin can induce severe EAM in Lewis rats (Nemoto et
0014-4800/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4800(03)00003-0
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243 Table 1 Muscle pathology in EAM rat Groupa
Histopathological grade 0
1
2a
2b
3a
3b
No. of EAM rats (of 6 rats) Biceps A B C Quadriceps A B C Gastrocnemius A B C a
239
sis and interstitial inflammation. We have designed a protocol of immunization with myosin in Lewis rats, which results in reproducible histological EAM while reducing the need for ancillary adjuvant. Myosin therefore remains an important potential autoantigen in human acquired inflammatory myopathy.
0 0 6
0 1 0
4 3 0
1 1 0
1 1 0
0 0 0
0 0 6
2 2 0
3 3 0
0 1 0
1 0 0
0 0 0
Animals
0 0 6
3 2 0
3 4 0
0 0 0
0 0 0
0 0 0
Six- to 8-week-old female Lewis rats (Charles River Laboratories, Wilmington, MA) were used in these experiments.
Materials and methods
A, WRM-treated; B, purified myosin-treated; C, normal saline-treated.
Antigens
al., 1993, 1994). Kojima et al. (1997) have also developed an immunization protocol in Lewis rats in which they could induce relatively severe and reproducible disease. That skeletal muscle myosin is the principal autoantigen in EAM has recently been challenged by showing that myosin-induced EAM in Lewis rats is mild, while the Cprotein-induced EAM is more severe (Kohayama and Matsumoto, 1999). In this study we extend our previous observation and demonstrate that skeletal muscle myosin induces severe clinical EAM associated with muscle necro-
Whole rabbit muscle extract (WRM) was obtained from rabbit pelvic skeletal muscle according to the method of Perry et al. (1955). Briefly, muscle tissues were collected free from nerves, connective tissue, or fascias, mixed with 30% weight-to-volume sucrose, and homogenized at 4°C. After centrifugation at 800Xg for 10 min, the supernatant was collected and either used immediately or stored at ⫺80°C and used within a month. Rabbit skeletal muscle myosin (Sigma Chemical Co., St. Louis, MO) was dialyzed against sterile phosphate-buffered saline (PBS) for 24 h and used for immunization.
Fig. 1. Inflammatory changes in Group A (WRM ⫹ CFA) showing muscle fiber necrosis plus endomysial, perimysial, and epimysial inflammation (⫻250) in biceps muscles using hematoxylin– eosin stain.
240
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
Fig. 2. Inflammatory changes in Group B (Myosin ⫹ CFA) showing (a) mononuclear cell infiltration and necrosis (⫻400) in quadriceps femoris muscles and (b) cellular infiltration in fibers plus perimysial and epimysial inflammation (⫻250) in biceps muscles. Hematoxylin– eosin stain.
Induction of EAM Rats from different groups were immunized as follows: Group A WRM in PBS (1 mg/ml) ⫹ complete Freund’s adjuvant (CFA) Group B myosin in PBS (100 g/ml) ⫹ CFA Group C PBS ⫹ CFA
On Day 0, rats were immunized subcutaneously with 1 ml of a 1:1 mixture of CFA containing M. tuberculosis 5 mg/ml (Difco, Detroit, MI) and antigen (1 mg/ml WRM or 100 g/ml myosin in PBS). Control rats received PBS in CFA only. The immunization was repeated on Days 7, 14, 21, and 28. Pertussis toxin (2 g/rat) was given intraperitoneally on Days 0 and 7.
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
241
dine. Cells were harvested and [3H]thymidine incorporation was measured in a beta-scintillation counter (Beckman LS8000). The proliferation index was read as the ratio of thymidine incorporation in the presence and absence of the stimulus in the cell culture condition.
Results Clinical signs During the observation period some rats developed some mild weakness and weight loss, but there were no significant differences between groups. Histopathology Fig. 3. The percentage of necrotic fibers in rat EAM. The percentage of necrotic fibers was determined by counting the number of necrotic fibers in the section and dividing by the total number of fibers. Three to eight thousand muscle fibers per muscle cross section were examined. In each muscle, 10 to 15 cross sections were analyzed.
Histology Rats were sacrificed by pentobarbital overdose at Day 35. Biceps femoris, quadriceps femoris, and gastrocnemius were collected from both sides, frozen in isopentane cooled with liquid nitrogen, and stored at ⫺80°C. Six-millimeterthick cryosections were cut, fixed in 70% ethanol, and stained with hematoxylin and eosin. To avoid variation in histological grading, muscle tissues were trimmed into 1 ⫻ 1-cm sections. Inflammation was scored as follows: Grade 0: no inflammation Grade 1: endomysial inflammation plus a minimum of 4 muscle fibers undergoing necrosis and/or regeneration. Grade 2a: focal perimysial inflammation ⫹ grade 1 Grade 2b: diffuse (⬎2 areas) perimysial inflammation ⫹ grade 1 Grade 3a: focal epimysial inflammation ⫹ grade 1 or 2 Grade 3b: diffuse epimysial inflammation ⫹ grade 1 or 2
Rats immunized with adjuvant only showed no abnormalities on histological evaluation. There were no statistically significant differences in histological score between groups A and B, which displayed similar degrees of muscle necrosis and regeneration (Table 1). Also, endomysial (grade 1), perimysial (grade 2) and epimysial (grade 3) inflammation were similar in groups A and B (immunized with WRM and myosin, respectively) (Figs. 1 and 2a and b). In the EAM rats (group A and B), the percentage of necrotic fibers was significantly higher in the biceps muscles than in the quadriceps or gastrocnemius muscles (P ⬍ 0.05). These percentages were, respectively, 0.22, 0.16, and 0.11 for group A and 0.20, 0.18, and 0.15 for group B. In the control group (C), all muscles showed less than 0.05% necrotic fibers (Fig. 3). Lymphocyte proliferation Lymphocytes from animals immunized with either myosin or WRM proliferated in response to myosin in a dosedependent fashion (Fig. 4). No proliferative response to myosin was seen in the lymphocytes from rats that had not been immunized with either muscle antigen (group C). Lymphocytes from all groups proliferated to Con A but not to ovalbumin (not shown).
Discussion Proliferation assay Lymphocytes were harvested from inguinal, axillary, submandibular and mesenteric lymph nodes, brought to single-cell suspensions in complete RPMI-1640 medium (BioWittaker, Walkersville, MD), and cultured at 106 cells/ml in the presence of rabbit myosin (31.2, 62.5, or 125 g/ml) for 72 h at 37°C under 5% CO2. Ovalbumin or Concanavalin A (Con A) (both from Sigma) were used as negative and positive controls, respectively. During the last 12 h of stimulation, the cells were pulsed with [3H]thymi-
This study shows that both WRM and purified myosin can induce EAM in the Lewis rat. Histological changes in the biceps, quadriceps, and gastrocnemius muscles were similar. Moreover, proliferation in response to myosin of lymphocytes taken from immunized rats was dose dependent and also comparable between the WRM and the myosin-induced EAM rats. We were the first to show that purified skeletal muscle myosin can induce severe EAM in Lewis rats (Nemoto et al., 1993, 1994). In this study we extend our earlier obser-
242
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
Fig. 4. Proliferation response of lymph node cells in rat EAM. Lymph node cells were harvested from inguinal, axillary, submandibular, and mesenteric lymph nodes and cultured in the presence of graded doses of antigens (106 cells/ml). Ovalbumin and Con A were used as negative or positive controls, respectively. After 60 h, cells were pulsed with [3H]thymidine (1 mCi/well) for the next 12 h, and then radioactivity was measured in a liquid beta-scintillation counter. The data represent the mean of six experiments in each group performed. Error bars indicate SD.
vations and demonstrate that skeletal muscle myosin induces severe clinical EAM associated with muscle necrosis and interstitial inflammation. Our results are in accord with those of Kojima et al. (1997), who demonstrated induction of EAM in Lewis rats by immunization with either partially or completely purified skeletal muscle myosin. Our immunization protocol resembles in some aspects that of Kojima et al. The difference was that we used pertussis toxin with the first and second immunizations only. Kojima et al. (1997) have shown rabbit myosin to be a suitable antigen, especially when combined with pertussis toxin for inducing EAM with high frequency. Myosin from other species such as guinea pig or human is capable of inducing more severe EAM. On the other hand, it is more difficult to prepare. Although EAM is inducible in rodents, it is not as easy to induce as other experimental autoimmune diseases. The use of pertussis as an ancillary antigen is required for disease of sufficient severity for EAM to serve as a suitable model for IMD. As in IMD, increasing evidence shows both humoral and cellular immune factors to be involved in the pathogenesis of EAM. Indeed, T cells reactive against muscle antigens including myosin have been shown in EAM. These T
cells are both CD4⫹ and CD8⫹ as are the cells identified in the inflammatory infiltrates in skeletal muscle in EAM (Nemoto et al., unpublished observations) and in IMD (Dalakas, 1991). Moreover, anti-myosin antibodies are present of sera of animals with EAM and of patients with polymyositis or dermatomyositis. The fact that pertussis toxin is an adjuvant stimulating both the humoral and the cellular arms of the immune response to coinjected antigen (Ryan et al., 1998) may explain its important role in inducing sufficiently severe EAM. Pertussis is also known to be required for a number of other experimental autoimmune diseases, in particular in mice (Constantinescu et al., 1998). In addition, pertussis can modify the natural resistance to induction of some experimental autoimmune diseases, as we have shown in SJL mice which, normally resistant to experimental autoimmune neuritis, become susceptible when immunized with peripheral nerve antigen plus pertussis (Calida et al., 2000). Our results confirm myosin as an important autoantigen in EAM. They do not exclude other potential autoantigens in the whole-muscle extract, such as C-protein, which, although a minor component of muscle, can induce severe EAM in Lewis rats (Kohayama and Matsumoto, 1999).
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
Identifying such autoantigens and creating reproducible animal models may lead to antigen-specific immune interventions, which if successful may be applicable to human inflammatory myopathies. In addition more insights into the pathogenesis of such conditions may be gained.
Acknowledgments This study was supported in part by Dr. Kakeshi Yanase’s grant to Toho University Medical School, Japan, and The Muscular Dystrophy Association.
References Calida, D.M., Kremlev, S.G., Fujioka, T., Hilliard, B., Ventura, E., Constantinescu, C.S., Lavi, E., Rostami, A.M., 2000. Experimental allergic neuritis in the SJL/J mouse: induction of severe and reproducible disease with bovine peripheral nerve myelin and pertussis toxin with or without interleukin-12. J. Neuroimmunol. 107, 1–7. Constantinescu, C.S., Hilliard, B., Fujioka, T., Bhopale, M.K., Calida, D., Rostami, A.M., 1998. Pathogenesis of neuroimmunologic diseases. Experimental models. Immunol. Res. 17, 217–227. Dalakas, M.C., 1991. Polymyositis, dermatomyositis, and inclusion body myositis. N. Engl. J. Med. 325, 1487–1498. Dawkins, R.I., 1965. Experimental myositis associated with hypersensitivity to muscle. J. Pathol. Bact. 90, 619 – 625. Esiri, M.M., MacLennan, I.C.M., 1974. Experimental myositis in rats: I. Histological and creatine phosphokinase changes and passive transfer to normal syngeneic rats. Clin. Exp. Immunol. 17, 139 –150. Esiri, M.M., MacLennan, I.C.M., 1975. Experimental myositis in rats: II. The sensitivity of spleen cells to syngeneic muscle antigen. Clin. Exp. Immunol. 17, 139 –150. Kakulas, B.A., 1966. In vitro destruction of skeletal muscle by sensitised cells. Nature 210, 1115–1118.
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Kalden, J.R., Williamson, W.G., Irvine, W.J., 1973. Experimental myasthenia gravis, myositis, and myocarditis in guinea pigs immunized with subcellular fractions of calf thymus or calf skeletal muscle in Freund’s complete adjuvant. Clin. Exp. Immunol. 13, 79 – 88. Kohayama, K., Matsumoto, Y., 1999. C-protein in the skeletal muscle induces severe autoimmune myositis in Lewis rats. J. Neuroimmunol. 98, 130 –135. Kojima, T., Tanuma, N., Aikawa, Y., Shin, T., Sasaki, A., Matsumoto, Y., 1997. Myosin-induced autoimmune polymyositis in the rat. J. Neurol. Sci. 151, 141–148. Mogan, G., Peter, J.B., Newbould, B.B., 1993. Experimental allergic myositis in rats. Arthritis Rheum. 14, 599 – 609. Nemoto, H., Schotland, D.L., Rostami, A., 1993. Myosin as an antigen for the induction of experimental autoimmune myositis. Ann. Neurol. 34, 274. Nemoto, H., Schotland, D.L., Rostami, A., 1994. Comparison of inflammatory cell phenotype in actively induced and passively transferred experimental autoimmune myositis. Muscle Nerve S1, S122. Neu, N., Rose, N.R., Beisel, K.W., Herskowitz, A., Gurri-Glass, G., Craig, S.W., 1987. Cardiac myosin induces myocarditis in genetically prediposed mice. J. Immunol. 139, 3630 –3636. Perry, S.V., 1955. Myosin adenosinetriphosphatase, in: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology, Academic Press, New York, pp. 582–588. Rosenberg, N.L., Ringel, S.P., Kotzin, B.L., 1987. Experimental autoimmune myositis in SJL/J mice. Clin. Exp. Immunol. 68, 117–129. Ryan, M., McCarthy, L., Rappuoli, R., Mahon, B.P., Mills, K.K., 1998. Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and statement of the co-stimulatory molecules B7-1, B7-2 and CD28. Int. Immunol. 10, 651– 662. Wada, J., Shintani, N., Kikutani, K., Nakae, T., Yamauchi, T., Takechi, K., 2001. Intravenous immunoglobulin prevents experimental autoimmune myositis in SJL mice by reducing anti-myosin antibody and by blocking complement deposition. Clin. Exp. Immunol. 124, 282–289. Wada, K., Ueno, S., Hazama, T., Ogasahara, S., Kang, J., Takahashi, M., Tarui., S., 1983. Radioimmunoassay for antibodies to human skeletal muscle myosin in serum from patients with polymyositis. Clin. Exp. Immunol. 52, 297–304.
Experimental and Molecular Pathology 74 (2003) 238 –243
www.elsevier.com/locate/yexmp
Skeletal muscle myosin is the autoantigen for experimental autoimmune myositis Hiroshi Nemoto,a Mahendra K. Bhopale,a Cris S. Constantinescu,b Donald Schotland,a and Abdolmohamad Rostamia,* a b
Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Division of Clinical Neurology, University Hospital, Queen’s Medical Centre, Nottingham NG7 2UH, UK Received 19 August 2002
Abstract Experimental autoimmune myositis (EAM) is a rodent model for human inflammatory muscle disease (IMD). It can be induced by immunization of rodents with skeletal muscle homogenate and adjuvant. The specific myositogenic autoantigen has not been clearly identified although some evidence points to skeletal muscle myosin. In this report we strengthen this evidence, showing that Lewis rats immunized with purified skeletal muscle myosin develop EAM with the same pattern and severity as EAM induced by whole rabbit skeletal muscle homogenate (WRM). Multiple inflammatory lesions are detected histopathologically in the biceps, quadriceps, and gastrocnemius muscles. Myosin-reactive T cells from animals immunized either with myosin or with WRM have similar patterns of antigen-induced proliferation. The results show that myosin, a component of skeletal muscle, is at least one autoantigen in EAM. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Experimental autoimmune myositis; Polymyositis; Dermatomyositis; Inflammatory muscle disease; Lewis rat; Complete Freund’s adjuvant; Pertussis toxin
Introduction Experimental autoimmune myositis (EAM) is an inflammatory autoimmune disease of skeletal muscle, which serves as a useful model for human inflammatory muscle disease (IMD), such as polymyositis and dermatomyositis, with which it shares similarities (Constantinescu et al., 1998). EAM can be induced in guinea pigs, mice, and rats by immunization with skeletal muscle homogenate (Dawkins, 1965; Kakulas, 1966; Kalden et al., 1973; Esiri and MacLennan, 1974, 1975; Rosenberg et al., 1987) or skeletal muscle protein (Kalden et al., 1973; Mogan et al., 1993). The resulting pathology consists of muscle fiber necrosis with vacuolation and infiltration with T cells (both
* Corresponding author. Department of Neurology, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, PA 19104, USA. Fax: ⫹1-215-573-2107. E-mail address: [email protected] (A. Rostami).
CD4⫹ and CD8⫹) and macrophages. There is up-regulation of statement of MHC class I and II molecules, adhesion molecules, immunoglobulins (Ig), and complement. All these features are similar to those of human IMD, therefore making EAM an attractive model system to investigate the pathogenesis of IMD. Further, it can be used for finding new therapies to suppress inflammation and muscle necrosis such as the successful treatment of EAM with intravenous gamma globulin (Wada et al., 2001). Cell-mediated and humoral immune mechanisms cooperate to produce pathology in IMD and EAM (Constantinescu et al., 1998; Dalakas, 1991). The identity of the inciting myositogenic autoantigen is not known with certainty. Myosin is an important protein component of skeletal muscle. Patients with IMD have anti-myosin autoantibodies, making myosin a strong candidate as an important autoantigen (Wada et al., 1983). We were the first to show that purified skeletal muscle myosin can induce severe EAM in Lewis rats (Nemoto et
0014-4800/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4800(03)00003-0
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243 Table 1 Muscle pathology in EAM rat Groupa
Histopathological grade 0
1
2a
2b
3a
3b
No. of EAM rats (of 6 rats) Biceps A B C Quadriceps A B C Gastrocnemius A B C a
239
sis and interstitial inflammation. We have designed a protocol of immunization with myosin in Lewis rats, which results in reproducible histological EAM while reducing the need for ancillary adjuvant. Myosin therefore remains an important potential autoantigen in human acquired inflammatory myopathy.
0 0 6
0 1 0
4 3 0
1 1 0
1 1 0
0 0 0
0 0 6
2 2 0
3 3 0
0 1 0
1 0 0
0 0 0
Animals
0 0 6
3 2 0
3 4 0
0 0 0
0 0 0
0 0 0
Six- to 8-week-old female Lewis rats (Charles River Laboratories, Wilmington, MA) were used in these experiments.
Materials and methods
A, WRM-treated; B, purified myosin-treated; C, normal saline-treated.
Antigens
al., 1993, 1994). Kojima et al. (1997) have also developed an immunization protocol in Lewis rats in which they could induce relatively severe and reproducible disease. That skeletal muscle myosin is the principal autoantigen in EAM has recently been challenged by showing that myosin-induced EAM in Lewis rats is mild, while the Cprotein-induced EAM is more severe (Kohayama and Matsumoto, 1999). In this study we extend our previous observation and demonstrate that skeletal muscle myosin induces severe clinical EAM associated with muscle necro-
Whole rabbit muscle extract (WRM) was obtained from rabbit pelvic skeletal muscle according to the method of Perry et al. (1955). Briefly, muscle tissues were collected free from nerves, connective tissue, or fascias, mixed with 30% weight-to-volume sucrose, and homogenized at 4°C. After centrifugation at 800Xg for 10 min, the supernatant was collected and either used immediately or stored at ⫺80°C and used within a month. Rabbit skeletal muscle myosin (Sigma Chemical Co., St. Louis, MO) was dialyzed against sterile phosphate-buffered saline (PBS) for 24 h and used for immunization.
Fig. 1. Inflammatory changes in Group A (WRM ⫹ CFA) showing muscle fiber necrosis plus endomysial, perimysial, and epimysial inflammation (⫻250) in biceps muscles using hematoxylin– eosin stain.
240
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
Fig. 2. Inflammatory changes in Group B (Myosin ⫹ CFA) showing (a) mononuclear cell infiltration and necrosis (⫻400) in quadriceps femoris muscles and (b) cellular infiltration in fibers plus perimysial and epimysial inflammation (⫻250) in biceps muscles. Hematoxylin– eosin stain.
Induction of EAM Rats from different groups were immunized as follows: Group A WRM in PBS (1 mg/ml) ⫹ complete Freund’s adjuvant (CFA) Group B myosin in PBS (100 g/ml) ⫹ CFA Group C PBS ⫹ CFA
On Day 0, rats were immunized subcutaneously with 1 ml of a 1:1 mixture of CFA containing M. tuberculosis 5 mg/ml (Difco, Detroit, MI) and antigen (1 mg/ml WRM or 100 g/ml myosin in PBS). Control rats received PBS in CFA only. The immunization was repeated on Days 7, 14, 21, and 28. Pertussis toxin (2 g/rat) was given intraperitoneally on Days 0 and 7.
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
241
dine. Cells were harvested and [3H]thymidine incorporation was measured in a beta-scintillation counter (Beckman LS8000). The proliferation index was read as the ratio of thymidine incorporation in the presence and absence of the stimulus in the cell culture condition.
Results Clinical signs During the observation period some rats developed some mild weakness and weight loss, but there were no significant differences between groups. Histopathology Fig. 3. The percentage of necrotic fibers in rat EAM. The percentage of necrotic fibers was determined by counting the number of necrotic fibers in the section and dividing by the total number of fibers. Three to eight thousand muscle fibers per muscle cross section were examined. In each muscle, 10 to 15 cross sections were analyzed.
Histology Rats were sacrificed by pentobarbital overdose at Day 35. Biceps femoris, quadriceps femoris, and gastrocnemius were collected from both sides, frozen in isopentane cooled with liquid nitrogen, and stored at ⫺80°C. Six-millimeterthick cryosections were cut, fixed in 70% ethanol, and stained with hematoxylin and eosin. To avoid variation in histological grading, muscle tissues were trimmed into 1 ⫻ 1-cm sections. Inflammation was scored as follows: Grade 0: no inflammation Grade 1: endomysial inflammation plus a minimum of 4 muscle fibers undergoing necrosis and/or regeneration. Grade 2a: focal perimysial inflammation ⫹ grade 1 Grade 2b: diffuse (⬎2 areas) perimysial inflammation ⫹ grade 1 Grade 3a: focal epimysial inflammation ⫹ grade 1 or 2 Grade 3b: diffuse epimysial inflammation ⫹ grade 1 or 2
Rats immunized with adjuvant only showed no abnormalities on histological evaluation. There were no statistically significant differences in histological score between groups A and B, which displayed similar degrees of muscle necrosis and regeneration (Table 1). Also, endomysial (grade 1), perimysial (grade 2) and epimysial (grade 3) inflammation were similar in groups A and B (immunized with WRM and myosin, respectively) (Figs. 1 and 2a and b). In the EAM rats (group A and B), the percentage of necrotic fibers was significantly higher in the biceps muscles than in the quadriceps or gastrocnemius muscles (P ⬍ 0.05). These percentages were, respectively, 0.22, 0.16, and 0.11 for group A and 0.20, 0.18, and 0.15 for group B. In the control group (C), all muscles showed less than 0.05% necrotic fibers (Fig. 3). Lymphocyte proliferation Lymphocytes from animals immunized with either myosin or WRM proliferated in response to myosin in a dosedependent fashion (Fig. 4). No proliferative response to myosin was seen in the lymphocytes from rats that had not been immunized with either muscle antigen (group C). Lymphocytes from all groups proliferated to Con A but not to ovalbumin (not shown).
Discussion Proliferation assay Lymphocytes were harvested from inguinal, axillary, submandibular and mesenteric lymph nodes, brought to single-cell suspensions in complete RPMI-1640 medium (BioWittaker, Walkersville, MD), and cultured at 106 cells/ml in the presence of rabbit myosin (31.2, 62.5, or 125 g/ml) for 72 h at 37°C under 5% CO2. Ovalbumin or Concanavalin A (Con A) (both from Sigma) were used as negative and positive controls, respectively. During the last 12 h of stimulation, the cells were pulsed with [3H]thymi-
This study shows that both WRM and purified myosin can induce EAM in the Lewis rat. Histological changes in the biceps, quadriceps, and gastrocnemius muscles were similar. Moreover, proliferation in response to myosin of lymphocytes taken from immunized rats was dose dependent and also comparable between the WRM and the myosin-induced EAM rats. We were the first to show that purified skeletal muscle myosin can induce severe EAM in Lewis rats (Nemoto et al., 1993, 1994). In this study we extend our earlier obser-
242
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
Fig. 4. Proliferation response of lymph node cells in rat EAM. Lymph node cells were harvested from inguinal, axillary, submandibular, and mesenteric lymph nodes and cultured in the presence of graded doses of antigens (106 cells/ml). Ovalbumin and Con A were used as negative or positive controls, respectively. After 60 h, cells were pulsed with [3H]thymidine (1 mCi/well) for the next 12 h, and then radioactivity was measured in a liquid beta-scintillation counter. The data represent the mean of six experiments in each group performed. Error bars indicate SD.
vations and demonstrate that skeletal muscle myosin induces severe clinical EAM associated with muscle necrosis and interstitial inflammation. Our results are in accord with those of Kojima et al. (1997), who demonstrated induction of EAM in Lewis rats by immunization with either partially or completely purified skeletal muscle myosin. Our immunization protocol resembles in some aspects that of Kojima et al. The difference was that we used pertussis toxin with the first and second immunizations only. Kojima et al. (1997) have shown rabbit myosin to be a suitable antigen, especially when combined with pertussis toxin for inducing EAM with high frequency. Myosin from other species such as guinea pig or human is capable of inducing more severe EAM. On the other hand, it is more difficult to prepare. Although EAM is inducible in rodents, it is not as easy to induce as other experimental autoimmune diseases. The use of pertussis as an ancillary antigen is required for disease of sufficient severity for EAM to serve as a suitable model for IMD. As in IMD, increasing evidence shows both humoral and cellular immune factors to be involved in the pathogenesis of EAM. Indeed, T cells reactive against muscle antigens including myosin have been shown in EAM. These T
cells are both CD4⫹ and CD8⫹ as are the cells identified in the inflammatory infiltrates in skeletal muscle in EAM (Nemoto et al., unpublished observations) and in IMD (Dalakas, 1991). Moreover, anti-myosin antibodies are present of sera of animals with EAM and of patients with polymyositis or dermatomyositis. The fact that pertussis toxin is an adjuvant stimulating both the humoral and the cellular arms of the immune response to coinjected antigen (Ryan et al., 1998) may explain its important role in inducing sufficiently severe EAM. Pertussis is also known to be required for a number of other experimental autoimmune diseases, in particular in mice (Constantinescu et al., 1998). In addition, pertussis can modify the natural resistance to induction of some experimental autoimmune diseases, as we have shown in SJL mice which, normally resistant to experimental autoimmune neuritis, become susceptible when immunized with peripheral nerve antigen plus pertussis (Calida et al., 2000). Our results confirm myosin as an important autoantigen in EAM. They do not exclude other potential autoantigens in the whole-muscle extract, such as C-protein, which, although a minor component of muscle, can induce severe EAM in Lewis rats (Kohayama and Matsumoto, 1999).
H. Nemoto et al. / Experimental and Molecular Pathology 74 (2003) 238 –243
Identifying such autoantigens and creating reproducible animal models may lead to antigen-specific immune interventions, which if successful may be applicable to human inflammatory myopathies. In addition more insights into the pathogenesis of such conditions may be gained.
Acknowledgments This study was supported in part by Dr. Kakeshi Yanase’s grant to Toho University Medical School, Japan, and The Muscular Dystrophy Association.
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