Experimental autoimmune myocarditis
Heart weight-to-body weight ratio
Left ventricular dimension in systole
Peak left ventricular pressure
Central venous pressure
Left ventricular end-diastolic pressure
- ± dP/dt:
First derivatives of left ventricular pressure
Stem cell factor
Progression of acute myocarditis involves a variety of inflammatory events. Mast cells have been implicated as the source of various cytokines, chemokines and histamine in acute inflammation and fibrosis. Interleukin (IL)-10 has well-known immunomodulatory actions that are exerted during the recovery phase of myocarditis. In this study, 9-week-old male Lewis rats were immunized with cardiac myosin. A plasmid vector expressing mouse IL-10 cDNA (800 μg per rat) was then transferred three times (7, 12 and 17 days after immunization) into the tibialis anterior muscles of the rats by electroporation. Microscopic examination of mast cells was carried out on toluidine blue-stained transverse sections of the mid ventricles. Mouse IL-10 gene transfer significantly reduced mast cell density, cardiac histamine concentration and mast cell growth, and prevented mast cell degranulation. Furthermore, improvement in both myocardial function and the overall condition of the rats was evident from the reduction in the heart weight-to-body weight ratio and inflammatory infiltration as well as improvement in hemodynamic and echocardiographic parameters. These findings suggest that IL-10 gene transfer by electroporation protected against myocarditis via mast cell inhibition.
Experimental autoimmune myocarditis (EAM) can be induced in rats by immunizing them with cardiac myosin together with complete Freund's adjuvant, providing a model that mimics the pathophysiology of human giant cell myocarditis 1, 2. Histological examination of hearts with EAM demonstrates infiltration of inflammatory cells with myocardial damage 3, 4. EAM in rats is induced 2 weeks after injecting cardiac myosin into the foot pads. Thereafter, myocarditis peaks around the 3rd week, and then gradually subsides during the 4th week. In the later stage, approximately after day 40, myocarditis progresses to dilated cardiomyopathy.
The inflammatory process underlying EAM is induced by a cellular immune response 5–8. T cells are reported to play an important role in EAM 6. Antibody against αβ T cell receptor blocks the induction of EAM in Lewis rats 9. EAM is also inducible in some strains of mice. Depletion of CD8+ T cells reduces the severity of cardiac myosin-induced EAM in mice 10. The precise pathogenesis of EAM may differ among species and strains of experimental animals. EAM in both rats and mice is a T cell-mediated autoimmune disease. Activated T cells secrete various chemokines and cytokines which recruit and activate other inflammatory cells, such as macrophages, neutrophils and mast cells.
Mast cells are resident cells in a variety of organ tissues, and have been implicated in the pathogenesis of certain inflammatory diseases, such as bronchial asthma 11, arthritis 12 and pancreatitis 13. These cells have also been demonstrated to be associated with various cardiovascular inflammatory conditions, including hypertensive cardiac hypertrophy 14, ischemia-reperfusion injury 15, ischemic cardiomyopathy 16 and cardiac allograft rejection 17. In 1968, Fernex 18 suggested that there may be a link between increased mast cell density and endomyocardial fibrosis. Recent reports have demonstrated a definite role of mast cells in other fibrogenic disorders 19–21. Paolocci et al. 22 showed that the degranulation of mast cells and eosinophils might induce myocardial damage and dysfunction in rats challenged with Trichinella spiralis. Those reports demonstrated an active role of mast cells in cardiac inflammation and fibrosis.
In rat EAM, active inflammation occurs at an early stage, and at a later stage (starting around day 28), fibrosis becomes prominent 4. Therefore, investigating this animal model may provide some additional information about mast cells. The mast cell is a multifunctional effector cell that expresses many receptors on its surface. Cytokine receptors such as CD2, CD121, CD18, CD50, CD54 and c-kit are expressed on mast cells, where they mediate adhesion, aggregation, degranulation and production of cytokines 23. Interleukin (IL)-10 is an anti-inflammatory cytokine that binds to specific IL-10 receptors located on mast cells and prevents the release of inflammatory mediators 24. This immunomodulatory cytokine plays an important role in the recovery from EAM by inhibiting pro-inflammatory T helper type 1 cells, macrophages and cytokines 25–27.
Among the non-viral techniques for in vivo gene transfer, electroporation is simple, safe and inexpensive. In earlier studies, it has been shown that gene transfer into muscles by electroporation can be used to deliver cytokines systemically 28, 29. In the present study, we investigated whether IL-10 gene transfer by electroporation protected rats from acute myocarditis and whether the effect of IL-10 involved the modulation of mast cells.
2.1 Heart weight-to-body weight ratio
The heart weight-to-body weight ratio (HW/BW) of group V (myosin-injected rats treated with empty plasmid) was increased compared to that of group N (normal rats). Administration of plasmid mouse IL-10 (myosin-injected rats treated with mouse IL-10 plasmid) significantly decreased HW/BW in group IL-10 (p<0.05; Table 1).
|Group N||Group V||Group IL-10|
|CVP (mm Hg)||–0.4±0.2||12.7±1.2*||5.8±0.6*,***|
|LVP (mm Hg)||110±1||65±4*||76±2*,**|
|LVEDP (mm Hg)||4.3±1.1||10.6±0.3*||5.9±0.6***|
|+ dP/dt (mm Hg/s)||5,837±224||4,902±408||5,564±298|
|– dP/dt (mm Hg/s)||5,898±248||5,127±354||5,872±331|
2.2 Serum mouse IL-10 and rat IL-10 levels
The levels of serum mouse IL-10 on day 21 in group V and group IL-10 were <5 pg/ml and 278±70 pg/ml, respectively. The serum rat IL-10 levels in group V and group IL-10 were 12±2 pg/ml and 10±3 pg/ml, respectively. In group N, both serum mouse IL-10 and rat IL-10 were below the level of detection.
2.3 Hemodynamic and echocardiographic parameters
In group V, both central venous pressure (CVP) and left ventricular end-diastolic pressure (LVEDP) were significantly increased, while peak left ventricular pressure (LVP) was significantly decreased, relative to the values in group N. A slight increase in heart rate (HR) and decrease in the first derivatives of left ventricular pressure (± dP/dt) were observed in group V in comparison to group N. A significant reduction in CVP and LVEDP and a significant increase in LVP were found in group IL-10, along with a slight increase in ± dP/dt and reduction in HR. The changes in ± dP/dt and HR were insignificant (Table 1).
The left ventricular dimension in systole (LVDs) was increased and ventricular fractional shortening (FS) was decreased significantly in group V compared to group N. In the case of group IL-10, LVDs was decreased (p<0.01) and FS was increased (p<0.01) significantly (Table 1).
2.4 Histamine concentration in the heart tissue and mast cell density
The concentration of histamine in the wet heart tissue of group N (1.6±0.3 μg/g of heart) was significantly lower than that of group V (3.4±0.6 μg/g of heart). In the IL-10 group, the histamine concentration (1.3±0.2 μg/g of heart) was significantly reduced compared to that of group N (Fig. 1A). The mast cell density in the myocardium of group V (12.8±2.8 cells/mm2) was higher than that of group N (8.4±0.4 cells/mm2). Administration of mouse IL-10 plasmid greatly reduced the mast cell density in group IL-10 (3.5±0.03 cells/mm2; Fig. 1B). The differences in mast cell density are shown in Fig. 2A–C, along with inflammatory infiltration (Fig. 2D–F).
2.5 Microscopy of mast cells
As shown in Fig. 1C, the median mast cell size in group V (10±0.6 μm) was larger than that in group N (5±0.4 μm), and this median mast cell size was decreased in group IL-10 (6.6±0.4 μm). The difference in mast cell size can be seen clearly in Fig. 3A–C. In addition to the enlargement of mast cells, mast cells with degranulation and partial degranulation were observed in group V (Fig. 3D, E). Degranulation was absent in the other two groups.
2.6 Immunohistochemical staining of stem cell factor or c-kit ligand
The immunoreactivity for stem cell factor (SCF) was stronger in group V than in group N, and it was reduced in group IL-10 (Fig. 4A–C).
The protective effect of mouse IL-10 gene transfer in the EAM model in rats was evident from the reduction in HW/BW and inflammatory infiltration. Hemodynamic and echocardiographic results also supported this conclusion. Gene transfer of viral IL-10 by electroporation also significantly improved the survival rate and reduced both cellular infiltration and the expression of interferon-γ and inducible nitric oxid synthase 30. In general, gene transfer by electroporation offers advantages over methods using viral vectors, such as the ability to perform repeated gene transfer without immunological interference by the vector, the negligible chance of recombination with the cell genome, and the easy procurement of purified DNA plasmid.
We found that cardiac mast cell density and histamine concentration of heart tissue were increased with acute myocarditis in rats. Increased histamine concentration in cardiac tissue also corresponds to increased mast cell density 16. In clinical situations, the presence of mast cells around and within the coronary blood vessels has been implicated in the pathophysiology of coronary artery disease 31–33. The role of mast cells has been established in myocardial fibrosis in Africans 18, and in dilated cardiomyopathy secondary to systemic sclerosis 34. The chronic stage of our animal model leads to dilated cardiomyopathy with pathophysiology similar to that of human dilated cardiomyopathy. The current investigation may shed some light on mast cells as a therapeutic target in these diseases.
We speculate that increased mast cell density in acute myocarditis may result from chemotaxis to cytokines released from T helper type 1 cells, in situ differentiation of mast cell precursors under inflammatory stimuli, replication of resident cardiac mast cells, and increased c-kit ligand or SCF. Our microscopic observations demonstrated that IL-10 gene transfer prevented degranulation and reduced the growth and density of mast cells. Based on the results of HW/BW, hemodynamics and echocardiography together with the prevention of mast cell degranulation and the reduction in mast cell density, cardiac histamine concentration and mast cell growth, one can suggest that inhibition of mast cells might have improved the cardiac performance of rats with acute myocarditis.
Long-term exposure to IL-10 down-regulates mast cell effector proteins c-kit and Fcϵ receptor I, and subsequently induces mast cell apoptosis 35. Likewise, gene transfer of IL-10 in our protocol may also have provided long-term exposure. As reported earlier, in EAM rats, mouse IL-10 levels are over 80 pg/ml until day 10 after a single administration of mouse IL-10 cDNA, which is greater than the peak value of serum rat IL-10 during the natural course of progression of acute myocarditis in this animal model (42±4 pg/ml) 36. Also in the present study, administration of mouse IL-10 cDNA on days 7, 12 and 17 resulted in elevation of the serum IL-10 level on day 21 compared to that in the group V animals (which received only empty plasmid on those days), which is again much greater than the peak value of serum rat IL-10 during the natural course of progression of acute myocarditis.
The concentration of c-kit ligand or SCF, which is known to induce differentiation, aggregation, growth and activation of mast cells 37–41, was reduced by IL-10 gene transfer when compared to that in the acute myocarditis group, as seen in the immunohistochemical staining. Therefore, IL-10 gene transfer may have prevented mast cell degranulation and decreased the growth and density of mast cells via these mechanisms. Moreover, mast cell-derived mediators are pro-inflammatory 42, 43 and fibrogenic 44, 45 in nature. Thus, the protective effect of IL-10 gene transfer may have been due to prevention of the release of these mediators. The present study did not focus on these molecular mechanisms. However, to the best of our knowledge, this is the first report about the effect of IL-10 on mast cells in acute myocarditis. In conclusion, a reduction in cardiac mast cell density and growth as well as the prevention of mast cell degranulation may have contributed to the protective effects of IL-10 gene transfer by electroporation in acute myocarditis.
4 Materials and methods
Nine-week-old male Lewis rats (Charles River Japan Inc., Kanagawa, Japan) were injected into the foot pads with antigen-adjuvant emulsion according to the procedure described previously 1, 46, 47. EAM morbidity was achieved in 100% of the rats immunized by this method 1, 46, 47. Throughout the studies, the rats were treated in accordance with the guidelines for animal experiments of our institute 47.
4.2 Construction of mouse IL-10 expression vector
Mouse IL-10 cDNA cloned after amplification by polymerase chain reaction was inserted into the unique Xho I site between the cytomegalovirus immediate early enhancer-chicken β-actin hybrid promoter and rabbit β-globin poly A site of the pCAGGS expression plasmid 28, 29. The resulting plasmid, pCAGGS-IL-10, was grown in Escherichia coli DH5α and prepared using plasmid purification columns (Qiagen, Tokyo, Japan). The purified plasmid DNA was dissolved in a buffer (10 mmol/l Tris-HCl and 1 mmol/l EDTA, pH 8.0), stored at –20°C and diluted to 4 μg/μl with phosphate-buffered saline (pH 7.4) immediately before use.
4.3 Intramuscular DNA injection and electroporation
Rats were anesthetized with diethyl ether. Aliquots of 50 μl of the plasmid DNA (pCAGGS-IL-10 or control pCAGGS) at 4 μg/μl in phosphate-buffered saline were injected four times (the total amount of DNA was 800 μg per rat) into the bilateral tibialis anterior muscles using a disposable insulin syringe with a 27-gauge needle 28. A pair of electrode needles with a gap of 5 mm was inserted into the muscle to a depth of 5 mm to encompass the DNA injection sites, and electrical pulses were delivered four times at 100 V using an electrical pulse generator (TR Tech. Co. Ltd., Tokyo, Japan) 28.
4.4 Estimation of mouse IL-10 serum level
On day 21, blood samples obtained from the inferior vena cava after hemodynamic and echocardiographic measurements were centrifuged (700 × g) for 20 min, the supernatant was collected, and mouse IL-10 and rat IL-10 were examined using an ELISA kit according to the manufacturer's instructions (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, UK).
4.5 Treatment protocol
Lewis rats were inoculated with pig myosin (day 0). pCAGGS-IL-10 (at a dose of 800 μg per rat) was administered three times (7, 12 and 17 days after immunization) to one group of rats (group IL-10, n=10), while controls received empty pCAGGS (group V, n=10). Lewis rats without any treatment were used as age-matched normal controls (group N, n=10).
4.6 Hemodynamic and echocardiographic studies
Rats were anesthetized with 2% halothane in O2 and subjected to surgical procedures to measure hemodynamic parameters on day 21. After instrumentation, the concentration of halothane was reduced to 0.5% to record steady-state hemodynamic data. Two-dimensional echocardiography was performed under 0.5% halothane using a 7.5-MHz transducer linked to an ultrasound system (Aloka, Tokyo, Japan). M-mode images were obtained to measure left ventricular dimension in diastole, LVDs and FS. Hemodynamic parameters such as LVP, CVP, LVEDP and ± dP/dt were recorded as previously described 47.
4.7 HW/BW and histopathology
After the measurement of hemodynamic and echocardiographic parameters, hearts were removed and weighed immediately, and HW/BW was calculated. The hearts were fixed with 10% formalin in phosphate-buffered saline and embedded in paraffin, and several transverse sections were cut from the mid ventricles and stained with hematoxylin/eosin. Infiltration of inflammatory cells was examined in the hematoxylin/eosin-stained slides viewed under a high-power light microscope.
4.8 Mast cell staining and quantitation
Histochemical staining with toluidine blue was performed to identify mast cells. For toluidine blue staining, slides of paraffinized sections of mid ventricles were dewaxed, rehydrated and incubated with 0.05% (w/v) toluidine blue for 30 min followed by counterstaining with 0.01% (w/v) eosin for 1 min. Mast cells were easily identified by metachromatic staining of their granules. Mast cell density was quantified by counting the number of toluidine blue-positive mast cells in 15 fields (100×). Mast cell density was expressed as cells/mm2.
4.9 Measurement of histamine concentration in heart tissue
Histamine was extracted from heart tissue and measured using a fluorometer according to the procedure described elsewhere 48. In brief, 0.5 g of cardiac tissue was disrupted in 1.0 ml of 0.4 N HClO4 using a homogenizer (Kinematica, Tokyo, Japan). The homogenate was mixed with 0.25 ml of water and centrifuged for 15 min at 15,000 rpm at 4°C. Then, 1.0 ml of supernatant was transferred to a small test tube containing 1.0 ml of water. The histamine in this diluted sample of tissue homogenate was extracted into isoamyl alcohol in multisteps. The extracted histamine formed a complex with orthopthaldialdehyde (Sigma, Tokyo, Japan). The fluorescence of the histamine-orthopthaldialdehyde fluorophor was maximized in the presence of citric acid and measured using a spectrofluorophotometer (Shimadzu, Kyoto, Japan) with 358 and 446 mm as the activation and fluorescence wavelengths, respectively. The concentration of cardiac histamine was expressed as μg/g of wet heart tissue.
4.10 Measurement of mast cell size
Using a micrometer, magnification fields for 1,000× and 400× magnification were standardized. Using the same specifications, the toluidine blue-stained slides were observed under a high-power light microscope to measure the size of toluidine blue-positive mast cells. The median mast cell size was expressed as μm.
4.11 Immunohistochemistry for SCF
Formalin-fixed, paraffin-embedded cardiac tissue sections were used for immunohistochemical staining. After deparaffinization and hydration, the slides were washed in Tris-buffered saline (TBS; 10 mmol/l Tris-HCl, 0.85% NaCl, pH 7.5) containing 0.1% bovine serum albumin. Endogenous peroxidase activity was quenched by incubating the slides in methanol and methanol/0.6% H2O2. In this study, the primary antibody used was goat polyclonal anti-SCF antibody (1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA). To perform antigen retrieval, the sections were pretreated with trypsin for 10 min at 37°C. After overnight incubation with primary antibody at 4ºC, the slides were washed in TBS buffer and biotinylated rabbit anti-goat secondary antibody was then added at room temperature for 45 min. The slides were washed in TBS buffer and incubated with diaminobenzidine tetrahydrochloride as the substrate and counterstained with hematoxylin. A negative control without primary antibody was included in the experiment to ensure the antibody specificity.
4.12 Statistical analysis
Data are presented as means ± SEM. Statistical comparison between groups was performed by one-way ANOVA, followed by Tukey's method.
We thank Mir I. I. Wahed, Gurusamy Narasimman, Ken Shirai, Kenichi Hirabayashi, Juan Wen, Yuichi Abe, Mayako Soga, Yusuke Nagai and Fadia A. Kamal for their assistance in this research work. This research was supported by grants from Yujin Memorial Grant; the Ministry of Education, Culture, Sports, Science and Technology of Japan; and Promotion and Mutual Aid Corporation for Private Schools of Japan.