Analysis of Heart-Infiltrating T-Cell Clonotypes in Experimental Autoimmune Myocarditis in Rats
Abstract Experimental autoimmune myocarditis (EAM) resembles the lethal giant cell myocarditis seen in humans, and the recurrent forms lead to dilated cardiomyopathy (DCM). EAM in rats induced by a subcutaneous injection of cardiac myosin has been shown to be a T cell–mediated autoimmune disease. αβ T cells have proved to be important by the observation that antibodies to αβ T-cell receptor (TCR) prevent disease progression. αβ T cells recognize antigenic peptides bound to major histocompatibility (MHC) molecules by αβ TCR, and complementarity determining region 3 (CDR3) is considered the most important region for antigen recognition. To elucidate the nature of this T cell–mediated myocarditis, we analyzed TCR Vβ chains of heart-infiltrating T cells. In the early stage of EAM, none of 22 TCR Vβ chain transcripts seemed to be dominant by reverse transcription–polymerase chain reaction analysis of total RNA and flow cytometric analysis. On the other hand, single-strand conformation polymorphism analysis of TCR Vβ8.2, Vβ8.5, Vβ10, and Vβ16 cDNA amplified by polymerase chain reaction encompassing the CDR3 revealed oligoclonal expansion in heart-infiltrating T cells isolated from animals at various disease stages. cDNA encoding Vβ CDR3 from heart-infiltrating and pericardial effusion T cells in rats with EAM revealed more restricted sequences than did cells from rats with normal spleens. Clones from distinct lesions of the same animal were identical, and clones from heart-infiltrating and pericardial effusion T cells from the same animal showed overlap. Thus, CDR3 of the TCR β chain may be important in rat EAM, and heart-infiltrating T cells are considered to recognize the specific antigen.
Experimental autoimmune myocarditis (EAM) is induced by injecting cardiac myosin into susceptible strains of rats1 and mice.2 Lewis rats develop myocarditis 14 days after injection of cardiac whole myosin or rod myosin. EAM in Lewis rats is characterized by cardiomegaly, pericardial effusion, and extensive myocardial necrosis as well as by massive infiltration of MNCs into the heart; some animals die of congestive heart failure. This model, which resembles lethal giant cell myocarditis in humans,3 was recently shown to develop recurrent episodes leading to DCM.4 EAM has been shown to be a T cell–mediated autoimmune disease by adoptive transfer.5 6 Because antibodies to the αβ TCR prevent the progression of myocarditis,7 αβ T cells are considered to play an important role in the disease. Heart-infiltrating and pericardial space–infiltrating αβ T cells are predominantly CD4+.8 Given that cardiac myosin–MHC class II complexes are present on resident antigen-presenting cells in the normal mouse heart,9 self-reactive T cells may recognize these antigen-presenting cells and trigger myocarditis in cardiac myosin–induced EAM.
αβ T cells recognize antigenic peptides in the context of MHC as a result of interaction of the peptide-MHC complex with the α and β chains.10 As is the case for immunoglobulins, the regions equivalent to CDR1 and CDR2 in TCR are encoded within the V gene itself, whereas the CDR3-equivalent region is formed by the conjunction of V and J (in TCR α or γ) or by V, D, and J (in TCR β and δ). CDR3 is considered the most important region for antigen recognition. Recently, several studies of animal models and human disease have examined TCR V gene expression11 12 13 14 15 and CDR3 sequences16 17 18 19 20 21 of infiltrating T cells. Seko et al22 analyzed the expression of TCR Vβ genes in infiltrating cells in the heart with acute myocarditis caused by coxsackievirus B3. In mouse and rat EAM, the expression of TCR Vβ genes and CDR3 sequences in heart-infiltrating T cells has not been reported. In the present study, we analyzed heart-infiltrating T-cell clonotypes in Lewis rat EAM.
Materials and Methods
Lewis rats were obtained from Charles River, Japan (Atsugi, Kanagawa, Japan) and were maintained in our animal facilities until 6 weeks of age.
Purification of Porcine Rod Cardiac Myosin
Whole cardiac myosin was prepared from the ventricular muscle of porcine hearts as previously described1 and was then digested with α-chymotrypsin at 20°C for 16 minutes in 0.12 mmol/L NaCl, 20 mmol/L imidazole-HCl (pH 7.0), and 1 mmol/L EDTA at an enzyme-to-substrate ratio of 1:200 (wt/wt). The preparation was then centrifuged at 100 000g for 1 hour. The pellet was dissolved in buffer A (0.6 mol/L NaCl and 50 mmol/L imidazole-HCl [pH 7.0]) and then mixed with 3 vol ice-cold ethanol. After centrifugation at 9000g for 10 minutes, the pellet was dissolved in and dialyzed against buffer A. The solution was then centrifuged at 100 000g for 1 hour, and the supernatant was collected and used as rod cardiac myosin.23
Induction of EAM
On day 0, Lewis rats were injected subcutaneously in the footpads with 0.3 mg of porcine rod cardiac myosin in complete Freund’s adjuvant supplemented with Mycobacterium tuberculosis at a concentration of 10 mg/mL.
Lymph node MNCs, spleen MNCs, and heart-infiltrating MNCs were isolated by forcing popliteal lymph node, spleen, and heart (digested with 0.015% trypsin and 0.015% collagenase for 15 minutes in MEM supplemented with 7.5 mmol/L HEPES and 2% newborn calf serum) through a 200-gauge stainless steel mesh. Red blood cells in the spleen cell preparation were lysed in 0.17 mol/L Tris supplemented with 0.83% NH4Cl. The heart cell preparation was washed with supplemented MEM, and MNCs were purified by passage through a column of glass wool (a 20-mL column packed with 0.8 g of glass wool). MNCs in peripheral blood were isolated by Ficoll-Faque (Pharmacia) density gradient centrifugation at 1000g for 20 minutes. MNCs in pericardial effusion were analyzed after hemolysis in 0.17 mol/L Tris supplemented with 0.83% NH4Cl.
RT-PCR Analysis of Vβ Expression
Total RNA was isolated from heart and from spleen MNCs by acid guanidium thiocyanate–phenol–chloroform extraction. cDNA was synthesized from 10 μg of total RNA with a TCR Cβ primer24 and murine Moloney leukemia virus reverse transcriptase (GIBCO-BRL) in a final volume of 20 μL. Heart and spleen Vβ-specific PCR products were generated with AmpliTaq polymerase (Toyobo), 22 Vβ-specific primers,24 and the same Cβ primer from 5 μL of heart cDNA and 0.2 μL of spleen cDNA, according to the following amplification profile: 30 cycles at 94°C for 60 seconds, 55°C for 90 seconds, and 72°C for 120 seconds. Amplified products were separated on 3% agarose gels and stained with ethidium bromide.
Flow Cytometry Analysis
The surface phenotype of fresh viable cells was determined by using monoclonal antibodies with two-color immunofluorescence. For detecting the expression of αβ TCR versus Vβ8.2, Vβ8.5, Vβ10, and Vβ16, MNCs were incubated first with unconjugated monoclonal antibodies to Vβ8.2 (R78), Vβ8.5 (B73), Vβ10 (G101), or Vβ16 (HIS42) (Pharmingen) and then, after washing, with phycoerythrin-conjugated antibodies to mouse IgG antibody (Vector Laboratories). After incubation with normal mouse serum, the cells were exposed to fluorescein isothiocyanate–conjugated mouse monoclonal antibody to αβ TCR (R73) (Serotec). Stained cells (1×104) were then analyzed with a FACScan flow cytometer (Becton Dickinson).
Amplified PCR products were diluted (2:3) in denaturing solution (95% formamide, 10 mmol/L EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol), heated at 96°C for 5 minutes, and then cooled on ice for 10 minutes. The diluted sample (10 μL) was adjusted to 50% glycerol, and 3 μL was subjected to SSCP analysis by electrophoresis on nondenaturing 5% polyacrylamide (ratio of acrylamide to bisacrylamide, 19:1) gels (14×14×0.1 cm) in 0.5× Tris-borate-EDTA containing 10% glycerol at 14 V/cm for 5 hours at room temperature. DNA fragments were detected with a silver staining kit (Daiichi).25
For the purposes of cloning and sequencing, Vβ-specific PCR products were purified with SUPREC-02 (Takara) and directly inserted into the pGEM-T vector (Promega). The recombinant plasmids were then used to transform Escherichia coli JM109 competent cells (Takara). Individual ampicillin-resistant colonies were isolated, and the plasmid DNA was sequenced with a TaqDyeDeoxy terminator cycle sequencing kit and a DNA sequencer (model 373A, Applied Biosystems).
RT-PCR Analysis of TCR Vβ Expression
Rats injected once with porcine rod cardiac myosin developed acute myocarditis 14 days later. Because a marked bias toward the expression of Vβ8.2 was apparent early during the onset of EAE induced by guinea pig MBP in Lewis rats,11 26 we compared TCR Vβ expression in the heart of two individual rats with myocarditis on day 14 (early stage) with that on MNCs from normal spleen. The hearts of two EAM rats killed on day 14 exhibited spotty macroscopic white lesions, from which RNA was extracted. PCR analysis revealed that the expression of 22 Vβ genes in the hearts of individual EAM rats was similar to that apparent in normal spleen (Fig 1⇓).
Flow Cytometry Analysis of Vβ8.2, Vβ8.5, Vβ10, and Vβ16 Expression of MNCs From Normal and EAM Rats
We also examined TCR Vβ expression by flow cytometry. All samples from two individual EAM rats and a normal rat were analyzed. Consistent with the RT-PCR results, two-color staining of αβ TCR plus Vβ8.2, Vβ8.5, Vβ10, or Vβ16 revealed that the expression of these Vβ chains on αβ T cells from spleen, peripheral blood, lymph node, and heart of EAM rats on day 14 was indistinguishable from that apparent on αβ T cells from normal rats (Fig 2⇓). αβ T cells expressing Vβ8.2, Vβ8.5, Vβ10, or Vβ16 constituted approximately one fourth of all αβ T cells from the heart of EAM rats on day 14.
PCR-SSCP Analysis of TCR β Chain CDR3
We examined the structure of PCR products corresponding to the CDR3 region of the TCR β chain by SSCP analysis. Heterogeneous PCR products corresponding to this region migrate as a smear on SSCP analysis, whereas homogeneous products migrate as bands.27 PCR products corresponding to Vβ8.2, Vβ8.5, Vβ10, and Vβ16 from normal spleen and from individual EAM heart on day 14, 16, or 19 migrated as a single band on ethidium bromide–stained agarose gels (Fig 3⇓). However, on SSCP analysis, PCR products from normal spleen produced a smear, whereas those from EAM heart generated bands within a smear (Fig 3⇓). These results show that TCR Vβ8.2, Vβ8.5, Vβ10, and Vβ16 cDNA from EAM heart was derived from oligoclonal αβ T cells. The positions of the bands in the smear of PCR products from EAM heart differed among the three time points, suggesting that the αβ T-cell clones varied with the stage of disease.
Sequence Analysis of TCR β Chain CDR3
CDR3 sequences of Vβ8.2, Vβ8.5, Vβ10, and Vβ16 cDNA from normal spleen did not reveal oligoclonal expansion (Fig 4⇓). In contrast, the corresponding CDR3 sequences of cDNA for EAM heart on days 14, 16, and 19 revealed clonal expansion at each stage (Fig 5⇓). For example, 11 of 14 and 2 of 14 Vβ8.2 cDNA CDR3 sequences from EAM heart on day 14 were identical. However, except for one common Vβ8.2 cDNA clone detected on days 14 and 19, the cDNA clones differed at each time point, consistent with the results of the PCR-SSCP analysis. Fig 6⇓ shows CDR3 sequences of Vβ8.2 cDNA clones from another gross lesion of the same EAM heart on day 14; the sequences were the same as the two shown in Fig 5A⇓, suggesting that the same clones expanded in each heart lesion of individual animals. The EAM rat whose heart was examined on day 16 showed moderate pericardial effusion; the CDR3 sequences of Vβ8.2, Vβ8.5, Vβ10, and Vβ16 cDNA from MNCs in the pericardial effusion showed an overlap with those from the corresponding rat heart (Fig 7⇓).
We have determined the clonotypes of heart-infiltrating αβ T cells in EAM induced by porcine rod cardiac myosin, because these cells were thought to play an important role in this disease. In the present study, CDR3 of the TCR β chain was important in rat EAM. Because CDR3 is the most important region for recognizing the antigen peptide, this finding shows that heart-infiltrating T cells are considered to recognize the specific antigen.
The etiology of DCM remains enigmatic, but the most widely accepted hypothesis is that DCM is initiated by a viral infection and perpetuated by immune factors.29 DCM is thought to be, at least in part, somewhat of an autoimmune disease.30 Anti-heart antibodies have been detected in individuals with DCM.31 Carlquist et al32 verified HLA-DR4 involvement in DCM. The class II MHC antigens are restriction elements for the interaction of the CD4+ T cells with antigens. In DCM, heart-infiltrating CD4+ αβ T cells may recognize the specific antigen in the context of MHC as a result of the interaction of the peptide-MHC complex with the α and β chains. Recently, rat EAM has been shown to develop into recurrent forms of myocarditis and to lead to DCM.4 T cells with the amino acid motif LRG in the CDR3 are found in EAE lesions in Lewis rats and in human multiple sclerosis lesions.20 The characteristic feature of T-cell clonotypes in rat EAM may also be applicable to human DCM, similar to EAE and human multiple sclerosis.
Vβ usage in the heart of EAM rats was similar to that in normal spleen, and none of the 22 TCR Vβ chain transcripts seemed to be dominant. The V-region disease hypothesis is controversial.33 In both rats and mice, molecular analyses of the T-cell populations reactive to MBP or its encephalitogenic peptide show them to be extremely limited with respect to TCR gene usage.11 34 In contrast, in EAE induced by PLP, heterogeneous TCR Vβ expression is apparent in central nervous system lesions, even when the immune response is initiated with a short peptide antigen or by a T-cell clone expressing a single TCR Vβ chain.13 35 36 In murine experimental autoimmune uveitis induced by interphotoreceptor retinoid-binding proteins,12 expression of the TCR β chain V gene is highly restricted at the site of inflammation. However, Vβ gene expression is not restricted in nonobese diabetic mice.37 Thus, whether TCR β chain V gene expression is restricted or not appears to depend on the species and the antigen of the autoimmune model.
In the present study, several T-cell clones were present at the site of inflammation at each stage of the disease. One reason may be that porcine rod cardiac myosin possesses several myocarditis-inducing epitopes. Our recent studies indicate the presence of several myocarditogenic epitopes in porcine rod cardiac myosin,23 and Wegmann et al38 identified several such epitopes in rat cardiac α-myosin heavy chain. In EAE, both MBP and PLP have several encephalitogenic epitopes. For example, both PLP-(178 to 191) and PLP-(139 to 151) peptides induce EAE, with the onset of disease being earlier for PLP-(178 to 191).39
In autoimmune diseases, T-cell clones responding to self peptides are thought to rearrange early during development and to fail to add nucleotides because of a lack of terminal deoxynucleotidyl transferase, as well as having a paucity of N-region nucleotide additions.40 N-region nucleotide additions are the random addition of nucleotides between the V, D, and J gene segments.10 Vβ8.2, Vβ8.5, Vβ10, and Vβ16 cDNA clones from normal spleen showed 1 to 16 N-region nucleotide additions (5.8±3.4 [mean±SD]), whereas those from heart or the pericardial space of EAM rats showed 1 to 20 N-region nucleotide additions (5.7±2.8). Thus, T-cell clones from inflammatory lesions showed the same number of N-region nucleotide additions as normal spleen.
A previous study showed that MNCs in pericardial effusion probably infiltrate the outer layer of the heart.8 The similarity of T-cell clonotypes in heart and pericardial effusion supports this conclusion. Clinically, myocarditis is usually accompanied by pericarditis and pericardial effusion. Clonotypes of MNCs in pericardial effusion could be sufficiently analyzed as synovial fluid cells in rheumatoid arthritis.
In conclusion, heart-infiltrating T cells in Lewis rat EAM induced by porcine rod cardiac myosin did not seem to show restricted TCR Vβ usage; however, they did show restricted CDR3 sequences of the TCR β chain. Thus, the study of the β chain CDR3 region is informative in rat EAM.
Selected Abbreviations and Acronyms
|EAE||=||experimental autoimmune encephalomyelitis|
|EAM||=||experimental autoimmune myocarditis|
|MBP||=||myelin basic protein|
|PCR||=||polymerase chain reaction|
|SSCP||=||single-strand conformation polymorphism|
This study was supported by a grant from the Study Group of Molecular Cardiology and grants-in-aid from the Niigata University Science Foundation and the Tsukada Medical Science Foundation.
- Received June 15, 1995.
- Accepted September 12, 1995.
- © 1996 American Heart Association, Inc.
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