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【摘要】
Repeated inflammation in the heart is one of the initiation factors of dilated cardiomyopathy (DCM). In a previous study, we established a new animal model for DCM by immunization of rats with recombinant cardiac C-protein fragment 2 (CC2). The present study examined factors involved in the development of DCM. Analysis using overlapping peptides revealed that the major carditogenic epitope resides only in the residue 615C647 . However, immunization with CC2P12 induced moderate inflammation without subsequent DCM. CDR3 spectratyping analysis of the T-cell repertoire demonstrated that Vß4-positive T cells were preferentially expanded in both CC2- and CC2P12-immunized rats. Although there was no significant difference in the T-cell characteristics, examinations of the B-cell epitope revealed that marked epitope spreading occurred in CC2-immunized but not CC2P12-immunized rats from 4 weeks after immunization. Consistent with this finding, immunization with CC2P12 and simultaneous transfer of anti-peptide antisera induced significantly more severe inflammation and fibrosis than CC2P12 immunization alone. However, the transfer of the antisera without CC2P12 immunization did not induce any pathology. These findings suggest that T-cell activation and B-cell epitope spreading in the CC2 molecule is a key step for the switch from myocarditis to the development of DCM.
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Dilated cardiomyopathy (DCM) is a serious and frequently fatal disorder and is a common cause of heart failure. The majority of DCM is sporadic, and mostly virus-induced immune mechanisms are suspected.1 Because the heart biopsy sometimes demonstrates the presence of inflammation, several immunosuppressive therapies have been tried to improve the status of DCM.2-4 However, significant progress has not been made, although these therapies have shown some improvements of the disease. Difficulties in finding effective therapies are mainly based on the fact that the pathogenesis of DCM is still poorly understood. The establishment of a suitable animal model that mimics human DCM and elucidation of pathogenesis of DCM will provide useful information for the development of effective therapies.
In a previous study, we demonstrated that cardiac C-protein, one of the myosin-binding proteins, induced severe experimental autoimmune carditis (EAC) and subsequent DCM in Lewis rats.5 Seventy-five percent of rats immunized with C-protein died by day 50, and all of the survivors developed DCM. Furthermore, it was revealed that cytokines and chemokines produced by T cells and macrophages were up-regulated in the heart lesions, mainly during the inflammatory phase of EAC. These findings suggest that pathogenic T cells and possibly B cells play an important role in the development of EAC and subsequent DCM.
In the present study, we first examined the carditogenic epitopes that reside in the cardiac C-protein fragment 2 (CC2) (corresponding to amino acid residues 317C647). Using overlapping peptides, we found that only peptide 12 (CC2P12) possessed the carditis-inducing ability in the CC2 molecule. Interestingly, CC2P12 induced nonfatal moderate EAC and did not develop DCM. Analysis of clonally expanded T cells in CC2- and CC2P12-immunized rats demonstrated that there was no significant difference between the two groups. In contrast, CC2-immunized rats exhibited marked B-cell epitope spreading 4 weeks after immunization and afterward, whereas CC2P12-immunized rats raised antibodies only against CC2P12 and CC2. Based on these findings, we performed transfer experiments and demonstrated that both activation of T cells and anti-peptide antibody elevation are required for the initiation and subsequent progression of the disease. The present study strongly suggests that B-cell epitope spreading is an essential step for the switch from myocarditis to DCM.
【关键词】 spreading critical c-protein-induced myocarditis cardiomyopathy
Materials and Methods
Animals and Proteins
Lewis rats were purchased from SLC Japan (Shizuoka) and bred in our animal facility. Seven- to 11-week-old male and female rats were used.
Preparation of Recombinant C-Protein Fragments and Synthetic Peptides
The preparation of recombinant C-protein was precisely described previously.5 Polymerase chain reaction (PCR) products corresponding to fragments 1, 2, 3, and 4 were inserted into a cloning vector, pCR4 Blunt-TOPO in the Zero Blunt TOPO kit (Invitrogen, Groningen, The Netherlands), and clones with correct sequences were subcloned into an expression vector, pQE30 (Qiagen, Tokyo, Japan). Then, recombinant C-protein fragments produced in transformed Escherichia coli were isolated under denaturing conditions and purified using Ni-NTA Agarose (Qiagen).
Synthetic peptides encompassing CC2, designated as CC2P1-CC2P12 (Table 1) , were synthesized using a peptide synthesizer (Shimadzu, Kyoto, Japan). All of the peptides used in this study were >90% pure as determined and were purified if necessary using HPLC.
Table 1. Amino Acid Sequences of Synthetic Peptides Encompassing CC2 Used in the Study
Conjugation of CC2P12 with KLH
To increase immunogenicity of CC2P12, the peptide was conjugated with keyhole limpet hemocyanin (KLH; Wako, Tokyo, Japan) as described previously.6 KLH (in 0.083 mol/L sodium phosphate, 0.9 mol/L NaCl, and 0.1 mol/L ethylenediamine tetraacetic acid, pH 7.2) and m-maleimidobenzoyl-N-hydrosuccinimide ester in dimethyl sulfoxide (MBS; Pierce, Chicago, IL) at concentrations of 10 and 20 mg/ml, respectively, were incubated at a ratio of 10:1 for 1 hour at room temperature. Then, excess MBS was removed on a HiTrap desalting column (Amersham Biosciences, Tokyo, Japan). Finally, the KLH-CC2P12 complex was formed by incubating MBS-KLH with CC2P12 for 2 hours at room temperature.
EAC Induction and Tissue Sampling
Lewis rats were immunized once on day 0 with the indicated antigen with complete Freund??s adjuvant (CFA) (2.5 mg/ml Mycobacterium tuberculosis) in the hind foot pads. At the time of immunization, rats received an intraperitoneal injection of 2 µg of pertussis toxin (PT; Seikagaku Corp., Tokyo, Japan). The numbers of rats used for experiments are shown in the
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作者单位：From the Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan