Department of Immunology and Rheumatology, Hôpital E. Herriot and
1 Department of Pathology, Centre Hospitalier Universitaire, Dijon, France
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Abstract |
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Methods. Biopsy samples of muscle from eight patients with inflammatory myopathies were studied for the presence of B19 DNA by polymerase chain reaction. Expression of VP1 and VP2 capsid proteins was evaluated by immunohistochemistry. Interleukin 6 (IL-6) production was measured in the supernatant of myoblasts following incubation with parvovirus B19.
Results. In seven samples, detection of B19 DNA was negative. The expression of VP1 and VP2 capsid proteins was not observed by immunohistochemistry. In one patient, detection was transiently positive but became negative despite a flare-up of muscle disease. In vitro, parvovirus B19 was not able to induce IL-6 production by myoblasts.
Conclusion. Our results do not support the direct implication of parvovirus B19 in the pathogenesis of myositis.
KEY WORDS: Myositis, Autoimmune disease, Parvovirus B19, Myoblast, Muscle biopsy.
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Introduction |
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We have previously reported the first case of dermatomyositis (DM), a muscle autoimmune disease, where B19 DNA was present in two sequential muscle biopsies [2]. Accordingly, the presence of parvovirus B19 in the muscle of our patient suggested the potential association between this viral infection and myositis.
Here we provide a follow-up of the same case where disease relapse was not associated with the presence of parvovirus B19 in a new muscle biopsy. Samples from additional patients were also negative.
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Case report |
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Patients and methods |
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Polymerase chain reaction (PCR)
The same procedure was used as in our previous study [2]. Viral DNA was extracted by the tissue protocol of the QIAamp® DNA Mini Kit (Qiagen), using ATL buffer and proteinase K solution. The first set of oligonucleotide primers consisted of a sense nucleotide 2445 to 2470 (5'-TGA GTA AAA AAA GTG GCA AAT GGT GG-3') and of an antisense nucleotide 2634 to 2608 (5'-GCA ACT AAG TCA AAC AGA GAG GAT GGG-3'). It amplifies a 189 bp segment, corresponding to the beginning of VP1 [4]. The reaction mixture for PCR amplification contained deoxynucleoside triphosphate with uracil, and heat-labile uracil-N-glycosidase (UNG Roche). Amplification was obtained with a Hybaid Omnigene system (35 cycles at an annealing temperature of 57°C). Amplified products were detected by ethidium bromide staining after agarose gel electrophoresis. Specific identification was performed with a 5'-biotin-labelled probe (5'-AAT ATT AAA AGA TCA TTA TAA TAT TTC TTT AGA TAA TCC CC-3', nucleotide 2560 to 2600) according to hybridization procedures of GEN-ETI-KTM (DEIA DiaSorin, Saluggia, Italy).
The second set of primers was selected to amplify a 208 bp fragment corresponding to NS1 coding sequence: sense nucleotide 1539 to 1560 (5'-GGT GGT CTG GGA TGA AGG TAT T-3'), antisense nucleotide 1747 to 1724 (5'-GCT CTT TTA AGG CTT TAG CAT GTA-3') [5]. Amplification used 40 cycles at an annealing temperature of 55°C. The specificity of the PCR positive products was confirmed with a 5'-biotin-labelled probe (5'-CGG GAA CAC TAC AAC AAC TG-3', nucleotides 1704 to 1723).
Immunohistochemistry
Paraffin-embedded sections of control and inflammatory muscle or placenta were treated in xylene and dipped in a gradient of ethanol (once in 99% ethanol, once in 95% ethanol, and once in water). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The sections were then incubated with a monoclonal antibody specific for human parvovirus B19 (VP1 and VP2 capsid proteins) obtained from Novocastra (Newcastle upon Tyne, UK) with 1% normal human serum. Antibody dilution was 1:20 as suggested by the manufacturer. After overnight incubation at 4°C and washing, the sections were incubated with biotinylated goat anti-mouse IgG antibody for 30 min at room temperature, followed by streptavidinperoxidase complex (Dako, Glostrup, Denmark) for 15 min and 3,3'-diaminobenzidine tetrahydrochloride (Dako) for 20 min. The sections were then counterstained with Mayer's haematoxylin.
Induction of IL-6 production assay
The possible effect of the parvovirus B19 infection on interleukin 6 (IL-6) production by myoblasts was studied in a biological assay. Normal skeletal myoblasts were obtained from muscle biopsy and used between passage 3 and 5. Myoblasts (104 cell/well) were incubated in 8-well LabTekTM chamberslides (Nunc, Rochester, NY) with 40 µl of a B19 PCR positive serum or a serum from a healthy donor with or without 10 pg/ml of human rIL-1ß (Sigma, St. Louis, MO) in a final volume of 200 µl of complete medium. After 72 h, supernatants were removed and IL-6 production analysed by enzyme-linked immunosorbent assay (ELISA) as previously described [6]. Myoblasts were then fixed and stained with the anti-B19 antibody.
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Results and discussion |
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We have reported the first case of DM associated with B19 DNA in muscle biopsies [2]. However, the presence of B19 DNA was not found in a third biopsy performed during a relapse associated with concordant muscle pathology findings. This dissociation did not support a direct link between myositis and B19 infection. Interestingly, the presence of B19 DNA has sometimes been suspected but rarely demonstrated in skeletal muscle (Table 1) [914].
To extend this single case report suggesting the absence of a link between parvovirus B19 and myositis, we studied seven other cases of myositis (Table 1). First, we analysed seven other muscle biopsies by PCR and immunohistochemistry. None of these was positive by PCR or by immunohistochemistry. In particular, there was no staining of muscle cells or inflammatory cells in any of these samples. Patients were not selected on the basis of positive antibodies since these are commonly observed in controls and patients in the absence of any muscle symptoms. Thus, a viral detection directly at the site of disease by PCR or Southern hybridization was necessary. Although the size of our population is small, our results suggest the absence of direct implication of the virus in the pathogenesis of myositis. However, parvovirus B19 could still initiate the myositis process but disappear in the chronic phase. Obviously the association between viral detection and disease could also be fortuitous. Details in Table 1
show the available clinical data from patients with myositis. No particular subset of patients with or without the presence of B19 DNA in muscle could be identified.
Parvovirus B19 infection has been found in several cell types including macrophages, follicular dendritic cells, T and B lymphocytes and endothelial cells, all potentially implicated in inflammatory muscle diseases. Surprisingly, infection of synoviocytes from RA patients following contact with parvovirus B19-infected cells induced or enhanced IL-6 production [8]. The mode of infection of these synoviocytes has not been elucidated [15], but in a recent study, parvovirus B19 induced invasive properties in normal human synoviocytes [16]. In addition, IL-6 production can be induced in human haemopoietic cell lines stably transfected with DNA encoding the NS1 protein [17].
These findings suggest the possible association between B19 infection and IL-6 production by mesenchymal cells. We tested the possible increase of IL-6 production by myoblasts stimulated with a B19-positive serum. IL-6 production was not increased in the presence of a B19-positive serum. Addition of IL-1, a pro-inflammatory monocyte-derived cytokine used as a positive control for IL-6 production, had no costimulatory effect. In the same conditions, the expression of VP1 and VP2 capsid proteins by normal myoblasts was not induced as determined by immunohistochemistry.
Our previous case of DM indicated a temporal association between parvovirus B19 infection and active myositis. Our present study and the published cases do not demonstrate the implication of parvovirus B19 in a particular phase of the disease or a subset of patients. If parvovirus B19 does contribute to the pathogenic process leading to myositis, it is probably through indirect mechanisms in patients with a particular genetic or risk factor.
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Acknowledgments |
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Notes |
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References |
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