Infrequency of detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of patients with rheumatoid arthritis

P. Gaudin, S. Ijaz, P. W. Tuke, F. Marcel2, A. Paraz2, J. M. Seigneurin2, B. Mandrand1, H. Perron3 and J. A. Garson

Department of Virology, Royal Free and University College Medical School, London, UK,
1 UMR 103 CNRS – bioMérieux, ENSL, Lyon,
2 Department of Virology, University Hospital, Grenoble and
3 bioMérieux STELHYS, Chemin de l'Orme, 69280 Lyon, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To determine whether the recently identified multiple sclerosis-associated retrovirus, MSRV, is detectable in the serum and synovial fluid of patients with rheumatoid arthritis (RA).

Methods. A reverse transcription–polymerase chain reaction (RT-PCR) assay was used to seek evidence of particle-associated MSRV/HERV-W RNA in the plasma and synovial fluid of patients with RA and controls. Stringent precautions were taken to avoid detection of contaminating human genomic DNA and cellular RNA sequences.

Results. Thirty-seven plasma samples were tested (20 from RA patients and 17 from controls) but none had detectable MSRV/HERV-W RNA. Synovial fluid samples were available from nine patients with RA and 10 controls. Particle-associated MSRV/HERV-W RNA was reproducibly detected in two of nine synovial fluid samples from RA patients and in one control sample. The identity of RT-PCR products was confirmed by sequencing.

Conclusion. MSRV/HERV-W RNA sequences are detectable in the synovial fluid of a small proportion of RA patients, but this phenomenon may not be specific to RA.

KEY WORDS: MSRV, Multiple sclerosis-associated retrovirus, Rheumatoid arthritis, Synovial fluid, Polymerase chain reaction.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA), which affects approximately 1% of the Caucasian population, is characterized by progressive destruction of the articular components, resulting over a period of several years in severe disability [1]. Within the affected joints, the synovial tissue becomes infiltrated by neutrophils, lymphocytes and macrophages. In association with this cellular infiltrate, there is increased production of metalloproteinases, such as collagenases and gelatinases, which contribute to the erosion of the extracellular matrix [2].

It is clear that autoimmune mechanisms play a major role in the pathogenesis of RA, although the precise cause of the disease remains unknown. Both genetic and environmental factors are thought to be involved, and several viral candidates, including rubella virus, parvovirus, herpesviruses and hepatitis viruses, have been proposed [35]. It has been suggested repeatedly that retroviruses, both exogenous and endogenous, might be involved in the causes of various autoimmune diseases, including Sjögren's syndrome, systemic lupus erythematosus, insulin-dependent diabetes, multiple sclerosis and RA [610]. Molecular mimicry has been proposed as a pathogenic mechanism in some cases and the involvement of an endogenous retroviral superantigen has recently been postulated in insulin-dependent diabetes [11], although this remains the subject of controversy [12]. In addition, a novel retrovirus (multiple sclerosis-associated retrovirus, MSRV), related to the human endogenous retrovirus-W (HERV-W) endogenous family, has been isolated recently from patients with multiple sclerosis [1316].

In a preliminary study designed to determine whether MSRV was associated with any autoimmune diseases other than multiple sclerosis, MSRV/HERV-W RNA was detected in approximately 50% of plasma samples from patients with RA [17]. However, the method employed for the preliminary study did not incorporate the rigorous measures now considered necessary to exclude the possibility of false positivity due to the copurification of related human genomic DNA and cellular RNA sequences [14]. The aim of the present study was to investigate the possible association between RA and MSRV using a more selective method [14] designed to detect only particle-associated MSRV/HERV-W RNA, indicative of virion production.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Synovial fluid and plasma samples were obtained, with ethics committee approval, from patients attending the Rheumatology Department of the Teaching Hospital of Grenoble. All patients with RA satisfied the diagnostic criteria of the American College of Rheumatology [18]. Non-RA controls included three patients with osteoarthritis, four with chondrocalcinosis, two with spondyloarthropathy and one with gout. Plasma was also obtained from 17 healthy adult controls.

Clinical samples
Following venesection and centrifugation, EDTA plasma was separated rapidly then stored in aliquots at -70°C. Synovial fluid was aspirated from joints during diagnostic or therapeutic arthrocentesis and centrifuged immediately at 10 000 g for 20 min at 4°C, and the cell-free supernatant was aliquoted and stored at -70°C. On thawing, the synovial fluid was incubated with hyaluronidase I-S (Sigma, Poole, UK) 40 U/ml for 15 min at 22°C in order to reduce the viscosity [19]. The sample was then centrifuged at 21 000 g for 20 min at 22°C to remove any residual cellular debris before RNA extraction.

Extraction of particle-associated MSRV/HERV-W RNA
The method employed was a modification of that described previously [14], and incorporated centrifugation, filtration, RNase and DNase digestion steps to ensure that only particle-associated MSRV/HERV-W RNA was extracted. Two hundred microlitres of sample (plasma or synovial fluid) was filtered through a 0.45 µm filter (Flowgen, Ashby de la Zouch, UK) and incubated with RNAse I (10 U for plasma, 30 U for synovial fluid) (Promega, Southampton, UK) for 30 min at 37°C. One hundred and fifty microlitres of the filtered, RNase-digested sample was processed to extract DNA-free viral RNA using a silica matrix spin-column technique (S.N.A.PTM reagents; Invitrogen, Leek, The Netherlands). The DNase step employed 30 U (plasma) or 60 U (synovial fluid) of RNase-free DNase I (Boehringer Mannheim, Lewes, UK). RNA was finally eluted in 105 µl of RNase-free water for immediate use in the reverse transcription–polymerase chain reaction (RT-PCR).

Reverse transcription and polymerase chain reaction
Combined RT-PCR was performed using 25 µl of the extracted RNA in a single-tube system (TitanTM reagents, Boehringer Mannheim). The MSRV pol-specific primers PTpol-A (5'-ggccaggcatcagcccaagacttga) and PTpol-F (5'-tgcaagctcatccct(g/c) (a/g)gacct) were used for the first round of amplification and primers PTpol-B (5'-gacttgagccagtcctcatacct) and PTpol-E (5'-ctttagggcctggaaagccact) for the second round [14]. The second round was performed using 1 µl of the first-round products and ExpandTM PCR reagents (Boehringer Mannheim). Thermal cycling parameters were as follows: reverse transcription, 30 min at 50°C then 30 min at 60°C; first-round PCR, 94°C for 2 min then 40 cycles of 94°C for 30 s, 60°C for 30 s, 68°C for 45 s and final extension at 68°C for 7 min; second-round PCR, 35 cycles of 94°C for 30 s, 60°C for 30 s, 68°C for 30 s and final extension at 68°C for 7 min. The 435-base pair (bp) second-round PCR product was identified by electrophoresis on a 2% agarose gel containing ethidium bromide. Bands of interest were cut out of the gel, and DNA was purified using QiaquickTM gel extraction kit reagents (Qiagen Ltd., Crawley, UK) and sequenced directly using automated sequencing apparatus (Vistra 725, Amersham International, Amersham, UK).

Experimental controls
Adequate sensitivity of the nested PCR was monitored by including a 10-fold dilution series of human genomic DNA in each run. For an experiment to be valid, a 435-bp band (MSRV-related HERV-W product [16]) had to be generated from 1 pg or less of human DNA. To ensure that the PCR products were derived from reverse-transcribed RNA rather than from traces of contaminating genomic DNA, a mock reaction without reverse transcriptase was performed. Any sample which generated a signal from this no-RT control was excluded from the analysis.

To exclude the possibility that PCR products were derived from copurified cellular RNA rather than from particle-associated RNA, the extracted RNA was also tested for the presence of the pyruvate dehydrogenase (PDH) RNA, a highly expressed cellular transcript, by RT-PCR. The bifunctional enzyme Tth (Perkin Elmer, Warrington, UK) was used in the first round with primers PDH1 (5'-gggtatggatgaggagctgga) and PDH2 (5'-tcttccacagccctcgactaa) [14]. RT was for 30 min at 50°C then 30 min at 60°C; first-round PCR involved denaturation at 94°C for 4 min then 35 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s and final extension at 72°C for 7 min. The second-round PCR was performed on 1 µl of the first-round product using Taq polymerase reagents (Perkin Elmer) with the semi-nested primers PDH2 and PDH3 (5'-cttggagaagaagttgcccagt) and 35 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s and final extension at 72°C for 7 min. PCR products were run on a 4% Nu-Seive (Flowgen) agarose gel to distinguish RNA-derived signals (61 bp) from DNA-derived signals (143 bp). The sensitivity of the PDH PCR system was monitored in each run by ensuring that one cell equivalent of human DNA (~10 pg) gave the expected 143-bp band resulting from amplification of the single-copy PDH gene. Any test sample that generated either an RNA-derived or a DNA-derived signal in the PDH RT-PCR assay was excluded from the analysis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MSRV/HERV-W RNA was not detected in any of the 20 plasma samples from patients with RA or in any of the 17 plasma samples from healthy adult subjects (Fig. 1Go).



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FIG. 1. Example of RT-PCR assay on plasma samples from patients with RA. Ethidium bromide-stained agarose gel of second-round RT-PCR products. Lanes 1–11 show no MSRV pol RT-PCR products detectable in plasma samples from RA patients. Lane 12 shows RT-PCR product derived from human cellular RNA-positive control. Lanes 13–15 show PCR products generated from DNA-positive control dilution series (100, 10 and 1 pg human DNA). Molecular weight marker (M) is HaeIII digest of phage {phi}X174 DNA. Arrow indicates the expected 435-bp size of an MSRV pol (or HERV-W) amplicon.

 
For the analysis of synovial fluid, the protocol described previously [14] had to be modified because the very high viscosity of the material initially caused difficulties with cellular nucleic acid contamination. The viscosity problem was overcome by pretreating the synovial fluid samples with hyaluronidase. In the analysis of synovial fluid samples, it was also found to be necessary to increase the amounts of RNase and DNase to 30 and 60 U/150 µl, respectively. Synovial fluid samples were available from nine patients with RA and from 10 controls with joint disease other than RA. Several synovial fluids were reactive on initial testing but were repeatedly negative on retesting, and were therefore not regarded as positive for the purpose of this analysis. However, three synovial fluid samples repeatedly gave positive reactions on retesting up to four times. Two of the samples repeatedly positive for MSRV/HERV-W RNA were obtained from patients with RA and one was from a patient with acute gout (Fig. 2Go). No correlation was observed between MSRV/HERV-W RNA positivity and clinical parameters such as age, sex, diagnosis, duration of disease, the presence of rheumatoid factor, C-reactive protein concentration, cellularity of the synovial fluid or treatment. Overall, approximately 10% of the samples tested were excluded from the analysis due to the presence of contaminating cellular DNA or RNA.



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FIG. 2. Example of RT-PCR assay of synovial fluid samples. Ethidium bromide-stained agarose gel of second-round RT-PCR products. Lanes 1–4, synovial fluids from RA patients (note 435-bp PCR product in lane 1). Lanes 5–8, synovial fluids from non-RA controls. Lane 9, RT-PCR product derived from human cellular RNA-positive control. Lanes 10–12, PCR products generated from DNA-positive control dilution series (100, 10 and 1 pg human DNA). Lanes 13–15, water-negative controls. Molecular weight marker (M) is HaeIII digest of phage {phi}X174 DNA. Arrow indicates the expected 435-bp size of the PCR product.

 
Sequencing of the RT-PCR products confirmed that they were MSRV-derived, and the degree of interpatient sequence heterogeneity excluded the possibility of cross-contamination. The sequences derived from the three repeatedly positive patients exhibited between 91 and 98% nucleotide homology with the prototype MSRV pol sequence [13].


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is now recognized that retroviruses are able to cause arthritis in a wide range of species; moreover, the inflammatory joint disease of goats induced by the retrovirus CAEV has been proposed as an experimental model of RA [7, 9, 20]. It is also clear that the human retroviruses HIV-1 and HTLV-1 are both associated with arthritis in a proportion of those infected [21, 22]. Furthermore, retrovirus-like particles have been visualized by electron microscopy in the synovial fluid of patients with RA, and there have been reports of RT activity in synoviocyte cultures from such patients [23, 24].

In view of this background, we were prompted to follow up preliminary observations which suggested a possible association between RA and the recently described retrovirus MSRV [17]. However, the results of the present study do not confirm such an association. The discrepancy probably relates to the use of an improved protocol [14] in the present study. The protocol described here includes a number of centrifugation, filtration and RNase digestion steps designed to ensure that all cellular debris and cellular RNA are completely removed from the clinical samples before lysis of the putative viral particles. Similarly, the DNase digestion step ensures that the extracted viral RNA is free of contaminating cellular DNA. These measures have been found to be essential in order to avoid false-positive results due to the presence of traces of cellular nucleic acids containing HERV-W sequences. The protocol is designed for the analysis of cell-free material and is therefore unsuitable for the testing of tissue biopsies, whole blood or cellular pellets.

Although particle-associated MSRV/HERV-W RNA was not detected in any of the plasma samples analysed, it was detected in two of nine (22%) synovial fluid samples from patients with RA and in one of 10 non-RA controls. However, it is possible that this prevalence may be an underestimate because the level of MSRV/HERV-W RNA in these samples was very close to the lower detection limit of the assay. It is noteworthy that Nakagawa et al. [25] detected transcripts of the MSRV-related endogenous sequence ERV-9, together with other endogenous retroviral transcripts, in cellular RNA from the synovium of patients with arthritis. Unlike the present study, however, in the study of Nakagawa et al. there was no attempt to select for cell-free RNA, which is indicative of virion production.

The detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of a patient with joint inflammation due to gout suggests that this phenomenon may not be limited to RA. Similarly, detection of the novel human retrovirus HRV-5 does not appear to be RA-specific since it has been found in several other forms of inflammatory joint disease, including osteoarthritis, reactive arthritis and psoriatic arthritis [9].

At present it is unclear whether the detection of MSRV sequences in this context simply represents an epiphenomenon (e.g. the abnormal expression of endogenous HERV-W or an unrelated co-infection) or whether it might play some part in the inflammatory cascade, possibly by affecting the expression of other genes, such as those encoding cytokines, matrix metalloproteinases or their inhibitors [26]. If RA represents the common clinical manifestation of an autoimmune process triggered by a variety of different aetiological agents, then the detection of MSRV in synovial fluid might be indicative of a particular aetiological subgroup. Clearly, further work is required to determine more precisely the prevalence and possible pathogenic significance of MSRV in RA and other diseases.


    Acknowledgments
 
This work was financially supported by bioMérieux SA, France and by the Wellcome Trust, UK (grant 057298/Z/99/Z/SRD).


    Notes
 
Correspondence to: Dr J. A. Garson, Department of Virology, Royal Free and University College Medical School, Windeyer Building, 46 Cleveland Street, London W1P 6DB, UK. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 27 July 1999; revised version accepted 17 February 2000.