No evidence of parvovirus B19, Chlamydia pneumoniae or human herpes virus infection in temporal artery biopsies in patients with giant cell arteritis

J. Helweg-Larsen, B. Tarp1, N. Obel1 and B. Baslund2

Department of Infectious Diseases, Hvidovre University Hospital,
1 Department of Infectious Diseases, Marselisborg Hospital, Aarhus University Hospital and
2 Department of Rheumatology, Rigshospitalet, Copenhagen, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. Recent studies have suggested that infective agents may be involved in the pathogenesis of giant cell arteritis (GCA), in particular Chlamydia pneumoniae and parvovirus B19. We investigated temporal arteries from patients with GCA for these infections as well as human herpes viruses using the polymerase chain reaction (PCR).

Methods. Thirty temporal artery biopsies from 30 patients suspected of having GCA within a period of 1 yr were examined. Thirteen patients had classical GCA, two had biopsy-negative GCA, 10 patients had polymyalgia rheumatica and five patients had other conditions. DNA was extracted from frozen biopsies and PCR was used to amplify genes from Chlamydia pneumoniae, parvovirus B19 and each of the eight human herpes viruses: herpes simplex viruses HSV-1 and 2, Epstein–Barr virus, cytomegalovirus, varicella zoster virus and human herpes viruses HHV-6, -7 and -8.

Results. In all 30 biopsies, PCR was negative for DNAs of parvovirus B19, each of the eight human herpes viruses and C. pneumoniae.

Conclusions. We found no evidence of DNA from parvovirus B19, human herpes virus or C. pneumoniae in any of the temporal arteries. These agents do not seem to play a unique or dominant role in the pathogenesis of GCA.

KEY WORDS: Giant cell arteritis, Temporal artery biopsy, Parvovirus B19, Chlamydia pneumoniae, HSV-1, HSV-2, EBV, CMV, VZV, HHV-6, HHV-7, HHV-8.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Giant cell arteritis (GCA) is a disease characterized by acute inflammation in medium to large arteries in elderly people. The disease has a predilection for branches of the temporal arteries. Typically, panarteritis is seen with granulomas composed of T cells, activated macrophages and multinucleated giant cells. The cause of GCA is unknown. Great variation in the incidence of GCA has been reported in different parts of the world, with a high incidence in Scandinavia [1], suggesting that environmental or genetic factors may contribute to the pathogenesis. Although certain HLA-DR alleles are correlated with GCA [2], genetic factors are insufficient to explain the marked variation in geographic prevalence and the temporal fluctuation in the incidence of the disease. Regular cyclical patterns in the incidence rate of GCA over time were shown in Minnesota, with a peak every 6–7 yr [3]. Similarly, a Danish study reported cyclic fluctuations that coincided with epidemics of Mycoplasma pneumoniae, parvovirus B19 and Chlamydia pneumoniae [4]. Furthermore, a retrospective study of 100 patients with GCA found that the prevalence of recent or current infections in patients with temporal arteritis was three times higher than that in matched control patients [5]. These findings suggest that infectious agents may be involved in the pathogenesis of the disease. Recently, possible associations between GCA and infection with parvovirus B19 or C. pneumoniae have been reported [6, 7]. In the present study we searched for evidence of these infections as well as infection with the human herpes viruses by using the polymerase reaction (PCR) on temporal artery biopsies obtained prospectively from patients suspected of having GCA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
In a one-year period from 1992–1993, temporal artery biopsies were taken from 30 consecutive patients suspected of having GCA or polymyalgia rheumatica, at the Department of Rheumatology, Hvidovre University Hospital. The diagnosis of GCA and polymyalgia rheumatica was based on the criteria of the American College of Rheumatology [8] and Bird et al. [9] respectively.

Specimen and DNA extraction
Fresh temporal artery biopsies were obtained from 30 patients suspected of having GCA. Tissue sections (1–2 cm) were transported in sterile saline, cryo-embedded in optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura, CA, USA) and stored at -70°C. Twenty-seven sections 2 µm thick were taken from each biopsy for routine histological staining. The remainder of each biopsy was used for DNA extraction. Each cryo-embedded section was thawed and the OCT compound removed by elution in sterile saline. DNA extraction was done with the QIAamp DNA mini-kit (Qiagen, Hilden, Germany). To increase DNA yield, glycogen as DNA carrier and three final elution steps were used according to the manufacturer's protocol. The DNA concentration was measured by spectrophotometric measurement of ultraviolet absorbance.

The DNA dilutions used for each PCR setup were between 80 and 100 µg/ml and their ratios of optical densities at 260 and 280 nm were between 1.7 and 1.9. To ensure that DNA had been extracted sufficiently and was non-degraded, dilutions of the DNA were tested by amplification of genes encoding the human genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) [10] and CCR5, a single-copy gene [11]. In all samples, strong PCR bands were observed down to a dilution of 1:105 of the original concentration. For samples that were negative to amplification, 1 µl of the sample was mixed with 1 µl of a PCR-positive sample and the PCR was repeated to control for inhibition.

PCR amplification of parvovirus B19
Primers were directed against the VP1 gene region and the non-structural protein (NSI)-coding region of B19 with PCR conditions according to previous reports [6, 12], with further optimization by the use of a touch-down procedure [13]. Tissue DNA (5 µl) was analysed in each PCR setup using reagents from the Amplitaq Gold kit (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) on a PE 9700 thermocycler (Perkin-Elmer Applied Biosystems).

PCR amplification of C. pneumoniae
Two different assays, one targeting the 16S rRNA gene and one targeting the outer surface membrane protein A (OMPA) gene, were used for the detection of C. pneumoniae. The 16S rRNA was amplified by single-round PCR as reported by Gaydos et al. [14]. The OMPA gene was amplified by nested PCR as reported by Balin et al. [15]. The PCR was set up using reagents from the Amplitaq Gold kit with cycling conditions as reported by Gaydos et al. and the use of a touch-down procedure on a PE 9700 thermocycler [16]. As a positive control we used dilutions of DNA extracted from C. pneumoniae cultured in Hep2 cells.

PCR amplification of human herpesviruses
PCR was performed for each of the eight human herpes viruses: herpes simplex viruses HSV-1 and 2, Epstein–Barr virus (EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and human herpes viruses HHV-6, -7 and -8. DNA was amplified from 2 µl of extracted DNA in a final volume of 21 µl. The reaction mixture contained 0.5 U Amplitaq Gold (Perkin-Elmer Applied Biosystems), 2 µl MgCl2 25 mmol/l, 2 µl dNTP 500 µmol/l, 2 µl PCR Buffer II (Perkin-Elmer Applied Biosystems) and 20 pmol of each primer (DNA Technology, Aarhus, Denmark). The primer sequences and amplification procedures used to detect the eight herpes viruses were as described previously [17].

Gel electrophoresis
A 10 µl aliquot of the PCR product was analysed by electrophoresis in a 2% agarose gel containing 0.5 µg/ml ethidium bromide and visualized under UV light. A negative control was run after each fifth sample and each group of nine samples was flanked by positive controls of the DNA concerned. Assays in which sensitivity of the positive controls was suboptimal were repeated.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Samples from a total of 30 patients were included in this study prospectively. The samples represented all temporal biopsies obtained from patients suspected of having GCA during a period of 1 yr. Among the 30 patients, 15 were diagnosed as having GCA (13 were biopsy-positive) and 10 patients with polymyalgia rheumatica. The remaining five patients were later diagnosed with other diseases (two patients had rheumatoid arthritis and three patients had malignancies).

All arteries were PCR-negative for C. pneumoniae, parvovirus B19 and each of the eight human herpes viruses. No samples showed inhibition in the PCR (Fig. 1Go).



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FIG. 1.  Representative PCR results from one GCA patient, showing positive PCR bands from amplification of human DNA controls (lanes 2 and 3), PCR bands from amplification of DNA from viral and bacterial controls (lanes 4–14) and negative PCR bands obtained by PCR amplification of each viral and bacterial gene, when amplifying temporal artery tissue DNA (lanes 15–26). Lane 1, DNA marker; lane 2, GAPDH; lane 3, CCR5; lanes 4 and 15, HSV-1; lanes 5 and 16, HSV-2; lanes 6 and 17, CMV; lanes 7 and 18, VZV; lanes 8 and 19, EBV; lanes 9 and 20, HHV-6; lanes 10 and 21, HHV-7; lanes 11 and 22, HHV-8; lanes 12 and 23, C. pneumoniae 16S RNA; lanes 13 and 24, C. pneumoniae OMPA; lanes 14 and 25, parvovirus VP1; lanes 15 and 26, parvovirus NSI; lane 27, water.

 
The detection limits of the PCR assays for the different human herpes viruses were less than 10 genome copies, the detection limits being determined as described previously [17]. The C. pneumoniae-positive control could be detected down to 1:105 of the original concentration with the single-round PCR and 1:106 with the nested PCR. The serum sample used as the positive control for parvovirus B19 was PCR-positive in dilutions to 1:104.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, no evidence of C. pneumoniae, parvovirus B19 or any of the eight human herpes viruses was found in temporal arteries.

An environmental infective trigger for GCA has long been suspected on the basis of the clinical presentation, as flu-like symptoms are often seen at the onset of disease and are followed by a marked acute-phase response [18]. In addition, molecular studies of GCA have suggested a possible link between infection and GCA. Sequence analysis of the T-cell receptor of tissue-infiltrating T cells in lesions of temporal arteritis indicates restricted clonal expansion, suggesting that an antigen residing in the arterial wall is recognized by a small fraction of T cells [19, 20]. It has been demonstrated recently that the vascular lesions of GCA can be maintained in human artery–mouse chimeras, indicating that all components necessary for the disease are present in the temporal artery [21]. These findings suggest that a specific antigen, located within affected arteries, elicits the disease. In this work, we chose to investigate temporal biopsies for evidence of infection with C. pneumoniae and parvovirus B19, which have recently been associated with GCA [6, 7]. As the disease has a predilection for elderly people, we hypothesized that reactivation of latent infection may play a role. The ability of herpes viruses to cause recrudescent or reactivated infection in the elderly and to provoke T-cell inflammation implicates these viruses also as possible disease triggers.

Although infection with C. pneumoniae primarily causes respiratory tract infection, the organism has recently been postulated to play a role in the pathogenesis of arteriosclerosis, in particular coronary heart disease. Several studies have detected C. pneumoniae DNA in atherosclerotic lesions and viable organisms have been recovered from atheromatous plaques [22, 23]. In vitro studies indicate that C. pneumoniae is able to infect and reproduce in human endothelial cells, smooth muscle cells and macrophages, producing inflammatory changes suggestive of arteriosclerosis [24, 25]. Along this line of evidence, Wagner et al. [7] recently searched for C. pneumoniae in temporal biopsies from patients with GCA. Temporal biopsies obtained from 17 patients were investigated by PCR and immunohistochemistry. In eight of nine GCA patients, C. pneumoniae was demonstrated, compared with one of three patients with polymyalgia rheumatica (immunohistochemistry alone) and in none of five control patients.

Infection with parvovirus B19 produces a variety of clinical manifestations, from asymptomatic infection to respiratory tract illness, rash, joint affection and bone marrow suppression. In addition, the infection has been noted in patients with systemic vasculitis and inflammatory arthritis. One case of GCA and persistent parvovirus B19 infection with high titres and virus in the blood was reported by Staud and Corman [26], but detection of virus in temporal biopsies was not attempted. Later, Gabriel et al. [6] prospectively examined temporal artery biopsy tissue from a consecutive series of 50 patients for the presence of parvovirus B19. Parvovirus B19 DNA was present in seven (54%) of the 13 biopsies from patients with GCA compared with only four (11%) of 37 biopsies from patients without GCA, suggesting the involvement of B19 in the pathogenesis.

In contrast to these studies, we were unable to find evidence of DNA from C. pneumoniae or parvovirus B19 in our samples. The negative PCR results could not be attributed to inhibition or lack of sensitivity. In all samples, DNA extraction was successful, as confirmed by the presence of sufficient and non-degraded human DNA, and no PCR inhibition was observed. Although it is difficult to exclude sampling errors totally, we believe that they are unlikely. In contrast to Gabriel et al. [6] and Wagner et al. [7], who extracted DNA from tissue sections of paraffin tissue samples, we used DNA extracted from whole blocks of arterial biopsies preserved in a cryopreservative made up from high molecular weight alcohols. Although no data comparing DNA from cryopreservation vs paraffin fixation are available, there is no reason to expect that the DNA yield and integrity obtained from cryopreserved tissue should be inferior to the yield from paraffin preserved tissue, in which DNA degradation and PCR inhibition may be a problem. Neither can differences in the PCR assay or sensitivity explain the discrepancy. To detect parvovirus B19, we used the same PCR assay as Gabriel et al. [6], targeting the NSI gene as well as an additional assay targeting the VP1 region. Similarly, we reproduced the two PCR assays used by Wagner et al. [7]. In addition, all PCR setups were optimized by the use of a touch-down hot-start procedure [13, 27].

So far, the observation that parvovirus B19 and C. pneumoniae infections are risk factors for GCA is based on only a few investigations. Our results do not confirm the findings of associations between these agents and GCA. In addition, we found no association between GCA and human herpes virus infection. To our knowledge, this study is the first to investigate temporal arteries for all eight human herpes viruses. Previously, only VZV has been investigated for a possible connection with GCA [28]. Further studies are required to establish whether microbial infection is central to the aetiology of GCA.


    Acknowledgments
 
We thank Dr Jørgen Skov Jensen of the State Serum Institute (SSI) for advice and the gift of C. pneumoniae DNA, Dr Anders Fomsgaard (SSI) for the parvovirus B19 control and Jesper Eugen-Olsen of Hvidovre Hospital for technical advice.


    Notes
 
Correspondence to: J. Helweg-Larsen, Copenhagen HIV programme, Department of Infectious Diseases 144, Hvidovre University Hospital, 2650 Hvidovre, Denmark. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 18 December 2000; Accepted 7 November 2001