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(Chest. 2000;118:923-927.)
© 2000 American College of Chest Physicians

Human Herpesvirus 8 Is Not Associated With Sarcoidosis in Japanese Patients*

Hiroyoshi Maeda, MD; Takashi Niimi, MD; Shigeki Sato, MD; Haruhiko Kawaguchi, MD; Yoshiki Sugiura, MD; Shigeo Mori, MD and Ryuzo Ueda, MD

* From the Second Department of Internal Medicine (Drs. Maeda, Niimi, Sato, Kawaguchi, Sugiura, and Ueda), Nagoya City University Medical School, Nagoya, Japan; and the Department of Pathology (Dr. Mori), Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Correspondence to: Dr. Hiroyoshi Maeda, Second Department of Internal Medicine, Nagoya City University Medical School, 1 Kawasumi, Mizuho-ku, Nagoya, Aichi 467-8601, Japan.


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: The etiology of sarcoidosis remains unknown, but recently it was reported that human herpesvirus 8 (HHV-8) may be detected in sarcoid tissue in a high proportion of patients. This study was performed to determine whether HHV-8 is implicated in sarcoidosis in Japan.

Materials and methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 100 patients with sarcoidosis and 100 healthy donors living in central Japan. Additionally, 19 samples of sarcoid tissue, 10 of tuberculous tissue, and 10 of lung cancers were examined. DNA was extracted from PBMC or tissue samples, and a hemi-nested polymerase chain reaction assay was performed for HHV-8 detection.

Results: In the PBMC study, the detection rates for HHV-8 in patients with sarcoidosis and in normal donors were 2% and 1%, respectively, the difference not being significant (p > 0.99). In the tissue study, HHV-8 was detected in 10.5% of sarcoid and in 15% of nonsarcoid tissues, again not a significant difference (p > 0.99).

Conclusions: From these results, we conclude that HHV-8 is not implicated in sarcoidosis in Japanese patients.

Key Words: human herpesvirus 8 • polymerase chain reaction • sarcoidosis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sarcoidosis is a systemic granulomatous disease. Its etiology is unknown, although it is thought to be a specific reaction of the host to some agent. In previous studies, mycobacteria, pine pollen, viruses, and mycoplasma have been investigated as candidates, but none have been proven to be a causative agent.1 2

Human herpesvirus 8 (HHV-8) is a {gamma}-herpesvirus that was discovered in Kaposi’s sarcoma tissues from patients with AIDS.3 The virus is present in all forms of Kaposi’s sarcoma,4 primary effusion lymphoma, and multicentric Castleman’s disease.5 Multiple myeloma may be associated with the virus, although this is still controversial.6

HHV-8 encodes a homolog of macrophage inflammatory protein-1 and of interleukin 6.7 Peripheral blood mononuclear cells (PBMCs) and T lymphocytes established from Kaposi’s sarcoma skin lesions, which contain HHV-8, are reported to produce large amounts of {gamma}-interferon.8

In sarcoidosis, the plasma monocyte inflammatory protein-1 {alpha} level correlates with disease activity,9 and alveolar macrophages in sarcoidosis patients produce high levels of interleukin 6.10 Elevation of {gamma}-interferon production by T lymphocytes in affected organs of sarcoidosis has also been reported.11 It is possible that these biological phenomena are associated with HHV-8.

Di Alberti et al12 detected HHV-8 DNA in a higher proportion of sarcoid than nonsarcoid tissues and therefore speculated on an involvement in sarcoidosis. However, subsequent studies in white patients by other investigators failed to show any correlation between sarcoidosis and HHV-8.13 14 In the present study, the aim was to assess whether HHV-8 is a causative agent for sarcoidosis in Japanese patients. We investigated HHV-8 DNA prevalence in PBMCs, comparing sarcoidosis patients with normal control subjects, because it is likely that HHV-8 infects PBMCs and that its cytokine homologs affect the onset of sarcoidosis. Samples of sarcoid tissue were also compared with control samples.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
We studied PBMCs from 100 patients with sarcoidosis living in central Japan. The sample consisted of 18 men and 82 women, whose average age (mean ± SD) was 58.3 ± 14.6 years. Sarcoidosis was diagnosed by clinical features or on the basis of noncaseous epitheloid granulomas in biopsy specimens. The chest radiographic stages were as follows: 41 patients with stage I, 7 patients with stage II, and 10 patients with stage III sarcoidosis. The remaining 42 patients were without radiographic findings. Among the 100 patients, 72 patients had histologically proven disease. The clinical features included bilateral hilar lymphadenopathy on chest radiography, elevation of serum angiotensin-converting enzyme level, Ga scintigraphic findings, elevation of CD4/CD8 ratio of lymphocytes obtained by BAL, and negative reaction to purified protein-derivative skin test. Tuberculosis was carefully excluded.

As normal control subjects, 100 healthy volunteers from the same area were investigated. They consisted of 40 men and 60 women, with a mean (± SD) age of 54.5 ± 19.5 years. They were subjected to both physical examinations and laboratory tests, the results of which indicated that they were free of any disease.

For the tissue study, materials were all formalin-fixed paraffin-embedded tissues, except for three frozen lymph nodes, obtained for diagnosis of sarcoidosis between 1981 and 1998 and stored at -80°C. Only samples of sufficient quality and quantity were selected for evaluation, and those consisted of one transbronchial specimen, six skin specimens, six lymph node specimens, one lung specimen, one muscle specimen, one liver specimen, and three frozen lymph node specimens.

Disease control materials were 10 tuberculous lymph nodes obtained for diagnosis and 10 samples of lung cancer tissue obtained by therapeutic resection. Skin biopsy samples from Kaposi’s sarcoma patients were tested as positive controls.

Informed consent was obtained from all subjects.

DNA Extraction
About 10 mL of heparinized peripheral blood was obtained from patients with sarcoidosis and from normal volunteers, and PBMCs were purified on Ficoll gradients. DNA was extracted from PBMCs and frozen biopsy materials, using proteinase-K digestion, phenol-chloroform extraction, and ethanol precipitation. Each paraffin-embedded pathologic sample was deparaffinized in xylene. Then DNA was extracted with a DNA extraction kit (DNA Isolator PS Kit; Wako Pure Chemical Industries; Tokyo, Japan). The DNA concentration was measured by reading optical densities at 260 nm (Gene Quant; Amersham Pharmacia Biotech AB; Uppsala, Sweden).

Polymerase Chain Reaction Amplification
For detection of HHV-8 DNA, a hemi-nested polymerase chain reaction (PCR) assay was performed. For the first-round PCR, primers KS1 (5'-AGCCGAAAGGATTCCACCAT-3') and KS2 (5'-TCCGTGTTGTCTACGTCCAG-3'), described by Chang et al3 were used. They amplify a 233-base-pair (bp) DNA fragment (KS330Bam233) from open reading frame 26 of the HHV-8 genome.

The PCR mixture contained 0.5 µg sample DNA (about 0.3 µg in the case of formalin-fixed paraffin-embedded tissues), 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, 200 mM primers, and 0.5 U Taq polymerase, and the total volume was 25 µL. PCR was performed with 35 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min. Before the 35 cycles, 2-min desaturation at 94°C was added, and the 35 cycles were followed by 5-min extension at 72°C (GeneAmp PCR System 2400; Perkin-Elmer; Norwalk, CT). An aliquot (1 µL) of the first round of PCR products was added to the second-round PCR mixture containing KS1 and NS2 (5'-AATGACACATTGGTGGTATA-3'), which amplify a 194-bp DNA fragment, in place of the primer set KS1 and KS2. The cycle conditions were the same as for the first round PCR, except that the annealing temperature was 57°C. BCP-1 cells 15 were used as positive controls, and a cell line of small cell lung cancer was used as a negative control. The hemi-nested PCR products were separated on 2.5% agarose gels and stained with ethidium bromide. The human ß-globin or human vitamin D receptor genes were amplified from samples to confirm that the samples were adequate for PCR.

Hemi-Nested PCR Sensitivity and Virus Load Estimation
The hemi-nested PCR sensitivity was determined following the method of Bigoni et al.16 First-round PCR was performed for HHV-8-positive DNA extracted from BCP-1 cells, then the 233-bp product was extracted from the gel, and serial dilutions were made and amplified by hemi-nested PCR. To estimate the virus load of HHV-8-positive samples, serial dilutions of the samples were performed.

Immunohistochemistry
To identify HHV-8-infected cells in the lytic cycle, an immunohistochemistry test using anti-ORF59 rabbit antibodies was performed for 11 of 16 paraffin-embedded sarcoid tissues. The conditions for the immunohistochemistry test were as described previously.17 Kaposi’s sarcoma tissues were employed as positive controls.

DNA Sequencing
The existence of HHV-8 DNA sequence variants is known.18 19 Di Alberti et al19 segregated the HHV-8 DNA sequences into four groups (A, B, C, and D) based on amino acid changes at codons 134 and 167 within open reading frame 26. DNA sequencing was carried out to ascertain which type of HHV-8 variant was present in each positive sample. For all positive samples, the hemi-nested PCR products were purified from the agarose gels, then subjected to DNA sequencing by the cycle sequence method (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and 373A DNA Sequencer; Perkin-Elmer; Norwalk, CT).

Statistical Analysis
Comparisons between the samples of sarcoidosis patients and normal control subjects or between the tissues of sarcoidosis patients and disease control tissues were made with the Fisher’s Exact Test. A p value of < 0.05 was considered significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hemi-Nested PCR Sensitivity
The hemi-nested PCR assay used in this study could detect at least 10 target sequences on ethidium bromide staining. With the first round PCR, >103 target molecules could be detected (Fig 1 ).



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  		Figure 1. Hemi-nested PCR sensitivity. Serial dilutions of the target molecules were amplified by first- and second-round PCR. More than 103 target molecules could be detected by the first-round PCR. The second-round PCR could detect >10 target molecules by ethidium bromide staining. Lane M: molecular size markers (Phai X 174, Hae III).

 
HHV-8 Detection in PBMC Samples
In PBMC samples, HHV-8 DNA was detected in 2 of 100 sarcoidosis patients and in 1 of 100 normal volunteers (Table 1 ), a difference of no statistical significance (p > 0.99). With 10-fold dilutions of HHV-8-positive PBMC samples, negative staining of agarose gels was apparent with ethidium bromide (Fig 2 ). Since 0.5 µg of template DNA corresponds to 7.5 x 104 diploid cells, these results show that one HHV-8 genome exists in about 7,500 cells of positive samples.


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  		Table 1. HHV-8 DNA in Sarcoidosis and in Nonsarcoidosis Specimens*

 


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  		Figure 2. HHV-8-positive samples, showing 194-bp bands. Left lanes contain nondiluted samples, and right lanes of each pair contain 10-fold diluted samples. Top, A: PBMC: samples 1 and 2, sarcoidosis; sample 3, normal control. Bottom, B: Tissue specimens: samples 4 and 5, lymph node and liver, respectively, from a sarcoidosis patient; sample 6, lung cancer; samples 7 and 8, tuberculous lymph nodes. Lane N: negative control, lane P: positive control.

 
HHV-8 Detection in Tissue Samples
Regarding the paraffin-embedded formalin-fixed tissues from sarcoidosis patients, one of six lymph node specimens and one liver sample were positive for HHV-8 DNA, and the others, including all three frozen lymph nodes from sarcoidosis patients, proved to be negative. Of the control tissue samples, 2 of the 10 paraffin-embedded tuberculous lymph nodes were positive, along with 1 of the 10 lung cancer specimens. Our results showed no significant difference between the sarcoidosis samples and disease control samples (p > 0.99). All 10-fold dilutions of positive samples gave negative staining on agarose gels (Fig 2) . In the two skin specimens from Kaposi’s sarcoma patients, HHV-8 DNA fragments were detected (Table 1) . All 11 sarcoidosis samples, including the 2 specimens that were positive by hemi-nested PCR, were shown to be negative by immunostaining using anti-ORF59 antibody (data not shown).

DNA Sequence Assay
For all samples that proved positive for HHV-8 by hemi-nested PCR, we obtained the expected sequence for the HHV-8 DNA fragment amplified by hemi-nested PCR used in this study. For both sarcoid and nonsarcoid samples, all the DNA sequences belonged to the B type described by Di Alberti et al.19


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the HHV-8 open reading frame 26 DNA fragment could be detected in PBMCs from both sarcoidosis patients and from normal control subjects, but the detection rate was low in both groups with no significant difference between the two. Moreover, no difference was found in HHV-8 detection between sarcoidosis-affected and nonaffected tissues. From these results, we can conclude there is no strong association between sarcoidosis and HHV-8 in Japan.

Epidemiologically, the prevalence of HHV-8 differs among areas and countries. In the United Kingdom and in the United States, HHV-8 DNA was not detected in PBMCs from healthy persons.20 21 However, Bigoni et al16 reported that 9% of healthy donors in Italy were positive for HHV-8 DNA. In this study, HHV-8 DNA in PBMCs was detected in only 1% of healthy persons in Japan, higher than the prevalence in the United Kingdom or the United States, but lower than that in Italy. In PBMCs from sarcoidosis patients, the detection rate of HHV-8 DNA was 2% and was similar to that in healthy control subjects. Additionally, the virus loads in HHV-8-positive samples were low in both normal control subjects and sarcoidosis patients.

In the tissue study, HHV-8 was positive in 20% of tuberculous lymph nodes and was detected in 10.5% of sarcoidosis tissues. In a previous study, HHV-8 was detected in about 29% of lymph nodes from cases of nonspecific lymphadenitis.12 In 9% of lymph nodes from reactive lymphadenopathies, HHV-8 was positive in another study, from which the authors speculated that the lymphoid system could represent a reservoir of latently infected cells.16 The HHV-8 detection rate in sarcoid tissue was not high compared with that in nonsarcoid tissue in this or previous studies.

The sensitivity of hemi-nested PCR was sufficiently high to detect 10 copies of HHV-8 DNA in this study. Moreover, the results of immunostaining showed negative findings in tissues that were positive by hemi-nested PCR. This may mean that the virus was not in the lytic cycle, because ORF59 protein is a lytic cycle-associated protein.22 Thus in tissues that are positive for HHV-8 by hemi-nested PCR, the virus might not be proliferating. We speculate that HHV-8 is present in local lesions only as an exogenous agent, not associated with the onset of sarcoidosis.

HHV-8 is an interesting virus that may be linked to various diseases, but we conclude that it is not implicated in the incidence of sarcoidosis in Japan. Research into the cause of sarcoidosis clearly must continue, with researchers concentrating their attention on both extrinsic agents and intrinsic immune responses.


   Acknowledgements
 
The authors thank Miss Asuka Kondo for kind assistance and Drs. Masayuki Suzuki, Munehiko Morishita, Yosiro Kato, Masahiko Yamamoto, Akimichi Morita, Shigeki Shimizu, Shoko Yoshida, and Kenji Akita for their expert advice.


   Footnotes
 
Abbreviations: bp = base-pair; HHV-8 = human herpesvirus 8; PBMC = peripheral blood mononuclear cell; PCR = polymerase chain reaction

This work was supported in part by grants from the Ministry of Health and Welfare, Japan, and by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and from the Culture, Japan, and Imanaga Medical Foundation.

Received for publication December 20, 1999. Accepted for publication April 13, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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