Absence of Kaposi's Sarcoma–associated Herpesvirus in Patients with Pulmonary Arterial Hypertension
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《美国呼吸和危急护理医学》
Institutes of Virology and Pathology, Department of Pulmonary Medicine, Hannover Medical School, Hannover, Germany
ABSTRACT
Rationale: In addition to Kaposi's sarcoma, Kaposi's sarcoma–associated herpesvirus (KSHV or HHV-8) has been associated with two other diseases: primary effusion lymphoma and the plasma cell variant of multicentric Castleman's disease. Recently, evidence of KSHV infection was reported in plexiform lesions of idiopathic pulmonary arterial hypertension (IPAH) as well as in adjacent parenchyma and bronchial epithelial cells.
Objectives: To further investigate a possible association of KSHV and pulmonary arterial hypertension.
Methods and Measurements: Twenty-six lungs explanted from German patients suffering from IPAH were tested for the presence of KSHV antigen and genomes by immunohistochemistry (IHC) and polymerase chain reaction (PCR).
Main Results: When stained with a commercial monoclonal antibody directed against the latency-associated nuclear antigen of KSHV, LANA-1, a positive signal reminiscent of the "speckled" nuclear pattern typical of latently KSHV-infected cells was found in 16 (61.5%) cases. Alveolar and bronchial epithelial cells in areas of unremarkable lung tissue, but not cells within the plexiform lesions, were the predominantly stained cell types. Different KSHV-PCR assays (based on orf26, orfK6, and orf72) performed on samples that had tested positively in IHC, however, could not confirm KSHV infection, indicating that the IHC signal was not due to KSHV infection. One IHC-negative patient tested positive by PCR. A PCR based on consensus degenerate hybrid oligonucleotide primers (CODEHOP) to detect yet unknown -herpesviruses did not reveal any specific sequences.
Conclusions: KSHV is unlikely to play a role in the pathogenesis of IPAH.
Key Words: etiology human herpesvirus-8 Kaposi's sarcoma–associated herpesvirus pathogenesis pulmonary arterial hypertension
Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease of unknown etiology that is characterized by progressively increasing pulmonary arterial pressure resulting from the obliteration of small pulmonary arteries. Histologically, a hallmark of IPAH are tumorlike lesions composed of compact, proliferating, spindle-shaped endothelial or smooth muscle cells with vascular slits. These so-called plexiform lesions are often but not exclusively found in IPAH biopsies and therefore not pathognomonic for this disease (1). The pathogenesis of IPAH remains unknown. Some cases of IPAH are associated with germ-line mutations of the bone morphogenetic protein receptor 2 (2, 3), but these mutations are neither necessary nor sufficient to cause IPAH and other—yet unidentified—factors must be involved.
Kaposi's sarcoma–associated herpesvirus (KSHV), also called human herpesvirus-8 or HHV-8, is the most recently discovered human oncogenic herpesvirus. It is a necessary cause of not only all epidemiologic forms of Kaposi's sarcoma (KS), from which this virus was originally isolated (4), but also of a rare, mainly AIDS-associated lymphoproliferative disease, primary effusion lymphoma (5). In addition, KSHV can be regularly found in the plasma cell variant of multicentric Castleman's disease (6). Since its discovery in 1994, KSHV infection has been tentatively linked with numerous malignant and nonmalignant diseases, including hemophagocytic syndrome (7) or bone marrow failure after transplantation (8), sarcoidosis (9), pemphigus (10), multiple myeloma (11), as well as other hematologic disorders, in particular in the immuncompromised patient. The etiologic relationship between KSHV infection and several of these disorders could, however, not be confirmed by others (12–14). Recently, KSHV latent nuclear antigen 1 (LANA-1), a viral nuclear protein essential for the replication and maintenance of latent viral genomes (15, 16), and KSHV genomic DNA were detected in lung tissue from 10 (62%) of 16 patients from the United States with IPAH, suggesting an association between KSHV and the pathogenesis of this disease (17). The present study investigated the presence of KSHV in lung tissue from 26 German patients with IPAH.
Some of the results of these studies have been previously reported in the form of an abstract (18).
METHODS
Lung tissue samples were obtained from 26 patients with pulmonary arterial hypertension (PAH), including 22 with classical IPAH, one with a familial form of PAH, and three with a veno-occlusive variant, who underwent transplantation from 1993 to 2003 in our hospital. Further information on subjects enrolled in this study is given in the online supplement.
Immunohistochemistry
Tissue samples were incorporated into three tissue microarrays (MaxArray system; Zymed Laboratories, Inc., South San Francisco, CA), each comprising up to 60 tissue cores. From each transplanted lung, three tissue cores comprising plexiform lesions were analyzed together with three tissue cores from areas with histologically normal lung tissue serving as controls. Tissue microarrays were manufactured, following the suppliers' instructions (19). Immunohistochemical stains were performed on the tissue microarrays applying a tyramine amplification technique (TSA-Renaissance kit; NenLifeScience, Boston, MA) as described elsewhere (20, 21). A supplementary description is given in online supplement. Cases with any specific nuclear signal, independently of its intensity, were regarded as positive. All other cases, including those with cytoplasmic or membrane stains, were regarded as negative. A KS biopsy served as positive control and stains with bovine serum instead of the primary antibody as negative controls.
Polymerase Chain Reaction
For polymerase chain reaction (PCR) analysis, DNA was extracted from whole paraffin blocks comprising tissue samples of 24 of 26 analyzed patients according to a protocol published elsewhere (22), with few modifications. In brief, tissue samples were incubated in proteinase K buffer (50 mM Tris HCl, pH 8.1; 1 mM ethylenediaminetetraacetic acid; 0.5% Tween; proteinase K, 1 mg/ml; Merck, Darmstadt, Germany) at 56°C for 25 h. The first lysate was obtained by collecting the supernatant after heating and centrifugation of these samples. This extract was used for PCR. In addition, DNA was recovered from this extract by phenol/chloroform treatment and subsequent DNA precipitation. Some PCRs were performed on both preparations (lysate and purified DNA) with a standard PCR protocol using the Amplitaq system (Perkin Elmer, Rodgan-Jügesheim, Germany). A sample was regarded as positive if one of the two different preparations revealed a positive product. Details of the experimental protocol as well as PCR primer sequences can be found in the online supplement. The presence of amplifiable DNA in the extracted samples was verified by amplification of a 289-bp fragment of the -globin gene (23). The DNA samples were then analyzed for the presence of KSHV genomes with different KSHV-specific PCR primer sets based on orf26 (24) and orf72 (25). In addition, a quantitative real-time PCR was used to amplify a fragment of the K6 gene of the KSHV genome. The technique was performed as described elsewhere (26). For the consensus degenerate hybrid oligonucleotide primers (CODEHOP) PCR, which was designed to detect a range of 2-herpesviruses, a triple, seminested protocol was performed essentially as described (27) using the primer gdtd1b for all rounds, whereas vygam served as reverse primer for first round, and clnia and vtqlga for the second and third rounds, respectively.
RESULTS
To investigate whether KSHV LANA-1 was present in lung tissue of 26 patients with IPAH, we performed an immunohistochemical stain with a monoclonal antibody directed against an epitope within the internal repeat region of the LANA-1 of KSHV. This antibody has been widely used to detect LANA-1 in tissue sections of KS, primary effusion lymphoma, and multicentric Castleman's disease by immunohistochemistry (IHC) (28, 29). All patient samples showed characteristic plexiform lesions. In 16 cases (61.5%), a mostly faint, but still in some cases typical, "speckled" nuclear LANA-1 staining signal was found (Table 1). Six of these 16 cases showed strong nuclear stains, as seen in KSHV-infected KS spindle cells (30). Alveolar (Figure 1A) and bronchial (Figure 1B) epithelial cells in areas of unremarkable lung tissue were the predominately stained cell type. Only in a single case were positive mesenchymal cells found adjacent to plexiform lesions (Figure 1C). A KS biopsy served as positive control (Figure 1D). To exclude the possibility that the use of tyramine amplification in the IHC protocol could have affected the results, histologic sections of all positive samples were also stained with the LANA-1 antibody using a Ventana automatic staining system (BenchmarkXT stainer; Ventana, Schwetzinger, Germany) with identical results (data not shown).
To complement these results, we extracted DNA from 24 of these 26 tested samples (insufficient tissue was available for the remaining two samples). For some samples, the different PCR systems were performed on two different extractions. A -globin PCR performed to check the DNA integrity amplified a product in 17 of 24 samples. Two different KSHV-specific PCR protocols based on orf72 and orf26 were performed. In the case of orf26, we used a nested PCR, as well as a modified single-round PCR with increased cycle numbers. Sensitivity of these assays, determined by a limiting dilution of a DNA preparation of a bacterial artificial chromosome (BAC) harboring the whole KSHV genome (31), were 1,000 BAC copies/reaction for orf72 PCR, 100,000 BAC copies/reaction for the modified version, and 10 BAC copies/reaction for the nested version of the orf26 PCR. None of these assays detected any specific PCR product. A real-time PCR targeting the orfK6 gene with a sensitivity of 10 BAC copies/reaction revealed one weakly positive sample (400 copies in the reaction). Serum of this patient (patient 10 in Table 1) was also found to contain antibodies to LANA-1 by immunofluorescence assay (data not shown). Interestingly, no cells expressing LANA-1 were seen in the lung tissue from this patient by IHC.
To exclude the possibility that tissue extracts could have inhibited the PCR reactions, extracts from all available tissue samples were spiked with a low copy number of KSHV K6 plasmid (4,000 copies/reaction) and analyzed by quantitative real-time PCR. The expected copy number was obtained in all but two samples, thus excluding a false-negative result due to PCR inhibition in 91.3 % of the tested samples. In one sample, from an IHC-negative patient, the K6 plasmid copy number was reduced 10-fold, indicating moderate inhibition (Table 1). In one IHC-positive sample (case 8), the PCR reaction failed to amplify the spiked plasmid DNA.
To address the possibility that the LANA-1 antibody could have reacted with a homologous protein of a yet unknown herpesvirus in the immunohistochemical stain, we screened 10 samples previously found to give a positive signal with the LANA-1 antibody with a seminested consensus primer PCR targeting the herpesviral DNA-polymerase (orf9) (27) of different - or -herpesviruses. However, no herpesviral sequences were detected in the IPAH samples.
DISCUSSION
This study investigated the presence of a latent KSHV antigen in lung tissue of 26 patients from Germany with IPAH and of KSHV DNA in 24 of these cases. In 16 (61.5%) lung tissue samples, nuclei of alveolar epithelial, bronchoepithelial, and/or mesenchymal cells displayed a positive signal when stained with a monoclonal antibody against the internal repeat region of LANA-1. In some cases, the nuclear staining pattern was reminiscent of the "speckled" nuclear pattern typical of KSHV-infected primary effusion lymphoma and endothelial cells (28, 30); in others, it was more diffuse. Similar staining results were obtained when an automated staining procedure not involving tyramine amplification was used (data not shown). However, none of the 16 samples showing this staining pattern were positive by PCR, despite using three different KSHV-specific PCR protocols with sensitivities of as low as 10 copies/reaction. Whether the positive antibody stain is really detecting KSHV LANA-1 in our samples is therefore doubtful. In only one patient was lung tissue weakly positive in the KSHV K6 real-time quantitative PCR. KSHV infection of this patient was supported by the detection of antibodies to LANA-1 in the patient's serum as measured by immunofluorescence (data not shown). The weak PCR signal could reflect the presence of KSHV genomes in infiltrating blood cells rather than in lung cells, because the immunohistologic LANA-1 stain was negative. Therefore, KSHV infection can occur in patients with IPAH, but it is unlikely to play an etiologic role in this condition.
Cross-reactivity of the LANA-1 antibody with a homologous protein of another virus or with a cellular protein involved in the pathogenesis of IPAH could be potential explanations for our observations. We therefore screened the samples with seminested CODEHOP PCR. This assay has successfully identified new 2-herpesviruses from African Green monkeys and chimpanzees (27, 32), an ungulate 2-herpesvirus (unpublished), as well as Epstein-Barr virus and cytomegalovirus (data not shown). Our PCR did not amplify a viral gene in 10 selected, immunhistochemically positive IPAH samples.
In accordance with our findings, two other Japanese groups recently failed to detect KSHV DNA in patients with IPAH from Japan (33, 34). In addition, Katano and colleagues (33), also looking at their samples by immunohistochemistry, did not see staining with an LANA-1 antibody. Furthermore, Brouchet and coworkers (35), who were searching for oncogenic virus genomes in lung carcinomas, saw an artifactual staining signal that could not be confirmed by PCR, when staining histologic slides for KSHV LANA-1 using the same antibody as in our study.
KSHV seroprevalence varies in different geographic regions. It is widespread in Africa with a seroprevalence between 30 and 60% and is also common in some Mediterranean countries with up to 35% seroprevalence. In the rest of the world, however, KSHV infection is quite uncommon, at least in the general, healthy population, with seroprevalence rates ranging below 10% (36). The incidence of classical KS outside of Africa mirrors the KSHV seroprevalence very well (37). Neither a geographic difference nor an ethnic predisposition is described for IPAH, although such an association might be concealed by a heterogeneous etiology of this disease. However, we have recently reported a small seroepidemiologic case-control study with 49 patients with IPAH, 17 patients with secondary causes of pulmonary hypertension, and 74 healthy control subjects, which did not reveal any difference in the KSHV seroprevalance between IPAH and control groups (38). A similar lack of association between IPAH and KSHV antibody positivity in a U.S. case-control study was recently reported (39).
Infection of mice with the closest murine homolog of KSHV, mouse herpesvirus 68, results in a productive infection of the lung (40), a fact that could suggest that the lung indeed is a physiologic site of KSHV-8 infection. Indeed, KSHV genomes have been reported in lung tissue not only in pulmonary KS but in other lung disease entities, like interstitial pneumonitis (41)/idiopathic pulmonary fibrosis (42) or inflammatory pseudotumors (43). Remarkably, in interstitial pneumonitis, KSHV is not only found in lymphoid cells (i.e., cells involved in the inflammatory process) but also in endothelial cells of the pulmonary vasculature and in pneumocytes (41).
Thus, a staining of alveolar and bronchial epithelial cells, as found in this study, would be in keeping with the biology of another 2-herpesvirus. However, in line with the conclusion reached by Daibata and colleagues (34), Katano and colleagues (33), and in particular Brouchet and coworkers (35), we believe that the LANA-1 staining of epithelial cells seen in our study does not reflect presence of KSHV. Taken together with the lack of association between KSHV antibodies and IPAH in German patients and patients from the United States, we conclude that KSHV is unlikely to play a role in the pathogenesis of IPAH cases occurring in KSHV-nonendemic countries.
FOOTNOTES
Supported by core funding from the Hannover Medical School.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200504-546OC on September 28, 2005
Conflict of Interest Statement: C.H.-G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.M. is one of the patent holders and licenser of the German patent for a system of the construction of tissue microarrays, used in the present study, and received 1,600 royalty from Zymed Laboratories in 2004. He has been reimbursed by Roche and Novartis for attending several conferences. M.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.F.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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ABSTRACT
Rationale: In addition to Kaposi's sarcoma, Kaposi's sarcoma–associated herpesvirus (KSHV or HHV-8) has been associated with two other diseases: primary effusion lymphoma and the plasma cell variant of multicentric Castleman's disease. Recently, evidence of KSHV infection was reported in plexiform lesions of idiopathic pulmonary arterial hypertension (IPAH) as well as in adjacent parenchyma and bronchial epithelial cells.
Objectives: To further investigate a possible association of KSHV and pulmonary arterial hypertension.
Methods and Measurements: Twenty-six lungs explanted from German patients suffering from IPAH were tested for the presence of KSHV antigen and genomes by immunohistochemistry (IHC) and polymerase chain reaction (PCR).
Main Results: When stained with a commercial monoclonal antibody directed against the latency-associated nuclear antigen of KSHV, LANA-1, a positive signal reminiscent of the "speckled" nuclear pattern typical of latently KSHV-infected cells was found in 16 (61.5%) cases. Alveolar and bronchial epithelial cells in areas of unremarkable lung tissue, but not cells within the plexiform lesions, were the predominantly stained cell types. Different KSHV-PCR assays (based on orf26, orfK6, and orf72) performed on samples that had tested positively in IHC, however, could not confirm KSHV infection, indicating that the IHC signal was not due to KSHV infection. One IHC-negative patient tested positive by PCR. A PCR based on consensus degenerate hybrid oligonucleotide primers (CODEHOP) to detect yet unknown -herpesviruses did not reveal any specific sequences.
Conclusions: KSHV is unlikely to play a role in the pathogenesis of IPAH.
Key Words: etiology human herpesvirus-8 Kaposi's sarcoma–associated herpesvirus pathogenesis pulmonary arterial hypertension
Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease of unknown etiology that is characterized by progressively increasing pulmonary arterial pressure resulting from the obliteration of small pulmonary arteries. Histologically, a hallmark of IPAH are tumorlike lesions composed of compact, proliferating, spindle-shaped endothelial or smooth muscle cells with vascular slits. These so-called plexiform lesions are often but not exclusively found in IPAH biopsies and therefore not pathognomonic for this disease (1). The pathogenesis of IPAH remains unknown. Some cases of IPAH are associated with germ-line mutations of the bone morphogenetic protein receptor 2 (2, 3), but these mutations are neither necessary nor sufficient to cause IPAH and other—yet unidentified—factors must be involved.
Kaposi's sarcoma–associated herpesvirus (KSHV), also called human herpesvirus-8 or HHV-8, is the most recently discovered human oncogenic herpesvirus. It is a necessary cause of not only all epidemiologic forms of Kaposi's sarcoma (KS), from which this virus was originally isolated (4), but also of a rare, mainly AIDS-associated lymphoproliferative disease, primary effusion lymphoma (5). In addition, KSHV can be regularly found in the plasma cell variant of multicentric Castleman's disease (6). Since its discovery in 1994, KSHV infection has been tentatively linked with numerous malignant and nonmalignant diseases, including hemophagocytic syndrome (7) or bone marrow failure after transplantation (8), sarcoidosis (9), pemphigus (10), multiple myeloma (11), as well as other hematologic disorders, in particular in the immuncompromised patient. The etiologic relationship between KSHV infection and several of these disorders could, however, not be confirmed by others (12–14). Recently, KSHV latent nuclear antigen 1 (LANA-1), a viral nuclear protein essential for the replication and maintenance of latent viral genomes (15, 16), and KSHV genomic DNA were detected in lung tissue from 10 (62%) of 16 patients from the United States with IPAH, suggesting an association between KSHV and the pathogenesis of this disease (17). The present study investigated the presence of KSHV in lung tissue from 26 German patients with IPAH.
Some of the results of these studies have been previously reported in the form of an abstract (18).
METHODS
Lung tissue samples were obtained from 26 patients with pulmonary arterial hypertension (PAH), including 22 with classical IPAH, one with a familial form of PAH, and three with a veno-occlusive variant, who underwent transplantation from 1993 to 2003 in our hospital. Further information on subjects enrolled in this study is given in the online supplement.
Immunohistochemistry
Tissue samples were incorporated into three tissue microarrays (MaxArray system; Zymed Laboratories, Inc., South San Francisco, CA), each comprising up to 60 tissue cores. From each transplanted lung, three tissue cores comprising plexiform lesions were analyzed together with three tissue cores from areas with histologically normal lung tissue serving as controls. Tissue microarrays were manufactured, following the suppliers' instructions (19). Immunohistochemical stains were performed on the tissue microarrays applying a tyramine amplification technique (TSA-Renaissance kit; NenLifeScience, Boston, MA) as described elsewhere (20, 21). A supplementary description is given in online supplement. Cases with any specific nuclear signal, independently of its intensity, were regarded as positive. All other cases, including those with cytoplasmic or membrane stains, were regarded as negative. A KS biopsy served as positive control and stains with bovine serum instead of the primary antibody as negative controls.
Polymerase Chain Reaction
For polymerase chain reaction (PCR) analysis, DNA was extracted from whole paraffin blocks comprising tissue samples of 24 of 26 analyzed patients according to a protocol published elsewhere (22), with few modifications. In brief, tissue samples were incubated in proteinase K buffer (50 mM Tris HCl, pH 8.1; 1 mM ethylenediaminetetraacetic acid; 0.5% Tween; proteinase K, 1 mg/ml; Merck, Darmstadt, Germany) at 56°C for 25 h. The first lysate was obtained by collecting the supernatant after heating and centrifugation of these samples. This extract was used for PCR. In addition, DNA was recovered from this extract by phenol/chloroform treatment and subsequent DNA precipitation. Some PCRs were performed on both preparations (lysate and purified DNA) with a standard PCR protocol using the Amplitaq system (Perkin Elmer, Rodgan-Jügesheim, Germany). A sample was regarded as positive if one of the two different preparations revealed a positive product. Details of the experimental protocol as well as PCR primer sequences can be found in the online supplement. The presence of amplifiable DNA in the extracted samples was verified by amplification of a 289-bp fragment of the -globin gene (23). The DNA samples were then analyzed for the presence of KSHV genomes with different KSHV-specific PCR primer sets based on orf26 (24) and orf72 (25). In addition, a quantitative real-time PCR was used to amplify a fragment of the K6 gene of the KSHV genome. The technique was performed as described elsewhere (26). For the consensus degenerate hybrid oligonucleotide primers (CODEHOP) PCR, which was designed to detect a range of 2-herpesviruses, a triple, seminested protocol was performed essentially as described (27) using the primer gdtd1b for all rounds, whereas vygam served as reverse primer for first round, and clnia and vtqlga for the second and third rounds, respectively.
RESULTS
To investigate whether KSHV LANA-1 was present in lung tissue of 26 patients with IPAH, we performed an immunohistochemical stain with a monoclonal antibody directed against an epitope within the internal repeat region of the LANA-1 of KSHV. This antibody has been widely used to detect LANA-1 in tissue sections of KS, primary effusion lymphoma, and multicentric Castleman's disease by immunohistochemistry (IHC) (28, 29). All patient samples showed characteristic plexiform lesions. In 16 cases (61.5%), a mostly faint, but still in some cases typical, "speckled" nuclear LANA-1 staining signal was found (Table 1). Six of these 16 cases showed strong nuclear stains, as seen in KSHV-infected KS spindle cells (30). Alveolar (Figure 1A) and bronchial (Figure 1B) epithelial cells in areas of unremarkable lung tissue were the predominately stained cell type. Only in a single case were positive mesenchymal cells found adjacent to plexiform lesions (Figure 1C). A KS biopsy served as positive control (Figure 1D). To exclude the possibility that the use of tyramine amplification in the IHC protocol could have affected the results, histologic sections of all positive samples were also stained with the LANA-1 antibody using a Ventana automatic staining system (BenchmarkXT stainer; Ventana, Schwetzinger, Germany) with identical results (data not shown).
To complement these results, we extracted DNA from 24 of these 26 tested samples (insufficient tissue was available for the remaining two samples). For some samples, the different PCR systems were performed on two different extractions. A -globin PCR performed to check the DNA integrity amplified a product in 17 of 24 samples. Two different KSHV-specific PCR protocols based on orf72 and orf26 were performed. In the case of orf26, we used a nested PCR, as well as a modified single-round PCR with increased cycle numbers. Sensitivity of these assays, determined by a limiting dilution of a DNA preparation of a bacterial artificial chromosome (BAC) harboring the whole KSHV genome (31), were 1,000 BAC copies/reaction for orf72 PCR, 100,000 BAC copies/reaction for the modified version, and 10 BAC copies/reaction for the nested version of the orf26 PCR. None of these assays detected any specific PCR product. A real-time PCR targeting the orfK6 gene with a sensitivity of 10 BAC copies/reaction revealed one weakly positive sample (400 copies in the reaction). Serum of this patient (patient 10 in Table 1) was also found to contain antibodies to LANA-1 by immunofluorescence assay (data not shown). Interestingly, no cells expressing LANA-1 were seen in the lung tissue from this patient by IHC.
To exclude the possibility that tissue extracts could have inhibited the PCR reactions, extracts from all available tissue samples were spiked with a low copy number of KSHV K6 plasmid (4,000 copies/reaction) and analyzed by quantitative real-time PCR. The expected copy number was obtained in all but two samples, thus excluding a false-negative result due to PCR inhibition in 91.3 % of the tested samples. In one sample, from an IHC-negative patient, the K6 plasmid copy number was reduced 10-fold, indicating moderate inhibition (Table 1). In one IHC-positive sample (case 8), the PCR reaction failed to amplify the spiked plasmid DNA.
To address the possibility that the LANA-1 antibody could have reacted with a homologous protein of a yet unknown herpesvirus in the immunohistochemical stain, we screened 10 samples previously found to give a positive signal with the LANA-1 antibody with a seminested consensus primer PCR targeting the herpesviral DNA-polymerase (orf9) (27) of different - or -herpesviruses. However, no herpesviral sequences were detected in the IPAH samples.
DISCUSSION
This study investigated the presence of a latent KSHV antigen in lung tissue of 26 patients from Germany with IPAH and of KSHV DNA in 24 of these cases. In 16 (61.5%) lung tissue samples, nuclei of alveolar epithelial, bronchoepithelial, and/or mesenchymal cells displayed a positive signal when stained with a monoclonal antibody against the internal repeat region of LANA-1. In some cases, the nuclear staining pattern was reminiscent of the "speckled" nuclear pattern typical of KSHV-infected primary effusion lymphoma and endothelial cells (28, 30); in others, it was more diffuse. Similar staining results were obtained when an automated staining procedure not involving tyramine amplification was used (data not shown). However, none of the 16 samples showing this staining pattern were positive by PCR, despite using three different KSHV-specific PCR protocols with sensitivities of as low as 10 copies/reaction. Whether the positive antibody stain is really detecting KSHV LANA-1 in our samples is therefore doubtful. In only one patient was lung tissue weakly positive in the KSHV K6 real-time quantitative PCR. KSHV infection of this patient was supported by the detection of antibodies to LANA-1 in the patient's serum as measured by immunofluorescence (data not shown). The weak PCR signal could reflect the presence of KSHV genomes in infiltrating blood cells rather than in lung cells, because the immunohistologic LANA-1 stain was negative. Therefore, KSHV infection can occur in patients with IPAH, but it is unlikely to play an etiologic role in this condition.
Cross-reactivity of the LANA-1 antibody with a homologous protein of another virus or with a cellular protein involved in the pathogenesis of IPAH could be potential explanations for our observations. We therefore screened the samples with seminested CODEHOP PCR. This assay has successfully identified new 2-herpesviruses from African Green monkeys and chimpanzees (27, 32), an ungulate 2-herpesvirus (unpublished), as well as Epstein-Barr virus and cytomegalovirus (data not shown). Our PCR did not amplify a viral gene in 10 selected, immunhistochemically positive IPAH samples.
In accordance with our findings, two other Japanese groups recently failed to detect KSHV DNA in patients with IPAH from Japan (33, 34). In addition, Katano and colleagues (33), also looking at their samples by immunohistochemistry, did not see staining with an LANA-1 antibody. Furthermore, Brouchet and coworkers (35), who were searching for oncogenic virus genomes in lung carcinomas, saw an artifactual staining signal that could not be confirmed by PCR, when staining histologic slides for KSHV LANA-1 using the same antibody as in our study.
KSHV seroprevalence varies in different geographic regions. It is widespread in Africa with a seroprevalence between 30 and 60% and is also common in some Mediterranean countries with up to 35% seroprevalence. In the rest of the world, however, KSHV infection is quite uncommon, at least in the general, healthy population, with seroprevalence rates ranging below 10% (36). The incidence of classical KS outside of Africa mirrors the KSHV seroprevalence very well (37). Neither a geographic difference nor an ethnic predisposition is described for IPAH, although such an association might be concealed by a heterogeneous etiology of this disease. However, we have recently reported a small seroepidemiologic case-control study with 49 patients with IPAH, 17 patients with secondary causes of pulmonary hypertension, and 74 healthy control subjects, which did not reveal any difference in the KSHV seroprevalance between IPAH and control groups (38). A similar lack of association between IPAH and KSHV antibody positivity in a U.S. case-control study was recently reported (39).
Infection of mice with the closest murine homolog of KSHV, mouse herpesvirus 68, results in a productive infection of the lung (40), a fact that could suggest that the lung indeed is a physiologic site of KSHV-8 infection. Indeed, KSHV genomes have been reported in lung tissue not only in pulmonary KS but in other lung disease entities, like interstitial pneumonitis (41)/idiopathic pulmonary fibrosis (42) or inflammatory pseudotumors (43). Remarkably, in interstitial pneumonitis, KSHV is not only found in lymphoid cells (i.e., cells involved in the inflammatory process) but also in endothelial cells of the pulmonary vasculature and in pneumocytes (41).
Thus, a staining of alveolar and bronchial epithelial cells, as found in this study, would be in keeping with the biology of another 2-herpesvirus. However, in line with the conclusion reached by Daibata and colleagues (34), Katano and colleagues (33), and in particular Brouchet and coworkers (35), we believe that the LANA-1 staining of epithelial cells seen in our study does not reflect presence of KSHV. Taken together with the lack of association between KSHV antibodies and IPAH in German patients and patients from the United States, we conclude that KSHV is unlikely to play a role in the pathogenesis of IPAH cases occurring in KSHV-nonendemic countries.
FOOTNOTES
Supported by core funding from the Hannover Medical School.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200504-546OC on September 28, 2005
Conflict of Interest Statement: C.H.-G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.M. is one of the patent holders and licenser of the German patent for a system of the construction of tissue microarrays, used in the present study, and received 1,600 royalty from Zymed Laboratories in 2004. He has been reimbursed by Roche and Novartis for attending several conferences. M.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.F.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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