Visualization of human herpesvirus type 8 in Kaposi's sarcoma by light and transmission electron microscopy
Orenstein, Jan M.1,6; Alkan, Serhan1; Blauvelt, Andrew2; Jeang, Kuan-Teh3; Weinstein, Mark D.4; Ganem, Don5; Herndier, Brian4
1The Department of Pathology, George Washington University, Washington, DC
2The Dermatology Branch, National Cancer Institute
3The Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
4The Department of Pathology, University of California, San Francisco, California, USA
5The Howard Hughes Medical Institute and Departments of Microbiology, Immunology, and Medicine, University of California, San Francisco, California, USA.
6Requests for reprints to: Dr Jan Marc Orenstein, Ross 502, Department of Pathology, George Washington University Medical Center, 2300 Eye Street NW, Washington DC 20037, USA.
Sponsorship: Supported by the National Institutes of Health grants NO1-DE-12585, RO1 CA73534.
Date of receipt: 10 December 1996; revised: 8 January 1996; accepted: 15 January 1996.
Background: Human herpesvirus type 8 (HHV-8) has been associated with Kaposi's sarcoma, body cavity-based lymphoma (BCBL), and multicentric Castleman's disease through DNA, in situ hybridization, and serologic studies. HHV-8 has been visualized only in HHV-8-positive/Epstein-Barr virus (EBV)-negative/cytomegalovirus (CMV)-negative BCBL cell lines, but not in HHV-8-positive/EBV-negative/CMV-negative Kaposi's sarcoma lesions.
Design: Kaposi's sarcoma of the skin, lymph node, and spleen from three patients with AIDS were analysed for HHV-8, EBV and CMV DNA by polymerase chain reaction (PCR), for HHV-8 RNA (T1.1 riboprobe) by in situ hybridization (ISH), for viral inclusions by light microscopy, and for herpesviruses by transmission electron microscopy (TEM). Sections were also labeled with T1.1 counterstained with CD34, an endothelial cell marker.
Results: The skin lesion was DNA PCR-positive for HHV-8 and CMV (nested, but not single PCR), the lymph node was positive for HHV-8 and EBV, and the spleen was positive for only HHV-8. TEM revealed infection by a virus displaying the typical morphology and cytopathicity of herpesviruses. Hexagonal nucleocapsids and mature enveloped virions were present in vasoformative spindle cells and mononuclear cells, often resembling lymphocytes. Extrapolating from TEM to standard light microscopy on hematoxylin and eosin-stained paraffin sections, eosinophilic, targetoid intranuclear inclusions were identified within spindle cells which often lined vascular lumina. The T1.1-riboprobe labeled CD34+ spindle cells containing intranuclear inclusions, as well as mononuclear cells within Kaposi's sarcoma and residual lymphoid tissue.
Conclusion: The herpesvirus visualized in Kaposi's sarcoma lesions has morphologic and cytopathic features typical of human herpesviruses, productively infects vasoformative spindle cells and mononuclear cells, and is consistent with HHV-8. It can also form intranuclear inclusions that are identifiable by light microscopy in hematoxylin and eosin sections and by ISH.
Human herpesvirus type 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, has been identified by in situ polymerase chain reaction (PCR) analysis in vascular and perivascular spindle cells of KS tumours [1–4]. The detection of HHV-8-specific DNA in KS, and the serologic correlation with its development, has suggested a putative role for this recently discovered human herpesvirus in the pathogenesis of KS [5–9]. Although herpesvirus-type virions have occasionally been visualized in KS specimens by transmission electron microscopy (TEM), their specific identity was undetermined [10–17]. Since all eight human herpesviruses are morphologically similar, it is important to exclude other herpesviruses [e.g. cytomegalovirus (CMV) and Epstein-Barr virus (EBV)] when attempting to assign the identity of herpesvirus particles in KS. In particular, CMV infection is ubiquitous in patients with AIDS and targets endothelium [17,18].
HHV-8 has been visualized by TEM only in cell lines derived from HHV-8-positive/EBV-negative/CMV-negative body cavity-based lymphomas (BCBL) of HIV-infected and non-infected patients [19–22]. Most of these cells are latently infected with HHV-8, although only a minor fraction (about 1%) are productively infected and exhibit cytopathic effects typical of herpesviruses. The cells can also be induced by phorbol esters, which leads to an increase in productively infected cells (about 10%) .
Although HHV-8 DNA is present in KS lesions, the nature of the association between the virus and tumour cells at the molecular level is unclear. Zhong et al.  have identified two major viral transcripts in KS, and in situ hybridization (ISH) studies of Staskus et al.  show that most spindle cells are latently infected and express one of these RNAs (T0.7), an mRNA which encodes a putative membrane protein. The other viral RNA, T1.1 (a nuclear transcript, now also called nut-1), is detected in a smaller subset of spindle cells and appears to be a gene that is expressed primarily during lytic growth. In BCBL cells (BCBL-1), T1.1 is transcribed after phorbol 12-tetradecanoyl 13-acetate induction and behaves like an early or intermediate-early viral gene . In KS, spindle cells expressing T1.1 also express mRNAs for lytic genes like the major capsid protein or glycoprotein H. These observations suggest that a lytic viral replication programme exists in some KS cells. In this context, it is paradoxical that few studies have reported herpesviral particles in KS specimens [10,16]. Herpesvirus particles have been observed in tissue cultures of KS, but their specific identity was unknown .
In the present study, we demonstrate intranuclear inclusions (INI), consistent with herpesviral cytopathic effects and abundant herpesvirus nucleocapsids and virions in vasoformative spindle cells and mononuclear cells from primary KS lesions. Such cells can be identified in specimens negative by PCR for CMV and EBV, and are associated with expression of HHV-8 T1.1/nut-1 RNA in the tumour. These observations strongly support the inference that lytic, as well as latent, infection is occurring in KS spindle cells.
Materials and methods
After obtaining informed consent, a nodular KS lesion in the right inguinal area of a homosexual man with AIDS was biopsied (March 1996) using a 6 mm punch. The patient had widespread cutaneous KS, a history of CMV colitis, and a CD4 T-cell count of 10 × 106/l.
The spleen and accessory spleen were removed (June 1990) from a homosexual male patient with AIDS, disseminated KS, and thrombocytopenia. The most recent CD4 T-cell count (3 months previously) was 218 × 106/l, at which time KS had been diagnosed in cervical lymph node and skin specimens. The patient died with widespread cutaneous and mucosal KS, including involvement of the upper respiratory tract, 2 months after splenectomy. He had a history of oral thrush, but not of symptomatic CMV infection.
Lymph nodes were removed (July 1987) from the right shoulder and axilla of a homosexual man with a 5-month history of AIDS, extensive oral and cutaneous KS, generalized lymphadenopathy, micronodular cirrhosis, and hepatosplenomegaly. The patient had no history of symptomatic CMV infection and no CD4 T-cell counts were available. He was lost to follow-up.
The EBV and CMV serologic status of these patients is unknown.
Tissue slices of all three specimens were fixed in buffered 10% formalin and embedded in paraffin. Five micrometre sections were cut and stained with hematoxylin and eosin (H&E).
Transmission electron microscopy
Small pieces of each specimen were immediately placed in neutral buffered 2.5% glutaraldehyde at room temperature and allowed to fix for at least 12 h . Tissue was post-fixed in 1% OsO4, dehydrated in graded ethanol and propylene oxide, and embedded in Spurr's epoxy. Semi-thin plastic sections (1 µm) were cut with glass knives and stained with combined methylene blue, azure II, and basic fuchsine stain. Thin sections were stained with uranyl acetate and lead citrate and examined on a Zeiss EM10 TEM operating at 60 kV.
DNA was isolated from 10 sections (5 µm) of formalin-fixed, paraffin-embedded tissue. Sections were treated twice each with xylene and ethanol, respectively. The deparaffinized tissue was resuspended in 200 µl DNA extraction buffer (100 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 1% Tween-20, 1% Nonidet P-40, and 0.5 mg/ml proteinase K) and incubated for 72 h at 56°C. The following day, additional proteinase K (0.5 mg/ml) was added to each sample. The proteinase K was inactivated by boiling for 10 min. The specimens were microcentrifuged, and 4 µl supernatant was used for PCR amplification.
DNA was also extracted from a piece of unfixed skin KS. Following liquefaction of the tumour using a tissue homogenizer, DNA was extracted using a Stratagene kit according to company protocols (Stratagene, La Salle, California, USA). DNA (1 µm), as measured by spectrophotometry, was used as template for PCR amplification.
The primer sets used for PCR and hybridization (5′-TCC GTG TTG TCT ACG TCC AG-3′ and 5′-AGC CGA AAG GAT TCC ACC AT-3′) have been described previously by Chang et al.  for the PCR amplification of KS330233 DNA sequence of HHV-8. Thirty cycles of PCR amplification were performed as previously described. The oligonucleotide primers for EBV included 5′-AGA AGG GGA GCG TGT GTT GT-3′ and 5′-GGC TCG TTT TTG ACG TCG GC-3′ . The amplification for CMV was performed by nested PCR in which the following primers were used: IEP2A, 5′-ATG GAG TCC TCT GCC AAG AG-3′; IEP4B, 5′-CAA TAC ACT TCA TCT CCT CG-3′; IEP3A, 5′-GTG ACC AAG GCC ACG ACG TT-3′ and IEP3B, 5′-TCT GCC AGG ACA TCT TTC TC-3′ .
The amplification products were separated on 1% Tris-boric acid-EDTA agarose gels and stained with 0.5 µg/ml ethidium bromide. The gel was photographed under ultraviolet light and transferred to a nylon membrane which was hybridized with terminal deoxynucleotide transferase-labeled oligonucleotide KS330233-specific probe.
In situ hybridization
T1.1 riboprobe derived from HHV-8 expression experiments was used for ISH . Digoxigenin-labeled riboprobes were made and applied to deparaffinized, formalin-fixed, paraffin-embedded tissue sections. In these experiments, acid wash and acetylation steps were found to be unnecessary and omitted. Detection of ISH was with alkaline phosphatase antidigoxigenin, followed by nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate substrate (Boehringer-Mannheim, Mannheim, Germany). ISH for T1.1 was combined with immunocytochemical staining for CD34.
Light microscopy of KS specimens
The skin biopsy contained foci of Kaposi's sarcoma occupying a relatively small portion of densely collagenized dermis, often associated with appendages, e.g. pilosebaceous units. The vascular lumina ranged from fine slits to small channels. Mitotic spindle cells, extravasated erythrocytes, hyaline droplets, and hemosiderin were also observed.
The spleen and accessory spleen contained KS plus some residual lymphoid areas with follicles and germinal centers. The KS mostly consisted of interlacing bundles of spindle cells with large, irregular, elongated nuclei, a fine salt and pepper-like chromatin pattern, and one or more small dark blue to red-staining nucleoli. Spindle cells had abundant eosinophilic cytoplasm and were observed in mitosis and undergoing degeneration. There was a background of interstitial erythrocytes (often fragmented), hyaline droplets, lymphocytes, and plasma cells. Atypical plasmacytoid cells with large nuclei, coarse heterochromatin, and prominent basophilic nucleoli were scattered throughout the KS lesion. Degenerating and necrotic mononuclear cells were common, and some appeared to be within loose cytoplasmic vacuoles of spindle cells.
The appearance of the KS in the right shoulder lymph nodes was very similar to that of the spleen, except for more sclerosis. There was some, mostly involuted, residual subcapsular lymphoid tissue containing a few small germinal centers. Sections of some blocks had peripheral areas with changes resembling Castleman's disease (hyalin-vascular type), with germinal centers having a central sclerosed vessel, surrounded by onion skin-like layers (single file) of lymphocytes. Right axillary nodes removed from this patient at the same time showed only follicular hyperplasia. Left cervical lymph nodes that had been removed exactly 18 months previously showed a mixed pattern of moderate follicular and interfollicular hyperplasia and follicular lysis with a small area of capsular/subcapsular KS, but no Castleman's disease-like changes.
Spindle cells with targetoid INI of the herpesviral type, were identified in all three KS specimens, but especially in the extensive spleen and lymph-node lesions. No intracytoplasmic inclusions were observed. Degenerating mononuclear cells were also found with amphophilic inclusions filling their nuclei.
TEM of KS specimens
Herpesvirus-infected cells were identified by TEM in all three KS specimens, and included vasoformative spindle cells and mononuclear cells (Figs 1–4). The typical KS spindle cell had an irregular elongated nucleus with fine chromatin and one or more small nucleoli, and a cytoplasm with varying amounts of rough endoplasmic reticulum, Golgi, and mitochondria, but no Weibel-Palade bodies.
Herpesvirus-infected spindle cells, like their uninfected counterparts, were joined to their neighbour by non-specific-type junctions, covered by stretches of external lamina, rich in pinocytotic vesicles, and associated with erythrocytes (Figs 1 and 2). The cytoplasm and nucleus of most infected spindle cells were swollen (Fig. 1). The infected nuclei typically contained flocculent material of varying density, intermixed with hexagonal viral nucleocapsids (Figs 1 and 2a, b). Depending on their plane of sectioning, these INI alternately appeared elongated (longitudinal section; Fig. 1c) or round (cross-section; Figs 1a, b and 2a, b). The INI were surrounded by a relatively clear zone containing few nucleocapsids (Fig. 1a, b), and then by a mixture of electron-dense material and nucleocapsids marginated against the nuclear membrane (Figs 1a, c and 2a).
The hexagonal intranuclear capsids measured about 110 nm in diameter and came in three forms: those that were totally empty, those containing an inner ring of about 60 nm in diameter, and others containing pleomorphic electron-dense cores (Figs 1b, d and 2b, d). Mature enveloped virions, measured about 140 nm in diameter and were seen within cytoplasmic cisternae and vacuoles (Figs 1e and 2d), but not in the extracellular space. There were no homogeneous, electron-dense cytoplasmic bodies characteristic of CMV-infected cells, and this herpesvirus was larger in size than EBV .
Degenerating and necrotic herpesvirus-infected cells were especially common in the spleen KS, sometimes within adjacent cells. Infected cells undergoing lysis were found in the interstitium and within cytoplasmic vacuoles of spindle cells that displayed junctions, external lamina, and pinocytotic vesicles (Fig. 3). Although the majority of dead cells were unclassifiable, herpesviral nucleocapsids and mature virions could still be identified within the cellular debris (Fig. 3c, e). However, some of the better preserved inviable cells closely resembled lymphocytes in appearance (Fig. 4). Unlike the infected spindle cell with its targetoid INI, the infected lymphocyte-like cell had a round nucleus with a central granular zone containing nucleocapsids, immediately surrounded (no halo) by a prominent irregular band of electron-dense material contiguous with the nuclear membrane [Fig. 3a (top), b and Fig. 4].
HHV-8-infected cells in H&E-stained paraffin sections
Extrapolating from the ultrastructural findings, it was apparent that the INI seen in the H&E-stained sections corresponded to the cytopathic changes induced by HHV-8 in the spindle and mononuclear cells (Figs 5 and 6). The INI appeared as central, rough-edged, straight-to-angled cylindrical, eosinophilic structures that on cross-section appeared target-like (Fig. 5a–c, e, f). The central inclusion was typically surrounded by a clear halo and then a distinct basophilic nuclear membrane-heterochromatin complex. Infected cells often had an edematous cytoplasm (Fig. 5a). At times, several INI-bearing cells were identified in a single medium-power field (e.g. ×20–25 objective and ×10 eye-piece), giving the impression of ‘foci’ of infection. The more uniform the H&E staining of a section, the easier it was to detect the INI.
Necrotic cells were located in the interstitium and within the cytoplasm of spindle cells. Whereas most lacked light microscopic features of viral infection, others had nuclei with an amphophilic-staining central area, surrounded by a prominent irregular basophilic rim (Fig. 6a, b). These necrotic cells often had the size, shape, and nuclear-cytoplasmic ratio of lymphocytes.
No other potential pathogens, viral or otherwise, were identified by light microscopy or TEM.
All specimens were positive for HHV-8 DNA by single PCR amplification/hybridization and nested PCR (Table 1, Fig. 7). Although all samples were negative for CMV DNA by non-nested PCR, nested PCR performed on DNA extracted from a portion of the fresh KS skin specimen (but not the paraffin-embedded tissue) disclosed CMV DNA. EBV DNA was detected only in the lymph-node specimen.
ISH results and immunohistochemistry
The T1.1 riboprobe identified INI-bearing spindle cells (Fig. 5d–f). Whereas most of the labeling was confined to the cell nucleus, some cells also showed varying amounts of cytoplasmic staining, possibly an indication of cell lysis. When the staining did not obscure the entire nucleus, it was seen to define the INI and the nuclear membrane (Fig. 5e, f). Dual CD34/T1.1 staining of vasoformative spindle cells was seen in the three KS specimens, and many of the co-stained cells had clear INI (Fig. 5f).
T1.1-positive mononuclear cells were seen in preserved lymphoid areas of the spleen and lymph-node sections, especially within germinal centers, where they resembled activated lymphocytes with round nuclei and one or two prominent nucleoli (Fig. 6c, d). Some of these cells showed both cytoplasmic and nuclear staining (Fig. 6d). T1.1-positive non-spindle cells were also seen scattered within the KS lesion.
In KS, we have demonstrated that a typical herpesvirus, consistent with HHV-8, which productively infects and is cytopathic for both spindle and non-spindle cells, produces INI that are visible in routine H&E-stained paraffin sections, and is morphologically similar to the herpesvirus of BCBL cell lines and to other human herpesviruses [14,19,30–32]. The herpesviruses identified in our KS specimens, which were from different sites of three individuals with AIDS, were identical in appearance, regardless of whether the specimens were also positive for EBV DNA or CMV DNA by PCR. That CMV DNA was detected in the fresh skin specimen by nested and not single PCR is perhaps not surprising, since the patient was known to have CMV colitis; CMV targets endothelium and can be detected in the blood of patients with CMV disease [33–35]. In our KS specimens, herpesvirus infection did not produce the cytoplasmic inclusions or the megaloblastic cellular changes typical of CMV, and the viruses were larger in size than EBV [12,26]. Although CMV can produce similar targetoid-appearing INI, the CMV INI in specimens from patients with AIDS often appear variegated, dark red to amphophilic, and fill the entire nucleus . The requirement for nested PCR for CMV detection indicates a very low copy number of CMV in the skin lesion, and is in contrast to the substantial copy number of HHV-8 typically found in KS lesions . Therefore, it is likely that the herpesvirus observed in the skin and lymph-node KS is the same HHV-8 observed in the KS of the spleen.
The hexagonal nucleocapsids of HHV-8 were associated with central, sausage-shaped, granulofibrillar INI which, when cut in cross-section resembled a bulls-eye. This represents the first identification and description of the nucleopathic changes likely to be caused by HHV-8 within KS tumours.
The ISH experiments with the latent HHV-8 marker T1.1 complemented the TEM and light microscopic findings . T1.1 labeling clearly defined the INI and the border of the nucleus, both sites of HHV-8 nucleocapsid concentration. T1.1 represents a nuclear RNA that probably does not serve any coding function; current speculations are that it may play a role in nuclear processing . The inviable non-spindle, mononuclear cells that labeled with the T1.1 riboprobe may have included atypical plasmacytoid cells, common to KS lesions, or cells of monocyte/macrophage origin, which have recently been shown to be infected with HHV-8 in KS lesions . The T1.1-positive mononuclear cells found in germinal centers in the same specimen with KS could represent activated B cells. HHV-8 DNA has been detected in multicentric Castleman's disease , with expression being colocalized to germinal centers (unpublished data). Unfortunately, the Castleman's disease-like changes seen focally in our lymph-node specimen were not present in the sections employed for ISH. HHV-8 DNA is found in peripheral blood mononuclear cells [38,39] and BCBL cells , which lyse during stimulation leading to virus production [8,9,20–22]. The cells of BCBL have the genotype and phenotype of activated B cells, and B cells have also been shown to be targets of HHV-8 infection in vitro [19,41]. Thus, HHV-8 appears to share endothelial cell tropism with CMV and EBV and B-cell tropism with EBV [41–43]. The absence of HHV-8-infected and uninfected mononuclear cells, and the cytokine milieu that they possibly provide, may help to explain why KS-derived spindle cell lines lack virus.
HHV-8 infection in KS appears to be associated with cell degeneration and necrosis. In the throes of productive infection, the intact spindle cells are edematous. These infected spindle cells shared TEM features with the vasoformative KS spindle cells and normal endothelial cells, including external lamina, intercellular junctions, and pinocytotic vesicles. Spindle cells appear to be capable of acting as facultative phagocytes internalizing cellular debris that can contain identifiable herpesvirus particles. The debris may represent remnants of lysed HHV-8-infected spindle cells or mononuclear cells. At both the light and electron microscopic levels, the nuclei of the infected lymphocyte-like mononuclear cells were different from those of the spindle cells. They were condensed and, instead of a targetoid INI, there was a thick band of dense material that immediately surrounded a less electron-dense area containing nucleocapsids. In H&E-stained sections, the central area appeared amphophilic. Surprisingly, it was possible to find multiple INI-bearing cells and remnants of infected cells in the same area (even more than one in the same spindle cell), as if there were foci of a spreading lytic infection.
The amount of virus and the level of viral maturation seen in two of our specimens suggests that, in some instances, it will be possible to specifically immunolabel infected cells for light microscopy (immunohistochemistry) and TEM (immunogold). In the future, specific antisera directed against HHV-8 proteins should identify virus-infected cells in formalin-fixed, paraffin-embedded specimens. Until then, we believe that this study represents the most specific and detailed morphologic description of HHV-8 in KS to date.
We acknowledge G. Turiansky and the expert technical assistance of S. Honig and C. Dye III.
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Kaposi's sarcoma-associated herpesvirus; human herpesvirus type 8; transmission electron microscopy; Kaposi's sarcoma; light microscopy; in situ hybridization
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