Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), has recently been identified within the bone marrow dendritic cells of multiple myeloma (MM) patients. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. HHV-8 utilizes α3β1 integrin as one of the receptors for its entry into the target cells via its gB interaction and induces the activation of focal adhesion kinase (FAK) (S. In the absence of HIV, HHV-8 DNA has been detected in T-cell PEL cells (Lechapt-Zalcman et al, 2001) and in lymphoma cells with plasmacytic differentiation, but not in cutaneous T- and B-cell lymphoma (Dupin et al, 1997) or in mycosis fungoides (Henghold et al, 1997). The mean follow-up was 4.2 years. B cells are an important target for viral infection (1, 12, 17), and latency is established in lymphoid cells. This review discusses the features of KSHV-associated lymphoproliferative disorders and the evidence supporting its role in the pathogenesis of these diseases.
Among these 32, 3 years after transplantation, graft survival was 72%, and KS prevalence was 28% (KS incidence: 8.2/yr/100 HHV-8+ recipients). Terms Related to the Moving Wall Fixed walls: Journals with no new volumes being added to the archive. However, it is the multifocal or multicentric form of the PC variant or PC-hyaline vascular combination that often arises in HIV-infected individuals and that has been frequently shown to contain HHV-8 DNA (24, 25, 28). Other genes previously implicated in the development of PAH are affected by HHV-8 infection, and cells infected with HHV-8 are resistant to apoptosis. Our data confirmed that HHV-8 is involved in the etiopathogenesis of all types of KS and that there is a correlation between HHV-8 DNA load and the severity and staging of this disease. Under this hypothesis, MCD and PEL would be the result of higher levels of vIL-6 expression in B cells than the lower levels in the spindle tumor cells in KS. In this report, we describe the results of an analysis of vIL-6 gene expression in defined cell types in KS, PEL, and MCD that provide strong evidence in support of this hypothesis.
A reduction in the number of virions would both directly and indirectly decrease the likelihood of transmission to cellular targets with the capacity to proliferate uncontrollably upon infection. The MCD specimen was from an HIV-infected individual with AIDS who also had a history of KS. This patient presented with a clinical and pathological picture beyond that which is seen in the usual HIV patient and which is consistent with CD. This included anemia, unexplained fevers and chills, severe myalgia, arthralgias, massive lymphadenopathy, and hepatosplenomegaly. Constitutional symptoms and lymphadenopathy dramatically decreased upon treatment with prednisone but recurred upon its discontinuation. This pattern was repeated upon multiple rounds of prednisone treatment. Histopathologically, the specimen has features of both the persistent generalized lymphadenopathy of HIV in the follicular involutional stage and of multicentric angiofollicular hyperplasia, i.e., enlarged lymph nodes involving significant follicular hyperplasia and perifollicular lymphoplasmacytic (predominantly plasmacytic) reaction, interfollicular plasmacytosis, and few immunoblasts (12).
Immunoperoxidase staining for the kappa and lambda light chains shows that the plasmacytic infiltrate is polyclonal. From these specimens, which are especially now rare and difficult to obtain since the introduction of HIV protease inhibitors for AIDS therapy, we derived and here present results that are representative of two cases of PEL, five cases of HHV-8-positive MCD, and numerous cases of KS that we have studied. © 2005 Wiley-Liss, Inc. Thin sections (6 to 8 μm) were cut and attached to silanized slides, dried, heated for 1 h at 60°C, deparaffinized with two 10-min incubations in xylene, cleared with two 10-min incubations in absolute ethanol, and dried. Most neoplastic lymphoid cells were strongly positive for CD30 and CD3 (Figure 1, B) and showed weak staining for CD45, whereas they were negative for CD19, CD20, and CD79a B-cell antigens. The hIL-6 subclone, HHV-8 nut-1 and T0.7 cDNA clones in pBluescript SK+ (33), and a PCR-generated HHV-8 vIL-6 DNA clone in pCR3.1 (Invitrogen) were linearized on either side of the insert to produce templates suitable for runoff transcription of antisense and sense RNAs. Radiolabeled RNAs with specific activities of ∼109 cpm/μg were synthesized with [35S]UTP and the Promega transcription system, and digoxigenin-labeled nut-1 antisense RNA was synthesized with the Boehringer Mannheim Genius transcription system.
All RNA transcripts were subjected to controlled alkaline hydrolysis to yield fragments with an average size of 350 ribonucleotides (9). The pretreatment and hybridization methods used have been described in detail elsewhere (15). Briefly, dried, deparaffinized slides were incubated for 30 min in 0.2 N HCl, neutralized with 0.15 M triethanolamine (pH 7.4), incubated in a 0.005% digitonin-containing solution for 5 min, treated with 5-μg/ml proteinase K in a CaCl2-containing buffer for 15 min at 37°C, acetylated, and dehydrated with graded alcohols. Hybridization mixtures containing 10% dextran sulfate, 50% deionized formamide, 20 mM HEPES (pH 7.4), 1 mM EDTA, 1× Denhardt’s medium, 1-mg/ml poly(A), 0.6 M NaCl, 100 mM dithiothreitol (DTT), 250-μg/ml yeast RNA, and 105-cpm/μl 35S-labeled riboprobe were applied to specimens, which were then covered with siliconized coverslips and sealed with rubber cement. Hybridization was carried out for 16 to 18 h at 45°C, after which time the coverslips were removed under 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at room temperature. The slides were washed with 5× SSC–10 mM DTT for 30 min at 42°C, with 2× SSC–50% formamide–10 mM DTT for 20 min at 60°C; twice in HWB (0.1 M Tris-HCl [pH 7.4], 0.4 M NaCl, 0.05 M EDTA) for 10 min each time at 37°C; with 25-μg/ml RNase A and 25-U/ml RNase T1 in HWB for 30 min at 37°C; and with HWB, 2× SSC, and 0.1× SSC for 15 min each at 37°C and dehydrated through graded alcohols containing 0.3 M ammonium acetate. The time interval between transplantation and onset of KS was relatively early compared to other skin tumors.
The transcript content of individual cells is directly proportional to the quantity of silver grains that develop in the layer of photographic emulsion covering cells that have bound a specific radiolabeled probe (15). To quantitate the silver grains, we captured video images of cells visualized by epipolarization microscopy and used Metamorph image analysis software (Universal Imaging, West Chester, Pa.) to enumerate the silver grains as previously described (16). When possible, grain counts were determined for at least 100 hybridization-positive cells from randomly selected fields of view for each specimen. Copy numbers were calculated based on transcript calibrations performed on HIV-infected cells (16). Reaction conditions were one cycle of 95°C for 2 min, thirty-five PCR cycles consisting of denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 30 s were run per round with a 7 min final extension at 72°C for both first round and nested PCR. DNase resistance of the HHV-8 PCR template DNA is indicative of the presence of virions. mRNA levels were 218 fold higher (CV: 45…391, n=100, p 10−14) in mECK36 than in Bac36-transfected NIH3T3 cells.
The slides were then placed in 10 mM sodium citrate, pH 6.0, exposed to microwaves for 10 min (medium-high setting, 1,200-W oven), allowed to cool slowly to room temperature, and acetylated. These two viruses are among the eight members of the herpes virus family to infect humans, causing a variety of illnesses ranging from cold sores to brain infection (encephalitis) to chickenpox to various cancers. During the study period ( January 1995 to June 2012), HHV-8 testing has been performed routinely for all CD cases, either at diagnosis or retrospectively. Lymphocyte subsets were measured by flow cytometry; HIV-1 viral load was measured using the Amplicor quantitative assay, version 1.5 (Roche Diagnostics). Coverslips were removed under 2× SSC, and the slides were washed as follows: twice in 2× SSC for 5 min each time at room temperature, in STE (0.5 M NaCl, 1 mM EDTA, 20 mM Tris-HCl [pH 7.5]) for 5 min at room temperature, in 2× SSC–50% formamide for 5 min at 50°C, in 1× SSC–0.1% sodium dodecyl sulfate for 10 min at 50°C, and in 0.5× SSC–0.1% sodium dodecyl sulfate for 15 min at 50°C. The slides were equilibrated for 5 min in 0.1 M Tris-HCl (pH 7.4)–0.15 M NaCl and then carried through the Boehringer Mannheim digoxigenin detection protocol using an alkaline phosphatase-conjugated antibody. Nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate substrate development was monitored under the microscope and terminated by incubating the slides for 3 min in TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA).
We chose to carry reactions out to 50 cycles-to-threshold for the purpose of detecting potentially very low viral loads in paraffin-embedded tissue blocks that had been in storage for up to 10 years. et al. For colocalization of two different viral transcripts, a hybridization mixture containing both 35S-labeled vIL-6 and digoxigenin-labeled nut-1 riboprobes was applied to slides pretreated for hybridization as described above. All five of the samples positive using the KSHV330233 primers were also positive with the primers for ORFK9 (Figure 5). Libi, D. However, only gBΔTM mediated the adhesion of target cells. Using a combination of ISH with a riboprobe specific for the HHV-8 T0.7 gene (open reading frame K12), which is transcribed during viral latency as well as productive infection (33), and IHC with an antibody to CD34, an antigen that is present on the KS spindle tumor cell and endothelial cells from which the tumor cell is thought to be derived (23), we demonstrated that most, if not all, of the tumor cells are infected, regardless of the stage of lesion development.
Rituximab therapy was imputed in a time-dependent manner. The other 12 replicates were subsequently tested for the five serial dilutions surrounding and including this dilution (one above and three below the last positive dilution). Conversely, liquid-phase lymphomas of the HIV-negative cohort are generally not of the null phenotype and can be shown to belong to B-cell or, in a few cases, T-cell lineages. Despite the HIV-negative background of our PEL specimen, we were unable to phenotype the tumor cell and consequently could not perform double-label ISH-IHC to simultaneously phenotype and quantitate those cells infected with HHV-8. ISH alone, however, with a probe to identify T0.7 transcripts revealed infection in a majority of the cells of the effusion, presumably the tumor cells (Fig. A). Hybridization with the nut-1-specific probe revealed that only a minority of these cells were potentially productively infected (Fig.
B). As we have previously shown for KS (29), there is a variable level of T0.7 expression across the population of infected cells, and those cells with the highest T0.7 content correspond to the nut-1-positive cells. Longer exposure times enhanced the signal over cells at the low end of T0.7 expression but did not reveal additional nut-1-positive cells (data not shown). The protein was expressed in bacteria and harvested as described above except that following incubation of clarified supernatant with glutathione-Sepharose 4B, the beads were pelleted and washed once with 5 ml of cleavage buffer (50 mM Tris [pH 7.0], 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol [DTT]). Thin sections of PEL (A and B) and MCD (C and D) specimens were hybridized with 35S-labeled riboprobes specific for HHV-8 genes expressed during latency (T0.7) and productive infection (nut-1). … In MCD, by contrast, with the same exposure time, there are a few cells in which T0.7 RNA can be detected and these contain relatively high levels of transcripts (similar to the high end of the spectrum in KS and PEL).
Increased exposure times enhanced the signal over these cells but did not reveal a significant number of additional cells with lower transcript content. These cells are located mainly within the follicular mantle of small lymphocytes surrounding the germinal centers with fewer cells in the germinal center and interfollicular plasma cell-containing region (Fig. C). The number and distribution of cells containing nut-1 RNA (Fig. D) or MCP RNA (data not shown) were similar, and double-label experiments with digoxigenin- and radiolabeled probes for T0.7, nut-1, and MCP (in various pair combinations) resulted in colocalization of these transcripts (data not shown). We interpret this observation as evidence that all of these cells are potentially productively infected. In this particular specimen, in the subcapsular sinus and extending into the interfollicular spaces, we also detected nests of cells with T0.7 RNA that, with respect to the abundance and cellular distribution of the hybridization signal, are reminiscent of KS.
Almost all of the cells within this area were T0.7 positive. There was a gradient of T0.7 transcript content across the population of these infected cells, and we detected nut-1 RNA in very few of these cells. Cells in this region of the MCD lymph node were spindle shaped with elongated nuclei, were colabeled with the nut-1 riboprobe and the antibody to CD34 (Fig. A and B), and represent an early stage of KS in the same tissue. Identification of HHV-8-infected cells in MCD. Combined ISH with a digoxigenin-labeled nut-1 riboprobe (dark purple nuclei) and IHC with a monoclonal antibody to human CD34 (brown peroxidase reaction product) reveals the presence of an infected spindle-shaped … Double-label experiments showed that many CD34-negative HHV-8-infected cells in the perifollicular region reacted with antibody to human lambda light chains (Fig.
C). In addition to these infected plasma cells, we detected a few infected T cells, which were identified with antibody to CD3 (Fig. D). There were a significant number of cells, however, that did not react with any of the antibodies tested (specific for B cells, T cells, plasma cells, κ, and λ light chains, dendritic cells, macrophages, and endothelial cells). We speculate that, like PEL, these may represent B cells in some stage of differentiation or transformation where detectable B-cell-associated antigens have been lost. A number of viral homologues of interesting human genes, including those for cytokines, growth factors, and receptors, have been identified in the HHV-8 genome. Among these is a homologue of the gene for IL-6 that has clearly been associated with clinical and pathological abnormalities of MCD (4, 32) and has been speculated to play a role in KS and PEL (11).
Therefore, HHV-8 could be regarded as a superior candidate due to constant immune stimuli triggered by the lifelong latency of itself. Indeed, the findings are supported further by the nucleotide sequence analyses of PCR products. In MCD (Fig. A), vIL-6 was highly expressed in cells corresponding in number and location to those containing both T0.7 (Fig. C) and nut-1 (Fig. In contrast, when comparing this series of 18 HIV-negative patients with HHV-8associated MCD to a series of 12 HIVnegative patients with HHV-8unrelated MCD, differences in demographics, clinical symptoms, and evolution could be observed between the 2 groups. All of the women in this analysis were from the couples cohort and were selected to evaluate HIV-1 transmission from a single HIV-positive male partner.
A, inset). vIL-6 expression in KS, PEL, and MCD. Hybridization of MCD with a 35S-labeled vIL-6 riboprobe reveals high levels of transcripts in cells localized mainly to the perifollicular lymphocyte layer of the specimen (A, 3-day exposure). These cells are similar … In PEL, the profiles of vIL-6 (Fig. B) and T0.7 (Fig. A) were similar.
BCBL-1 cells (about 3 × 108) were cultured for 6 days in the presence of TPA. Briefly, confluent HFF monolayers in 24-well plates were washed and blocked for 30 min at 4°C with phosphate-buffered saline (PBS) containing 1% FBS, 5 mM bovine serum albumin (BSA), and 0.1 mM CaCl2. B). Other MCD characteristics were also not associated with the risk of NHL. For subjects 1 and 4, enough cells were available to repeat the assay with 22 instead of 12 replicates; for subject 2, the assay was repeated with 17 replicates. In this central part of the lesion, filled with spindle tumor cells that contain detectable levels of T0.7 (not shown), these cells were about as infrequent as the lytic nut-1-containing cells, and we predicted that vIL-6 and nut-1 would colocalize. The collections of HHV-8-infected cells in the subcapsular sinus of the MCD specimen that hybridized to T0.7- and nut-1-specific probes in a manner similar to KS also produced a KS-like hybridization pattern with vIL-6.
Specificity of hybridization to the viral homologue of IL-6 was confirmed by lack of hybridization of the hIL-6 probe to a subjacent section of PEL (Fig. D). Quantitative image analysis of appropriately exposed slides confirmed the visual similarities and differences in single-cell levels of viral transcripts among the three diseases. Single-cell levels of T0.7 within the populations of latent or putative productively infected cells were similar in KS, PEL, and MCD. The latent populations in KS and PEL ranged from 30 to 300 copies of T0.7 per cell, and the high-end cells in all three diseases contained an average of 400 copies per cell. There were dramatic differences in the levels of vIL-6 among the three diseases. To each vial was added 4 ml of ScintiVerse Bio-HP scintillation cocktail (Fisher Scientific).
). Frequency distribution of vIL-6 copy number in MCD, PEL, and KS. The number of copies of vIL-6 RNA in individual infected cells was determined by using computerized image analysis to count grains over randomly selected cells. Grain counts were converted … We had found previously that virtually all of the KS spindle tumor cells in the KS lesion were infected with HHV-8 but that only a minor population had a transcript profile expected of lytic and productive infections. Similarly, in this study, we found evidence of HHV-8 gene expression in most cells of the PEL. Few of these cells contained detectable levels of the nut-1 gene and other genes, such as that for MCP, that are expressed in the lytic cycle.
In dramatic contrast to KS and PEL, MCD appears to contain relatively few infected cells within the lesion and most of these are potentially lytic infections. In this study, the identification and quantitation of infected cells are based on our ability to detect T0.7 RNA, a transcript that has been shown to be expressed in a majority, if not all, of the latently infected cells in both KS lesions and PEL-derived cell lines. It is possible, however, that in MCD, HHV-8 exhibits an alternative program of latency that is characterized by much lower and undetectable levels of T0.7 and that many more cells of the lesion are infected than we describe here. This will be clarified by the refinement and application of a reliable single-cell method for low-copy DNA detection. Other striking differences that we documented in these pathological states are the types of cells infected and the levels of expression of vIL-6. We were not able to phenotype the PEL, but we determined that in MCD, the majority of infected cells were positive for immunoglobulin light chains and were therefore of B-lymphocyte lineage. We also found HHV-8 infection of a minor population of T cells in MCD.
The average levels of vIL-6 RNA in the populations of infected cells in both PEL and MCD was greater by at least an order of magnitude than the low but detectable levels found in a subpopulation of the total KS spindle tumor cells. These data are generally consistent with those of Moore et al. (21) and Parravicini et al. (25), who used a specific antibody to detect relatively high levels of HHV-8 vIL-6 in PEL and MCD, respectively. However, they were unable to consistently detect the vIL-6-containing subpopulation of spindle tumor cells in KS lesions, most likely due to the limited sensitivity of their technology. The morphological characterization of the vIL-6-positive cells of MCD by Parravicini et al. is also consistent with our predominant phenotypic data, but they did not detect vIL-6 in CD3-positive cells as we have shown here.
One clear difference between our two studies that warrants further investigation in this regard is the HIV status of the donors. Lastly, of considerable interest is the difference in vIL-6 levels between the latently infected populations of KS and PEL. This finding may signify the existence of at least two cell type-specific programs of latency for HHV-8, a precedent for which exists in Epstein-Barr virus. Kempf W , Perniciaro C , Adams V , Müller B , Burg G : Human herpesvirus 8 (HHV-8) in familial Kaposi’s sarcoma . For example, PEL is frequently coinfected with Epstein-Barr virus. IL-6 is expressed in many types of B-cell lymphomas (4, 11), and we believe that expression of vIL-6, perhaps in conjunction with other potential oncogenes, such as the viral homologue of cyclin D, could play an important role in tumor formation. The elevated levels of IL-6 clinically described for MCD are thought to contribute to the characteristic polyclonal plasmacytosis, hypergammaglobulinemia, and follicular hyperplasia (17, 32).
The high levels of vIL-6 in a relatively few B cells documented in this study could act by a paracrine mechanism to drive proliferation and differentiation of B cells in HHV-8-associated MCD. More generally, differential expression of HHV-8 genes in different cell types could be responsible for the heterogeneity of pathological states in these and perhaps as yet undiscovered diseases. 1. Ansari M Q, Dawson D B, Nador R, Rutherford C, Schneider N R, Latimer M J, Ricker L, Knowles D M, McKenna R W. Primary body cavity-based AIDS-related lymphomas. Am J Clin Pathol. 1996;105:141–143.
15. Haase A T. Analysis of viral infections by in situ hybridization. In: Valentino K L, Eberwine J H, Barchas J D, editors. Since only about 20% of TPA-induced BCBL-1 cells expressed HHV-8 lytic cycle proteins (2, 46), the yield of purified gB by affinity chromatography was insufficient for functional studies. New York, N.Y: Oxford University Press; 1987. Gérard, J.-M.M, and E.O.
Thus, even low elevations of the mean virus load, such as the threefold elevation seen in our BCBL-1 experiments, can represent substantial active virus replication in a small subset of cells.