Abstract

Kaposi's sarcoma (KS) is an angioproliferative disease associated with infection by the human herpesvirus-8 (HHV-8). HHV-8 possesses genes including homologs of interleukin-8 (IL-8) receptor, Bcl-2, and cyclin D, which can potentially transform the host cell. However, the expression of these genes in KS tissues is very low or undetectable and HHV-8 does not seem to transform human cells in vitro. In addition, KS may not be a true cancer at least in the early stage. This indicated that besides its transforming potential, HHV-8 may act in KS pathogenesis also through indirect mechanisms. Evidence suggests that KS may start as an inflammatory-angiogenic lesion mediated by cytokines. However, little is known on the nature of the inflammatory cell infiltration present in KS, on the type of cytokines produced and on their role in KS, and whether this correlates with the presence of HHV-8. Here we show that both acquired immunodeficiency syndrome (AIDS)-KS and classical KS (C-KS) lesions are infiltrated by CD8+ T cells and CD14+/CD68+monocytes-macrophages producing high levels of γ-interferon (γIFN) which, in turn, promotes the formation of KS spindle cells with angiogenic phenotype. γIFN, in fact, induces endothelial cells to acquire the same features of KS cells, including the spindle morphology and the pattern of cell marker expression. In addition, endothelial cells activated by γIFN induce angiogenic lesions in nude mice closely resembling early KS. These KS-like lesions are accompanied by production of basic fibroblast growth factor, an angiogenic factor highly expressed in primary lesions that mediates angiogenesis and spindle cell growth. The formation of KS-like lesions is upregulated by the human immunodeficiency virus Tat protein demonstrating its role as a progression factor in AIDS-KS. Finally, γIFN and HLA-DR expression correlate with the presence of HHV-8 in lesional and uninvolved tissues from the same patients. As HHV-8 infects both mononuclear cells infiltrating KS lesions and KS spindle cells, these results suggest that HHV-8 may elicit or participate in a local immune response characterized by infiltration of CD8+ T cells and intense production of γIFN which, in turn, plays a key role in KS development.

KAPOSI'S SARCOMA (KS) is a proliferative disease of vascular origin particularly frequent and aggressive in human immunodeficiency virus (HIV-1)–infected homosexual men (acquired immunodeficiency syndrome [AIDS]-KS) as compared with classical KS (C-KS) that is rare and indolent.1-3 However, these forms have the same histopathology. In the very early stages, KS is characterized by inflammatory cell infiltration, endothelial cell activation, and angiogenesis. This is followed by the appearance of the typical spindle-shaped cells that represent a heterogeneous population dominated by activated endothelial cells mixed with macrophages and dendritic cells.4-7 In advancing lesions, spindle cells tend to become the predominant cell type, although angiogenesis remains always a prominent feature.

A new herpesvirus, termed KS-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8), has been recently identified in KS tissues.8 In contrast with other viruses, HHV-8 has been consistently detected in all forms of KS,9-13 in peripheral blood mononuclear cells (PBMC) from the same patients, and, at a lower frequency, in AIDS patients without KS and in normal individuals, particularly in geographical areas at high risk for KS14-17(and G. Rezza et al, submitted). Other studies showed that HHV-8 infection can be predictive of KS development.18-20Thus, evidence suggests that HHV-8 may have an important role in KS pathogenesis.

HHV-8 possesses several genes acquired from the host, including homologs of macrophage inflammatory protein (MIP) (v-MIP–I and v-MIP–II), interleukin-6 (IL-6) (v-IL–6), IL-8 receptor (v-IL–8R), Bcl-2 (v-Bcl–2), and cyclin D (v-cyclin D).21-29 Most of these genes have been shown to be functional and v-IL–8R has in vitro transforming activity.21,23-26,29 This suggested that they may participate in KS cell transformation or in the induction of basic fibroblast growth factor (bFGF),23 an angiogenic factor that has a key role in KS pathogenesis.30-32 However, to exert a role in KS cell transformation, these viral genes should be expressed in the transformed cells as found in primary effusion lymphomas (PEL) and PEL-derived cell lines.21-23 In contrast, the expression of these viral genes in KS tissues is low or undetectable,21-23,26,28,33 whereas the human bcl-2 and IL-6 are expressed at very high levels in primary lesions.34,35 In addition, the only two tumor cell lines derived from KS do not contain HHV-8,36 and spindle cells derived from the lesions are latently infected, but they lose the virus on culture37-39 (and our unpublished data). Finally, HHV-8 does not seem to transform human endothelial cells, the precursors of KS spindle cells, or B cells in vitro.40 

Recent findings show that, in addition to primary B cells,41 HHV-8 is present in circulating monocytes42 (and S. Colombini et al, submitted), in spindle-like macrophagic cell progenitors of the blood of KS patients43,44 and in infiltrating mononuclear cells of KS lesions including monocytes and macrophages where the virus yields a productive infection.42,45,46Altogether these observations suggest that HHV-8 may act through different mechanisms in KS pathogenesis.

Previous studies by us and others showed that KS behaves as a cytokine-mediated disease, and that, at least in early stages, KS is not a true cancer, but a hyperplastic-proliferative disease.47-49 bFGF and vascular endothelial cell growth factor (VEGF) are highly expressed in spindle cells of both AIDS-KS and C-KS, and they mediate the spindle cell growth, angiogenesis, and edema of KS30-32,50,51 (and F. Samaniego et al, submitted). Other data showed that the extracellular HIV-1 Tat protein released by infected cells can increase synergistically the angiogenensis and spindle cell growth mediated by bFGF.33,52-56 As extracellular Tat is present in AIDS-KS lesions and its receptors are highly expressed by vessels and spindle cells,32,57 this suggested that Tat may increase the frequency and aggressiveness of KS in infected individuals acting as a progression factor.

Further data suggested that inflammatory cytokines (IC) may trigger these events. Conditioned media (CM) from activated T cells (TCM) induce the production and release of bFGF and VEGF in cultured KS and/or endothelial cells58,59 (and F. Samaniego et al, submitted). In addition, TCM-treated endothelial cells acquire features similar or identical to KS cells and, as observed with AIDS-KS cells, they become responsive to the growth, invasion, and adhesion effects of Tat.54-57 TCM contain a variety of IC including tumor necrosis factor (TNF), IL-1, IL-6, oncostatin M (OM), and γ-interferon (γIFN) (see Materials and Methods section),53-59 and some of them (TNFα, IL-6, IL-1, OM) have been found in KS,60-62 suggesting a role for the immune system and in particular of host IC in the induction and progression of KS. In particular, the presence of HHV-8 productively infected cells in KS tissues suggests that the virus may trigger an inflammatory response and the expression of cytokines. However, little or nothing is known on the type of inflammatory cell infiltration, specific cytokine production, and role of these cytokines in KS pathogenesis or whether the presence of these cytokines correlates with that of HHV-8. Here we show that (1) KS lesions from both AIDS-KS and C-KS contain a prevalent CD8 T-cell infiltration and infiltration with monocytes-macrophages; (2) these cells produce IC and in particular γIFN; (3) γIFN is required to induce endothelial cells to acquire the phenotypic and functional features of KS spindle cells, both in vitro and in vivo, and to induce angiogenic KS-like lesions in nude mice whose frequency and intensity is increased by the HIV-1 Tat protein; and (4) γIFN and HLA-DR expression in tissues is associated with the presence of HHV-8.

MATERIALS AND METHODS

Immunostaining of tissues and cells.

Frozen sections from KS and uninvolved tissues were fixed in cold acetone and single- or doubly-stained by the alkaline phosphatase antialkaline phosphatase (APAAP) method alone or combined with the peroxidase antiperoxidase (PAP) method. For APAAP single method, monoclonal antibodies (MoAbs) were used directed against γIFN (1:25, Genzyme Diagnostics, Cambridge, MA), TNFα (1:200), IL-1β (1:200), HLA-DR (1:20), CD4 (1:20), CD8 (1:100), CD14 (1:100), CD68 (1:200), CD20 (1:200) (all from DAKO, Golstrup, Denmark). For double-staining experiments, the APAAP and the PAP methods were used by combining the antibodies described above with a rabbit polyclonal antibody directed against γIFN (1:200, Genzyme) as described previously.32All incubations were performed at room temperature. Briefly, for single-staining, the slides were incubated with the MoAb for 30 minutes. After washing with Tris-buffered solution (TBS), the rabbit antimouse IgG (1:25; Dako) was applied for 20 minutes and after additional washing with TBS, the slides were incubated with APAAP (mouse) complex (1:25; Dako) for 20 minutes. The second and the third steps were repeated to amplify the reactions. The reaction was developed with the Fast Red Substrate System (Dako) and slides counterstained with Mayer's hematoxylin solution (Sigma Chemical Co, St Louis, MO). For double-staining (APAAP/PAP)32 the endogenous peroxidase activity was suppressed by using Peroxidase Blocking Reagent (Dako) for 5 minutes. To reduce the background, slides were incubated with normal swine serum (1:5, Dako) for 20 minutes, followed by the addition of MoAbs. After 30 minutes of incubation and washing in TBS, the rabbit polyclonal antibody was added for 30 minutes. After washing in TBS, the slides were incubated with goat antimouse (1:25; Dako) for 30 minutes, rinsed again, and swine antirabbit (1:100; Dako) was applied for an additional 30 minutes. The slides were washed again and APAAP (mouse) complex was applied for 30 minutes, rinsed, and then PAP (rabbit) (1:400; Sigma) was applied for 30 minutes. The APAAP reaction was developed in Fast Red Substrate System (Dako) for 20 minutes and the PAP reaction was developed with 3'3 diaminobenzidin (DAB) solution for 5 minutes. The slides were counterstained as described above. The percentage of red (APAAP) or yellow-brown (PAP) positive cells alone or combined were counted separately in duplicate samples for each experiment and in five high power microscopic fields (HPMF) per slide.

bFGF staining was performed on frozen tissue sections from the sites of inoculation of mice injected with untreated, TCM-treated, or γIFN-treated human umbilical vein endothelial (HUVE) cells by using a rabbit polyclonal anti-bFGF antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) diluted 1:20 or 1:40 in 1% phosphate-buffered saline-bovine serum albumine (PBS-BSA) and the PAP method as described above, but by using the peroxidase-antiperoxidase from Dako (1:100 dilution) as described previously.31,59 

The staining of HUVE cells for CD34, vascular endothelial (VE)-cadherin, FVIII-RA, EN-4, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecular-1 (ICAM-1), endothelial leukocyte adhesion molecule (ELAM), and HLA-DR was performed by the APAAP method with cytospin preparations or cells grown on gelatine-coated slides. Slides were fixed in cold acetone for 10 minutes, air dried, and then stained as described for tissue staining. The primary antibodies were applied for 20 minutes. The percentage of positive cells in duplicate samples for each experiment and in five HPMF per slide was then evaluated. The specificity and derivation of the primary antibodies used in these experiments are shown in Table 1.

Table 1.

Cell Marker Expression in HUVE Cells Before or After Treatment With TCM, RTCM Lacking or Containing γIFN, or γIFN Alone

Antibody Specificity % Positive Cells
HUVE TCMHUVE RTCM-HUVE (−γIFN)RTCM-HUVE (+γIFN) γIFNHUVE
CD34 Hematopoietic precursors cells, vascular endothelial cells, fibroblasts, smooth muscle cells; monoclonal (Monosan)  75 ± 5 68 ± 12  73 ± 8  62 ± 2  73 ± 10 
VE-cadherin  Endothelial cells; monoclonal-150 76 ± 9 84 ± 8  93 ± 1  86 ± 5  87 ± 3 
FVIII-RA  Endothelial cells; monoclonal (Dako)  61 ± 8 13 ± 4  28 ± 16  27 ± 14  18 ± 13  
EN-4 Endothelial cells, platelets, monocytes, granulocytes, B cells; monoclonal (Monosan)  89 ± 6  52 ± 6  72 ± 13 58 ± 10  40 ± 5  
VCAM-1  Activated endothelial cells, monocytes, dendritic cells, myoblasts; monoclonal (Amac) 11 ± 4  42 ± 12  55 ± 6  42 ± 5 45 ± 18  
ICAM-1  Activated endothelial cells, monocytes, T and B cells, dendritic cells, epithelial cells; monoclonal (Amac) 23 ± 12  66 ± 13  96 ± 3  94 ± 3 40 ± 1  
ELAM-1  Activated endothelial cells; monoclonal (Amac)  2 ± 1  54 ± 8  65 ± 3  53 ± 12 30 ± 1  
HLA-DR  Activated endothelial cells, B cells, monocytes, macrophages, activated T and NK cells; monoclonal (Dako) Neg  Neg  Neg  2 ± 1 60 ± 4 
Antibody Specificity % Positive Cells
HUVE TCMHUVE RTCM-HUVE (−γIFN)RTCM-HUVE (+γIFN) γIFNHUVE
CD34 Hematopoietic precursors cells, vascular endothelial cells, fibroblasts, smooth muscle cells; monoclonal (Monosan)  75 ± 5 68 ± 12  73 ± 8  62 ± 2  73 ± 10 
VE-cadherin  Endothelial cells; monoclonal-150 76 ± 9 84 ± 8  93 ± 1  86 ± 5  87 ± 3 
FVIII-RA  Endothelial cells; monoclonal (Dako)  61 ± 8 13 ± 4  28 ± 16  27 ± 14  18 ± 13  
EN-4 Endothelial cells, platelets, monocytes, granulocytes, B cells; monoclonal (Monosan)  89 ± 6  52 ± 6  72 ± 13 58 ± 10  40 ± 5  
VCAM-1  Activated endothelial cells, monocytes, dendritic cells, myoblasts; monoclonal (Amac) 11 ± 4  42 ± 12  55 ± 6  42 ± 5 45 ± 18  
ICAM-1  Activated endothelial cells, monocytes, T and B cells, dendritic cells, epithelial cells; monoclonal (Amac) 23 ± 12  66 ± 13  96 ± 3  94 ± 3 40 ± 1  
ELAM-1  Activated endothelial cells; monoclonal (Amac)  2 ± 1  54 ± 8  65 ± 3  53 ± 12 30 ± 1  
HLA-DR  Activated endothelial cells, B cells, monocytes, macrophages, activated T and NK cells; monoclonal (Dako) Neg  Neg  Neg  2 ± 1 60 ± 4 

Marker expression in untreated HUVE cells or in cells treated with TCM, RTCM in the presence or absence of γIFN (4 U/mL), or γIFN alone (103 U/mL). HUVE cells were cultured for 5 to 6 days and stained by immunohistochemistry as described in Materials and Methods. The results shown are the average of the percentage of positive cells from 4 independent experiments ± standard deviations.

Abbreviations: Neg, negative; NK, natural killer; HUVE, untreated HUVE cells; γIFN-HUVE, γIFN-treated HUVE cells; RTCM-HUVE, HUVE cells treated with RTCM (+γIFN, 4 U/mL) or RTCM (−γIFN); TCM-HUVE, TCM-treated HUVE cells.

F0-150

Donated by E. Dejana (Institute Mario Negri, Milan, Italy).

PCR and Southern blot analysis or liquid hybridization.

High molecular weight DNA was extracted from frozen tissues using a standard phenol/chloroform procedure. Polymerase chain reaction (PCR) analysis was performed with the following sets of primers. Two sets of primers derived from the published sequence8 were used to amplify HHV-8 sequences. Set 1: (700-810) 5′ TAG CCG AAA GGA TTC CAC CAT 3′, and (1207-1228) 5′ GGA TCC GTG TTG TCT ACG TC 3′; Set 2: (112-130) 5′ TGC GAT CTG TTA GTC CGGA 3′, and (430-453) 5′ ATT CGC CAA GGA CGT ACA GCA 3′. The probe was a 45mer (nucleotides 980-1025 of the published sequences) for primers set 1, and a 51mer (nucleotides 181-232 of the published sequence) for primers set 2, respectively. Primers used for Epstein-Barr virus (EBV) amplification were: 5′ AGG CTG CCC ACC CTG AGG AT 3′, and 5′ GCC ACC TGG CAG CCC TAA AG 3′ and the probe was the internal oligonucleotide 5′ GTT GCC GCC AGG TGG CAGC 3′. Primers used for HHV-6 amplification were: 5′ GCG TTT TCA GTG TGT AGT TCG GCA G 3′ and 5′ TGG CCG CAT TCG TAC AGA TAC GGA GG 3′, and the probe was 5′ GCT AGA ACG TAT TTG CTG CAG AAC G 3′. Primers used for HHV-7 amplification were (1-26): 5′TAT CCC AGC TGT TTT CAT ATA GTA AC 3′ and (186-161) 5′ GCC TTG CGG TAG CAC TAG ATT TTT TG 3′, and the probe was (82-111) 5′ CCT AAT GAA GGC TAC TTT GAA GTA CAA ATG 3′. Primers used for cytomegalovirus (CMV) were: (2038-2057) 5′ GGT GCT CAC GCA CAT TGA TC 3′ and (2300-2281) 5′ AGA CCT TCA TGC AGA TCT CC 3′ and the probe was (2133-2162) 5′ TGA TGA CCA TGT ACG GGG GCA TCT CTC TCT 3′. Primers used for β-globin were (54-73) 5′ CAA CTT CAT CCA CGT TCA CC 3′ and (-195 through -176) 5′ GAA GAG CCA AGG ACA GGT AC 3′. PCR was performed under standard buffer conditions (1 mmol/L MgCl2) using the ampliTAQ PCR amplification kit (Perkin-Elmer-Cetus, Norwalk, CT) according to the manufacturer's instructions. After an initial denaturation of 5 minutes at 94°C, 35 cycles of denaturation (92°C for 1 minute), annealing (55°C for 2 minutes) and extension (72°C for 2 minutes) were performed on a DNA thermal cycler 480 (Perkin-Elmer-Cetus). All PCRs were subjected to a final extension of 7 minutes at 72°C. PCR products were analyzed by agarose gel fractionation and Southern blot hybridization or by liquid hybridization using internal oligonucleotides as32P-end–labeled probes. For liquid hybridization, 10 μL of amplified DNA was mixed with 1 μL of 32P-labeled oligonucleotide and 5 μL of OH1.1 buffer (66.7 mmol/L NaCl and 44 mmol/L EDTA). The sample was then overlaid with mineral oil and subjected to 5 minutes of denaturation at 94°C and 15 minutes annealing at 55°C in a Perkin Elmer Thermal Cycler 480. The product was then loaded in a 10% TB acrylamide minigel (Novex, San Diego, CA) and exposed with Kodak XOMAT film for 1 hour to 12 hours. All cases negative by Southern blot analysis were tested again by liquid hybridization.

Preparation of TCM and reconstituted TCM (RTCM).

TCM were prepared from human T-lymphotropic virus type II-infected/transformed (nonvirus-producing) CD4+ T cells as previously described.53-55 These CM contain the same cytokines produced by mitogen-activated PBL or enriched T cells from normal donors and do not contain viral proteins.53 The average concentration of these cytokines as determined by enzyme-linked immunosorbent assay (ELISA) is: IL-1α (0.5 ng/mL), IL-1β (3.5 ng/mL), IL-2 (0.3 ng/mL), IL-6 (35 ng/mL), TNFα (0.2 ng/mL), TNF-β (50 pg/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.4 ng/mL), OM (0.5 to 1 ng/mL) and γIFN (150 pg/mL, corresponding to 3 to 4 U/mL of the recombinant γIFN [Boehringer Mannheim, Indianapolis, IN] used in these experiments). No bFGF is present in the CM. RTCM were prepared by combining recombinant cytokines at the concentrations described above. OM was purchased by R & D Systems (Minneapolis, MN) or obtained by B.C. Nair (Advanced BioScience Laboratories, Inc, Kensington, MD). All of the other cytokines were purchased from Boheringer Mannheim.

Cell cultures.

HUVE cells (passage 5 to 10) were cultured as previously described30-32 on gelatinized flasks in complete medium composed of RPMI 1640, 15% fetal bovine serum (FBS), 45 μg/mL of endothelial cell growth supplement (ECGS) (Collaborative Products, Bedford, MA) and 30 μg/mL of heparin (Sigma), 1% nutridoma HU (100 × solution) (Boehringer Mannheim), 1% essential amino acids (50 × solution) (GIBCO, Grand Island, NY), 1% nonessential amino acids (100 × solution) (GIBCO), 1 mmol/L of sodium pyruvate (GIBCO), 100 U/mL penicillin G-sodium, 100 mg/mL streptomycin sulfate, 0.25 mg/mL amphotericin B (GIBCO). Cytokine-treatment was performed by culturing HUVE cells for 5 to 6 days in the presence of TCM, RTCM, or γIFN.

Animal experiments.

γIFN-treated (102 U/mL), TCM-treated (1:4 dilution), or untreated HUVE cells (3 × 106 cells in 200 μL of media), were injected subcutaneously into the lower back (right side) of Balb/c nu/nu athymic mice, in the presence or in the absence of Tat (10 μg) as described previously.32 The negative control (media in which the cells were resuspended) was injected into the left side of the same mice, as previously described.31,32 Cells or media were mixed with an equal volume (200 μL) of Matrigel (Collaborative Biomedical Products) before inoculation.32Mice were killed 6 days later and the sites of injection were evaluated for the presence of macroscopic vascular lesions. Tissue samples were taken from all inoculated sites and fixed in formalin for histologic examination after hematoxylin and eosin (H & E) staining. The histologic changes observed at the site of injection, blood vessel formation, spindle cell proliferation, and edema were evaluated by comparison with the negative controls and graded according to intensity from 1 to 8 with the minimal alteration observed given a value of 1 (intensity value), as described previously.32 

RESULTS

CD8+ T cells and monocytes-macrophages are the predominant inflammatory cell types of KS and produce γIFN.

To analyze the type of infiltrating immune cells and the prevalent IC production in KS, immunohistochemical analyses were performed on frozen sections from both AIDS-KS and C-KS lesions and uninvolved tissues from the same patients by using antibodies specific for CD4, CD8, CD14, CD68, CD20, and HLA-DR and for the IC IL-1β, TNFα, and γIFN.

A prevalent CD8+ T-cell infiltration and infiltration with CD14+ and CD68+ monocytes-macrophages were detected in all lesions examined and were more evident in early stage lesions (Fig 1). A variable proportion of CD4+ T cells was also found, whereas B cells (CD20+) were few or absent in all lesions examined (data not shown). This cell infiltration was associated with a detectable expression of IL-1β and TNFα (data not shown), as described previously,60-62 but particularly with a high level of expression of γIFN (Fig 1). γIFN was found expressed in 100% (19/19) AIDS-KS and in 100% (3/3) C-KS lesions examined. Anti-γIFN antibodies stained mostly mononuclear cells, but also spindle-shaped cells (Fig 1). In contrast, in uninvolved tissues, only some mononuclear cells were stained (Fig 1). HLA-DR, which was evaluated to estimate the cell activation and the biologic activity of γIFN, was expressed in all of the γIFN+ KS lesions and tissues tested, and, at a low level, in one uninvolved tissue in which γIFN was not detected. Positivity for HLA-DR was also strong in vessels and endothelial cells of KS lesions.

Fig. 1.

Expression of γIFN, CD8, CD14, and CD68 in a representative KS lesion (lower panels, original magnification × 400) and uninvolved tissue (upper panels, original magnification × 1,000) by immunohistochemistry (APAAP) as described in Materials and Methods. Positivity for γIFN (red stain) was mostly observed in mononuclear cells, but also in spindle-shaped cells, as compared with uninvolved tissues. A strong increase in CD8+ and CD14+/CD68+ infiltrating cells, often with a subendothelial localization, was also observed in KS lesions, whereas these infiltrating cells are rare in uninvolved tissues. Similar results were obtained in all KS lesions analyzed, but were prevalent in early stage lesions.

Fig. 1.

Expression of γIFN, CD8, CD14, and CD68 in a representative KS lesion (lower panels, original magnification × 400) and uninvolved tissue (upper panels, original magnification × 1,000) by immunohistochemistry (APAAP) as described in Materials and Methods. Positivity for γIFN (red stain) was mostly observed in mononuclear cells, but also in spindle-shaped cells, as compared with uninvolved tissues. A strong increase in CD8+ and CD14+/CD68+ infiltrating cells, often with a subendothelial localization, was also observed in KS lesions, whereas these infiltrating cells are rare in uninvolved tissues. Similar results were obtained in all KS lesions analyzed, but were prevalent in early stage lesions.

To identify the cell types producing γIFN, double-staining experiments were performed with anti-γIFN and anti-CD8, -CD4, -CD14, -CD68, or anti-FVIII–RA antibodies. The results indicated that the cells producing γIFN (double positive cells) were mostly of the CD8+ phenotype (Fig 2). Spindle-shaped cells producing γIFN had a CD14+ or CD68+ phenotype, whereas endothelial cells (FVIII-RA+) in vessels did not stain for γIFN (Fig 2).

Fig. 2.

Expression of γIFN by CD8+, CD14+, or CD68+ cells infiltrating KS lesions. Examples of double-staining by immunohistochemistry (APAAP/PAP) of γIFN and CD8, γIFN, and CD14 or CD68, γIFN, and FVIII-RA in a representative KS lesion. Similar results were obtained with other specimens. CD8+ cells (red stain) coexpressing γIFN (brown stain) had a mononuclear morphology (upper right panel), whereas anti-CD14 or anti-CD68 antibodies (red stain), recognizing monocytes-macrophages, costain γIFN+ (brown stain) spindle-shaped cells (lower left and right panels). In contrast, anti-γIFN antibodies (brown stain) do not stain FVIII-RA+ vessels (red stain, upper right panel), but γIFN+ cells are often localized in the proximity of vessels.

Fig. 2.

Expression of γIFN by CD8+, CD14+, or CD68+ cells infiltrating KS lesions. Examples of double-staining by immunohistochemistry (APAAP/PAP) of γIFN and CD8, γIFN, and CD14 or CD68, γIFN, and FVIII-RA in a representative KS lesion. Similar results were obtained with other specimens. CD8+ cells (red stain) coexpressing γIFN (brown stain) had a mononuclear morphology (upper right panel), whereas anti-CD14 or anti-CD68 antibodies (red stain), recognizing monocytes-macrophages, costain γIFN+ (brown stain) spindle-shaped cells (lower left and right panels). In contrast, anti-γIFN antibodies (brown stain) do not stain FVIII-RA+ vessels (red stain, upper right panel), but γIFN+ cells are often localized in the proximity of vessels.

γIFN is the principal inducer of the KS spindle cell phenotype.

Previous observations indicated that KS spindle cells represent a heterogeneous population dominated by activated endothelial cells.4-7,63-68 IC and in particular γIFN are known to have profound effects on endothelial cells and to induce changes generally described as activation,69-71 which are the same phenotypic changes found in KS lesions. To investigate the role of IC and of γIFN in these changes, specific endothelial cell markers and activation molecules were analyzed after culture of HUVE cells in the presence of TCM that contain the same IC expressed in KS tissue. These experiments were also performed with RTCM that was obtained by adding together recombinant cytokines at the same concentration measured in TCM with or without γIFN, or in the presence of γIFN alone (103 U/mL) (Table 1).

Similar levels of CD34 and VE-cadherin were observed in HUVE cells independently from the treatment. On the contrary, all cytokine combinations (TCM, RTCM) and γIFN alone downregulated FVIII-RA and EN-4 expression (Table 1). This same pattern of marker expression, including FVIII-RA downregulation, is found in KS cells both in vivo and in vitro,55,68 and both IL-1 and γIFN can downregulate FVIII-RA expression.55,71,72 TCM and RTCM also increased the expression of VCAM-1, ICAM-1, and ELAM-1 as found for KS cells.55,67,68 γIFN alone had a similar effect, although less intense than in the presence of combined cytokines (Table 1). In addition, γIFN-treated cells expressed HLA-DR, another marker found to be expressed in spindle cells and vessels of KS; in contrast, cells exposed to TCM or RTCM stained negative due to the counteraction of IL-1.73 

Finally, as previously found with other cell types74,75TCM-treated or γIFN-treated HUVE cells acquired a typical spindle morphology indistinguishable from that of KS cells (Fig 3). Thus, γIFN can induce phenotypic changes similar or identical to those found in spindle cells in vitro and in most spindle cells of the lesions. However, this effect is maximal in the presence of other IC that contribute to these changes directly or by increasing γIFN function.69,71 

Fig. 3.

γIFN and TCM induce HUVE cells to acquire a spindle morphology. Shown are HUVE cells (left panels, original magnification × 40; right panels, original magnification × 100) cultured under standard conditions (HUVE) or after 6 days of culture in the presence of TCM (TCM-HUVE) or 103 U/mL of γIFN (γIFN-HUVE).

Fig. 3.

γIFN and TCM induce HUVE cells to acquire a spindle morphology. Shown are HUVE cells (left panels, original magnification × 40; right panels, original magnification × 100) cultured under standard conditions (HUVE) or after 6 days of culture in the presence of TCM (TCM-HUVE) or 103 U/mL of γIFN (γIFN-HUVE).

γIFN induces endothelial cells to acquire angiogenic properties and to induce KS-like lesions in nude mice.

Inoculation of cultured KS spindle cells in nude mice induces vascular lesions of mouse cell origin closely resembling early KS.31,32,76 These KS-like lesions develop in response to the cytokines produced by AIDS-KS spindle cells, such as bFGF, which mediates angiogenesis and spindle cell growth, and VEGF, which synergizes with bFGF in inducing angiogenesis and edema30-32,50 (and F. Samaniego et al, submitted). Angiogenic cytokine production, on the other hand, is induced in vitro in KS cells or endothelial cells by TCM or IC50,58,59 (and F. Samaniego et al, submitted). To determine whether γIFN alone could induce normal endothelial cells to acquire the capability to promote KS-like lesions, untreated, TCM-treated, or γIFN-treated HUVE cells were inoculated in nude mice (Fig 4 [see page 959], Table 2). γIFN-treated or TCM-treated HUVE cells induced macroscopic vascular lesions in 41% and 59% of the inoculated mice, respectively. Histologic alterations typical of KS such as angiogenesis, spindle cell growth, and edema were present in 70% to 82% and 100% of the mice inoculated with γIFN-treated or TCM-treated cells, respectively. In contrast, no lesions were induced by untreated HUVE cells (Fig 4A, Table 2). Tissue samples from the sites inoculated with TCM-treated, γIFN-treated cells, or from untreated cells were then analyzed for bFGF expression by immunohistochemistry (Fig 4B). No bFGF was detected in sites inoculated with untreated HUVE cells, in contrast, high levels of bFGF were found in sites inoculated with both TCM-treated or γIFN-treated HUVE cells. These results indicated that γIFN induces normal endothelial cells to acquire angiogenic, KS-promoting activity due to induction of bFGF whose production is activated by γIFN in cultured endothelial cells.59 In addition, the presence of other IC as in TCM increased the angiogenic effect of γIFN consistent with in vitro data of synergistic effects of combined γIFN, IL-1, and TNF on bFGF production.58,59 As both γIFN and bFGF are expressed in KS lesions (from all forms of KS), these data suggest that these mechanisms are operative in vivo.

Fig. 4.

γIFN-treated or TCM-treated HUVE cells induce KS-like lesions in nude mice. Lesion formation is associated with expression of bFGF. (A) Shows examples of the histopathology (H & E staining, original magnification × 400) and (B) shows bFGF expression by immunohistochemistry in the same tissues from mice inoculated with untreated HUVE cells (HUVE), TCM-treated HUVE cells (TCM-HUVE), or γIFN-treated HUVE cells (γIFN-HUVE), respectively.

Fig. 4.

γIFN-treated or TCM-treated HUVE cells induce KS-like lesions in nude mice. Lesion formation is associated with expression of bFGF. (A) Shows examples of the histopathology (H & E staining, original magnification × 400) and (B) shows bFGF expression by immunohistochemistry in the same tissues from mice inoculated with untreated HUVE cells (HUVE), TCM-treated HUVE cells (TCM-HUVE), or γIFN-treated HUVE cells (γIFN-HUVE), respectively.

Table 2.

γIFN-Treated or TCM-Treated HUVE Cells Induce in Nude Mice Vascular Lesions Closely Resembling KS That Are Increased by the HIV-1 Tat Protein

Cells Treatment Lesion*AngiogenesisSpindle CellsEdema
HUVE  —  0% (12)  0% (0) 0% (0)  0% (0)  
HUVE  TCM  59% (17) 100% (5)  100% (6)  100% (5)  
HUVE  γIFN 41% (17)  70% (2)  82% (3)  82% (4)  
HUVE TCM + Tat  100% (10)  100% (8)  100% (8) 100% (5)  
HUVE  γIFN + Tat  60% (15)  73% (3) 100% (4)  100% (5) 
Cells Treatment Lesion*AngiogenesisSpindle CellsEdema
HUVE  —  0% (12)  0% (0) 0% (0)  0% (0)  
HUVE  TCM  59% (17) 100% (5)  100% (6)  100% (5)  
HUVE  γIFN 41% (17)  70% (2)  82% (3)  82% (4)  
HUVE TCM + Tat  100% (10)  100% (8)  100% (8) 100% (5)  
HUVE  γIFN + Tat  60% (15)  73% (3) 100% (4)  100% (5) 

Reported is the percentage of mice developing macroscopic vascular lesions and the percentage of mice developing histologic alterations. HUVE cells were treated with γIFN (102 U/mL) or TCM (1:4) and 3 × 106 cells were inoculated in nude mice in the presence or in the absence of Tat (10 μg) as described in Materials and Methods. Mice were killed 6 to 7 days after inoculation and tissue slides examined after H & E staining and graded as described previously.32 Inoculation of Tat alone did not induce lesions or histologic alterations, as described previously.32 

*

Percentage of mice developing macroscopic vascular lesions (size ranging from 4 × 5 to 7 × 7 mm). Parenthesis show the number of inoculated mice.

Percentage of mice developing histologic alterations. Parenthesis show the average “intensity value” for each histopathologic feature observed per each experimental condition.

HIV-1 Tat protein increases the KS-like forming activity of endothelial spindle cells induced by γIFN or TCM.

Previous data indicated that the Tat protein of HIV-1 can increase the frequency and aggressiveness of KS in HIV-1–infected individuals32 and may act as a progression factor. In fact, inoculation of mice with Tat alone has little or no effect. However, when Tat is injected in the presence of suboptimal amounts of bFGF, synergistic angiogenic KS-promoting effects are observed and a higher number of mice develop lesions as compared with injection of bFGF alone.32 These are due to the enhancement by Tat of endothelial cell growth, migration, and invasion induced by bFGF and to bFGF-induced expression of the integrins α5β1 and αvβ3 that function as the receptors for Tat.32,57 Thus, bFGF is required for the in vivo effect of Tat. Because TCM or γIFN induce production of bFGF (Fig 4B) and expression of the same integrins57 (and data not shown), this suggested that Tat could exert its effect on KS lesion formation. To investigate this, mice were inoculated with γIFN-treated or TCM-treated cells in the presence of Tat. As shown in Table 2, Tat increased the number of mice developing lesions from 41% to 60% with γIFN-treated cells and from 59% to 100% with TCM-treated cells, respectively. In addition, Tat enhanced each histologic alteration induced by treated cells (Table 2). Thus, Tat can augment the angiogenic activity of TCM- or γIFN-treated endothelial cells and this results in enhancing effects on KS lesion formation. This effect is maximal in the presence of other IC as shown by the more potent effect of Tat on KS-like lesions induced by TCM-treated cells. As Tat is relesased by HIV-1 infected cells52,56,57,77 and is present in AIDS-KS lesions,32 these data support its role as a progression factor for HIV-1–infected individuals and indicate that γIFN alone or, more efficiently, in combination with other IC renders the tissues responsive to the effect of Tat.

HHV-8 infection and expression of γIFN and HLA-DR in AIDS-KS and C-KS.

Herpesviruses are known to activate CD8 T cells and to induce production of γIFN.78-82 Thus, the presence of HHV-8 in all forms of KS suggested that it may induce or contribute to the local immune response and inflammatory cell infiltration that leads to production of cytokines and in particular of γIFN. To verify whether HHV-8 is detectable in the KS lesions and uninvolved tissues examined for γIFN and whether it is the prevalent infectious agent as compared with other herpesviruses, the same tissues were analyzed by regular PCR and Southern blot or liquid hybridization for the presence of HHV-8, EBV, HHV-6, HHV-7, and CMV. β-Globin was used to confirm that the DNA extracted from tissues was amplifiable. As shown in Table 3, HHV-8 was detected in the majority (86%) of both forms of KS analyzed (18/21). Specifically, HHV-8 sequences were amplified in 89% of AIDS-KS (16/18) and 67% (2/3) of C-KS lesions examined. In addition, 40% (2/5) of the uninvolved tissues examined were positive for HHV-8 specific amplification. When the PCR data were compared with the results of the γIFN and HLA-DR staining, we observed that 100% of HHV-8+ KS lesions examined expressed γIFN (18/18) and HLA-DR (14/14), respectively. In contrast, only 14% (3/21) and 12% (2/16) of the γIFN+and HLA-DR+ lesions, respectively, were negative for HHV-8 sequences.

Table 3.

Expression of γIFN and HLA-DR in AIDS-KS, C-KS Lesions, and Uninvolved Tissues and Correlation With the Presence of HHV-8 and EBV

Specimen Average % (range) γIFNPositive Cells HLA-DR*PCR
HHV-8EBV
AIDS-KS  
  1 skin 45 (41-49)  73 (70-75)  ND  ND  
  2 skin 32 (27-36)  50 (48-52)  +  +  
  3 skin 11 (10-11)  62 (58-65)  +  −  
  4 skin 42 (33-50)  80 (75-84)  +  −  
  5 skin 50 (34-50)  80 (80-81)  +  −  
  6 skin 30 (28-32)  55 (49-61)  +  −  
  7 skin 28 (26-30)  59 (55-62)  +  −  
  8 skin 43 (41-44)  47 (36-65)  +  +  
  9 skin 29 (19-39)  45 (43-64)  +  −  
 10 skin 12 (7-17)  45 (43-64)  +  −  
 11 skin 15 (11-21)  ND  −  −  
 12 skin 39 (38-40)  35 (29-41)  −  −  
 13 skin 9 (3-15)  ND  +  −  
 14 skin 45 (40-50)  64 (58-70)  +  +  
 15 skin 40 (40-40)  ND  +  ND  
 16 skin 36 (25-49)  44 (38-50)  +  ND  
 17 skin 35 (29-41)  47 (38-56)  +  ND  
 18 skin 28 (13-49)  ND  +  ND  
 19 lymphnode 40 (39-40)  69 (66-72)  +  +  
Classical KS  
 1 skin  43 (41-60)  30 (30-31)  −  +  
 2 skin 26 (25-27)  19 (17-21)  +  −  
 3 skin  5 (5-7) ND  +  ND  
Uninvolved  
 1 skin  2 (2-2) 23 (21-25)  −  ND  
 2 skin  5 (5-5)  19 (19) −  ND  
 3 skin  10 (10-11)  32 (28-37)  ND  
 4 skin  12 (7-17)  41 (32-51)  +  ND  
 5 skin  0   11 (11-11)  −  ND 
Specimen Average % (range) γIFNPositive Cells HLA-DR*PCR
HHV-8EBV
AIDS-KS  
  1 skin 45 (41-49)  73 (70-75)  ND  ND  
  2 skin 32 (27-36)  50 (48-52)  +  +  
  3 skin 11 (10-11)  62 (58-65)  +  −  
  4 skin 42 (33-50)  80 (75-84)  +  −  
  5 skin 50 (34-50)  80 (80-81)  +  −  
  6 skin 30 (28-32)  55 (49-61)  +  −  
  7 skin 28 (26-30)  59 (55-62)  +  −  
  8 skin 43 (41-44)  47 (36-65)  +  +  
  9 skin 29 (19-39)  45 (43-64)  +  −  
 10 skin 12 (7-17)  45 (43-64)  +  −  
 11 skin 15 (11-21)  ND  −  −  
 12 skin 39 (38-40)  35 (29-41)  −  −  
 13 skin 9 (3-15)  ND  +  −  
 14 skin 45 (40-50)  64 (58-70)  +  +  
 15 skin 40 (40-40)  ND  +  ND  
 16 skin 36 (25-49)  44 (38-50)  +  ND  
 17 skin 35 (29-41)  47 (38-56)  +  ND  
 18 skin 28 (13-49)  ND  +  ND  
 19 lymphnode 40 (39-40)  69 (66-72)  +  +  
Classical KS  
 1 skin  43 (41-60)  30 (30-31)  −  +  
 2 skin 26 (25-27)  19 (17-21)  +  −  
 3 skin  5 (5-7) ND  +  ND  
Uninvolved  
 1 skin  2 (2-2) 23 (21-25)  −  ND  
 2 skin  5 (5-5)  19 (19) −  ND  
 3 skin  10 (10-11)  32 (28-37)  ND  
 4 skin  12 (7-17)  41 (32-51)  +  ND  
 5 skin  0   11 (11-11)  −  ND 

Frozen sections from involved or uninvolved skin and a lymph node of AIDS-KS and C-KS patients were stained by the APAAP method using MoAbs directed against γIFN and HLA-DR. Reported are the average percent of positive cells from 5 HPMF (100× magnification) per slide and in parenthesis is shown the range of positivity from different areas of the lesions. In KS tissues γIFN positive cells were of mononuclear and spindle-shaped morphology. In control tissues γIFN positivity was in mononuclear cells. In KS tissues HLA-DR was strongly expressed in vessels, spindle cells and mononuclear cells. A total of 1 to 10 μg of tissue DNA was analyzed by PCR for HHV-8 and EBV DNA sequences. Each case was tested at least twice. Negative cases resulted negative also by using 10 μg DNA and different tissue sections. HHV-8 negative cases were consistently negative independently from the amount of DNA used and were early KS lesions. All the PCR negative samples were positive for β-globin amplification. PCR analysis was also performed for HHV-6, HHV-7, and CMV. These herpesviruses resulted absent or present at a low frequency (HHV-6 [5%] or CMV [8%]) in association with HHV-8.

Abbreviation: ND, not done.

*

Positivity for HLA-DR was also present in vessels.

EBV was detected in 4/14 AIDS-KS (28%) and in 1/3 C-KS (33%) lesions examined (Table 3). For AIDS-KS, all cases positive for EBV were also positive for HHV-8 and expressed both γIFN and HLA-DR. Surprisingly, for C-KS, the case positive for EBV was consistently negative for HHV-8 by unnested PCR, but it was still positive for γIFN or HLA-DR expression. No other herpesviruses except for a small percentage of HHV-6 (5%) and CMV (8%) were detected in the lesions examined and they were associated with the presence of HHV-8 (data not shown).

Finally, γIFN and HLA-DR were both found to be expressed in 2/2 (100%) HHV-8+ uninvolved tissues. In addition, γIFN was expressed in 2/3 (66%) and HLA-DR in 3/3 (100%) HHV-8- uninvolved tissues, respectively. However, the levels of expression of both markers and particularly of γIFN were lower than in HHV-8+ tissues (Table 3). Thus, γIFN is highly expressed by CD8+ and CD14+/CD68+ cells infiltrating the lesions from both AIDS-KS and C-KS that contain HHV-8 and/or EBV specific sequences and these events are associated with endothelial cell activation, as shown by HLA-DR staining of spindle cells and vessels, which are also positive for other activation markers such as ELAM-1, ICAM-1, and VCAM-1.68 

DISCUSSION

The consistent presence of HHV-8 in tissues from patients with all forms of KS8-12 and the high rate of infection in individuals at risk of KS indicate that this virus plays an important role in KS development.18,83-85 The specific role of HHV-8 in KS pathogenesis, however, has not yet been delineated. HHV-8 possesses genes encoding for potential transforming proteins, in particular cyclin D, IL-8R, and bcl-2. Other viral genes encode for proteins, like v-IL–6, that can act with paracrine mechanisms on neighbor cells.21-23,86 These viral genes have been shown to be functional and some display a transforming activity in vitro. In addition, their homologs are present in herpesviruses with known or suggested transforming activities, like EBV, herpesvirus Sahiri (HVS) and CMV.87-89 These observations suggested that these viral products may play a key role in the pathogenesis of KS. However, HHV-8 cyclin D, IL-8R, and IL-6 are expressed at very low levels or are undetectable in KS tissues.21-23,26,28,33 In addition, it is unclear whether these genes contribute to the transforming activity of EBV and HVS whose transforming functions are encoded by other genes.90-92 Furthermore, EBV-bcl–2 is expressed in cells undergoing lytic infection after reactivation from latency.93 Consistent with this, HHV-8 viral homologs are induced at high levels in PEL cell lines after reactivation of lytic infection by TPA.21,33 Because only a small percentage of HHV-8–infected cells of monocytic-macrophagic origin undergo viral lytic infection in KS tissues,33,42,45 the low levels of expression of v-IL–8R, v-cyclin D, and v-IL–6 are consistent with these functions being expressed in this small percentage of cells. In contrast, the human IL-6 and bcl-2 are expressed at high levels in KS.34,35 In addition, HHV-8 is absent in the only two KS tumor cell lines obtained to date,36 and although endothelial spindle cells present in KS lesions are latently infected with HHV-8, they lose the virus on culture33,37-39,94-96(and our unpublished data). Finally, HHV-8 does not seem to transform cells in vitro.40 

It appears therefore conceivable that HHV-8 may act in KS pathogenesis also through indirect mechanisms, for example by activating the expression of host molecules able to induce the KS spindle cell phenotype and the cascade of events leading to KS lesion formation. This is also in agreement with experimental and clinical data indicating that, at least in early stage, KS is not a true cancer, but an hyperplastic angiogenic proliferation that may regress.47-49 These considerations prompted us to identify host factors triggering KS formation and to investigate their correlation with HHV-8 infection.

Our results show that γIFN may play a pivotal role in KS development. γIFN is strongly expressed in both AIDS-KS and C-KS lesions and in HHV-8+ uninvolved tissues from the same patients. γIFN expression is associated with endothelial cell activation, as indicated by the expression of HLA-DR and adhesion molecules in vessels and spindle cells of the lesions. Although it is mostly produced by infiltrating CD8+ T cells, γIFN is also expressed by spindle cells of macrophage origin with a subendothelial localization, in agreement with previous studies with macrophages.97,98Data from the report by Sirianni et al99further support these findings by showing that a prevalent CD8+ T-cell infiltration is present in both AIDS-KS and C-KS lesions, and that PBMC, tumor infiltrating lymphocytes, and spindle cells of macrophage origin cultured from KS lesions produce prevalently γIFN.

γIFN induces the formation of spindle cells with angiogenic activity. Specifically, γIFN promotes phenotypical and functional changes and activities in endothelial cells closely resembling KS spindle cells including the angiogenic activity and the angiogenic synergy with Tat. In fact, γIFN alone is sufficient to induce a modulation of marker expression that is similar or identical to the pattern of markers expressed by KS spindle cells and it is accompanied by the acquisition of the typical spindle morphology. In addition, when normal endothelial cells are treated with TCM or γIFN alone, they acquire the capability of inducing KS-like lesions and histologic alterations of mouse cell origin, which are indistinguishable from those induced by KS cells. As for KS cells, this is associated with the upregulation of bFGF production in the inoculated mice, as shown in vitro58,59and it is consistent with the presence of high levels of expression of bFGF in all forms of KS.32 Finally, these effects are increased synergistically by Tat demonstrating its role as a progression factor in AIDS-KS, in fact, bFGF and Tat are both present in AIDS-KS lesions and Tat increases the KS-forming activity of bFGF.32 Further, Tat can amplify HHV-8 viral load100 and can activate a further increase of IC (reviewed in Chang et al101). All of the effects of γIFN are potentiated by the presence of additional IC that increase γIFN function or synergize with γIFN. For example, IL-1 and TNF synergize with γIFN in activating bFGF expression and release.58,59Similar enhancing effects are observed for adhesion molecule expression and spindle morphology.69-72,74,75 Thus, although γIFN alone is sufficient, other IC present in KS, such as IL-1 and TNFα, cooperate with γIFN to induce the histological and phenotypical features of the lesion.

γIFN and HLA-DR expression correlate with the presence of HHV-8 in involved or uninvolved tissues. Data from the accompanying paper by Sirianni et al99 show that the infiltration of CD8+ T cells and the presence of macrophages producing γIFN is associated with that of HHV-8 in PBMC, KS tissues, and macrophagic spindle cells cultured from KS lesions of the same patients. The blood of these patients contains spindle-like macrophagic cells that are infected by HHV-8.43,44 This is consistent with recent data indicating HHV-8 lytic infection in mononuclear cells infiltrating KS lesions.42,45 These data therefore suggest that an immune response to HHV-8–infected cells present in tissues triggers or amplifies KS development through induction of γIFN. It is remarkable that one early C-KS lesion expressing both γIFN and HLA-DR was negative for HHV-8 (or contained viral DNA in such a low amount to prevent its detection by unnested PCR), but it was infected by EBV. In fact, EBV, as well as other herpesviruses, are known to activate CD8+ T cells and to induce γIFN production.78-82 Indeed, an oligoclonal expansion of CD8 T+ cells has been described following EBV infection in patients with HIV and in macaques infected with the simian immunodeficiency virus.82,102,103 Although γIFN and HLA-DR expression are increased in HHV-8+ versus HHV-8- tissues, a low production of γIFN is also found in HHV-8- uninvolved tissues from KS patients, suggesting that γIFN expression may precede a detectable HHV-8 infection. As γIFN has also been shown to act as a major mediator in recruiting cells into the skin,104 this may represent a process to increase the tissue localization of HHV-8–infected cells, and it may explain why γIFN detection can precede HHV-8 detection. This would be in agreement with recent evidence that HHV-8 prevalence and viral load can increase with lesion progression.37,105 

In conclusion, although other IC may cooperate in the pathogenesis of KS, γIFN seems to play a major role in KS development and this may be in response to HHV-8 infection. In support of this concept are studies that have shown that unstimulated106 or activated107 purified blood mononuclear cell cultures from HIV-1–infected homosexual men with documented early phase infection produce more γIFN than the seronegative controls. Furthermore, CD8 activation, generally accompanied by γIFN production, is higher in HIV-1 seronegative homosexual men than in healthy donors, as documented by the increased serum levels of CD8 and soluble ICAM in these individuals.108,109 These patients are also infected by HHV-8 and are at high risk of KS development. Finally, the administration of γIFN to KS patients has resulted in disease progression.110,111 Although it remains to be determined whether HHV-8 itself can induce a CD8+ T-cell activation and consequent γIFN production, this may represent a pathway by which this virus can trigger or amplify the development of KS.

ACKNOWLEDGMENT

We thank E. Dejana (Istituto di Ricerche Farmacologiche “Mario Negri,” Milano, Italy) for the anti–VE-cadherin MoAbs; B.C. Nair (Advanced BioScience Laboratories, Inc, Kensington, MD) for OM; P. Secchiero (Institute of Human Virology, Baltimore, MD) for the HHV-6 and HHV-7 PCR primers; V. Kao (Laboratory of Tumor Cell Biology [LTCB], NCI) for technical help; G. Barillari (Department of General Pathology, II University of Rome), P. Verani (Laboratory of Virology, Istituto Superiore di Sanità, Rome) for helpful discussions, and Angela Lippa for editorial assistance.

Supported in part by a grant from Associazione Italiana Ricerca sul Cancro (AIRC; Milan) and from the IX AIDS Project from the Ministry of Health, Rome, Italy. V.F. was partially supported by Associazione Nazionale per la Lotta contro l'AIDS, Ministry of Health in Rome, Italy.

Address reprint requests to Barbara Ensoli, MD, PhD, Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

1
Friedman-Kien
AE
Disseminated Kaposi's sarcoma syndrome in young homosexual men.
J Am Acad Dermatol
5
1981
468
2
Gottlieb
GJ
Ackerman
AB
Kaposi's sarcoma: An extensively disseminated form in young homosexual men.
Hum Pathol
13
1982
882
3
MacNutt
NS
Fletcher
V
Conant
MA
Early lesions of Kaposi's sarcoma in homosexual men. An ultrastructural comparison with other vascular proliferations in skin.
Am J Pathol
111
1983
62
4
Ruszczak
Z
Mayer-Da Silva
A
Orfanos
CE
Kaposi's sarcoma in AIDS. Multicentric angioneoplasia in early skin lesions.
Am J Dermatopathol
9
1987
388
5
Regezi
SA
MacPhail
LA
Daniels
TE
DeSouza
YG
Greenspan
JS
Greenspan
D
Human immunodeficiency virus-associated oral Kaposi's sarcoma: Heterogeneous cell population dominated by spindle-shaped endothelial cells.
Am J Pathol
143
1993
240
6
Zhang
YM
Bachmann
S
Hemmer
C
Van Lunzen
D
Vn Stemm
A
Kern
P
Fietrich
M
Ziegler
R
Waldmerr
R
Nawroth
PP
Vascular origin of Kaposi's sarcoma: Expression of leucocyte endothelial adhesion molecule-1, thrombomodulin, and tissue factor.
Am J Pathol
144
1994
51
7
Kaaya
EE
Parravicini
C
Ordonez
C
Gendelman
R
Berti
E
Gallo
RC
Biberfeld
P
Heterogeneity of spindle cells in Kaposi's sarcoma: Comparison of cells in lesions and culture.
J AIDS Hum Retrov
10
1995
295
8
Chang
Y
Cesarman
E
Pessin
MS
Lee
F
Culpepper
J
Knowles
DM
Moore
PS
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science
266
1994
1865
9
Moore
PS
Chang
Y
Detection of herpesvirus-like sequences in Kaposi's sarcoma in patients with and those without HIV infection.
N Engl J Med
332
1995
1181
10
Schalling
M
Ekman
M
Kaaya
E
Linde
A
Bieberfeld
P
A role for a new herpesvirus (KSHV) in different forms of Kaposi's sarcoma.
Nature Med
1
1995
707
11
Huang
YQ
Li
JJ
Kaplan
MH
Poisez
B
Katabira
E
Zhang
WC
Feiner
A
Friedman-Kien
AE
Human herpesvirus-like nucleic acid in various forms of Kaposi's sarcoma.
Lancet
345
1995
759
12
Dupin
N
Grandadam
M
Calvez
V
Gorin
I
Aubin
JT
Havard
S
Lamy
F
Leibowitch
M
Huraux
JM
Escande
JP
Agut
H
Herpesvirus-like DNA sequences in patients with Mediterranean Kaposi's sarcoma.
Lancet
345
1995
761
13
Foreman
KE
Friborg
J
Kong
W
Woffendin
C
Polverini
PJ
Nickoloff
BJ
Nabel
GJ
Propagation of a human herpesvirus from AIDS-associated Kaposi's sarcoma.
N Engl J Med
336
1997
163
14
Bigoni
B
Dolcetti
R
de Lellis
L
Carbone
A
Boiocchi
M
Cassai
E
Di Luca
D
Human herpesvirus 8 is present in the lymphoid system of healthy persons and can reactivate in the course of AIDS.
J Infect Dis
173
1996
542
15
Blackbourn
DJ
Ambroziak
J
Lennette
E
Adams
M
Ramachandran
B
Levy
JA
Infectious human herpesvirus 8 in a healthy north American blood donor.
Lancet
349
1997
609
16
Kedes
DH
Ganem
D
Ameli
N
Bacchetti
P
Greenblatt
R
The prevalence of serum antibody to human herpesvirus 8 (Kaposi sarcoma-associated herpesvirus) among HIV-seropositive and high-risk HIV-seronegative women.
JAMA
277
1997
478
17
Humphrey
RW
O'Brian
TR
Newcomb
FM
Nishihara
H
Wyvill
KM
Ramos
GA
Saville
MW
Goedert
JJ
Straus
SE
Yarchoan
R
Kaposi's sarcoma (KS)-associated herpesvirus-like DNA sequences in peripheral blood mononuclear cells: Association with KS and persistence in patients receiving anti-herpesvirus drugs.
Blood
88
1996
297
18
Whitby
D
Howard
M
Tenant-Flowers
M
Brink
N
Copas
A
Boshoff
TH
Suggett
FAE
Aldam
DM
Denton
AS
Miller
RF
Weller
I
Weiss
R
Tedder
R
Schulz
T
Detection of Kaposi's sarcoma associated herpersvirus in peripheral blood of HIV-1 infected individuals and progression to Kaposi's sarcoma.
Lancet
346
1995
799
19
Moore
PS
Kingsley
LA
Holmberg
SD
Spira
T
Gupta
P
Hoover
DR
Parry
JP
Conley
LJ
Jaffe
HW
Chang
Y
Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma.
AIDS
10
1996
175
20
Gao
S
Kingsley
L
Hoover
DR
Spira
TJ
Rinaldo
CR
Saah
A
Phair
J
Detels
R
Parry
P
Chang
Y
Moore
P
Seroconversion to antibodies against Kaposi's sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi's sarcoma.
N Engl J Med
335
1996
233
21
Moore
PS
Boshoff
C
Weiss
RA
Chang
Y
Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV.
Science
274
1996
1739
22
Cesarman
E
Nador
RG
Bai
F
Kaposi's sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposi's sarcoma and malignant lymphoma.
J Virol
70
1996
8218
23
Arvanitakis
L
Geras-Raaka
E
Varma
A
Gershengorn
MC
Cesarman
E
Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation.
Nature
385
1997
347
24
Neipel
F
Albrecht
JC
Ensser
A
Huang
YQ
Li
JJ
Fridman-Kien
AE
Fleckenstein
B
Human herpesvirus 8 encodes a homolog of interleukin-6.
J Virol
71
1997
839
25
Li
M
Lee
H
Yoon
D-W
Albrecht
J-C
Fleckenstein
B
Neipel
F
Jung
JU
Kaposi's sarcoma-associated herpesvirus encodes a functional cyclin.
J Virol
71
1997
1984
26
Guo
H-G
Browning
P
Nicholas
J
Hayward
GS
Tschachler
E
Jiang
Y-W
Sadowska
M
Raffeld
M
Colombini
S
Gallo
RC
Reitz
MS
Characterization of a chemokine receptor-related gene in human herpesvirus 8 and its expression in Kaposi's sarcoma.
Virology
228
1997
371
27
Nicholas
J
Ruvolo
V
Zong
J
Ciufo
D
Guo
H-G
Reitz
MS
Hayward
GS
A single 13-kilobase divergent locus in the Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genome contains nine open reading frames that are homologous to or related to cellular proteins.
J Virol
71
1997
1963
28
Sarid
R
Sato
T
Bohenzky
RA
Russo
JJ
Chang
Y
Kaposi's sarcoma-associated herpesvirus encodes a functional Bcl-2 homologue.
Nature Med
3
1997
293
29
Chang
Y
Moore
PS
Talbot
SJ
Boshoff
CH
Zarkowska
T
Godden-Kent
D
Paterson
H
Weiss
RA
Mittnacht
S
Cyclin encoded by KS herpesvirus.
Nature
382
1996
410
30
Ensoli
B
Nakamura
S
Salahuddin
SZ
Biberfeld
P
Larsson
L
Beaver
B
Wong-Staal
F
Gallo
RC
AIDS-Kaposi's sarcoma-derived cells express cytokines with autocrine and paracrine growth effects.
Science
243
1989
223
31
Ensoli
B
Markham
P
Kao
V
Barillari
G
Fiorelli
V
Gendelman
R
Raffeld
M
Zon
G
Gallo
RC
Block of AIDS-Kaposi's sarcoma (KS) cell growth, angiogenesis, and lesion formation in nude mice by antisense oligonucleotide targeting basic fibroblast growth factor.
J Clin Invest
94
1994
1736
32
Ensoli
B
Gendelman
R
Markham
P
Fiorelli
V
Colombini
S
Farreld
M
Cafaro
A
Chang
HK
Brady
JN
Gallo
RC
Synergy between basic fibroblast growth factor and human immunodeficiency virus type 1 Tat protein in induction of Kaposi's sarcoma.
Nature
371
1994
674
33
Zhong
W
Wang
H
Herndier
B
Ganem
D
Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus-8) genes in Kaposi's sarcoma.
Proc Natl Acad Sci USA
93
1996
6631
34
Morris
BC
Gendelman
R
Marrogi
AJ
Lu
M
Lockyer
JM
Alperin-Lea
W
Ensoli
B
Immunohistochemical detection of bcl-2 in AIDS-associated and classical Kaposi's sarcoma.
Am J Pathol
148
1996
1055
35
Miles SA, Rezai AR, Salazar-Gonzalez JF, VanderMeyden M, Stevens RH, Logan DM, Mitsuyasu RT, Taga T, Hirano T, Kishimoto T, Martinez-Maza O: AIDS Kaposi sarcoma-derived cells produce and respond to interleukin 6. Proc Natl Acad Sci USA 87:4068, 1990
36
Lunardi-Iskandar
Y
Gill
P
Lam
VH
Zeman
RA
Michaels
P
Mann
DL
Reitz
MS
Kaplan
M
Bernman
ZN
Carter
D
Isolation and characterization of an immortal neoplastic cell line (KS Y-1) from AIDS-associated Kaposi's sarcoma.
J Natl Cancer Inst
87
1995
977
37
Stürzl
M
Blasig
C
Schreier
A
Neipel
F
Hohenadl
C
Cornali
E
Ascherl
G
Esser
S
Brockmeyer
NH
Ekman
M
Kaaya
EE
Tschachler
E
Biberfeld
P
Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma (KS).
Int J Cancer
72
1997
68
38
Staskus
KA
Zhong
W
Gebhard
K
Herndier
B
Wang
H
Renne
R
Beneke
J
Pudney
J
Anderson
DJ
Ganem
D
Haase
AT
Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells.
J Virol
71
1997
715
39
Lebbé C, de Crémoux P, Rybojad M, Costa da Cunha C, Morel P, Calvo F: Kaposi's sarcoma and new herpesvirus. Lancet 345:1180 1995
40
(abstr)
Boshoff
C
Talbot
S
Whitby
D
Reeves
J
Weiss
RA
AIDS associated tumors and HHV8.
J AIDS Hum Retrovirol
14
1997
S9
41
Ambroziak
JA
Blackbourn
DJ
Heindier
BG
Glogan
RG
Gullett
JH
Mc Donald
AR
Lennette
ET
Levy
JA
Herpes-like sequences in HIV-infected and uninfected Kaposi's sarcoma patients.
Science
268
1995
582
42
Blasig
C
Zietz
C
Haar
B
Neipel
F
Esser
S
Brockmeyer
NH
Tschachler
E
Colombini
S
Ensoli
B
Stürzl
M
Monocytes in Kaposi's sarcoma lesions are productively infected by human herpesvirus-8.
J Virol
10
1997
7963
43
Browning
PJ
Sechler
JMG
Kaplan
M
Washington
RH
Gendelmann
R
Yarchoan
R
Ensoli
B
Gallo
RC
Identification and culture of Kaposi's sarcoma-like spindle cells from pheripheral blood of HIV-1 infected individuals and normal controls.
Blood
84
1994
2711
44
Sirianni
MC
Uccini
S
Angeloni
A
Faggioni
A
Cottoni
F
Ensoli
B
Circulating spindle cells: Correlation with human herpesvirus-8 (HHV-8) infection and Kaposi's sarcoma.
Lancet
349
1997
225
45
Orenstein
JM
Alkan
S
Blauvelt
A
Jeang
K-T
Weinstein
MD
Ganem
D
Herndier
B
Visualization of human herpesvirus type 8 in Kaposi's sarcoma by light and transmission electron microscopy.
AIDS
11
1997
35
46
Decker
LL
Shankar
P
Khan
G
Freeman
RB
Dezube
BJ
Lieberman
J
Thorley-Lawson
DA
The Kaposi's sarcoma-associated herpesvirus (KSHV) is present as an intact latent genome in KS tissue but replicates in the peripheral blood mononuclear cells of KS patients.
J Exp Med
184
1996
283
47
Levy
JA
Ziegler
JL
Acquired immunodeficiency syndrome is an opportunistic infection and Kaposi's sarcoma results from secondary immune stimulation.
Lancet
2
1983
78
48
Ensoli
B
Gallo
RC
Growth factors in AIDS-associated Kaposi's sarcoma: Cytokines and HIV-1 Tat protein.
AIDS Updates
7
1994
1
49
Stürzl
M
Brandstetter
H
Roth
WK
Kaposi's sarcoma: A review of gene expression and ultrastructure of KS spindle cells in vivo.
AIDS Res Hum Retroviruses
8
1992
1753
50
Cornali
E
Zietz
C
Benelli
R
Weninger
W
Masiello
L
Breier
G
Tshachler
E
Albini
S
Stürzl
M
Vascular endothelial growth factor regulates angiogenesis and vascular permeability in Kaposi's sarcoma.
Am J Pathol
149
1996
1851
51
Xerri
L
Hassoun
J
Planche
J
Guigon
V
Grob
JJ
Parc
P
Biornbaum
D
de Lapeyviere
O
Fibroblast growth factor gene expression in AIDS-Kaposi's sarcoma detected by in situ hybridization.
Am J Pathol
138
1991
9
52
Ensoli
B
Barillari
G
Salahuddin
SZ
Gallo
RC
Wong-Staal
F
The Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients.
Nature
344
1990
84
53
Barillari
G
Buonaguro
L
Fiorelli
V
Hoffman
J
Michaels
F
Gallo
RC
Ensoli
B
Effects of cytokines from activated immune cells on vascular cell growth and HIV-1 gene expression.
J Immunol
149
1992
3727
54
Albini
A
Barillari
G
Benelli
R
Gallo
RC
Ensoli
B
Angiogenic properties of human immunodeficiency virus type 1 Tat protein.
Proc Natl Acad Sci USA
92
1995
4838
55
Fiorelli
V
Gendelman
R
Samaniego
F
Markham
PD
Ensoli
B
Cytokines from activated T cells induce normal endothelial cells to acquire the phenotypic and functional features of AIDS-Kaposi's sarcoma spindle cells.
J Clin Invest
95
1995
1723
56
Chang HC, Samaniego F, Nair BC, Buonaguro L, Ensoli B: HIV-1 Tat protein exits from cells via la leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. AIDS (in press)
57
Barillari
G
Gendelman
R
Gallo
RC
Ensoli
B
The Tat protein of HIV-1, a growth factor for AIDS-Kaposi's sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.
Proc Natl Acad Sci USA
90
1993
7941
58
Samaniego
F
Markham
PD
Gallo
RC
Ensoli
B
Inflammatory cytokines induce AIDS-Kaposi's sarcoma (KS)-derived spindle cells to produce and release basic fibroblast growth factor and enhance KS-like lesion formation in nude mice.
J Immunol
154
1995
3582
59
Samaniego
F
Markham
PD
Gendelman
R
Gallo
RC
Ensoli
B
Inflammatory cytokines induce endothelial cells to produce and release basic fibroblast growth factor and to promote Kaposi's sarcoma-like lesions in nude mice.
J Immunol
158
1997
1887
60
Oxholm A, Oxholm P, Permin H, Bendtzen L: Epidermal tumor necrosis factor α and interleukin 6-like activities in AIDS-related Kaposi's sarcoma. APMIS 97:533, 1989
61
Cai
J
Gill
PS
Masood
P
Chandrasoma
P
Jung
B
Law
RE
Radka
SF
Oncostatin-M is an autocrine growth factor in Kaposi's sarcoma.
Am J Pathol
145
1984
75
62
Stürzl
M
Brandstetter
H
Zietz
C
Eisenburg
B
Raivich
G
Gearing
D
Speiser
B
Brockmeyer
NH
Hofschneider
PH
Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: A combined in vitro and in vivo analysis.
Oncogene
10
1995
2007
63
Najdi
M
Morales
AR
Zigler-Weissman
J
Pehneys
NS
Kaposi's sarcoma: Immunohistologic evidence for an endothelial origin.
Arch Pathol Lab Med
105
1981
274
64
Guarda
LG
Silva
EG
Ordonez
NG
Smith
JL
Factor VIII in Kaposi's sarcoma.
Am J Clin Pathol
76
1981
197
65
Rutgers
JL
Wieczorek
R
Bonetti
F
Kaplan
KL
Pasnett
DN
Friedman
KAE
Knowles
DM
The expression of endothelial cell surface antigens by AIDS-associated Kaposi's sarcoma evidence for a vascular endothelial cell origin.
Am J Pathol
122
1986
493
66
Kraffert
C
Planus
L
Penneys
NS
Kaposi's sarcoma: Further immunohistologic evidence of a vascular endothelial origin.
Arch Dermatol
127
1991
1734
67
Yang
J
Xu
Y
Zhu
C
Hagan
MK
Lawley
T
Hoffermann
MK
Regulation of adhesion molecules expression in Kaposi's sarcoma cells.
J Immunol
152
1994
223
68
(suppl 3)
Gendelman
R
Fiorelli
V
Kao
V
Gallo
RC
Ensoli
B
Spindle cells from both AIDS-associated and classical Kaposi's sarcoma (KS) lesions are of endothelial cell origin and present an activated phenotype.
AIDS Res Hum Retroviruses
10
1994
S101
69
Cotran RS, Pober JS: Endothelial activation: Its role in inflammatory and immune reaction, in Simionescu N, Simionescu M (eds): Endothelial Cell Biology. Plenum, New York, NY, 1988 p 335
70
Pober
JS
Cytokine-mediated activation of vascular endothelium. Physiology and pathology.
Am J Pathol
133
1988
426
71
Holzinger
C
Weissinger
E
Zuckermann
A
Imhof
M
Kink
F
Schollhammer
A
Kopp
C
Wolner
E
Effects of interleukin 1, 2, 4, 6, interferon-gamma and granulocyte/macrophage colony stimulating factor on human vascular endothelial cells.
Immunol Lett
35
1993
109
72
Tannenbaum
SH
Gralnich
HR
γ-interferon modulates von Willenbrand factor release by cultured human endothelial cells.
Blood
75
1990
2177
73
Watanabe
Y
Lee
S
Allison
AC
Control of the expression of a class II major histocompatibility gene (HLA-DR) in various human cell types: Down-regulation by IL-1 but not by IL-6, prostaglandin E2, or glucocortisoids.
Scand J Immunol
32
1990
601
74
Gresser
I
Metamorphosis of human amnion cells induced by preparations of interferon.
Proc Natl Acad Sci USA
47
1961
1817
75
Montesano
R
Orci
L
Vassalli
P
Human endothelial cell cultures: Phenotypic modulation by leucocyte interleukins.
J Cell Physiol
122
1985
424
76
Salahuddin
SZ
Nakamura
S
Biberfeld
P
Kaplan
MH
Markham
PD
Larsson
L
Gallo
RC
Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro.
Science
242
1988
430
77
Ensoli
B
Buonaguro
L
Barillari
G
Fiorelli
V
Gendelman
R
Morgan
RA
Wingfield
P
Gallo
RC
Release, uptake, and effects of extracellular HIV-1 Tat protein on cell growth and viral transactivation.
J Virol
67
1993
277
78
Levin
MJ
Murray
M
Rotbart
HA
Zerbe
GO
White
CJ
Hayward
AR
Immune response of elderly individual to live attenuated varicella vaccine.
J Infect Dis
166
1992
253
79
YamamotoT, Osaki T, Yoneda K, Ueta E: Immunological investigation of adult patients with primary herpes simplex virus-1 infection. J Oral Pathol Med 22:263, 1993
80
Aurelius
E
Andersson
B
Forsgren
M
Skoldenberg
B
Strannegard
O
Cytokines and other markers of intrathecal immune response in patients with herpes simplex encephalitis.
J Infect Dis
170
1994
678
81
Glimaker
M
Olcen
P
Anderson
B
Interferon-γ in cerebrospinal fluid from patients with viral and bacterial meningitis.
Scand J Infect Dis
26
1994
141
82
Callan
MFC
Steven
N
Krausa
P
Wilson
JDK
Moss
PAH
Gillespie
GM
Bell
JI
Rickinson
AS
McMichael
AJ
Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.
Nature Med
2
1996
906
83
Simpson
GR
Schulz
TF
Whitby
D
Cook
PM
Boshof
CF
Rainbow
L
Howard
MR
Gao
SJ
Bohenzky
RA
Simmonds
P
Lee
C
Ruiter
A
Hatzakies
A
Tedder
RS
Weller
IV
Weiss
RA
Moore
PS
Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen.
Lancet
349
1996
1133
84
Kedes
DH
Operskalski
E
Busch
M
Kohn
R
Fllod
J
Ganem
D
The seroepidemiology of human herpesvirus-8 (Kaposi's sarcoma-associated herpesvirus): Distribution of infection in KS risk groups and evidence for sexual transmission.
Nature Med
2
1996
918
85
Lennette
ET
Blackbourn
DJ
Levy
JA
Antibodies to human herpesvirus type-8 in the general population and in Kaposi's sarcoma patients.
Lancet
348
1996
858
86
Russo
JJ
Bohenzky
RA
Chien
MC
Chen
J
Yan
M
Maddalena
D
Parry
JP
Peruzzi
D
Edelman
IS
Chang
Y
Nucleotide sequence of the Kaposi's sarcoma-associated herpesvirus (HHV-8).
Proc Natl Acad Sci USA
93
1996
14862
87
Henderson
S
Huen
D
Rowe
M
Dawson
C
Johnson
G
Rickinson
A
Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death.
Proc Natl Acad Sci USA
90
1993
8479
88
Nicholas
J
Cameron
KR
Honess
RH
Herpesvirus saimiri encodes homologues of G protein-coupled receptors and cyclins.
Nature
355
1992
362
89
Chee
MS
Satchwell
C
Preddie
E
Weston
KM
Barrell
E
Human cytomegalovirus encodes three G protein-coupled receptor homologues.
Nature
344
1990
774
90
Kieff E: Epstein-Barr virus and its replication, in Fields BN, Knipe DM, Howely PM (eds): Virology (vol 2). Lippingott-Raven, 1996, p 2343
91
Desrosiers
RC
Bakker
A
Kamine
J
Falk
LA
Hunt
RD
King
NW
A region of the herpesvirus saimiri genome required for oncogenicity.
Science
228
1985
184
92
Jung
JU
Desrosiers
RC
Identification and characterization of the herpesvirus saimiri oncoprotein STP-C488.
J Virol
65
1991
6953
93
Hardwick
JM
Lieberman
PM
Hayward
D
A new Epstein-Barr virus transactivator, R, induces expression of a cytoplasmic early antigen.
J Virol
62
1988
2274
94
Boshoff
C
Schulz
TF
Kennedy
MM
Graham
AK
Fisher
C
Thomas
A
McGee
LOD
Weiss
RA
O'Leary
JJ
Kaposi's sarcoma-associated herpesvirus infects endothelial and spindle cells.
Nature Med
1
1995
1274
95
Li
JJ
Huang
YQ
Cockerell
CJ
Friedman-Kien
AE
Localization of human herpes-like virus type 8 in vascular endothelial cells and perivascular spindle-shaped cells of Kaposi's sarcoma lesions by in situ hybridization.
Am J Pathol
148
1996
1741
96
Dictor
M
Rambech
E
Way
D
Witte
M
Bendsoe
N
Human herpesvirus 8 (Kaposi's associated herpesvirus) DNA in Kaposi's sarcoma lesions, AIDS Kaposi's sarcoma cell lines, endothelial Kaposi's sarcoma stimulators, and the skin of immunosoppressed patients.
Am J Pathol
148
1996
2009
97
Nugent
KM
Monick
J
Hunninghake
MM
Stimulated human alveolar macrophages secrete interferon-γ.
Am Rev Respir Dis
131
1985
714
98
Robinson
BW
McLemore
TL
Cristal
RG
γ-interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis.
J Clin Invest
75
1985
1488
99
Sirianni MC, Vincenzi L, Fiorelli V, Topino S, Scala E, Uccini S, Angeloni A, Faggioni A, Cerimele D, Cottoni F, Aiuti F, Ensoli B: γ-Interferon production in peripheral blood mononuclear cells and tumor infiltrating lymphocytes from Kaposi's sarcoma patients: Correlation with the presence of human herpes virus-8 in peripheral blood mononuclear cells and lesional macrophages. Blood 91:xxx, 1998 (in press)
100
Harrington W, Sieczkowski L, Sosa C, Chan-a-Sue S, Cai JP, Cabral L, Wood C: Activation of HHV-8 by HIV-1 tat. Lancet 349:774, 1997
101
Chang
H-K
Gallo
RC
Ensoli
B
Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 Tat protein.
J Biomed Sci
2
1995
189
102
Pantaleo
G
Demarest
JF
Soudeyns
H
Graziosi
C
Denis
F
Adelsberger
W
Borrow
P
Saag
MS
Shaw
GM
Sekaly
RP
Major expansion of CD8+ T cells with a predominant Vβ usage during the primary immune response to HIV.
Nature
370
1994
463
103
Chen ZW, Kou ZC, Lekutis C, Shen L, Zhou D, Malloran M, Li U, Sodrosky U, Lee-Parritz D, Lei NC: T cell receptor Vβ repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus. J Exp Med 182:21 1995
104
Colditz
IG
Watson
DL
The effect of cytokines and chemotactic agonists on the migration of T lymphocytes into skin.
Immunology
76
1992
272
105
Noel
JC
Kaposi's sarcoma and KSHV.
Lancet
346
1995
1359
106
Fan
J
Bass
HZ
Fahey
JL
Elevated IFN-γ and decreased IL-2 gene expression are associated with HIV-1 infection.
J Immunol
152
1993
5031
107
Caruso
A
Gonzales
R
Stellini
R
Scalzini
A
Peroni
L
Turano
A
Interferon-γ marks activated T lymphocytes in AIDS patients.
AIDS Res Hum Retroviruses
6
1990
899
108
Schlessinger
M
Nian Chu
F
Badamchian
M
Jiang
JD
Robox
JP
Goldstein
A-L
Bekesi
JG
A distinctive form of soluble CD8 is secreted by stimulated CD8+ cells in HIV-1 infected and high risk individuals.
J Clin Immunol Immunopathol
73
1994
252
109
DePauli
P
Caffau
C
D'Andrea
M
Tavio
M
Tirelli
U
Santini
G
Serum levels of intercellular adhesion molecule 1 in patients with HIV-1 related Kaposi's sarcoma.
J Acquir Immune Defic Syndr
7
1994
695
110
Kriegel
RL
Odajnyk
CM
Laubenstaein
LJ
Ostreicher
R
Wernz
V
Vilcek
V
Rubinstein
P
Friedman-Kien
AE
Therapeutic trial of interferon-γ in patients with epidemic Kaposi's sarcoma.
J Biol Response Mod
4
1985
358
111
Albrecht
H
Stellbrink
HJ
Gross
G
Berg
B
Helmchen
V
Mensing
H
Treatment of typical leishmaniasis with interferon-γ resulting in progression of Kaposi's sarcoma in an AIDS patient.
Clin Investigator
72
1994
1041