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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4044-4058
Reactivation and Persistence of Human Herpesvirus-8 Infection in B
Cells and Monocytes by Th-1 Cytokines Increased in Kaposi's
Sarcoma
By
Paolo Monini,
Sandra Colombini,
Michael Stürzl,
Delia Goletti,
Aurelio Cafaro,
Cecilia Sgadari,
Stefano Buttò,
Marina Franco,
Patrizia Leone,
Stefano Fais,
Pasqualina Leone,
Gianna Melucci-Vigo,
Chiara Chiozzini,
Francesca Carlini,
Gudrun Ascherl,
Emmanuelle Cornali,
Christian Zietz,
Eric Ramazzotti,
Fabrizio Ensoli,
Massimo Andreoni,
Patrizio Pezzotti,
Giovanni Rezza,
Robert Yarchoan,
Robert C. Gallo, and
Barbara Ensoli
From the Laboratory of Virology, Istituto Superiore di Sanità,
Rome, Italy; the Institute of Human Virology, University of Maryland at
Baltimore, Baltimore, MD; GSF-National Research Center for Environment
and Health, Institute of Molecular Virology, Neuherberg, Germany;
Max-Planck-Institut für Biochemie, Abteilung für
Virusforschung, Martinsried, Germany; Pathologisches Institut der
LMU-München, München, Germany; the Department of Allergy
and Clinical Immunology, University of Rome "La Sapienza," Rome,
Italy; the Chair of Infectious Disease, University of "Tor
Vergata," Rome Italy; the Laboratory of Epidemiology and
Biostatistics, Istituto Superiore di Sanità, Rome, Italy; and the
HIV and AIDS Malignancy Branch, National Cancer Institute, National
Institutes of Health, Bethesda, MD.
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ABSTRACT |
Patients with Kaposi's sarcoma (KS) have a human herpesvirus-8
(HHV-8) load higher than patients without KS and present a CD8+ T-cell activation with production of Th1-type
cytokines both in tissues and peripheral blood mononuclear cells
(PBMC). Because in tissues of KS patients detection of inflammatory
cytokines (IC) can precede detection of HHV-8 DNA and because signs of
immunoactivation and/or dysregulation can precede KS development, we
investigated the effect of IC on HHV-8 infection. To achieve this goal,
PBMC and purified cell populations from 45 patients with KS and 45 patients at risk of KS were analyzed for HHV-8 DNA and/or gene expression and for cell survival, growth, and phenotype before or after
culture with or without the IC increased in KS. The results indicate
that PBMC that are polymerase chain reaction (PCR)-positive at day 0 generally loose the virus upon culture. However, the presence of IC
maintains HHV-8 DNA load in cultured cells. In addition, IC increase
viral load to detectable levels in PBMC from serologically positive
patients that were PCR-negative before culture.  Interferon is
sufficient for these effects, whereas tumor necrosis factor and
interleukin-6 have little or no activity. The increase of HHV-8 DNA by
IC is observed after short-term (7 days) or long-term (28 days) culture
of the cells and occurs in one or both of the two circulating cell
types that are infected in vivo: B cells and monocytes. In both cases
it is associated with lytic gene expression, suggesting that virus
reactivation is one of the most likely mechanisms for the effect of IC
on virus load. However, IC have also effects on the cells target of
HHV-8 infection, because they increase B-cell survival and induce the growth and differentiation of monocytes into KS-like spindle cells with
markers of endothelial macrophages. Because cells with markers of
endothelial macrophages are present in blood and lesions from KS
patients and are infected by HHV-8, these data may explain the high
HHV-8 load associated with KS development and suggest that infected
monocytes may carry the virus to tissues, transmit the infection, or
differentiate in loco in spindle cells with endothelial macrophage markers.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN HERPESVIRUS-8 (HHV-8) is associated
with Kaposi's sarcoma (KS)1 and its presence in
individuals at high risk of KS can predict disease
development.2,3 In KS patients, HHV-8 load is higher than
in infected individuals without KS4 and the virus is also
detected in secretions and uninvolved tissues.5-7 Similarly, HHV-8 load increases during lesions progression to the
nodular stage.8,9 Thus, virus persistence and replication is associated and, perhaps, required for KS development. However, patients without KS and normal blood donors can also be infected by
HHV-8, particularly in geographical areas with a high incidence of KS
(eg, Mediterranean countries and Africa).10-15
This finding suggested that additional factors cooperate with HHV-8
in KS development.
KS lesions are characterized by angiogenesis, inflammatory cell
infiltration, and the presence of spindle-shaped cells that are
considered to be the tumor cells of KS. Recent evidence indicates that
spindle cells are a mixed cell population dominated by activated endothelial cells and macrophages.16-19 In addition,
lesional macrophages express vascular-endothelial cadherin
(VE-cadherin), an endothelial cell marker typically expressed by the
so-called endothelial macrophages of lymphatic
organs.18,20,21
Spindle cells are latently infected by HHV-8,9,22,23
whereas lesional mononuclear cells can be lytically
infected.24,25 HHV-8 has also been detected in B
cells26,27 and in KS-like spindle cell progenitors that are
increased in the blood of patients with all forms of KS and in
individuals at high risk for KS.28,29 Upon culture, spindle
cells of endothelial origin loose the virus,30 whereas
macrophagic spindle cells from the lesions and KS-like spindle cell
progenitors from the blood are persistently infected.19,29
Inflammatory cells, including CD8+ T cells and
CD14+/CD68+ monocytes/macrophages, are abundant
in early stage KS lesions and produce inflammatory cytokines
(IC).7,16-19,31 These include interferon (IFN),
tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, and others.
High levels of Th1-type cytokines, such as IFN, are also produced by
activated peripheral blood mononuclear cells (PBMC) of patients with
the acquired immunodeficiency syndrome (AIDS)-associated KS (AIDS-KS)
or classical KS (CKS) as compared with patients with other
dermatological disorders.19 Finally, IFN can be detected
in early lesions and uninvolved tissues from KS patients even before
HHV-8 detection by the polymerase chain reaction (PCR).7
These findings and the progressive increase of viral load in
individuals developing KS suggested that immunodysregulation and
production of IC may modify HHV-8 replication, spread, and persistence.
We therefore analyzed the effect of IC increased in KS or in
individuals at high risk of KS on HHV-8 infection of PBMC from patients
with all the epidemiological forms of KS and from individuals at high
risk of KS, including homosexual men with AIDS or asymptomatic and
posttransplanted individuals that are positive or negative for HHV-8
infection by serology.
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MATERIALS AND METHODS |
Patients.
Human immunodeficiency virus (HIV)-infected homosexual men with AIDS-KS
or with AIDS without KS (NKS-AIDS) or asymptomatic (HIV+),
HIV-seronegative patients with CKS or posttransplant KS (PT-KS), or
posttransplant patients without KS (PT) were studied. Patients with
NKS-AIDS or AIDS-KS were treated with combinations of AZT, D4T, 3TC,
ddC, ddI, granulocyte-monocyte colony-stimulating factor (GM-CSF),
 IFN, vincristin, bleomycin, or Taxol. Patients with CKS were treated
with IFN or cortisone or were not under therapy. Posttransplant
patients with or without KS were treated with a combination of
cyclosporin and cortisone. All patients gave their informed consent to
participate in the study.
Cytokines and cell cultures.
Conditioned media from activated T cells (TCM) were prepared as
previously described.7,19,32-34 The average concentration of cytokines in TCM as determined by enzyme-linked immunosorbent assay
(ELISA) is as follows: IL-1 (0.5 ng/mL), IL-1 (3.5 ng/mL), IL-6
(35 ng/mL), TNF- (0.2 ng/mL), TNF- (50 pg/mL), GM-CSF (0.4 ng/mL), oncostatin M (0.5 to 1 ng/mL), and IFN (150 pg/mL).
Reconstituted in vitro TCM (RTCM) were prepared by combining
recombinant cytokines (Boehringer Mannheim, Mannheim, Germany) at the
same concentration described above. Oncostatin M was purchased from R&D
Systems (Minneapolis, MN).
PBMC were isolated by Ficoll-Hypaque density gradient centrifugation
and seeded in 6-well culture plates (3 to 4 × 106
cells/well). Total PBMC were cultured in RPMI 1640 containing 15%
fetal calf serum with or without TCM or RTCM (1:4 dilution) or single
cytokines. A half volume of fresh medium was added at day 3. TCM or
RTCM were added at day 3 and single cytokines were added at day 2 or 4, as specified. For long-term culture experiments, a half volume of fresh
medium was added at day 3 every week, as was performed for the
short-term cultures. In addition, half of the culture medium was
replaced with fresh medium with or without RTCM at the end of each week
of culture. Cells eventually present with the medium removed were
harvested by low speed centrifugation and readded to the culture. At
the end of the coculture, cells were harvested as a bulk or by
separating floating and adherent cells counted, and cell viability
determined by trypan blue dye exclusion. Adherent cells were all viable
at the time of harvesting, whereas bulk PBMC and floating cells showed
some level of cell death. The average percentage of dead cells was
comparable in PBMC cultured in the absence or presence of TCM or RTCM
(unfractionated PBMC: 6% [±4.9%] without TCM or RTCM, 7%
[±6.6%] with TCM or RTCM; floating cells: 17% [±19.1%]
without TCM or RTCM, 17% [±14.5%] with TCM or RTCM).
Statistical analysis of the data (see below) showed that the viability
of unfractionated PBMC or floating cells cultured with TCM or RTCM did
not differ from that of cells cultured with medium alone (Wilcoxon
signed-rank test; unfractionated PBMC, P = .679; floating
cells, P = .884).
Primary effusion lymphoma (PEL) cell lines.
Exponentially growing BCBL-135,36 cells (106
cells/mL) were collected, suspended in growth medium (5 × 105 cell/mL), activated with 20 ng/mL of
phorbol-12-myristate-13-acetate (TPA; Sigma, St Louis, MO), and
prepared for immunofluorescence assay (IFA), as described below.
Cell purification.
B cells were isolated from PBMC with anti-CD19 and further purified
with anti-CD4, anti-CD8, and anti-CD14 antibody-coated beads (Dynal,
Oslo, Norway), according to the manufacturer's instructions. After
removal of B cells, T cells were purified with anti-CD4 and anti-CD8
beads. T cells were further purified with a cocktail of anti-CD14 and
anti-CD19 beads. Monocytes were isolated from the residual cells by 1 hour of adherence at 37°C on tissue culture plates. Adherent cells
were then scraped and further purified with anti-CD4, anti-CD8, and
anti-CD19 beads. Cell purification was always monitored by
fluorescence-activated cell sorting (FACS) analysis. Freshly isolated
PBMC and purified cell populations were counted and viability was
determined by trypan blue dye exclusion. Both freshly isolated PBMC and
purified cell populations were all viable after isolation.
PCR analysis.
PBMC or derived cell fractions (floating or adherent cells or purified
cell populations) were counted and suspended at the same cell density
(107 cells/mL) in lysis buffer containing 0.001% Triton
(Sigma), 0.0001% sodium dodecyl sulfate (SDS; Sigma), 0.6 mg/mL
proteinase K (Sigma), or, alternatively, 0.1% polyoxyethylene
10 lauryl ether (Sigma), and 0.1 mg/mL proteinase K,
incubated at 56°C or 65°C, respectively, for 2 hours and
heat-inactivated at 94°C for 15 minutes. Because dead cells also
contain amplifiable DNA, total cells (viable and dead) were normalized
with lysis buffer.
Amounts of lysates corresponding to 105 cells were
amplified with primer set 1 (nucleotides 790-810 and 1207-1228 in the
KS330 BAM sequence)37 or set 2 (nucleotides 112-130 and
430-453 in the KS631 BAM sequence)37 in 34 consecutive
patients. Similarly, primer set 3 (nucleotides 987-1006 and 1200-1219 in the KS330 BAM sequence)37 was used in 56 consecutive
patients. Oligonucleotides internal to the amplified sequences were
used as probes for PCR product detection. -Globin primers were
5'-CAA CTT CAT CCA CGT TCA CC-3' and 5'-GAA GAG CCA
AGG ACA GGT AC-3'. PCR conditions with primer set 1 and 2 were as
follows: 5 minutes at 94°C, 35 to 45 cycles of denaturation
(92°C for 1 minute), anealling (55°C for 2 minutes), and
extension (72°C for 2 minutes); 1 mmol/L MgCl2 was
included in the reaction mixture. PCR conditions with
primer set 3 (35 to 45 cycles) were as described.37 PCR
products were blotted on nylon membranes or subjected to liquid
hybridization. For liquid hybridization, 10 µL of amplified DNA was
mixed with 1 µL of 32P-labeled oligonucleotide and 5 µL
of a solution containing 66.7 mmol/L NaCl and 44 mmol/L EDTA; the
samples were then subjected to 5 minutes of denaturation at 94°C
and 15 minutes of anealling at 55°C. Products were loaded onto 10%
nondenaturing acrylamide gels and exposed for 1 to 12 hours.
Hybridization of blotted PCR products were performed by standard techniques.
For semiquantitative PCR analysis, cell extracts were serially diluted
in a buffer containing 10 mmol/L Tris-HCl (pH 7.8), 0.1 mmol/L EDTA,
and highly purified sonicated salmon sperm DNA (50 µg/mL; all from
Sigma) as described.36 Dilution factors are indicated in
the text. To ascertain that the same relative amount of cells was
analyzed, the same extracts were analyzed by serial dilution PCR with
primers for -globin.36
Reverse transcription-PCR (RT-PCR) analysis.
Total RNA was extracted with the RNA assay Mini kit (Qiagen, GmbH,
Germany) and further purified with pancreatic DNAse I (Boehringer Mannheim), and purified RNA (0.5 µg) was retrotranscribed with the
reverse transcription system kit (Promega, Madison, WI) by incubating the reactions with hexanucleotide random primers for 10 minutes at room temperature, 30 minutes at 42°C, and 30 minutes at
53°C. After heat inactivation of RT, one third of each reaction was
subjected to 45 cycles of PCR for VP23 or T0.7, whereas amplification of -actin was performed with 1/15 of RT-reactions and 40 PCR cycles.
Primers set 3 was used for VP23 amplification, and primers RT-22A (CAC
CAT TCC TCT CCG CAT TA) and RT-22B (GTC TGC CGA AGT CAG TGC CA) were
used for T0.7 amplification with the same cycling conditions. -Actin
was amplified with primers BA1 (CAT GTG CAA GGC CGG CTT CG) and BA4
(GAA GGT GTG GTG CCA GAT TT).
In situ hybridization (ISH).
Cultured PBMC or BCBL-1 cells were harvested, centrifuged, washed
twice, suspended in phosphate-buffered saline (PBS), seeded onto
silan-coated slides, air-dried, and fixed in 4% buffered paraformaldehyde as described.9 ISH was performed under
high stringency conditions with strand-specific
35[S]-radiolabeled VP23 RNA hybridization probes
(specific activity,  109 cpm/µg) transcribed from the
plasmid p557-19 as described previously.25
FACS analysis.
Cells were analyzed by FACS38 with mouse monoclonal
antibodies conjugated with fluorescein isothiocyanate (FITC) or
phycoerythrin (PE): anti-CD3-FITC + anti-CD8-PE or anti-CD20-FITC + anti-CD14-PE (Becton Dickinson, Bedford, MA). Cells stained with FITC-
or PE-conjugated isotype-matched antibodies directed against irrelevant
epitopes served as negative controls. Living cells were gated based on forward and side scatter parameters. Cells positive for the isotypic controls (ie, nonspecifically stained) were excluded from the gate.
Immunocytochemistry.
PBMC were plated in gelatin-coated 8-well chamber slides (5 × 105 cells/well; Nunc Inc, Naperville, IL) and grown with or
without TCM or RTCM (1:4). After 6 or 7 days, floating cells were
harvested and adherent cells were washed with PBS without
Ca2+ and Mg2+ and fixed (10 minutes) at 4°C
with ethanol (80% vol/vol). Floating cells were analyzed by FACS or
plated on polylysine-coated chamber slides and fixed as described
above. Slides were stained using the alkaline phosphatase antialkaline
phosphatase (APAAP) method as described39 with monoclonal
antibodies directed against CD4 (1:20), CD8 (1:100), CD19 (1:20), CD20
(1:200), CD14 (1:20), CD31 (1:250), CD68 (1:200), CD45 (1:300), CDla
(1:20), VE-cadherin (1:20), FVIII-RA (1:100), and CD34 (1:20) (all from
Dako [Glostrup, Denmark], except for CD1a [Becton
Dickinson] and VE-cadherin [Coulter Immunotech, Marseille, France]).
The antibody directed against CD45 (leukocyte common antigen [LCA])
reacts with all CD45 isoforms. Slides were incubated with the antibody
for 2 hours at room temperature or 12 hours at 4°C, washed with
Tris-buffered solution, incubated (30 minutes) with rabbit antimouse
IgG (1:25; Dako), washed, and incubated (45 minutes) with the APAAP
(mouse) complex (1:40; Dako). The reaction was developed with the Fast
Red Substrate System (Dako) and slides counterstained with Mayer's
hematoxylin solution (Sigma). The percentage of positive cells in at
least 3 high power microscopic fields per slide was expressed as the
average and the range of the minimal and maximal values.
Anti-HHV-8 serology.
BCBL-1 cells were treated for 48 hours with TPA (20 ng/mL). Ten
microliters of a suspension of 4 × 106 cells/mL was
smeared on coverslips, rapidly air-dried, and fixed in acetone/methanol
solution for 10 minutes. Fixed smears were incubated successively in
two steps of 30 minutes each at 37°C with serially diluted serum
samples (in duplicate) and with fluorescein-labeled affinity-purified
goat antibodies to human IgG (KPL Lab Inc, Gaithersburg, MD). All of the microscopic examinations were conducted by two different investigators on coded samples in a blinded fashion. An
inverse titer of 20 or more was considered positive in the presence of
a bright cytoplasmic staining. No correlation was found between
Epstein-Barr virus (EBV) and HHV-8 antibody titers by this
assay.3,15 Serum samples from 8- to 12-month-old babies and
HIV-seronegative KS patients were used as negative and positive controls, respectively.
Statistical analysis.
Ninety-five percent confidence intervals (95%CI) for HHV-8 DNA
prevalence in PBMC and HHV-8 seroprevalence were estimated using
binomial distribution. The estimated prevalence of HHV-8 DNA or
anti-HHV-8 antibodies in KS patients was compared with the prevalence
found in non-KS patients using the test on equality of proportions.
The percentages of dead cells (unfractionated PBMC or floating cells)
after culture with RPMI or in the presence of RTCM were compared with
the Wilcoxon matched-pairs signed-ranks test. This test was used also
to evaluate the induction of adherent cell growth and floating cell
survival by IC. This was performed by comparing the number of total
adherent or viable floating cells present at day 7 of culture in PBMC
cultured with TCM/RTCM versus PBMC cultured without IC. In addition,
the adherent cell growth induced by IC was estimated after 7 days of
culture as the ratio of the number of adherent cells from PBMC cultured
with TCM or RTCM and the number of adherent cells from PBMC cultured
with medium alone. The increase of survival of floating cells by IC was
similarly evaluated as the ratio between the percentage of alive
floating cells after 7 days of culture in PBMC cultured in the presence
of TCM or RTCM as compared with cells cultured without IC. These ratios
were calculated for KS patients and patients at risk of KS and compared
throughout the Mann-Whitney test. This test was used also to compare
the levels of adherent cell growth among patients that showed
(responders) or did not show (nonresponders) an increased PCR signal or
conversion to PCR positivity upon culture with RTCM for 7 days.
The Mc-Nemar test for matched dychotomous data was used to calculate
the probability that the responses to TCM (see Table 2) or RTCM (see
Table 3) were obtained by chance.
One way analysis of variance was applied to analyze the expression of
VE-cadherin in adherent cells from PBMC cultured with or without TCM or RTCM.
All statistical analyses were performed using STATA, version 5.0 package (StataCorp Stata Statistical software [release 5.0], College
Station, TX).
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RESULTS |
Effect of cytokines increased in KS on HHV-8 load in PBMC from patients
with KS or at risk of KS.
PBMC from 45 patients with KS and 45 individuals at risk of KS were
analyzed by PCR with primers amplifying 3 different HHV-8 DNA regions.
Sixty-four of these patients were HIV+ homosexual men with
AIDS-KS (32 patients), or NKS-AIDS (25 patients) or asymptomatic
(HIV+, 7 individuals) not undergoing therapy with
HIV-protease inhibitors; and 26 were HIV individuals
with CKS (11 patients), PT-KS (2 patients), or PT (13 patients).
HHV-8 DNA was detected by PCR in 29 (64%) of the 45 KS patients (22 [69%] of the 32 with AIDS-KS, 6 [54%] of the 11 with CKS, and 1 [50%] of the 2 with PT-KS) and in 4 (9%) of the 45 individuals at
risk of KS (2 [8%] of the 25 NKS-AIDS patients, 2 [15%] of the 13 PT patients, and in none of the 7 HIV+ individuals;
Table 1). Fifty-nine of these patients for
whom sera were available were also analyzed for anti-HHV-8 antibodies by IFA and 47 (80%) of the 59 analyzed were found to be positive with
the highest seroprevalence (100%) in KS patients (Table 1). The
prevalence of both viral DNA and specific antibodies was significantly higher in patients with KS as compared with patients at risk of KS
(test on equality of proportions; P < .001; Table 1).
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Table 1.
Detection of HHV-8 DNA in PBMC and Presence of
Anti-HHV-8 Antibodies in Sera of Patients With KS or at Risk of KS
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To analyze the effect of IC on HHV-8 infection, PBMC from KS and risk
individuals were cultured for 6 to 7 days with or without TCM. TCM
contain the same IC increased in KS patients and they have been
previously used to mimic the IC combination found in the
lesions.7,19,32,33 The addition of TCM to PBMC induced a
dramatic enhancement of both the intensity of the HHV-8-specific PCR
signals and the detection of viral DNA in TCM-cultured PBMC as compared
with cells cultured in its absence
(Fig
1A and Table 2).
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| Fig 1.
(A) Increase of HHV-8 DNA load in PBMC from 2 AIDS-KS
patient after culture (days 6 to 7) in the presence of TCM or RTCM or
in its absence (RPMI). Shown are the autoradigrams of serial-dilution
PCR experiments performed with primers specific for HHV-8 ORF 26 (VP23)
and for the human -globin gene used as a control of the amount of
the genomic DNA analyzed. PCR products were hybridized with specific
oligonucleoutide probes. Numbers above lanes represent dilution factors
(for VP23) or aliquots of cell extracts corresponding to the indicated
number of cells (for -globin), respectively. Cell extracts were
diluted in salmon sperm DNA as described in Materials and Methods. (C) Amplification of HHV-8 DNA from PBMC (day
0) and long-term cultures (21 or 28 days) of PBMC from 4 patients with
KS (2 AIDS-KS, 2 CKS) and a PT patient. Floating (F) and adherent (A)
cells were separately harvested at day 21 or 28 from PBMC cultured in
the presence (RTCM) or absence (RPMI) of IC and the same number of
cells (105) were analyzed with primer set 3. Negative
controls (NC) are PCR reactions performed without DNA template or
aliquots of salmon sperm DNA processed with PBMC. Positive controls
were made with the indicated numbers of molecules (Mol) of plasmid
p557-19. PCR products were transferred to nylon membranes and
hybridized to a 32[P]-labeled oligonucleotide probe
internal to the amplified sequences. Ethidium bromide stainings showed
amplification of -globin gene sequences from the same specimens.
PBMC from the 2 CKS patients and the PT patient were analyzed also at
day 7 and 14 with negative results. (D) PCR analysis of HHV-8 DNA with PBMC
cultured with or without RTCM, IFN, TNF, or IL-6. The same cell
number was analyzed with primer set 3. NC is the negative control,
consisting of either salmon sperm DNA processed with the specimens or
PCR reactions lacking DNA template. (a) NKS-AIDS patient; (b through e)
AIDS-KS patients; (f) 50 and 5 molecules of a positive control plasmid.
PBMC from the patient shown in (a) were cultured for 11 days; PBMC from
the other patients were cultured for 3 to 5 days. Fresh RTCM was added
at days 0 and 3 of culture; single cytokines were added at days 0 and 2 for the patients shown in (a) and (b) and at days 0 and 4 for the other
patients, respectively. lFN was used at a concentration of 10, 50, or 100 IU/mL, as indicated in parenthesis. TNF was used at 30 ng/mL and
IL-6 was used at 100 IU/mL, respectively. Experiments repeated with TNF
or IL-6 (at 100 or 1,000 IU/mL) on 2 other patients that responded to
RTCM gave similar results. All samples were positive for -globin
amplification, as shown by ethidium bromide staining of the PCR
products. (B) Detection of HHV-8 DNA in PBMC (day 0) and
in floating (F) or adherent (A) cells from 4 AIDS-KS patients
(AIDS-KS), an asymptomatic homosexual man (HIV+), and a
PT patient (PT) cultured (6 to 7 days) in the presence of TCM or RTCM
or in its absence (RPMI). The same cell number (105)
was analyzed with primer set 3. PC are positive controls made
with the indicated numbers of molecules of plasmid p557-19. NC is
negative control made without adding template DNA. PCR products were
transferred to nylon membranes and hybridized to a
32[P]-labeled oligonucleotide probe internal to the
amplified sequences. Ethidium bromide staining shows amplification of
-globin gene sequences from the same specimens.
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The same effect was also obtained by adding together the same
(recombinant) IC at the same concentration as found in TCM (RTCM; Table 3).
Patients whose PBMC showed an enhancement of the PCR signal or a
conversion to PCR positivity with TCM or RTCM (Tables 2 and 3) were
defined as responders. In total, 24 (39%) of the 62 patients analyzed
were found to be responders. These included 19 (65%) of the 29 AIDS-KS
patients analyzed (9 showed an increased PCR signal and 10 showed a
conversion to PCR positivity); 1 (17%) of the 6 CKS patients; and 4 (15%) of the 26 patients without KS (2 NKS-AIDS, 1 HIV+,
and 1 PT patients) examined (all of these patients showed a conversion
to PCR positivity; Tables 2 and 3). In contrast, all of the other
patients analyzed remained PCR-negative except for 1 AIDS-KS patient
that was PCR-negative after culture with RTCM but positive in its
absence. The enhancement of the PCR-signal and the conversion to
PCR-positivity were found to be significantly associated with the
exposure to IC (Mc-Nemar test for matched dychotomous data; TCM:
P = .025; RTCM: P < .01; Tables 2 and 3).
The increase of the PCR signal was quantitated by serial dilution PCR
in 2 AIDS-KS patients. This showed in both patients a 10-fold increase
of HHV-8 DNA load in cells cultured with RTCM as compared with cells
cultured in its absence (Fig 1A). In addition, the sera from the
patients that were found to convert to PCR positivity and that were
available for the analysis were found to be all positive for
anti-HHV-8 antibodies (Table 3).
These data indicated that IC increase HHV-8 load and allow virus
detection in cultured PBMC.
Increase of HHV-8 viral load induced by IC in floating and adherent
cells from short-term and long-term PBMC cultures.
To further examine the effect of IC on HHV-8 infection and to gain
information on the target cell types, viral load was analyzed in fresh
PBMC (day 0) and in cells cultured for 6 to 7 days with or without IC
that were harvested in toto or after separation in floating and
adherent cells. This analysis was performed for 33 HHV-8-seropositive
patients with or without KS that were either PCR-positive or
PCR-negative at day 0 (Table 4).
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Table 4.
Maintenance of the PCR Signal or Induction of
PCR-Conversion Upon Culture With TCM or RTCM in PBMCs or in
PBMC-Derived Adherent or Floating Cells From PCR-Positive or -Negative
Patients
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Without IC, a dramatic reduction or loss of the HHV-8 PCR signal
occurred after culture of unfractionated PBMC or in both floating or
adherent cells from patients whose PBMC were positive at day 0. By
contrast, cultures treated with IC maintained HHV-8 DNA in
unfractionated PBMC or in adherent or floating cells (Fig 1B and Table
4). It is noteworthy that IC increased viral DNA load and allowed virus
detection in PBMC and adherent or floating cells from seropositive
patients whose PBMC were negative at day 0 (Fig 1B and Table 4). Only a
few or no CD14+ cells were present in the floating
populations at the time of harvesting (see Fig 4); therefore, it is
highly unlikely that detached adherent cells accounted for the results
obtained with the floating cell fraction.
To determine whether IC can maintain the virus in culture for a
prolonged period of time, PBMC from 5 HHV-8 seropositive patients (2 AIDS-KS, 2 C-KS, and 1 PT patient) were cultured with or without RTCM
for 3 to 4 weeks, and adherent and floating cells were separately analyzed for the presence of HHV-8 DNA at day 21 or 28. For those patients that had enough cells, PCR was also performed at earlier time
points. Three of these patients (1 with AIDS-KS and the 2 CKS patients)
were PCR-positive at day 0 and the other 2 were negative. Two of the 3 patients that were PCR-positive at day 0 (both CKS patients) turned
negative at day 7 and day 14; however, they became positive again in
the adherent or floating cells after long-term culture (28 days) in the
presence of RTCM but not in its absence (Fig 1C). One of the 2 patients
negative at day 0 (the PT patient) remained negative at day 7 and 14 but turned positive in the adherent cell fraction after long-term
culture with RTCM but not in its absence. For the other patients, the virus was lost (1 AIDS-KS) or remained undetectable (Fig 1C).
These data suggested that IC can reactivate HHV-8 upon short-term or
long-term culture, that this can occur in both floating or adherent
cells, and that it may require a chronic exposure to IC for detection.
Detection of HHV-8 DNA in PBMC after culture with IFN.
To identify the cytokine(s) responsible of the effect of TCM or RTCM on
HHV-8 infection, IFN, TNF, or IL-6, which are the most prominent
cytokines present in TCM and increased in KS patients, were added to
PBMC of patients with or without KS. The addition of IFN at 10 or 50 IU/mL but not at higher concentrations augmented viral DNA load in all
4 of the patients analyzed (Fig 1D, a through d), mimicking the effect
of TCM or RTCM. Three of these patients showed a conversion to PCR
positivity and 1 showed an increased PCR signal, respectively (Fig 1D).
In contrast, TNF and IL-6 had little or no activity at the
concentrations used (Fig 1D). Thus, IFN plays a key role in the
effects of TCM or RTCM on HHV-8 infection.
Induction of virus reactivation by IC.
To determine whether the effect of IC on HHV-8 viral load was due to
virus reactivation, latent (T0.7) and lytic (VP23) viral gene
expression were analyzed in 6 patients with AIDS-KS at both day 0 and
upon 2 days of culture with or without IC. Three of the 6 patients were
negative for RNA expression by RT-PCR in all samples, including that
from day 0, although viral DNA was present in fresh PBMC (day 0). The
other 3 patients analyzed showed expression of T0.7 but not VP23 at day
0, and 2 of these showed both lytic and latent viral gene expression
upon culture. This was found to be augmented or induced by IC in both
floating or adherent cells
(Fig 2A). In
particular, both patients showed lytic VP23 gene expression in adherent
cells in the presence of RTCM but not in its absence. Floating cells
from 1 patient showed lytic gene expression in RPMI; however, the
expression was augmented by IC (Fig 2A). For an additional patient with
a very high viral load, it was possible to perform an in situ
hybridization analysis using a probe specific for the lytic VP23
mRNA.25 This was found to be expressed in cells with the
typical morphology of lymphocytes and monocytes but only upon culture
with RTCM (Fig 2B). Thus, IC appear to promote viral reactivation in
both floating or adherent cells (Fig 2A and B).
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| Fig 2.
(A) Detection of latent and lytic viral gene
expression in total PBMC at day 0 and in floating or adherent cells
cultured with or without IC from 2 patients with AIDS-KS. (a) RT-PCR
and hybridization for T0.7; (b) RT-PCR and hybridization for VP23; (c)
RT-PCR and hybridization for T0.7 in the absence of reverse
transcriptase; (d) RT-PCR and ethidium bromide staining for human
-actin. NC are negative controls, including Jurkat cells and PCR
reactions without DNA template. RT indicates -actin
RT-PCR reactions performed from the same samples of RNA in the absence
of reverse transcriptase. The same number of RTCM-treated and untreated
cells were processed and RNA normalized by spectrophotometric
determination. (B) ISH detection of HHV-8 lytic (Vp23)
gene expression in PBMC from an AIDS-KS patient after culture with
RTCM. ISH of (A) PBMC cultured for 72 hours without
RTCM (original magnification × 25) and (B) corresponding dark
field; (C) PBMC cultured for 72 hours with RTCM (original magnification × 25), arrows point to ISH-positive cells and (D) corresponding dark
field; (E) PBMC from a patient not infected by HHV-8 (original
magnification × 25) and (F) corresponding dark field; (G) higher
magnification showing positive cells with monocytic morphology; a cell
with lymphocyte morphology is negative. Several microscopic fields were
analyzed and a similar density of positive cells was present in
cells cultured in the presence of RTCM. In contrast, cells
cultured in the presence of RPMI were always negative. Hybridization to
a latency-associated HHV-8 gene probe could not be performed, because
cells were not sufficient.
|
|
Identification of the blood cells infected in vivo by HHV-8.
The effect of IC on HHV-8 infection may be due to direct effects on the
virus but also to effects on the cells target of HHV-8 infection.
Therefore, experiments were designed to identify the cell types
infected in vivo. In the previous experiment, PBMC from the AIDS-KS
patient that was analyzed by ISH showed viral gene expression mostly in
cells with a bean-shaped nucleus that is typical of
monocyte-macrophages (Fig 2B). However, infected lymphocytes were also
detected. Therefore, B cells, T cells, and monocytes were purified from
PBMC of 5 patients (2 AIDS-KS, 2 NKS-AIDS, and 1 HIV+) and
analyzed by PCR. HHV-8 DNA was detected in B cells and monocytes from
HIV-infected homosexual men with or without KS or AIDS and in T cells
from a late stage AIDS-KS patient (Fig 3).
HHV-8 DNA was also detected in monocytes or B cells from a NKS-AIDS
patient and an HIV+ homosexual man, respectively, whose
PBMC were negative by PCR (Fig 3 and data not shown). In total, 3 (60%) of 5 B-cell fractions, 3 (60%) of 5 monocyte fractions, and 1 (20%) T-cell fraction from the 5 patients were PCR-positive. Thus,
both B cells and monocytes can be infected by HHV-8 in vivo.
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| Fig 3.
Detection of HHV-8 DNA in purified cell populations from
PBMC of homosexual men with HIV infection. Aliquots of cell lysates
(105 cells) were subjected to PCR with primer set 3.
PCR products were analyzed by Southern blot hybridization with a
32P-labeled oligonucleotide probe internal to the amplified
sequences. Shown are the results with B cells (B), monocytes (M), or T
cells (T) from 2 AIDS-KS and 2 NKS-AIDS patients. Cell populations were
purified as described and purification was monitored by FACS.
All samples were positive for -globin amplification (data not
shown). An HIV+ patient was positive in B cells (data not
shown). The NKS-AIDS and the HIV+ patients were positive
for anti-HHV-8 Ab by IFA.
|
|
Effect of cytokines increased in KS on the survival and growth of the
cells target of HHV-8 infection.
To evaluate the effects of IC on the cell types infected by HHV-8 in
vivo, PBMC were cultured for 6 to 7 days with or without TCM or RTCM.
Floating and adherent cells were separately harvested, counted, and
analyzed by FACS or immunocytochemistry and compared with the PBMC of
origin (day 0).
The number of viable (survived) floating cells after culture for 6 or 7 days in the presence or absence of IC did not differ (Wilcoxon
signed-rank test; P = .345), however, FACS analysis of PBMC
(day 0) and of floating cells from the cultured PBMC indicated that IC
increased B-cell survival
(Fig 4A). In
contrast, CD8+ and CD4+ T cells,
CD8+ natural killer cells, and non-T cells decreased with
time and at a similar rate with or without IC (Fig 4A and data not
shown).
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| Fig 4.
(A) FACS analysis of PBMC at day 0 and PBMC-derived
floating cells cultured for 6 days without TCM (RPMI, day 6) or with
TCM (TCM, day 6). T cells were doubly stained for CD3 and CD8 to obtain
the percentage of CD8+ T cells
(CD3+/CD8+), CD4+ T cells
(CD3+/CD8), CD8+ NK cells
(CD3/CD8+), and non-T cells
(CD3/CD8). Staining with anti-CD14 and
anti-CD20 antibodies was used to identify monocyte and B cells,
respectively (left side). Shown are representative examples from 2 patients (upper and lower panels, respectively). Similar results were
also obtained with floating cells from other 3 AIDS-KS patients and 1 asymptomatic HIV+ individual analyzed by
immunocytochemistry (data not shown). (B) Phenotypic characterization of adherent
cells obtained from PBMC of an AIDS-KS patient cultured for 6 days with
TCM and analyzed by immunohistochemistry (APAAP method). (a) A
representative negative control (isotype-matched control Ab) of
adherent cells counterstained by hematoxylin with the typical spindle
morphology is shown (original magnification × 400). (b through d)
Representative examples of staining for CD68 (b; original magnification × 400), CD14 (c; original magnification × 1,000), and VE-cadherin
(d; original magnification × 400) are presented. Nearly 100% of the
cells present in these fields showed a specific red staining. The
average percentage of positive cells from different fields for all the
patients analyzed is shown in Table 5.
|
|
IC had even more striking effects on adherent cells, because they
induced the development of spindle-shaped cells (Fig 4B, a) in the
majority of the cultures, although in AIDS-KS patients they were
already detectable without IC. IC also induced the growth of these
cells (Wilcoxon signed-rank test; P < .0001). Adherent cells
from patients with KS or AIDS showed the highest proliferative response
(average of 3.1- ± 2.0-fold growth induction by day 6 of culture
for 16 KS patients versus 3.0- ± 4.0-fold for 14 NKS-AIDS patients,
1.6- ±0.59-fold for 4 HIV+ individuals, and 1.4- ± 0.89-fold for 7 PT patients, respectively).
To verify whether the effect of IC on HHV-8 DNA load in adherent cells
was or was not related to the proliferation of latently infected cells,
the growth of adherent cells from these patients was compared with the
PCR results in both the presence and absence of IC. However, no
significant association of cell growth with the maintenance of the PCR
signal or the conversion to PCR positivity was detected (data not
shown; Mann-Whitney test; all patients, P = .241; KS patients,
P = .257). This confirmed that the increase of viral load
observed in this cell population is due to virus reactivation and not
to the proliferation of latently infected cells.
IC induce circulating spindle cell progenitors to differentiate in
endothelial macrophages that are present in KS lesions.
As expected, adherent cells induced by IC did not express markers of T
and B lymphocytes (CD8, CD19, and CD20), whereas they expressed the
leukocyte common antigen (LCA/CD45), markers of tissue macrophages such
as CD68 (Fig 4B, b), and markers of monocytic cells (CD31 and CD14; Fig
4B, c), both with or without IC (Table 5).
In addition, CD1a, a marker of dendritic cells, was detected in 1 HIV+ patient and its expression was increased upon culture
with IC (Table 5). It is noteworthy that untreated cells from AIDS-KS patients, but not from the other groups, also expressed VE-cadherin, a
marker of endothelial cells and endothelial macrophages (Fig 4B, d, and
Table 5). In addition, VE-cadherin expression was induced by IC in
adherent cells from all groups of patients (Table 5). Because staining
for endothelial cell markers such as factor VIII-related antigen
(FVIII-RA) and CD3432 remained negative (Table 5), these
cells were identified as endothelial macrophages.21 Thus,
PBMC-derived adherent cells are of monocytic origin and IC induce their
proliferation and differentiation toward tissue macrophages and
spindle-like endothelial macrophages. It is noteworthy that these same
cells have been found to be increased in the blood of KS
patients20,28,29 and that they are present in KS
lesions18,19; in both cases, they are infected by HHV-8 and
maintain the virus upon culture.19,29
| |
DISCUSSION |
Previous evidence suggested that IC, particularly of the Th1-type, may
act as triggering factors in KS development. IC, and in particular
IFN, appear to initiate KS development. In fact, they induce
endothelial cells to acquire the features of KS spindle cells cultured
in vitro or present in primary lesions and promote the formation of
KS-like lesions after injection in mice.7,32,34,40-42 The
development of KS-like lesions is mediated by basic fibroblast growth
factor and vascular endothelial growth factor, two angiogenic factors
that are highly expressed in KS and whose production is induced by
IC.34,39,41-43 IC also promote the expression of the receptors for the HIV-l Tat protein that acts as a progression factor
in KS development33,39,44 and increases HHV-8
load.45 IC also support the establishment and the long-term
growth of lesional KS spindle cells of both endothelial and macrophage
origin.7,19,34,40
Consistent with these data, the administration to KS patients of
IFN, TNF, or IL-2 has resulted in KS progression or
onset.46,47 In agreement with these findings are also data
indicating that immunoactivation or immunodysregulation and production
of IC are common in individuals at a high risk of KS, including
homosexual men even before HIV-1 infection,7,19,32,40,48,49
African individuals from areas at high incidence of
KS,49,50 or elderly men of the Eastern Mediterranean area
that are preferentially affected by CKS.51,52 All of these
individuals have a high HHV-8 seroprevalence3,11,15 and
present a CD8 T-cell activation.7,19,32,40,49,52 CD8+ T cells are one of the most important source of IFN
and other IC in KS patients.7,19
We have shown here that the same IC maintain or increase HHV-8 DNA load
in cultured PBMC from patients with KS or at risk of KS. These effects
occur on both B cells and monocytes that are the two main circulating
cell types infected in vivo.
Specifically, IC can maintain HHV-8 DNA in PBMC that are PCR-positive
at day 0 or can increase viral DNA load to detectable levels in PBMC
that are PCR-negative before culture. One or the other of these two
effects were observed in 24 (39%) of 62 patients analyzed; hence,
many, although not all, patients can respond to IC. Quantitation of
HHV-8 DNA load indicates that IC can increase viral load up to 10-fold
as compared with cells cultured in their absence.
The results of the long-term culture experiments also show that HHV-8
can be maintained at undetectable levels for long periods of time in
PBMC cultured with IC, but viral load increases to detectable levels
after chronic exposure to IC. Furthermore, IC induce the expression of
HHV-8 lytic genes in cultured PBMC to levels detectable by RT-PCR or ISH.
These effects of IC are observed in both floating and/or adherent cells
from cultured PBMC. Consistent with these data, B cells and monocytes
were found to be the two main cell types infected in vivo. The data,
therefore, indicate that IC increase or maintain HHV-8 infection and
have similar effects in both cell types.
Although other cytokines may contribute to the viral effects of TCM or
RTCM on PBMC, IFN appears to be sufficient to maintain and to
increase HHV-8 load. Because IFN is the earliest and most abundant
IC detectable in KS,7 these data suggest that it may be key
to both HHV-8 load and persistence and to the development of KS.
The mechanisms responsible for the increase of viral load induced by IC
may be several, including the reactivation of HHV-8 infection and
effects on the survival of B cells and on the growth and
differentiation of monocyte/macrophages or to a combination of these
effects. However, several lines of evidence suggest that the major
mechanisms of IC is the reactivation of HHV-8 infection in these cell types.
Although IC induced the growth of adherent cells, this was not
significantly associated with the increase of the PCR signal or with
the conversion to PCR positivity. These data strongly argue against the
preferential growth of a pre-existing population(s) of latently
infected cells. In addition, both adherent and/or floating cells turned
PCR-positive upon several weeks of culture in the presence of IC but
the same cells were found to be PCR-negative at day 0 and/or after
culture for shorter periods of times. This is strongly suggestive of
viral reactivation induced by the chronic exposure to IC, and it is
similar to the reactivation of human cytomegalovirus (HCMV) observed in
PBMC after a prolonged allogeneic stimulation.53
Moreover, IC induced the expression of the lytic VP23 RNA in both
floating cells (lymphocytes) and adherent cells (monocytes). Adherent
cells from all the patients analyzed expressed VP23 only in the
presence of IC; in contrast, although IC increased lytic gene
expression also in floating cells, reactivation was already observed
simply by culturing the cells. In contrast, only the latency-associated
T0.7 mRNA was detected in PBMC at day 0, in agreement with previous
work showing that PBMC from KS patients can harbor either lytic or
latent HHV-8 genomic forms.4 Although additional studies
are required, these data indicate that IC induced HHV-8 lytic
replication in cells from PBMC. Work is in progress to study in a
greater detail the effect of IC on the expression of HHV-8
latency-associated and lytic genes. The effects of IC on HHV-8 gene
expression appear to be specific for PBMC, because they were
undetectable in chronically infected PEL cell lines.35,54 In fact, no significant differences in latent or lytic viral gene expression were observed in BCBL-1 cells cultured for 2 days in the
presence or absence of RTCM or single cytokines, including IFN,
TNF, and IL-6, by both RT-PCR and in situ hybridization (data not
shown). Moreover, in contrast to PBMC, RTCM inhibited in a
dose-dependent fashion the expression of the lytic T1.1 HHV-8 nuclear
RNA expressed by cells undergoing spontaneous virus reactivation in
BC-1 cells, a PEL cell line doubly infected by HHV-8 and EBV (data not shown).
The effects of IC on B-cell survival and adherent cell growth and
differentiation may suggest that specific changes induced by IC in the
phenotype of the infected cells may be required for the reactivation of
HHV-8 infection in PBMC. In particular, the differentiation of adherent
cells toward the endothelial-macrophage phenotype may be key for HHV-8
reactivation not only in vitro but also in vivo. In fact, these cells
are expanded in the blood of KS patients and are present in KS
lesions.18,20,28,29
These and recent data on monocyte-macrophages in KS
lesions7,18,24,25 also suggest a crucial role of this cell
type for the localization of the virus into tissues. In particular, because B cells are rare or absent in KS lesions, circulating monocytes
may recruit the virus into tissues and, upon exposure to IC, they may
undergo lytic infection and transmit the virus to neighbor cells.
Alternatively, they may differentiate into macrophages and spindle-like
endothelial macrophages with a latent infection and a high viral load
as observed in vivo in lesional spindle cells.9,22,23 The
recruitment of these cells in tissues and in KS lesions is driven by
the expression of adhesion molecules in the vascular endothelium that
is activated by IC.7,17,19,32,39,40,55 Therefore, it is
tempting to speculate that the link between immunoactivation, IC
production, and HHV-8 infection in KS development is related to the
recruitment, growth, and differentiation of HHV-8-infected circulating monocytes.
| |
ACKNOWLEDGMENT |
The authors thank E. Trwniszewska, H.S. Chang, R. Humphrey, T. Merced-Galindez (National Institutes of Health, National Cancer Institute), and A. Schreier (Max-Planck-lnstitut für Biochemie) for technical help and A. Lippa and F.M. Regini for editorial assistance.
| |
FOOTNOTES |
Submitted July 13, 1998; accepted March 31, 1999.
P.M. and S.C. contributed equally to this work.
Supported by Italian grants from the Associazione Italiana per la
Ricerca sul Cancro (AIRC), Progetto Sangue, and the IX AIDS project
from the Ministry of Health; by the European Concerted Action
"Pathogenesis of AIDS-KS"; and by a grant from the Deutsche Forschungsgemeinschaft (SFB 464).
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.
Address reprint requests to Barbara Ensoli, MD, PhD, Laboratory of
Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy; e-mail: ensoli{at}virus1.net.iss.it.
| |
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TOP
ABSTRACT
INTRODUCTION