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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 968-976
 -Interferon Production in Peripheral Blood Mononuclear Cells and
Tumor Infiltrating Lymphocytes From Kaposi's Sarcoma Patients:
Correlation With the Presence of Human Herpesvirus-8 in Peripheral
Blood Mononuclear Cells and Lesional Macrophages
By
Maria Caterina Sirianni,
Laura Vincenzi,
Valeria Fiorelli,
Simone Topino,
Enrico Scala,
Stefania Uccini,
Antonio Angeloni,
Alberto Faggioni,
Decio Cerimele,
Francesca Cottoni,
Fernando Aiuti, and
Barbara Ensoli
From the Departments of Allergy and Clinical Immunology and
Experimental Medicine and Pathology, University of Rome "La
Sapienza," Rome; the Department of Dermatology, Catholic University
of Rome, Rome; the University of Sassari, Sassari; and the Laboratory
of Virology, Istituto Superiore di Sanità, Rome, Italy.
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ABSTRACT |
Evidence indicates that, at least in the early stage, Kaposi's
sarcoma (KS) is a cytokine-mediated disease and that it is consistently
associated with a novel herpesvirus termed human herpesvirus-8 (HHV-8).
To gain insights into the mechanisms by which cytokines and HHV-8 may
cooperate in disease pathogenesis, we examined the phenotype, the Th1
( -interferon [IFN]) and Th2 (interleukin-4 [IL-4]) cytokine
profile and the presence of HHV-8 in peripheral blood mononuclear cells
(PBMC), tumor-infiltrating lymphocytes (TIL), and spindle cell cultures
derived from skin lesions of patients affected by classical KS (C-KS)
and acquired immunodeficiency syndrome (AIDS)-associated KS (AIDS-KS).
TIL and spindle cell cultures were examined at day 0 or after culture in conditioned media from activated T cells (TCM) that contain the same
cytokines increased in KS tissues. No differences were found in the
immunophenotype of PBMC from C-KS patients versus controls, except for
AIDS-KS patients who showed a T-CD8+ expansion. However,
a preferential infiltration of T-CD8+ cells was found in
all KS lesions examined, which was maintained after culture of TIL in
TCM. IFN production was found in both PBMC and cultures derived from
all KS examined; some IL-4 positive supernatants were found only in
three AIDS-KS cases. Uninvolved skin did not show appreciable
lymphocyte infiltration or cytokine production. The culture conditions
of the lesional skin allowed also the appearance of adherent,
spindle-like cells bearing markers of tissue macrophages. Finally, most
or all of the PBMC, lesions, and macrophagic cell cultures from the
skin lesions were found to be positive for HHV-8 infection by nested
polymerase chain reaction (PCR). These findings indicate that patients
with KS express a Th1 phenotype with a prevalent IFN production,
likely accounted for by the local T-CD8+ infiltration. By
analogy with other viral infections (ie, Epstein-Barr virus), this
suggests that in loco recruitment of lymphoid cells and the subsequent
IFN production may be in response to or elicited by HHV-8 that was
found in both PBMC and macrophagic cell cultures from the lesions of
the same patients.
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INTRODUCTION |
KAPOSI'S SARCOMA (KS) is an
angioproliferative disease involving the skin and mucosas that affects
elderly men of Mediterranean origin (classical KS, [C-KS]), as well
as transplantanted patients; it is endemic in Africa (endemic
KS),1-5 and it is the most common neoplasm of human
immunodeficiency virus type 1 (HIV-1) infected homo-bisexual men
(acquired immunodeficiency syndrome [AIDS]-KS).6,7 In
these patients, KS acquires a more aggressive course than C-KS. However, all of these forms have the same histopathology, including spindle-shaped cells, considered to be the tumor cells of KS, angiogenesis, inflammatory cell infiltration, and edema.1
The spindle-shaped cells are composed by an heterogeneous cell
population of endothelial cells with an activated cell phenotype,
macrophages, and dendritic cells.8,9
Recently, DNA sequences from a new virus called Kaposi-Sarcoma
herpesvirus or human herpesvirus-8 (HHV-8), have been found in the
majority of the lesions from patients with all forms of KS, and the
virus has been propagated from a KS skin lesion, suggesting an
epidemiologic association of the virus with KS,10-18
although its role in KS pathogenesis is yet unknown. HHV-8 has also
been found in peripheral blood mononuclear cell (PBMC) from these
patients, as well as in other pathologic conditions.19-21
Recent data also indicate that HHV-8 is persistently present in
circulating B cells22 and in blood-derived spindle-like
cells that are found in KS patients or in HIV-1-infected homosexual
men.23,24 In addition, productively infected mononuclear
cells including monocytes/macrophages25,26 and latently
infected endothelial and spindle cells27-29 are present in
KS lesions. The virus, however, is rapidly lost by cultured endothelial
spindle cells from KS lesions28,29 (and B.E., unpublished data, June 1995).
The immune system seems to play a major role in KS development. For
example, homosexual men, the group of HIV-1-infected individuals at
highest risk for KS, show increased signs of immunoactivation, including soluble CD8 and soluble intercellular adhesion molecule (ICAM) even before HIV-1 infection, and KS can represent the first sign
of AIDS.30-35 Recent studies indicate that KS itself
behaves as a cytokine-mediated disease, since the first observation
that conditioned media (CM) derived from retroviruses-infected
CD4+ T cells or CM from activated primary T cells or PBMC
(TCM), rich in tumor necrosis factor (TNF), -interferon (IFN),
interleukin-1 (IL-1), IL-6, and Oncostatin M (OM) were able to support
the growth of KS-spindle cells.36-39 KS cells themselves
produce a variety of cytokines and angiogenic factors, including IL-1,
IL-6, IL-8, basic fibroblast growth factor (bFGF), and vascular
endothelial growth factor (VEGF), that mediate the formation of KS-like
lesions induced by inoculation of these cells in nude
mice40-44 (and F. Samaniego et al, submitted).
KS-like cells with characteristics similar to endothelial and
macrophagic spindle cells derived from the lesions have also been found
in blood from KS patients with all forms of the
disease.23,24,45 Finally, the inflammatory cell
infiltration found in KS lesions, particularly in the early stage, is
associated with the production of the same inflammatory cytokines found
in TCM.42,46-49,49a TCM promote normal
endothelial cells to acquire the features of the KS cell phenotype,
including the responsiveness to the HIV-1 Tat molecule,37,49-51 that can increase the frequency and
aggressiveness of AIDS-KS.43
Despite this body of evidence for the involvement of the immune system
in KS development, little or nothing is known on the type of cells
infiltrating the lesions and on the Th1 and Th2 cytokine profile in
patients with KS and of its correlation with the presence of HHV-8.
Similarly, little or no information is currently available on the
effects of the cytokines increased in KS on tumor-infiltrating
lymphocytes (TIL) and spindle cells present in KS.
Th1 cytokines (IL-2 and IFN) are involved in the development of
delayed type hypersensitivity reactions, whereas Th2 cytokines (IL-4,
IL-5, IL-10) are the main regulators of immunoglobulin production.52
In this study, we report that IFN is found in supernatants derived
from both skin lesions and PBMC cultures of all C-KS patients and from
the majority of AIDS-KS patients examined. The concomitant immunophenotyping of TIL indicates that the majority of these cells are
lymphocytes of the T-cell lineage that are maintained in culture by
TCM. TCM also allow the establishment in culture of spindle-shaped
cells with markers of tissue macrophages. Finally, the majority of the
cultures examined, both at the skin and at the PBMC level, harbor
HHV-8, suggesting that T-CD8+ cells producing IFN may be
recruited in KS lesions in response to HHV-8 infected cells.
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MATERIALS AND METHODS |
Patient population.
Eighteen patients affected by C-KS (men; mean age, 60 years) as well as
22 patients affected by AIDS-KS (21 men and one woman; mean age, 30 years; 18 homosexuals and four heterosexuals) were studied. Patients
with C-KS presented a cutaneous involvement only, whereas patients with
AIDS-KS presented a cutaneous and visceral (in 16 cases) involvement.
In all cases, the diagnosis of KS was supported by conventional
histology. A group of 20 normal volunteers was studied as control for
immunologic phenotyping of PBMC and a group of 34 patients affected by
skin disorders other than KS, such as psoriasis and chronic dermatitis,
were studied as control of cytokine production. All of the patients were free of cytokine therapy. Fourteen patients with HIV-1 infection were under antiretroviral therapy with AZT, whereas eight patients were
studied at the time of the first KS diagnosis and had refused any prior
antiretroviral therapy. Blood samples were obtained after written
informed consent.
Isolation of PBMC.
PB from patients was collected by venipuncture and heparinized (10 IU/mL, Liquemin, Hoffman-La Roche Co, Rome, Italy). PBMC were separated
by a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient. Cells were
washed twice in Hanks' balanced salt solution, resuspended in RPMI
1640 (GIBCO Laboratories, Grand Island, NY) containing 20% fetal calf
serum (FCS) (GIBCO) and plated at a concentration of 106
cells/mL in 24-well plates (Falcon, Becton Dickinson, Lincoln Park,
NJ). Cells were stimulated with phytohemagglutinin (PHA) (PHA-L, Sigma Chemical, St Louis, MO) at a final dilution of 1 µm/mL
for 72 hours at 37°C to assess cytokine production. Control cultures consisted of PBMC plated in the presence of medium alone.
Cultures from KS skin biopsies.
Punch biopsies were performed at the site of cutaneous KS lesions and
at the site of apparently uninvolved skin after obtaining from the
patients a written informed consent to the biopsy. Tissues were
mechanically minced, fragments were washed in medium, and the released
lymphoid cells (TIL) were recovered and phenotyped immediately (day 0).
The tissue fragments were then cultured in gelatin-coated flasks (1.5%
final dilution of gelatin; bovine skin albumin, Sigma) with a culture
medium previously used to culture KS spindle cells from the
lesions.37 Growth medium consisted of RPMI 1640 medium
supplemented with 20% FCS, 1% Nutridoma-HU (100× solution)
(Boehringer Mannheim, Milan, Italy), 1% nonessential amino acids
(100× solution) (Sigma), 1 mmol/L sodium pyruvate (Sigma), 100 U/mL penicillin G-sodium and 100 mg/mL streptomycin sulfate (Sigma) and
20% CM prepared as described previously from normal donors' PBMC
after stimulation with PHA-L for 72 hours (TCM).37,49 A
bulk stock of TCM was prepared and used for all of the cultures. TCM
contained 96,551 pg/mL of IL-2, 841 pg/mL of IFN, 234 pg/mL of
IL-1 , 60 pg/mL of IL-6, and 0 pg/mL of IL-4 and bFGF. This TCM has
differences as compared with previously reported
TCM.23,37,49 Specifically, it has a higher content of IL-2
and IFN and a lower content of IL-6. The culture medium was changed
every 3 to 6 days. Supernatants from these passages were filtered and
immediately stored at -80°C until cytokine measurements. Some
cultures were also grown in the absence of TCM and the supernatants from these cultures were also tested. At day 3 after culture with or
without TCM, floating cells (containing the lymphoid population) were
harvested and subjected to immunophenotyping by fluorescence-activated cell sorting (FACS) analysis. The adherent cells were maintained in
culture for an additional 4 weeks and then assessed for the presence of
several cell markers by immunohistochemistry.
Cell flow cytometry.
For double and triple fluorescence analysis, 2 × 105 cells (PBMC or TIL) were examined after staining with
the appropriate amounts of monoclonal antibodies (MoAbs) (20 µL of
phycoerythrin [PE], fluorescein isothiocyanate [FITC], or peridinin
chlorophyle protein [per CP]-conjugated) for 30 minutes on ice. After
two washings, cells were resuspended in phosphate-buffered saline (PBS)
and analyzed by flow cytometry (Cytoron, Ortho Diagnostics, Raritan,
NJ) after electronic gating on lymphocytes. The following MoAbs were
used: PE-conjugated CD8, PerCP-conjugated CD4, FITC-conjugated TCR , CD16, CD19, TCR , CD25, LeukoGate (anti-CD14 and
anti-CD45). All MoAbs were purchased from Becton Dickinson
Immunocytometry System, (Mountain View, CA). TIL were
immunophenotyped for cell surface marker analysis immediately after the
tissue mincing (day 0) or after 3 days expansion in TCM.
Immunohistochemistry.
Immunohistochemical analyses were performed on adherent cells obtained
after 4 weeks of culture in TCM. Cells were detached by ice-cold
trypsin-EDTA (GIBCO), as per manufacturer's protocol, resuspended in
RPMI 1640, at a concentration of 2 × 106 and then
cytocentrifuged smears were made. These were incubated with the
following MoAbs: CD68 (Dakopatts, Dako, Golstrup, Denmark), LeuM3
(anti-CD14, Becton-Dickinson), OKT4 (anti-CD4, Ortho), OKT8 (anti-CD8,
Ortho), Leu11 (anti-CD16, Becton-Dickinson), CD19 (anti-B, Dakopatts),
anti-CD45 (Dakopatts), 11C81 (ICAM-1, British Biotechnology), HLA-DR
(Dakopatts), vWF/FVIII-RA (Dakopatts). The sections were then treated
with a biotin-conjugated horse anti-mouse Ig MoAbs and later with
avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA)
and with 0.06% 3,3 diaminobenzidine (Sigma) and 0.03% hydrogen
peroxide.
Cytokine measurement.
Cytokines were measured on supernatants from PHA-stimulated PBMC or
skin cultures by enzyme-linked immunosorbent assay (ELISA) using
manifacturer's protocol (R&D Systems, Minneapolis, MN).
Detection of HHV-8 nucleic acid sequences.
Total DNA was extracted with a Microturbogen DNA extraction kit
(Invitrogen, San Diego, CA) from skin specimens as well as from
cultures derived from KS skin lesions or uninvolved skin cultured for a
period of 4 weeks. Some PBMC samples were also examined. A total of 100 ng of DNA was then amplified for HHV-8 nucleic acid sequences by nested
PCR by using as primers KS4 and KS5 for the first round of
amplification, followed by a second round with the primers KS1 and KS2,
previously described.10 Amplification was performed as
follows: 94°C for 3 minutes (1 cycle); 94°C for 1 minute,
58°C for 1 minute, 72°C for 1 minute (35 cycles); 72°C for
5 minutes (1 cycle). Each PCR reaction was performed with 50 pmol of
each primer, 1 U of Taq polymerase (Perkin-Elmer, Branchburg, NJ), 200 µmol/L of each deoxyribonucleotide triphosphates (dATP, dTTP, dGTP,
dCTP), Perkin-Elmer buffer 1×, 1.5 mmol/L MgCl2, in a
final volume of 50 µL. Amplifications were performed with a
Perkin-Elmer 9600 Thermocycler. PCR products were analyzed by Southern
blot hybridization using the HHV-8 specific probe KS330Bam 32P-end-labeled.10 Hybridization was performed
at 65°C. The positive control was the DNA extracted from a KS skin
biopsy and the negative control was DNA extracted from PBMC previously
known to be negative for HHV-8 DNA sequences.
Statistical analysis.
Analysis of variance was performed by the ANOVA test. A multiple
comparison of variance was performed by the Newman-Keuls test.
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RESULTS |
Clinical features of KS patients.
The main clinical features of patients with C-KS and AIDS-KS, as well
as data from healthy controls are reported in
Table 1. Most of the patients with KS were
men. Patients with C-KS were middle- or late-aged in comparison to
AIDS-KS patients and presented no risk factors for HIV-1 infection. The
majority of AIDS-KS patients were male homosexuals, with occasional
experience of intravenous drug use. Healthy controls had no risk
factors for HIV-1 infection.
Immmunological analysis of PBMC and TIL from KS patients: Local
infiltration of T-cell receptor
(TCR)/+CD8+
and
TCR/+CD4-CD8-
lymphocytes in KS lesions.
The analysis of lymphocyte subpopulations was performed on freshly
isolated PBMC and TIL obtained from biopsies of lesional and uninvolved
skin. No statistically significant difference was found among the study
groups in regard to circulating CD3+, CD16+,
and CD19+ cells (Table 1). As expected, the absolute number
of CD4+ cells was significantly reduced in AIDS-KS patients
as compared with controls (F = 79.11, P < .001) or C-KS
patients (F = 27.02, P < .001). Similarly, no difference was
found between healthy controls and C-KS patients. The absolute number
of CD8+ cells was significantly increased in AIDS-KS
patients as compared with controls (F = 74.47, P < .001) and
to C-KS patients (F = 34.84, P < .001), whereas no
differences were found between normal controls and C-KS. In contrast,
when TIL were phenotyped, immediately after the biopsy and tissue
mincing (day 0), a prevalent infiltration of CD8+ T cells
was found in the majority of KS patients, irrespective of their
seropositivity for HIV-1. The remaining lymphoid population was
represented by CD4+ cells. No or few B cells were found
(Table 2). No significant lymphoid
infiltration was found in uninvolved skin. These findings were
confirmed by immunohistochemical stainings of the lesions and they are
consistent with those reported in by Fiorelli et al.49a
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Table 2.
Immunophenotyping of TIL From KS Lesions and Uninvolved
Tissues at Day 0 and After 3 Days Expansion in TCM
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To expand and further characterize infiltrating lymphoid cells,
biopsies from involved and uninvolved skin from the same patients were
then cultured in the presence or absence of TCM. TCM contain mainly Th1
cytokines that are the same found to be increased in KS
lesions.42,46-49,49a This system allowed the growth of an
adherent cell population with a spindle-shaped morphology, described
later, and of a floating cell population with a lymphoid morphology
(Fig 1). The latter cells, easily removed
by pipetting, were phenotyped after a short-term growth (3 days) in the
presence or absence of TCM. Nearly all of the floating cells were found
to be lymphocytes, as determined by flow cytometry after electronic
gating on lymphocytes and then by the use of a combination of MoAbs to
CD14 (recognizing cells of the monocyte lineage) and to CD45
(recognizing cells of leukocyte origin) that distinguish lymphocytes
from monocytes and debris (Fig 2A). These
cells were mainly of a CD8+ phenotype and were found both
in C-KS and AIDS-KS. CD4+ cells were also detected, but to
a lesser extent. Very few or no B cells (CD19+) were found
(Fig 2B; Table 3). The values of
CD4+ and CD8+ cells were variable in different
lesions, and this was due to the different stages of the lesions
examined, being CD8+ cells detected more easily in early
stage lesions.
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| Fig 1.
Phase photomicrograph of cultured cells from a KS skin
biopsy. Fragments of skin were minced and grown for 4 weeks in culture media containing TCM, as described in Materials and Methods (phase contrast 200×). A double population was obtained: one adherent with a
spindle-shaped morphology and another of floating cells with a lymphoid
morphology.
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| Fig 2.
Immunophenotyping of TIL from a C-KS lesion (nodular
type). Cells were recovered and cultured in the presence of TCM, as
reported in Materials and Methods. Floating cells were phenotyped after 3 days of culture in TCM. (A) Nearly all of the cells (92%) are represented by lymphocytes, as assessed by the LeukoGATE system, which
allows the discrimination between these cells, other mononuclear cells,
and debris. (B) No CD19+ B cells were found in cultured
lesions. Shown is a single immunofluorescence from a representative
case. (C) Triple immunofluorescence from a representative case showing
that the majority of the cells coexpress the / chain of the TCR
as well as the CD4+ (24%), the CD8+(39%),
or both CD4+CD8+ (13%) markers. Notably
24% of TCR/+ cells are
CD4-CD8-. The figure represents the CD4 and CD8
staining on / positive gated cells. (D) Apoptotic figures are
observed by morphologic analysis for orthogonal (y-axis) versus forward
(x-axis) light scatter. A, apoptotic region; B, viable lymphocytes.
{/CAPT;;;left;stack}
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Table 3.
Th1 and Th2 Cytokine Profile in Supernatants From PBMC,
Cultured Lesional, and Uninvolved Skin From C-KS and AIDS-KS Patients and From PBMC of Patients With Dermatologic Diseases Other Than KS
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The majority of the T cells detected were
TCR/+CD8+ or
TCR/+CD4+, although a high percentage
(about one quarter of total TCR /+ cells) of
TCR/+CD8-CD4- cells was also
found by triple immunofluorescence (Fig 2C). Very few cells were
bearing the / chains of the T-cell receptor and, after three days
activation, a small percentage of the CD4+ and
CD8+ cells acquired the IL-2 receptor.
Finally, the FACS analysis showed the presence of a population of
lymphoid cells (up to 60% of total lymphocytes) with morphologic characteristics of apoptotic cells, as shown by an increased orthogonal light scatter and a low forward scatter (Fig 2D), which were prevailing in the CD8+ T-cell fraction (data not shown).
Control cultures set up in the absence of TCM did not show viable
cells, and the characterization of uninvolved skin did not show
appreciable presence of lymphocytes, as assessed by the number of
CD4+ or CD8+ cells (Table 2).
Immunohistochemical analysis of KS-derived spindle cells: Adherent
cells bear markers of the macrophage lineage.
The adherent cells derived from the skin lesions were grown for 4 weeks
in the presence or absence of TCM. These cells acquired a
spindle-shaped morphology (Fig 1) and the large majority of them was
strongly positive for CD68, HLA-DR, and ICAM-1
(Fig 3), but negative for markers of
leukocyte/lymphoid origin, including CD45, CD4, and CD8 (data not
shown). The endothelial cell marker vWF/FVIII-related antigen was low
or undetectable (data not shown). Thus, the growth conditions allowed
the expansion of spindle cells with an immunophenotype of tissue
macrophages. Uninvolved skin did not show the appearance of significant
amounts of these cells.
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| Fig 3.
Immunohistochemical analysis of cultured spindle-like
cells derived from skin lesions. The cells were removed from the flask using a mild trypsinization cytocentrifuged and immunostained with
MoAbs directed against CD68 (A), HLA-DR (B), and ICAM-1 (C) and
counterstained with hematoxylin. The staining pattern for CD68 was
cytoplasmic and diffuse (A), for HLA-DR, it was mainly granular (B),
and for ICAM-1, it was more evident at the cell surface (C). The large
majority of the cultured KS cells were HLA-DR positive. Magnifications
are (A) ×630, (B) ×700, and (C) ×800.
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Cytokine profile: The majority of KS patients preferentially release
IFN on stimulation.
The Th1 (IFN) and Th2 (IL-4) cytokine profile was assessed in
supernatants from both PHA-stimulated PBMC and skin cultures from KS
and uninvolved tissues cultured in the presence or absence of TCM.
Cultures established in the absence of TCM did not show appreciable
levels of any cytokine tested. In contrast, both C-KS and AIDS-KS
patients showed a preferential expression of IFN, in both PBMC and
TCM-cultured skin (Table 3). There was no statistical difference
between C-KS and AIDS-KS patients regarding IFN production (P = .278 for PBMC; P = .968 for cultured skin).
However, AIDS-KS patients showed also the emergence of IL-4-producing
cells in both PBMC after PHA stimulation and in supernatants derived
from cultured skin (Table 3), and this was statistically significant in
comparison to C-KS patients (P = .01 for PBMC; P = .003 for cultured skin). No detectable levels of cytokines were found in cultures from uninvolved skin from the same patients (Table 3). IL-2,
as well as IL-12, were not found in supernatants from skin cultures
from both groups of KS patients (data not shown).
IL-4 and IFN were also tested in supernatants derived from PBMC
obtained from 34 patients with other skin disorders, such as psoriasis
and chronic dermatitis, and cultured in the same TCM used to expand TIL
from KS patients. In these cases, IL-4 was never found, whereas IFN
was found, but at levels much lower than in KS patients (Table 3).
HHV-8 is detected in PBMC, lesions, and in spindle-like macrophagic
cell cultures of lesional skin from KS patients.
Because among all of the infectious agents proposed to play a role in
KS, HHV-8 appears to be the only one consistently associated, the
presence of HHV-8 DNA sequences was searched by nested PCR in our
experimental systems. Viral sequences were consistently detected in
100% (9/9) of the lesions from C-KS patients and in 100% (5/5) of the
lesions from AIDS-KS patients, as well as in 88% (8/9) and 66% (6/9)
of the PBMC from C-KS and AIDS-KS patients, respectively, but not in
the uninvolved skin far from the lesions (Table 4). Some perilesional skin was also
found to be positive. In addition, HHV-8 was detected in 88% (8/9) and
in 85% (6/7) 4-week-old adherent spindle cell cultures with a
macrophage phenotype derived from lesional skin of C-KS and AIDS-KS
patients, respectively and established in the presence of TCM
(Fig 4; Table 4).
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| Fig 4.
Representative Southern Blot analysis of a nested PCR by
using KS1 and KS2 primers to amplify HHV-8 DNA sequences. (A) Lane 1, positive control; lanes 2 to 4 and 6 to 7, lesional skin from C-KS and
AIDS-KS patients, respectively; lane 5, uninvolved skin; lane 8, negative control. (B) Lesional skin cultures from C-KS patients: lane
1, positive control; lane 2, negative control; lanes 3 and 4, cultured
skin from 2 C-KS patients. (C) PBMC: lane 1, positive control; lane 2, negative control; lanes 3 to 6, PBMC from four C-KS patients; lane 4, PBMC from an AIDS-KS patient.
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DISCUSSION |
Experimental observations have demonstrated a role of inflammatory and
angiogenic cytokines in KS development. These cytokines are increased
in KS lesions from both AIDS-KS and C-KS patients and stimulate the
growth of KS cells and the angiogenesis found in
KS.36-44,46-49
We report here on the prevalent production of IFN in PBMC and in
cultures established from lesional skin of both AIDS-KS and C-KS
patients. In lesional skin, IFN production is associated with the
presence of lymphoid T cells. In contrast, uninvolved skin from the
same patients, which did not show a relevant lymphoid infiltration, was
negative for cytokine production.
The immunophenotyping of TIL at day 0 or after 3 days in culture with
TCM showed that the majority of them are of the T-cell lineage, in
particular TCR /+CD8+ cells. These cells
are consistently found in both AIDS-KS and C-KS. In patients with C-KS,
no signs of immunodeficiency or any unbalance of the lymphocyte
subpopulations are found at the PBMC level, thus arguing for a local
recruitment of T cells in lesional tissue. These data are in agreement
with two previous studies53,54 and with the findings of
Fiorelli et al49a indicating that IFN production is high
in KS and it is mostly produced by T-CD8+ cells. In
addition, an unusual subpopulation of T cells, bearing the TCR
/+CD4 CD8
phenotype is evident after TCM-culture of TIL. This T-cell
subpopulation generally represents a very minor (<2%) subset of
normal lymphoid cells that is rarely found expanded in the peripheral
blood.55 These cells have been associated with the
development of autoimmune diseases56,57 and in the
extrathymic maturation and differentiation of
lymphocytes,58 even if their precise role remains to be
determined. Interestingly, a study describing the emergence of this
cell population in a primary immunodeficiency patient, who developed a
graft-versus-host disease, has shown that these cells are able of
producing IFN.59
Altogether, our data suggest the presence of a local immune response in
KS lesions. This resembles that reported in the peripheral blood during
acute viral infections like acute cytomegalovirus infection and
infectious mononucleosis60,61 and, locally, in the
liver.58 In the latter case, the appearance of TCR
/+CD4CD8 and of
TCR /+CD8+ cells, with apoptotic figures
as those observed by us in the skin, have also been found. Th1
cytokines, contained in the TCM used by us to culture TIL, may have
amplified the apoptosis of cells primed to die, like host immune cells
involved in an antiviral response.62 This has already been
described in cultures of lymphocytes from lymphocytic choriomeningitis
virus-infected mice expanded in the presence of IL-2.63
Several infectious agents have been associated with KS; however,
epidemiologic findings indicate that only HHV-8 is stringently associated with the disease.10-19,22 Recently, a molecular
mimicry of human cytokines and cytokines response pathway genes by
HHV-8 has been reported and it has been proposed that HHV-8 may
interfere with the host immune response.64 We have found
HHV-8 in PBMC, KS lesions, and in the short-term (4 weeks) cultures
from KS lesions in the majority of the patients studied. Among
hematopoietic cells, HHV-8 has been previously detected in B
cells22,65 and rarely in T cells.66 However,
more recent evidence indicates that the virus is present in circulating
monocytes (Colombini et al, submitted) and in mononuclear
cells including monocytes/macrophages infiltrating KS
lesions.25,26 In addition, peripheral blood-derived KS-like spindle cells with macrophagic cell markers,23,45 which are also detected in KS lesions,67 were recently found in all
forms of KS and they were found to be infected by HHV-8.24
In comparison to KS-derived spindle cell lines bearing markers of
activated endothelial cells,49 our culture conditions have
preferentially allowed the growth of T-TIL, as well as of adherent
cells bearing markers of tissue macrophages.68 This is
likely due to the higher amount of Th1 cytokines contained in the TCM
used to expand our cultures in comparison to the TCM used to establish
KS cells with an endothelial phenotype cited in other
reports.23,36-43,49 In particular, our TCM is richer in
IL-2, and this may account for the stimulation of T-CD8+
cells to produce IFN, which is a powerful activator of
macrophages.69 Moreover, activation of the T cells may have
induced growth of the adherent cell layer, as also reported in
peripheral blood in a similar system, aimed at the long-term culture of
human T lymphocytes.70
Our results, therefore, suggest that macrophages present in the
adherent layer of our cultures carry HHV-8. In fact, although endothelial and spindle cells of the lesions are latently infected by
HHV-8, they lose the virus on culture in the presence or absence of TCM
(B.E., unpublished data, June 1995). Because KS lesions contain few or
no B cells, but are rich in monocytes macrophages49a that
are productively infected with HHV-8,25,26 it is likely that this cell type recruits the virus into tissues. In this context, the presence of TIL and their production of IFN may represent a
physiologic response to HHV-8, as found for other viral infections, including Epstein-Barr virus, a closely associated herpesvirus. In
addition, the possibility that HHV-8 encoded chemokines, like viral
macrophage inflammatory proteins-I and -II,64 may be
chemoattractant for lymphoid cells cannot be ruled out.
Production of IFN by infiltrating cells may play a pivotal role in
KS pathogenesis as described in the accompanying manuscript by Fiorelli
et al.49a This cytokine is able to induce normal endothelial cells to acquire a spindle morphology and to express markers similar to those of KS spindle cells. Moreover, among the
cytokines detectable in TCM and in KS lesions, only IFN is capable
of inducing alone the release of biologically relevant amounts of
bFGF,71,72 promoting the development of KS-like lesions in
nude mice (see Fiorelli et al49a). Finally, this cytokine increases the expression of the CD40 antigen on endothelial and spindle
cells of KS and this may amplify the tissue inflammation.73 It must be underlined that the majority of the adherent cells growing
in our skin cultures stained positive for HLA-DR, that is a biological
marker of IFN activity similar to in situ findings presented in the
report by Fiorelli et al.49a Finally, it has been
demonstrated that IFN-treated endothelial cells acquire HLA-DR
expression, favor proliferation of CD8+ cells that secrete
Th1 cytokines, and may acquire antigen-presenting function.74
Our observations should be taken into account when designing clinical
trials aimed at treating KS patients, especially if concomitantly
affected by AIDS, where a shift to a Th1 response is often desired. In
KS patients, the recruitment of lymphoid cells and the release of
IFN may represent a physiologic response to a foreign antigen, such
as HHV-8. However, the hyperactivation of these cells may result in
disease progression, as already described in an HIV-1 seropositive
patient, who developed KS and visceral Leishmaniasis. The
administration of IFN together with anti-Leishmanial therapy, in
fact, led to KS progression.75 Similarly, treatment of KS
patients with IL-2 and IFN induced progression of KS.76
| |
FOOTNOTES |
Submitted May 20, 1997;
accepted September 26, 1997.
Supported by grants from the IX AIDS Project from the Italian Ministry
of Health (Rome), Associazione Italiana per la Ricerca sul Cancro
(AIRC, Milan), and Ministero Università Ricerca Scientifica (MURST, Rome).
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.
| |
ACKNOWLEDGMENT |
We thank Dr G. Barillari (Department of Experimental Medicine,
University of "Tor Vergata," Rome, Italy) and Dr Paolo Monini (Laboratory of Virology, Isituto Superiore di Sanità, Rome,
Italy) for helpful discussion and A. Lippa for editorial assistance.
| |
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Inflammatory Cytokines Synergize with the HIV-1 Tat Protein to Promote Angiogenesis and Kaposi's Sarcoma Via Induction of Basic Fibroblast Growth Factor and the {alpha}v{beta}3 Integrin
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IFN-{gamma} Induces Endothelial Cells to Proliferate and to Invade the Extracellular Matrix in Response to the HIV-1 Tat Protein: Implications for AIDS-Kaposi's Sarcoma Pathogenesis
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Abstract
Introduction
Abstract