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Identification and Culture of Kaposi's Sarcoma-Like Spindle Cells
From the Peripheral Blood of Human Immunodeficiency
Virus-l -Infected Individuals and Normal Controls
By Philip J. Browning, Joan M.G. Sechler, Mark Kaplan, Robyn H. Washington, Rita Gendelman,
Robert Yarchoan, Barbara Ensoli, and Robert C. Gallo
We examined 26 patients with human immunodeficiency
virus-l (HIV-l)-associated Kaposi's sarcoma IKS), and 76
HIV-l-infected (HIV-I+) people without KS or uninfected
(HIV-I-) controls for the
presence of circulating KS-like spindle cells. Adherent cells that had spindle morphology and
several characteristics of spindle cells of KS lesions (KS cells)
were identifiedin the peripheral blood mononuclear cell fraction only after culture in the presence of conditioned medium (CM) from activatedlymphocytes.
The peripheral
blood-derived spindle cells(PBsc) expressed a variety of endothelial cellmarkers, such as UIex europaeus I lectin, EN4,
EN2/3, EN7144, CD13, CD34 CD36, CD54, ELAM-l, and HLADR. However, they were negative for CD2, CD19, PalE, and
factor VIII-related antigen. The PBsc produced angiogenic
factors as evidenced by the ability
of CM from these cells t o
promote growth of normal
vascular endothelial cells. In
addition, subcutaneously injected PBsc stimulated angiogenesis in vivo in athymic nude mice. We determined that
the number ofPBsc grown from the
peripheral blood of HIVIfpatients with KS or at high risk t o develop KS were increased by 78-fold ( P = .OOOl) and 18-fold ( P = .005), respectively, when compared with HIV-1- controls. The number of
spindle cells cultured from the HIV-1' patients at low risk
for developing KS, eg, HIV-I+ injection drugusers, showed
no statistical increase when compared with HIV-1- controls.
The presence of increased PBsc with characteristics of KS
cells in HN-1' KS patients or patients at highrisk for developing KS gives insights into the originof KS cells and may
explain the multifocal nature
of the disease. In addition, this
may be useful in predicting therisk of KS development.
0 1994 by The American Societyof Hematology.
UMAN IMMUNODEFICIENCY virus type 1' (HIV1)-associated Kaposi's sarcoma (KS)'" is a multifocal proliferative disorder characterized by proliferating spindle-shaped cells (KS-spindle cells) of probable vascular endothelial cell origin and angiogenesis. KS is the most
common tumor found in patients with the acquired immunodeficiency syndrome (AIDS).5.6HIV- l-infected patients at
high risk for developing KS are more likely to be homosexual or bisexual men or female sexual partners of bisexual
men, rather than HIV- 1 -infected heterosexual men, women,
injection drug users (IDU), and children.' It is unknown why
AIDS-associated KS is a multifocal disease. Possibilities include that KS lesions develop de novo, a finding compatible
with histologic evidence that uninvolved skin in KS patients
has angioproliferative changes'"'; or that multifocal KS lesions are the result of blood stream metastasis, although
there is no evidence to support this hypothesis.
Our laboratory established an in vitro system for growing
spindle cells of KS lesions by culturing the cells with conditioned medium (CM) from activated T cells.",'* These
CM".'3 contain the same inflammatory cytokines increased
in HIV-1 -infected individuals, particularly homosexual
Further studies in our laboratory suggested that KS
is a cytokine-mediated disease, at least in early stages, and
that inflammatory and angiogenic ~ytokines"~"~'~
and the
HIV-1 Tat p r ~ t e i n ' ~ . 'may
~ . ' ~cooperate in its induction and
progression in HIV-1 -infected susceptible individuals.
Using this system, we tested the hypothesis that the KSspindle cell or cells closely related to them circulate in the
peripheral blood. Our objectives were (1) to determine
whether spindle cells could be cultured from the peripheral
blood using activated lymphocyte CM; ( 2 ) to determine the
phenotypic and functional characteristics of the peripheral
blood-derived spindle cells (PBsc); and (3) to determine
whether the PBsc are increased in HIV-1 -infected patients
with KS and in those HIV-l-infected patients who are at
high risk to develop KS. In this study, we report the establishment of an in vitro system for culturing and characteriz-
ing circulating cells (PBsc) that closely resemble KS-spindle
cells.
H
Blood, Vol 84, No 8 (October 15), 1994 pp 2711-2720
MATERIALS AND METHODS
Patients. PB samples were obtained from 76 HIV-l-infected
patients with and without KS who had been observed in the National
Institutes of Health (NIH) Clinical Center (Bethesda, MD) and North
Shore University Hospital (Manhasset, NY), and from 26 HIV-1infected NIH Clinical Center Blood Bank volunteers. The clinical
data regarding the patients and normal volunteers studied are described in Table l.
Preparation of CM from mitogen activated peripheral blood lymphocytes. PB mononuclear cells (PBMC) from normal HIV-1uninfected volunteers were isolated using lymphocyte separation
medium (Organon Teknika Corp, Durham, NC), as per the manufacturer's protocol. This separation medium contains 6.2 g of ficoll and
9.4 g of sodium diatrizoate per 100 mL. Using this medium, the
granulocytes and red blood cells are separated from the mononuclear
cells, eg, lymphocytes, monocytes, dendritic cells, etc, by density
centrifugation. Activated-lymphocyte CM was prepared by culturing
the PBMC in RPM1 1640 containing 10%fetal bovine serum (FBS),
and 2 ,ugh" of Bacto-phytohemagglutinin P (DIFCO Laboratories,
Detroit, MI) at 2 X lo6 cells/mL. After 24 hours, cells were spun
down and cultured at 1 X lo6 cellslmL in RPM1 1640, containing
FromThe Laboratory of Tumor Cell Biology and Medicine
Branch, National Cancer Institute, National Institutes of Health,
Bethesda, MD; and The Department of Internal Medicine, Division
of Infectious Diseases and Immunology, North Shore University Hospital, Manhasset, NY.
Submitted August 16, 1993; accepted June 22, 1994.
Address reprint requests to Robert C. Gallo, MD, Laboratory of
Tumor Cell Biology, NCI, NlH, Bldg. 37, Room 6A09.9000 Rockville
Pike, Bethesda. MD 20892.
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.
0 1994 by The American Society of Hematology.
0006-4971/94/8408-0001$3.00/0
271 1
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BROWNING ET AL
271
Table 1. Clinical Characteristics of HIV-l-Infected Patients and Normal Controls
Characteristic
HIV-1'KS
n
Sex (Mff)
Age (yr)
Mean
Range
Homosexual, no IDU
Homosexual, IDU
Heterosexual, no IDU (M/F)
Heterosexual, IDU (Mff)
WBC (1.000 cells/pL)
ALC (cells/pL)
AMC (cells/pL)
CD4 (cellshL)
HIV-l-KS-
PValue'
P Valuet
HIV-1'KS'
26
25/1
26
15/11
50
38/12
39.5
24-64
NA
NA
NA
NA
4.9 2 1.2
1.447 2 507
286 -t 115
ND
35.3
19-51
27
0
14 (4/10)
9 (7/2)
3.3 2,139
1.0
1,112 2 552
300 2 147
395 -C 264
39.6
28-49
24
0
1
1 (0/1)
.074 4.2 ? 2.0
.029
878 2 1,050
.23 389 2 248
171 2 179
.346
.23
.005
ND
Abbreviations: NA, not available; WBC, white blood cell count; ALC, absolute lymphocyte count; AMC, absolute monocyte count; ND, not
determined; IDU, injection drug user.
* HIV-1'KS' v HIV-1'KS- patients, two-tailed Student's t-test.
t HIV-l+-KS+v HIV-1-KS- controls, two-tailed Student's t-test.
10% FBS, and 20 U/mL interleukin-2 (Boehringer Mannheim, Indianapolis, IN). After 72 hours, the C M was collected, filtered through
a 0.45-pm filter (Coming, Coming, NY), aliquoted, and stored at
-20°C. Before use in these studies, the C M was tested for its ability
to stimulate the in vitro growtho f AIDS-KS-derived cells. Activated
lymphocyte C M that stimulated a threefold increase o f the AIDSKS cell number after 7 days was used in this study.
Cell isolation and culrure of PBsc. PBMC were isolated as described above and then cultured atan initial density o f 1.5 X IO5
cells/mL in 25-cm2 culture flasks (Falcon, Franklin Lakes, NJ) that
Fig 1. Phase photomicrograph ofPB cultures. HIV-l-infected patient withoutKS (A) and with KS (B). The PBMC were isolatedas described
in the Materials andMethods, grown for10 days in culture media containing 2096 activated lymphocyteCM. The flasks containing cells were
photographed using a Zeiss phase contrast microscope. Bar = 100 pm.
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PERIPHERALBLOODSPINDLECELLS
IN AIDS-KS
had been precoated with 1.5% gelatin (Sigma, St Louis, MO). PBMC
were cultured in RPM1 1640 (GIBCO-BRL, Gaithersburg, MD) containing 15% FBS (GIBCO-BRL), supplemented with 1 0 0 pmoVL
nonessential amino acids (GIBCO-BRL), 10 pmoVL essential amino
acids (GIBCO-BRL), 1 mmoVL sodium pyruvate (GIBCO-BRL),
20% activated-lymphocyte CM, and
molL tetrahydrocortisone
(Sigma). One-half of the culture medium was changed every 3 or 4
days. A confluent monolayer of cells formed after 10 to 14 days of
culture. These cells could be passaged 1:2 using cell dissociation
buffer-low trypsin (GIBCO-BRL), as per the manufacturer’s protocol.
Antibodies and
histochemical
srains.
Monoclonal antibodies
were purchased from the following companies: mouse IgM, rat IgG,
ENW3,’’ and EN7144’’ from Accurate Scientific (Westbury, NY);
CD1,I9 CD34,2”.2’ CD36;’CD54;’
factor VIII-related an tiger^;^.^
4D10,26 cy-smooth muscle actin (clone 1A4).27 and vimentin2’ from
AMAC (Westbrook, ME); mouse IgG,, mouse IgG2, CD2?9 CD4,”’
CD13,”’ CD14,”’ CD19,”’ CD23,3’.” and HLA-DR3’*’6 from
Becton Dickinson (Mountain View, CA); c y t ~ k e r a t i nfrom
~ ~ DAKO
(Glostrup, Denmark); EN43*4 from Catlag (South San Francisco,
CA); and PalE4’.42and Ufex europaeus lectin4’ from Pierce (Rockford, IL).
Fluorescenceanalysis. Primary antibodies were added to live
cells in suspension and incubated at 4°C for 30 minutes. Cells were
thenwashed twice using a solution of phosphate-buffered saline
(PBS; GIBCO-BRL) that contained 0.1% sodium azide (Sigma),
and 0.5% bovine serum albumin (BSA; Sigma). Fluorescent-labeled
secondary antibody was added and the cells were incubated for 30
minutes at 4°C. The cells were washed twice with a PBS solution
2713
containing 0.1% sodium azide and 0.5% BSA, fixed with 1% paraformaldehyde (Sigma), and analyzed on the FACScan (Becton Dickinson). Controls were (1) stained cells known to be negative, (2)
PBsc stained with secondary antibody only, and (3) stained cells
known to be positive. In addition, each experiment contained, as a
negative control, PBsc incubated with a nonspecific mouse or rat
isotype primary antibody. After washing as described above, the
cells were incubated with the conjugated secondary antibody specific
for the primary isotype control antibody. There was no difference
in the shift of the mean fluorescence between the cells treated in
this way compared with unstained cells. The PBsc usedin this
analysis were obtained from HIV-I -infected individuals at high risk
to develop KS (n = 4).
Hisrochemicafanalysis.
Immunocytochemical analysis of the
PBsc was performed on cells that were grown on gelatin-coated
tissue culture slides, either by double-indirect immunoperoxidase
staining or by the alkaline phosphatase-antialkaline phosphatase
(APAAP) method. The slides were fixed in cold acetone or acetone
and chloroform (1: l), washed in tris-buffered saline, and incubated
(1 hour at room temperature or overnight at 4°C) with the primary
antibodies. The secondary antibodies were diluted in PBS-I% BSA
and applied to the slides. Slides were then incubated overnight at
4°C. For the double-indirect immunoperoxidase method, slides were
subsequently incubated for 1 hour at room temperature with a rabbit
antimouse peroxidaseconjugated antibody (1: 100; Dakopatts) and
with a swine antirabbit peroxidase-conjugated antibody (1: 100, Dakopatts) and developed with diaminobenzidine in PBS containing
hydrogen peroxide. For the APAAP method, the secondary antibodies were applied after the APAAP reaction and slides were incubated
Fig 2. Wright-Geimsastain of PBMC from a patient with AIDS-KS. The PBMC were grown as described in the Materials and Methods. After
10 days in culture, the cells were removed from the flask using nonenzymatic cell dissociation buffer, cuttured on gelatin-coated slide flask
for 24 hours, washed with PBS, and then stained with Wright-Giemsa stain.
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2114
BROWNING
AL
Table 2. Phenotypic Comparison ofthe PBsc to the AIDS-KSSpindle Cells
Monoclonal Antibody or
Histochemical Stain
Dendritic
Anti-CDl”
Anti-CD2”
Anti-CD430
Helper
Anti-CD133’
Anti-CD143‘
Anti-CD1932
Anti-CD34”.”
Anti-CD5423
EN2/3”
~~438.40
Proliferating
Vascular
EN7/44“
PalE4’.42
H”DR35.36
Factor VIII-related
0-Napthyl acetate esterase4‘
UEA-1
Low molecular weight ~ytokeratin~~
Vimentin”
rY-Smooth muscle actinz7(1A4)
Cell Reactivity
and Langerhan cells
T lymphocytes
T lymphocytes, monocytes, endothelial cells
Monocytes, granulocytes, high endothelial venules
Monocytes, macrophages, follicular dendritic cells, high
endothelial venules
B lymphocytes
Hematopoietic precursor and endothelial cells
Endothelial cells and many cells upon activation
Proliferating endothelial cells
Vascular endothelial cells
endothelial cells
endothelial cells
Antigen presenting cells, activated B and T lymphocytes
Endothelial cells, megakaryocytes
Monocytes, macrophages, histiocytes, endothelial cells
Endothelial cells
Mesenchymal cells
Fibroblast, smooth muscle, endothelial cells
Smooth muscle, endothelial, and monocytic cells
PBsc
Cells
AIDS-KS-Spindle
-
-
-
+++
+
++
-
-
+
+++
++
+
++
-
+++
+l-
+++
+++
++
+++
++
++
++
++
+
+
-
-
-
++
+++
+++
+++
++
Abbreviations: -, negative; +/-,very weak or nonconsistently detected;
FACS or immunohistochemistry; ND, not done.
+, weak staining; ++, moderate staining; +++, strong staining using
for 1 hour at room temperature. An alkaline phosphate substrate
containing naphthol AS-MX phosphate, fast red TR, and levamisole
was used to visualize the reaction. For both methods, the slides were
contrastained in Mayer’s solution for 1 to 2 minutes, rinsed in water,
and air-dried. The controls used were ( l ) secondary antibody only,
(2) PBS, and (3) known positive tissues and primary cells. The PBsc
used in this analysis were cultured from HIV-I -infected individuals
with KS (n = 3) and HIV-l-infected individuals at high risk to
develop KS (n = 2).
Enzyme cytochemistry. The a-naphthyl esterase staina was performed using the reagents and protocol obtained from Sigma. Monocyte-macrophages were used as positive controls, and peripheral
blood lymphocytes were used as negative controls. Positive staining
could be suggestive of cells derived from a myeloid progenitor cell.
This stain was performed on the PBsc cultured from HIV-I -infected
individuals with KS (n = 6).
Phagocytosis. Phagocytosis was evaluated using boiled opsonized Candida albicans as the target. Adherent and phagocytosed
particles were distinguished using Trypan blue, as previously de~cribed.4~
This assay was performed on the PBsc cultured from HIVl-infected individuals with KS (n = 6).
Preparation of CM from the PBsc. CM from PBsc (SC-CM)
was prepared after culturing the cells for 12 to 14 days. At this time,
cells were washed and media replaced with RPM1 1640 (GIBCO)
containing 1 mmoVL sodium pyruvate (GIBCO) and lo-‘ m o m
tetrahydrocortisone (Sigma). The cells were cultured in this serumfree medium for 24 hours. The SC-CM was collected, centrifuged,
filtered through a 0.22-pm filter unit (Coming), aliquoted, and stored
at -20°C until use.
Proliferation of PBsc with recombinant purified HIV-1 Tat protein. These assays were performed with PBsc cultured from HIV1-infected patients with KS (n = 2) as previously described for the
AIDS-KS-derived cell strains.’3.’6.’7
Culture of human umbilical vein endothelial cells (HUVEC) and
growthassays.
HUVEC were established and cultured as previously described.” For the growth assays, cells (3 X lo3 cells/well)
were plated in gelatin-coated 24-well plates (Falcon and Becton
Dickinson) in RPM1 1640 containing 10% FBS and serial dilutions
of SC-CM. On day 7, the cells were counted using a Coulter Zm
counter (Coulter Corp, Hialeah, FL).
In vivoangiogenesisassays.
PBsc cultured from an HIV- 1 infected patient with KS were trypsinized, suspended in PBS, mixed
1:1 with Matrigel (Becton Dickinson), and injected subcutaneously
into the lower back (left side) of two athymic nude mice (2 X 10’
cells per mouse). As a negative control, the opposite side was injected with an equal volume of Matrigel mixed with PBS (1:l).
After 10 days, mice were killed and the sites of injection were
examined for angiogenesis. Biopsies were fixed in 10% formalin,
mounted in parafin, and then stained with hematoxylin-eosin.
Determination of the percentage of PBsc. After 14 days of culture, an adherent cell monolayer was present in most cultures. The
adherent cells consisted of two populations: oval-shaped cells with
large cytoplasmic to nuclear ratio characteristic of monocyte-macrophages, and spindle-shaped cells with scant cytoplasm. At this time,
the flasks were washed twice with PBS and fixed with 10% neutral
buffered formalin solution (Sigma), and spindle cells were counted
in four random microscopic fields using a Zeiss phase contrast microscope (10 X objective). On the average, 100 to 200 total cells were
counted in each high powered field (hpf). The percentage of PBsc
was then computed according to the following formula: % PBsc =
(no. of spindle cells/hpf)/(total no. of adherent celldhpf) X 100.
Statistical analysis. All statistical analysis was performed using
the two-tailed Student’s t-test.
RESULTS
PBMC were cultured from 50 HIV-1-infected patients
without KS, 26 HIV-1-infected patients with a diagnosis of
KS, and 26 HIV-1 -uninfected controls, using the CM from
activated lymphocytes as described in Materials and Methods. At the time of plating, there were no differences in the
total number of monocyte-macrophages (Table 1 ) within the
groups studied. There also were no differences in the total
number of adherent cells observed within 12hours after
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PERIPHERALBLOODSPINDLECELLS
IN AIDS-KS
2715
c
4
.
:
j
ry
Fig 3. Immunohistochemistry ofPBsc. The PBMC were grown as described. Afterl 0 days in culture, the cells were removed fromthe flask
using nonenzymatic cell di-iation buffer, cultured on gelatin-coated slide flask for
24 hours, washed with PBS, and then processed for
immunohistochemistry as described in the Materials end Methods. (A) UEA-I, (B) vimentin, (C) a-smooth muscle actin, and (D) factor VIIIrelated antigen. The figure is representativeof five experiments.
plating, suggesting equivalent plating efficiencies. However, Phenotypic analyses (Table 2) of the PBsc showed characspindle-like cells appeared after 2 to 3 days of culture. SpinteristicssimilartotheKS-spindlecellsgrownfrom
skin
dle-like cells were not detected if PBMC were cultured in
lesions or pleural effusions ofAIDS-KS patients (AIDSthe absence of activated lymphocyte CM.After10to14
KS-cells)." The PBsc expressed low molecular weight cydays of culture, the adherent cells consistedof two distinct
t~keratin?~
a-smooth muscle actin," vimentin,% the endotheliallectin Ulex europueus I ( U E A - I ) 4 3 (Fig3),and
populations. One population had the typical morphology of
monocyte-macrophages (Fig 1A) and the other ahad
spindle- EN4?'4 UEA-143 does not bind to B andT lymphocytes or
like morphology (Fig 1B). Thesetwo cell phenotypes were
tomonocyte-macrophages,butbindstomostendothelial
also evident after Wright-Giemsa staining and light micros- cells. As with AIDS-KS cells," the PBsc did not express the
copy (Fig 2). The first cell population (Fig 1A) was difficult vascular endothelial cell markers PalE41*42 and factorVIIIto remove from the culture flask using trypsin and, therefore,
related antigen.'**u In addition,thedendriticcellantigen
could not be passaged in culture. On the contrary, the spin- CDl," and T- and B-cell antigens
CD2;' CD4,30 and CD1g3*
dle-shaped cells (Fig1B) could easily be removed by gentle were not expressed by the PBsc (Table 2). These data suggest
trypsinization, grown, and passaged for a limited period of
that the PBsc are of mesenchymal origin, and that they may
be derived from the endothelium.
time always in the presence of activated lymphocyte CM.
Spindle-shaped cells (Fig 1B) were characteristic of the cul- The PBsc were also analyzed by fluorescence-activated
cell sorter (FACS) analysis (Fig 4). The PBsc were positive
tures obtained from HIV-1 -infected patients with KS or
for antigens expressed by proliferating endothelial cells (Tathose HIV-l-infected patients at high risk for developing
KS, such as homosexual and bisexual men.
In contrast, cells ble 2) and recognized by the antibodies EN U3"'and EN
with typical macrophage morphology (Fig 1A) were charac-7/44." TheCD34antigen,recognizedbytheQ-END/lO
teristic of the cultures obtained from HIV-l-infected paantibody,"" and the endothelial leukocyte adhesion moletients at low risk for developing KS, such
cule (ELA"l),& recognized by the antibody 4D10," were
as women, heteroweakly expressed on a small percentage of PBsc (Fig 4).In
sexual men, IDU, or HIV-l-uninfected normal volunteers.
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2716
BROWNING ET AL
301
Q-END
0
“1
0
I
=l
CD62
111
CD23
I
a
W
0
3
z
J
J
w
0
0
461
L
601
I
EN2/3
900
101
102
103
EN7144
104
900
101
102104 103
RELATIVE FLUORESCENCE INTENSITY
these cultures, cells that morphologically resembled monocyte-macrophages did not express these endothelial-specific
antigens (data not shown). In addition, the PBsc strongly
expressed CD13,3LCD23,33*”CD36,” and CD5423(Fig 4),
which are expressed by both endothelial cells and monocytemacrophages. Unfortunately, only four cultures derived from
HIV-l-infected homosexual men without KS yielded sufficient numbers of adherent cells for FACS analysis. FACS
analyses were also performed with uncultured PBMC derived from 40 patients and normal controls; however, positive cells were not identified using these endothelial-specific
markers (data not shown). This finding suggests that the
circulating spindle cell is a rare cell that cannot be detected
in the whole blood, or that this cell type may only arise in
culture through differentiation from another cell.
There were also functional similarities between the PBsc
and the AIDS-KS-spindle cells previously grown in our
laboratory and the KS-spindle cells in vivo. Erythrophagocytosis is a pathologic hallmark of KS-spindle cells in vivo.
The PBsc phagocytosed opsonized candida after stimulation
with y-interferon. Both the PBsc and the AIDS-KS-spindle
cells showed fluoride-sensitive nonspecific esterase activity,
Fig 4. Fluoreacence analysis of cultured PBsc on
day 10 from an HIV-l-infected patient without KS.
The adherentcells were removed from theflask using nonenzymatic d l diuod.tlon buffer. Cell susp e n d with
~~~
greater than 98% viability by Trypen
blue exclusion were uaed
in them anatyam. The experimental details are describedthe
in Materials and
Methods.
proliferated in response to exogenous HIV-l Tat protein13J6.17 (data not shown), secreted factors that promoted
the growth of HUVEC (Fig 5A), and induced angiogenic
lesions in athymic nude mice (Fig 5B). The endothelial cell
growth-promoting activity in the SC-CM may suggest that
this activity is produced by these cells, although this hypothesis was not tested in these studies. These data showed that
the PBsc share both phenotypic and functional properties
with the AIDS-KS-spindle cells previously cultured in our
laboratory.’1~L2~16
To determine whether the PBsc were increased in HIVl-infected individuals, the number of these cells was
determined in 102 individuals, consisting of HIV-1 -infected
patients and HIV-l-uninfected individuals (Fig 6). In the
cultures derived from normal people, 0.7% of the adherent
cells were spindle-shaped and there was no difference based
on age or sex. To the contrary, the percentage of spindle
cells was increased 78-fold in the cultures from HIV-1infected patients with KS, compared with HIV-1-uninfected
normal controls ( P = .OOOl). Similarly, when PBMC from
the 50 HIV-l-infected patients without KS were cultured,
the percentage of spindle cells was 17-fold higher when
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PERIPHERALBLOODSPINDLECELLS
m
IN AIDS-KS
2717
'"p
Spindle Cell Conditioned Medium (%)
Fig 5. Angiogenic activity in vitro and in vivo. PBsc were cultured from an HIV-l-infected patient with KS. (A) CM from the spindle cell
stimulate growth of human umbilical vein endothelial cells (HUVEC). SC-CM was
culture was obtained and used t o test f o r t h e a b i l i i t o
collected as described in the Materials and Methods. HUVEC (3 x lo3 cellslwelll were plated in triplicate and cultured in the presence of
increasing serial dilutions of the SC-CM. After 7 day, each well containing cells was counted. These results are representative of three
experiments. (B) Cultured spindlecells from an HIV-1-infected patient with KS were removedfrom theflask using nonenzymatic cell dissociation buffer. Cell viability was determined using
Trypan blue exclusion. Cells with greater than 98% viability were used in these experiments.
After resuspending the cells in PBS, the cell suspension was mixed with an equal volume of matrigel. PBsc (2 x lo5)were subcutaneously
nulnu mice. The mice werealso subcutaneously injected into the right
flank with an equal volume
injected into the left flank of Balblc athymic
and concentration of
matrigel. After 10 days, the sitesof injection wereexamined histologically forevidence of neoangiogenesis. Angiogenesis
was not observed in the negative control (matrigel alone). Bar = 100 pm. The figure is representative of two experiments.
compared with HIV-l-uninfected normal controls ( P =
.0003). When these data were analyzed based on the HIVl-infected risk groups, we found an18-fold increase of
spindle cells in the cultures from HIV- 1-infected patients
at high risk to develop KS, such as HIV-1-infected homosexual men as compared with HIV-I -uninfected controls ( P
= .005). Overall, the HIV-l-infected patients at low risk
for developing KS, such as heterosexual men, women, and
IDU had little (4-fold) increase in spindle cell numbers compared with the HIV-l-uninfected controls ( P = .27).
DISCUSSION
In this study, we showed that cells closely related to spindle cells from KS lesions of AIDS patients can be cultured
from the peripheral blood using CM from activated lymphocytes. These PBsc possess phenotypic and functional similarities with AIDS-KS-spindle cells in vitro and in vivo (Table
2). Their percentage is increased in the peripheral blood of
HIV- 1-infected patients with KS or those HIV- 1-infected
patients at highrisk to develop KS, eg, homosexual and
bisexual men.
The spindle-shapedmorphologywas
used as thecriteria
type. was different both
for identifyingthePBsc.Thiscell
morphologicallyandphenotypicallyfrom
the other type of
adherent cells normally cultured from the PB, such as monocyte-macrophage and dendritic cells.The number of PBsc was
expressed as a percentage because the number of macrophages
were the same in all patient groups. The PBsc could not be
identified directly in blood or in cultures grown without using
activated lymphocyteCM. In addition, these cells were identified only after culturing, suggesting that the cell that gives rise
to the PBsc is a rare circulating cell even in KS patients.
The PBsc expressed markers of mesenchymal cells, endothelial- and monocyte-macrophage-specific markers,
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2718
BROWNING
ET
AL
To reconcile the duel expression on the PBsc of markers
found on monocyte-macrophages and endothelial cells, one
possible interpretation is that the PBsc are derived from
progenitor cells that can differentiate into the other mesenI
chymal cell types depending on the culture conditions (eg,
the pericyte). Peri~ytes~'.'~
express the intermediate filament
vimentid' and a-smooth muscle actin.52Proposed functions
of pericytes include regulation of permeability, modulation
of endothelial cell growth, and maintenance of vascular integrity.50,51356
Pericytes are progenitors of o ~ t e o b l a s t s , a~ ~ . ~ ~
macrophage-like cell that functions in bone remodeling. In
addition, pericytes have been reported to differentiate into
endothelial cells or smooth muscle cells depending on the
culture conditions.'6 We suggest that the PBsc result from
HIV-1'
HIV-l+
Normals HIV-l*
HIV-l+
the differentiation of a circulating progenitor cell, possibly
Low
Hlgh
NoKS
KS
a pericyte.
(n=27)
(n =50) (n=26)
(n =26)
(n=23)
Compared with healthy donors, the PBsc were increased
by 78-fold in HIV-infected patients with KS, 18-fold in HIVFig 6. Quantification of PBsc. IDU, injection drugUsers; Low, HIVI -infected homosexual men without KS, and fourfold in
l-infected patients with a low risk to develop KS;High, HIV-l-infected patients at high risk to develop KS. The percentage of PBsc
other HIV-1-infected patients without KS, eg, heterosexual
was determined as described in the Materials and Methods.
men, IDU, and women, compared with the HIV-l-uninfected controls. Although the increase in the latter group is
not statistically significant in comparison with the HIV-Iwhereas they were negative for T, B, fibroblast, and epitheuninfected controls, the finding merits closer scrutiny belial antigens. These results indicate that the PBsc are of
cause it may have biologic significance.
mesenchymal origin within the reticuloendothelial lineage.
The incidence of KS is increased in all HIV- 1-infected
Possibilities include monocyte-macrophage cells, dendritic
patients, with the greatest risk found in homosexual men.3-5J7
cells, pericytes, endothelial cells, or a progenitor for these
Through the release of the Tat protein,'6s17 HIV-lis known
cell types.
to promote growth of activated normal vascular endothelial
Dendritic cells have been described aascomponent of early
cells13 and the KS-spindle cells in ~ i t r o . ~ 'Inflammatory
~'~~'~
and late KS lesions.4' In vitro, dendritic cells have a limited
cytokines are increased in HIV-1 -infected individuals and
capacity to proliferate such as the p B ~ c . 4Dendritic
~
cells dethey render endothelial and smooth muscle cells responsive
rived from the PB have been reported to strongly express CD13
and CD36, whereas they weakly express CD14.48,49 However, to the proliferative effects of extracellular Tat.'? This may
dendritic cells have
not been reported to express the endothelial cause a generalized mesenchymal cell hyperplasia in vivo.
Supportive evidence for this in vivo is the recent observation
antigens' EN4,384 ENU3,"and EN7/44I8or the 65-kD metalin HIV- 1-infected children who have an increased incidence
loprotein that binds to UEA-I."3 Because of the coexpression
of leiomyosarcomas as well as KS.58,59
of myeloid, endothelial, and smooth muscle cell antigens,it is
KS appears to be a disease of the reticuloendothelial sysunlikely that PBsc cells are dendritic cells.
Studies elsewhere in our laboratory have led us to
The evidence suggesting a macrophage origin for the PBsc
propose that the pathogenesis of one form of KS, HIV-1includes (1) limited in vitro proliferation; (2) fluoride-sensiassociated KS, involves a combination of chronic overprotive nonspecific esterase enzyme activityu; (3) Fc-dependent
duction of inflammatory cytokines and release of the HIV
phagocytosis; (4)expression of the antigens for CD13,3'
Tat protein driving proliferation of primitive endothelial
CD14,31and CD3622;and (5) elaboration of growth factors
cells, which in turn release angiogenic factors, in particular
that stimulated the in vitro proliferation of endothelial cells.
basic fibroblast growth factor that results in the KS leThe evidence suggesting an endothelial origin for the PBsc
sions~13.1S-17For this reason, we hypothesize that the cells
include (1) fluoride-sensitive nonspecific esterase enzyme
described in this report are likely to be the progenitors of
activity@; (2) expression of the antigens CD34,20.2' CD36,"
the spindle-like cells found in KS lesions.
EN4,38s-40
EN7/44,I8 EN2/3," ELAM-l,& and UEA-143; and
In summary, we show that spindle-like cells are found in
(3) Fc-dependent phagocytic activity. The in vitro growth
the
peripheral blood of healthy HIV-1 -uninfected normal
properties of the PBsc were limited and not typical of large
individuals, and are increased in HIV-l-infected patients
or small vessel endothelial cells, or ofthe AIDS-KS-spindle
with KS and in those HIV-l-infected patients at high risk
cells similarly cultured from AIDS-KS lesions. However, it
for developing KS. It is possible that these observations
is unlikely thatthe PBsc are of monocyte-macrophageorigin,
will also prove useful in predicting KS development and for
although they have similar growth properties in vitro, bemonitoring the efficacy of anti-KS therapy.
cause the PBsc express endothelial-specific antigens that are
not normally expressed by monocyte-macrophages. At the
ACKNOWLEDGMENT
same time, we do not think that these cells are typical endothelial cells because they lack proliferative properties of enWe thank G. Barillari for the Tat assays; F. Samaniego, 2.
Berneman, S. Arya,and M. Klotman for helpful discussions and
dothelial cells.
T
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
PERIPHERAL BLOOD SPINDLE CELLS IN AIDS-KS
critical review of this manuscript; L. Shay for collecting clinical
laboratory data on the patients studied; and L. Anderson for editorial
assistance.
REFERENCES
1. Gallo RC, Salahuddin SZ, Popovic M, Shearer GM, Kaplan
M, Haynes BF, Palker TJ, Redfield R, Oleske J, Safai B, White G,
Foster P, Markham PD: Frequent detection and isolation of cytopathic retroviruses (HTLV-111) from patients with AIDS and at risk
for AIDS. Science 224:500, 1984
2. Gross DJ, Safai B: Kaposi’s sarcomdAIDS cofactors. Int J
Dermatol 28571, 1989
3. Safai B: Pathophysiology and epidemiology of epidemic
Kaposi’s sarcoma. Semin Oncol 14:7, 1987 (erratum 14viii, 1987)
4. Safai B, Johnson KG, Myskowski PL, Koziner B, Yang SY,
Cunningham-Rundles S, Godbold JH, Dupont B: The natural history
of Kaposi’s sarcoma in the acquired immunodeficiency syndrome.
Ann Intern Med 103:744, 1985
5. Rutherford GW, Payne SF, Lemp GF The epidemiology of
AIDS-related Kaposi’s sarcoma in San Francisco. J Acquir Immune
Defic Syndr 3:S4, 1990
6. Farizo KM, Buehler JW, Chamberland ME, Whyte BM, Froelicher ES, Hopkins SG, Reed CM, Mokotoff ED, Cohn DL, Troxler
S, Phelps AF, Berkelman RL: Spectrum of disease in persons with
human immunodeficiency virus infection in the United States. JAMA
267:1798, 1992
7. Beral V, Peterman TA, Berkelman RL, Jaffe HW: Kaposi’s
sarcoma among persons with AIDS: A sexually transmitted infection? Lancet 335: 123, 1990
8. Schwartz JL, Muhlbauer JE, Steigbigel RT: Pre-Kaposi’s sarcoma. J Am Acad Dermatol 11:377, 1984
9. Ruszczak Z, Mayer da SA, Orfanos CE: Angioproliferative
changes in clinically noninvolved, perilesional skin in AIDS-associated Kaposi’s sarcoma. Dermatologica 175:270, 1987
IO. Ruszczak Z, Mayer-Da Silva A, Orfanos CE: Kaposi’s sarcoma in AIDS. Multicentric angioneoplasia in early skin lesions.
Am J Dermatopathol 9:388, 1987
11. Nakamura S, Salahuddin SZ, Biberfeld P, Ensoli B, Markham
PD, Wong-Staal F, Gallo RC: Kaposi’s sarcoma cells: Long-term
culture with growth factor from retrovirus-infected CD4+ T cells.
Science 242426, 1988
12. 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
242:430,1988
13. 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. Implications for AIDS-Kaposi’s sarcoma pathogenesis. J Immunol
149:3727, 1992
14.Fan J, Bass HZ, Fahey JL: Elevated IFN-gamma and decreased IL-2 gene expression are associated with HIV infection. J
Immunol 151:5031, 1993
15. Ensoli B, Nakamura S, Salahuddin SZ, Biberfeld P, Larsson
L, Beaver B, Wong-Staal F, Gallo RC: AIDS-Kaposi’s sarcomaderived cells express cytokines with autocrine and paracrine growth
effects. Science 243:223, 1989
16. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal
F Tat protein of HIV-I stimulates growth of cells derived from
Kaposi’s sarcoma lesions of AIDS patients. Nature 345234, 1990
17. Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman
R, Morgan RA, Wingfield P, Gallo RC: Release, uptake, and effects
of extracellular human immunodeficiency virus type 1 Tat protein
on cell growth and viral transactivation. J Virol 67277, 1993
2719
18. Hagemeier HH, Vollmer E, Goerdt S, Schulze-Osthoff K,
Sorg C: A monoclonal antibody reacting with endothelial cells of
budding vessels in tumors and inflammatory tissues, and non-reacrive with normal adult tissues. Int J Cancer 38:48I, 1986
19. Martin LH, Calabi F, Milstein C: Isolation of CD1 genes: A
family of major histocompatibility complex-related differentiation
antigens. Proc Natl Acad Sci USA 83:9154, 1986
20. Anthony PP, Ramani P: Endothelial markers in malignant
vascular tumours of the liver: Superiority of QB-END/lO over von
Willebrand factor andUlex europaeus agglutinin 1. J Clin Pathol
44:29, 1991
21. Schlingemann RO, Rietveld FJ, de Waal RM, BradleyNJ,
Skene AI, Davies AJ, Greaves MF, Denekamp J, Ruiter DJ: Leukocyte antigen CD34 is expressed by a subset of cultured endothelial
cells and on endothelial abluminal microprocesses in the tumor
stroma. Lab Invest 62:690, 1990
22. Greenwalt DE, Lipsky RH, Ockenhouse CF, Ikeda H, Tandon
NN, Jamieson GA: Membrane glycoprotein CD36: A review of its
roles in adherence, signal transduction, and transfusion medicine.
Blood 80: 1105, 1992
23. Makgoba MW, Sanders ME, Ginther Luce GE, Dustin ML,
Springer TA, Clark EA, Mannoni P, Shaw S: ICAM-l a ligand for
LFA-l-dependent adhesion ofB, T andmyeloid cells. Nature
331236, 1988
24. Wagner DD, Marder VJ: Biosynthesis of von Willebrand protein by human endothelial cells: Processing steps and their intracellular localization. J Cell Biol 99:2123, 1984
25. Wagner DD, Olmsted JB, Marder VJ: Immunolocalization of
von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol 95:355, 1982
26. Goerdt S, Zwadlo G, Schlegel R, Hagemeier HH, Sorg C:
Characterization and expression kinetics of an endothelial cell activation antigen present in vivo only in acute inflammatory tissues.
Exp Cell Biol 55:117, 1987
27. Gabbiani G, Schmid E, Winter S, Chaponnier C, de Ckhastonay C , Vandekerckhove J, Weber K, Franke W: Vascular smooth
muscle cells differ from other smooth muscle cells: predominance
of vimentin filaments and a specific alpha-type actin. Proc Natl Acad
Sci USA 78:298, 1981
28. Osborn M, Debus E, Weber K: Monoclonal antibodies specific for vimentin. Eur J Cell Biol 34:137, 1984
29. Howard FD, Ledbetter JA, Wong J, Bieber CP, Stinson EB,
Herzenberg LA: A human T lymphocyte differentiation marker definedbymonoclonal
antibodies that block E-rosette formation. J
Immunol 126:2117, 1981
30. Engleman EG, Benike CJ, Glickman E, Evans R L Antibodies
to membrane structures that distinguish suppressorkytotoxic and
helper T lymphocyte subpopulations block themixed leukocyte reaction in man. J Exp Med 154:193, 1981
31. Griffin JD, Ritz J, Nadler LM, Schlossman S F Expression
of myeloid differentiation antigens on normal and malignant myeloid
cells. J Clin Invest 68:932, 1981
32. Nadler LM, Ritz J, Griffin JD, Todd RF, Reinherz EL,
Schlossman SF: Diagnosis and treatment of human leukemias and
lymphomas utilizing monoclonal antibodies. Prog Hematol 12:187,
1981
33. Kikutani H, Inui S, Sato R, Barsumian EL, Owaki H, Yamasaki K, Kaisho T, Uchibayashi N, Hardy RR, Hirano T, Tsunasawa
S, Sakiyama F, Suemura M, Kishimoto T: Molecular structure of
human lymphocyte receptor for immunoglobulin E. Cell 47:657,
1986
34. Kikutani H, Suemura M, Owaki H, Nakamura H, Sat0 R,
Yamasaki K, Barsumian EL, Hardy RR, Kishimoto T: Fc epsilon
receptor, a specific differentiation marker transiently expressed on
mature B cells before isotype switching. J Exp Med 164:1455, 1986
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2720
35.Brodsky FM, Parham P, Barnstable CJ, Crumpton MJ,
Bodmer WF: Monoclonal antibodies for analysis of the HLA system.
Immunol Rev 47:3, 1979
36. Charron DJ, McDevitt HO: Analysis of HLA-D region-associated molecules with monoclonal antibody. Proc Natl Acad Sci
USA 76:6567, 1979
37. Moll R, Franke W, Schiller DL, Geiger B, Krepler R: The
catalog of human cytokeratins: Patterns of expression in normal
epithelia, tumors and cultured cells. Cell 31:11, 1982
38. Holden CA, Spaull J, Williams R, Spry CJ, Jones RR, Jones
EW: The detection of endothelial cell antigens in cutaneous tissue
using methacarn and periodate lysine paraformaldehyde fixation. J
Immunol Methods 91:45, 1986
39. Cui YC, Tai PC, Gatter KC, Mason DY, Spry CJ: A vascular
endothelial cell antigen with restricted distribution in human foetal,
adult and malignant tissues. Immunology 49:183, 1983
40. Jones RR, Spaull J, Spry C, Jones EW: Histogenesis of
Kaposi’s sarcoma in patients withand without acquired immune
deficiency syndrome (AIDS). J Clin Path01 39:742, 1986
41. Nadimi H, Saatee S , Armin A, Toto PD: Expression of endothelial cell markers PAL-E and EN-4 and Ia-antigens in Kaposi’s
sarcoma. J Oral Path01 17:416, 1988
42. Schlingemann RO, Dingjan GM, Emeis JJ, Blok J, Warnaar
SO, Ruiter DJ: Monoclonal antibody PAL-E specific for endothelium. Lab Invest 52:71, 1985
43. Miettinen M, Holthofer H, Lehto VP, Miettinen A, Virtanen
I: Ulex europaeus I lectin as a marker for tumors derived from
endothelial cells. Am J Clin Path01 79:32, 1983
44. Yam LT, Li CY, Crosby WH: Cytochemical identification of
monocytes and granulocytes. Am J Clin Path01 55:283, 1971
45. Sechler JM, Warren MK, Gallin JI: Human non-transformed
monocyte-derived macrophage cell lines. J Immunol Methods
119:277, 1989
46. Bevilacqua M P , Stengelin S , Gimbrone MA Jr, Seed B: Endothelial leukocyte adhesion molecule 1: An inducible receptor for
neutrophils related to complement regulatory proteins and lectins.
Science 243:1160, 1989
47. Regezi JA, MacPhail LA, Daniels TE, DeSouza YG,
BROWNING ET AL
Greenspan JS, Greenspan D: Human immunodeficiency virus-associated oral Kaposi’s sarcoma. A heterogenous cell population dominated by spindle-shaped endothelial cells. Am J Pathol 143:240,
1993
48. Thomas R, Davis LS, Lipsky PE: Isolation and characterization of human peripheral blood dendritic cells. J Immunol 150:821,
1993
49. Buckley PJ: Phenotypic subpopulations of macrophages and
dendritic cells in human spleen. Scanning Microsc 5:147, 1991
50. D’Amore PA: Capillary growth: a two-cell system. Semin
Cancer Biol 3:49, 1992
5 1. Laaf H, Vandscheidt W, Weiss JM, Schaefer HE, Schoepf E:
Immunohistochemical investigation of pericytes in chronic venous
insufficiency. J Vasc Dis 20:323, 1991
52. Nehls V, Drenckhahn D: Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin. J Cell Biol l13:147, 1991
53. Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR,
Woodbury RA: The pericyte as a possible osteoblast progenitor cell.
Clin Orthop 275:287, 1992
54. Dim-Flores L, Gutierrez R, Lopez-Alonso A, Gonzalez R,
Varela H: Pericytes as a supplementary source of osteoblasts in
periosteal osteogenesis. Clin Orthop 275280, 1992
55. Frey A, Meckelein B, Weiler-Guttler H, Mockel B, Flach R,
Gassen HG: Pericytes of the brain microvasculature express gammaglutamyl transpeptidase. Eur J Biochem 202:421, 1991
56. Madri JA, Bell L, Marx M, Merwin JR, Basson C, Prinz C:
Effects of soluble factors and extracellular matrix components on
vascular cell behavior in vitro and invivo: Models of de-endothelialization and repair. J Cell Biochem 45:123, 1991
57. Peterman TA, Jaffe HW, Beral V: Epidemiologic clues to the
etiology of Kaposi’s sarcoma. AIDS 7:605, 1993
58. Orlow SJ, Kamino H, Lawrence RL: Multiple subcutaneous
leiomyosarcomas in an adolescent with AIDS. AmJ Pediatr Hematol
Oncol 14:265, 1992
59. DiCarIo FJ Jr, Joshi VV, Oleske JM, Connor EM: Neoplastic
diseases in children with acquired immunodeficiency syndrome.
Prog AIDS Pathol 2:163, 1990
60. Dayan AD, Lewis PD: Origin of Kaposi’s sarcoma from the
reticuloendothelial system. Nature 213:889, 1967
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1994 84: 2711-2720
Identification and culture of Kaposi's sarcoma-like spindle cells from
the peripheral blood of human immunodeficiency virus-1-infected
individuals and normal controls [see comments]
PJ Browning, JM Sechler, M Kaplan, RH Washington, R Gendelman, R Yarchoan, B Ensoli and
RC Gallo
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