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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1747-1754
Human Immunodeficiency Virus-1 (HIV-1)-Tat Protein Promotes Migration
of Acquired Immunodeficiency Syndrome-Related Lymphoma Cells and
Enhances Their Adhesion to Endothelial Cells
By
Renato G.S. Chirivi,
Giulia Taraboletti,
Maria Rosa Bani,
Luca Barra,
Giampiero Piccinini,
Mauro Giacca,
Federico Bussolino, and
Raffaella Giavazzi
From Laboratory of the Biology and Treatment of Metastasis and
Laboratory of Cellular and Molecular Biology of the Immune Response and
Auto Immunity, Mario Negri Institute for Pharmacological Research,
Bergamo, Italy; International Centre for Genetic Engineering and
Biotechnology (ICGEB), Trieste, Italy; and Institute for Cancer
Research and Treatment (I.R.C.C.), School of Medicine, University of
Turin, Candiolo, Italy.
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ABSTRACT |
Human immunodeficiency virus-1 (HIV-1)-Tat, the transactivating gene
product of HIV-1, has been shown to interact with different cell types,
inducing gene expression, altering their growth and migratory behavior.
In this study we examined whether Tat might affect functions of
acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's
lymphoma (NHL), relevant to the in vivo dissemination. Our results show
that Tat significantly augmented the motility of the two AIDS-related
Burkitt's lymphoma cell lines (AS283 and PA682PB) and AIDS-primary
effusion lymphoma cell line (HBL-6-AIDS-PEL). Mutations in RGD or basic
domain of Tat (KGE-MBP and LxI-MBP, respectively) sharply reduced
migration compared with wild type, suggesting that both domains are
required for migration. In contrast, a Tat protein
mutation outside the active domains (NH2-TAT-GST) did not
reduce lymphoma cell migration. The treatment of lymphoma cells with
Tat did not influence their adhesion to matrix proteins or to human
vascular endothelial cells, but endothelial cells treated with Tat
became more adhesive to lymphoma cells. Flow cytometric analysis showed
that treatment of endothelial cells with Tat induced the cell surface
expression of the adhesion molecules vascular cell adhesion molecule-1
(VCAM-1) and E-selectin and increased the expression of intercellular
adhesion molecule-1 (ICAM-1). Only antibodies against
VCAM-1 on endothelial cells or against the VLA-4 integrin expressed on
AS283 cells inhibited the increment of adhesion, indicating the
relevance of this pathway in the adhesion of lymphoma cells to vascular
endothelium. In our work, we show for the first time that Tat can
enhance the migration of lymphoma cells and their adhesion to
endothelial cells, two processes that may contribute to the malignant
behavior of NHL in patients with AIDS.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NON-HODGKIN'S LYMPHOMA (NHL) represents
one of the most frequent complications in acquired immunodeficiency
syndrome (AIDS).1,2 The majority of
AIDS-related NHLs are diffused aggressive B-cell lymphomas with
extranodal involvement of the central nervous system in
particular.1,3 Their histology includes four categories,
namely small noncleaved-cell (including Burkitt's lymphoma),
large-cell immunoblastic plasmocytoid, anaplastic-large cell, and
AIDS-related body-cavity-based lymphoma.1,4 Small noncleaved-cell lymphomas are characterized by activating mutations of
c-myc, frequent alterations of p53, and occasional
Epstein-Barr virus infection.5,6 Large cell subtype
lymphomas are strictly associated with Epstein-Barr virus infection and
with rearrangements of c-myc and BCL-6.5,7
Finally, AIDS-related body-cavity-based lymphoma is consistently
associated with the coinfection of human herpesvirus type-8 and
Epstein-Barr virus,8 whereas the anaplastic subtype has not
been studied extensively. A feature common to the four AIDS-NHL
subtypes is the presence of mutations in the noncoding region of
BCL-6 in the proximity of the gene promoter.9
The mechanism by which AIDS-NHL cells home into the central nervous
system is largely unknown and could involve signals from endothelial
cells of blood-brain barrier infected by human immunodeficiency virus-1
(HIV-1)10,11 or specific chemoattractants for lymphoma cells. Microvascular endothelial cells originated from the brain or the
bone marrow and infected by HIV-1 support the growth and the adhesion
of neoplastic B cells through the surface expression of CD40 that binds
CD40 ligand present on lymphoma cells.12 Tat is a potent
inducer of the expression of endothelial cell adhesion molecules,
including vascular cell adhesion molecule-1 (VCAM-1)13
that is involved in the migration of B cells to and within the germinal
center of lymphonodes.14
Tat is a viral protein that transactivates viral genes and increases
the replication rate of virion.15 Tat is also able to
regulate the expression of host genes, including tumor necrosis factors
 and  , interleukin-2 (IL-2), IL-6, major histocompatibility complex of class I, IL-2 receptor, p53 tumor suppressor gene, c-fos, and superoxide dismutase in T cells.16-22
Tat can also be released by infected cells, regardless of whether these
cells are alive or not,23,24 and extracellular Tat acts as
a cytokine-like molecule with a broad range of activities on
mesenchymal cells.13,25-33 One of the most striking effects
of Tat on vascular endothelial cells is the induction of functions
related to angiogenesis and inflammation, including migration,
proliferation, and expression of adhesion
molecules.13,31,34-36
Furthermore, it has been recently shown that Tat activates the
migration and the tissue invasion of monocytes.27,29
Finally, Huang et al have reported that Tat regulates the expression of Fas in B cells.32
In light of the fact that the infection is a key event in the
progression of AIDS-NHLs12 and the viral product Tat can
influence the functions of B cells32 and vascular
endothelial cells,13,31,34-36 we have investigated whether
Tat regulates the homing mechanisms of AIDS-NHL cells.
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MATERIALS AND METHODS |
Cells.
The AS283 and PA682PB AIDS-related Burkitt's lymphoma and KD488
Burkitt's lymphoma cell lines were obtained through the courtesy of Dr
I.T. Magrath (National Cancer Institute, Bethesda, MD) and are
described by Kiwanuka et al.37 The Namalwa and
BJAB Burkitt's lymphoma, the diffuse histocytic
lymphoma-4 (DHL-4) follicular lymphoma, and the Capo Epstein-Barr virus
(EBV)-transformed lymphoblastoid cell lines were all obtained from Dr
J. Golay (Mario Negri Institute, Milan, Italy). We also
used the HBL-6-AIDS-primary effusion lymphoma cell line
(HBL-6-AIDS-PEL) obtained from Dr G. Gaidano (University of Piemonte
Orientale "A. Avogadro", Turin, Italy). All these lines were maintained in suspension in RPMI 1640 supplemented with 2 mmol/L glutamine and 10% fetal calf serum (FCS) 3T3-NIH mouse
fibroblasts (American Type Cell Collection, Bethesda, MD) and human
vascular smooth muscle cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 2 mmol/L glutamine and 10% FCS.
Endothelial cells (EC) were isolated from human umbilical vein as
previously described38 and maintained in medium 199 supplemented with 10% FCS, 10% newborn calf serum, 50 µg/mL
endothelial growth supplement (crude extract from bovine brain), 100 µg/mL heparin, and 20 mmol/L HEPES. All culture reagents were from
GIBCO (Paisley, Scotland).
Preparation and biological activity of HIV-1-Tat molecules.
The 358-bp Pst I-BamHI fragment of Tat gene
containing exon I and II was kindly donated by Dr A. Caputo (Institute
of Microbiology, University of Ferrara, Ferrara, Italy) and has been
subcloned in the pMAL-c2 vector (New England Biolabs, Beverly, MA) and
then expressed in Escherichia coli according to the
manufacturer's instructions. Tat fused to the maltose binding protein
(Tat-MBP) was purified by affinity chromatography on amylose resin and
used as fusion proteins. The purified protein gave a unique band
detected by silver staining (Gelcode; Pierce Chemical Co, Rockford, IL) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); 10%) and was able to induce transcriptional activation of
HIV-1 long terminal repeat (LTR) in HL3T1 cells that contain the
bacterial gene of chloramphenicol acetyltransferase (CAT) directed by
HIV-1 LTR.39 Tat-MBP dose dependently stimulated CAT
expression. Briefly, in prewarmed phosphate-buffered saline (PBS), our Tat-MBP preparation was added to confluent
HL3T1 cells in a 100-mm diameter dish. Cells were immediately scraped
from the plastic surface, then resuspended in fresh medium and
centrifuged. The cells were again plated in CO2 incubator,
and a CAT assay was performed after 6 hours according to Ausubel et
al.40 In our experimental condition, just scraping of the
cells in absence of Tat-MBP had no effect on LTR-directed CAT gene
expression (125 ± 56 [3H]acetylated
chloramphenicol, n = 3). However, CAT gene expression was markedly
elevated in cells that received Tat-MBP (0.5 µg/mL: 423 ± 86
[3H]acetylated chloramphenicol; 1 µg/mL:
2,345 ± 167 [3H]acetylated chloramphenicol; 5 µg/mL: 6,543 ± 567 [3H]acetylated chloramphenicol).
Tat-MBP was also active in terms of the migration of Kaposi's sarcoma
cells, human vascular endothelial cells, and human
monocytes.27,31,41 For mutagenesis, cDNA Tat was subcloned
in the pALTER-Ex1 vector (Promega, Madison, WI). Side-direct
mutagenesis was performed with the altered sites mutagenesis kit
(Promega) by using mutant oligonucleotides (synthesized by TibMol-Biol,
Genova, Italy) to introduce specific mutations (KGE-MBP
[Tat-R78K/D80E]: 5' CCC GAG GGG AAC CGA CAG GCC 3' and 5' ACC TCC CAA
TCC AAA GGG GAA CCG AC 3'; LXI-MBP [Tat-G49/I50]: 5' CTC CTA TGG CGG
GAT CAA GCG GAG ACA 3' and 5' CGG GAT CAA GCT AAT ACA GCG ACG AAG 3').
Specific mutations induced in the cDNA were verified by DNA sequencing by using Sequenase method (Amersham, Buckinghamshire, UK). The mutated
cDNAs (356-bp salI-BamHI fragments), subcloned into the pMAL-c2 vector, expressed in E coli as fused protein and
purified as described above. NH2-TAT-GST, lacking the first
21 NH2 amino acids, and GST protein were prepared as
described.42
Wild-type Tat-MBP, the mutated proteins, and MBP were
lipolysaccharide-free, assessed by Limulus assay (Sigma Chemical Co, St
Louis, MO). Proteins were stored at  80°C in the dark, in
buffered-phospate saline containing 0.1 mmol/L ZnCl2, 0.1%
human serum albumin (Farma Biagini, Lucca, Italy), and 1 mmol/L dithiothreitol.
Reagents.
Human recombinant IL-1 (IL-1; specific activity, 107
U/mg) was obtained from BASF, Bioresearch Corp (Cambridge, MA).
Monoclonal antibodies to VCAM-1 (CD106; clone 4B2), E-selectin (CD62A;
clone 13D5), intercellular adhesion molecule-1 (ICAM-1) (CD54; clone 11C8/1), and 41 (VLA-4;CD49d/CD29; clone 2B4) were obtained from
British Biotech Pharmaceuticals Ltd (Oxford,
UK). Anti-51 (CD49e/CD29; clone
SAM-1) was obtained from Immunotech S.A. (Marseille, France), and
monoclonal antibodies to v3 (CD51/CD61; clone LM609) and v5
(CD51/CD-; clone P1F6) were purchased from Chemicon (Temecula, CA).
Monoclonal antibodies to L2 (lymphocyte function-associated antigen-1 [LFA-1]; CD11a/CD18) were a kind gift from
Dr A. Rambaldi (Ospedali Riuniti, Bergamo, Italy). Recombinant soluble
VCAM-1 and E-selectin were obtained from Dr J. Clements (British
Biotech Pharmaceuticals Ltd). Heparin was purchased from Sigma.
Monoclonal antibody to Tat (clone 15.1) was from Intracel (London, UK).
Chemotaxis.
Chemotaxis was conducted in the Boyden chamber, as described
previously.43 Different dilutions of Tat-MBP, KGE-MBP,
LxI-MBP, MBP, NH2-Tat-GST, and GST with 0.5 µg/mL heparin
were added to the lower compartment of the chamber. To
block the chemotactic effect of Tat, Tat proteins were mixed with
monoclonal antibodies to Tat (dilution 1:50), or Tat proteins were
boiled for 10 minutes and then added to the lower compartment of the
chamber. Eight-micrometer pore size polyvinylpyrrolidone (PVP)-free
polycarbonate Nucleopore filters (Costar, New York, NY) were coated
with gelatin by immersing them overnight in a solution of 100 µg/mL
gelatin in 0.1% acetic acid and then air
dried. The filter separated the
attractants from the upper part of the chamber in which AS283 cells
were added. After 4 hours of incubation at 37°C, filters were stained
with Diff-Quick (Merz-Dade, Dudingen, Switzerland), and the migrated cells in 20 high-power fields were counted. Checkerboard analysis of
the chemotactic response was performed by varying the concentrations of
Tat-MBP in the upper and lower compartment of the Boyden chamber, as
described previously.43
Adhesion assay.
EC grown to confluent monolayers in 96-well plates were activated for 4 hours with the indicated concentrations of Tat-MBP, KGE-MBP, LxI-MBP,
or MBP diluted in culture medium. IL-1 at 2 ng/mL was used as a
reference control to activate EC.44 After incubation,
monolayers were washed twice, and wells were refilled with 50 µL
medium 199 containing 1% bovine serum albumin (BSA; test medium).
AS283 cells were radiolabeled with
[125I]-Iododeoxyuridine (Amersham), as described
previously.45 Fifty microliters of the
cell suspension (3 × 104 cells) was added to each well
and incubated at 37°C for 30 minutes. After incubation, nonadherent
cells were removed by careful aspiration and three washes with test
medium. Wells were incubated for 10 minutes with 100 µL of 0.1 mol/L
NaOH, and the lysate was counted in a gamma counter.
To test the adhesion of AS283 cells on purified VCAM-1, 96-well plates
(Falcon Pro-bind; Becton Dickinson Labware, Franklin Lakes, NJ) were
coated with soluble VCAM-1 by adding 50 µL of protein (5 µg/mL) and
incubating them overnight at 4°C. After two washes
in PBS, the coated surfaces were incubated for 1 hour with PBS
containing 1% BSA and then used for the adhesion assay according to
the procedure described above.
To test the involvement of adhesion molecules expressed on EC and AS283
cells in the adhesion assays, activated EC were preincubated with test
medium alone or containing 20 µg/mL monoclonal antibody (MoAb)
anti-VCAM-1, anti-E-selectin, or anti-ICAM-1. Lymphoma cells were
preincubated with test medium with or without 20 µg/mL MoAb
anti-VLA-4 or anti-LFA-1 for 30 minutes at 37°C before the adhesion assay.
Adhesion was expressed as the percentage of attached tumor cells
relative to the total added cells.
Flow cytometric analysis.
EC and AS283 cells were treated for 4 hours with Tat-MBP (20 ng/mL),
equimolar concentrations of MBP or IL-1 (2 ng/mL). The expression of
adhesion molecules on EC and AS283 cells was measured by indirect
immunofluorescence using a FACSort (Becton Dickinson). After incubation
with the appropriate primary antibody (1:50 dilution) in PBS with 2.5%
FCS for 30 minutes at 4°C, cells were washed and incubated with an
affinity-purified fluorescein isothiocyanate (FITC)-labeled goat
antimouse Ig antiserum for 30 minutes at 4°C (Tecno Genetics, Italy).
Cells were washed again and fixed with PBS containing 1% formalin.
Results were expressed as percentage positive cells and the mean of the
channel fluorescence intensity.
Statistics.
The Student's t-test was used to determine statistical
differences. Statistical significance was set at P < .05.
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RESULTS |
Induction of AS283 lymphoma cell migration by Tat-MBP.
Tat-MBP (2 to 200 ng/mL) used as chemoattractant stimulate the motility
of AS283 cells in a concentration-dependent manner (Fig
1). At the optimal concentration of 20 ng/mL Tat-MBP, the number of migrated AS283 cells augmented from 14.5 ± 0.7 in the control wells to 50.5 ± 4.5 in wells with Tat-MBP
(3.5-fold increase). The fusion protein MBP alone did not affect
migration (Fig 1), nor did heparin alone (data not shown). Similar
results were obtained with the AIDS-related lymphoma lines PA682PB and
HBL-6-AIDS-PEL, in which 20 ng/mL Tat-MBP induced a 3.1-fold and a
2-fold increase in migration, respectively (Table
1). In contrast, the migration of the
Burkitt's lymphomas (KD488, Namalwa, and BJAB), the follicular lymphoma (DHL-4), and the EBV-transformed lymphoblastoid cell line
(Capo) was not increased by Tat-MBP (Table 1). Fibroblasts and vascular
smooth muscle cells used as a normal cell control did not respond to
Tat-MBP (Table 1).
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| Fig 1.
Induction of AS283 lymphoma cell motility by Tat-MBP.
Motility response of AS283 lymphoma cells to increasing amounts of
Tat-MBP (2 to 200 ng/mL). Control medium, Tat-MBP, and MBP were used as
attractants in the presence of heparin (0.5 µg/mL). Data are
expressed as the number of migrated cells in 20 high-power fields.
Columns, mean of three replicates and representative of 1 out of 3 experiments; bars, SD. *P < .001 compared with control.
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Checkerboard analysis showed that Tat-MBP mainly induced a directional
motility response of AS283 lymphoma cells, although a chemokinetic
component was also present (Fig 2).
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| Fig 2.
Checkerboard analysis of AS283 motility response to
Tat-MBP. Different gradient conditions were obtained by adding the
indicated concentrations of Tat-MBP to the upper and lower compartments
of the Boyden chamber. Cells, in the upper compartment, were exposed to
conditions of null gradient (same concentration of Tat-MBP in the upper
and lower compartments, diagonal), positive gradients (higher
concentrations of Tat-MBP in the lower compartment, below the
diagonal), or negative gradients (higher concentrations of Tat-MBP in
the upper compartment, above the diagonal). Data are expressed as the
number of migrated cells in 20 high-power fields (mean and SD of
triplicates).
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To identify the molecular domain of Tat responsible for the stimulation
of AS283 motility, we tested two mutated Tat-MBP proteins, one mutated
in the RGD domain (KGE-MBP) and one in the basic domain (LxI-MBP). Figure 3A shows
that KGE-MBP and LxI-MBP have drastically lost the ability to induce
the migration of AS283 cells. Even higher concentrations of the two
mutated Tat-MBP proteins did not influence the migration of lymphoma
cells (data not shown). These data indicate that the chemotactic
properties of Tat-MBP might be dependent on a collaboration between the
RGD and the basic domains. A Tat protein mutated in the apparently
noninvolved N-terminal domain was also used (NH2-Tat-GST).
NH2-Tat-GST showed similar activity on AS283 cell migration
as did Tat-MBP (Fig 3B). The fusion protein GST alone had no effect at
all. The specificity of Tat-MBP has been confirmed by the finding that
neutralizing antibodies against Tat and heat inactivation blocked its
activity on AS283 cell motility (Fig 4A).
Similar results were obtained using NH2-Tat-GST (Fig 4B).
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| Fig 3.
Chemotactic activity of mutated Tat proteins. (A)
Inhibition of AS283 lymphoma cell motility by mutated Tat-MBP proteins.
KGE-MBP and LxI-MBP proteins are mutated, respectively, in the RGD and
basic domain of Tat. (B) AS283 lymphoma cell motility induced by a Tat
protein mutated in the N-terminal domain. Each protein was tested at
the concentration of 20 ng/mL. MBP or GST alone were used at the
equimolar concentration. Data are expressed as the number of migrated
cells in 20 high-power fields. Columns, mean of three replicates and
representative of 1 out of 3 experiments; bars, SD. *P < .05 compared with corresponding control.
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| Fig 4.
Inactivation of chemotactic activity of Tat. Monoclonal
antibodies against Tat (dilution 1:50) were mixed with Tat proteins, or
Tat proteins were boiled for 10 minutes and then added to the lower
compartment of the chamber. Tat-MBP and NH2-Tat-GST were
used in (A) and (B), respectively. Each protein was tested at the
concentration of 20 ng/mL. Data are expressed as the number of migrated
cells in 20 high-power fields. Columns, mean of 3 replicates (and
representative of 1 out of 2 experiments done); bars, SD. *P < .001 compared with Tat-MBP without treatment, **P < .05 compared with NH2-Tat-GST without treatment.
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Induction of EC adhesiveness by Tat-MBP.
The adhesion of AS283 lymphoma cells was significantly increased on EC
activated with Tat-MBP for 4 hours with maximal stimulation at 20 ng/mL
Tat-MBP (Fig 5). Tat-MBP induced
approximately a 1.6-fold increase of adhesion, reproducible in all the
experiments performed. MBP alone had no effect.
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| Fig 5.
Adhesion of AS283 lymphoma cells to endothelial cells
activated with Tat-MBP. AS283 lymphoma cell adhesion was tested on EC
activated with different concentrations of Tat-MBP. EC were treated
with medium alone (control), Tat-MBP (20 to 200 ng/mL), or equimolar
concentration of MBP. EC were activated for 4 hours, and adhesion of
AS283 lymphoma cells was evaluated after 30 minutes. Results show
attached cells as a percentage of total added cells. Columns, mean of
three replicates and representative of 1 of 3 experiments; bars, SD.
*P < .01 compared with control.
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We therefore investigated by fluorescence-activated cell sorter (FACS)
analysis whether the increased adhesiveness was sustained by an
increment of adhesion molecules. Twenty nanograms/milliliter Tat-MBP
induced the expression of E-selectin and VCAM-1 and increased the
expression of ICAM-1 after 4 hours of activation (Table
2). MBP alone had no significant effect on
the expression of adhesion molecules. The increment of lymphoma cell
adhesion to EC treated with Tat-MBP was lower compared with
IL-1-activated EC, used as a positive control (3.1-fold increase of
adhesion). The treatment of EC together with Tat-MBP did not have any
additional effect with regards to enhancing AS283 cell adhesion (data
not shown). Accordingly, Tat-MBP was less potent than IL-1 in
augmenting the expression of adhesion molecules on EC (Table 2).
Increasing the concentration of Tat-MBP had no further effect on the
expression of these adhesion molecules. Treatment of AS283 cells with
Tat-MBP did not increase their adhesion to EC (data not shown). Indeed, adhesion molecules such as ICAM-1, LFA-1, and VLA-4 expressed on AS283
cells did not change after Tat-MBP (20 ng/mL) treatment (data not
shown). Similar to that of the AS283 cells, the adhesion of PA682PB
cells was also augmented on EC activated by Tat-MBP (data not shown).
To identify the adhesion molecules involved in AS283/EC adhesion, EC
were treated with antibodies against VCAM-1, E-selectin, and ICAM-1.
Only antibodies against VCAM-1 blocked the increased adhesion of AS283
cells to EC (Fig 6A). Antibodies against
E-selectin and ICAM-1 had no effect on the adhesion to EC (Fig 6A). To
confirm that AS283 cell adhesion on Tat-MBP-activated EC depended on
the VCAM-1/VLA-4 interaction, we found that AS283 cells selectively attached themselves to VCAM-1-coated plastic, and this adhesion could
be blocked by antibodies against VLA-4 to tumor cells (Fig 6B).
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| Fig 6.
(A) Inhibition of AS283 lymphoma cell adhesion to
Tat-activated EC. EC were treated for 4 hours with medium alone
(control) or Tat-MBP (20 ng/mL) and thereafter incubated with
antibodies against the adhesion molecules E-selectin, VCAM-1, and
ICAM-1. Adhesion of AS283 lymphoma cells was evaluated after 30 minutes. (B) AS283 lymphoma cell adhesion to VCAM-1. Wells were coated
with 5 µg/mL VCAM-1 or buffer only (control). AS283 cells treated or
not with antibodies against VLA-4 were incubated on soluble
VCAM-1-coated plastic for 30 minutes. Results show the attached cells
as a percentage of total cells added. Columns, mean of three replicates
and representative of 1 of 3 experiments; bars, SD. *P < .05 compared with EC treated with Tat-MBP without MoAb (A) or
to VCAM-1 (B).
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DISCUSSION |
It has been found that biological functions of mesenchymal cells, like
monocytes and endothelial cells, can be modulated by the HIV-1-Tat
protein.20,29,31,34,36 Recently, it has been described that
Tat can upregulate Fas expression in B lymphocytes,32 suggesting that Tat may contribute to B-cell hyperactivation during HIV
infection. Polyclonal hyperactivation and proliferation of B cells has
been associated with the development of NHL in AIDS patients.46 In this study, we show that Tat induces the
migration of two AIDS-related Burkitt's lymphoma cell lines (AS283 and
PA682PB)37 and increases their adhesiveness to human
endothelium, two processes that are required for the spreading of
highly malignant AIDS-NHL cells. We also tested other kinds of lymphoma
cells and found that only the HBL-6-AIDS-PEL lymphoma cell line
migrated against Tat-MBP (Table 1), thus suggesting a selective role
for the Tat protein in the migration of lymphomas derived from
HIV-infected patients. An extensive study on primary lymphoma cells
from patients with AIDS is likely to be necessary to address this hypothesis.
Tat protein has been found extracellularly, and induction of lymphoma
cell migration was observed at concentrations that have been detected
in AIDS patients.26 Similar concentrations of Tat have been
reported to induce monocyte29 and EC31 motility and also to upregulate FAS expression in B cells.32 We
therefore propose that secreted Tat can promote lymphoma migration and
extravasation into tissues. Tat concentrations detected in the serum of
AIDS patients are approximately 1 to 2 ng/mL.31 In our
experiments, we observed that concentrations between 2 and 20 ng/mL are
able to significantly stimulate cell motility. It is possible that the
phenomenon of lymphoma cell recruitment by Tat becomes relevant at
sites of Tat production and release, where concentrations are presumably higher than those in the circulation.
Extracellular activities of Tat can be mediated through the
RGD-containing region or through the basic domain.31,47 To investigate the role of Tat domains in the induction of lymphoma cell
motility, two mutated Tat-MBP proteins were used: the KGE-MBP protein
that is mutated in the RGD domain, and the LxI-MBP protein that is
mutated in the basic domain. We found that neither the RGD nor the
basic domain-mutated Tat protein had any effect on lymphoma cell
migration. A third Tat protein, NH2-Tat-GST, which is
deleted of the first 21 amino acids and does not contain the cysteine-rich domain, had similar activity on lymphoma migration as
Tat-MBP, and the data obtained exclude a role for this domain in
Tat-induced lymphomas. These data indicate that the chemotactic properties of Tat for AIDS-related lymphoma cells might require both
the RGD and the basic domain of Tat. In contrast, the cysteine-rich domain, which has been recently implicated in monocyte chemotaxis induced by Tat,48 seems to be irrelevant for the motility
of lymphoma cells. At variance with our findings, it has been shown that the motility of EC was equally stimulated by synthetic peptides containing both the basic or the RGD domain of Tat,47
though angiogenesis in vivo was only induced by the basic
peptide.31 The fact that AS283 cells did not express the
integrin receptors v3 and 51 that recognize the RGD
sequence of Tat,35 nor the integrin v5 that binds the
basic domain of the Tat protein49 (data not shown),
suggests that other ligands are involved in the interaction of Tat with
AS283 cells. Altogether, these data indicate that different cell types
respond to different domains of Tat. Furthermore, it is also possible
that unique Tat domains are responsible for different physiological
functions, such as migration in vitro and angiogenesis in vivo on the
same cell type.31,47
We have recently shown that Tat-induced migration of endothelial cells
and monocytes occurs through the activation of the high affinity
tyrosin kinase receptors for vascular endothelial growth factor (VEGF),
VEGFR-2/KDR/Flk-1, and VEGFR-1/Flt-1, respectively.27,31 Here we found that VEGF (20 ng/mL) induced AS283 cell migration and
that pretreatment of AS283 cells with VEGF resulted in an inhibition of
a subsequent response to Tat (data not shown). This result is
reminiscent of the observation that VEGF is an autocrine/paracrine growth factor for acute myeloid cells.50 Although these
findings indicate that AS283 cell migration induced by Tat and VEGF
depended on the same receptor, VEGFR-1/Flt-1 and VEGFR-2/Flk-1/KDR
receptors were not expressed on AS283 cells as analyzed by Northern
blot analysis (data not shown). This may indicate the presence of
another receptor on AS283 cells. Recently, a third VEGF coreceptor,
identical to human neuropilin-1, has been described on endothelial
cells and different types of tumor cells.51 Whether this or
another receptor is responsible for Tat-induced lymphoma cell motility needs further investigation.
Another important event in the lymphoma dissemination is the
interaction between tumor cells and vascular endothelial cells. We show
that the treatment of EC with Tat enhances their adhesiveness to
lymphoma cells. The upregulation of ICAM-1, VCAM-1, and E-selectin on
EC after Tat treatment has been previously shown.13 The
role of VCAM-1 and ICAM-1 in the adhesion of B lymphocytes has been shown.52,53 Here we show that the augmented adhesion of
AS283 lymphoma cells to Tat-activated EC is mainly associated with an increased expression of VCAM-1, and this is abolished only by antibodies against anti-VCAM-1, but not against ICAM-1. Accordingly, two different reports have shown that lymphocyte adhesion (including Burkitt's lymphoma cell) to cytokine-activated EC is preferentially mediated by the upregulation of VCAM-1.54,55 The lack of
adhesion of Burkitt's lymphoma to ICAM-1 expressed on endothelial
cells is explained by Patarroyo et al.56 They compared a
panel of 10 lymphoblastoid cell lines with 10 Burkitt's lymphoma for
the expression of LFA-1 and found that Burkitt's lymphoma expresses only low levels of this integrin, whereas lymphoblastoid cell lines,
representing various stages of B-lymphocyte development, express
considerable levels of LFA-1. Although we found that by FACS analysis
our AS283 cell line expressed LFA-1, its adhesion to Tat-activated EC
was not abolished by antibodies against LFA-1, supporting further the
preferential use of the VCAM-1/VLA-4 pathway in the adhesion to
Tat-activated EC. The VCAM-1/VLA-4-dependent adhesion of AS283 has
been further shown by completely blocking its adhesion to soluble
VCAM-1-coated plastic and by treating AS283 cells with antibodies
against VLA-4.
It has been shown that anti-CD40 stimulates the heterotypic adhesion of
normal and transformed B cells to EC, and this is mainly mediated by
VCAM-1/VLA-4 pathway in alternative to ICAM-1/LFA-1.53 Furthermore, it has been found that HIV-infected microvascular EC
promotes the attachment and growth of malignant B-cell
lymphoma.12 It is worth noting that HIV infection of
microvascular EC stimulates the expression of CD40, resulting in
preferential induction of VCAM-1 after CD40 triggering,12
once again showing the importance of the VCAM-1/VLA-4 pathway. However,
we did not find CD40 expression on endothelial cells treated with Tat
(data not shown), suggesting that this pathway is not involved in our
experimental model.
In conclusion, AIDS-related lymphomas frequently involve extranodal
sites, particularly in the gastrointestinal tract and the central
nervous system, which represent the primary sites of involvement in a
significant fraction of the cases.1-3,5 We showed in this
work that HIV-1-Tat can enhance the migration of AIDS-related lymphoma
cells and their adhesion to endothelial cells. In AIDS patients, this
may contribute to the homing and growth of malignant lymphomas at these
extranodal sites.
| |
ACKNOWLEDGMENT |
The authors thank Dr L.T. Magrath (National Cancer Institute, Bethesda,
MD), Dr G. Golay (M. Negri Institute for Pharmacological Research,
Milan, Italy), and Dr G. Gaidano (University of Piemonte Orientale
"A. Avogadro", Turin, Italy) for providing some of the lymphoma
cell lines used in this study.
| |
FOOTNOTES |
Submitted June 22, 1998; accepted April 26, 1999.
Supported by grants from the Istituto Superiore di Sanità (AIDS
Project and Program on Tumor Therapy), and Associazione Italiana per la
Ricerca sul Cancro (AIRC).
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 Raffaella Giavazzi, PhD,
Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy; e-mail: giavazzi{at}irfmn.mnegri.it.
| |
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ABSTRACT
INTRODUCTION