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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1077-1085
Basic Fibroblast Growth Factor Is Expressed by CD19/CD11c-Positive
Cells in Hairy Cell Leukemia
By
Gerhard Gruber,
Josef D. Schwarzmeier,
Medhat Shehata,
Martin Hilgarth, and
Rudolf Berger
From the Ludwig Boltzmann Institute for Cytokine Research and the
Department of Internal Medicine I, Division of Hematology, University
of Vienna Medical School, Vienna, Austria.
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ABSTRACT |
Several features are characteristic for hairy cell leukemia (HCL).
Among those are pancytopenia, bone marrow fibrosis, and the appearance
of a defined tumor cell phenotype in peripheral blood (PB), bone marrow
(BM), and spleen. Hairy cells (HC) coexpress antigens specific for B
lymphocytes and monocytes/macrophages and thus the malignant cell does
not seem to be restricted to a defined lineage. When serum or bone
marrow aspirate was screened by enzyme-linked immunosorbent assay
(ELISA) for basic fibroblast growth factor (bFGF), specimen derived
from HCL (serum: mean value, 29 pg/mL; BM aspirate: mean value, 641 pg/mL) contained significantly higher levels than those from healthy
subjects. To study whether peripheral blood mononuclear cells (PBMC)
derived from patients suffering from HCL and healthy donors (HD) were
capable of producing bFGF, culture supernatant (conditioned medium,
[CM]) was tested for the presence of this cytokine. While bFGF was
not detectable in cell cultures from HD, HCL-derived CM contained
relatively high levels of bFGF. CM was successfully used for
stimulation of mesenchymal cell proliferation, which could be inhibited
by a neutralizing anti-bFGF antibody. Cellular activation by pokeweed mitogen (PWM) or the combination of
12-o-tetradecanoyl-phorbol-13-acetate (TPA) plus calcium ionophore
(Ca-Ip) led to an enhanced mRNA expression. Results of Western blot
experiments showed that HC synthesize at least three isoforms
(approximately 18, 23, and 25 kD), but only the 23-kD isoform is
exported. To assess the nature of the producer cell, double
immunofluorescence analysis using a bFGF-specific and an anti-CD11c
monoclonal antibody (MoAb) was undertaken. The majority of cells
scoring positive for CD11c were also reactive with the anti-bFGF MoAb.
Furthermore, enrichment of CD19/CD11c-positive cells correlated with
enhanced bFGF levels, thereby supporting the argument for HC being the
producer cells of bFGF. A biological function of bFGF in HCL might be
mediation of chemoresistance, as 2-chlorodeoxyadenosine
(2-CdA)-induced inhibition of cell proliferation can be reversed by
bFGF. Endogenous bFGF production by HC is not affected by this purine
analogue and 2-CdA-induced apoptosis is diminished in bFGF-producing
HC as compared with normal PBMC. Therefore, bFGF expression by HC might
be important for resistance to chemotherapy and survival of the
malignant cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BASIC FIBROBLAST growth factor (bFGF), a
pleiotropic cytokine, is important for embryogenesis, angiogenesis,
wound healing, and mesodermal development. During these physiologic processes, its expression is tightly regulated.1,2 Due to its stimulatory capacity on the formation of new blood vessels, bFGF is
suggested to play a major role for growth and dissemination of tumors.
In fact, a high expression has been reported in various tumors.3-6
The producer cells of bFGF are mainly of mesenchymal origin. However,
cells of the hematopoietic lineage have also been found to express and
respond to this cytokine.7-9 Most of these studies have
used permanent cell lines of myeloid origin.10,11 In
primary cells derived from hematopoietic malignancies, bFGF, as well as its receptors (bFGF-R), seem to be preferentially produced by myeloid
cells.12,13 More recently, however, intracellular bFGF has
been detected in CD34+ cells,14 in B cells
derived from chronic lymphocytic leukemia (CLL),15 and has
been found in culture fluids of serum-starved, long-term T-cell
cultures.16,17
Hairy cell leukemia (HCL) represents a distinct and rare form of
chronic leukemias. Besides a characteristic morphology, the malignant
cells coexpress both the B-cell-specific antigen, CD19, and the
myeloid-specific antigen, CD11c.18 A functional feature invariably associated with HCL is bone marrow (BM)
fibrosis.19 The mechanisms underlying this pathologic
process are not completely understood. Because bFGF seems to be
involved in fibrinogenic diseases such as liver fibrosis,20
fibrosis in gastric carcinoma,21 or in systemic
scleroderma,22 it is reasonable to speculate that it also
plays a role in the development of BM fibrosis in HCL. Moreover, the
production of bFGF by hairy cells (HC) would argue for a functional
resemblance with myelomonocytic cells and would be compatible with the
expression of CD11c on the surface of CD19-positive cells.
In the present study, we report on the capability of peripheral blood
mononuclear cells (PBMC) derived from HCL to produce biologically
active bFGF. Furthermore, we show that mRNA expression can be modulated
by activators of protein kinase C (TPA+Ca-Ip), that bFGF is released by
CD19/CD11c-positive cells as a high molecular weight isoform, and that
bFGF mediates resistance to 2-chlorodeoxyadenosine (2-CdA)-induced apoptosis.
| |
MATERIALS AND METHODS |
Patients.
Seven patients suffering from HCL and seven healthy donors were
investigated. HCL was diagnosed on the basis of cell morphology, double
immunofluorescence staining using monoclonal antibodies (MoAb) against
CD11c and CD19 and BM and splenic histology.
Cell culture.
NIH-3T3 mouse fibroblasts were obtained from American Type Culture
Collection (ATCC; Rockville, MD). D15-7 (established in our own laboratory) are human skin fibroblasts from an 8-year-old healthy donor. The erythroleukemic cell line, K562, was kindly provided
by Dr G. Klein (Karolinska Institute, Stockholm, Sweden).
PB was taken after informed consent from patients with HCL and healthy
volunteers. PBMC were separated by density gradient centrifugation
using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Cells banding at the
interphase were washed twice in phosphate-buffered saline (PBS),
resuspended in culture medium, and seeded in 6-well plates at 2 × 106 cells/mL. In certain experiments, T lymphocytes were
depleted by a rosetting technique using neuraminidase-treated sheep red blood cells.
PBMC were grown in RPMI 1640 supplemented with 2 mmol L-glutamine,
penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% heat
inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere containing 5% CO2. For certain experiments,
cells were activated by pokeweed mitogen (PWM, 10 µg/mL) or
12-o-tetradecanoyl-phorbol-13-acetate (TPA, 10 ng/mL) plus
calcium-ionophore A23187 (Ca-Ip, 10 ng/mL).
Production of conditioned medium (CM).
If not stated otherwise, culture supernatant was harvested from
unstimulated cultures after 48 hours, centrifuged at 200g for
10 minutes, sterile filtered, and stored at  20°C until assayed.
Reagents and antibodies.
Cell culture media (RPMI 1640, MCDB 104), penicillin-streptomycin,
L-glutamine, and FCS were obtained through GIBCO, Life Technologies
(Paisley, UK). PWM, TPA, and Ca-Ip were purchased by Sigma Chemicals
(St Louis, MO). 2-CdA was provided by Ortho Biotech (Raritan, NJ).
Monoclonal antibodies against CD3, CD5, CD11c, CD14, CD19, and the
respective isotype controls, as well as ABComplex for Western blot
analysis, were obtained through Dako (Copenhagen, Denmark), a
Cy3-labeled anti-CD11c MoAb was purchased from Sigma Chemicals. A
neutralizing mouse anti-bFGF MoAb and recombinant human bFGF (rhbFGF)
were purchased from R&D Systems (Minneapolis, MN) and a biotin-labeled
goat anti-mouse antibody was obtained through Accurate Chemicals
(Westbury, NY). For Western blotting experiments, a mouse anti-bFGF
MoAb (clone Ab-3, Oncogene Science, Uniondale, NY) was used.
Immunofluorescence and fluorescence-activated cell scanning (FACS)
analysis.
Immunofluorescence analysis using a Cy3-labeled anti-CD11c and an
anti-bFGF MoAb (R&D) was performed on aceton-fixed cells. Incubation
steps with a biotinylated goat antimouse antibody and conjugates
supplied in the TS amplification kit (Du-Pont-NEN, Boston, MA) were
performed following instructions of the manufacturer.
MoAbs directed against CD3, CD5, CD11c, CD14, CD19 were used to analyze
the cellular composition of the PBMC. For FACS analysis, 2 × 105 cells in PBS/bovine serum albumin (BSA)
(1%) containing 0.1% sodium azide were stained with the fluorescein
isothiocyanate (FITC)-labeled MoAb or isotype control for 30 minutes at
4°C. Samples were analyzed on a FACScan (Becton Dickinson, San
Jose, CA). Gates for data analysis were set using the respective
isotype staining as negative control. Quantitative analysis of
apoptotic cells was performed using an Annexin V-FITC detection kit
obtained from Pharmingen (San Diego, CA).
Detection of cytokines.
Sera, BM aspirates, cell lysates, and CM from cultures were tested for
presence of bFGF by a commercially available ELISA-system (Bio-Trak;
Amersham, Backinghamshire, UK) following the manufacturer's protocol.
Samples were tested in duplicates, optical density was measured, and
bFGF values were calculated with a computer assisted plate reader
(Dynatech, Guernsey Channel Islands, UK). The detection limit of the
test is 1 pg/mL.
Western blot analysis.
Freshly isolated PBMC (107 cells) from a patient with HCL
(66% CD19+/CD11c+ cells) and a healthy donor
were cultured as described above. Supernatants from controls and
PWM-stimulated cultures were passed through a 0.2-µm filter and
incubated with 300 µL heparin-coated beads (AF-Hep 650M; Toso Haas,
Tokyo, Japan) for 1 hour at room temperature (RT). rhbFGF
was treated identically serving as positive control. Beads were washed
twice in 1 mmol/L Tris/HCl, 0.2 mol NaCl, and bound proteins were
eluted by boiling for 3 minutes in 300 µL electrophoresis buffer
(0.125 mmol/L Tris/HCl; pH 6.8; 1% sodium dodecyl sulfate [SDS]). A
total of 2 × 106 cells was resuspended in 1 mL
electrophoresis buffer and lysed by boiling for 3 minutes. Unsoluble
material was removed by centrifugation and aliquoted samples were
stored at 20°C. Thirty-microliter samples were separated on
homogenous gels under reducing conditions and transferred onto
nitrocellulose by semidry blotting. Western blot analysis was performed
using a mouse anti-bFGF MoAb followed by a
biotin-streptavidin-horseradish-peroxidase incubation step. Positive
reactions were visualized by enhanced chemiluminescence (Super Signal;
Pierce, Rockford, IL) on ECL-Hyperfilm (Amersham, Backinghamshire, UK).
Proliferation assays.
NIH-3T3 fibroblasts and primary human dermal fibroblasts were cultured
under serum-free conditions for 24 hours before addition of CM from
PBMC of HCL patients or healthy donors. In a separate set of
experiments, CM was preincubated for 1 hour at 37°C with a
neutralizing anti-bFGF antibody (10 µg/mL) before addition to cultures. After 2 or 4 days, fibroblasts were pulsed with 1 µCi of
3H-thymidine (3H-TdR; NEN) for 8 hours,
harvested, and 3H-TdR uptake was determined using a
Beta-Plate Scintillation Counter (Wallac-Pharmacia, Uppsala, Sweden).
Cell cultures were performed in triplicates using 96-well plates.
To measure proliferation of PBMC, 2 × 105 cells/well
were cultured in 96-well plates for 72 hours with and without 2-CdA and recombinant human bFGF, pulsed with 3H-TdR for 8 hours, and
thymidine uptake was determined in a liquid scintillation counter.
mRNA-isolation and reverse transcription-polymerase chain reaction
(RT-PCR).
Cells were harvested 48 hours after onset of cultures, washed twice in
ice-cold PBS, and counted automatically (Coulter Counter, Coulter
Electronics Ltd, Luton, UK). A total of 5 × 106 cells
were subjected to RNA isolation by the
guanidium-isothiocyanate-phenol-chloroform extraction procedure. cDNA
was synthesized using 1 µg RNA as a template and a commercially
available kit (Pharmacia). Polymerase chain reaction (PCR) was
performed in a final volume of 50 µL in the presence of 0.25 mmol/L
of each dNTP, 1 U Taq polymerase (Finnzyme, Helsinki,
Finland), and 100 pmol/L of either bFGF- or  -actin-specific
primers. For -actin PCR (upstream primer: 5'-AGG CCG GCT TCG
CGG GCG AC-3'; downstream primer: 5'-CTC GGG AGC CAC ACG
CAG CTC-3'),23 30 cycles were performed at 94°C for 1 minute (denaturing), 60°C for 90 seconds (annealing), and 72°C for 90 seconds (extension). The final extension was performed at 72°C for 3 minutes. Each experiment was repeated at least two times and results showed no difference in -actin expression, indicating equal amounts of mRNA. For bFGF-specific PCR (upstream primer: 5'-ACC ACG CTG CCC GCC TTG CCC-3'; downstream
primer: 5'-CTT ATA GCC AGG TAA CGG TTA
GCA-3'),12 40 cycles of amplification were performed
(denaturation at 92°C/1 minute, annealing at 62°C/90 seconds,
extension at 72°C/1 minute, final extension step at 72°C/5 minutes). Amplified DNA fragments were visualized by agarose gel electrophoresis. Southern blotting, hybridization to a digoxigenin (DIG)-labeled bFGF-specific cDNA probe (by courtesy of Dr N. Frazer, University of Oxford, Oxford, UK) and exposure to x-ray
films was performed following standard procedures.
| |
RESULTS |
Detection of bFGF in serum and BM aspirate from patients with HCL.
Serum derived from seven patients with HCL and seven healthy donors
(HD) was screened by a bFGF-specific ELISA (detection limit, 1 pg/mL)
(Table 1).
Figure 1A indicates that in serum from
all patients with HCL, bFGF was detectable, ranging from 1 to 90 pg/mL
with a mean of 29 pg/mL. With the exception of one individual (4.6 pg/mL), no bFGF could be found in the serum from HD. Because BM is a
primary site of HC appearance, BM aspirates were also tested. As
indicated in Fig 1B, the mean concentration of bFGF in HCL-derived BM
(641 pg/mL) was more than 16 times higher than in samples from HD
(mean, 39 pg/mL).
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| Fig 1.
bFGF levels in sera and BM aspirates from healthy donors
and HCL-patients. Sera from EDTA-drawn blood samples (n = 7) and BM
aspirates (n = 7) were tested for bFGF contents by ELISA (limit of
detection: 1 pg/mL). HCL-derived sera (range, 1 to 90 pg/mL; mean, 29 pg/mL) (A) and BM aspirates (range, 230 to 1,005 pg/mL; mean, 641 pg/mL) (B) contained significantly elevated amounts of bFGF. In HD,
only one of seven serum samples was weakly positive (4.6 pg/mL) and BM
samples showed a mean value of 39 pg/mL, range, 1 to 83 pg/mL bFGF.
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PBMC from patients with HCL release bFGF.
Because serum- or BM-derived bFGF might originate from nonhematopoietic
cells or from cells that are disrupted during BM aspiration, we
investigated whether PBMC from HD and HCL patients are capable of
producing bFGF. Cells were cultured for 48 hours and culture supernatants were analyzed for bFGF by ELISA
(Fig 2). While in cultures of unstimulated
cells from HD no bFGF was detected, significant concentrations were
found in HCL (mean value, 55 pg/mL). Stimulation with PWM led to a
minute release of bFGF in only two of seven HD (2.4 pg/mL and 1.9 pg/mL). In HCL, however, an increase of up to 100% was seen (mean
value, 110 pg/mL).
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| Fig 2.
In vitro bFGF production by PBMC from patients with HCL
and HD. A total of 2 × 106 cells were cultured in 12-well
plates for 48 hours with and without PWM. Supernatants were assayed by
a bFGF-specific ELISA. Unstimulated cultures from HD did not contain
detectable amounts of bFGF in contrast to HCL-derived samples (mean, 55 pg/mL; range, 4 to 161 pg/mL). PWM stimulation led to an increased bFGF
release in HCL (mean, 110 pg/mL; range, 8 to 262 pg/mL), while only two
of seven HD showed weak positive reaction (2.4 pg/mL and 1.9 pg/mL).
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Stimulation of fibroblast proliferation by HCL-derived bFGF.
To examine if HCL-derived bFGF induces mesenchymal cell growth, human
dermal fibroblasts were incubated with CM obtained from PBMC of an HCL
patient and an HD. After 48 and 96 hours, cell proliferation was
assessed by 3H-TdR uptake. As shown in
Fig 3, CM from HCL induced a much higher increase in fibroblast proliferation than CM derived from HD. The
addition of a neutralizing bFGF-specific MoAb markedly reduced the
growth stimulatory effect of CM from HCL, but not that from HD.
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| Fig 3.
Proliferative response of fibroblasts to CM from HD and
HCL patients. Human dermal fibroblasts (D15-7) were cultured in the
presence of 25% CM derived from PBMC of an HD and from an HCL patient
(open bars). In a separate experiment, CM was preincubated (1 hour,
37°C) with a neutralizing anti-bFGF antibody (10 ng/mL) before
addition to fibroblasts (solid bars). After 48 and 96 hours,
proliferation was assessed by 3H-TdR incorporation.
Proliferation of cells without CM was considered as 100%.
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Because binding to heparin is a feature of bFGF, CM from HCL patients
was incubated with heparin beads for 30 minutes at RT. Heparin
treatment of CM clearly reduced fibroblast proliferation triggered by
HCL-derived CM or by 10 ng rhbFGF (data not shown).
Kinetics and regulation of bFGF mRNA expression in PBMC from HCL.
To test whether bFGF production results from constitutive expression or
from in vitro activation, PBMC from HDs and patients with HCL were
subjected to mRNA isolation immediately after blood donation and
gradient centrifugation. Results of a RT-PCR are shown in
Fig 4A. bFGF mRNA was highly expressed in
HCL, while no or only minute mRNA expression could be detected in PBMC
from HDs.
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| Fig 4.
(A) Expression of bFGF-specific mRNA. PBMC from HDs (n
= 4) and patients suffering from HCL (n = 4) were subjected to RNA
isolation immediately after separation by gradient centrifugation.
After RT-PCR amplification using bFGF-specific primers and Southern
blotting, positive reactions were visualized by hybridization with a
DIG-labeled bFGF cDNA probe followed by autoradiography. Negative
control: no cDNA added; K562 mRNA served as positive control. In
parallel experiments, -actin expression was analyzed by RT-PCR to
ensure that identical amounts of cDNA are used (not shown). (B)
Synthesis of bFGF protein. PBMC derived from HCL patients (n = 3) and
HD (n = 3) were analyzed for intracellular bFGF. A total of 2 × 106 cells were lysed and bFGF was determined by ELISA. Data
are shown as mean values (HD, 7 pg/mL; HCL, 420 pg/mL). (C) Activation
of bFGF-specific mRNA. PBMC from HDs (n = 2) and HCL patients (n = 2) were cultured for 48 hours in medium or stimulated with either PWM
(10 µg/mL) or TPA (10 ng/mL) + Ca-Ip A23187 (10 ng/mL)
(TPA+Ca-Ip). After RNA isolation, RT-PCR, Southern blotting, and
hybridization with a DIG-labeled bFGF, cDNA-positive reactions were
visualized by autoradiography (A). K562 mRNA served as
positive control. (B) Shows results of -actin-specific
RT-PCR.
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To examine if the expression of bFGF mRNA is paralleled by the
synthesis of bFGF protein, lysates from freshly isolated cells from
both HD and patients with HCL were subjected to bFGF-ELISA. As shown in
Fig 4B, high amounts of intracellular bFGF were detectable in
HCL-derived cells only.
Stimulation of PBMC for 48 hours by a mitogen (PWM) or activators of
protein kinase-C (TPA+Ca-Ip) (Fig 4C) enhanced bFGF-specific mRNA
expression. In HCL, the basic expression of bFGF mRNA was enhanced by
both PWM and TPA+Ca-Ip. In HDs, treatment with either substances led to
weak mRNA expression.
The concentration of extracellular bFGF protein in unstimulated HCL
cultures did not change significantly over a period of 6 hours, but
then gradually declined. In contrast, stimulation by TPA+Ca-Ip resulted
in upregulation of bFGF after 24 hours, possibly indicating a
correlation between cellular activation via protein kinase C and bFGF
production (data not shown).
High-molecular-weight isoforms of bFGF are expressed and released by
PBMC from HCL.
bFGF is expressed in several isoforms.24 To investigate if
a particular isoform is predominantly produced in HCL, immunoblotting was performed. Samples were separated on SDS gels under reducing conditions (see Materials and Methods), blotted, and analyzed using
an anti-bFGF MoAb. The pattern of bFGF protein expression detected in
lysates derived from PBMC of HCL patients showed three distinct
isoforms of approximately 18, 23, and 25 kD, while a lysate from an HD
was negative (Fig 5A). In a second set of
experiments, PBMC-derived cell-free CM from an HD and an HCL-patient
were subjected to heparin chromatography. Bound proteins were eluted
and analyzed as above. As demonstrated in Fig 5B, CM obtained from an
HD again was essentially devoid of bFGF. In sharp contrast to lysates, the supernatant derived from a patient with HCL contained only the
23-kD high-molecular-weight isoform.
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| Fig 5.
(A) Intracellular bFGF expression pattern of PBMC from
HCL patients. A total of 30 µL of cell lysates (2 × 106
cells/mL) were separated by SDS-PAGE, blotted, and analyzed by Western
blotting. PBMC from HCL expressed three bFGF isoforms (approximately
18, 23, and 25 kD), while no bFGF could be detected in HD. rhbFGF was
used as control. (B) Release of a 23-kD bFGF isoform. CM from HD and
HCL cultures was harvested after 48 hours, subjected to heparin
chromatography, and bulk eluted fractions were separated by SDS-PAGE.
Proteins were transferred onto nitrocellulose and analyzed by Western
blotting. rhbFGF served as a positive control.
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bFGF is produced by CD19+/CD11c+
cells.
Double immunofluorescence analysis was performed to confirm whether HC
are the producers of bFGF. PBMC from a patient with 66%
CD19+/CD11c+ cells were examined. A typical
reaction pattern of a bFGF- and a CD11c-specific MoAb is shown in
Fig 6, showing that most of the
CD11c-positive cells also express bFGF. In contrast, PBMC from an HD
were negative for bFGF (data not shown). Because these results strongly
suggested that CD19+/CD11c+ cells might be the
producers of bFGF, we investigated the capacity of this particular cell
population to produce bFGF. PBMC from the same patient and from an HD
were investigated for coexpression of CD11c and CD19 by FACS analysis.
While 66% of the PBMC from the patient expressed CD19, as well as
CD11c, only 1.9% of the PBMC from the HD were double-positive for
these antigens. When cells were cultured for 48 hours, only PBMC from
the patient with HCL released bFGF (272 pg/mL;
Fig 7A). In a parallel set of experiments, PBMC were separated from E-rosetting cells by gradient centrifugation. While in the case of HCL, this procedure yielded 91%
CD19+/CD11c+ cells (<2% CD3 or CD5 and
<1% CD14-positive cells), the non-T-cell population from the
healthy donor contained 5% CD19+/CD11c+ cells
only (<5% CD3, CD5, and CD14-positive cells, Fig 7B). Again, non-T
cells from HD did not produce any detectable bFGF. In contrast, CM from
PBMC enriched for CD19+/CD11c+ cells of the HCL
patient contained high levels of bFGF (539 pg/mL). Therefore,
enrichment for CD19+/CD11c+ cells leads to an
increase of almost 100% (272 pg/mL to 539 pg/mL).
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| Fig 6.
Double-immunofluorescence staining of PBMC from an HCL
patient. Cell smears were fixed in acetone and incubated with a
Cy3-labeled mouse anti-CD11c MoAb and a mouse anti-bFGF MoAb followed
by the TS amplification system (see Materials and Methods). Green (A,
anti-bFGF) and red (B, anti-CD11c) fluorescent signals from the same
field were recorded sequentially. Experiments using isotype control
antibodies or PBMC from an HD were negative (not shown).
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| Fig 7.
Production of bFGF by
CD19+/CD11c+ cells. PBMC from HD and HCL
were enriched for CD19+/CD11c+
cells by T-cell depletion using a rosetting technique. FACS
staining showed <3% CD3- and CD5-positive cells, respectively. Cells
were cultured for 48 hours and supernatants were screened by ELISA.
(A) Represents FACS profiles and bFGF production of unseparated
cells, and (B) shows results of T-cell-depleted populations.
Fluorescence-1 (FL1) and fluorescence-2 (FL2) represent
CD19+ and CD11c+ populations,
respectively.
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bFGF mediates resistance of PBMC to 2-CdA.
Endogenous bFGF production has a beneficial effect on cell
proliferation and rescue from apoptotic cell death in various cell types, as has been reported earlier.25-28 To test whether
bFGF exerts a protective effect on HC, cultures were set up in the presence of 2-CdA, a drug that induces apoptosis in HC29
and is successfully used for treatment of HCL.30 As can be
seen from Fig 8A, bFGF-producing cells from
HCL could only be partially inhibited by the addition of 2-CdA. In
contrast, proliferation of PBMC derived from an HD was completely
abrogated. A possible explanation for the weak inhibition of HC might
be that these cells express bFGF spontaneously and therefore are
protected from the cytopathic effects of 2-CdA. This assumption is
supported by results of experiments in which cultures treated with
2-CdA were supplemented with rhbFGF. Despite the presence of 2-CdA, exogenous rhbFGF restored the proliferative capacity of PBMC from a
patient with HCL and an HD, suggesting that bFGF protects PBMC from the
cytopathic action of 2-CdA. In vivo, such protection would require a
constitutive expression of this cytokine even in the presence of 2-CdA.
Thus, we examined if 2-CdA has an influence on bFGF release by HC. PBMC
derived from two patients suffering from HCL were cultured for 48 hours
and supernatants were screened for bFGF by ELISA. Indeed, PBMC from
patient no. 1 produced high levels of bFGF, regardless of the presence
of 2-CdA, while PBMC from patient no. 2 were inhibited by 22% only
(Fig 8B). In this regard, it is interesting to note that patient no. 1 is resistant to therapy with 2-CdA.
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| Fig 8.
Effect of 2-CdA on proliferation, bFGF release, and
apoptosis of HC. (A) A total of 2 × 105 cells/well were
cultured in medium or medium supplemented with bFGF (100 pg/mL) with or
without 2-CdA (1 µg/mL). After 72 hours, cells were pulsed with 5 µCi/mL 3H-TdR and incorporation was measured by liquid
scintillation counting. Mean values of triplicate cultures are shown.
(B) A total of 2 × 106 PBMC from two patients with HCL
were cultured in 6-well plates in the absence or presence of 2-CdA (1 µg/mL). After 48 hours, culture supernatants were screened by an
ELISA specific for human bFGF. Results are expressed as mean values of
triplicate cultures. (C) A total of 2 × 106 PBMC from a
healthy donor and a patient with HCL were cultured in medium or medium
containing 2-CdA (1 µg/mL, 0.1 µg/mL). Cells were harvested after
48 hours and assayed for FITC-labeled Annexin binding by FACS. Shown
are results of a representative experiment.
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In the next set of experiments, we tested whether bFGF expression in HC
is paralleled by a reduced sensitivity for 2-CdA-mediated apoptosis.
Cells were cultured for 48 hours and analyzed for Annexin-V binding, a
feature of cells undergoing apoptosis. Figure 8C shows that 2-CdA
induced Annexin-V binding in 52% of PBMC from a healthy donor, while
only 26% of bFGF-producing HC bound Annexin-V. These results might
indicate resistance to 2-CdA via bFGF.
| |
DISCUSSION |
The present study reports on the detection of elevated levels of bFGF
in serum and BM from patients with HCL. This study also indicates that in HCL, bFGF is released in a biologically active form
by the malignant cells, as CM derived from HC was shown to induce a
proliferative response on fibroblasts, which could be diminished by
treatment with a neutralizing anti-bFGF antibody. Stimulation of PBMC
with PWM or TPA plus Ca-Ip led to an increase in bFGF mRNA expression
in HCL, substantiating that bFGF expression can be upregulated by
cellular activation. In cell lysates, three different isoforms can be
detected by immunoblotting, but only a single bFGF isoform of
approximately 23 kD is released by HC. Double-immunofluorescence and
cell separation studies showed that the cytokine is produced and
exported by CD19+/CD11c+ cells. Finally, bFGF
expression is not blocked by 2-CdA and seems to mediate resistance to
2-CdA-mediated apoptosis.
Expression of bFGF and its receptors has been shown in few other
hematologic malignancies, and the cytokine was implicated to play a
potential pathological role in these diseases. Thus, in patients with
myelofibrosis, an increased production of bFGF has been correlated with
a possible involvement with the fibrotic process.14 In
chronic lymphocytic leukemia, elevated bFGF levels were found to
correlate with the stage of disease activity and resistance to
fludarabine.15
HCL, a rare form of chronic leukemia, is characterized by several
unique and unusual features (reviewed in Hess31). Some of
them might be explained by abnormalities in cytokine
production32 leading to BM insufficiency or deregulation of
adhesion molecule expression.33
It is important to note that release of bFGF seems to be a feature of
tumor cells, as transformed cells derived from breast carcinoma or
fibrosarcomas export bFGF, while in nontransformed fibroblasts, bFGF
remains cell-associated.34-36 In addition, acquisition of a
signal peptide by molecular cloning converts bFGF to induce transformation of mouse fibroblasts, which are tumorigenic and metastatic in nude mice.37
The bFGF detected in culture supernatants from HCL patients was capable
of inducing proliferation of primary human fibroblasts. This might
implicate that the cytokine exerts important effects also on cells in
the close proximity to HC, ie, on mesenchymal cells, or molecules
deposited within the extracellular matrix, eg, heparan sulfate
proteoglycans (HSPG). Complex formation of HSPG with bFGF facilitates
binding to FGF receptors and thereby triggers cellular
activation.38,39
In HCL, bFGF might also be operative through other growth factors.
Among these transforming growth factor-1 (TGF-1) is a possible
candidate. Interactions between bFGF and TGF-1 have been described
in several biological systems40-42 and may also be relevant
for BM fibrosis seen in HCL. In this context, bFGF might contribute to
BM fibrosis via activation of latent TGF-1 in the BM
microenvironment and consequently induction of extracellular matrix
(ECM) synthesis and accumulation.43
Indeed, both active and latent forms of TGF- are highly elevated in
HCL, particularly in BM (M. Shehata, in preparation).
One possible mechanism by which bFGF might exert a direct role in
leukemogenesis could be through the prevention of apoptosis. There is
evidence presented by Menzel et al15 who showed that bFGF
accumulates intracellularly in malignant B cells from chronic lymphocytic leukemia (B-CLL) and delays fludarabine-induced cell death.
Similarily, experiments described in this report showed that
recombinant bFGF was capable of protecting HD-derived, as well as
HCL-derived PBMCs, from the cytopathic effects of 2-CdA. Moreover,
production and release of endogenous bFGF was not affected by 2-CdA.
Therefore, overexpression and export of bFGF may mediate resistance to
therapy and contribute to tumor progression.
Besides a possible direct effect on the survival and growth of
CD19+/CD11c+ B cells, bFGF may also influence
immune surveillance. In vitro and in vivo studies have shown that bFGF
inhibits adhesion of natural killer (NK) cells to endothelial
cells.33 Provided that bFGF functions in a similar way on
HC, it may exert a protective mechanism for the malignant cells in the
presence of NK cells. Evidence for a reduced NK cell activity in HCL
has been provided earlier.44,45
In primary mesenchymal cells, bFGF is produced as an 18-kD glycoprotein
without a signal sequence.46,47 In addition to the 18-kD
form, at least three N-terminal extended bFGF isoforms of approximately
22, 23, and 25 kD molecular weight have been described. They originate
from unusual translational initiation at CUG codons upstream of the
original AUG initiation codon.48 While the 18-kD form of
bFGF is present in the cytosol, these high molecular weight forms are
predominantly associated with the nucleus due to a nuclear localization
signal in the alternatively expressed N-terminal region.49
Interestingly, bFGF derived from HCL displayed a molecular weight
(MW) of 23 kD, an isoform that is characterized by its
nuclear localization. It is feasible that high MW forms of bFGF also
control gene expression because the presence of a high MW form within
the nucleus may facilitate interaction with transcription factors. It
has been shown by Kevil et al50 that the translation
initiation factor eIF-4 preferentially increases translation of CUG-1
isoforms, resulting in release of large quantities of the 23-kD
isoform. Whether or not transcription factor-mediated mechanisms are
also responsible for the elevated bFGF expression seen in HCL remains
to be elucidated.
Both production and distribution of a particular isoform may be
instrumental for cell proliferation, as well as development and
progression of tumors. Therefore, bFGF may play a role in the
development of pathological conditions in HCL and hence be involved in
myelofibrosis, prevention of apoptosis, and proliferation of HC.
| |
FOOTNOTES |
Submitted July 14, 1997; accepted April 6, 1999.
Supported in part by Grants No. 9217 and 11210 from the Austrian
Science Foundation.
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 correspondence to Rudolf Berger, PhD, Ludwig Boltzmann
Institute for Cytokine Research, PO Box 16, A-1097 Vienna, Austria;
e-mail: rudolf.berger{at}akh-wien.ac.at.
| |
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ABSTRACT
INTRODUCTION