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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2595-2604
Mechanism of flt3 Ligand Expression in Bone Marrow Failure:
Translocation From Intracellular Stores to the Surface of T
Lymphocytes After Chemotherapy-Induced Suppression of Hematopoiesis
By
Elena Chklovskaia,
Wendy Jansen,
Catherine Nissen,
Stewart D. Lyman,
Christoph Rahner,
Lukas Landmann, and
Aleksandra Wodnar-Filipowicz
From the Department of Research, University Hospital Basel,
Switzerland; IMMUNEX Corp, Seattle, WA; and the Institute of Anatomy,
University of Basel, Basel, Switzerland.
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ABSTRACT |
The flt3 ligand (FL) is a growth factor for primitive hematopoietic
cells. Serum levels of FL are inversely related to the number and
proliferative capacity of early hematopoietic progenitors. We sought to
elucidate the molecular mechanism underlying this regulation.
Expression of FL was examined in peripheral blood (PB) and bone marrow
(BM) cells under normal steady-state hematopoiesis and during transient
BM failure induced by chemoradiotherapy in 16 patients with
hematological malignancies. Using anti-FL antibodies in Western
analysis, flow cytometry, and confocal microscopy, we detected high
levels of preformed FL inside but not on the surface of T lymphocytes
in steady-state hematopoiesis. Intracellular FL colocalized with
giantin and ERGIC-53, indicating that it is stored within and close to
the Golgi apparatus. After chemotherapy-induced hematopoietic failure,
FL rapidly translocated to the surface of T lymphocytes and the levels
of FL released to serum increased approximately 100-fold. Expression of
FL mRNA was enhanced only about sevenfold; a similar, twofold to
sixfold increase in mRNA was observed in the thymus and BM of mice with
irradiation-induced aplasia. Upregulation of FL mRNA was delayed when
compared with the appearance of cell surface-associated and soluble
protein isoforms. The described changes in FL expression in response to chemotherapy-induced aplasia were observed in all patients,
irrespective of the diagnosis and treatment regimen. Our data
demonstrate that mobilization of preformed FL from intracellular stores
rather than de novo synthesis is responsible for increased FL levels in
BM failure.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS is a multistep cell
proliferation and differentiation process sustained by a pool of
hematopoietic stem cells located primarily in the bone marrow (BM).
Pathways that lead from individual stem cells to the various terminally
differentiated blood cells are tightly regulated and involve complex
interactions between soluble and membrane-bound growth factors and
their corresponding cell surface receptors.1,2 The flt3
ligand (FL) belongs to the small family of hematopoietic cytokines,
including also stem cell factor (SCF) and macrophage colony-stimulating
factor (M-CSF), that are specific for tyrosine kinase
receptors.3-5 The flt3 receptor is predominantly
expressed on primitive hematopoietic progenitors, an indication that
signaling through this receptor is important in the very early steps of
hematopoiesis.6 Indeed, FL has proven to be a potent
cytokine for in vitro expansion and in vivo mobilization of stem cells
(reviewed in Lyman and Jacobsen7). It also increases the
yield of dendritic cells generated from hematopoietic
precursors8,9 and effectively enhances antitumor immune
responses in mice.10 So far, most studies have focused on
elucidating the biological properties of FL and less attention has been
paid to the mechanisms regulating expression of the cytokine in human
hematopoiesis. In contrast to the restricted distribution of
flt3 receptors, FL mRNA is ubiquitously expressed in
hematopoietic and nonhematopoietic tissues.4,11 Analysis of
multiple splice variants of human FL mRNA indicates that the
predominant form of FL is a cell surface transmembrane protein, which
presumably generates soluble FL upon proteolytic
cleavage.12 The protease responsible for this cleavage has
not yet been identified, and neither is it known whether the two FL
isoforms have different functions in hematopoiesis.
Serum levels of FL increase dramatically, from an average of 14 up to
7,000 pg/mL, in diseases characterized by a deficiency of primitive
hematopoietic progenitors, such as congenital and acquired aplastic
anemia, and during transient pancytopenia induced by high-dose
chemotherapy.13,14 FL levels do not increase in diseases
affecting single blood lineages,13 in contrast to
lineage-specific growth factors such as erythropoietin, thrombopoietin,
and granulocyte colony-stimulating factor, which are elevated in pure
red blood cell aplasia, congenital thrombocytopenia, and severe
congenital neutropenia, respectively.15-17 Moreover,
response to multilineage hematopoietic damage is specific for FL,
because serum levels of a related early acting cytokine, SCF, do not
change in chemotherapy-induced aplasia or aplastic
anemia.18,19 These results suggest that FL has a
nonredundant role in the compensatory hematopoietic response in BM
failure aimed at replenishment of the stem cell compartment and
restoration of normal hematopoiesis.
The mechanism responsible for the dramatic increase in
circulating FL levels in patients with BM failure has not been
identified. In this study, we investigated the regulation of FL
expression in peripheral blood (PB) and BM cells during
chemotherapy-induced transient aplasia. The results indicate
that, under physiological steady-state conditions, FL is produced
constitutively but retained intracellularly within and close to the
Golgi apparatus. In hematopoietic failure, FL is rapidly translocated
to the cell surface of T lymphocytes. The existence of such a
regulatory mechanism securing a rapid supply of FL in hematopoietic
deficiency argues for the importance of this cytokine in reconstitution
of BM function in primary and iatrogenic BM failure.
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MATERIALS AND METHODS |
Patients and controls.
Sixteen patients with different hematological malignancies who
underwent myeloablative therapy were included in the study; 7 patients
underwent chemotherapy and 9 patients were transplanted. For the
detailed characteristics of the disease and therapy, see Table 1. All PB and BM samples were
obtained with informed consent in compliance with the guidelines of the
Ethical Committee of the University Hospitals of Basel (Basel,
Switzerland). Control PB and BM from normal donors were used after
informed consent was received.
Cell purification.
Mononuclear cells from heparinized PB or BM (PBMC and BMMC,
respectively) were isolated by Ficoll-Hypaque density gradient centrifugation (d = 1.077). For flow cytometry (fluorescence-activated cell sorting [FACS]), confocal microscopy, RNA
preparation, and protein extraction (see below), cells were used
immediately without prior freezing. T cells were purified from PBMC of
normal donors. For confocal microscopy, they were rosetted with sheep
erythrocytes. For in vitro cultures, negative selection using
super-paramagnetic MACS MicroBeads (Miltenyi Biotech, Auburn, CA) was
used. Purified T cells were cultured in Iscove's modified Dulbecco's
medium under serum-free conditions, in the absence or presence of 10 µg/mL of brefeldin A and cycloheximide (both from Sigma
Immunochemicals, St Louis, MO).
Preparation of PB serum and measurement of soluble FL.
Serum from patients undergoing chemotherapy was collected daily from
native PB after centrifugation for 10 minutes at 3,000 rpm. Samples
were aliquoted and stored at  70°C. Soluble FL was measured
by an enzyme-linked immunosorbent assay (ELISA); the limit of detection
was 100 pg/mL.13
FACS analysis of membrane-bound FL.
PBMC and BMMC, washed in a FACS buffer containing phosphate-buffered
saline (PBS), 0.5% bovine serum albumin (BSA), and 0.02% NaN3, were incubated for 20 minutes on ice with monoclonal
antibody (MoAb) M5 against human FL (rat IgG2a13), followed
by fluoresceine isothiocyanate (FITC)-conjugated goat antirat IgG
(Caltag Lab, Burlingame, CA). M5 antibody specifically recognizes an
epitope in the extracellular domain of FL.13 For
double-color FACS analysis, MoAb M5 followed by goat antirat IgG-FITC
with minimal species cross-reactivity (Jackson Immunoresearch
Laboratories, West Grove, PA) and phycoerythrin (PE)-labeled mouse
antihuman CD3 MoAb (Becton Dickinson, San Jose, CA) were used. Control
staining was with goat antirat IgG-FITC (Jackson Immunoresearch
Laboratories) and PE-labeled mouse IgG1 (Becton Dickinson). Acquisition
was performed with FACScan (Becton Dickinson). The lymphocyte gate was
set according to forward and sideward light scatter; dead cells were
stained with propidium iodide and excluded from the analysis. Ten
thousand to 25,000 events were acquired. Analysis was performed using
CellQuest software (Becton Dickinson).
FACS analysis of intracellular FL.
The analysis was performed as decribed.20 Briefly, 2 × 105 PBMC or BMMC were washed in a FACS buffer and
preincubated with an excess of MoAb M5 (100 µg/mL for 1 hour) to
prevent further detection of any membrane-bound FL. After fixation in
4% paraformaldehyde, cells were permeabilized for 1 hour in PBS
containing 0.5% BSA and 0.1% saponin (PBS-S buffer) and stained with
MoAb M5 that had been conjugated with FITC using FluoroTag FITC
conjugation kit (Sigma Immunochemicals). To confirm the specificity of
detection of intracellular FL, control experiments were performed using FITC-conjugated MoAb M5 preincubated with a 100-fold molar excess of
recombinant human FL (rhFL) or BSA. Five thousand to 10,000 events were
acquired and analyzed as described above.
Confocal microscopy.
PBMC or T cells purified from PBMC by rosetting were allowed to settle
onto Poly-L-Lysin-coated coverslips (Sigma), fixed with 4%
paraformaldehyde, and permeabilized with 0.1% saponin. Unspecific
staining was blocked with 0.5% sodium borohydride (Merck, Darmstadt,
Germany) and 5% goat serum. Cells were incubated for 1 hour at room
temperature with MoAb M5 or control rat IgG2a (PharMingen, San Diego,
CA) and mouse antihuman CD3, washed in PBS-S, and stained for 1 hour in
the dark with secondary antibodies (goat antirat IgG-FITC [Jackson
Immunoresearch Laboratories] and goat antimouse IgG-Cy3 [Amersham,
Pittsburgh, PA]). For intracellular localization of FL, T cells were
stained with MoAb M5 as described above and with antibodies against
human giantin, ERGIC-53, transferrin receptor, lamp-1 (all mouse IgG1;
gift from Hans-Peter Hauri, Biozentrum, Basel,
Switzerland) or rabbit antihuman cathepsin D (Accurate Chemicals, San
Francisco, CA), followed by goat antimouse or goat antirabbit IgG-Cy3
(Amersham). Slides were mounted with Mowiol (Hoechst, Frankfurt,
Germany) containing 20 mg/mL 1,4-diazabicyclooctane (DABCO; Sigma).
Confocal microscopy was performed with TCS4D (Leica, Glattbrugg,
Switzerland) operating in the sequential acquisition mode with 488 (FITC) and 568 (Cy3) excitation lines. Images were adjusted for
brightness and contrast and were analyzed for colocalization using the
IMARIS (Bitplane AG, Zürich, Switzerland) software package.
Induction of aplasia in mice.
Ten-week-old C57BL/6 mice were subjected to 8 Gy total body
irradiation. Groups of three mice were killed at 0, 3, 6, and 10 days
and tissue RNA was prepared after determination of BM cellularity. The
experiments were approved by the local animal welfare committee.
RNA preparation and reverse transcription-polymerase chain rection
(RT-PCR).
Total cellular RNA was extracted.21 Murine tissues were
rinsed in PBS upon excision, immediately transferred to guanidinum isothiocyanate, and homogenized using Polytrone PT 1200 (Kinematica AG,
Luzern, Switzerland). One microgram of RNA was reverse transcribed into
complementary DNA (cDNA) in 20 µL reactions using 2.5 µmol/L random
hexamers (Perkin Elmer, Branchburg, NJ) as primers and 2.5 U/µL
Superscript II RNase H -reverse transcriptase (GIBCO,
Gaithersburg, MD). Four microliters of the cDNA product and 0.15 µmol/L of each oligonucleotide primer specific for FL or  -actin
sequences were amplified by PCR using AmpliTaq DNA polymerase at 0.025 U/µL (Perkin Elmer). A control reaction containing water instead of
RNA was performed. The following programs were used for both FL and
-actin: denaturing at 94°C for 1 minute, annealing at 60°C
for 1 minute, and extending at 72°C for 1 minute; for 25 cycles
(human probes) or 27 cycles (murine probes) in Perkin Elmer Cetus DNA
termal cycler 8930 (Norwalk, CT). Sense and antisense primers
corresponded to the following nucleotide positions: human FL, 60-81 and
345-366 in exons 2-411; murine FL, 139-160 and 411-431 in
exons 2-422; human -actin, 2038-2058 and
2447-246623; and murine -actin, 530-553 and
738-761.24 PCR products were separated in 1.2% agarose
gels, examined by staining with ethidium bromide, transferred onto
nylon membranes, and hybridized with the
( -32P)ATP-labeled oligonucleotide probes corresponding
to the internal sequences of the amplified PCR products: human FL,
173-190; murine FL, 202-222; human -actin, 2058-2075, and murine
-actin, 639-659. Radioactivity in each band was quantified in
PDUnits using Phosphoimager analysis (Bio-Rad, Hercules, CA). FL mRNA
levels were determined based on the ratio of signals given by FL and
-actin PCR products. Autoradiography was performed using X-OMAT
Kodak x-ray film and amplifying screens (Eastman Kodak, Rochester, NY).
Protein extraction and Western analysis.
PBMC or BMMC were lysed in extraction buffer containing 1% Triton-X,
50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 3 mmol/L
phenylmethylsulfonylfluoride, 20 µg/mL aprotinin, and 50 µg/mL
leupeptin (all protease inhibitors from Boehringer Mannheim, Mannheim,
Germany). Extracts from 1 × 106 cells or 5 ng of rhFL
in Laemmli's sample buffer were electrophoresed on reducing sodium
dodecyl sulfate (SDS)-polyacrylamide gels (12%) and transferred to
nitrocellulose. Western analysis of FL was performed with MoAb M5
followed by secondary horseradish peroxidase-conjugated rabbit antirat
IgG (Southern Biotechnology, Birmingham, AL), and detection was with
enhanced chemiluminesce (Pierce, Rockford, IL). For the control
immunoblotting, isotype-matched rat IgG2a (PharMingen) or secondary
antibody only were used. For the control antibody-blocking experiments,
MoAb M5 was preincubated with 50-fold molar excess of rhFL for 1 hour
on ice.
Statistical analysis.
The Student's t-test was used to compare the time course of
expression of FL mRNA and FL protein isoforms. Spearman Rank
Correlation test was used to analyze the relationship between FL serum
levels and PB leukocyte counts.
| |
RESULTS |
Expression of FL during normal hematopoiesis.
In humans, the highest levels of constitutively expressed FL mRNA have
been reported in PBMC,4,11 but expression of FL protein in
these cells has not been analyzed. Western analysis of PBMC and BMMC
showed multiple FL-specific signals with some heterogeneity in their
pattern in cells from individual healthy donors
(Fig 1A, lanes 2 through 4 and 8 through
10). The size of detected proteins was larger than that of the
recombinant soluble FL. The major products of about 30 and 36 kD were
similar in size to proteins expressed in COS cells transfected with FL
cDNA, which are thought to represent the intermediate and fully
glycosylated transmembrane forms of FL, respectively.25
Additional bands may correspond to heterogenously glycosylated proteins
and/or products of alternatively spliced variants of FL mRNA.
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| Fig 1.
Expression of FL in PBMC or BMMC. (A) Immunoblot analysis
of FL. Recombinant human CHO-derived FL (rhFL; 5 ng; lanes 1 and 7)
or cell lysates from 1 × 106 PBMC (lanes 2 through 4)
or BMMC (lanes 8 through 10) from 3 healthy donors were analyzed by
Western blotting with anti-FL MoAb M5. Controls: lanes 5 and 6, PBMC
and rhFL, respectively, analyzed with rat IgG2a (control Ab); lanes 11 and 12, BMMC and rhFL, respectively, analyzed with M5 preincubated with
50-molar excess of rhFL before probing (M5 Ab-blocked). Migration of
protein size markers is indicated; arrowheads point to 30- and 36-kD
proteins. The amount of FL can be estimated as approximately 1 to 5 ng/106 mononuclear cells, because signals are weaker than
those given by 5 ng of rhFL (lanes 1 and 7) and no signal is given by 1 ng of loaded rhFL (not shown). (B) Flow cytometric analysis of cell
surface FL. PBMC or BMMC were stained with FITC-conjugated MoAb M5
(shaded area) or with FITC-conjugated control rat IgG2a (broken line).
(C) Flow cytometric analysis of intracellular FL. PBMC or BMMC were
preincubated with MoAb M5 to block cell surface FL,
permeabilized with 0.1% saponin, and stained with FITC-conjugated
M5 (shaded area). Control staining was with FITC-conjugated M5
preincubated for 30 minutes with a 100-fold excess of rhFL (solid line)
or BSA (dotted line).
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Although detected in Western analysis, FL levels were unmeasurable or
very low on the surface of BM and PB cells when analyzed by flow
cytometry (Fig 1B). However, immunostaining for FL was positive after
permeabilization of cells with saponin (Fig 1C). The intracellular
signal was abrogated after preincubation of anti-FL antibody with
recombinant human FL, but not with bovine serum albumin, confirming
that recognition of the ligand inside the cells was specific.
Dual-staining of permeabilized cells with anti-FL antibodies and
antibodies against mononuclear cell surface markers detected FL inside
peripheral CD3+ and CD19+ lymphocytes and in
CD34+ hematopoietic progenitors purified from BM, but not
in purified PB monocytes (data not shown). These results of the Western
and FACS analyses demonstrate that, during normal steady-state
hematopoiesis, FL is produced by PB and BM cells but is undetectable or
very sparse on their surface.
Expression of FL during aplasia.
We have previously demonstrated that stem cell deficiency
in cancer patients receiving high-dose chemoradiotherapy leads to an
approximately 100-fold increase in serum levels of FL.14 To
examine the mechanism by which FL levels are upregulated, 16 patients
with various hematological malignancies were recruited for the study
(Table 1). From day 8 or 9 after initiation of chemotherapy, all
patients developed severe aplasia lasting for at least 2 weeks, with
peripheral leukocyte counts decreasing from 5 to 10.0 × 109/L to as little as 0.1 × 109/L.
As in cells from healthy donors, surface expression of FL in patients'
cells before treatment was very low or undetectable (Fig 2A). However, 3 to 5 days after
initiation of therapy, membrane-bound FL appeared on the cell surface
and remained high throughout the period of aplasia. Surface expression
of the ligand then gradually declined to barely detectable pretreatment
levels along with hematopoietic recovery. The kinetics of the
appearance of membrane-bound FL paralleled changes in serum levels of
soluble FL (Fig 2B). As measured by ELISA, FL was undetectable in serum
before treatment, increased to 200 to 700 pg/mL after 3 to 5 days and
to 2,500 pg/mL during severe aplasia, and then decreased upon
normalization of blood cell counts. It should be emphasized that the
initial increase in both membrane-bound and soluble FL occurred when
leukocyte counts were still within the normal range (see Fig 2A). To
identify the cell population expressing surface FL, antibodies specific for mononuclear cell surface markers were used in flow cytometry. In
severe aplasia, the vast majority of CD3+ cells in both PB
and BM expressed high levels of cell surface-bound FL
(Fig 3). Persistent cell surface expression
of FL in T cells during aplasia was observed with all analyzed patients
undergoing myeloablative chemotherapy, independent of the underlying
disease and treatment regimen.
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| Fig 2.
Expression of cell surface and soluble FL in
chemotherapy-induced aplasia. (A) Flow cytometric analysis of
cell surface FL. PBMC were stained with FITC-conjugated MoAb M5
(shaded area) or with FITC-conjugated control rat IgG2a (broken line).
Analysis was performed with 9 patients; representative results for 3 patients are shown. Days of treatment and peripheral leukocyte count
are indicated. (B) Serum concentrations of soluble FL (sFL) determined
by ELISA. *Below the detection limit of 100 pg/mL. Results refer to the
same 3 patients and same days as the FACS data shown in (A). The
standard deviation is indicated. Correlation between sFL levels and
leukocyte count was .51 (P < .0001).
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| Fig 3.
T lymphocytes express FL on the cell surface in
chemotherapy-induced aplasia. Flow cytometric analysis of
membrane-bound FL in PBMC or BMMC stained with PE-conjugated anti-CD3
MoAb and FITC-conjugated anti-FL MoAb M5. Analysis was performed with 4 patients; representative results for patients B.G. and K.B. are
shown.
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Confocal microscopy was used to visualize changes in FL distribution
during steady-state and compromised hematopoiesis. In normal peripheral
T cells, FL was detected intracellularly as a strong clustered signal
(Fig 4B). In T cells from patients during aplasia, FL signals became scattered in the typical vesicular distribution pattern of a secreted protein, visible also in the outer
rim of the cytoplasm, partly overlapping with staining of the CD3
surface antigen (Fig 4C). These results confirm that FL is present
inside T cells during steady-state hematopoiesis and is translocated to
the cell surface in hematopoietic failure.
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| Fig 4.
Two-color immunofluorescence confocal microscopy analysis
of FL expression in T lymphocytes. PBMC were settled on the slides,
fixed with paraformaldehyde, permeabilized with 0.1% saponin, and
stained with anti-FL MoAb M5 followed by FITC-labeled goat antirat IgG
and with anti-CD3 antibody followed by Cy3-labeled goat antimouse IgG.
(a) Donor cells, staining with anti-CD3 (red) and control rat IgG2a
followed by secondary FITC-labeled goat antirat IgG (no signal). (b)
Donor cells stained for CD3 (red) and FL (green). (c) Patient's cells
(M.T.) stained for CD3 (red) and FL (green). CD3 staining of patient's
cells was partly destroyed during handling due to drug-related
fragility of cell membranes. Areas of overlap are highlighted in
yellow.
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Intracellular localization of FL in T cells.
To define more precisely the localization of intracellular FL in T
lymphocytes during normal hematopoiesis, we performed dual immunofluorescence confocal microscopy using anti-FL antibodies and
antibodies recognizing organelle-specific proteins
(Fig 5). FL colocalized to a large degree
with giantin, a Golgi marker, and ERGIC-53, a marker for the
endoplasmic reticulum/Golgi intermediate compartment. FL did not
colocalize with the transferrin receptor, a marker for early endosomes;
lamp-1, a marker for late endosomes and lysosomes; or cathepsin D, a
marker for lysosomes (reviewed in Farquhar and Hauri26 and
Rothman and Wieland27). These results indicate that
preformed FL in peripheral T lymphocytes is localized in the Golgi
apparatus and in nearby structures of the endoplasmatic reticulum.
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| Fig 5.
Localization of intracellular FL (Flt3L) in T
lymphocytes. Two-color immunofluorescence confocal microscopy of T
cells purified from PB of a healthy donor was performed after staining
with anti-FL MoAb M5 followed by FITC-labeled goat antirat IgG (green)
and with antibodies against giantin, ERGIC-53, transferrin receptor
(TfR), lamp-1 (all mouse IgG1), and cathepsin D (rabbit IgG), followed
by secondary Cy3-labeled goat antimouse IgG or goat antirabbit IgG, as
appropriate (red signals). Areas of exact overlap of green FL signal
with red signals of giantin or ERGIC-53 are highlighted in yellow.
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To verify that it is preexisting FL that is translocated from the Golgi
to the cell surface, T cells were treated with brefeldin A, a reagent
that blocks protein secretion by disrupting the Golgi apparatus,28 or with cycloheximide to inhibit translation.
In vitro cultures of purified T lymphocytes were established and analyzed by flow cytometry (Fig 6). After
72 hours in serum-free conditions, T cells spontaneously expressed FL
on their surface, indicating that mere exposure to in vitro conditions
is sufficient to induce externalization of the ligand. Importantly, the
surface appearance of FL in cultured cells was fully prevented by
brefeldin A, but not by cycloheximide. The clearly pronounced
difference between the effects of the two drugs demonstrates the
requirement for a functional protein transport machinery, but not de
novo protein synthesis, and confirms that the control of FL expression is fundamentally posttranslational.
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| Fig 6.
Effect of brefeldin A and cycloheximide on expression of
FL by cultured T lymphocytes. T lymphocytes, purified from PB of a
healthy donor, were cultured for 72 hours in serum-free medium in the
absence or presence of brefeldin A or cycloheximide (both at 10 µg/mL). Flow cytometric analysis of cell surface FL was performed
with anti-FL MoAb M5 followed by FITC-labeled goat antirat IgG. (Shaded
area) FL expression in untreated cells; (solid line) FL expression in
brefeldin A- or cycloheximide-treated cells, as indicated; (broken
line) control staining with FITC-labeled goat antirat IgG.
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FL mRNA expression in aplasia.
To examine if transcriptional regulation contributes to the increase of
FL in aplasia, we measured FL mRNA levels in PBMC collected during
chemotherapy (Fig 7A). Using
semiquantitative RT-PCR under conditions of the linear accumulation of
PCR products (Fig 7B), FL-specific transcripts were detected before
treatment, and their level increased approximately sevenfold during
severe aplasia and returned to pretreatment values upon hematopoietic recovery. An increase in mRNA level was observed between days 8 and 10 after the start of chemotherapy. Thus, the initial appearance of FL on
the cell surface and in serum (see Fig 2) preceded the initial increase
of FL mRNA (4.6 ± 0.7 and 4.3 ± 1.4 v 9.0 ± 1.5 days, respectively; P < .05). Furthermore, mRNA levels
increased only when pancytopenia was very severe, as judged by an at
least 50-fold reduction in white blood cells, whereas externalization of FL started when leukocyte counts were still within the normal range.
In parallel with a delayed increase in FL mRNA levels, an approximately
threefold increase of total cell content of FL protein was observed by
Western analysis (not shown). These data argue in favor of rapidly and
massively elevated membrane-bound and soluble forms of FL being derived
from the protein present in patients' cells before the beginning of
treatment rather than from de novo expression.
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| Fig 7.
Expression of FL mRNA during aplasia. (A)
Semiquantitative RT-PCR analysis in PBMC from patients during
chemotherapy treatment. Twenty-five PCR cycles were performed under
conditions of the linear accumulation of FL and -actin PCR products
(B). Southern blots were hybridized with 32P-labeled
internal probes, and radioactivity in each band was quantified by
phosphorimaging analysis. Autoradiography was for 4 hours (FL) and 30 minutes (-actin). Three analyses were performed for each of 4 analyzed patients; representative results for 2 patients are shown.
Days of treatment are indicated above and peripheral leukocyte counts
below the autoradiograms. Methods more accurate than RT-PCR could not
be applied given the extremely limited availability of patients' cells
at the time of treatment. (C) Semiquantitative RT-PCR analysis in mouse
tissues during irradiation-induced aplasia. Mice received 8.0 Gy total
body irradiation. RNA was isolated before irradiation (day 0), during
aplasia (days 3 and 6), and at the beginning of recovery (day 10), and
FL mRNA expression was analyzed as described above. Three analyses were
performed for 2 animals; representative results are shown. The BM
nucleated cell count per femur plus tibia is indicated below the
autoradiogram.
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To test the possibility that BM failure leads to an increase in FL mRNA
levels in other tissues, we induced transient aplasia in mice by
sublethal irradiation with 8.0 Gy and isolated RNA from several
hematopoietic and nonhematopoietic organs. FL mRNA increased
moderately, up to sixfold, only in the BM and thymus (Fig 7C), but
remained unchanged in spleen, kidney, lymph nodes, brain, liver, lung,
and heart (not shown). It thus appears unlikely that organs other than
the lymphohematopoietic system contribute significantly, at least by
assessment of mRNA content, to the increase in FL levels seen in
response to chemotherapy and irradiation in humans.
| |
DISCUSSION |
The restricted range of hematopoietic cells carrying flt3
receptors6 in conjunction with widespread expression of FL
mRNA4,11 led to the suggestion that expression of the
receptor and not the ligand is the key to understanding the role of FL
in hematopoiesis. In the work described here, FL expression was
examined by Western analysis, flow cytometry, and confocal microscopy
in PB and BM cells from healthy donors and patients with multilineage
BM failure caused by high-dose chemoradiotherapy. Our results show that
the supply of FL for hematopoiesis in humans is tightly regulated by a
process specific for this cytokine. This regulation is based on
intracellular retention of preformed FL and its release from intracellular stores, dependent on the status of the stem cell compartment.
During normal hematopoiesis, FL is produced constitutively but little
of the cytokine is released by cells: it is undetectable or very sparse
on the surface of cells and in the circulation. Instead, high levels of
preformed FL are present inside T and B lymphocytes and
CD34+ hematopoietic precursors. In T cells, intracellular
FL colocalized with ERGIC-53 and giantin, the proteins resident in the
endoplasmic reticulum/Golgi intermediate compartment and Golgi
apparatus, respectively.27 This finding is surprising
because, as a cytokine designed for an extracellular function, FL is
devoid of amino acid sequences serving as typical intracellular
retrieval/retention signals.11 The intracellular
accumulation of FL argues for the existence of a negative regulator
controlling the availability of the ligand in steady-state
hematopoiesis. This mechanism of intracellular retention of FL does not
function when cells are maintained outside their natural
microenvironment, because T lymphocytes cultured in vitro spontaneously
express FL at their surface. In vivo, release of the ligand from
intracellular stores may be triggered by stem cell deficiency in BM
failure. Indeed, the highest expression of membrane-bound FL after
myeloablative therapy was observed in patients with profound aplasia
(Fig 2A). The nature of inductive signal(s) generated by BM failure
remains unknown. In analogy to a heme-containing oxygen sensor involved
in feedback regulation of expression of erythropoietin,28 a
putative stem cell sensor may be controlling the level of FL by
counteracting the retention signal and mobilizing FL in response to
stem cell deficiency.
Intracellular storage and release upon demand distinguish FL regulation
from known mechanisms regulating the production of hematopoietic
cytokines by T cells and other blood cell types. Expression levels of
interleukin-2, interleukin-3, interferon-, and
granulocyte-macrophage colony-stimulating factor are primarily determined by the rate of mRNA transcription and/or
decay,29-31 and these factors have not been found to
accumulate in human PB cells.32 In contrast,
transcriptional control plays a secondary role in regulating the
expression of FL. FL mRNA is expressed constitutively, and the increase
in its levels in chemotherapy-induced marrow failure is less pronounced
and delayed compared with the increase in FL protein levels. No
difference in the proportion of the FL splice variants encoding
transmembrane and soluble FL in patients' as compared with normal PB
cells was found (results not shown). Transcript stability is not likely
to play a major regulatory role either, because FL mRNA is highly
stable, with a half-life of about 24 hours (results not shown).
Intracellular storage of the preformed protein also distinguishes FL
from SCF and M-CSF, two other ligands for hematopoietic tyrosine kinase receptors. SCF and M-CSF, produced predominantly by BM stroma cells,
are constitutively expressed but also rapidly secreted, resulting in
remarkably high (in nanograms per milliliter) serum levels of these
cytokines during normal hematopoiesis as well as hematopoietic
failure.18,19,33,34 Interestingly, expression of ligands
for Fas and CD40 may be regulated in some cell types in a manner
similar to FL. In primary resting human monocytes and platelets and in
Jurkat T cells, prestorage of these ligands by an unknown mechanism and
secretion in response to cell-specific stimuli have been recently
reported.35-37 Therefore, posttranslational control by
prestorage and regulated release, rather than de novo synthesis, is
emerging as a novel mechanism regulating the availability of
biologically relevant molecules.
During chemotherapy, surface expression of membrane-bound FL and serum
levels of FL increase in parallel. The mechanism by which soluble FL is
generated awaits identification of a putative protease that, by analogy
to the metalloproteinase-processing tumor necrosis
factor- ,38,39 may release FL from the membrane. The
simultaneous increase of both ligand forms in response to BM failure
and their simultaneous decrease upon hematopoietic recovery suggest
that circulating FL in serum is derived from cleavage of the ligand
translocated to the T-cell surface from intracellular stores. The
amounts of preformed intracellular FL, estimated as 1 to 5 ng/106 cells (see Fig 1, legend), would be sufficient to
elevate serum FL to the observed concentrations, if secreted by T cells
that are still present in normal numbers during the first days of
treatment. Alternative cell or tissue sources of FL cannot be ruled
out, although we could show that, in mice with experimentally induced aplasia, levels of FL mRNA increased only in BM and thymus, suggesting that lymphohematopoietic organs are the major site of FL upregulation in response to BM failure also in humans.
Our results point to a role for T-cell surface-associated FL in the
reconstitution of hematopoiesis. During myeloablation caused by the
severe toxicity of chemotherapy, T lymphocytes remain as a predominant
population of circulating cells. Membrane-bound FL, expressed in high
amounts throughout aplasia, may stimulate and enhance the level of
hematopoietic progenitors by direct cell-to-cell contact. Signaling by
membrane-bound and soluble FL isoforms may not be redundant, in analogy
to signaling by membrane-bound SCF on stroma cells having an essential
function for normal hematopoiesis.40,41 Cell-surface
expression of FL by T lymphocytes in aplasia suggests that not only
interactions between stroma and progenitor cells, but also those
between immune and hematopoietic cells may be involved in a
compensatory growth factor response to overcome BM failure.
| |
ACKNOWLEDGMENT |
The authors thank A. Gratwohl, J. Passweg, I. Turkalij, and C. Pino for
patients' material; A. Rolink for irradiated mice; H.-P. Hauri for
MoAbs towards organellar markers; M. Bürk for help with
intracellular FACS; and N. Hynes, A. Lanzavecchia, G. De Libero, and
R.C. Skoda for critically reviewing the manuscript.
| |
FOOTNOTES |
Submitted October 6, 1998; accepted December 14, 1998.
Supported by grants from the Swiss National Science Foundation
(32-045926.95 and 7GUPJ041426), Basler Krebsliga (17/96),
Schweizerische Akademie der Medizinischen Wissenschaften, and Stiftung
zur Krebsbekämpfung to C.N., A.W.-F., and E.C.
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 Aleksandra Wodnar-Filipowicz, PhD,
Department of Research, University Hospital Basel, Hebelstr. 20, CH-4031 Basel, Switzerland; e-mail: Filipowicz{at}ubaclu.unibas.ch.
| |
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ABSTRACT
INTRODUCTION