From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Plenary paper
Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells
from spontaneous apoptosis through stromal cell–derived factor-1
Jan A. Burger, Nobuhiro Tsukada, Meike Burger, Nathan J. Zvaifler, Marie Dell’Aquila, and Thomas J. Kipps
A subset of blood cells from patients with
B-cell chronic lymphocytic leukemia (CLL)
spontaneously differentiates in vitro into
large, round, or fibroblast-like adherent
cells that display stromal cell markers,
namely vimentin and STRO-1. These cells
also express stromal cell–derived factor-1 (SDF-1), a CXC chemokine that ordinarily is secreted by marrow stromal cells.
Leukemia B cells attach to these bloodderived adherent cells, down-modulate
their receptors for SDF-1 (CXCR4), and
are protected from undergoing spontaneous apoptosis in vitro. Neutralizing antibodies to SDF-1 inhibit this effect. Moreover, the rapid deterioration in the survival
of CLL B cells, when separated from such
cells, is mitigated by exogenous SDF-1.
This chemokine also results in the rapid
down-modulation of CXCR4 and activation of p44/42 mitogen-activated proteinkinase (ERK 1/2) by CLL B cells in vitro. It
is concluded that the blood of patients
with CLL contains cells that can differen-
tiate into adherent nurse-like cells that
protect leukemia cells from undergoing
spontaneous apoptosis through an SDF1–dependent mechanism. In addition to
its recently recognized role in CLL B-cell
migration, SDF-1–mediated CLL B-cell activation has to be considered a new
mechanism involved in the microenvironmental regulation of CLL B-cell
survival. (Blood. 2000;96:2655-2663)
© 2000 by The American Society of Hematology
Introduction
B-cell chronic lymphocytic leukemia (CLL), the most common
adult leukemia in the Western hemisphere, is characterized by the
relentless accumulation of long-lived, mature, monoclonal B cells
in the blood, secondary lymphoid tissues, and marrow.1 Circulating
leukemia cells primarily are arrested in the G0/G1 phase of the cell
cycle and are resistant to undergoing programmed cell death.2,3
This is hypothesized to contribute to the noted resistance of CLL
cells to standard chemotherapy.4 Understanding the mechanism(s)
that contribute to the resistance of CLL cells to apoptosis could lead
to new and more effective therapeutic strategies for patients with
this disease.
Despite their longevity in vivo, CLL cells often undergo
spontaneous apoptosis under conditions that support the growth of
human B-cell lines in vitro.2,5 This implies that such ex vivo
conditions lack essential survival factors and that the resistance to
apoptosis is not intrinsic to the CLL cell.5-8 It recently has been
shown that CLL B cells, but not normal CD5⫹ B cells, may be
rescued from spontaneous or corticosteroid-induced apoptosis
when cultured with human marrow stromal cells.6,9 In patients with
CLL, the marrow invariably is infiltrated with leukemia cells.
Furthermore, the extent of marrow infiltration correlates with
clinical stage and prognosis.10,11 These observations indicate that
regulatory signals in the marrow microenvironment, particularly
contact with accessory cells, such as marrow stromal cells, may be
important for the prolonged survival of CLL cells in vivo.
The marrow is a complex tissue containing hematopoietic
progenitor cells and their progeny in close contact with a connective tissue network of mesenchymal-derived cells collectively
referred to as stroma.12 During B-cell development in the marrow,
programmed cell death is a physiologic regulator of homeostasis,
diverting a large fraction of B-lineage cells into an apoptotic death
pathway to eliminate functionless or potentially harmful cells.13,14
Critical factors for the survival of selected B cells are interactions
with stromal cells in the marrow microenvironment, expression of
surface immunoglobulin molecules, and expression of apoptosisregulatory proteins, such as bcl-2.15,16
A major advance for studies on the regulation of B lymphopoiesis by stromal cells was the development of the long-term B-cell
culture system by Whitlock and Witte.17 In these cultures, B-cell
lymphopoiesis is supported by adherent stromal cells that develop
into a layer in long-term marrow cultures. B cells adhere to or
migrate under this layer of marrow stromal cells,18 which is similar
to the spontaneous migration of CLL B cells beneath marrow
stromal cells (pseudo-emperipolesis) that we recently characterized.19 Based on these observations it has been proposed that
stromal cell contact and short-range growth factors are critical
determinants for B lymphopoiesis.12,20
The chemokine stromal cell–derived factor-1 (SDF-1) plays an
important role in B-cell development. High levels of SDF-1 are
produced by stromal cells within the marrow, the primary site of
early B-cell differentiation.21,22 SDF-1–deficient, or SDF-1–receptordeficient, mice display severe defects in the generation of B cells
but not of T cells.23-26 SDF-1 regulates B lymphopoiesis by
retaining B-cell precursors in close contact with stromal cells
within the supportive hematopoietic microenvironment,27 preventing their premature release into the circulation. Moreover, SDF-1
From the Department of Medicine, Division of Hematology/Oncology, and the
Division of Rheumatology, Allergy, and Immunology, University of California at
San Diego; and the Department of Immunology, The Scripps Research
Institute, La Jolla, CA.
Reprints: Thomas J. Kipps, Division of Hematology/Oncology, School of
Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla,
CA 92093-0663; e-mail: [email protected].
Submitted March 20, 2000; accepted June 15, 2000.
Supported in part by Deutsche Krebshilfe grant D-96-17136 (J.A.B.), Deutsche
Forschungsgemeinschaft grant SA 623/2-1 (M.B.), and National Institutes of
Health grants 5R37-CA49870-11 and PO1-CA81534 (T.J.K.).
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
2655
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2656
BURGER et al
may also function as a B-cell growth factor. Initially, SDF-1 was
designated pre–B-cell growth-stimulating factor (PBSF), because
recombinant SDF-1 supported the proliferation of a stroma celldependent B-cell line (DW34).21 More recent studies also indicated
that SDF-1 can have a direct effect on the growth of B-lineage
cells.28,29 Most recently, we found that CLL B cells also express
functional receptors for SDF-1 and migrate to stromal cells that
secrete this chemokine.19
To gain insight into the regulation of CLL cell survival by
stromal cells and their products, in particular SDF-1, we characterized the conditions necessary for the survival of CLL cells in
long-term cultures of peripheral blood mononuclear cells (PBMC)
from patients with CLL.
Materials and methods
Cell purification, cell lines
After obtaining informed consent, blood samples were collected from
patients fulfilling diagnostic and immunophenotypic criteria for common
B-cell CLL at the University of California at San Diego Medical Center.1
The patients were not previously treated and had not received recombinant
growth factors or exogenous cytokines. Peripheral blood mononuclear cells
were isolated by density-gradient centrifugation over Ficoll-Hypaque
(Pharmacia, Uppsala, Sweden). Cells were used fresh or viably frozen in
fetal calf serum (FCS) containing 5% dimethyl sulfoxide for storage in
liquid nitrogen. The viability of the CLL cells was always greater than 85%
at the time of initiation of the CLL PBMC cultures, as determined by
staining the cells with 5 ␮g/mL propidium iodide (PI; Molecular Probes,
Eugene, OR) for 15 minutes at 37°C. All CLL PBMC samples examined
contained more than 90% CLL B cells, as determined by FACS analysis
with anti-CD19, anti-CD5, and anti-CD3 monoclonal antibodies (mAb).
The murine marrow stromal cell line M2-10B4 was purchased from the
American Type Culture Collection (Rockville, MD). CLL cells and cell
lines were cultured at 37°C, 5% CO2 in RPMI 1640 supplemented with 10%
FCS and penicillin–streptomycin–glutamine (Gibco BRL, Rockville, MD;
complete RPMI medium).
Chemokine, antibodies, flow cytometry
Synthetic human SDF-1␣ (1-67) was purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibodies used for flow cytometry that
were specific for CXCR4 (12G5), CD3, CD19, or isotype controls were
purchased from PharMingen (San Diego, CA). The mAb STRO-130 was
purchased from the Developmental Studies Hybridoma Bank, The University of Iowa (Iowa City, IA). The mAbs specific for Vimentin (V9) or CD68
mAb (EBM11) were purchased from DAKO A/S (Glostrup, Denmark), and
those used for immunohistochemistry that were specific for CD14 or
CD106 and isotype controls were purchased from PharMingen. For
inhibition of the SDF-1–mediated “nursing” function of nurse-like cells
(NLC), anti–SDF-1 polyclonal goat IgG was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). To remove sodium azide, we used the
Ultrafree-0.5 Centrifugal Filter Device (Millipore, Bedford, MA). For flow
cytometry, the cells were adjusted to a concentration of 5 ⫻ 106 cells/mL in
RPMI 1640 with 0.5% bovine serum albumin (FACS buffer). 5 ⫻ 105 cells
were stained with saturating antibody concentrations for 30 minutes at 4°C,
washed 2 times, and analyzed on a FACSCalibur (Becton Dickinson,
Mountain View, CA). Flow cytometry data were analyzed using the FlowJo
2.7.4 software (Tree Star, San Carlos, CA).
Long-term cultures of PBMC from patients with CLL
To examine the survival of CLL cells in long-term cultures with or without
marrow stromal cells, PBMC from patients with CLL were suspended in
RPMI 1640 supplemented with 10% FCS and penicillin–streptomycin–
glutamine (Gibco BRL) to a final concentration of 1.5 ⫻ 107 cells/mL.
These cells were assessed for viability and expression of CD19, CD3, and
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
CXCR4 before they were plated in tissue culture-treated 6-well plates with
or without stromal cells (4 mL/well). For cocultures with marrow stromal
cells, M2-10B4 stromal cells were seeded the day before initiation of CLL
cultures into 6-well plates at a concentration 2 ⫻ 105 cells per well. The
CLL cells then were cultured at 37°C in 5% CO2 in air for 14 days, during
which 300-␮L aliquots were removed at the indicated time points from
alternative wells for viability assays, as described below. At the same time,
cultures were examined by phase-contrast microscopy for the outgrowth of
adherent NLC, and the outgrowth of these cells was measured by counting
the adherent cells in 3 different visual fields per patient sample at 200⫻
magnification.
After 14 days, the nonadherent CLL cells were harvested by vigorously
pipetting the contents of the well and subsequently rinsing the plates with
complete RPMI medium. Harvested cells were washed and then suspended
to a concentration of 2.5 ⫻ 106 cells/mL in complete RPMI medium. Cells
then were plated onto 125-cm2 tissue culture plates and incubated for 2
hours at 37°C in 5% CO2 in air to remove any adherent cells by plastic
adherence. In the meantime, an aliquot of the harvested cells was examined
for expression of CD19, CD3, and CXCR4 and for viability. Afterward the
CLL cells again were harvested and suspended in complete RPMI medium
to a concentration of 1 ⫻ 107 cells/mL. Then CLL cells from each patient
sample were divided in half and either plated back onto the adherent NLC
or plated onto a fresh 6-well plate without NLC. Aliquots were removed at
the indicated time points for viability assays from both culture conditions.
The supernatants from CLL PBMC long-term cultures were harvested on
day 14 and used as conditioned medium from NLC. Conditioned medium
from M2-10B4 cells was generated as described.19 To determine effects of
these conditioned media on CLL cell survival, the CLL cells that were
separated from NLC on day 14 were cultured without NLC either in fresh
complete RPMI medium or complete RPMI medium mixed 1:1 with
conditioned medium.
Fluorescence in situ hybridization
We cultured PBMC from a patient with leukemia cells noted to have
trisomy 12 in chambered slides for 14 days. Cultures were rinsed with
sterile saline, fixed twice in 3:1 methanol:acetic acid, and air-dried.
Pretreatment and hybridization were performed by a modification of a
previously described protocol.31 Briefly, slides were pretreated in 0.75%
pepsin solution, dehydrated in ethanol series, and denatured in 70%
formamide. Hybridization was performed with directly labeled CEP 12
spectrum orange alpha satellite DNA probes (Vysis, Downers Grove, IL) for
15 to 18 hours, washed, and counterstained with 4⬘,6-diamine 2⬘phenylindole dihydrochloride (DAPI II) (Vysis). Nurse-like cells (large nuclei) and
CLL cells (small nuclei) were scored for number of fluorescent signals per
nucleus for 500 interphase nuclei.
SDF-1 expression of NLC from the blood of patients with CLL
PBMC from patients with CLL were isolated and suspended in complete
RPMI medium to a concentration of 1.5 ⫻ 107 cells/mL and incubated in
75-cm2 tissue culture flasks (Falcon) for 14 to 21 days. After this time,
nonadherent lymphoid cells were vigorously washed off; this was followed
by 2 washing steps. The complete removal of lymphocytes from the layer of
NLC was verified by phase-contrast microscopy.
NLC were lysed in the culture flask, and this was followed by RNA
extraction with the Qiagen RNeasy kit as described by the manufacturer
(Qiagen, Santa Clarita, CA). RNA then was used for first-strand cDNA
synthesis with the SuperScript preamplification system (Gibco BRL,
Rockville, MD), according to the manufacturer’s instructions. The following human SDF-1␤–specific primers were used: 5⬘ primer, GAG AAT TCA
TGA ACG CCA AGG TCG TG; 3⬘ primer, GAT CTA GAT CAC ATC TTG
AAC CTC TTG. A sequenced plasmid containing the human SDF-1␤
cDNA was used as a positive control. The annealing temperature was 58°C,
and the reaction proceeded for 35 cycles. To normalize for the amount of
RNA, we performed RT-PCR for human glyceraldehyde-3-phosphate
dehydrogenase (GA3PD), as described.32 To exclude that cDNA from
residual CLL B cells contributed to the amplification signal, CLL B cells
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
from 4 representative patients were purified with CD19-Dynabeads according to the manufacturer’s instructions (Dynal AS, Oslo, Norway); then
followed RNA extraction, cDNA synthesis, and RT-PCR for human SDF-1,
as described above.
Rescue of CLL cell viability by synthetic SDF-1␣
To determine the effect of exogenous SDF-1 on the survival of CLL B cells,
we replaced NLC in long-term cultures of CLL B cells 14 days after
initiation of the cultures with 500 ng/mL synthetic SDF-1␣ (Upstate
Biotechnology). Subsequently, the viability was monitored and compared
to that of the same CLL B cells in wells with or without NLC, as
described below.
Measurement of cell death
Determination of CLL cell viability in this study is based on the analysis of
mitochondrial transmembrane potential (⌬␺m) by 3,3⬘ dihexyloxacarbocyanine iodine (DiOC6) and cell membrane permeability to PI, as described.33,34 For viability assays, 300 ␮L CLL cell suspension was collected
at the indicated time points and transferred to FACS tubes containing 300
␮L of 60 nmol/L DiOC6 (Molecular Probes) and 10 ␮g/mL PI (Molecular
Probes) in FACS buffer. Cells then were incubated at 37°C for 15 minutes
and analyzed within 30 minutes by flow cytometry using a FACSCalibur
(Becton Dickinson). Fluorescence was recorded at 525 nm (FL-1) for
DiOC6 and at 600 nm (FL-3) for PI. For comparison, CLL cell viability was
also examined using the different relative size and granularity (forward
scatter and side scatter) characteristics of vital and dead cells.
Immunophenotyping of nurse-like cells
CLL PBMC isolated by density-gradient centrifugation were cultured at
37°C and 5% CO2 for 14 days in sterile 4-well tissue culture-treated
microchamber slides (Falcon). For controls, PBMC from healthy donors
were purified from buffy coat cells obtained from The San Diego Blood
Bank (San Diego, CA). The cells were seeded at a concentration of 1.5 to
2 ⫻ 107 cells/mL in RPMI 1640 supplemented with 10% FCS and
penicillin–streptomycin–glutamine (1-mL cell suspension per well). After
14 days, the slides were washed with phosphate-buffered saline (PBS) to
remove nonadherent cells, and the adherent cells were fixed in ice-cold 4%
paraformaldehyde solution for 20 minutes. Immunohistochemical staining
was performed according to the manufacturer’s instructions using the
VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA).
Briefly, after quenching of endogenous peroxidase activity with 0.3% H2O2,
the slides were incubated with diluted normal blocking serum from the
same species as the secondary antibody. Then the slides were incubated
with the primary mAb at 4°C overnight, washed 3 times in PBS, and
incubated at room temperature for 1 hour with biotinylated secondary
antibody (antimouse IgG or IgM mAb from Vector Laboratories or
PharMingen, respectively). The antibody–biotin conjugates were detected
with an avidin–biotin–peroxidase complex, applied for 30 minutes at room
temperature. A color reaction was developed using 3,3⬘-diaminobenzidine
(DAB), and, finally, specimens were lightly counterstained with hematoxylin (Vector). For controls, slides were incubated with an isotype control Ig
of irrelevant specificity, eg, MOPC21 (PharMingen). The slides were
examined on a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY),
and digital images were captured with a Hamamatsu cooled CCD color camera (Hamazatsu, Bridgewater, NJ).
CLL BLOOD-DERIVED NURSE-LIKE CELLS
2657
body Thr202/Tyr204 (New England BioLabs, Beverly, MA) that specifically recognizes the phosphorylated (active), but not the nonphosphorylated, form of p44/42 MAPK protein. Immunoreactive bands were visualized
using horseradish peroxidase–conjugated goat-antirabbit secondary antibody (New England BioLabs) and the enhanced chemiluminescence system
(ECL; Amersham).
Data analysis, statistics
Results are shown as mean ⫾ SD or SEM of at least 3 experiments each.
For statistical comparison between groups, the Student paired t test or the
Bonferroni t test was used. Analyses were performed using the Biostatistics
software developed by Stanton A. Glantz (University of California at San
Francisco, CA). Flow cytometry data were analyzed using the FlowJo
software (Tree Star).
Results
Viability of CLL cells on marrow stromal cells and outgrowth
of adherent cells from the blood of patients with CLL
To study the effect of marrow stromal cells on spontaneous
apoptosis of CLL cells in vitro, we examined the viability of CLL
cells in cultures with or without murine marrow stromal cells
(M2-10B4) over time. CLL cells plated onto mouse stromal cells
retained their initial viability throughout the 14 days of the study
(Figure 1A). In comparison, CLL cells cultured in flasks without
murine stromal cells had an initial decrease (18% ⫾ 9%) in
viability over the first 24 hours. Any further reduction in viability
over time was minimal (1%-2% decrease; Figure 1A). Concomitantly, we noted the outgrowth of adherent cells in such cultures, to
which many of the CLL cells were attached. These adherent cells
were observed after 3 days in culture, and their numbers increased
in the first 5 days (Figure 1B). Thereafter, the number of adherent
cells did not significantly increase. However, the cells increased in
size and then formed a layer of large, round, adherent, or
fibroblast-like cells after 14 days.
Nurse-like cells from the blood of patients protect CLL cells
from in vitro apoptosis
Separation of CLL cells from NLC 14 days after the initiation of
CLL PBMC long-term cultures resulted in a subsequent continuous
Phospho-p44/42 mitogen-activated protein kinase assay
The p44/42 mitogen-activated protein kinase (MAPK) assays were performed as described.35 Briefly, CLL cells were serum-starved for 2 hours,
and then lysates from 1 ⫻ 107 CLL cells per sample were prepared after
stimulation with 200 ng/mL SDF-1␣ at the indicated time points. Protein
content was determined using the Pierce (Rockford, IL) Coomassie Protein
Assay Reagent. Equal amounts of protein were separated by polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose
membranes (Bio-Rad Laboratories, Richmond, CA). Western blot analysis
was performed using the phospho-p44/42 MAPK rabbit polyclonal anti-
Figure 1. Survival of CLL B cells and outgrowth of NLC in vitro. (A) Marrow
stromal cells protect CLL B cells from spontaneous apoptosis in vitro. Presented is
the mean relative viability (⫾SEM) of CLL B cells from 6 patients cultured in the
presence (boxes) or absence (diamonds) of the murine marrow stromal cell line
M2-10B4. The viability of the CLL cells was determined at the time points indicated.
This is displayed as the normalized percentage viability relative to that noted at the
initiation of the culture (day 0). (B) Outgrowth of adherent NLC from PBMC of patients
with CLL. Cultures of PBMC from 4 representative patients with CLL were examined
microscopically for the number of adherent cells at the indicated time points. Lines
connect the mean number (⫾SEM) of adherent cells from each patient counted at
200⫻ magnification in 3 different visual fields at each time point.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2658
BURGER et al
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
decline in the viability of CLL cells from each of 12 patients. These
CLL cells showed a reduction in mitochondrial membrane potential
(⌬␺m), which is a characteristic early event during apoptosis.34
However, a reduction in ⌬␺m alone only can be observed for a short
time during cell death; therefore, most of the CLL cells undergoing
apoptosis had a decreased ⌬␺m and an increased cell membrane
permeability to PI, whereas vital cells exclude PI and have a high
⌬␺m (Figure 2A). For comparison, the fraction of CLL cells that
had a size and granularity characteristic of vital cells was determined. This was found to be similar to the fraction of vital cells, as
determined by DiOC6/PI staining in all cases (Figure 2B).
Before the assessment of CLL cell viability, the immunophenotype of the cells was determined using anti-CD19 and anti-CD3
mAbs on the initial day of the long-term cultures (d0), and 2 weeks
later (d14), when CLL cells were removed from the adherent NLC.
Figure 2C displays the viability of CLL cells from a representative
experiment, in which CLL cell viability was monitored for 12 days
either after their separation from the NLC (Figure 2C, diamonds;
n ⫽ 6) or after they were replated onto NLC (Figure 2C, boxes;
n ⫽ 6). Cells recovered from long-term cultures were predominantly CLL B cells (97.4% ⫾ 1.8%; n ⫽ 6), with only a few
detectable T cells (1.7% ⫾ 1.7%; n ⫽ 6). Therefore, it can be
assumed that the viability data presented in Figure 2C reflect the
viability of the CLL B cells. The viability of CLL cells replated
onto NLC remained stable over time (Figure 2C, boxes), whereas
the same CLL cells without NLC had a continuous decrease in
viability. Mean relative viability (⫾SEM) by day 12 in cultures
without NLC decreased to 21% ⫾ 7%, compared to the viability of
the CLL cells at the beginning of the culture, whereas the mean
relative viability (105% ⫾ 6%) in cocultures with NLC did not
decrease during this time. Conditioned media from CLL PBMC
cultures did not improve the viability of CLL cells separated from
NLC (n ⫽ 6) at any time point examined (data not shown).
Immunophenotyping of nurse-like cells from the blood
of patients with CLL
Nurse-like cells derived from the blood of patients with CLL
uniformly bound mAb specific for the stromal cell marker vimentin
(Figure 3A,E), whereas no staining was observed for samples
incubated with a control mAb of irrelevant specificity, MOPC-21
(Figure 3B). Moreover, the NLC weakly stained with the STRO-1
IgM mAb. This mAb recognizes stromal cells that have the
capacity to recapitulate the hematopoietic microenvironment in
vitro30 (Figure 3C). In contrast, NLC were negative for CD3,
CD19, CD83, and VCAM-1 (CD106) (data not shown). Nurse-like
cells express CD68, a member of a family of acidic, highly
glycosylated, lysosomal-associated membrane proteins (data not
shown). The morphology and immunophenotype of adherent cells
from the blood of healthy donors were different from the adherent
NLC from CLL blood samples. Here, a population of smaller cells
that were strongly positive for CD14 accounted for most of the
adherent cells, whereas cells with the morphology and phenotype
of NLC were infrequent (Figure 3D). Figure 3E demonstrates the
attachment of vimentin-negative CLL cells to vimentin-positive NLC.
Figure 2. Blood NLC protect CLL B cells from spontaneous apoptosis in vitro.
(A) Determination of CLL B-cell viability by staining with DiOC6 and PI. Presented are
contour maps of CLL B cells from a representative patient defining the relative green
(DiOC6) and red (PI) fluorescence intensities of the leukemia cells on the horizontal
and vertical axes, respectively. The vital cell population (DiOC6bright, PI exclusion) was
determined for CLL cells cultured in the presence (left box) or absence (right box) of
NLC. Vital cells were gated as indicated by the polygons with the broken lines.
Relative percentage numbers of vital cells are displayed above each of these gates.
(B) Determination of CLL B-cell viability as assessed by light scatter. These contour
plots show the relative forward-light scatter (or FSC) or granularity (side-light scatter,
or SSC) of CLL cells from the same patient sample as displayed in A, cultured with
(left box) or without (right box) NLC. Vital cells were gated as indicated by the circles
with the broken lines, and the relative percentage numbers of vital cells are indicated
above each of these gates. (C) Viability of CLL cells in the presence or absence of
NLC. CLL cells from 6 patients were removed from NLC in long-term CLL B-cell
cultures 14 days after seeding, and the leukemia cells were plated in either wells with
NLC (squares) or without NLC (diamonds). Displayed are the mean percentage
viability values (⫾SEM) of leukemia cells assessed at the indicated time points
relative to those noted at initiation of culture on day 0.
Fluorescence in situ hybridization for trisomy 12
Leukemia cells of one patient had trisomy 12, allowing us to
examine whether this genetic abnormality also was present in the
population of NLC. NLC and CLL cells from this patient were
examined by fluorescence in situ hybridization (FISH) analysis,
using chromosome 12–specific alpha satellite DNA probes on the
nuclei of cells in long-term culture. CLL and NLC cells could be
distinguished from each other by nuclear size and morphology, the
NLC displaying a 2-fold larger nuclear diameter and a more oblong
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
Figure 3. Phenotype of NLC derived from the blood of patients with CLL. CLL B
cells from representative patients were cultured for 14 to 21 days, and nonadherent
cells were vigorously (A-D) or carefully (E) removed before immunohistochemical
examination. Cells were stained with antibody–biotin conjugates that subsequently
were developed with an avidin–biotin–peroxidase complex and DAB, resulting in a
brown-red stain for positive cells. Specimens were lightly counterstained with
hematoxylin (Vector) and were photographed at 400⫻ magnification. Displayed are
pictures of NLC derived from the blood of patients with CLL (A-C, E) or adherent cells
derived from the blood of a normal adult donor (D). Adherent NLC from the blood of
patients with CLL expressed stromal cell markers vimentin (A, E) and STRO-1 (C).
CLL B cells, on the other hand, did not express vimentin (E). Control staining with the
MOPC-21 mAb of irrelevant specificity was negative (B). In contrast, most adherent
cells derived from the blood of normal adult donors (D) were smaller and strongly
positive for CD14. The close physical interaction of NLC and CLL B cells is
demonstrated in (E), where vimentin-negative CLL cells are attached to vimentinpositive NLC. This is typically seen in long-term cultures of CLL B cells, suggesting a
close, symbiotic relationship of these 2 cell types.
appearance (Figure 4). Trisomy 12 was clearly detectable in the
CLL population by FISH analysis and was not observed in the
overwhelming majority of NLC from the same patient (Figure 4).
Evaluation of the signal distribution in 500 small nuclei revealed
75.6% had 3 fluorescent signals, 18.8% had 2 signals, 3.8% had 1
signal, and 1.4% had no observable signal. Evaluation of 500 larger
nuclei revealed 4.6% had 3 signals, 89.4% had 2 signals, 4.2% had
1 signal, and 0.6% had no signal. These findings show that the NLC
and CLL populations do not share identical chromosomal complements, indicating that the NLC are not part of the CLL clone.
CLL BLOOD-DERIVED NURSE-LIKE CELLS
2659
Figure 4. Fluorescence in situ hybridization for trisomy 12. Depicted are the
nuclei of NLC (large ovals) and CLL cells (small circles) examined for trisomy 12 by
FISH. The 2 large NLC nuclei have only 2 bright fluorescence signal spots, whereas
the 4 CLL cell nuclei each have 3 bright signal spots, reflecting the presence of
trisomy 12.
cells from NLC allowed the return of CXCR4 expression to levels
comparable to pretreatment levels.
Nurse-like cells express mRNA for stromal cell–derived factor-1
SDF-1 is a potent chemoattractant for CLL B cells and an important
mediator in cellular interaction of CLL cells with marrow stromal
Nurse-like cells induce down-modulation of CXCR4
on CLL cells in vitro
We evaluated the surface immunophenotype of CLL B cells before
and after 14 days of coculture with NLC. Leukemia cells were
noted to retain the expression of B-cell surface antigens such as
CD19. However, there was marked reduction in the staining of
CLL B cells for CXCR4 after 14 days in culture. The mean
fluorescence intensity ratio (MFIR) of CLL B cells in long-term
culture with NLC was significantly lower (MFIR, 18.6 ⫾ 4; n ⫽ 8)
than that of CLL B cells at the time of initiation of the cultures
(MFIR, 303 ⫾ 49; n ⫽ 8). Figure 5 displays anti-CXCR4 and
isotype control stains of CLL B cells from 2 representative patients
before and after coculture with NLC. However, the removal of CLL
Figure 5. Down-regulation of CXCR4 on CLL B cells cultured with NLC.
Histograms of leukemia B cells from 2 representative patients with CLL, indicating the
relative red (anti-CXCR4) fluorescence intensity of CLL cells before (shaded) or after
coculture with NLC (bold lines). Thin lines represent the isotype control staining.
MFIR values are displayed next to each histogram.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2660
BURGER et al
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
The viability of CLL cells cultured without NLC was significantly greater in cultures supplemented with SDF-1␣ than in
cultures without this chemokine (Figure 7A). The mean relative
viability of CLL cells without NLC 1 day after separation from the
NLC (d1) was 71.1% ⫾ 2.7% (n ⫽ 6), whereas the mean viability
of the same samples in cultures supplemented with SDF-1␣ was
84.5% ⫾ 3.1% (n ⫽ 6; P ⫽ .009). However, the addition of SDF-1␣
to cultures without NLC did not completely protect CLL cells from
apoptosis, compared to CLL cells plated back onto the NLC
(93.5% ⫾ 2.9% at d1; P ⫽ .06).
Antibody to SDF-1 inhibits the protection of CLL B cells
from spontaneous apoptosis by NLC
Figure 6. SDF-1 mRNA expression by NLC and p44/42 mitogen-activated
protein kinase (Erk 1/2) activation in CLL B cells by SDF-1␣. (A) cDNA from
purified NLC from 4 patients with CLL was examined for the expression of SDF-1
mRNA by RT-PCR. The specific 230-bp PCR fragment is visible in each of the 4 NLC
samples (lanes 2-5), and in the positive control, namely an SDF-1–containing
plasmid (lane 6). Lanes 7-11 display PCR fragments of the expected size using
GA3PD primers for the NLC samples (lanes 7-10) but not for the SDF-1 plasmid
control. Lanes 1 and 12 display the separation of the 100-bp marker DNA, and the
200, 600, and 1000 bp bands are marked on the left side. (B) p44/42 MAPK activation
in CLL B cells treated with SDF-1␣ at 200 ng/mL. CLL cell lysates were obtained at
the time points indicated on the horizontal axis and examined for phospho-p44/42
MAPK protein by Western blot analysis. Protein bands of the expected sizes of 42
and 44 kd, as indicated on the left, were prominent after the stimulation of CLL cells
with SDF-1␣, whereas they were only weakly apparent before activation by SDF-1
(time 0).
We examined whether antibodies to SDF-1 could inhibit the effect
of NLC on the survival of CLL B cells in vitro. CLL B cells from
long-term cultures were separated from NLC, as described above,
and replated into multiple wells that did or did not contain NLC. At
the same time, anti–SDF-1 antibody or control immunoglobulin
was added to separate wells, and viability was examined at the time
points indicated (Figure 7B).
The viability of CLL cells cultured on NLC was significantly
reduced by the addition of anti–SDF-1 but not the control antibody
in all cases. In contrast, the addition of anti–SDF-1 antibody did not
significantly change the viability of CLL cells in samples without
NLC (Figure 7B).
cells.19 CXCR4 is the only receptor for this chemokine. SDF-1 is
expressed and secreted by marrow stromal cells19,21,22 but not by
blood leukocytes.36,37 We found that NLC isolated from the cells of
each of 4 different patients with CLL expressed SDF-1 mRNA
(Figure 6A). In contrast, we did not detect SDF-1 expression in
cDNA from purified CLL B cells from any of 4 patients.
SDF-1␣ induces activation of the p44/42 MAPK signal
transduction pathway
Engagement of CXCR4 by SDF-1␣ induces the rapid transient
activation of the p44/42 MAPK signaling pathway in CXCR4expressing cell lines.38,39 A rapid and robust activation of p44/42
MAPK was observed on the addition of SDF-1␣ to CLL cells from
each of 6 patients. Figure 6, panel B shows a representative time
course of p44/42 MAPK activation after stimulation with 100
ng/mL SDF-1␣.
Synthetic SDF-1␣ rescues CLL B cells from apoptosis in vitro
We examined whether SDF-1 participates in mediating survival
signals from NLC to CLL B cells. Experiments were performed in
which the NLC were replaced with synthetic SDF-1␣. We initiated
the long-term cultures of CLL PBMC, as described above. On day
14, nonadherent CLL cells were removed from the adherent NLC.
The CLL cells were divided into equal parts and then incubated
under 3 different conditions. They were either plated back onto the
NLC, plated into wells without NLC, or plated into wells without
NLC but supplemented with 500 ng/mL synthetic human SDF-1␣.
Cell viability was observed thereafter at the times indicated.
Figure 7. Protection of CLL B cells from spontaneous apoptosis in vitro is
partially mediated by SDF-1. (A) Synthetic SDF-1␣ could protect CLL cells from
spontaneous apoptosis. Displayed is the mean relative viability of CLL cells from
each of 6 representative CLL patients cultured with (squares) or without (circles and
diamonds) NLC. Cultures without NLC were supplemented with SDF-1␣ at 500
ng/mL on day 0 (circles) or were cultured in medium alone (diamonds). Viability was
assessed at the times indicated on the horizontal axis. Without NLC, leukemia cells
cultured with SDF-1␣ had significantly higher viability than did leukemia cells cultured
without SDF-1 (P ⬍ .005; Student t test). Nonetheless, the viability of CLL cells
cultured without NLC and SDF-1␣ (circles) was less than that of CLL cells cultured
with NLC (boxes). (B) Anti–SDF-1 antibody inhibits the survival of CLL B cells in
cultures with NLC. CLL cells were separated from NLC as described and were
replated into wells with (solid symbols) or without (open symbols) NLC. To wells with
or without NLC, anti–SDF-1 antibody at 10 ␮g/mL (circles) or 1 ␮g/mL (triangles), or a
control IgG (squares), was added at day 0. Viability was subsequently determined for
each of these conditions at the time points indicated on the horizontal axis. Displayed
are the mean (⫾SD) viability values of samples from each of 3 representative patients.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
Discussion
The initial aim of this study was to determine the factors involved
in regulating the survival of CLL cells in vitro when cultured on
marrow stromal cells. CLL cells cultured on murine marrow
stromal cells retain their high viability for 14 days (Figure 1A).
This is similar to the findings of previous studies using human
marrow stromal cells to protect CLL cells from spontaneous
apoptosis.6,9 Our observation that murine stromal cells protect CLL
cells from apoptosis indicates that factors responsible for this
protective effect can function across species barriers. This excludes
several hematopoietic factors, such as interleukin-4 and granulocyte–
macrophage colony-stimulating factor, which have speciesrestricted activities, from being essential to the protective effects of
marrow stromal cells for leukemia B cells in vitro.
When mononuclear cells from the blood of patients with CLL
were cultured without stromal cells, we were surprised to observe a
relatively stable viability after an initial decrease in the first 24 to
48 hours (Figure 2C). This was associated with an outgrowth of
adherent cells to which CLL B cells became attached and that
protected the leukemia cells from spontaneous apoptosis in vitro.
When the CLL cells were removed from these cells, the CLL B
cells experienced a rapid decline in viability, whereas the same
CLL B cells retained their initial viability when replated onto the
NLC. Because the adherent cells supported the survival of CLL
cells in vitro and CLL B cells became attached to them, we called
them nurse-like cells, or NLC.
Nurse cells were first recognized in situ in the thymus, where
they form characteristic complexes with immature T lymphocytes
and play an important role in thymocyte maturation and differentiation.40 This cellular interaction is characterized by the active
invasion into thymic nurse cells by thymocytes (emperipolesis). In
vitro, it has been recognized that T- or B-lineage cells can
spontaneously migrate beneath adherent cells derived from longterm marrow cultures41,42 or dermal tissue43 or from the synovium
of patients with rheumatoid arthritis.44 Although lymphocytes
crawl under these cells, they do not become internalized. As such,
this process is called pseudo-emperipolesis, and the supporting
cells are termed nurse-like cells. The close physical interaction and
the capacity to support the survival and differentiation of lymphocytes are the main characteristics of NLC. These 2 features were
also noted for the interaction between CLL B cells and the NLC
described in this study. However, whereas many of the CLL B cells
became attached to the NLC, they did not display the characteristic
appearance of pseudo-emperipolesis by phase-contrast microscopy.
This may be owing to the observed lack of CD106 (VCAM-1) on
NLC derived from the blood of patients with CLL because
interaction between CD106 and its respective ligand on lymphocytes (VLA-4 or CD49d) plays an important role in mediating
pseudo-emperipolesis.41,44
When cultured, the PBMC from patients with CLL developed
abundant numbers of NLC that became the predominant population
of adherent cells. In contrast, PBMC of healthy donors rarely
generated such NLC when cultured under identical conditions. This
may be because of a difference between the blood of patients with
CLL and that of healthy donors in the relative proportion of cells
that can give rise to such NLC. Patients with CLL may have greater
numbers of circulating NLC progenitor cells, possibly secondary to
the infiltration of the marrow by leukemia B cells. Alternatively,
CLL cells may elaborate factors, such as transforming growth
factor-beta (TGF-␤), that could support the outgrowth of NLC in
CLL BLOOD-DERIVED NURSE-LIKE CELLS
2661
vitro. TGF-␤ is secreted by CLL cells at high concentrations45 and
is known to be a potent differentiation factor for stromal cells.46,47
As such, the CLL cells may contribute to the distinct phenotype of
the adherent cells in CLL long-term cultures as a result of a
symbiotic relationship between NLC and CLL cells in vitro. In any
case, we cannot explain the abundance of NLC in CLL blood
cultures with a model proposing that such cells are clonally related
to the CLL cells or are derived from a common transformed
precursor cell. In addition to the finding that NLC do not express
B-cell differentiation antigens, NLC do not share cytogenetic
abnormalities with the CLL B-cell clone, as demonstrated by our
studies on the patient with leukemia cells that had trisomy 12
(Figure 4).
Phenotypic characterization of CLL NLC suggests they are
related to marrow stromal cells. These cells lack expression of
B-cell or T-cell differentiation antigens and do not express CD83, a
marker of mature dendritic cells.48 On the other hand, NLC are
noted to express stromal cell markers, such as vimentin and
STRO-1. Moreover, NLC express mRNA for SDF-1 and support
CLL B-cell survival through the action of this chemokine. Marrow
stromal cells are known to be an important source of SDF-1,21,26
whereas PBMC do not constitutively express this chemokine.36,37
Moreover, it also is recognized that stromal cells derived from the
marrow17,18,49-51 or extramedullary sites44 can support B-cell survival and differentiation. In view of these reports, it is tempting to
speculate that the blood-derived NLC described in this study are
derived from circulating immature stromal cells. However, further
studies are required to establish this relationship.
The initial decrease in CLL cell viability in the first 24 hours
and the lack of microscopically identifiable NLC during the initial
2 to 3 days of culture suggest that NLC are functionally immature
in the bloodstream and gain their capacity to “nurse” CLL cells
during in vitro differentiation. Therefore, mature counterparts of
NLC are likely to play a role in protecting CLL B cells from
apoptosis in distinct lymphoid (and nonlymphoid) tissue microenvironments rather than in the bloodstream. Trafficking of CLL B
cells between the blood and the marrow or the lymphoid tissues is a
new concept that now is receiving increased attention. The finding
that CLL B cells express functional chemokine receptors19,52,53
implies that CLL B cells can actively migrate to the marrow and to
secondary lymphoid tissues, where an interaction with NLC may
occur. In such microenvironments, CLL B cells are likely to
encounter surface-bound SDF-1 on marrow stroma21 or reticulum
cells in secondary lymphoid tissues.54 CLL cells would be expected
to down-regulate the expression of CXCR4 receptors within these
tissue microenvironments rather than when they are circulating
freely in the blood.19,55
We recently characterized the chemokine SDF-1 as a potent
chemoattractant for CLL cells that is required for the spontaneous
migration of CLL cells beneath marrow stromal cells in vitro.19 The
attachment of CLL B cells to the surface of NLC, the expression of
SDF-1 mRNA by NLC, and the down-modulation of CXCR4 on
CLL cells cultured on NLC suggest that SDF-1, made by NLC,
plays a role in the interaction between these cell types. From our
earlier studies, it appears that the role of SDF-1 lies in attracting
CLL cells, leading to the characteristic picture of NLC surrounded
by CLL cells (Figures 1C, 3E). However, the current study
demonstrates that SDF-1 also can function as a CLL B-cell survival
factor that may play a role in the microenvironmental regulation of
resistance to apoptosis.
SDF-1 is a highly conserved chemokine that has 99% homology
between mouse and human, which allows for its action across
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2662
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
BURGER et al
species barriers. Because it also is a growth factor for stromal
cell–dependent B-lineage cells,21,29 we hypothesized that SDF-1
may play a dual role in the interaction between NLC and CLL B
cells, functioning not only as a chemoattractant but also as a
“nursing” factor. Lagneaux et al6 earlier noted that CLL cell
survival on marrow stromal cells was dependent on close contact
because CLL B cells underwent apoptosis when separated from
stromal cells by a micropore filter. These authors reasoned that
hematopoietic cytokines might not play a major role in protecting
CLL cells from apoptosis in such cultures. However, soluble
factors were not rigorously excluded in these experiments, because
cytokines can be retained on the surfaces of stromal cells and thus
may be effective only within a short range. Growth factors bound to
heparan sulfate on the surfaces of marrow stromal cells are the
biologically relevant forms of hematopoietic cytokines in the
marrow microenvironment.56 SDF-1 is a highly basic protein that
binds through a cluster of basic amino acids in the first ␤-strand to
cell membrane heparan sulfates, leaving the N-terminal signaling
domain exposed for interaction with its receptor, CXCR4.57 As
such, a proteoglycan-bound SDF-1, immobilized on the surface of
cells, appears to be the functional form of this chemokine in vivo.57
This may explain why conditioned medium from NLC cells did not
significantly improve the survival of CLL cells in vitro, as also
noted earlier for stromal cell–conditioned medium.6 Nevertheless,
the observations that synthetic SDF-1␣ could inhibit the spontaneous apoptosis of CLL cells that were separated from NLC in vitro
and that antibodies against SDF-1␣ inhibited the protective effect
of NLC for CLL cells indicate that SDF-1 can function as a survival
factor for CLL cells in vitro.
Consistent with this, we found that the stimulation of CLL cells
with synthetic SDF-1␣ induced rapid, transient activation of
p44/42 MAPK (ERK1/2), a key signaling pathway for promoting
cell survival through transcription-dependent and -independent
mechanisms.58,59 Through MAPK-activated effector molecules
(Rsks), activation of the MAPK promotes cells survival directly by
inactivating the pro-apoptotic BAD protein, and indirectly by
activating the transcription factor CREB, which is important for the
transcriptional up-regulation of the anti-apoptotic Bcl-2 gene.60
These observations suggest that SDF-1 on stromal cells or NLC
engages CLL B cells through CXCR4 and thereby affects components of the cell death machinery, leading to the noted resistance of
CLL cells to apoptosis.3,4,16
Future studies will have to define whether additional factors,
such as integrin receptors, have a role in mediating adhesion or
survival signals between B lymphocytes and respective stromal cell
ligands, such as fibronectin and VCAM-1 (CD106). Signals from
integrin receptors can synergize with those induced by cytokines,
such as SDF-1, in regulating the organization of the cytoskeleton,
transcriptional activation, and cell survival or proliferation.61
In summary, this study demonstrates that CLL cell survival in
vitro can be regulated by blood-derived NLC that protect CLL B
cells from apoptosis through an SDF-1–dependent mechanism. In
this symbiotic system, the chemokine SDF-1 functions not only as
a CLL cell chemoattractant but also as a survival factor for CLL
cells. As such, this study provides a new mechanism by which
accessory cells can regulate the survival of neoplastic B cells even
outside the marrow microenvironment. Future studies may define
whether substances that inhibit interactions between NLC and CLL
B cells affect the survival of CLL cells in vitro and in vivo. Such
approaches could lead to new therapeutic avenues for patients with
B-cell CLL.
Acknowledgments
We thank Dr James R. Feramisco for assistance with the preparation of the photomicrographs. We also thank Diane A. Nguyen and
T. A. Johnson for their excellent technical assistance.
References
1. Kipps TJ. Chronic lymphocytic leukemia and related diseases. In: Beutler E, Lichtman MA, Coller
BS, Kipps TJ, eds. Williams Hematology. New
York: McGraw-Hill; 1995:1017-1039.
2. Rai KR, Patel DV. Chronic lymphocytic leukemia.
In: Hoffmann R, Benz EJ, Shattil SJ, et al, eds.
Hematology—Basic Principles and Practice. New
York: Churchill Livingstone; 1999:1350-1363.
3. Kitada S, Andersen J, Akar S, et al. Expression of
apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in
vivo chemoresponses. Blood. 1998;91:33793389.
4. McConkey DJ, Chandra J, Wright S, et al. Apoptosis sensitivity in chronic lymphocytic leukemia
is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX.
J Immunol. 1996;156:2624-2630.
5. Collins RJ, Verschuer LA, Harmon BV, Prentice
RL, Pope JH, Kerr JF. Spontaneous programmed
death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro. Br J
Haematol. 1989;71:343-350.
6. Lagneaux L, Delforge A, Bron D, De Bruyn C,
Stryckmans P. Chronic lymphocytic leukemic B
cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood. 1998;91:2387-2396.
7. Holder MJ, Wang H, Milner AE, et al. Suppression
of apoptosis in normal and neoplastic human B
lymphocytes by CD40 ligand is independent of
Bc1–2 induction. Eur J Immunol. 1993;23:23682371.
8. Jurlander J. The cellular biology of B-cell chronic
9.
10.
11.
12.
13.
14.
15.
lymphocytic leukemia. Crit Rev Oncol Hematol.
1998;27:29-52.
Panayiotidis P, Jones D, Ganeshaguru K, Foroni
L, Hoffbrand AV. Human bone marrow stromal
cells prevent apoptosis and support the survival
of chronic lymphocytic leukaemia cells in vitro.
Br J Haematol. 1996;92:97-103.
Han T, Barcos M, Emrich L, et al. Bone marrow
infiltration patterns and their prognostic significance in chronic lymphocytic leukemia: correlations with clinical, immunologic, phenotypic, and
cytogenetic data. J Clin Oncol. 1984;2:562-570.
Pangalis GA, Roussou PA, Kittas C, et al. Patterns of bone marrow involvement in chronic lymphocytic leukemia and small lymphocytic (welldifferentiated) non-Hodgkin’s lymphoma: its
clinical significance in relation to their differential
diagnosis and prognosis. Cancer. 1984;54:702708.
Dorshkind K. Regulation of hemopoiesis by bone
marrow stromal cells and their products. Annu
Rev Immunol. 1990;8:111-137.
Osmond DG, Rico-Vargas S, Valenzona H, et al.
Apoptosis and macrophage-mediated cell deletion in the regulation of B lymphopoiesis in mouse
bone marrow. Immunol Rev. 1994;142:209-230.
Melchers F, Rolink A, Grawunder U, et al. Positive
and negative selection events during B lymphopoiesis. Curr Opin Immunol. 1995;7:214-227.
Lu L, Lejtenyi D, Osmond DG. Regulation of cell
survival during B lymphopoiesis: suppressed apoptosis of pro-B cells in P53-deficient mouse
bone marrow. Eur J Immunol. 1999;29:24842490.
16. Hanada M, Delia D, Aiello A, Stadtmauer E, Reed
JC. bcl-2 gene hypomethylation and high-level
expression in B-cell chronic lymphocytic leukemia. Blood. 1993;82:1820-1828.
17. Whitlock CA, Witte ON. Long-term culture of B
lymphocytes and their precursors from murine
bone marrow. Proc Natl Acad Sci U S A. 1982;79:
3608-3612.
18. Witte PL, Robinson M, Henley A, et al. Relationships between B-lineage lymphocytes and stromal cells in long-term bone marrow cultures. Eur
J Immunol. 1987;17:1473-1484.
19. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4
chemokine receptors that mediate spontaneous
migration beneath bone marrow stromal cells.
Blood. 1999;94:3658-3667.
20. Jacobsen K, Osmond DG. Microenvironmental
organization and stromal cell associations of B
lymphocyte precursor cells in mouse bone marrow. Eur J Immunol. 1990;20:2395-2404.
21. Nagasawa T, Kikutani H, Kishimoto T. Molecular
cloning and structure of a pre-B-cell growthstimulating factor. Proc Natl Acad Sci U S A.
1994;91:2305-2309.
22. Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti
A, Springer TA. A highly efficacious lymphocyte
chemoattractant, stromal cell-derived factor 1
(SDF-1). J Exp Med. 1996;184:1101-1109.
23. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor
CXCR4 in haematopoiesis and in cerebellar development. Nature. 1998;393:595-599.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
24. Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract [see comments]. Nature. 1998;393:591-594.
25. Ma Q, Jones D, Borghesani PR, et al. Impaired
B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1deficient mice. Proc Natl Acad Sci U S A. 1998;
95:9448-9453.
26. Nagasawa T, Hirota S, Tachibana K, et al. Defects
of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine
PBSF/SDF-1. Nature. 1996;382:635-638.
27. Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is required for the retention of B
lineage and granulocytic precursors within the
bone marrow microenvironment. Immunity. 1999;
10:463-471.
28. Kawabata K, Ujikawa M, Egawa T, et al. A cellautonomous requirement for CXCR4 in long-term
lymphoid and myeloid reconstitution. Proc Natl
Acad Sci U S A. 1999;96:5663-5667.
29. Nishii K, Katayama N, Miwa H, et al. Survival of
human leukaemic B-cell precursors is supported
by stromal cells and cytokines: association with
the expression of bcl-2 protein. Br J Haematol.
1999;105:701-710.
30. Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a
novel monoclonal antibody, STRO-1. Blood.
1991;78:55-62.
31. Anastasi J, Le Beau MM, Vardiman JW, Fernald
AA, Larson RA, Rowley JD. Detection of trisomy
12 in chronic lymphocytic leukemia by fluorescence in situ hybridization to interphase cells: a
simple and sensitive method. Blood. 1992;79:
1796-1801.
32. Hanauer A, Mandel JL. The glyceraldehyde 3
phosphate dehydrogenase gene family: structure
of a human cDNA and of an X chromosome
linked pseudogene: amazing complexity of the
gene family in mouse. EMBO J. 1984;3:26272633.
33. Leoni LM, Chao Q, Cottam HB, et al. Induction of
an apoptotic program in cell-free extracts by
2-chloro-2’-deoxyadenosine 5’-triphosphate and
cytochrome c. Proc Natl Acad Sci U S A. 1998;95:
9567-9571.
34. Zamzami N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an
early irreversible step of programmed lymphocyte
death in vivo. J Exp Med. 1995;181:1661-1672.
CLL BLOOD-DERIVED NURSE-LIKE CELLS
and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics.
1995;28:495-500.
37. Pablos JL, Amara A, Bouloc A, et al. Stromal-cell
derived factor is expressed by dendritic cells and
endothelium in human skin. Am J Pathol. 1999;
155:1577-1586.
38. Ganju RK, Brubaker SA, Meyer J, et al. The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-proteincoupled CXCR-4 receptor and activates multiple
signal transduction pathways. J Biol Chem. 1998;
273:23169-23175.
39. Dutt P, Wang JF, Groopman JE. Stromal cell-derived factor-1 alpha and stem cell factor/kit ligand
share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis. J Immunol. 1998;161:
3652-3658.
2663
the T-cell areas of lymphoid organs. Immunol
Rev. 1997;156:25-37.
49. Manabe A, Murti KG, Coustan-Smith E, et al. Adhesion-dependent survival of normal and leukemic human B lymphoblasts on bone marrow stromal cells. Blood. 1994;83:758-766.
50. McGinnes K, Quesniaux V, Hitzler J, Paige C.
Human B-lymphopoiesis is supported by bone
marrow-derived stromal cells. Exp Hematol.
1991;19:294-303.
51. Merville P, Dechanet J, Desmouliere A, et al.
Bcl-2⫹ tonsillar plasma cells are rescued from
apoptosis by bone marrow fibroblasts. J Exp
Med. 1996;183:227-236.
52. Jones D, Benjamin RJ, Shahsafaei A, Dorfman
DM. The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a
marker of B-cell chronic lymphocytic leukemia.
Blood. 2000;95:627-632.
40. Wekerle H, Ketelsen UP, Ernst M. Thymic nurse
cells: lymphoepithelial cell complexes in murine
thymuses: morphological and serological characterization. J Exp Med. 1980;151:925-944.
53. Trentin L, Agostini C, Facco M, et al. The chemokine receptor CXCR3 is expressed on malignant
B cells and mediates chemotaxis. J Clin Invest.
1999;104:115-121.
41. Miyake K, Hasunuma Y, Yagita H, Kimoto M. Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath
stromal cells in culture. J Cell Biol. 1992;119:653662.
54. Bleul CC, Schultze JL, Springer TA. B lymphocyte
chemotaxis regulated in association with microanatomic localization, differentiation state, and B
cell receptor engagement. J Exp Med. 1998;187:
753-762.
42. Hiai H, Shisa H, Nishi Y, et al. Symbiotic culture of
mouse leukaemias: regulation of cell interaction
by an activity of serum. Virchows Arch. 1980;32:
261-279.
55. Mohle R, Failenschmid C, Bautz F, Kanz L. Overexpression of the chemokine receptor CXCR4 in
B cell chronic lymphocytic leukemia is associated
with increased functional response to stromal
cell-derived factor-1 (SDF-1). Leukemia. 1999;13:
1954-1959.
43. Iwagami S, Furue S, Toyosaki T, et al. Establishment and characterization of nurse cell-like
clones from human skin: nurse cell-like clones
can stimulate autologous mixed lymphocyte reaction. J Immunol. 1994;153:2927-2938.
44. Shimaoka Y, Attrep JF, Hirano T, et al. Nurse-like
cells from bone marrow and synovium of patients
with rheumatoid arthritis promote survival and
enhance function of human B cells. J Clin Invest.
1998;102:606-618.
45. Lotz M, Ranheim E, Kipps TJ. Transforming
growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells. J Exp
Med. 1994;179:999-1004.
46. Robledo MM, Ursa MA, Sanchez-Madrid F,
Teixido J. Associations between TGF-␤1 receptors in human bone marrow stromal cells. Br J
Haematol. 1998;102:804-811.
35. Hunter T. Protein kinases and phosphatases: the
yin and yang of protein phosphorylation and signaling. Cell. 1995;80:225-236.
47. Takai H, Kanematsu M, Yano K, et al. Transforming growth factor-beta stimulates the production
of osteoprotegerin/osteoclastogenesis inhibitory
factor by bone marrow stromal cells. J Biol Chem.
1998;273:27091-27096.
36. Shirozu M, Nakano T, Inazawa J, et al. Structure
48. Steinman RM, Pack M, Inaba K. Dendritic cells in
56. Roberts R, Gallagher J, Spooncer E, Allen TD,
Bloomfield F, Dexter TM. Heparan sulphate
bound growth factors: a mechanism for stromal
cell mediated haemopoiesis. Nature. 1988;332:
376-378.
57. Amara A, Lorthioir O, Valenzuela A, et al. Stromal
cell-derived factor-1␣ associates with heparan
sulfates through the first beta-strand of the chemokine. J Biol Chem. 1999;274:23916-23925.
58. Nebreda AR, Gavin AC. Cell survival demands
some Rsk. Science. 1999;286:1309-1310.
59. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38
MAP kinases on apoptosis. Science. 1995;270:
1326-1331.
60. Bonni A, Brunet A, West AE, Datta SR, Takasu
MA, Greenberg ME. Cell survival promoted by
the ras-MAPK signaling pathway by transcriptiondependent and -independent mechanisms [In
Process Citation]. Science. 1999;286:1358-1362.
61. Kumar CC. Signaling by integrin receptors. Oncogene. 1998;17:1365-1373.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2000 96: 2655-2663
Blood-derived nurse-like cells protect chronic lymphocytic leukemia B
cells from spontaneous apoptosis through stromal cell−derived factor-1
Jan A. Burger, Nobuhiro Tsukada, Meike Burger, Nathan J. Zvaifler, Marie Dell'Aquila and Thomas J.
Kipps
Updated information and services can be found at:
http://www.bloodjournal.org/content/96/8/2655.full.html
Articles on similar topics can be found in the following Blood collections
Apoptosis (747 articles)
Chemokines, Cytokines, and Interleukins (564 articles)
Neoplasia (4182 articles)
Plenary Papers (495 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.