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PLENARY PAPER
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.
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
blood-derived 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 protein-kinase (ERK 1/2) by CLL
B cells in vitro. It is concluded that the blood of patients with CLL
contains cells that can differentiate into adherent nurse-like cells
that protect leukemia cells from undergoing spontaneous apoptosis
through an SDF-1-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) 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 apoptosis-regulatory 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-receptor-deficient, 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 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 cell-dependent 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.
Cell purification, cell lines
Chemokine, antibodies, flow cytometry
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 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-µL 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 Rescue of CLL cell viability by 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 µL CLL cell
suspension was collected at the indicated time points and transferred
to FACS tubes containing 300 µL of 60 nmol/L DiOC6
(Molecular Probes) and 10 µg/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).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 antibody 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).
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 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.
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 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.
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 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 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 the rapid transient
activation of the p44/42 MAPK signaling pathway in CXCR4-expressing 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 . 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.
The viability of CLL cells cultured without NLC was significantly
greater in cultures supplemented with SDF-1
Antibody to SDF-1 inhibits the protection of CLL B cells from spontaneous apoptosis by NLC 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).
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 species-restricted 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 long-term 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- 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 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 Consistent with this, we found that the stimulation of CLL cells with
synthetic SDF-1 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.
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.
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.).
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.
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: tkipps{at}ucsd.edu.
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M. Burger, T. Hartmann, M. Krome, J. Rawluk, H. Tamamura, N. Fujii, T. J. Kipps, and J. A. Burger Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells Blood, September 1, 2005; 106(5): 1824 - 1830. [Abstract] [Full Text] [PDF]
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A. Petlickovski, L. Laurenti, X. Li, S. Marietti, P. Chiusolo, S. Sica, G. Leone, and D. G. Efremov Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells Blood, June 15, 2005; 105(12): 4820 - 4827. [Abstract] [Full Text] [PDF]
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S. Deaglio, T. Vaisitti, L. Bergui, L. Bonello, A. L. Horenstein, L. Tamagnone, L. Boumsell, and F. Malavasi CD38 and CD100 lead a network of surface receptors relaying positive signals for B-CLL growth and survival Blood, April 15, 2005; 105(8): 3042 - 3050. [Abstract] [Full Text] [PDF]
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A. J. Johnson, L. L. Smith, J. Zhu, N. A. Heerema, S. Jefferson, A. Mone, M. Grever, C.-S. Chen, and J. C. Byrd A novel celecoxib derivative, OSU03012, induces cytotoxicity in primary CLL cells and transformed B-cell lymphoma cell line via a caspase- and Bcl-2-independent mechanism Blood, March 15, 2005; 105(6): 2504 - 2509. [Abstract] [Full Text] [PDF]
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H. El-Daly, M. Kull, S. Zimmermann, M. Pantic, C. F. Waller, and U. M. Martens Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532 Blood, February 15, 2005; 105(4): 1742 - 1749. [Abstract] [Full Text] [PDF]
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F. D. Arditti, A. Rabinkov, T. Miron, Y. Reisner, A. Berrebi, M. Wilchek, and D. Mirelman Apoptotic killing of B-chronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-alliinase conjugate Mol. Cancer Ther., February 1, 2005; 4(2): 325 - 332. [Abstract] [Full Text] [PDF]
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R. S. Abraham, K. V. Ballman, A. Dispenzieri, D. E. Grill, M. K. Manske, T. L. Price-Troska, N. G. Paz, M. A. Gertz, and R. Fonseca Functional gene expression analysis of clonal plasma cells identifies a unique molecular profile for light chain amyloidosis Blood, January 15, 2005; 105(2): 794 - 803. [Abstract] [Full Text] [PDF]
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K. Felix, S. Gerstmeier, A. Kyriakopoulos, O. M. Z. Howard, H.-F. Dong, M. Eckhaus, D. Behne, G. W. Bornkamm, and S. Janz Selenium Deficiency Abrogates Inflammation-Dependent Plasma Cell Tumors in Mice Cancer Res., April 15, 2004; 64(8): 2910 - 2917. [Abstract] [Full Text] [PDF]
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 G. Monaco, M. Konopleva, M. Munsell, C. Leysath, R.-Y. Wang, C. E. Jackson, M. Korbling, E. Estey, J. Belmont, and M. Andreeff Engraftment of Acute Myeloid Leukemia in NOD/SCID Mice Is Independent of CXCR4 and Predicts Poor Patient Survival Stem Cells, March 1, 2004; 22(2): 188 - 201. [Abstract] [Full Text] [PDF]
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 I. M. Ghobrial, N. D. Bone, M. J. Stenson, A. Novak, K. E. Hedin, N. E. Kay, and S. M. Ansell Expression of the Chemokine Receptors CXCR4 and CCR7 and Disease Progression in B-Cell Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Mayo Clin. Proc., March 1, 2004; 79(3): 318 - 325. [Abstract] [PDF]
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E. Albesiano, B. T. Messmer, R. N. Damle, S. L. Allen, K. R. Rai, and N. Chiorazzi Activation-induced cytidine deaminase in chronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone Blood, November 1, 2003; 102(9): 3333 - 3339. [Abstract] [Full Text] [PDF]
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L. Granziero, P. Circosta, C. Scielzo, E. Frisaldi, S. Stella, M. Geuna, S. Giordano, P. Ghia, and F. Caligaris-Cappio CD100/Plexin-B1 interactions sustain proliferation and survival of normal and leukemic CD5+ B lymphocytes Blood, March 1, 2003; 101(5): 1962 - 1969. [Abstract] [Full Text] [PDF]
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M. J. Keating, N. Chiorazzi, B. Messmer, R. N. Damle, S. L. Allen, K. R. Rai, M. Ferrarini, and T. J. Kipps Biology and Treatment of Chronic Lymphocytic Leukemia Hematology, January 1, 2003; 2003(1): 153 - 175. [Abstract] [Full Text] [PDF]
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 A. Ptasznik, E. Urbanowska, S. Chinta, M. A. Costa, B. A. Katz, M. A. Stanislaus, G. Demir, D. Linnekin, Z. K. Pan, and A. M. Gewirtz Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells J. Exp. Med., September 2, 2002; 196(5): 667 - 678. [Abstract] [Full Text] [PDF]
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I. M. Pedersen, S. Kitada, L. M. Leoni, J. M. Zapata, J. G. Karras, N. Tsukada, T. J. Kipps, Y. S. Choi, F. Bennett, and J. C. Reed Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1 Blood, August 13, 2002; 100(5): 1795 - 1801. [Abstract] [Full Text] [PDF]
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F. Bertolini, C. Dell'Agnola, P. Mancuso, C. Rabascio, A. Burlini, S. Monestiroli, A. Gobbi, G. Pruneri, and G. Martinelli CXCR4 Neutralization, a Novel Therapeutic Approach for Non-Hodgkin's Lymphoma Cancer Res., June 1, 2002; 62(11): 3106 - 3112. [Abstract] [Full Text] [PDF]
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Y. Lee, A. Gotoh, H.-J. Kwon, M. You, L. Kohli, C. Mantel, S. Cooper, G. Hangoc, K. Miyazawa, K. Ohyashiki, et al. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines Blood, May 29, 2002; 99(12): 4307 - 4317. [Abstract] [Full Text] [PDF]
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M. Barragan, B. Bellosillo, C. Campas, D. Colomer, G. Pons, and J. Gil Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells Blood, April 15, 2002; 99(8): 2969 - 2976. [Abstract] [Full Text] [PDF]
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J.-J. Lataillade, D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, and M.-C. Le Bousse-Kerdiles Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism Blood, February 15, 2002; 99(4): 1117 - 1129. [Abstract] [Full Text] [PDF]
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N. Tsukada, J. A. Burger, N. J. Zvaifler, and T. J. Kipps Distinctive features of "nurselike" cells that differentiate in the context of chronic lymphocytic leukemia Blood, February 1, 2002; 99(3): 1030 - 1037. [Abstract] [Full Text] [PDF]
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K. Balabanian, A. Foussat, L. Bouchet-Delbos, J. Couderc, R. Krzysiek, A. Amara, F. Baleux, A. Portier, P. Galanaud, and D. Emilie Interleukin-10 modulates the sensitivity of peritoneal B lymphocytes to chemokines with opposite effects on stromal cell-derived factor-1 and B-lymphocyte chemoattractant Blood, January 15, 2002; 99(2): 427 - 436. [Abstract] [Full Text] [PDF]
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C. Hernandez-Lopez, A. Varas, R. Sacedon, E. Jimenez, J. J. Munoz, A. G. Zapata, and A. Vicente Stromal cell-derived factor 1/CXCR4 signaling is critical for early human T-cell development Blood, January 15, 2002; 99(2): 546 - 554. [Abstract] [Full Text] [PDF]
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N. E. Kay, T. J. Hamblin, D. F. Jelinek, G. W. Dewald, J. C. Byrd, S. Farag, M. Lucas, and T. Lin Chronic Lymphocytic Leukemia Hematology, January 1, 2002; 2002(1): 193 - 213. [Abstract] [Full Text]
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A. Bernal, R. D. Pastore, Z. Asgary, S. A. Keller, E. Cesarman, H.-C. Liou, and E. J. Schattner Survival of leukemic B cells promoted by engagement of the antigen receptor Blood, November 15, 2001; 98(10): 3050 - 3057. [Abstract] [Full Text] [PDF]
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 J. L. Gabrilove Hematologic Malignancies: An Opportunity for Targeted Drug Therapy Oncologist, October 1, 2001; 6(2008): 1 - 3. [Full Text] [PDF]
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J. L. Gabrilove Angiogenic Growth Factors: Autocrine and Paracrine Regulation of Survival in Hematologic Malignancies Oncologist, October 1, 2001; 6(2008): 4 - 7. [Abstract] [Full Text] [PDF]
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J. Kijowski, M. Baj-Krzyworzeka, M. Majka, R. Reca, L. A. Marquez, M. Christofidou-Solomidou, A. Janowska-Wieczorek, and M. Z. Ratajczak The SDF-1-CXCR4 Axis Stimulates VEGF Secretion and Activates Integrins but does not Affect Proliferation and Survival in Lymphohematopoietic Cells Stem Cells, September 1, 2001; 19(5): 453 - 466. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-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
Basic Principles and Practice. New York: Churchill Livingstone; 1999:1350-1363.