|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
From the Servicios de Inmunología y
Hematología, Hospital Ramón y Cajal, Madrid, Spain.
Tumoral lymphocytes from patients with B-chronic lymphocytic
leukemia (B-CLL) are long-lived cells in vivo, but they die rapidly by
apoptosis in vitro. Here, it is reported that endothelial cells (ECs)
inhibit the apoptosis of B-CLL cells, as determined by 4 different flow
cytometric methods, and that this antiapoptotic effect is mediated
mainly by soluble factor(s), as can be deduced from the following
findings. First, EC-conditioned medium (ECCM) inhibited the apoptotic
rate in B-CLL to approximately 50% of control. Second, the
antiapoptotic effect mediated by EC/B-CLL cell contact was more
apparent than real; using a fluorescence-based phagocytosis assay, it
was demonstrated that this effect was due to the phagocytic capacity of
ECs, which internalized apoptotic cells. Third, the protective effect
of ECCM was associated neither with proliferation nor differentiation
signals. Fourth, the survival factor was a dimeric form of IL-6 because
anti-IL-6 antibodies completely neutralized the antiapoptotic effect
mediated not only by the crude ECCM but also by the 45- to 55-kd active
fractions obtained after gel filtration, which contained high levels of IL-6. These IL-6 dimers (IL-6D) were noncovalently
associated. Sixth, human recombinant IL-6D
(hrIL-6D) inhibited B-CLL apoptosis, whereas hrIL-6
monomers (hrIL-6M) did not. Binding and functional competition experiments showed not only that monomers and dimers had
similar affinity for the IL-6R, but also that hrIL-6M
inhibited the antiapoptotic activity of hrIL-6D. These data
suggest that IL-6D derived from ECs promote the survival of
B-CLL cells.
(Blood. 2001;97:242-249) B-cell chronic lymphocytic leukemia (B-CLL)
is characterized by sustained lymphocytosis in the peripheral blood
(PB) of monoclonal CD5+ B-lymphocytes. These cells are
frozen in the G0/G1 phase of the cell cycle and
are long-lived in vivo.1,2 Indeed, it has been suggested
that the prolonged lifespan is the main factor for the accumulation of
the tumoral cells.1 However, in culture, PB B-CLL cells
undergo rapid and spontaneous apoptosis,3 suggesting that
programmed cell death in cultured B-CLL cells may be the result of the
absence of several humoral factors, cellular factors, or both present
in vivo. Thus, the prevention of apoptosis in B-CLL cells by several
cytokines, such as interferon (IFN)- There is growing evidence that all cells are constantly supported in
vivo by survival factors produced by other cells.9 Therefore, regulation of the size of the B-CLL tumoral population is
likely to depend directly on signals external to the cell that determine whether the cell-death program is engaged. As such, the
factors that can modulate B-CLL survival include not only cytokines but
also cell-cell contacts, extracellular matrix (ECM) components, or
both. In this sense, 2 recent reports10,11 suggest that
cell-cell interactions mediated by bone marrow stromal cells prevent
B-CLL apoptosis, though the adhesion molecules mediating this effect
have not been characterized.
It is well known that B-CLL lymphocytes are recirculating cells that
probably interact with vascular endothelial cells (ECs), the cell
population that covers the inner surfaces of blood
vessels.12 However, ECs are absent or rarely present in
venous blood extraction. Because B-CLL cells undergo apoptosis within
hours of removal from the body, in contrast to their prolonged survival
in vivo, we hypothesized that ECs may affect the viability of
circulating B-CLL cells. Our results indicate that IL-6 dimers
(IL-6D) contained in the conditioned medium produced by ECs
(ECCM), but not by close cell-cell interactions, are potent factors for
preventing B-CLL cells from entering apoptosis in culture.
Patients
Antibodies and cytokines
Cell purification Peripheral blood mononuclear cells (PBMCs) from patients or healthy subjects were obtained by density gradient centrifugation of heparinized blood on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Non-B cells were removed using magnetic beads (Dynabeads; Dynal, Oslo, Norway) coated with antibodies CD2 and CD14. B-CLL preparations contained 97% ± 1% CD19+ cells, as determined by flow cytometry (FACScan; Becton Dickinson). Total tonsillar B cells were prepared as previously described.14EC cultures and preparation of ECCM Human umbilical vein endothelial cells (HUVECs) were obtained through a previously described method15 and used at the second or third passage. Human umbilical cord-derived ECV-304 cells (American Type Culture Collection, Rockville, MD) were plated at a cell density of 0.1 × 106/mL and cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) (complete media; CM). The cells were passaged every 3 to 4 days by trypsinization.After cultivation for different time intervals, the conditioned medium
from HUVECs or ECV-304 cells was collected and filtered (0.22 µm),
and the supernatant was frozen at Cell cultures Purified tumoral B-lymphocytes and normal PBMCs and tonsillar B cells were cultured at a final concentration of 0.5 × 106 cells/mL in a 24-well plate (Costar, Cambridge, MA). In parallel experiments, tumoral B-lymphocytes (0.3 × 106 cells/well) were cocultured either in direct contact with ECs or physically separated from ECs using a transwell insert (Costar) with a 0.4-µm microporous membrane that permits the passage of soluble factors but prevents direct B-CLL-endothelium contact.Adhesion blocking assay and preparation of ECM The following blocking mAbs were incubated with B-CLL cells for 30 minutes at 4°C before the cell-cell contact assay: TS2/16 (anti-CD29), TS1/18 (anti-CD18), BU75 (anti-CD44), and LAM 1-3 (anti-CD62L) were purchased from Serotec (Oxford, United Kingdom), Endogen (Woburn, MA), Ancell (Bayport, MN), and Coulter, respectively. ECM was prepared from ECs cultured for 1, 2, or 3 days by the method described by Cavender et al.16Fixation of the EC monolayers ECV-304 cell monolayers were washed twice in phosphate-buffered saline (PBS) and then incubated for 15 minutes at 37°C with 1% paraformaldehyde. To remove residual fixative, the monolayers were washed 5 times with PBS solution and incubated with RPMI and 10% FCS for 3 days at 37°C before use.IL-6 content and neutralization of IL-6 activity in culture supernatants IL-6 was measured in the ECCM using an ELISA assay (Genzyme) according to the manufacturer's instructions. To neutralize IL-6 activity, aliquots of ECCM were incubated with a polyclonal rabbit antihuman IL-6 antibody at final dilutions capable of neutralizing 0.1 to 50 ng hIL-6. Rabbit serum was used as a control. The reaction was carried out for 1 hour at 37°.Immunofluorescent staining and cytometric analysis of apoptotic cells Flow cytometry for surface staining was performed according to the published procedure.14 Apoptosis of lymphocytes was assessed by 4 flow cytometric methods annexin-V binding and propidium
iodide (PI) staining, as described17,18; whole-cell
staining with PI, as described19; Nick translation assay
(TUNEL), according to the manufacturer's instructions (Boehringer
Mannheim, Mannheim, Germany); and modifications in scatter
properties (FSC/SSC), as described.20 FSC-annexin-V
fluorescence dot plot was used to discriminate debris from apoptotic
cells. An appropriate scattering gate was then set around viable,
early, and late apoptotic cells.
Phagocytosis assay Purified nonapoptotic B-CLL cells (5 × 106) were labeled with 50 µg viable dye TAMRA 5-(&6)-carboxy tetramethylrhodamine, succinimide ester; Molecular Probes, Eugene, OR), washed and then added to monolayers of ECV-304 cells as previously described.21 In the FSC/SSC dot plot, ECs were localized on the basis of their medium-to-high FSC and medium-to-high SSC characteristics.Flow cytometric assay of IL-6 receptors and competition experiments We determined IL-6R expression by using the relative binding of Fluorokine IL-6 biotin and streptavidin-FITC, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN), performed with fresh B-CLL cells. By preliminary titration experiments, 10 ng biotin IL-6 saturated IL-6R of all B-CLL samples tested, and this concentration was used for all experiments. Competition experiments were performed after preincubating cells for 30 minutes at 4°C with different amounts (0-100 ng) of IL-6 dimers or monomers.Heat inactivation and size fractionation of ECCM (S-200) Aliquots of ECCM were heated at either 60°C or 90°C for 15 minutes to assess the effect of heat treatment on inhibitory activity.To characterize the size of the inhibitory factor(s) present in the ECCM, we concentrated this medium 30-fold with an XM50 Amicon membrane (Amicon, Beverly, MA) before chromatography. A 1.5-mL aliquot of concentrated supernatant was layered onto a Sephacryl S-200 (Pharmacia LKB Biotechnology, Uppsala, Sweden) column (500 mL; 2.5 cm × 100 cm). The column was run at a constant flow rate of 60 mL/h. Fractions of 5 mL were collected. Protein elution was monitored at 280 nm, and fractions were stabilized with 10% FCS (vol/vol). Analysis of activity was measured in every fraction. The presence of IL-6 was determined by ELISA. hrIL-6 preparations derived from E coli or CHO cells (10 µg hrIL-6/200 µL) were applied onto a Sephacryl S-200 column (20 mL; 0.8 cm × 40 cm) and eluted at a constant flow rate of 5 mL/h. Fractions (200 µL) were stabilized with FCS (10% vol/vol) and analyzed for IL-6 content by ELISA. Immunoprecipitation of metabolically labeled IL-6 ECV-304 cells were cultured for 2 days at 105/mL in CM in a 24-well plate (Costar). Cells were washed twice in methionine-free DMEM medium and resuspended in the same medium supplemented with dialyzed FCS. After 1 hour, 25 µCi/mL 35S-methionine was added to each well. Methionine-labeled supernatants were collected after 20 hours of incubation. Protease inhibitors (phenylmethylsulfonyl fluoride 200 µM; EDTA 2 mM; pepstatin A 0.7 µg/mL; leupeptin 0.5 µg/mL) were added. Supernatants were then incubated with 5 µL normal rabbit serum (Sigma). After 2 hours, 10 µL protein G-Sepharose (Pharmacia) was added to each one. Samples were then incubated for 1 hour at 4°C with gentle agitation and centrifuged for 5 minutes at 2000g. Precleared supernatants were mixed with a polyclonal rabbit antihuman IL-6 (10 µg/mL) (Genzyme) for 3 hours at 4°C. The immune complexes were adsorbed onto protein G-Sepharose for 1 hour at 4°C and centrifuged at 12 000 rpm. The beads were sequentially washed 3 times and boiled in reducing (10% -mercaptoethanol) or nonreducing
(without -mercaptoethanol) sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) sample buffer. SDS-PAGE and
autoradiography were used to analyze the material eluted from
the beads.
Statistical evaluation The data are presented as mean ± SEM of n experiments. Statistical significance was evaluated using paired Student t test for comparisons between 2 means and analysis of variance for more than 2 means. P < .05 indicated statistical significance.
ECCM inhibits apoptosis of B-CLL cells To determine whether conditioned medium derived from HUVEC had antiapoptotic activity, B-CLL cells were cultured for 1, 2, or 3 days in CM or ECCM. For this purpose, we examined cell death by double staining with annexin-V and PI. As shown in Figure 1A, the percentage of apoptotic cells was substantially reduced in ECCM throughout the culture period. Because the isolation and culture of fresh HUVEC is laborious and their expansion and maintenance are limited, medium conditioned by the human umbilical cord-derived ECV-304 cell line was tested for its ability to inhibit B-CLL apoptosis. As can be seen in Figure 1B, ECCM derived from ECV-304 cells supported the survival of B-CLL cells with efficiency at least similar to that of conditioned medium derived from fresh HUVEC. Consequently, all future experiments were performed with ECCM derived from ECV-304 cells. Thus, in the presence of ECCM, the percentage of apoptotic B-CLL cells was significantly reduced from 35% ± 6% to 17% ± 4% on day 1 (n = 30, P < 10 7),
57% ± 8% to 32% ± 8% on day 2 (n = 30;
P < 106), and 73% ± 9% to
49% ± 9% on day 3 (n = 28; P < 105).
The ECCM-mediated prevention of in vitro apoptosis was observed in all
patients tested regardless of their age, Rai stage, white cell count,
or lymph node involvement. In contrast to B-CLL cells, the apoptotic
kinetics of normal PBMCs and tonsillar B cells were not modified by the
ECCM (data not shown).
Moreover, the incidence of hypodiploid and TUNEL+ cells and
of B-CLL lymphocytes with morphologic alterations agreed closely with
the results of annexin-V/PI analysis (Figure
2). In comparison, however, the annexin-V
assay was able to detect early phases of B-CLL apoptosis. For this
reason, the annexin-V/PI method was chosen to quantify apoptotic
leukemic cells. These results suggest that ECCM transduces a survival
signal to B-CLL cells.
Antiapoptotic effect mediated by EC/B-CLL cell contacts is more apparent than real We next performed experiments to determine the role of direct contact with endothelium versus the effect of soluble factors produced by ECs in mediating this B-CLL apoptosis inhibition. As can be seen in Figure 3, apoptosis was almost completely inhibited when B-CLL cells were plated in direct contact with the endothelium. This antiapoptotic effect was also observed when B-CLL cells were physically separated from ECs by transwell inserts, though to a lesser extent than that observed in endothelium-contact cultures. It should be noted that the viability observed in endothelium-prevented contact cultures was commonly lower than that detected with conditioned medium, at least in the first 2 days of culture (Figure 3A). After 3 days of culture, however, the transwell system was more effective in inhibiting B-CLL apoptosis than ECCM (Figure 3B). These results appear to indicate the consumption of soluble factor(s) in the ECCM after 3 days of culture. Taken together, these data also apparently suggest that B-CLL cells were protected from spontaneous apoptosis not only by soluble factor(s) but also through cell-cell interactions.
To determine whether adhesion proteins or their cognate ligands (ECM)
were involved in this process,22 neutralizing mAb antiadhesion molecules expressed by B-CLL cells were added to endothelium-contact cultures. As shown in Figure
4, the addition of blocking mAbs, alone
or in combination, against CD29, CD18, CD44, or CD62L did not interfere
with the protective capacity of ECs to prevent apoptosis in leukemic
cells. A complex ECM obtained by endothelial-cell lysis with 0.5%
sodium deoxycholate after 3 days of culture was also unable to inhibit
tumoral B-cell apoptosis (Figure 4). Interestingly, paraformaldehyde
fixation of ECs abrogated their ability to prevent B-CLL apoptosis:
the percentages of apoptotic cells after 1 and 3 days of culture
were 30% ± 7% and 70% ± 8% in the presence of fixed ECs
versus 32% ± 9% and 69% ± 9% in CM (n = 4). A synergistic
effect mediated by physical interactions and soluble factors was ruled
out because fixed ECV-304 cells with ECCM prevented B-CLL apoptosis in
a manner similar to that observed with ECCM alone (data not shown).
These results indicate 2 apparently contradictory facts: the direct
contact with ECs seemed to be involved in the prevention of B-CLL
apoptosis (Figure 3), and the protective role of ECs was not mediated
by adhesion molecules or by ECM proteins (Figure 4). With regard to
this paradox, we observed that significantly fewer B-CLL cells were
recovered from cultures in which leukemic cells were cultured in
contact with endothelium than when these cells were cultured either
separated from endothelium by a transwell insert or with ECCM (data not
shown). Because the role of ECs in recognizing and phagocytosing
apoptotic cells is well established,23,24 we predicted
that apoptotic B-CLL cells would be engulfed by ECs. To test this
hypothesis, the percentage of ECs with phagocytosed apoptotic cells was
determined at different time intervals using a fluorescence-based
phagocytosis assay in which fresh B-CLL cells were previously labeled
with TAMRA.21 As shown in Figure
5 from a representative experiment, the
percentage of ECs labeled with TAMRA after 24 hours of coculture with
B-CLL cells was 26% ± 8%, and this increased to 45% ± 6% and
95% ± 4% after 48 and 72 hours, respectively (n = 5). Moreover,
most ECs were TAMRA+/CD45
ECCM does not affect either proliferation or differentiation of B-CLL cells Our next objective was to find out whether the inhibitory effect of ECCM on the apoptosis of B-CLL cells was mediated by proliferative signals. For this purpose, we measured viability and the cell-cycle state of B-CLL cells that had been cultured with ECCM after 48 hours in comparison with fresh B-CLL cells. Results from 5 experiments showed that monoclonal B-lymphocytes rescued from apoptosis were mostly noncycling tumor cells after 2 days of culture with ECCM (98% ± 2% of B-CLL cells in G0/G1 phase of the cell cycle versus 97% ± 2% of fresh tumoral B cells; P > .05). Therefore, the persistence of B-CLL cells cultured in ECCM was not mediated by an increase in cell proliferation. We also confirmed that the antiapoptotic effect observed with the ECCM was not mediated by a differentiation signal because the percentage of more mature cells (CD38++) was undetectable after 2 days of culture (1% ± 1%; n = 5) and because the phenotypic profile was identical to that observed in freshly isolated B-CLL cells (CD20low, CD23+, CD5+, surface Iglow).Antiapoptotic factor contained in the ECCM is a dimeric form of IL-6 To identify the antiapoptotic soluble factor(s) produced and released by ECV-304 cells, we performed 2 parallel sets of experiments. First, the effect of neutralizing polyclonal antibodies against several well-known soluble factors produced by human ECs was tested. Second, concentrated ECCM was analyzed by Sephacryl S-200 gel filtration to investigate the approximate molecular weight of the active soluble factor(s). As can be seen in Figure 6, antiapoptotic activity in the crude ECCM was completely neutralized by anti-IL-6 polyclonal antibodies in a dose-dependent manner. In contrast, the addition of anti-GM-CSF, anti-IL-4, anti-IL-8, or anti-IL-13 neutralizing antibodies did not modify the kinetics of entry of the B-CCL cell into apoptosis in these cultures (n = 5; P > .05). These results were in close agreement with the fact that the highest antiapoptotic activity was always found in the ECCM collected from ECV-304 monolayer after 3 days of cultureIL-6
content was 1 ± 0.4 ng/mL at day 1 and 3 ± 1 ng/mL at day 2, peaking at day 3 (11 ± 7 ng/mL) and declining thereafter, as
measured by ELISA. Moreover, fractionation of concentrated ECCM (the
eluate obtained after size separation by Amicon ultrafiltration had no
antiapoptotic activity; data not shown) showed that the
apoptosis-inhibiting agent was a molecule(s) with a relative molecular
mass in the range of 45 to 55 kd (Figure
7). Human IL-6 is a protein with a
molecular mass ranging from 21 to 28 kd, depending on the cellular source and preparation.25 On the basis of the apparent
molecular weight of the soluble factor identified in the above
experiment, however, ECs secreted a form of IL-6 with a molecular mass
ranging from 45 to 55 kd. To confirm this hypothesis, we used a
sensitive ELISA to detect the IL-6 eluting from the column. As is
apparent from Figure 7, one major immunoreactive peak was detected,
corresponding to 45- to 55-kd fractions, whereas a minor peak was
detected between fractions 44 and 47 (22 to 25 kd) that had no effect
as survival factor. The dimer-monomer ratio was 10:12/1 (n = 3).
Thus, the elution of bioactivity correlates with the elution of IL-6
immunoreactivity, confirming the findings of previous
reports26 demonstrating that the native IL-6 exists as a
dimer. It is of note that the concentration of IL-6 in the 22- to 25-kd
fractions was much lower than that in the 45- to 55-kd fractions.
Consequently, the failure of IL-6M to prevent B-CLL cell
apoptosis could be due to insufficient amounts of monomer. To exclude
this possibility, the 22- to 25-kd fractions and the 45- to 55-kd
fractions were pooled and further concentrated to explore their
antiapoptotic activity. As shown in Figure 7, IL-6D
inhibited apoptosis in a dose-dependent manner. In contrast, identical
concentrations of IL-6M added to B-CLL cultures were
inactive. In addition, the survival effect mediated by
IL-6D was fully inhibited by anti-IL-6 neutralizing
antibodies: the percentages of annexin-V+ cells after 2 days of culture were 53% ± 8%, 27% ± 6%, and 51% ± 8% in
CM, IL-6D (10 ng/mL), and IL-6D + anti-IL-6 antibodies, respectively (n = 3).
Moreover, IL-6D contained in the ECCM were heat stable because heat treatment at 60°C or 90°C for 15 minutes did not alter their antiapoptotic activity: the percentages of apoptotic cells after 3 days of culture were 68% ± 10%, 44% ± 7%, and 47% ± 8% in CM, ECCM, and heat-treated ECCM, respectively (n = 5). To determine the electrophoretic behavior of this IL-6, ECV-304
endothelial cells were initially labeled with
35S-methionine, and IL-6 products were immunoprecipitated
from conditioned medium. Subsequent separation of this
immunoprecipitate by SDS-PAGE led to the appearance of a single band
corresponding to Mr 25 000 under both nonreducing and reducing
conditions (Figure 8, lanes 2 and 4). In
contrast, no band corresponding to the dimeric form detected in gel
filtration analysis was found. These results suggested that
IL-6D contained in the ECCM could be formed by a
noncovalent association.
Ability of various recombinant IL-6 (rIL-6) proteins to prevent B-CLL apoptosis Having determined the antiapoptotic effect mediated by natural IL-6D produced by ECs, we determined whether other IL-6 protein preparations showed a similar effect with regard to their ability to inhibit B-CLL apoptosis. Crude hrIL-6 preparations derived from E coli had little, if any, effect on B-CLL apoptosis (percentages of apoptotic cells after 2 days of culture were 56% ± 8% and 50% ± 6% in CM and CM+hrIL-6, respectively; n = 9; P > .05). For this reason, hrIL-6 was fractionated, as has been described previously for endothelial IL-6. When subjected to gel filtration, this IL-6 preparation, eluted in 2 peaks, distributed in a manner similar to that observed in the ECCM, indicating that it has similar molecular sizes in solution (Figure 9A). The dimer-monomer ratio was 2:6/1 (n = 3). To investigate the ability of both forms of hrIL-6 to prevent B-CLL apoptosis, fractions containing either monomers or dimers were pooled: hrIL-6D prevented in a dose-dependent manner the apoptosis of B-CLL cells, whereas monomers did not (Figure 9B). This hrIL-6D-mediated effect was reversed by the addition of blocking anti-IL-6 antibodies (percentage of apoptotic cells, 94% ± 5%; n = 3); hrIL-6D derived from CHO cells prevented B-CLL apoptosis in a concentration-dependent manner, similar to that observed in response to hrIL-6D derived from E coli (data not shown).
To further understand the mechanism responsible for the increased effect of hrIL-6D, compared with hrIL-6M, on B-CLL apoptosis, we tested the capacity of both monomeric and dimeric forms of hrIL-6 derived from E coli for binding to IL-6R (CD126). Initially, we performed experiments on the binding of biotin-IL-6 to human B-CLL cells. The percentage of leukemic cells capable of binding IL-6 was 83% ± 13% (n = 10). The specificity of the binding was demonstrated by the achievement of more than 95% inhibition of the binding by preincubating biotin-IL-6 with an inhibitory antihuman IL-6 antibody. Both monomers and dimers were then examined for their ability to inhibit binding of biotin-IL-6 to B-CLL cells. The IC50 values for either hrIL-6M or hrIL-6D (ie, concentration of competitor inhibiting binding of biotin-IL-6 by 50%) were similar and ranged from 75 to 100 ng/mL (n = 3). These results not only suggest that monomers and dimers had similar affinity for the IL-6R but also that the bioactivity of the 2 forms of IL-6 did not correlate with their receptor-binding ability. Combining the data from the binding studies with the lack of activity of crude hrIL-6 preparations, we interpret that the monomers would interfere with the antiapoptotic effect mediated by the dimers. To confirm this possibility, varying concentrations of hrIL-6M were incubated for 48 hours with B-CLL cells in the presence of an optimal concentration (10 ng/mL) of hrIL-6D. As shown in Figure 9C, hrIL-6M inhibited, in a dose-dependent manner, the antiapoptotic effect of hrIL-6D on B-CLL cells. These results clearly suggest that IL-6M specifically inhibits the survival effect mediated by IL-6D.
This study shows for the first time that IL-6D secreted by ECs promotes B-CLL cell survival in vitro. B-CLL is characterized by the accumulation in the blood, bone marrow, lymph nodes, and spleen of a clonal population of B-lymphocytes. Interestingly, ECs cover the inner surface of blood vessels and are an important component of all of these sites in vivo.12 Therefore, the ability of ECs to support B-CLL viability may be relevant in the pathogenesis of this disease. In contrast to our results, previous studies using the pre-established EA.hy 926 endothelial cell line found that its conditioned medium was unable to prevent B-CLL apoptosis.27 Although the mechanism of this difference remains to be elucidated, a possible explanation is the different nature of these cell lines: ECV-304 is a spontaneously transformed human umbilical endothelial cell line,28 whereas EA.hy 926 is a cell line produced by hybridizing HUVECs with the human epithelial cell line A549.27 Indeed, it has been published that EA.hy 926 cells appear to have lost the ability to respond to IL-4 or IFN stimulation,29 whereas ECV-304 cells have not.28 It is possible, therefore, that these cell lines differ not only in their response to several cytokines but also in the synthesis and secretion of survival factors. Our initial results suggested that contact-mediated events were also responsible for the antiapoptotic effect of ECs on B-CLL cells. We have demonstrated that this apparent survival effect was due to the phagocytic capacity of ECs, which rapidly internalized apoptotic cells. On the one hand, the fact that ECs, and other nonprofessional phagocytes, engulf apoptotic cells is not surprising because this phenomenon has already been described by several groups of researchers.15,16,30 On the other hand, the use of live feeder cells in assays to determine cell-contact-dependent survival effects has been widely studied.31,32 Indeed, 2 reports have shown that ECs are useful as feeder cells in contact-mediated survival systems for B-CLL cells.27,33 Many of these system cocultures, however, are performed without excluding the probable contribution to this phenomenon of the phagocytic activity displayed by these cells. Therefore, it is likely that, at least in some cases, the use of cell-contact-dependent assays greatly overestimates the antiapoptotic effect mediated by the feeder cell owing to its phagocytic activity. Tumorigenesis can be induced by activating cell proliferation, by inhibiting metabolic pathways regulating programmed cell death, or both.34 However, it is difficult to separate the antiapoptotic activity mediated by soluble factors from their ability to promote proliferation.35 Our results consistently showed that most malignant cells remained in the G0/G1 phase after 2 days of culture. Moreover, we also discarded the possibility that EC triggered a differentiation signal, on receipt of which the B-CLL cells were transformed into plasmacytoid-like cells. In this sense, our results also demonstrate that the signal mediated by ECCM is integrated into survival but not proliferation or differentiation pathways. Here we present evidence that the antiapoptotic factor contained in the ECCM is a dimeric form of IL-6. The evidence that IL-6 forms dimers in solution at physiological concentrations is widely accepted.26,36,37 We also observed that the monomer association in IL-6 derived from ECs is noncovalent and does not involve disulfide linkage. This was not an unexpected finding because previous works have demonstrated that IL-6D derived from different sources is not stable under denaturing conditions.26,37 In our hands, crude commercially available hrIL-6 preparations had little, if any, effect on our culture system. In contrast, hrIL-6D obtained after size exclusion chromatography showed antiapoptotic activity, confirming that dimer formation is essential in the prevention of B-CLL apoptosis. Moreover, we also demonstrate that the monomeric form functions as a competitive inhibitor of the dimeric form. These results may explain why there is considerable confusion regarding the role of IL-6 in B-CLL cell apoptosis: most researchers have found no effect,5,6 whereas Reittie et al38 have described the inhibition of apoptosis by IL-6. Because all the recombinant IL-6 proteins appear as 2 components on gel filtration, though the abundance of the 2 forms does vary between preparations, the dimer-monomer ratio could be related to the in vitro activity of hrIL-6. Alternatively, other disparities in the hrIL-6 proteins may account for the varying abilities to inhibit the apoptosis of B-CLL cells and may be unique for each protein expression system.39-41 This study also shows that IL-6D contained in the ECCM was clearly more active than rIL-6D derived from E coli. Although it is uncertain from the current work whether the difference in the magnitude of survival effect mediated by EC (natural) and E coli-derived IL-6D could result from their glycosylated and unglycosylated nature, respectively, this hypothesis is unlikely to be correct. Indeed, glycosylated hrIL-6D obtained from CHO cells was active in inhibiting B-CLL apoptosis with a dose-response curve similar to that of hrIL-6D derived from E coli (data not shown). Differences in the mode of dimerization that affects the stability of the IL-6D can be predicted to decrease the mediation of survival activity, as has been described for the noncovalently associated hrSCF dimer, which can undergo spontaneous rapid monomer dissociation.42 Accordingly, it is also possible that IL-6D derived from ECV-304 cells exhibit more stability than hrIL-6D derived from E coli. Indeed, IL-6D contained in the ECCM were heat stable. This is in contrast to hrIL-6D derived from E coli, which are rapidly dissociated into monomers at 37°C.43 The most intriguing question raised by this work is why dimeric, but not monomeric, form of IL-6 shows antiapoptotic activity. Our results are, indeed, in contrast to those of May et al,26 who have reported that IL-6M is more active than or as active as IL-6D in the B9 hybridoma growth factor and Hep3B hepatocyte-stimulating factor assays, respectively. It is well known that IL-6 exerts its biologic activities through the IL-6-specific receptor complex that consists of the IL-6-binding subunit (IL-6R) and the signal transducing subunit (gp130).44 Moreover, several reports demonstrate that clustering of membrane-bound cytokine receptors is important in establishing a downstream signal. In fact, dimerization of several cytokine receptors is sufficient to elicit a biologic response.45,46 Because our competition-binding experiments demonstrate that both monomeric and dimeric forms of hrIL-6 bind IL-6R with comparable affinity, it was reasonable to assume that IL-6M could induce only IL-6R occupancy, not receptor clustering, and could exhibit partial signaling in comparison to the complete signaling generated by IL-6D. This hypothesis, however, is unlikely because monomeric IL-6 is dimerized at least at the receptor site and can induce intracellular signal transduction pathways by inducing the homodimerization of gp130, as previously described.26 As a possible explanation, Ward et al37 demonstrate that IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay, suggesting that both IL-6 forms mediate different intracellular signals. Moreover, Van den Berghe et al47 have found recently that although monomers and dimers of the fibroblast growth factor-2 (FGF-2), another well-known dimeric soluble factor, show similar receptor binding capacity, their biologic capacities are different. Interestingly, monomeric FGF-2 induced cell differentiation but not cell proliferation. In contrast, dimeric FGF-2 was capable of inducing both biologic responses. The authors thus demonstrate the uncoupling between proliferation and differentiation FGF-2 signaling pathways and the essential role of dimerization in the mitogenic activity of this factor. It is of note that our results demonstrate that dimeric IL-6 form inhibits apoptosis but does not affect proliferation or differentiation status of B-CLL cells. This analogous behavior led us to speculate that similar uncoupled signals may operate in our system. Moreover, neither Bcl-2 nor Bax expression correlated with the ability of ECCM to prevent B-CLL cell death (not shown). Because Bcl-xL or Mcl-1 proteins correlate with survival of human peripheral blood B-lymphocytes better than Bcl-2,48 experiments are in progress to determine whether the survival signal provided by the ECCM in human B-CLL cells could be mediated through the up-regulation of survival genes such as Bcl-xL or Mcl-1.
We thank Drs A. Silva, A. García-Pardo, and M. T. de la Fuente (Centro de Investigaciones Biologicas) and Drs A. Durantez, J.A. Vargas and R. Castejon (Clínica Puerta de Hierro) for some helpful discussions. We also thank David Weston for the critical reading of the manuscript.
Submitted September 7, 1999; accepted August 26, 2000.
Supported by the Comunidad Autónoma de Madrid (grants 07/029/96 and 08.1/0012/97) and by Fondo de Investigaciones Sanitarias de la Seguridad Social (grant 96/1555).
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: Ernesto Roldán Santiago, Servicio de Inmunología, Hospital Ramón y Cajal, Carretera Colmenar Km 9.100, 28034 Madrid, Spain.
1.
Dighiero G, Travade P, Chevret S, et al.
B-cell chronic lymphocytic leukemia: present status and future directions.
Blood.
1991;78:1901-1914
2.
Dameshek W.
Chronic lymphocytic leukemia: an accumulative disease of immunologically incompetent lymphocytes.
Blood.
1967;29:566-584 3. Collins RJ, Verschuer LA, Harmon BV, Prentice RL, Pope JH, Kerr JFR. Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro. Br J Haematol. 1989;71:343-350[Medline] [Order article via Infotrieve].
4.
Panayiotidis P, Ganeshaguru K, Jabbar S, Hoffbrand A.
Alpha-interpheron (
5.
Dancescu M, Rubio-Trujillo M, Biron G, Bron D, Delespesse G, Sarfati M.
Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates bcl-2 expression.
J Exp Med.
1992;176:1319-1326
6.
Buschle M, Campana D, Carding SR, Richard C, Hoffbrand AV, Brenner MK.
Interferon gamma inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.
J Exp Med.
1993;177:213-218
7.
Chaouchi N, Wallon C, Goujard C, et al.
Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis.
Blood.
1996;87:1022-1029
8.
Di Celle PF, Mariani S, Riera L, Stacchini A, Reato G, Foa R.
Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion.
Blood.
1996;87:4382-4389 9. Raff MC. Social control on cell survival and cell death. Nature. 1992;356:397-400[CrossRef][Medline] [Order article via Infotrieve]. 10. Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand V. Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukemia cells in vitro. Br J Haematol. 1996;92:97-103[CrossRef][Medline] [Order article via Infotrieve].
11.
Lagneaux L, Delforge A, Bron D, De Bruyn C, Stryckmans P.
Chronic lymphocytic leukemia B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells.
Blood.
1998;91:2387-2396 12. Risau W. Differentiation of endothelium. FASEB J. 1995;9:926-933[Abstract].
13.
Rai K, Sawitsky A, Cronkite E, Chanana AB, Leey RN, Pasternak BSA.
Clinical staging of chronic lymphocytic leukemia.
Blood.
1975;46:219-234
14.
Rodriguez C, Roldan E, Navas G, Brieva JA.
Essential role of tumor necrosis factor- 15. Meagher L, Mahiouz D, Sugars K, et al. Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction. J Immunol Methods. 1994;175:237-246[CrossRef][Medline] [Order article via Infotrieve]. 16. Cavender DE, Cearns J, Barrus CQ, Dunaway-Piccioni D. T-cell adhesion to extracellular matrix molecules secreted by endothelial cells cultured in a substrate of type IV collagen. J Immunol Methods. 1991;144:185-196[CrossRef][Medline] [Order article via Infotrieve].
17.
Lens SMA, Tesselaar K, Den Drijver BFA, Van Oers MHJ, Van Lier RAW.
A dual role for both CD40-ligand and TNF- 18. Vermes I, Haanen C, Richel DJ, Schaafsma MR, Kalsbeek-Batenburg E, Reutelingsperger CPM. Apoptosis and secondary necrosis of lymphocytes in culture. Acta Haematol. 1997;98:8-13[Medline] [Order article via Infotrieve]. 19. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991;139:271-279[CrossRef][Medline] [Order article via Infotrieve]. 20. Swat W, Ignatowitcz L, Kisielow P. Detection of apoptosis of immature CD4+CD8+ thymocytes by flow cytometry. J Immunol Methods. 1991;137:79-87[CrossRef][Medline] [Order article via Infotrieve]. 21. Hess KL, Babcock GF, Askew DS, Cook-Mills JM. A novel flow cytometric method for quantifying phagocytosis of apoptotic cells. Cytometry. 1997;27:145-152[CrossRef][Medline] [Order article via Infotrieve]. 22. Meredith JE, Fazeli B, Schwartz A. The extracellular matrix as a cell survival factor. Mol Biol Cell. 1993;4:953-961[Abstract]. 23. Dini L, Lentini A, Diez-Diez G, et al. Phagocytosis of apoptotic bodies by liver endothelial cells. J Cell Sci. 1995;108:967-973[Abstract]. 24. Hess KL, Tudor KSRS, Johnson JD, Osati-Ashtiani F, Askew DS, Cook-Mills JM. Human and murine high endothelial venule cells phagocytose apoptotic leukocytes. Exp Cell Res. 1997;236:404-411[CrossRef][Medline] [Order article via Infotrieve]. 25. Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993;54:1-78[Medline] [Order article via Infotrieve].
26.
May LT, Santhanam U, Sehgal PB.
On the multimeric nature of natural interleukin-6.
J Biol Chem.
1991;266:9950-9955
27.
Long BW, Witte PL, Abraham GN, Gregory SA, Plate JR.
Apoptosis and interleukin 7 gene expression in chronic lymphocytic leukemia cells.
Proc Natl Acad Sci U S A.
1995;92:1416-1420 28. Takahasi K, Sawasaki Y. Rare spontaneously transformed human endothelial cell line provides useful research tool. In Vitro Cell Dev Biol. 1992;28A:380-382. 29. Thornhill MH, Li J, Haskard DO. Leukocyte endothelial cell adhesion: a study comparing human umbilical vein endothelial cell line EA-hy 926. Scand J Immunol. 1993;38:279-286[CrossRef][Medline] [Order article via Infotrieve]. 30. Hall SE, Savill JS, Henson PM, Haslett C. Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin. J Immunol. 1994;153:3218-3227[Abstract].
31.
Manabe A, Coutan-Smith E, Behm FG, Raimondi SC, Campana D.
Bone marrow-derived stromal cells prevent apoptotic cell death in B-lineage acute lymphoblastic leukemia.
Blood.
1992;79:2370-2377 32. Hardin JA, Yamaguchi K, Sherr DH. The role of peritoneal stromal cells in the survival of sIgM+ peritoneal B lymphocyte populations. Cell Immunol. 1995;161:50-60[CrossRef][Medline] [Order article via Infotrieve]. 33. Jewell AP, Yong KL. Regulation and function of adhesion molecules in B-cell chronic lymphocytic leukaemia. Acta Haematol. 1997;97:67-72[Medline] [Order article via Infotrieve]. 34. Williams GT. Programmed cell death: apoptosis and oncogenesis. Cell. 1991;65:1097-1098[CrossRef][Medline] [Order article via Infotrieve]. 35. Shi Y, Wang R, Sharma A, et al. Dissociation of cytokine signals for proliferation and apoptosis. J Immunol. 1997;159:5318-5328[Abstract]. 36. Wijdenes J, Clement C, Klein B, et al. Human recombinant dimeric IL-6 binds to its receptor as detected by anti-IL-6 monoclonal antibodies. Mol Immunol. 1991;28:1183-1192[CrossRef][Medline] [Order article via Infotrieve].
37.
Ward LD, Hammacher A, Hawlett GJ, et al.
Influence of interleukin-6 (IL-6) dimerization on formation of the high affinity hexameric IL-6-receptor complex.
J Biol Chem.
1996;271:20138-20144 38. Reittie JE, Yong KL, Panayiotidis P, Hoffbrand V. Interleukin-6 inhibits apoptosis and tumour necrosis factor induced proliferation of B-chronic lymphocytic leukaemia. Leuk Lymphoma. 1996;22:83-90[Medline] [Order article via Infotrieve]. 39. Hirano T, Yasukawa K, Harada H, et al. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature. 1986;324:73-76[CrossRef][Medline] [Order article via Infotrieve]. 40. Zilberstein A, Ruggieri R, Korn JN, Revel M. Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines. EMBO J. 1986;5:2529-2537[Medline] [Order article via Infotrieve].
41.
May LT, Ghrayeb J, Santhanam U, et al.
Synthesis and secretion of multiple forms of 42. Lu HS, Chang WC, Mendiaz EA, Mann MB, Langley KE, Hsu YR. Spontaneous dissociation-association of monomers of the human stem cell factor dimer. Biochem J. 1995;305:563-568. 43. Matthews JM, Hammacher A, Howlett GJ, Simpson RJ. Physicochemical characterization of an antagonistic human interleukin-6 dimer. Biochemistry. 1998;37:10671-10680[CrossRef][Medline] [Order article via Infotrieve]. 44. Taga T, Hibi M, Hirata Y, et al. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell. 1989;58:573-581[CrossRef][Medline] [Order article via Infotrieve]. 45. Klemm JD, Schreiber SL, Crabtree GR. Dimerization as a regulatory mechanism in signal transduction. Annu Rev Immunol. 1998;16:569-592[CrossRef][Medline] [Order article via Infotrieve]. 46. Spivak-Kroizman T, Lemmon MA, Dikic I, et al. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell. 1994;79:1015-1024[CrossRef][Medline] [Order article via Infotrieve]. 47. Van den Berghe L, Mortier I, Zanibellato C, et al. FGF-2 dimerization involvement in growth factor mediated cell proliferation but not cell differentiation. Biochem Biophys Res Comm. 1998;252:420-427[CrossRef][Medline] [Order article via Infotrieve].
48.
Lomo J, Smeland EB, Krajewski S, Reed JC, Blomhoff HK.
Expression of the Bcl-2 homologue Mcl-1 correlates with survival of peripheral blood B-lymphocytes.
Cancer Res.
1996;56:40-43
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  N. Kolliputi and A. B. Waxman IL-6 cytoprotection in hyperoxic acute lung injury occurs via PI3K/Akt-mediated Bax phosphorylation Am J Physiol Lung Cell Mol Physiol, July 1, 2009; 297(1): L6 - L16. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ruiz, Y. Krupnik, M. Keating, J. Chandra, M. Palladino, and D. McConkey The proteasome inhibitor NPI-0052 is a more effective inducer of apoptosis than bortezomib in lymphocytes from patients with chronic lymphocytic leukemia. Mol. Cancer Ther., July 1, 2006; 5(7): 1836 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Trikha, R. Corringham, B. Klein, and J.-F. Rossi Targeted Anti-Interleukin-6 Monoclonal Antibody Therapy for Cancer: A Review of the Rationale and Clinical Evidence Clin. Cancer Res., October 15, 2003; 9(13): 4653 - 4665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Taverner, N. E. Hall, R. A. J. O'Hair, and R. J. Simpson Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry J. Biol. Chem., November 22, 2002; 277(48): 46487 - 46492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Longo, J. A. Kennell, M. J. Ochocinska, S. E. Ross, W. S. Wright, and O. A. MacDougald Wnt Signaling Protects 3T3-L1 Preadipocytes from Apoptosis through Induction of Insulin-like Growth Factors J. Biol. Chem., October 4, 2002; 277(41): 38239 - 38244. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
,
20°C until use. Maximal
antiapoptotic activity in the ECCM was observed when HUVECs or ECV-304
cells were grown to near confluence (7 and 3 days, respectively). ECCM
and cytokines were added at the onset of the culture.
), 2 (
), or 3 (
) days of culture, B-CLL cells were removed from the well by
pipetting and were stained with annexin-V-FITC and PI for flow
cytometry analysis. Conditioned medium from HUVEC or ECV-304 cells was
collected after 7 and 3 days of culture, respectively. Results are
expressed as the mean ± SEM of 5 independent experiments and
represent the percentage of apoptotic cells.
) was measured as
described in "Materials and methods." The Mr standards are
indicated by arrows; 
, A280; ×, annexin-V. A representative
experiment of 3 similar elution profiles is shown. The inset shows the
antiapoptotic activity of monomeric and dimeric forms of endothelial
IL-6. B-CLL cells were incubated with various concentrations of
IL-6D (
) or IL-6M (
