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PLENARY PAPER
From the Department of Internal Medicine and
Oncology, Department of Biomedical Sciences and Human Oncology,
Section of Internal Medicine and Clinical Oncology, University of Bari,
Italy.
Highly malignant myeloma cells up-regulate their Fas-ligand (Fas-L)
to escape immune surveillance by Fas+ cytotoxic cells. Here
it is demonstrated that this abnormality is involved in the
pathogenesis of the severe anemia associated with progression of
multiple myeloma (MM). By measuring Fas and Fas-L in plasma cells and
erythroblasts from 19 MM patients and 5 with monoclonal gammopathies of
undetermined significance (MGUS), it was found that both
Fas-L+ myeloma cells and Fas+ erythroid
progenitors were significantly increased in patients with stage III MM
whose erythroblasts, cultured in the presence of autologous plasma
cells or their supernatant, underwent prompt apoptosis as evaluated by
propidium iodide staining, the TUNEL assay, and detection of the
APO2.7-reactive mitochondrial antigen. Flow cytometry of fresh
erythroblasts revealed a considerable expression of the caspases CPP32
and FLICE in both their constitutive proenzymatic forms and in cleaved
subunits. By contrast, their intracytoplasmic expression was defective
in patients with inactive disease and MGUS controls. The evidence that
Fas-L+ myeloma clones directly prime erythroblast
apoptosis in vivo was further supported by the occurrence of
fluorescein isothiocyanate-TUNEL+ erythroblasts
juxtaposed to myeloma cells in bone marrow smears. These results
strongly suggest that the deregulated apoptosis in myeloma clones plays
an active role in the progressive destruction of the erythroid matrix
by a cytotoxic mechanism based on up-regulation of Fas-L.
(Blood. 2001;97:1155-1164) Fas-ligand (Fas-L) is a type 2 glycoprotein1 expressed by activated lymphocytes and
cytotoxic cells2,3 but is also detectable in a number of
cell types4-6 and in tumors of different lineages.7 By cross-linking its natural membrane
coreceptor, namely Fas (Apo-I/CD95),8 Fas-L induces
sequential activation of caspases, and this results in the apoptotic
cell death of Fas+ cells.9 The expression of
Fas-L by nonlymphoid cells in a few organs such as
testis,10 uterus,11 and the anterior chamber of the eye is thought to maintain a state of immune privilege by
eliminating Fas+ infiltrating lymphocytes.12 A
similar immune escape mechanism has also been proposed for several
tumor cell types whose Fas-L counterattacks the Fas+
effector cells committed to immune surveillance.
In addition to solid tumors such as melanoma,13
astrocytoma,14 and colon carcinoma,15 other
neoplasms of T-cell origin have been reported to overexpress
Fas-L.16 Previous studies also showed that
Fas-L+ plasma cell lines extinguish Fas+ T
lymphoblasts in vitro17 and suggested that this mechanism operates in multiple myeloma (MM) to escape the suppressive control of
cytotoxic cells. We have recently provided evidence that both overexpression and secretion of Fas-L in vitro by a number of myeloma
cell clones reflect a high degree of malignancy, namely the ability to
proliferate in the absence of interleukin-6 as well as the poor
Fas-mediated apoptosis.18 By contrast, a variable sensitivity to cell death was present in additional myeloma cell lines,
one of which showed little or undetectable production of Fas-L either
on its cell membrane or in soluble form.
Clinical manifestations of MM include skeleton colonization by
malignant plasma cells and a normochromic/normocytic anemia, both of
which influence its progression.19 A number of different mechanisms have been postulated to account for the severity of the
anemia frequent in hematologic malignancies. Chronic derangement of the
cytokine network at the bone marrow level as well as a persistent
defect of erythropoietin (EPO), particularly in patients with kidney
failure, are believed to play a pivotal role.20-22
Recent studies have also shown that the Fas/Fas-L system is actively
involved in the regulation of erythropoiesis.23 Fas is
strongly up-regulated in hematopoietic progenitor cells24 during chronic hematologic disorders by the increased levels of both
tumor necrosis factor (TNF) and interferon (IFN)- In this study, we explored the extent of apoptosis in bone marrow
erythroblasts of MM patients with respect to Fas-L overexpression by
the plasma cell clones. We found that the deregulated apoptosis in
highly malignant plasma cell lines is powerfully detrimental to bone
marrow erythroid growth and maturation and that this mechanism is
engaged in the expansion of the myeloma clone within the bone marrow.
Patients
Bone marrow cell phenotyping and cultures
Aliquots of bone marrow samples were also processed to prepare cultures of both plasma cell and erythroid lineages. Briefly, myeloma cells were enriched by passing the mononuclear cells previously obtained by Ficoll-Hypaque sedimentation over a discontinuous density gradient of a Percoll solution at 90% to 30%. Plasma cells collected at both 60% and 45% gradient concentration were enriched to more than 80%. Then, 1 × 106 cells were cultured in complete medium (RPMI 1640 plus 10% fetal calf serum and 2 mM glutamine) in the presence of 500 U/mL interleukin-6 (Genzyme, Cambridge, MA). Cultures were maintained in a CO2-humidified incubator up to 3 weeks to establish continuous proliferation. Enrichment in malignant plasma cells was monitored weekly by CD38+ bright cytofluorimetric detection. Erythroblast proliferation was also stimulated in parallel aliquots of bone marrow samples by preparing an Iscove's modified Dulbecco's medium supplemented with 20% fetal calf serum, 500 U/mL ampicillin (Sigma, St Louis, MO), 100 U/mL rHu-EPO (Janssen-Cilag, Schaffausen, Switzerland), 10 µg/mL insulin, and 5% sterile human AB serum, as described.28 Cultures were maintained up to 9 days and used for coculturing experiments. Cocultures of erythroblasts with plasma cells Mixed cultures of erythroblasts and plasma cells were established to investigate the potential cytotoxic effect of Fas-L+ cells within each culture. Briefly, 2 × 105 to 5 × 105 of 8-day cultured erythroblasts were incubated overnight in 24-well plates at 37°C in the presence of plasma cells at a 1:1 ratio. In most instances erythroblasts were cultured in the presence of enriched autologous myeloma cells, whereas parallel experiments included treatment of erythroblasts for an equivalent time with the conditioned supernatant (SN) from the relative plasma cell culture.A number of control experiments included inhibition of the interaction between Fas and Fas-L in culture to assess the specificity of erythroblast suppression. Separate erythroblast preparations from selected patients were preincubated with increasing amounts of nonagonist Fas-reactive IgG1-UB2 (Immunotech) MoAb (0.01, 0.1, 1.0, 10, and 100 µg/mL) to saturate the receptor binding. These cells were cocultured with myeloma cells, and the extent of their apoptosis was measured. In parallel, alternative experiments included the pretreatment of myeloma cells with similar levels of the recombinant Fas-Fc chimeric protein (Pharmingen) to block their Fas-L linkage sites. These cell preparations were then used to measure erythroblast apoptosis. In additional experiments we prepared enriched erythroblast populations from the bone marrow of a few patients by magnetic cell-sorting procedures (Miltenyi Biotec, Bergisch Gladbach, Germany). These methods used the positive selection of erythroblasts by magnetic GpA-coupled microbeads and provided adequate amounts of purified erythroblasts according to the manufacturer's instructions. We used these cells for further control experiments measuring their spontaneous apoptosis in the absence of either cocultured myeloma cells or their conditioned SN. Measurement of apoptosis Three different approaches were adopted to detect apoptosis in cocultured erythroblasts in comparison with control unstimulated cells from each patient. We first used propidium iodide (PI) cell staining, which included overnight treatment of cells with 100 µg PI following their preparation with 70% ethanol at 4°C. Measurement of the subdiploid DNA-containing erythroblast population in each coculture was then completed by flow cytometry on the relative GpA+ subset.Erythroblast apoptosis was also assessed by the TUNEL assay
(Boehringer-Mannheim, Mannheim, Germany), which measures the extent of nuclear DNA fragmentation by labeling the fluorescent nucleotides at
the 3' ends with terminal deoxynucleotidyl transferase (TdT). We used
this method both in a cytofluorimetric assay and in fresh cells from
bone marrow smears to evaluate the occurrence of erythroblast apoptosis
in vivo. Cells were treated with 4% paraformaldehyde for 20 minutes
prior to their incubation with an anti-GpA MoAb in parallel with
anti- Lastly, an additional attempt was devoted to exploring the expression of a 38-kd mitochondrial antigen recognized by the APO2.7 MoAb (Immunotech) and considered as an early marker of apoptosis.32 Its amplitude was also estimated by flow cytometry in double fluorescence experiments. Measurement of Fas-L secretion Two mouse MoAbs to unrelated epitopes of Fas-L, namely NOK-1 and NOK-2 (Pharmingen), were used in a standardized enzyme-linked immunosorbent assay (ELISA)33,34 to measure relative soluble Fas-L (sFas-L) concentrations in SNs from plasma cell cultures. Briefly, plates were coated with 10 µg/mL NOK-2 in carbonate buffer, pH 9.6, and then supplemented with SNs for 5 hours. After washing, plates were incubated with biotinylated NOK-1, subsequently with avidin-conjugated peroxidase (Sigma), and developed with O-phenylendiamine solution. Each determination was assessed in triplicate and referred to a standard control value obtained with 2 µg/mL recombinant Fas-L (Pharmingen). The function of Fas-L secreted by cultured myeloma cells was also investigated in relation to its cytocidal property in the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. This procedure is a colorimetric method for measuring cell viability and is based on cleavage of the yellow tetrazolium salt by active mitochondria to form a dark-blue formazan product. Thus, it was used as an indirect method to measure the percent of killing of mouse lymphoblasts overexpressing human Fas, namely the WC8 cell line35 along with its Fas parental
cell line WR19L as control (kindly provided by Dr S. Nagata, Osaka,
Japan). The assay included a 40-hour incubation of
1 × 105 target cells with each myeloma-conditioned SN
and, in parallel, in the presence of control unconditioned medium.
Therefore, 100 µL MTT solution (2 mg/mL) was added to each well.
After 4 hours at 37°C, the formazan crystals were dissolved by adding
100 µL dimethyl sulfoxide (Sigma), and the 96-well plates were read
in a spectrophotometer (Bio-Rad Laboratories, Irvine, CA). Dead cells were determined as reported.18
Evaluation of caspase activity Several experiments were performed to measure caspase expression and activation in fresh erythroblasts. We used double fluorescence analysis to measure the constitutive expression of caspase 1 (ICE) and caspase 3 (CPP32) by rabbit polyclonal antisera (Alexis, Vinci, Italy), whereas caspase 8 (FLICE) was assessed by a MoAb (Biosource, Camarillo, CA). Although several of these reagents were optimized by their manufacturers for immunoblotting methods, we demonstrated their reliability in flow cytometry assay. Conversely, cleavage of the CPP32 proenzyme form, namely the classical hallmark of apoptosis activation throughout the Fas pathway, was investigated by detection of both its 12-kd cleaved subunit and the proteolytic cleavage of poly (ADP) ribose polymerase (PARP) by rabbit polyclonal reagents (Pharmingen, and New England Biolabs, Hitchin, UK, respectively). Briefly, the cells were incubated with the anti-GpA reagent and then fixed, permeabilized, and separately treated with specific anticaspase reagents to their intact proenzyme forms, the CPP32 activated subunit or PARP. Lastly, the assay was completed by a secondary FITC-conjugated antibody prior to flow cytometry. These analyses were carried out along with parallel tests using Jurkat cells as a control-established Fas+ cellular model to measure both caspase constitutive expression and cleavage following the Fas pathway activation. Therefore, Jurkat cells were evaluated by flow cytometry before and after Fas stimulation with the myeloma-conditioned SN from patient no. 16, which showed high concentration of sFas-L.Statistical analysis Differences between means of groups of data were calculated using Student t test, whereas the correlation of data was assessed by Spearman's Rho nonparametric method.
Discrepancy in Fas and Fas-L expression in relation to the progression of MM As shown in Table 1, the clinical stages of MM were associated with different expressions of Fas and Fas-L in bone marrow plasma cells and erythroblasts, along with an independent relation to its progression in the case of plasma cells. The mean values (Figure 1) show that stage III MM plasma cells up-regulate their Fas-L expression in the presence of a variable modulation of Fas. This pattern of weak expression of the native Fas on highly malignant plasma cells was in agreement with a previous description of their insensitivity to Fas stimulation.18 Comparison between patients with stage III MM and those with MGUS revealed a significant difference in each receptor expression (P < .02 in both instances) and indicated that advanced MM is associated with abnormalities in myeloma cell apoptosis.
Bone marrow erythroblasts also up-regulated their Fas expression in
advanced MM (P < .01 as compared with the other groups), whereas Fas-L was defectively expressed (P < .02),
pointing to a prevalence of Fas-L-negative or immature erythroblasts.
To validate this interpretation, we measured the percent distribution
of basophilic erythroblasts on bone marrow smears as compared with
other mature erythroid progenitors in each group of patients. The mean
values from this analysis (Table 2) point
to a progressive deficiency of both polychromatophilic and
orthochromatic erythroblasts as the disease progresses. We also
compared both Fas and Fas-L positivities in erythroblast and plasma
cell populations, and Figure 1B plots their values against the
hemoglobin (Hb) levels. Spearman's Rho revealed an inverse
relationship of Hb with the CD38+ bright/Fas-L+
and GpA+/Fas+ subsets
(r =
Plasma cell and erythroblast cultures Culture experiments were carried out to investigate the extent of defective apoptosis with respect to Fas/Fas-L deregulation in expanded plasma cell and erythroid populations and to obtain enriched cell profiles for autologous coculturing tests as an in vitro model of the mixed proliferation of myeloma cells with erythroid progenitors within the bone marrow.We established both types of cultures from 9 patients. Aliquots of
cells were harvested and analyzed by double fluorescence in the FACScan
by gating the cells positive for CD38 or GpA antigens. Figure
2A illustrates the distribution of both
Fas-L and Fas in each culture of plasma cells and erythroblasts,
respectively. A striking difference was observed in the phenotype
expansions, in that the Fas-L+ subset of myeloma cells was
widely expanded in plasma cell cultures from stage III patients and
rose to about 90% in one (patient no. 16). A concurrent, though lower,
pattern of Fas+ erythroid cell expansion was detected in
their parallel erythroblast cultures. Median values of both expansions
were significantly higher (P < .02 in all instances) when
compared with the variable, though weak, phenotype expression in the
other groups. Figure 2B shows representative patterns of this diversity
between an MGUS subject and MM patient no. 16. Both CD38+
bright/Fas-L+ plasma cell and
GpA+/Fas+ erythroblast populations were greatly
expanded in the MM patient, in keeping with the high replication rate
of Fas-L+ myeloma clones observed in stage III
MM.18
Functional sFas-L is variably secreted by myeloma cell cultures Further studies explored the extent of Fas-L solubilization by malignant plasma cells in culture. We first adopted an optimized ELISA to measure its quantitative release in SNs and then tested their cytocidal property by a functional assay. Figure 3 shows these results. The number of myeloma cultures differed because in several instances no established cell lines were obtained. A significant secretion of sFas-L was nonetheless detected in cultures from stage III patients (patients no. 12, 13, and 16-18) as compared with the controls (P < .02). This pattern was confirmed by a substantial major cytotoxic effect on Fas+ target cells in the MTT test, whose positive control included 2 µg/mL recombinant Fas-L. Therefore, at least in SNs from myeloma cultures of patients no. 16 and 17, the amount of secreted Fas-L was higher than that of the reference value. In addition, the remarkable cytocidal effect induced on WC8 cells by most SNs agreed with the ELISA measurement and suggested an apparent concordance between the 2 methods. Further support for the major release of sFas-L by cells from 6 stage III patients (patients no. 12-14 and 16-18) was obtained when the MTT test using the control Fas WR19L cells as target failed to demonstrate the
ability of those SNs to induce an evaluable extent of cell death. These
results provided further evidence that highly malignant myeloma cells are up-regulated in both expression and solubilization of Fas-L in
vitro and that sFas-L is functional.
Myeloma cells prime erythroblast apoptosis in cocultures Coculturing experiments of erythroblasts with autologous myeloma cells were carried out to resemble in vivo conditions within the bone marrow and assess the susceptibility of Fas+ cultured erythroblasts to apoptosis in response to Fas-L presentation either by myeloma cells or in its secreted form in culture medium. Therefore, the analysis investigated erythroblast apoptosis by PI staining, TUNEL, and APO2.7-reactive marker detection in 6 cultures from patients no. 2, 8, 12, and 16-18 and in that from MGUS in patient no. 2. The results for patient no. 16 are presented in Figure 4A. Despite the low levels of spontaneous apoptosis in control unstimulated erythroblast cultures, as also confirmed in cells sorted by magnetic GpA microbeads, a dramatic variation of those parameters was recorded in the erythroblast population incubated with either the autologous myeloma cells or the relative SN. The extent of subdiploid cells (M1) was greatly enhanced up to more than 3 times, and there was a parallel, though lower, increase in the erythroblast suspension treated with the conditioned SN. Similarly, the experiments measuring the extent of erythroblast apoptosis by TUNEL detection (M2) as well as the expression of the mitochondrial marker (M2) showed a variable increment of both parameters in cocultured erythroblasts (Figure 4Aii,iii). This activation pattern of cell death was detected in most experiments, though with slight variations. The highest apoptogen effect (TUNEL M2 > 45%) occurred in erythroblast populations whose autologous myeloma cells were major producers of Fas-L (patients no. 16 and 17). In contrast, cultured erythroblasts from the control MGUS subject no. 2 were not clearly affected by the autologous plasma cell-conditioned SN, because no evident variations in the extent of apoptosis were detected (data not shown). A few inhibitory experiments were also completed on cell preparations from patients no. 18 and 19 and from the MGUS patient no. 3 to prove involvement of the Fas/Fas-L pathway. Both erythroblast and plasma cell populations were separately preincubated with UB2 anti-Fas and Fas-Fc construct, respectively, to prevent their Fas/Fas-L interaction. Figure 4B shows the dose effect of these inhibiting tests. We observed a progressive decline of erythroblast apoptosis by both procedures in relation to their inhibitor accretion. In both MM patients, Fas erythroblast saturation with UB2 MoAb almost completely prevented the apoptogen potential of the plasma cells by more than 1 µg/mL of antibody, whereas Fas-Fc had to be added at a nearly 10-fold higher concentration to obtain a comparable effect. As expected, very little inhibition was observed in the MGUS patient no. 3 by incubating erythroblasts with more than 10 µg/mL of UB2 (data not shown). These inhibition tests provided a clear-cut validation that apoptosis was directly activated in erythroblasts by their immediate interaction with Fas-L of myeloma cells.
Erythroblast apoptosis is activated in vivo in MM patients A number of experiments were devoted to exploring whether erythroblasts undergo apoptosis in vivo following their putative stimulation by Fas-L+ myeloma cells. Two approaches were adopted. In 2 MM patients with severe anemia (patients no. 16 and 17) and 1 with MGUS (patient no. 5), the constitutive caspases on bone marrow fresh erythroblasts were measured by double fluorescence analysis (Figure 5A). There was a remarkable expression of CPP32 and FLICE in both MM patients. Erythroblasts from patient no. 16 showed the most significant values: 96% and 44.2% of positive cells, respectively, versus 29.1% and 20.8% for the MGUS subject (P < .02 in both analyses). However, values related to ICE in fresh erythroblasts were only moderately enhanced (19.8% vs 13.2%) whereas, as expected, the native expression of 3 caspases in control Jurkat cells was also considerably represented (ICE: 30.8%; CPP32: 80.8%; and FLICE: 67.5% of positive cells). This result pointed to a prevalent up-regulation of both constitutive proenzyme forms of CPP32 and FLICE in fresh erythroblasts from MM patients with severe anemia.
To determine the occurrence of activated CPP32 resulting in its cleaved 12-kd subunit and the 89-kd active PARP, we used the relative rabbit reagents in further flow cytometry assays. Figure 5B shows the increment of those caspase subunits (M2) in fresh erythroblasts from patient no. 16: the cleaved form was detectable in about 70% of GpA+ erythroblasts, and more than 50% of these cells also expressed the 89-kd PARP subunit. By contrast, control fresh erythroblasts from the MGUS patient showed a remarkably lower expression of the activated caspases, whereas Jurkat cells underwent prompt cleavage of both CPP32 and PARP following incubation with the Fas-L-enriched SN from plasma cell culture of patient no. 16. Therefore, the high presence of those cleaved subunits in fresh erythroblasts from patients with severe anemia supported the hypothesis that the biochemical transduction of death signals promoted by cleavage of CPP32 was operative in vivo. The second approach included a double fluorescence TUNEL assay on bone
marrow cells to demonstrate the occurrence of apoptotic erythroblasts adjacent to myeloma cells. Figure 5C illustrates the peculiar pattern occasionally detected in smears from patient no.
16. The PE-GpA+ erythroblast (Figure 5C, top) was found to
be in apoptosis by the TUNEL test (Figure 5C, bottom) and adherent to a
FITC-
The present study focused on the overexpression of Fas-L by highly malignant plasma cells as a major pathogenetic mechanism of bone marrow invasion. Because the clinical progression of MM includes both severe anemia and skeleton infiltration by tumor masses, we postulated that Fas-L up-regulation in myeloma clones plays a role in the destruction of the erythroid matrix observed in advanced disease. By exploring Fas/Fas-L expression in MM patients at different clinical stages, we corroborated the indications by others17 and ourselves18 of a peculiar recurrence of Fas-L+ myeloma clones in patients with aggressive disease. This pattern was apparently independent of the concurrent presence of Fas, which was prevalently down-regulated in bone marrow plasma cells. Their defective expression of the membrane-bound isoform of the receptor was associated in previous molecular cloning and complementary DNA sequencing analysis18 with the presence of a splicing variant, namely Fas Exo4,6Del,36 thus explaining the relative resistance of highly malignant myeloma cells to Fas-mediated apoptosis by the prototypic agonist anti-Fas MoAb from clone CH11.18 Erythroblasts from most stage III MM patients underwent prompt apoptosis when cultured in the presence of either autologous Fas-L+ myeloma cells or, to a lesser extent, their culture medium containing the soluble form of the ligand. The small differential activity of the plasma cell membrane-bound Fas-L versus the soluble form was probably associated with the presence of accessory molecules,37 including intercellular adhesion molecule-1, as recently suggested,38 although this aspect was not investigated. In addition, cytofluorimetric analysis of fresh erythroblasts showed a remarkable presence of cleaved subunits of both CPP32 and FLICE, namely the caspases enrolled in the Fas pathway, whereas only moderate activation of ICE, a predominantly TNF receptor (TNF-R)-related caspase, was detected. Further experiments aimed at exploring the occurrence of apoptosis in vivo also demonstrated fluorescent TUNEL+ erythroblasts in close contact or juxtaposed to myeloma cells in bone marrow smears. Taken together, these results emphasize that Fas-L expressed by the malignant plasma cell clones plays a primary cytotoxic role in the chronic deterioration of hematopoiesis, which leads to the severe anemia of MM. Anemia of hematologic malignancies is commonly associated with chronic
derangement of the cytokine network. Circulating levels of TNF- Direct involvement of Fas in the inhibition of human erythroid colonies
has been attributed to IFN- Data from the present study strongly support the contention that, in addition to these pathogenetic mechanisms, a deregulated Fas-L expression by malignant plasma cell clones plays a central role in the disturbance of erythropoiesis. Further confirmation of this pathogenetic mechanism of anemia in MM comes from recent studies describing the Fas/Fas-L-regulated apoptosis of erythroblasts in the control of erythropoiesis.23,28 In normal subjects, immature erythroblasts, particularly at the basophilic stage, lack Fas-L on their cell membrane and are driven to apoptosis by mature Fas-L+ erythroblasts23 owing to their high Fas sensitivity during erythroid differentiation. This negative regulatory feedback of erythropoiesis is physiologically active and paralleled by inadequate levels of EPO, which acts as a major growth factor for immature erythroid progenitors and regularly prevents their apoptosis.52 Therefore, because in MM immature Fas-L However, the susceptibility to apoptosis is apparently up-ruled only in
immature erythroblasts, which exhibit a number of apoptogen receptors
including the TNF-R type 1, both TRAIL receptors 1 and
2,57 and Fas.23 These erythroid progenitors
are Fas-L Another interesting aspect of our study concerns the basic mechanism of myeloma clone expansion within the bone marrow. Progression of MM implies an increasing substitution of bone marrow by nests of malignant plasma cells, which contribute to both destruction of the erythroid matrix and the appearance or extension of osteolytic lesions. Interaction of myeloma clones with marrow stromal cells by a number of adhesion molecules boosts the replication of malignant plasma cells,58 though the extensive replacement of bone marrow suggests concurrent elimination of other cells. We believe that a major cytotoxic mechanism promoted by the Fas-L of myeloma clones drives such a devastating expansion to the detriment of the erythroid progenitors. Both colonization and progression of bone lesions could also account for a similar pathogenic mechanism of induction of apoptosis in osteoblasts, because these cells are sensitive to Fas stimulation.59 In preliminary experiments we found that primary normal osteoblast cultures underwent prompt apoptosis following their incubation with the myeloma culture SN from patient no.16. Although these data need to be expanded in additional research, they suggest that a similar mechanism could be involved in osteolytic bone resorption. Insufficient erythropoiesis in MM patients has also been ascribed to defective EPO production, particularly in those with myeloma renal failure.60 A number of clinical trials have been carried out on the use of rHu-EPO to treat anemia of MM.19,61,62 However, these studies and our own experience63,64 show that only a few stage III patients respond and that this treatment seems more effective in less advanced stages. This unresponsiveness has been attributed inter alia to the effect of neutralizing autoantibodies to the recombinant hormone.65-67 Our present results indicate that Fas-L cytotoxicity on erythroid progenitors is an additional, and possibly primary, pathogenetic mechanism of MM-associated anemia.
The authors are indebted to Dr S. Nagata for providing the WR19L cell line and the transfected control.
Submitted January 27, 2000; accepted October 8, 2000.
Supported by a grant from the Ministry for the Universities and Scientific and Technological Research and by the National AIDS Research Project of the Italian Ministry of Health, Istituto Superiore di Sanità, Rome, Italy.
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: Franco Silvestris, DIMO, Department of Biomedical
Sciences and Human Oncology, Section of Internal Medicine and
Clinical Oncology, University of Bari, Piazza G. Cesare, 11
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© 2001 by The American Society of Hematology.
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  R. Greil, G. Anether, K. Johrer, and I. Tinhofer Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools J. Leukoc. Biol., September 1, 2003; 74(3): 311 - 330. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, which also
inhibit the erythroid colony-forming units (CFU-E) from bone marrow
CD34+ cells.
(IgG1
light chain antisera. The test was completed with
the fluorescent nucleotide mixture in the presence of TdT, and the
slides were examined under a fluorescence microscope.
0.7848 and 
) and TUNEL
(
) measurements of the apoptogen potential of myeloma cells by
preincubation of either erythroblasts or plasma cells with a nonagonist
MoAb to Fas (UB2) or with the Fas-Fc construct, respectively. These
inhibiting tests of erythroblast apoptosis were completed in 2 patients
with advanced MM. Pretreatment with both reagents induced a progressive
saturation of the binding sites of Fas on erythroblasts and,
alternatively, of Fas-L on plasma cells, leading to exhaustion of the
apoptosis induction through Fas/Fas-L pathway. This effect was
dose-dependent and promptly induced by blocking Fas on erythroblasts,
with amounts of UB2 higher than 1 µg/mL in culture medium. By
contrast, saturation of Fas-L on plasma cells required a nearly 10-fold
higher concentration of Fas-Fc to produce a similar effect in cultured
erythroblasts from both patients.
are
increased in MM
70124
Bari, Italy; e-mail: