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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 1031-1043
By
From INSERM U365 and Service d'Hématologie, Institut Curie,
Paris; Laboratoire virus, neurone et immunité, UFR Kremlin
Bicêtre, Kremlin Bicêtre, Service d'Hématologie,
Hôpital de l'Hôtel Dieu, Paris; Fractales Biotech,
Hôpital Saint Joseph, Paris; and INSERM U462, Hôpital Saint
Louis, Paris.
The expression of different isoforms of nitric oxide synthase (NOS)
was investigated in B-cell chronic lymphocytic leukemia (B-CLL) to
delineate a possible role for nitric oxide (NO) in the control of
apoptosis of the tumoral cells. By reverse transcription-polymerase chain reaction (RT-PCR), all B-CLL cells were found to express spontaneously inducible NOS (iNOS) mRNA, whereas endothelial
constitutive NOS (ecNOS) mRNA was undetectable. The iNOS protein was
detected by immunofluorescence in the cytoplasm of permeabilized
leukemic cells and identified by Western blotting, using different
anti-iNOS antibodies, as a protein of 135 kD in B-CLL
cytoplasmic extracts. B-CLL cell lysates also displayed basal NOS
enzymatic activity, as measured by the conversion of
14C-labeled L-arginine into 14C-L-citrulline.
Ligation of CD23, expressed on the vast majority of B-CLL cells,
resulted in increased iNOS expression and activity. The NO released
exerted an anti-apoptotic effect on B-CLL cells that was counteracted
by NOS inhibitors and engagement of the APO-1/Fas pathway. Therefore,
the existence of a functional iNOS in B-CLL cells will provide further
insights into the mechanisms that control proliferation and apoptosis
in these tumor cells.
© 1998 by The American Society of Hematology.
B-CELL CHRONIC lymphocytic leukemia
(B-CLL) is characterized by a progressive accumulation of monoclonal
CD5+ B cells arrested in the G0/G1
phase of the cell cycle1,2 and which overexpress the CD23
(Fc The capacity to stimulate the expression of Bcl-2 or to
slow its decrease in B-CLL cells is correlated with the anti-apoptotic effect of IL-4,17-20 IFN- Therefore, it has been proposed that a defective apoptosis, caused by
Bcl-2 overexpression, may explain why B-CLL cells accumulate in
G0.25-27 Although the Bcl-2 gene was first
shown to be dysregulated in lymphomas associated with a t(14;18)
chromosomal translocation, its overexpression in most B-CLL cells is
not associated with a gene rearrangement. The anti-apoptotic activity
of Bcl-2 is antagonized by the homologous protein Bax, which
accelerates the rate of cell death, and an increased Bcl-2/Bax ratio
was observed in B-CLL, particularly in patients unresponsive to
chemotherapy.28 The enhanced apoptosis triggered by
anti-IgM in B-CLL and its reversal by CD6 ligation are also correlated
with modulations in the Bcl-2/Bax ratio.29 Gottardi et
al30 have studied the expression of the various members of
the Bcl-2 family (Bcl-2, Bcl-xL, Bcl-xS, and Bax) in leukemic
CD5+ B cells and found that the pattern of expression of
these genes was skewed toward prevention of apoptosis, favoring the
accumulation of the leukemic cells.
Nitric oxide (NO) plays a role in the control of apoptosis in various
cell types, including tumoral cells. The effect of NO on the apoptotic
pathway is complex, probably due to the "double-edged sword"
aspect of its action, depending on its concentration and on the redox
potential of the neighboring cells where it is released.31 NO, described as a potent immunoregulator,32,33 is
synthesized from L-arginine by NO synthases (NOS).34
Recently, Epstein-Barr virus (EBV)-transformed human B lymphocytes and
Burkitt's lymphoma B cells have been reported to express low levels of
inducible nitric oxide synthase (iNOS) and the NO produced appeared to
inhibit apoptosis and EBV virus reactivation.35 In
addition, endothelial constitutive NOS (ecNOS) mRNA and NOS protein
have been reported in subpopulations of human normal B
lymphocytes.36
These data prompted us to study whether these NOS isoforms were present
and functional in B-CLL cells. Inasmuch as most B-CLL cells display
membrane CD23,37 and as we have shown previously that CD23
ligation results in iNOS activation in human monocytes,38 we decided also to investigate the effects of CD23 engagement on NOS
expression by B-CLL cells.
Our present work indicates that whereas ecNOS is undetectable, B-CLL
cells spontaneously express a functional iNOS that is upregulated after
ligation of membrane CD23 and which has an anti-apoptotic role.
Patients
Cell Isolation
Reagents The two NOS inhibitors NG-monomethyl-L-arginine (L-NMMA) and nitro L-arginine methyl ester (L-NAME) were purchased from Sigma (St Louis, MO). The characterization of the different MoAbs directed against the CD23 antigen (135 and MHM-6, both of IgG1 isotype) has been reported previously39; the MOPC-21 MoAb (Sigma) is an isotype-matched (IgG1) control antibody. The phycoerythrin-conjugated anti-CD23 MoAb was from Becton Dickinson (Grenoble, France) and the anti-APO-1/Fas MoAb from Immunotech for the clone CH-11 (IgM) and from Biomol (Plymouth Meeting, PA) for the clone BG27 (IgG2a). 14C-labeled L-arginine was from Amersham (Amersham, UK). Isotype-matched control or fluorescent-labeled MoAbs were from Becton Dickinson. The strong acid cation exchange resin, binding to L-arginine but not to L-citrulline (AG 50W-X8, 100-200 mesh, H form) was from Biorad Laboratories (Richmond, CA). All other reagents and chemicals were from Sigma.Determination of Nitrite Concentrations To assess the amount of NO produced, the culture supernatants were assayed for the stable end product of NO oxidation, nitrite, using the Griess reagent as previously described.40 Supernatants were tested as such or after treatment with nitrate reductase to reduce nitrate to nitrite. Briefly, 100 µL of the various supernatants was dispensed in 96-well microplates, followed by the addition of 100 µL of a reactive solution containing 1% sulfanilamide in 30% CH3COOH and with 0.1% N-1-naphthyl ethylenediamine dihydrochloride in 60% CH3COOH (1 vol/1 vol). The standard calibration curve was constructed using sodium nitrite diluted in complete medium. Optical densities (OD) were measured at 540 nm using an ELISA plate reader (Titertek Multiskan; Flow Laboratories, Eflab Oy, Helsinki, Finland).Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for the Detection of iNOS and ecNOS mRNA in B-CLL Cells RNA preparation. The expression of iNOS and ecNOS mRNA was investigated by RT-PCR, according to previously described techniques.41 Briefly, total RNAs were prepared from 1 to 10 × 106 cells by a modified single-step guanidinium isothiocyanate and phenol/chloroform extraction method,42 using the Trizol reagent (GIBCO) according to the specifications of the manufacturer. The RNA pellets were then washed with 75% ethanol, vacuum-dried, and resuspended in diethylpyrocarbonate-treated water. cDNA synthesis. Aliquots of 1 to 2 µg from each RNA sample were mixed with 250 µmol/L of each deoxynucleotide triphosphate (dNTP; Pharmacia), 1 µg random hexamer (PdN6; Boehringer Mannheim, Meylan, France) in a 67-mmol/L Tris-HCl pH 8.8 buffer containing 6.7 mmol/L MgCl2, 16.6 mmol/L (NH4)2SO4, in a final volume of 20 µL. The mixture was incubated at 65°C for 5 minutes. After cooling, 200 U of Moloney murine leukemia virus reverse transcriptase (MMLV-RT: GIBCO-BRL, Cergy-Pontoise, France) and 125 U of human placenta ribonuclease inhibitor (HPRI; Amersham France, Les Ulis) were added for a 30-minute incubation at 42°C. The RT was then heat-inactivated at 65°C for 5 minutes, and the 20-µL cDNA synthesis reaction mixtures were diluted to 500 µL in RNAse-free water for use in PCR amplification. PCR.
Ten microliters of diluted RT product (cDNA) were dispensed in PCR
tubes, then 90 µL of a mixture containing 250 µM of each deoxynucleotide triphosphate, dNTP (Pharmacia), 50 µmol/L of each specific upstream and downstream oligonucleotide primer, reaction buffer consisting of 67 mmol/L Tris-HCl pH 8.8 containing varying concentrations of MgCl2 (1.7 or 2.7 mmol/L), 16.6 mmol/L
(NH4)2SO4, and finally 1.25 U of
Taq polymerase (Boehringer). Tubes were overloaded with 50 µL of
mineral oil (Sigma) and PCR was performed in a thermal cycler (GenAmp
9600; Perkin Elmer, Norwalk, CT). After an initial
denaturation step at 94°C for 30 seconds, a step cycle program was
set as follows for iNOS and
Detection of iNOS and ecNOS protein by fluorescence-activated cell
sorting (FACS) analysis.
The presence of iNOS and ecNOS was analyzed by flow cytometry of
permeabilized cells, using the Cytoperm permeabilization and fixation
kit (Serotec, Oxford, UK). Briefly, 106 cells in 50 µL of
phosphate-buffered saline (PBS) were treated at room temperature for 15 minutes with 100 µL of reagent A. After washing, the cell pellet was
resuspended in 100 µL of reagent B and 10 µL of either monoclonal
anti-iNOS (macNOS, clone 54; Transduction Laboratories, Lexington, KY),
or anti-human endothelial constitutive NOS (anti-ECNOS, clone 3;
Transduction Laboratories), or their control isotype (IgG1)
from Coulter France (Margency) were added for a 30-minute incubation at
room temperature. After new washing, the pellet was resuspended in 50 µL of reagent B and 10 µL of an F(ab Immunocytochemistry. Freshly isolated human B-CLL cells were cytocentrifuged on glass slides and fixed with acetone. The slides were then incubated in a humidified chamber, either with a 1/100 dilution of the mouse anti-macNOS (clone 54) MoAb, or with isotype-matched IgG1 MoAb (Becton Dickinson) as a control. Cells were then incubated with a biotinylated anti-mouse IgG antibody and the labeling was revealed using the Vectastain ABC procedure with avidin coupled to horseradish peroxidase (Vector Laboratories). Quantification of apoptosis.
Apoptosis experiments were performed selectively with B-CLL cells
purified from freshly collected blood samples and only in a few
instances with B-CLL cells isolated from frozen PBMC samples but, in
the latter cases, dead cells and debris were removed by an additional
centrifugation on Ficoll after the separation steps and only
populations with a viability Determination of NOS enzymatic activity.
[14C] L-arginine conversion into [14C]
L-citrulline was evaluated according to a modification of a technique
described elsewhere.43 Briefly, 107 cells per
experimental point were lysed by 4 cycles of freezing on liquid
nitrogen and thawing in a 50 mmol/L Tris-HCl buffer pH 7.5, containing
the following protease inhibitors: 1 µg/mL leupeptin, 1 µg/mL
pepstatin, 40 µg/mL bestatin, 10 µg/mL chymostatin, 50 µg/mL TLCK
(all from Sigma), 1 mmol/L AEBSF (Calbiochem), and 5 mmol/L
dithiothreitol (DTT) (Sigma). Cell extracts were then centrifuged at
14,000g for 15 minutes at 4°C to remove debris. Supernatants (crude homogenates) were collected and their protein concentration was determined by the Bradford technique.48
Twenty microliters of these samples (in triplicate) were then incubated for 15 minutes at 37°C with 100 µL of a 100 mmol/L HEPES, 1 mmol/L MgCl2, pH 7.5 buffer containing 1 mmol/L
CaCl2, 1 µmol/L calmodulin, 300 µmol/L
Detection of iNOS protein in B-CLL extracts by Western blotting.
Cytosolic extracts from unstimulated or stimulated B-CLL cells were
prepared by submitting the leukemic cells to four cycles of
freeze-thawing in a homogenization buffer consisting of 50 mmol/L Tris,
1 mmol/L EDTA, pH 7.4, containing a mixture of protease inhibitors (10 µg/mL leupeptin, 100 µg/mL phenylmethyl sulfonyl fluoride (PMSF), 10 µg/mL antipain, 10 µg/mL pepstatin, 100 µg/mL chymotrypsin/trypsin inhibitor, and 10 µg/mL chymostatin), and 5 mmol/L DTT. After adjustment of the concentration, the different samples were submitted to electrophoresis on an 8% acrylamide gel
containing 0.1 % sodium dodecyl sulfate (SDS). Rainbow markers (Amersham, Les Ulis, France) were run in parallel to estimate the
molecular weights. After electrophoresis, the gel was blotted onto a
membrane of nitrocellulose (0.45 µmol/L; Schleicher and Schuel,
Dassel, Germany) using a wet transfer technique. The
efficiency of the transfer was checked by staining the membrane with
Ponceau red. After destaining, the membrane was incubated overnight at 4°C in a blocking buffer of 10 mmol/L Tris, 50 mmol/L NaCl, 2.5 mmol/L EDTA pH 7.5, containing 5% nonfat milk. The membrane was rinsed
with a 15 mmol/L Tris, 150 mmol/L NaCl, 0.2% Tween-20 buffer containing 1% BSA, then incubated for 2 hours at room temperature with
agitation with a 1/500 dilution of a specific anti-iNOS MoAb (macNOS,
IgG1, clone 54; Transduction Laboratories), directed against the
carboxy-terminal portion of the murine macrophage iNOS
and cross-reacting with human iNOS. After washing, the membrane was
incubated for 90 minutes at room temperature with a 1/5,000 dilution of
a peroxidase-conjugated rabbit anti-mouse IgG (ECL kit; Amersham).
Development was performed by incubating the membrane, after washing,
with the ECL reagent substrate. Detection was visualized by
autoradiography after 1 minute of contact of the membrane with a
Biomax-MR1 film (Eastman Kodak, Rochester, NY). As a positive control
for iNOS expression, we used either a lysate of IFN-
Detection of iNOS mRNA Expression in Human B-CLL Cells by RT-PCR Total RNA was extracted from purified B-CLL leukemic B cells obtained from frozen or freshly collected blood samples. Inasmuch as NOS mRNA in human hematopoietic cells is generally difficult to detect by Northern blot, RT-PCR analysis was performed. When total RNAs were reverse transcribed and amplified in the presence of a couple of amplimers specific for the human iNOS gene, in comparison with the positive A549 control cell line, a 259-bp fragment was regularly observed for all 22 B-CLL cell samples tested by this technique (no differences were observed between fresh and frozen samples), as exemplified in Fig 1. The size of the amplified fragment is in agreement with the expected size deduced from the sequence of the human iNOS gene. Similar results were obtained with the second couple of primers (not shown), which was used previously to show the induction of iNOS in human monocytes following the engagement of CD2343 and whose sequence of the amplified fragment was identical to that of human hepatocyte iNOS.44 No signal was detected in the absence of RNA or of Taq polymerase and, when tested by RT-PCR, the U937 monocytic and RPMI-8226 myeloma cell lines were negative for the presence of iNOS mRNA (not shown). In contrast, endothelial constitutive NOS (ecNOS) mRNA was not detectable in the different B-CLL cells tested, whereas in U937 cells used as a positive control of ecNOS expression a 277-bp fragment was visualized, as previously reported46 (not shown).
Detection of iNOS Protein in B-CLL Cells B-CLL cells were analyzed for the intracytoplasmic expression of ecNOS and iNOS proteins by indirect immunofluorescence FACS analysis on permeabilized cells. All B-CLL cells tested were significantly labeled with the anti-mac NOS (clone 54), as shown in Fig 2. The percentage of positive cells was in the range of 13% to 82% (mean, 54%; n = 13). This result was further confirmed by direct immunofluorescence on permeabilized cells using another anti-iNOS MoAb (clone 6) directly conjugated to FITC, and also by immunocytochemistry (not shown). In contrast, B-CLL cells were essentially negative for the expression of ecNOS (range of positive cells: 0% to 12%; mean: 4.3%; n = 13), as depicted in Fig 3.
Detection by Western Blotting of the iNOS Protein in B-CLL Cell Lysates As seen in Fig 4, iNOS was detected by Western blot in the lysates of two different B-CLL cell samples, using the anti-macNOS MoAb (clone 54) for the visualization. The protein detected in the B-CLL lysates had a slightly lower molecular weight than that detected in the lysate of the murine RAW 264.7 macrophage cell line, used as a positive control, but was found to be identical to that detected in lysates of human A549 cells (not shown). Western blotting was repeated with two other MoAbs against the human iNOS (21C10-D10 and 1E8 D8), which were kindly provided by Dr R. Webber (R & D, Minneapolis, MN). A protein of 135 kD was again detected by Western blot in the B-CLL lysates (not shown).
Ligation of CD23 on B-CLL Cells Stimulates iNOS Expression The vast majority of B-CLL cells, unlike their normal counterparts, the mature resting B lymphocytes, spontaneously displayed a high level of membrane CD23 expression. We and others have reported previously that the engagement of CD23, induced in human monocytes by IL-4 pretreatment, resulted in the activation of iNOS in these cells.38,40,43 This prompted us to examine the effect of CD23 ligation on iNOS mRNA expression and protein level in B-CLL cells. Treatment of B-CLL cells with an anti-CD23 MoAb (MoAb 135) stimulated the expression of iNOS mRNA, as detected by RT-PCR (Fig 5). In five different experiments, the level of iNOS mRNA, estimated in comparison with that of 2-microglobulin, was significantly increased following
ligation of CD23 (range, 179% to 543%). The expression of the iNOS
protein was also increased in B-CLL cells after the engagement of CD23,
as seen in Fig 6. In contrast, ligation of
CD23 did not induce ecNOS expression in these cells, as shown in
Fig 7.
NOS Activity in B-CLL Cell Extracts and Intact Cells NOS activity was determined in cytosolic extracts of different B-CLL cells, unstimulated or incubated with anti-CD23 MoAb or IgG1 as a control, by measuring the conversion of labeled 14C-L-arginine into 14C-L-citrulline. These homogenates were found to display a catalytic activity of about 20 pmol/min/mg, which was increased about three times after stimulation with anti-CD23 (Table 1A). NOS activity was also quantified in intact 14C-L-arginine-loaded B-CLL cells by measuring the release of radiolabeled 14C-L-citrulline in the culture supernatants. A significant basal NOS activity was detected, as estimated by the L-NMMA-inhibitable release of labeled citrulline in supernatants from control B-CLL cultures. Addition of anti-CD23 MoAb resulted in increased activity, whereas isotype-matched IgG1 had little effect (Table 1B). Attempts to detect the release of nitrite/nitrate in the supernatants of B-CLL cells stimulated or not with anti-CD23 MoAb were hampered by large interindividual variations. Nonetheless, when paired samples were compared, L-NAME was found to reduce the basal nitrite release, whereas treatment with anti-CD23 resulted in a significant increase in nitrite production over basal release. The latter increase was largely reduced by L-NAME (Table 2).
Anti-Apoptotic Role of NO in B-CLL Cells Cells undergoing apoptosis display an early modification in the distribution of their membrane phospholipids, characterized by an exposure of PS at the outer leaflet. The latter can be quantified by the use of FITC-labeled annexin, which has a high affinity for PS. Therefore, freshly collected B-CLL cells were stained with fluorescent annexin, either at day 0 or after 2 days of culture with medium alone or in the presence of anti-CD23 MoAb. When the dot plots (side scatter/forward scatter) of these cells were compared, untreated cells at day 0 displayed a single distribution (region R1: 79% of total cells), as shown in Fig 8, and were negative (<5%) for the labeling with FITC-annexin V. After 2 days of culture in medium alone, a marked change in the dot-plot distribution was apparent. Although some cells retained the same pattern as day 0 cells (region R1: 27% of total cells), a new population was detected, characterized by heterogeneous granulometry (region R2: 41% of total cells). Most of these cells (about 85%) were positively labeled with annexin V, confirming that they were entering apoptosis, whereas the population in the region R1 consisted of "intact" cells, mostly negative for this marker (0% to 11%), as shown in Fig 9. B-CLL cells incubated in the presence of anti-CD23 MoAb also displayed a bimodal dot-plot distribution, but the percentage of the R1 population (48%) versus the R2 population (22%) was much higher than for incubated control cells. Interestingly, the inhibitory effect of CD23 ligation was partially reversed in the presence of L-NMMA, an inhibitor of the NOS pathway. When global B-CLL cell populations were examined for the expression of the annexin V marker, a significant reduction in the positive cells was observed after incubation of these cells with anti-CD23, as shown in the examples presented in Fig 10. The CD23-driven reduction in the percentage of annexin V+ cells was largely reversed by addition of L-NMMA, as shown in Table 3.
Our data indicate that iNOS-specific mRNA can be detected by RT-PCR in
purified leukemic B lymphocytes from B-CLL patients. The presence of
this mRNA is unlikely to be due to a contamination by other cell types,
inasmuch as blood samples were collected from hyperleukocytic patients
and similar results were observed when rigorous depletion of residual
contaminating cells was performed. In addition, the presence of iNOS in
unstimulated human T lymphocytes and monocytes has not been reported.
In some instances, an amplification fragment corresponding to the iNOS
was observed with RNA extracted from blood-derived B cells, but the
intensity of the bands was weak when compared with those of B-CLL
cells. Therefore, the presence of iNOS mRNA in resting mature small B
lymphocytes cannot be totally excluded, but our previous attempts to
detect an NO-dependent cGMP production in normal B lymphocytes or in B
lymphocytes triggered by CD23 ligation were unsuccessful.40
Submitted December 3, 1997;
accepted March 31, 1998.
The help of Nicole Romquin for the RT-PCR techniques, Danny Rouillard
for the flow cytometry analysis, and David Marsh for the English style
was greatly appreciated.
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Abstract
Introduction
Abstract
R2) antigen.
(IFN-
,
95% for both cell types.
CGG TGC TGT ATT TCC
TTA CGA GGC GAA GAA GG 3
dpm = dpm (test sample ± EGTA) 
dpm (test sample + EGTA+L-NMMA), Total dpm = total radioactivity in dpm,
Q = total amount of arginine (unlabeled + 14C-labeled) in nanomoles, Vr = volume of the
reaction in mL, Vc = counted volume in mL, T = time of
reaction in min, Vs = volume of sample in mL, and C = concentration of protein in the cell lysate in milligrams per
milliliter.
