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PHAGOCYTES
From the Hematology Section, Department of Medicine,
Institute of Medical Microbiology and Department of Virology,
Sahlgren's University Hospital, Göteborg, Sweden.
Natural killer (NK) cells are deficient in patients with chronic
myelogenous leukemia (CML), but the mechanisms responsible for the
dysfunction are not completely understood. This study reports that CML
cells effectively inhibit the baseline and interleukin-2 (IL-2)-induced
NK cell cytotoxicity against a CML cell-derived line (K562). A sizable
fraction of NK cells subsequently acquired features characteristic of
programmed cell death/apoptosis. The CML cell-mediated inhibition of NK
cells required triggering of reduced nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase-mediated formation of reactive oxygen species
(ROS) and was prevented by catalase, a scavenger of ROS, and by
histamine, acting via H2-receptor-mediated inhibition of
ROS production in CML cells. In contrast, nonmalignant neutrophilic
granulocytes inhibited NK cells via ROS production without the
requirement of exogenous NADPH oxidase-triggering stimuli. We propose
that paracrine production of ROS may contribute to the dysfunction of
NK cells in CML and that histamine may serve as an autocrine inhibitor
of ROS formation in leukemic granulocytes.
(Blood. 2000;96:1961-1968) Chronic myelogenous leukemia (CML) is a fatal
myeloproliferative disease characterized by massive expansion of
hematopoietic progenitor cells with the appearance of cells of the
granulocyte (GR) lineage in the peripheral blood. Natural killer (NK)
cells, a subset of lymphocytes endowed with spontaneous cytotoxicity against tumor cells,1,2 have been proposed to participate in surveillance of the malignant clone in CML.3 However,
NK cells decrease in number and function during the course of
CML; typically, the absolute number of NK cells is profoundly
reduced in patients with late-stage disease as compared with
healthy control subjects, and a reduction of NK cell inducibility and
proliferation accompanies disease progression.3 The
NK cell dysfunction is apparently unrelated to prior cytotoxic therapy
and triggered by an as yet undefined inhibitory signal in the malignant
microenvironment.4
Understanding the mechanisms of the NK cell dysfunction in CML
could be useful in elucidating how this disease develops and in
identifying therapeutic strategies. Earlier studies have revealed that
cells of the monocyte/macrophage lineage effectively inhibit NK cell
function; thus, monocytes suppress NK cell cytotoxicity, proliferation,
and cytokine gene transcription5,6 and render NK cells
resistant to activating cytokines such as interleukin-2 (IL-2) or
interferon- Malignant CML cells carry a functional membrane NADPH oxidase and
are thus endowed with the capacity to produce ROS.8 We therefore explored whether ROS generated by CML cells alter functions of NK cells in vitro, and compared these properties of CML cells with
those of nonmalignant neutrophilic granulocytes (GR). We report that
CML cells profoundly inhibit the baseline and cytokine-induced cytotoxicity of NK cells and trigger significant apoptosis in NK cells.
Histamine, a constituent of CML cells, and catalase almost completely
rescue NK cell cytolytic activity and protect NK cells from CML
cell-induced apoptosis. In contrast to nonmalignant GR, CML cells
require an external stimulus to inhibit NK cells. It is hypothesized
that paracrine production of ROS may contribute to the NK cell
dysfunction in CML and that histamine may serve as an autocrine
regulator of ROS production in leukemic granulocytes.
Separation of lymphocytes, GR, and CML cells
The MNC fraction was enriched for NK cells using counter-current
centrifugal elutriation, as described in detail
elsewhere.6 Briefly, the MNC were resuspended in
elutriation buffer containing 0.5% bovine serum albumin (BSA) and
0.1% EDTA in buffered NaCl and fed into a Beckman J2-21
ultracentrifuge with a JE-6B rotor at 2100 rpm. A lymphocyte fraction
enriched for NK cells (with CD3 To enrich GR, the red blood cell (RBC)/neutrophil pellet recovered
after Ficoll-Hypaque separation was immediately mixed with 100 mL
elutriation medium (ie, Krebs-Ringer phosphate buffer; KRG, pH 7.3; 120 nmol/L NaCl, 5 mmol/L KCl, 1.7 mmol/L KH2PO4, 8.3 mmol/L NaHPO4, and glucose 10 mmol/L, not supplemented
with Ca++ to minimize spontaneous activation and adhesion
of neutrophils to tubing). At a flow rate of 20 mL/min, the cells were
fed into a Beckman ultracentrifuge with a JE-6B rotor spinning at 2150 rpm at 4°C. Under these conditions, GR remain in the elutriation chamber. After 25 minutes the flow rate was increased to 40 mL/min and
neutrophilic GR were consistently recovered at a purity of more than
98%. Freshly isolated GR were thereafter resuspended in KRG buffer
supplemented with Ca++ (1 mmol/L) and Mg++ (1.5 mmol/L), and kept on ice until used either in the cytotoxic or
apoptosis assays or for measurement of ROS production.
The same elutriation procedure was used to enrich CML cells from a
RBC/neutrophil pellet. The elutriated cell fraction contained cells
with the morphology of mature neutrophilic granulocytes (95%),
monocytes (1%), bands (1%), and eosinophils (3%). The CML cells were
recovered from patients (n = 9; age 18-63) with newly diagnosed,
untreated CML in chronic phase. Chromosome analysis of cells revealed
100% translocation 9:22 (Ph+) in all patients. Manual
differential counts at the time of cell sampling showed a median of 0%
(range, 0-0.5) blasts, 0.5% (0-1.5) promyelocytes, 4% (0-7.5)
myelocytes, 1.5% (0-12) metamyelocytes, 4% (3-9) bands, and 65%
(58-77) mature neutrophilic granulocytes.
Target cells and microcytotoxicity assay
The NK cell-enriched lymphocytes and target cells were incubated in
sextuplicate in microplates in a total volume of 200 µL in the
presence or absence of GR or CML cells. The compounds used were added
at the onset of incubation with the exception of N-formyl-Met-Len-Phe (fMLF), which was added at 15 minutes. After incubation at
37°C for 16 hours, supernatant fluids were collected by a tissue
collecting system (Amersham) and assayed for radioactivity in a
In accordance with earlier studies,5,6 more than 90% of the lymphocyte cytotoxicity against K562 cells was depleted by the removal of CD56+ NK cells (by use of anti-CD56-coated Dynabeads) from the effector lymphocyte preparation; in contrast, removal of CD3+ T cells (by use of anti-CD3-coated Dynabeads) did not significantly reduce cytotoxicity. Detection of surface antigens One million cells were incubated with appropriate fluorescein isothiocyanate (FITC)-and phycoerythrin (PE)-conjugated monoclonal antibodies (Becton & Dickinson, Stockholm, Sweden; 10 µL/106 cells) on ice for 30 minutes. The cells were washed twice in PBS and resuspended in 500 µL sterile filtrated PBS and analyzed by use of flow cytometry on a FACSort with a Lysys II software program (Becton & Dickinson). Lymphocytes were gated on the basis of forward and right angle scatter. The flow rate was adjusted to less than 200 cells/s and at the least 5 × 103 cells were analyzed for each sample.Apoptosis in NK cells Three methods were used to analyze the frequency of lymphocytes with apoptotic features after contact with GR or CML cells. Apoptotic morphology was detected by use of flow cytometry. A gate was set to comprise lymphocytes with reduced forward scatter and increased right angle scatter characteristic of apoptosis.7 Two FACS-based methods were used to confirm apoptosis in these cells, detection of extracellular annexin V (Annexin V-FITC Apoptosis Detection Kit I; PharMingen, San Diego, CA),10 and analysis of intracellular content of reduced intracellular glutathione (Cell Tracker, Eugene, OR).11,12Chemiluminescence measurement Chemiluminescence measurement was performed at 37° in a 6-channel Biolumat LB 9505 (Berthold Co, Wildbad, Germany) by using disposable 4-mL polypropylene tubes, as described.13 The reaction mixture contained 0.8 mL elutriated CML cells (5 × 106/mL) in a balanced salt solution (Krebs Ringer glucose) also containing horseradish peroxidase (HRP; 4 U) and luminol (20 µmol/L). The tubes were allowed to equilibrate for 5 minutes at 37° before fMLF (0.1 µmolL, final concentration) was added, and light emission was recorded (results given as cpm, and 7.2 × 107 cpm equals 1 nmole of superoxide anion, as measured with a cytochrome C-reduction technique).13Compounds Histamine dihydrochloride (Sigma Chemicals, St Louis, MO), the H2R antagonist ranitidine hydrochloride (Glaxo, Mölndal, Sweden), AH202399AA (a chemical control to ranitidine, kindly provided by Glaxo), catalase (Boehringer Mannheim, Mannheim, Germany), human recombinant IL-2 (EuroCetus, Amsterdam, The Netherlands), and fMLF (Sigma Chemical) were used. MNC treated with culture medium served as controls.
Nonmalignant GR inhibit NK cell function The effects of benign GR on NK cell function were determined by adding GR to lymphocytes enriched for NK cells in microplates for assay of cytotoxicity against CML cell-derived target cells (K562). The GR/effector cell ratios used were 1:1 to 1:10 (100 000 NK cell-enriched lymphocytes admixed with 100 000-10 000 GR).Granulocytes effectively depressed NK cell-mediated cytotoxicity
against K562 cells without the apparent requirement of additional exogenous stimuli. Figure 1 shows a
representative experiment in which GR inhibited the spontaneous
cytotoxicity of heterologous NK cells in a 16-hour 51Cr
assay, and similar results were obtained using GR and NK cells recovered from 9 blood donors. In most experiments, GR/lymphocyte ratios of 1:2 or 1:4 were sufficient to significantly reduce
cytotoxicity (Figure 1), and significant inhibition of NK cell
cytotoxicity was observed also in 4-hour 51Cr assays (data
not shown). Autologous and heterologous GR were equally effective in
reducing NK cell cytotoxicity, and the degree of inhibition was similar
after the removal of CD3+ T cells (by use of
anti-CD3-coated Dynabeads) from the effector cell preparations (data
not shown). We also explored whether GR affected the inducibility of NK
cells by concomitantly treating mixtures of enriched NK cells and GR
with IL-2, a prototypical NK cell activator.1 The presence
of GR effectively reduced the NK cell activation induced by IL-2 (Table
1).
Role of ROS To investigate the putative contribution by ROS for the observed GR-induced NK cell inhibition, we first added fMLF, a chemotactic tripeptide known to rapidly trigger NADPH-dependent ROS formation in GR.14 fMLF did not alter NK cell-mediated cytotoxicity in the absence of GR, but effectively reinforced the GR-induced inhibition of NK cells. A total of 7 experiments were performed in which fMLF was found to boost the GR-induced inhibition at GR/effector cell ratios of 1:8, 1:4, 1:2, and 1:1 (P < .01 for all ratios examined, Mann-Whitney U test). Figure 1 (inset) shows NK cell inhibition by a GR/effector cell ratio of 1:8. At this low density, unstimulated GR did not significantly affect NK cell cytotoxicity, whereas fMLF-treated GR reduced NK cell cytotoxicity by more than 50%.Secondly, we introduced scavengers of ROS to corroborate the role of
NADPH oxidase products for the GR-induced inhibition and to determine
which species of ROS mediated the inhibitory signal. Superoxide
dismutase, a scavenger of superoxide anion,5,14 did not
reverse the spontaneous or fMLF-induced inhibition of NK cells, when
used at concentrations sufficient to scavenge more than 99% of
superoxide anion (200 U/mL; data not shown). Catalase, a scavenger of
hydrogen peroxide,5,6,14 was found to significantly rescue
NK cell function in the presence of suppressive GR (Figure 2) with an ED50 of
approximately 20 U/mL (Figure 2, inset), and to restore the NK cell
response to IL-2. Catalase also prevented a major part of the
fMLF-induced inhibition (Figure 1, inset).
Granulocytes produce reactive nitrogen intermediates (NO).15 To study whether NO induction in GR contributed to the observed NK cell inhibition, we used a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester [L-NAME]).15 This compound, used at final concentrations of 1 to 100 µmol/L, did not affect the GR-induced suppression of NK cells (data not shown). Regulation by histamine via H2R Histamine has earlier been shown to inhibit NADPH oxidase-dependent formation of ROS in neutrophilic GR and in other phagocytes.5 Histamine was found to protect NK cells from GR-induced inhibition with an efficacy similar to that of catalase (Figure 2), and significantly reversed the inhibition induced by fMLF-activated GR (Figure 1, inset). The effect of histamine was dose dependent with an ED50 of approximately 2 µmol/L (Figure 2, inset). To determine which subtype of histamine receptors mediated the protective effect, we used ranitidine, an antagonist at H2R. Ranitidine, used at concentrations equimolar to histamine, completely blocked the histamine effect (Figure 3). To exclude that nonspecific effects of ranitidine accounted for its blocking properties, we used AH202399AA, a ranitidine analogue in which a thioether group has been replaced by an ether, thereby reducing H2R antagonism more than 50-fold.5,12 This chemical control, used at concentrations equimolar to ranitidine, did not block the NK cell-protective effects of histamine (Figure 3, inset).
Stimulus-dependent inhibition of NK cell function by CML cells In contrast to nonmalignant GR, CML cells did not constitutively inhibit NK cell function (Figure 4, left). However, CML cells induced to generate ROS by treatment with fMLF effectively inhibited the cytotoxicity of heterologous NK cells against K562 cells (Figure 4, right). The inhibition of NK cell cytotoxicity was observed at a CML cell/lymphocyte ratio of 1:1 using CML cells from all of the 9 patients examined, but the degree of inhibition was variable; fMLF-activated CML cells from some patients significantly suppressed NK cell cytotoxicity also at lower CML cell/lymphocyte ratios (data not shown). The inhibition of NK cell cytotoxicity was prevented by catalase and histamine (Figure 4, right). Ranitidine, used at concentrations equimolar to histamine, blocked the protective effect of histamine (data not shown). Figure 5A summarizes the effect of GR or CML cells from 9 blood donors or 9 CML patients on NK cell function and its regulation by histamine. Figure 5B shows results obtained in parallel experiments in which NK cells were activated by IL-2.
Because histamine is frequently a constituent of CML cells,17 we explored the possibility that release of histamine from CML cells in vitro could account for the lack of constitutive inhibition of NK cell cytotoxicity by CML cells. In these experiments, we added ranitidine, at 0.1 to 100 µmol/L, to mixtures of CML cells and NK cell-enriched lymphocytes, with the aim to antagonize extracellular histamine released from CML cells. Ranitidine did not alter NK cell-mediated cytotoxicity (against K562 target cells) in these cell mixtures (data not shown), indicating that release of histamine from CML cells was not a major mechanism responsible for the inability of resting CML cells to inhibit NK cells. Histamine inhibits ROS formation in CML cells The finding that catalase prevented the CML cell-induced inhibition of NK cells suggested that ROS generated by CML cells significantly contributed to the inhibition. We therefore investigated whether histamine regulated ROS formation in CML cells using fMLF as the inducer. fMLF induced a prompt production of ROS in CML cells. This response was inhibited in a dose-dependent manner by histamine with an ED50 of approximately 2 µmol/L. The inhibitory effect of histamine was blocked by ranitidine but not by its chemical control, AH202399A, indicating that the effect of histamine was specifically transduced by H2-type receptors on CML cells (Figure 6).
Benign and malignant GR induce apoptosis in NK cells These results imply that GR, by producing ROS, are potent inhibitors of baseline and IL-2-induced NK cell cytotoxicity against a CML-derived target cell line, and that fMLF-activated CML cells share the NK cell-inhibitory properties of nonmalignant GR. In a series of experiments, we studied the fate of NK cells after contact with benign or malignant GR. We estimated the frequency of lymphocytes with morphologic features characteristic of apoptosis, that is, a reduced forward scatter and increased right angle scatter,7,12 after overnight incubation with GR or CML cells. To confirm apoptosis in the gated lymphocytes, 2 additional cytometric methods were used detection of extracellular annexin V and intracellular
glutathione. These methods were chosen because they reflect early
changes associated with apoptosis,10,11 and therefore
allow a concomitant analysis of lymphocyte phenotype.
Apoptotic cells translocate the membrane phosphatidylserine from the inner leaflet of the plasma membrane to the outer, and phosphatidylserine is thereby exposed to the extracellular environment, and annexin V binds to externalized phosphatidylserine.10 Simultaneous analyses of annexin V and morphology revealed a corresponding degree of annexin V staining and the appearance of lymphocytes with apoptotic morphology (data not shown). Oxidative-induced apoptotic cell death is also accompanied by depletion of intracellular stores of glutathione.11,12 The vast majority of lymphocytes with apoptotic morphology (> 95%) had intracellular glutathione levels, measured fluorimetrically as mean fluorescence intensity, which were 10- to 100-fold lower than those of lymphocytes with normal forward and side angle scatter. We observed an accumulation of CD56+ NK cells with
apoptotic morphology and a parallel striking reduction of intracellular glutathione in NK cells after incubation with GR. These apoptotic features were barely detectable 4 hours after the onset of incubation with GR and reached a maximum at 16 to 24 hours. The GR-induced apoptotic morphology in NK cells was prevented by catalase and by
histamine, and the effect of histamine was completely reversed by
ranitidine (Figure 7). The protective
effects of catalase and histamine were confirmed using annexin V
staining and measurement of intracellular glutathione. Superoxide
dismutase (200 U/mL) or L-NAME (1-100 µmol/L) did not prevent
GR-induced apoptosis in NK cells (not shown).
We next examined whether CML cells induced apoptosis in NK cells
in a fashion similar to GR. In contrast to GR, CML cells did not
constitutively induce apoptosis in lymphocytes, but a sizable fraction
of NK cells became apoptotic after overnight incubation with
fMLF-activated CML cells. The CML cell-induced apoptosis in NK cells
was prevented by histamine (Tables 2 and 3). The protective effect of histamine
was mimicked by catalase and completely reversed by ranitidine, but not
by AH202399AA (data not shown).
Lymphocyte subsets differ in sensitivity to GR or CML cell-induced apoptosis We compared the degree of apoptosis in CD56+ NK cells and in other lymphocyte subsets. GR as well as CML cell-induced apoptosis was significantly more pronounced in CD56+ NK cells than in CD3+ T cells. Thus, at a GR density of 2.5 × 104/well, approximately 40% (mean of 5 donors; Figure 7) of all gated CD56+ NK cells had acquired apoptotic morphology, whereas less than 25% of gated T cells were apoptotic, and this difference attained statistical significance (P < .01, Student t test for pairs; Figure 7, inset). A similar difference in propensity to apoptosis was observed when GR were replaced by fMLF-activated CML cells (Tables 2 and 3).
In the first part of this study, we show that normal GR constitutively and effectively inhibit NK cell function, render NK cells resistant to activation by IL-2, and induce apoptosis in NK cells. These GR-triggered events were reduced or inhibited by a scavenger of hydrogen peroxide (catalase), but not by a scavenger of superoxide anion (superoxide dismutase) or a NO synthase inhibitor (L-NAME), suggesting that hydrogen peroxide, or NADPH oxidase-dependent ROS formed downstream of this compound, were the main mediators of the GR-induced suppression. Thus, GR share the NK cell-inhibitory properties previously reported for monocytes/macrophages,6,12 and use a similar inhibitory mechanism. Several investigators have reported that GR inhibit the cytotoxicity of NK cells, but the mechanism responsible for the inhibition has been a matter of controversy. Shau and Golub reported that resting neutrophils inhibited lymphokine-activated killer cell-mediated cytotoxicity when added to IL-2-activated lymphocytes and target cells. The inhibition was resistant to scavengers of ROS such as superoxide dismutase or catalase.18 Lala and coworkers reported that long-term incubation (1-4 days) of NK cells with polymorphonuclear neutrophils resulted in the formation of NK cell-suppressive prostaglandins.19 In our hands, GR-derived ROS were the predominant suppressive mediators, although the contribution by other inhibitory factors could not be ruled out. For example, even optimal concentrations of catalase or histamine could not entirely eliminate the GR-induced suppression. In this regard, GR differ from monocytes/macrophages, because the inhibition of NK cells induced by these cells under similar experimental conditions is almost completely prevented by catalase and NADPH oxidase inhibitors.5,12 A second part of this study was devoted to effects of CML cells on NK
cell function and viability. Our interest in this area was inspired by
the numerous reports claiming that NK cells contribute in surveillance
of the malignant clone in CML. Thus, CML cells are frequently
susceptible to the lytic activity of NK cells; early studies
demonstrated that allogeneic NK cells activated by IFN- In addition to the proposed role of NK cells in surveillance of malignant cells in CML, it seems well established that NK cells are deficient in number and function in this disease. Early studies showed that peripheral blood lymphocytes recovered from CML patients were less cytotoxic for conventional NK cell-sensitive target cells in vitro as compared with lymphocytes recovered from healthy donors,27 or from patients with other chronic leukemias.28 In subsequent studies, it was shown that the absolute number of circulating NK cells and the cell cycle proliferation of NK cells of patients with CML are subnormal, and that a reduction of the number and inducibility of NK cells accompanies disease progression.4,29 Much effort has been devoted to the characterization and functional
significance of the NK cell defect in CML, but the mechanism underlying
the dysfunction has remained largely unknown.3 In brief,
the results of this study reveal a complex interplay between CML cells
and NK cells, which may be of relevance to the NK cell inhibition in
CML. Thus, ROS produced by activated CML cells inhibited the baseline
and cytokine-induced cytotoxicity of NK cells, and a significant
fraction of NK cells from healthy blood donors became apoptotic after
incubation with activated CML cells. These CML cell-triggered
events However, our data also reveal that CML cells are less effective
inhibitors of NK cells than normal GR. Thus, CML cells, but not GR,
depended on an NADPH oxidase-triggering stimulus, fMLF, to inhibit NK
cells. Whether CML cell-derived ROS inhibit NK cells in vivo and
whether factors that trigger NADPH oxidase activity are required for
such inhibition remains to be established. Two arguments may be put
forward in support for the hypothesis that CML cell-derived ROS
contribute to the NK cell dysfunction in this disease. First, in CML,
the malignant cells are frequently present in high numbers in blood and
in bone marrow and even a moderate production of ROS by these cells may
have profound effects on adjacent cells. Second, recent data suggest
that lymphocytes from CML patients may be subjected to oxidative
inhibition. Buggins and coworkers demonstrated that NK cells and other
lymphocytes in the peripheral blood of patients with CML at various
stages of disease display a pronounced reduction of the expression of CD3 We also report that histamine, acting via H2R, inhibited fMLF-induced ROS production by CML cells and, thereby, prevented the CML cell-induced inhibition of cytotoxicity and protected NK cells from apoptotic cell death. In earlier studies, H2-receptors transducing inhibition of ROS formation have been demonstrated on normal GR and on leukemic cell lines derived from phagocytes,15 but our report is the first to demonstrate functional histamine H2-receptors on freshly recovered CML cells. Whether the CML cell content of histamine significantly alters ROS synthesis in vivo is not known, but a role for histamine is suggested by the high serum levels of histamine and the occasional symptoms related to hypersecretion of gastric acid and pepsin in CML patients, suggesting that H2R can be targeted by CML cell-derived histamine in vivo.33 Our data suggest that histamine may serve as an autocrine inhibitor of ROS production in CML cells, thereby preventing functional inhibition and apoptosis in adjacent lymphocytes. However, if we accept that ROS production is a mechanism by which CML cells can escape NK cells or other lymphocytes with antineoplastic function, it seems reasonable to also assume that the production of ROS-inhibitory histamine by the CML cells is not sufficient to prevent inhibition of NK cells, because NK cells undoubtedly deteriorate in function and number during progression of the disease.3,4 This assumption is supported by our finding that CML cells did not constitutively release histamine in amounts sufficient to protect NK cells or T cells. Thus, a putative balance between ROS production and histamine in CML is probably shifted in favor of ROS production. The frequency of apoptosis in lymphocytes after contact with activated CML cells or nonmalignant GR was considerably higher in CD56+ NK cells than in T cells. These findings are consonant with earlier studies demonstrating that NK cells are more prone than T cells to acquiring apoptotic features after exposure to hydrogen peroxide,7,12 and suggest that T cells are, at least in part, protected against oxidatively-induced apoptosis. The mechanisms underlying this difference in apoptotic propensity between NK cells and T cells should be the focus of further investigation. Our data are suggestive of novel mechanisms by which CML cells affect
functions of NK cells. A balance may be at hand in CML, because CML
cells are producers of NK cell-inhibitory ROS, and contain histamine,
which can function as an autocrine inhibitor of ROS formation and,
thereby, protect NK cells from CML cell-derived inhibition. These
interactions are schematically depicted in Figure 8.
We are indebted to Marie-Louise Landelius for expert technical assistance and to Bo Nilsson and Örjan Strannegård for critical review of this manuscript.
Submitted September 8, 1999; accepted May 2, 2000.
Supported by the Swedish Society Against Cancer (Cancerfonden), the Foundations of Jubileumskliniken at Sahlgren's University Hospital, the Assar Gabrielsson Foundation, the Faculty of Medicine, University of Göteborg, and Maxim Pharmaceuticals, Inc, San Diego, CA.
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: Kristoffer Hellstrand, Department of Virology, Sahlgren's University Hospital, S-413 46 Göteborg, Sweden; e-mail: kristoffer.hellstrand{at}microbio.gu.se.
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H. Nakajima, R. Zhao, T. C. Lund, J. Ward, M. Dolan, B. Hirsch, and J. S. Miller The BCR/ABL Transgene Causes Abnormal NK Cell Differentiation and Can Be Found in Circulating NK Cells of Advanced Phase Chronic Myelogenous Leukemia Patients J. Immunol., January 15, 2002; 168(2): 643 - 650. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(IFN-
/56+
phenotype) and T cells (CD3+/56
-counter. Maximum 51Cr-release was determined in target
cell cultures treated with Triton X-100. NK cell cytotoxicity was
calculated as cell lysis percent according to the following formula:
100 x [(experimental release 
spontaneous release)/(maximum
release 
) or fMLF (0.1 µmol/L, 
), added at 15 minutes after the onset of incubation. Data are cell lysis percent ± SEM of sextuplicate determinations. The inset shows a corresponding
experiment in which cells were treated with culture medium (med),
histamine (H; 10 µmol/L), or catalase (C; 100 U/mL).
), catalase (100 U/mL, 
), or culture
medium (
)
or histamine (10 µmol/L, 
) and histamine + ranitidine (10 µmol/L + 10 µmol/L, 
,