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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1456-1464
PHAGOCYTES
From the Division of Physiological Chemistry II, Department of
Medical Biochemistry and Biophysics, and the Division of Hematology,
Department of Internal Medicine, Danderyd Hospital at Karolinska
Institutet, Stockholm, Sweden, and the Division of
Hematology, Department of Internal Medicine, Karolinska Hospital at
Karolinska Institutet, Stockholm, Sweden.
Elevated leukotriene (LT)C4 synthase
activity was observed in peripheral blood granulocyte suspensions from
patients with chronic myeloid leukemia (CML). Magnetic cell sorting
(MACS) with CD16 monoclonal antibodies (mAbs), which were used to
fractionate granulocytes from CML patients and healthy individuals,
yielded highly purified suspensions of CD16+ neutrophils.
The purity of these cell fractions was verified by extensive
morphologic examination. Reverse transcriptase-polymerase chain
reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4
messenger RNA (IL-4 mRNA), further confirmed the negligible
contamination of eosinophils in these fractions. Notably, purified CML
CD16+ neutrophils from all tested patients transformed
exogenous LTA4 to LTC4. These cells also
produced LTC4 after activation with ionophore A23187 or
the chemotactic peptide fMet-LeuPhe
(N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of
LTC4 synthase mRNA in CML CD16+
neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses
consistently demonstrated expression of LTC4 synthase at
the protein level in CML CD16+ neutrophils, whereas
expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC4 synthase activity
or expression of the protein could not be demonstrated in
CD16+ neutrophil suspensions from any of the healthy
individuals. Instead, these cells, as well as CML CD16+
neutrophils, transformed LTA4 to LTB4. The
results indicate that aberrant expression of LTC4 synthase
is a regular feature of morphologically mature CML CD16+
neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC4 synthesis in
vivo. Such overproduction may be of pathophysiological relevance
because LTC4 has been demonstrated to stimulate
proliferation of human bone marrow-derived myeloid progenitor cells.
(Blood. 2000;95:1456-1464)
Chronic myeloid leukemia (CML) is characterized by
markedly accelerated myelopoiesis, with increased numbers of immature
and mature myeloid cells in peripheral blood and bone marrow. A
characteristic feature of the disease is a specific chromosome
abnormality, the t9;22 reciprocal translocation, which
creates the Philadelphia (Ph) chromosome. The Ph chromosome is the
product of a molecular rearrangement between the c-ABL
proto-oncogene situated on chromosome 9 and the breakpoint cluster
region (BCR) gene on chromosome 22. The BCR-ABL fusion
gene encodes a 210-kd protein, which is an aberrant highly active
tyrosine kinase. It is currently believed that the initiation,
maintenance, and progression of CML are consequences of
BCR-ABL-induced activation of multiple signal transduction pathways. This leads to, for example, cytokine-independent uncontrolled proliferation of myeloid cells in the bone marrow and to resistance to
apoptosis.1
We have previously reported that suspensions of peripheral white blood
cells from patients with CML possess a markedly elevated capacity to
produce leukotriene (LT)C4 from endogenous arachidonic acid.2 Furthermore, overproduction of LTC4 was
observed also in granulocyte suspensions2 and unfractioned
bone marrow cells3 from CML patients. These
findings are of potential interest, since LTs, which are endogenously
formed mediators of inflammation and asthma,4,5 may also
play a role in the regulation of human myelopoiesis. Thus
LTC4 and, to a lesser extent, LTB4 have been demonstrated to potentiate granulocyte-macrophage colony-stimulating factor-induced (GM-CSF-induced) myeloid progenitor cell
(CFU-GM) proliferation.6,7 In addition,
inhibition of LT formation attenuated CFU-GM growth, an effect that
could be counteracted by the addition of exogenous LTC4 or
LTB4.6 Accordingly, 5-lipoxygenase (5-LO)
inhibitors have been observed to suppress the proliferation and DNA
synthesis of normal CFU-GM and myeloid leukemic cell
lines.8-11 Furthermore, inhibition of LT synthesis has been
reported to induce programmed cell death in U937 cells
and in CML blast cells.12,13
Cells of myeloid origin are the main producers of LTs in humans. Upon
cell activation, the LT biosynthesis is initiated by phospholipase
A2-dependent release of arachidonic acid from membrane phospholipids.14 Subsequently, 5-LO translocates to the
nuclear membrane, where it cooperates with the 5-LO activating protein (FLAP) in the conversion of arachidonic acid to the unstable epoxide LTA4.15 This intermediate can be metabolized
either by a cytosolic LTA4 hydrolase to the
leukocyte-activating dihydroxy acid LTB416 or
by a membrane-bound LTC4 synthase,17 which
specifically catalyzes conjugation of LTA4 with the
tripeptide glutathione, thereby yielding LTC4. Once formed,
LTC4 is actively exported to the extracellular space, a
process involving the multidrug resistance-associated protein
(MRP).18-20 Extracellularly, LTC4 is converted
by Among human blood cells, neutrophils, eosinophils, and basophils, as
well as monocytes, possess the enzymatic machinery needed for LTA4
production from endogenous substrate and are thus capable to
produce LTs after cell stimulation. However, the subsequent metabolism
of LTA4 differs among these cell types. Thus, neutrophils, equipped with LTA4 hydrolase but lacking LTC4
synthase, produce LTB4 but not LTC4, whereas
the reverse applies for eosinophils and basophils.21,22
Monocytes express both these enzymes, consequently producing both
LTB4 and LTC4.23 In addition,
platelets24-26 and erythrocytes27 lack
5-LO/FLAP but express LTC4 synthase and LTA4
hydrolase, respectively. Recent findings demonstrate that activated neutrophils release the main part of produced
LTA4 extracellularly upon stimulation with ionophore A23187
or physiological stimuli.28,29 These results clearly
indicate that platelets and erythrocytes may participate in LT
biosynthesis in vivo via transcellular
mechanisms.30
LTC4 synthase is a highly specific glutathione
S-transferase that comprises 2 subunits (18 kd each) and is active as a
homodimer.17 This enzyme has been purified from several
sources,31,26 and cloning of the gene, as well as
expression of the protein, has been reported.32,33
LTC4 synthase is a member of the membrane-associated proteins involved in the eicosanoid and glutathione metabolism (MAPEG)
protein family, which also includes FLAP, microsomal glutathione S-transferase (MGST) 1-334 and prostaglandin (PG)
E2 synthase.66 Among these proteins, MGST2 and
MGST3 have also been shown to possess LTC4 synthase
activity.35,36 The activity of LTC4 synthase has been demonstrated to be regulated by phosphorylation-dependent mechanisms.37-41 A recent report describing
LTC4 synthase deficiency in relation to a fatal
developmental syndrome further suggests that the enzyme may be of
importance also in areas not related to allergy and
inflammation.42
The gene encoding human LTC4 synthase has been mapped to
chromosome 5, long arm, band 35 (5q35) and contains several
transcription factor-binding motifs in the 5' flanking region,
suggesting a regulated mode of expression.43,44 In
agreement, the transcription of the LTC4
synthase gene was recently demonstrated to be up-regulated by
the transforming growth factor- In this report we address the abnormal LTC4 synthase
activity in CML granulocytes. The results, obtained after magnetic cell sorting (MACS) with monoclonal antibodies (mAbs), demonstrate a
regularly occurring aberrant expression of active LTC4
synthase in highly purified CD16+ neutrophils from CML patients.
Materials
Patients and healthy control subjects
Granulocyte isolation Peripheral venous blood was drawn into blood collection tubes containing ethylenediaminetetraacetic acid (EDTA). After centrifugation at 200g for 15 minutes, the platelet-rich plasma was removed, and granulocytes were isolated from the remaining lower phase by dextran sedimentation, hypotonic ammonium chloride lysis, and sodium metrizoate centrifugation, as previously described.49Magnetic cell sorting The granulocyte fraction obtained after centrifugation was resuspended in phosphate-buffered saline (PBS, pH 7.4) supplemented with 0.1% bovine serum albumin (BSA) and 2 mmol/L EDTA (buffer A) to a cell concentration of 10 × 107 cells/mL and incubated with anti-CD16 microbeads at 8°C for 30 minutes. The cells were applied to an LS+ MACS column, and the CD16 eosinophil-enriched cell fraction was eluted
with 9 mL buffer A. Thereafter, the column was washed with 3 mL buffer
A, removed from the magnetic field, and eluted with 5 mL buffer A in
order to collect CD16+ neutrophils.
Morphologic examination Duplicate aliquots of the cell fractions were subjected to cytocentrifugation and May-Grünwald-Giemsa staining. Thereafter, the slides were numbered by code and evaluated blindly by an experienced hematology morphologist (S. Widell). Two hundred cells were counted on each slide.Preparation of subcellular fractions CD16+ neutrophils were centrifuged at 300g for 10 minutes, resuspended in 0.125 mol/L potassium phosphate buffer (2.5 × 107 cells/mL, pH 7.4), and sonicated at 0°C for 5 × 15 seconds prior to centrifugation at 1400g for 15 minutes. The supernatant was collected and further centrifuged at 100 000g for 60 minutes. After collection of the supernatant (cytosolic fraction), the pellet (microsomal fraction) was resuspended in 0.125 mol/L potassium phosphate buffer (equal volume as cytosolic fraction) prior to assay of enzyme activity in the fractions, as described below.Determination of LTC4 synthase activity Intact cell preparations were centrifuged at 300g for 10 minutes and resuspended in PBS (containing 0.9 mmol/L calcium chloride and 0.03% human serum albumin [HSA], pH 7.4) to a final concentration of 1.5 × 107 or 0.3 × 107 (CD16
eosinophil-enriched fraction) cells/mL. The metabolism of
LTA4 in intact cells was determined by incubation with 10 µmol/L LTA4 at 37°C for 5 minutes. Subcellular
fractions of CD16+ cells were incubated with 60 µmol/L
LTA4 and 5 mmol/L reduced glutathione at 20°C for 10 minutes in the presence of 0.05% BSA. The capacity of normal and CML
CD16+ neutrophils to produce LTC4 from
endogenous LTA4 was determined by incubation with 1 µmol/L A23187 or 1 µmol/L fMLP in combination with 8 µmol/L
arachidonic acid at 37°C for 5 minutes. All incubations were
stopped by the addition of 5 volumes of ethanol containing prostaglandin B2 as an internal standard.
Prior to analysis, the samples were centrifuged, evaporated, dissolved
in 250 µL high-pressure liquid chromatography (HPLC) mobile phase,
and recentrifuged. Thereafter, LTs were analyzed by reversed-phase HPLC
as described,39 using a 3.9 × 150-mm column
(Nova-Pak C18; Waters Associates, Milford,
MA), and eluted with a mixture of acetonitrile, methanol, water, and
acetic acid at a ratio of 27:18:54:0.8, vol/vol (apparent pH 5.6). Eluted compounds were quantified using a variable
wavelength ultraviolet (UV) detector (LDC Spectromonitor
III, Stone, England) connected to an integrator (LDC/Milton Roy
CI-4000, Laboratory Data Control). The compounds were identified by
cochromatography with authentic standards and on-line UV spectroscopy.
RNA isolation, cDNA synthesis, and polymerase chain reaction Total RNA was extracted from isolated cells by the guanidinium-phenol-chloroform extraction technique50 (Ultraspec II, Biotecx). The yield and purity of RNA were examined by spectrophotometrical measurements at 260 and 280 nm. For the preparation of cDNA, 1 µg RNA was used in a 20 µL cDNA synthesis reaction with 20 units avian myeloblastosis virus RT, 3.2 µg random hexamer primers, 10 mmol/L Tris (tris[hydroxymethyl aminomethane]), 50 mmol/L potassium chloride (KCl), 5 mmol/L magnesium dichloride (MgCl2), deoxynucleotide (dNTP) mix (1 mmol/L each), and 50 units ribonuclease (RNase) inhibitor. The reaction mixture was incubated for 10 minutes at 25°C (primer annealing), 60 minutes at 42°C (transcription), and 5 minutes at 99°C (inactivation of RT). Thereafter, 1 µL of the cDNA synthesis reaction was added to a PCR reaction mixture (total volume 50 µL) containing 1.25 units Thermus aquaticus (Taq) polymerase, 10 mmol/L Tris-hydrochloride, 50 mmol/L KCl, 1.5 mmol/L MgCl2, dNTP mix (0.2 mmol/L each) and 0.3 µmol/L primers. The following primers were used: LTC4 synthase: 5'-ACCTGGGCTCGGTAGAC-3' and 5'-GAGTCCTGCTGCAAGCCTACTTC-3'; -actin:
5'-GAGGAGCACCCC GTGCTGCTGA-3' and
5'-CTAGAAGCATTTGCGGTGG 3'; IL-4:
5'-CGGCAACTTTGACCACGGACACAAGTGCGATA-3' and 5'-ACGTA
CTCTGGTTGGCTTCCTTCACAGGACAG-3'. The expected sizes of the DNA
fragments amplified with these primers were 119 base pairs (bp) for
LTC4 synthase, 784 bp for -actin, and 344 bp for IL-4.
Immunoblotting Normal and CML CD16+ neutrophils, as well as normal CD16 eosinophil-enriched cell
preparations, were isolated as described above and suspended to a cell
concentration of 15 × 106 cells/mL in lysis buffer
(PBS without Ca2+ and Mg2+, supplemented with 1 × protease inhibitor cocktail) (Complete, Boehringer Mannheim).
Thereafter, cells were sonicated at 0°C for 3 × 10 seconds
prior to centrifugation at 1000g for 15 minutes. The
supernatant was removed and further centrifuged at 100 000g for 60 minutes. The pellet (microsomal fraction) was resuspended in
lysis buffer, mixed with 1 volume 2 × loading buffer (containing 125 mmol/L Tris, 20% glycerol, 4% SDS [sodium dodecyl sulfate], 10% 2-mercaptoethanol, and 0.002% bromophenol blue; pH 6.8), and boiled for 5 minutes. Protein levels were determined, and 20 µg of
protein was separated on 14% SDS-PAGE (polyacrylamide gel
electrophoresis). Proteins were electroblotted onto a nitrocellulose
membrane followed by blocking of the membrane with 5% milk powder in
100 mmol/L Tris (containing 0.9% NaCl and 0.1% Tween
20, pH 7.5) for 60 minutes. After blocking, the membranes were washed
and incubated for 60 minutes with antibodies against LTC4
synthase or MGST2 (both 1:1000 dilution) in 100 mmol/L Tris (containing
0.9% NaCl, 0.05% Tween 20, and 2% milk powder; pH 7.5) prior to
being washed and incubated with secondary antirabbit
antibody1:1000 in 100 mmol/L Tris (containing 0.9% NaCl,
0.05% Tween 20, and 2% milk powder; pH 7.5) for 60 minutes. Enhanced
chemiluminescence (ECL plus, Amersham) was used for detection per
manufacturer instructions.
Statistics The 2-sided Student t test for unpaired samples was employed to compare LT production in leukemic and normal cells.
Morphological and RT-PCR analyses of granulocyte preparations Morphological analyses demonstrated that normal and CML granulocyte suspensions obtained after density gradient centrifugation contained approximately 90% neutrophils and 6%-7% eosinophils together with minute amounts of lymphocytes (Table 1). Since eosinophils are potent producers of LTC4, MACS was employed in order to eliminate eosinophil contamination. After immunomagnetic separation of the granulocyte suspensions with CD16 mAbs, CD16+ cell suspensions from CML patients and controls contained more than 99% neutrophils (Table 1). The mean eosinophil content in these fractions was estimated to be less than 0.04%. The presence of platelets was evaluated in these cell fractions, and platelet contamination in the normal and CML CD16+ neutrophil preparations was always less than 1 platelet per 20 neutrophils. The CD16 cell preparations contained
predominantly eosinophils together with other cell types, mostly
neutrophils and lymphocytes. In addition, the CML
CD16 cell preparations contained immature myeloid
cells, particularly myelocytes (Table 1). Messenger RNA (mRNA) encoding
the cytokine IL-4 was present in normal and CML CD16
fractions, as demonstrated by RT-PCR amplification of a DNA fragment of
the expected size (344 bp) (Table
2). In contrast, IL-4 expression could not
be observed in normal or CML CD16+ neutrophil fractions.
The integrity of the RNA was verified by amplification with
-actin-specific primers, and an IL-4 cDNA was used as a positive
control to verify specific amplification with the IL-4 primers (results
not shown). The findings are in accordance with the negligible
contamination of eosinophils in the CD16+ fractions,
because IL-4 mRNA has been demonstrated to be expressed in eosinophils
but not in neutrophils.51-53
Leukotriene C4 synthase activity in granulocyte suspensions The metabolism of exogenous LTA4 was investigated in unfractionated granulocyte suspensions that were not subjected to MACS. Granulocytes from CML patients and normal controls transformed LTA4 to LTC4 and LTB4. Notably, the levels of LTC4 were almost 4 times higher in CML granulocyte suspensions compared with normal granulocytes, as measured by the mean ± SEM (standard error of the mean) (44.4 ± 7.7 pmol/106 cells [n = 13] versus 12.0 ± 1.5 pmol/106 cells [n = 11]; P = .0007), although the eosinophil content was similar in these suspensions (Table 1). In contrast, the production of LTB4 was unaltered in the CML suspensions (Figure 1). These data indicate increased LTC4 synthase activity in CML granulocytes.
Leukotriene C4 synthase activity in CD16+
neutrophils and CD16 eosinophil-enriched cell preparations
isolated by MACS was investigated. Notably, CML CD16+
neutrophils from all patients tested transformed LTA4 to
LTC4, (range, 8.1-44.2 pmol/106 cells; mean ± SEM 27.7 ± 5.1 pmol/106 cells [n = 9])
(Figure 2A). In contrast and as
anticipated, normal CD16+ neutrophils failed to produce
significant amounts of LTC4 from LTA4 (range,
0-2.9 pmol/106 cells; 1.3 ± 0.3 pmol/106
cells [n = 11]; P < .0001). The capacity to convert
LTA4 to LTB4 was similar in CML and normal
CD16+ neutrophils (Figure 2A). Expectedly, the
CD16 eosinophil-enriched cell preparations from
leukemic patients and normal individuals efficiently transformed
LTA4 to LTC4 (176.7 ± 56.9 and
291.7 ± 72.8 pmol/106 cells, respectively) and minor
amounts of LTB4 (Figure 2B). Determination of
LTA4 metabolism in subcellular fractions obtained from CML CD16+ neutrophil suspensions revealed that the
LTC4 synthase activity was almost exclusively confined to
the microsomal fraction (Figure 3), in
accordance with the known subcellular distribution of LTC4 synthase.17 In contrast and as expected,54 the
LTA4 hydrolase activity was essentially located to the
cytosolic fraction. Formation of LTD4 or LTE4
was not observed in any of the cell fractions.
Endogenous formation of LTC4 in CML CD16+ neutrophils The capacity of CML CD16+ neutrophils to produce LTC4 from endogenous arachidonic acid was investigated by incubation with 1 µmol/L ionophore A23187 for 5 minutes. Again, these cells readily produced LTC4 (24.8 ± 8.9 pmol/106 cells [n = 4]) (Figure 4). Furthermore, CML CD16+ neutrophils also produced LTC4 (9.6 ± 5.2 pmol/106 cells [n = 4]) when activated with the chemotactic tripeptide fMLP in the presence of low amounts of exogenous arachidonic acid (8 µmol/L) for 5 minutes. Again, further conversion of LTC4 to LTD4 or LTE4 could not be observed. In addition, these cells produced LTB4 after stimulation with ionophore A23187 or fMLP plus arachidonic acid. In comparison, normal CD16+ neutrophils only produced LTB4 when stimulated with these agents (Figure 4). LT production was not observed after the addition of 8 µmol/L arachidonic acid to normal or CML CD16+ neutrophils (results not shown).
Expression of LTC4-producing enzymes in CML CD16+ neutrophils Expression of LTC4 synthase protein in CML and normal CD16+ neutrophils was investigated by immunoblot analysis. Protein samples obtained from normal CD16
eosinophil-enriched cell preparations were used as positive controls. LTC4 synthase was consistently expressed at intermediate to
high levels in CML CD16+ neutrophils (Figure
5). In contrast, LTC4 synthase
immunoreactivity was not observed in normal CD16+
neutrophils. The 2 CML CD16+ neutrophil samples displaying
the highest levels of LTC4 synthase also contained
detectable amounts of MGST2. However, in the 2 additional CML
samples, as well as in the samples obtained from normal controls, MGST2
immunoreactivity could not be observed. The positive
control for MGST2 was prepared from MGST2-transfected Sf9 cells, as
described previously.48
In previous studies we reported increased production
of LTC4 in myeloid cells from patients with
CML.2,3 The LTC4 overproduction, consistently
observed after ionophore A23187 stimulation of unfractionated leukocytes, bone marrow cells, or granulocyte preparations strongly indicated enhanced LTC4 synthase activity in CML. In the
current investigation, elevated LTC4 synthase activity in
CML granulocyte suspensions, as compared with the enzyme activity in
normal granulocytes, was confirmed by significantly enhanced
LTC4 formation after incubation of unstimulated intact
cells with exogenous LTA4, the immediate enzyme substrate.
Notably, cautious morphological analyses demonstrated that the number
of eosinophils was not essentially higher in CML granulocyte
suspensions than in control preparations. These findings exclude the
possibility that the increased LTC4 synthase activity was
related to the number of eosinophils. However, these data could not
conclude whether the increased LTC4 synthesis was due to
elevated intrinsic LTC4 synthase activity in CML
eosinophils or to an aberrant expression of the enzyme in the
leukemic neutrophils.
We thank Dr Robert Zipkin, Biomol Research Laboratories (Plymouth
Meeting, PA), for the generous gift of LTA4 methyl ester.
Submitted June 28, 1999; accepted October 12, 1999.
Supported by grant 2663 from the Swedish Cancer Society
(Stockholm, Sweden), grants 03X-6805 and 03X-12573 from
the Swedish Medical Research Council (Stockholm, Sweden),
and research funds from the Karolinska Institutet (Stockholm, Sweden).
Reprints: Jan Åke Lindgren, Division of Physiological
Chemistry II, Department of Medical Biochemistry and Biophysics, Scheele Laboratory, Karolinska Institutet, S-171 77 Stockholm, Sweden;
e-mail: jan.ake.lindgren{at}mbb.ki.se.
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.
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|
Top
Abstract
Introduction
-glutamyl transpeptidase to the biologically active
LTD4 and is further metabolized by dipeptidase to
LTE4 via successive elimination of a 
interactions with granulocyte-macrophage colony-stimulating factor.
Blood.
1993;80:352