Aberrant expression of active leukotriene C₄ synthase in CD16⁺ neutrophils from patients with chronic myeloid leukemia

The following materials were used in this study: blood collection tubes (Vacutainer; Becton Dickinson, Rutherford, NJ); sodium metrizoate (Lymphoprep; Nyegaard, Oslo, Norway); paramagnetic microbeads conjugated to mouse antihuman CD16 mAbs, magnetic stands, and LS⁺ MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany); ionophore A23187 (Calbiochem-Boehring, La Jolla, CA); arachidonic acid (Nu-Check Prep, Elysian, MN); fMet-Leu-Phe (N-formylmethionyl-leucyl-phenylalanine [fMLP]) and agarose (Sigma, St. Louis, MO); LTA₄ methyl ester, saponified before use as previously described39 (gift from Dr Robert Zipkin, Biomol Research Laboratories, Plymouth Meeting, PA); LTB₄, LTC₄, and PGB₂(Biomol Research Laboratories); nitrocellulose membranes, precast polyacrylamide gels, and protein standards (Novex, San Diego, CA); LTC₄ synthase and MGST2 antibodies, developed as previously described47 48 (gift from Merck Frosst, Montreal, Quebec, Canada); horseradish peroxidase–coupled antirabbit secondary antibodies as well as a chemoluminescence detection kit (ECL plus; Amersham, Buckinghamshire, England); film (Biomax MR; Kodak, Rochester, NY); RNA extraction kit (Ultraspec II; Biotecx, Houston, TX); first strand cDNA synthesis kit, protease inhibitor cocktail, and amplification mixture (Complete, PCR Master; Boehringer Mannheim, Mannheim, Germany); LTC₄ synthase PCR primers (Scandinavian Gene Synthesis, Köping, Sweden); IL-4 PCR primers, β-actin PCR primers, and IL-4 complementary DNA (cDNA) (Clontech, Palo Alto, CA); and a PCR cloning kit (pCR-Script Amp SK+; Stratagene, La Jolla, CA).

Fresh blood samples were obtained from a total of 17 patients in the chronic phase of Ph chromosome-positive (Ph⁺) CML and 15 healthy medication-free volunteers. All blood samples were collected with the informed consent of involved individuals in accordance with the approval of the project from the Ethics Committee of Karolinska Institutet (Stockholm, Sweden). The peripheral white blood cell differential counts of the patients were routinely analyzed at the time of blood collection.

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.49 

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.

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.

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.

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.

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′; β-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 β-actin, and 344 bp for IL-4.

Temperature cycling (Mastercycler 5330; Eppendorf, Hamburg, Germany) was performed as follows: First cycle, denaturation at 94°C for 60 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds; subsequent cycles: denaturation at 94°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds. In total, 30 cycles were performed for β-actin, 35 cycles for LTC₄ synthase, and 40 cycles for IL-4. PCR products were analyzed by agarose gel electrophoresis. To verify specific LTC₄ synthase amplification, the PCR product was ligated into a plasmid vector (pCR-Script Amp SK+, Stratgene) following manufacturer instructions. Bacteria were transformed, and double-stranded plasmid DNA was isolated from recombinant clones and sequenced according to standard protocols.

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 000gfor 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 LTC₄synthase 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 antibody^1:1000 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.

The 2-sided Student t test for unpaired samples was employed to compare LT production in leukemic and normal cells.

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) (Table2). 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 β-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 

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.

The metabolism of LTA₄ 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/10⁶cells [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 LTC₄synthase.17 In contrast and as expected,54 the LTA₄ hydrolase activity was essentially located to the cytosolic fraction. Formation of LTD₄ or LTE₄was not observed in any of the cell fractions.

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 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 (Figure5). 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 

Expression of mRNA encoding LTC₄ synthase was demonstrated in CML CD16⁺ neutrophils by RT-PCR using samples from normal CD16⁻ eosinophil-enriched cells as positive controls (Figure 6). To verify RNA integrity, amplifications with β-actin–specific primers were also performed. The identity of the LTC₄ synthase amplification product was confirmed by cloning and sequencing (results not shown).

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.

To elucidate this question, MACS methodology based on CD16 antigen immunoselection was applied in order to obtain eosinophil-free, highly purified neutrophil preparations. Expression of the CD16 surface antigen on neutrophils but not on eosinophils has been demonstrated.55 Furthermore, CD16 expression on CML neutrophils has been confirmed.56 Using the MACS technique, we obtained CD16⁺ neutrophil preparations with more than 99% purity and no detectable eosinophil contamination, as judged by morphological examination. The high purity of these cells was further indicated by negative RT-PCR analysis of mRNA encoding IL-4, which is known to be expressed in lymphocytes and eosinophils but not in neutrophils.51-53 57 CML CD16⁺cells were as potent as the corresponding control cells in transforming LTA₄ to LTB₄, indicating unaltered LTA₄ hydrolase activity in the cells. Since LTA₄ hydrolase expression is a characteristic of neutrophils, these findings further confirm the identity of the leukemic CD16⁺ neutrophils. Interestingly, however, all CD16⁺ neutrophil preparations from CML patients also expressed LTC₄ synthase activity, as judged by the ability of these cells to convert LTA₄ to LTC₄.

Experiments with subcellular fractions demonstrated that the enzyme activity was solely distributed to the microsomal fraction, in agreement with the known intracellular localization of LTC₄synthase.17 In contrast, the LTC₄ synthase activity in normal CD16⁺ neutrophil suspensions was negligible. The contamination of platelets, a potential source of LTC₄ synthase,24-26 was always low in both normal and CML CD16⁺ neutrophil fractions, with less than 50 000 platelets per 10⁶ neutrophils. In an earlier study using platelet preparations from 44 normal donors, platelet LTC₄ formation under conditions identical to those used in the present study was approximately 3 pmol/10⁶platelets.39 Furthermore, we have observed a similar LTC₄ synthase activity in CML and normal platelets (Lindgren et al, unpublished observation). This excludes the possibility that the LTC₄ synthase activity observed in CML CD16⁺ neutrophil preparations was the result of platelet contamination.

Further investigation of the LTC₄-producing capacity in CML CD16⁺ neutrophils revealed that these cells also synthesized LTC₄ from endogenous substrate upon challenge with calcium ionophore A23187. Similarly, LTC₄ synthesis was provoked in these cells by the chemotactic tripeptide fMLP. Again, normal CD16⁺ neutrophils failed to produce LTC₄under these conditions, whereas the synthesis of LTB₄ was similar in normal and CML neutrophils. The findings demonstrate that the LTC₄ synthase activity in CML neutrophils is efficiently coupled to the enzymatic machinery providing endogenous LTA₄ and strongly suggest that these cells are capable of LTC₄ synthesis in vivo.

The expression of mRNA encoding LTC₄ synthase in CML CD16⁺ neutrophils was demonstrated by RT-PCR, indicating that the gene is transcriptionally active in these cells. Moreover, expression of LTC₄ synthase protein in all CML CD16⁺ neutrophil preparations tested was verified by immunoblot analysis. In contrast, LTC₄ synthase immunoreactivity could not be observed in protein extracts from normal CD16⁺ neutrophils.

Messenger RNA encoding MGST2 was previously found to be highly expressed in K-562, a myeloid leukemia cell line,35 suggesting expression of this enzyme in myeloid leukemia. It was therefore of interest to investigate the expression of this enzyme on the protein level in freshly isolated CD16⁺neutrophils from CML patients. Interestingly, CD16⁺neutrophil preparations from some of the CML patients displayed MGST2 immunoreactivity. However, significant expression of this enzyme could not be detected in CD16⁺ neutrophils from other CML patients despite the fact that these cells always possessed LTC₄ synthase activity. Thus, these data suggest that the LTC₄ synthase activity observed in CML neutrophils depends on the aberrant expression of LTC₄ synthase and not the expression of MGST2. Whether abnormal expression of MGST2 is a common feature of CML neutrophils or other myeloid cells and whether this can contribute in any degree to the abnormal LTC₄synthase activity need to be further investigated. In addition, our data suggest that MGST2 expression is not a regular feature of neutrophils, because MGST2 immunoreactivity could not be observed in any of the normal CD16⁺ neutrophil preparations.

The present findings provide new insights regarding the mechanisms behind the previously reported elevated LTC₄ synthase activity in myeloid cells from CML patients.2,3Furthermore, the observed aberrant LTC₄ synthase expression in highly purified mature CML neutrophils strongly indicates correlation between the abnormal LTC₄ synthase activity and the malignant transformation. Interestingly, however, the LTC₄ overproduction was much less pronounced in the neutrophils than in unfractionated white blood cell suspensions from CML patients.2 Thus, after ionophore A23187 stimulation, the LTC₄ production was twice that of LTB₄ in CML white blood cells, whereas this proportion was reversed in CML CD16⁺ neutrophils. This strongly suggests that less mature leukemic myeloid cells contribute to the overproduction of LTC₄ in CML. Presently, we are investigating the expression and activity of LTC₄ synthase immature myeloid cells from CML patients.

The CD16⁻ cell fractions were highly enriched in eosinophils (63% and 78% in CML and control preparations, respectively) but also contained other cell types. No attempt was made to further purify the eosinophils. These cell suspensions efficiently produced LTC₄ when incubated with LTA₄, in agreement with a high LTC₄ synthase activity in eosinophils. Notably, there was no sign of elevated LTC₄synthase activity in CML eosinophils compared with normal cells. The CD16⁻ eosinophil-enriched cell suspensions also transformed LTA₄ to LTB₄, reflecting the presence of contaminating LTA₄ hydrolase-expressing cells such as neutrophils and lymphocytes58 in these cell preparations.

It can be approximated from the present data that the mean LTC₄ synthase activity was roughly 10-fold higher in CML eosinophils than in CML CD16⁺ neutrophils. However, in CML the normal myelopoiesis is gradually replaced by the production of malignant cells. It has been demonstrated that normal myeloid cells coexist with leukemic cells in the bone marrow as well as in the peripheral blood during the chronic phase of the disease.59 60 Since it can be speculated that normal neutrophils within the CML CD16⁺ cell fractions lack expression of LTC₄ synthase, it may be assumed that the determined enzyme activity in these cell fractions is an underestimate of the real LTC₄ synthase activity in leukemic neutrophils. Furthermore, since the number of neutrophils in bone marrow and peripheral blood is usually more than 10 times higher than the number of eosinophils, it is probable that leukemic neutrophil LTC₄ synthase activity accounts for a substantial part of the in vivo LTC₄ synthesis and that this may lead to a significant overproduction of this compound in patients with CML.

Studies of LTC₄ synthase expression and activity in differentiating myeloid cells have suggested up-regulation of LTC₄ synthase expression during eosinophilic differentiation.47,61 Furthermore, LTC₄synthase activity or expression could not be detected in CD34⁺ cord blood stem cells.61 In contrast, we have recently obtained data demonstrating LTC₄ synthase activity in MACS-purified immature progenitor cells from normal human bone marrow (Sjölinder et al, to be published). Accordingly, the LTC₄ synthase activity in white blood cell suspensions obtained from patients with acute myeloid leukemia62 was highly correlated to the proportion of circulating blast cells. Thus, it may be speculated that LTC₄ synthase is expressed in stem cells and progenitors in the bone marrow and that the expression is down-regulated before differentiation of the cells into mature neutrophils. It may be further speculated that this down-regulation is disturbed in CML neutrophils, leading to aberrant expression of LTC₄ synthase.

The present results demonstrate that abnormal expression of LTC₄ synthase is a regular feature in CML. Elevated expression of LTC₄ synthase has recently been demonstrated in aspirin-sensitive asthma.63 However, in this condition the increase was merely quantitative, because the altered expression was observed in eosinophils, which normally express LTC₄synthase. Thus, the present paper is the first report in which aberrant expression of LTC₄ synthase can be coupled to a specific disease.

Several cellular abnormalities in CML have been directly associated to the tyrosine kinase activity possessed by the BCR-ABL fusion protein.64 Tyrosine kinase is known to influence the expression of several genes via a wide range of signal transduction pathways. Therefore it will be of interest to elucidate whether this abnormal enzyme is involved in the mechanisms leading to aberrant expression of LTC₄ synthase in CML neutrophils. Notably, we have previously reported a normal LT biosynthesis in white blood cell suspensions from patients with polycythemia vera.2 This Ph chromosome-negative chronic myeloproliferative disorder is typically characterized by excessive erythrocytosis in addition to an accumulation of myeloid cells. Furthermore, various neutrophil abnormalities, such as impaired stimulus-induced activation of phospholipase D and oxidative metabolism, have been described.65 

It may be speculated that the aberrant LTC₄ synthase gene expression in CML neutrophils may lead to overproduction of LTC₄ in the bone marrow of these patients. This may be of potential interest because LTs may play a role in myelopoiesis.6 Further studies are in progress to investigate the pathophysiological importance of the increased LTC₄ production in CML.

We thank Dr Robert Zipkin, Biomol Research Laboratories (Plymouth Meeting, PA), for the generous gift of LTA₄ methyl ester.