Stem Cells 2002;20:394-401
www.StemCells.com
© 2002 AlphaMed Press
Membrane and Intracellular Platelet-Activating Factor Receptor Expression in Leukemic Blasts of Patients with Acute Myeloid and Lymphoid Leukemia
M. Donnard,
L. Guglielmi,
P. Turlure,
C. Piguet,
M.J. Couraud,
D. Bordessoule,
Y. Denizot
UMR CNRS 6101, Service d’Hématologie Clinique, Service de Pédiatrie, CHU Dupuytren, Limoges, France
Key Words. AML • ALL • PAF receptor • Blast cells • CD34+ cells
Y. Denizot, Ph.D., UMR CNRS 6101, Faculté de Médecine, 2 rue Dr. Marcland, 87025 Limoges, France. Telephone: 33-5-5543-5896 Fax: 33-5-5543-5897; e-mail: yves.denizot{at}unilim.fr
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ABSTRACT
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Platelet-activating factor (PAF), a phospholipid mediator with a wide range of actions on mature leukocytes, acts through PAF-receptors (PAF-Rs) on the membranes of responsive cells. No results are available concerning the putative presence of PAF-Rs on leukemic blasts. Using multiparameter flow cytometry, we assessed intracellular and membrane PAF-Rs on blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients. Membrane PAF-Rs were documented in 7/15 cases of ALL and 0/28 cases of AML. Putative intracellular PAF-Rs were found in blasts of 8/8 ALL and 13/13 AML patients. Vitamin D3 and dimethyl sulfoxide that induced the expression of PAF-Rs on the membrane of the human promyelocytic leukemia cell line, HL60, failed to induce their expression on the membranes of CD34+ AML blasts. The lack of membrane PAF-Rs on the membranes of AML blasts confirms that these receptors represent a marker of mature cells and that their membrane induction is a consequence of cell maturation and differentiation.
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INTRODUCTION
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Platelet-activating factor (PAF) is a phospholipid mediator that sparks off a wide range of immunoregulatory actions on various blood cell types, such as monocytes and B lymphocytes. Indeed, PAF is well known in enhancing the immunoglobulin secretion by B lymphocytes [1] and in stimulating the production of various cytokines by circulating monocytes [2]. The PAF-receptor (PAF-R), a 7-transmembrane molecule that belongs to the G-protein-coupled family, is present on the membranes of responsive cells [3]. Indeed, functional PAF-Rs are detected on mature monocytes [4] and B tonsillar lymphocytes [5]. While the HL-60 promyelocytic leukemia cell line did not express membrane PAF-Rs, their differentiation toward the monocyte/ macrophage and granulocyte phenotype was associated with induction of PAF-R gene expression [6,7], thus suggesting that this receptor might represent a marker of mature cells, and its induction might be a consequence of cell maturation and differentiation. So far, few results are available concerning the presence of PAF-Rs on immature leukocytes. In view of the potentially important role of PAF in processes of leukocyte maturation and function [1,8], we investigated, using multiparameter flow cytometry, the presence of intracellular and/or membrane PAF-Rs on blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients.
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PATIENTS AND METHODS
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Patients
Blood samples were obtained from patients at diagnosis over a period of 18 months. Samples were first used for routine laboratory investigations. The remaining blood was then used for PAF-R investigation. Blood recovered on EDTA was obtained from 28 AML patients (11 men and 17 women, mean age 69 years) and 15 ALL patients (11 men and four women, mean age 41 years) according to the Helsinki recommendations. The AML population (graded according to the French-American-British classification) consisted of 15 AML0, five AML1, and eight AML2 patients. The ALL population consisted of five pre-B1 ALL, seven pre-B2 ALL, and three pre-B3 ALL patients.
Flow Cytometry Analysis
Fresh cells were used, and membrane PAF-Rs were assessed as previously described [9]. Briefly, for membrane PAF-Rs 100 µl of blood were incubated for 10 minutes in the presence of 10% serum anitbody (AB) (ETS; Nantes, France) at 37°C (serum AB was used to reduce the unspecific background). After washing, cells were incubated with anti-human PAF-R mouse IgG antibodies (Spi Bio; Massy, France; http://www.spibio.com) or irrelevant mouse IgG antibodies, as an isotype control (Immunotech; Marseille, France) for 30 minutes at 4°C. After washing, cells were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse antibodies ([GAM] Dakopatts; Glostrup, Denmark; http://www.dakocymation.com). For AML patients, cells were then labeled with phycoerythrin (PE)-labeled anti-CD33 or SpectralRed (PC5)-labeled anti-CD34 antibodies (Immunotech). For ALL patients, cells were then labeled with PE-labeled anti-CD10 or PC5-labeled anti-CD34 antibodies. Cell suspensions were then submitted to flow cytometric analysis (XL II; Coulter; Margency, France; http://www.coulter.com). For intracellular PAF-R assessment, 50 µl of blood were fixated and permeabilized with the IntraPrepTM permeabilization reagent (Immunotech) according to the manufacturer’s recommendations prior to incubation with anti-human PAF-R mouse antibodies or irrelevant mouse antibodies.
In Vitro Expression of PAF-Rs from Blast Cells
In a first set of experiments, blood cells from four AML patients (one AML0, one AML1, and two AML2) were harvested by centrifugation, washed in Hanks’ balanced salts solution and incubated up to 3 hours at 37°C in a PAF-free medium (RPMI 1640). The presence of membrane PAF-Rs on CD34+ AML blasts was then assessed as described above. In a second set of experiments, PAF-Rs were investigated on CD34+ blast cells from two AML2 patients before and after 4 days of growth in liquid medium (RPMI 1640 with 10% fetal calf serum, 10% 5637-conditioned medium, as source of colony-stimulating factor, and antibiotics) with 0.1 µM vitamin D3 (Sigma; Saint Quentin Fallavier, France; http://www.sigmaaldrich.com), 0.5% dimethyl sulfoxide (DMSO), or 50 U/ml of macrophage-colony-stimulating factor (M-CSF) at 37°C in 5% C02 in air [10].
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RESULTS
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Results indicated no membrane PAF-Rs on blast cells of 28 different AML patients. Thus, CD34+CD33- blasts (five AML0 and two AML2), CD34+CD33+ blasts (eight AML0, two AML1, and five AML2), and CD34-CD33+ blasts (two AML0, three AML1, and one AML2) did not express membrane PAF-Rs (Table 1
, Figure 1A). In contrast, blasts from 13/13 AML patients (six AML0, two AML1, and five AML2) contained intracellular PAF-Rs (Table 1, Figure 1B). For these experiments, the means (± standard deviation [SD]) of fluorescence obtained with PAF-R antibodies (2.5 ± 0.3, n = 13) were higher (p < 0.01, t test for paired data) than those obtained with control antibodies (0.7 ± 0.1, n = 13).
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Figure 1. Membrane and intracellular PAF-R of AML blasts. A) A representative result from CD33+CD34+ blasts from one AML0 patient. B) A representative result from CD33+CD34+ blasts from one AML1 patient. C) A representative result from CD33-CD34+ blasts from one AML2 patient. Left panel: Blasts gated on physical parameters were analyzed for the presence of membrane PAF-Rs (FITC labeling), CD33 (PE labeling), and CD34 (PC5 labeling). Right panel: Blasts were fixed, permeabilized, and stained with a control antibody (unshaded area) or specific anti-PAF-R antibody followed by an FITC-conjugated secondary antibody (shaded area).
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Results indicated that membrane PAF-Rs were present on blast cells of seven ALL patients but absent on blast cells of eight other ALL patients (Table 2). Flow cytometry analysis showed heterogeneity in the distribution of PAF-Rs on the ALL blasts obtained from a single patient (Figure 2A). Intracellular PAF-Rs were found in blast cells of 8/8 ALL patients (Table 2, Figure 2B). For these experiments, the means (± SD) of fluorescence obtained with PAF-R antibodies (5.1 ± 1.7, n = 8) were higher (p < 0.01, t test for paired data) than those obtained with control antibodies (0.6 ± 0.1, n = 8).
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Figure 2. Membrane and intracellular PAF-Rs of ALL blasts. A) A representative result from CD10-CD34+ blasts from one pre-B1 ALL patient. B) A representative result from CD10+CD34+ blasts from one pre-B2 ALL patient. C) A representative result from CD10-CD34+ blasts from one pre-B3 ALL patient. Left panel: Blasts gated on physical parameters were analyzed for the presence of membrane PAF-Rs (FITC labeling), CD10 (PE labeling), and CD34 (PC5 labeling). Right panel: Blasts were fixed, permeabilized, and stained with a control antibody (unshaded area) or specific anti-PAF-R antibody followed by an FITC-conjugated secondary antibody (shaded area).
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Incubation of washed CD34+ AML blasts up to 3 hours in a PAF-free medium did not induce the expression of PAF-Rs on CD34+ cell membranes (Figure 3A). As shown in Figure 3B, 4 days of treatment with vitamin D3, M-CSF, and DMSO (data not shown) did not induce the expression of PAF-Rs on the membranes of CD34+ AML2 blasts.
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Figure 3. Analysis of membrane PAF-Rs on CD34+ AML blasts after washing (A) and after 4 days of culture (B). A) AML blasts were labeled with PC5-conjugated anti-CD34 antibodies and anti-PAF-R antibodies associated with FITC-conjugated GAM as a secondary fluorochrome before and after washing, followed by 1 or 3 hours in PAF-free medium. One representative experiment out of four is shown. B) AML2 blasts were labeled with PC5-conjugated anti-CD34 antibodies and anti-PAF-R antibodies associated with FITC-conjugated GAM as a secondary fluorochrome after 4 days of growth with and without 0.1 µM of vitamin D3 or 50 U/ml of M-CSF. One representative experiment out of two is shown.
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DISCUSSION
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Previous results reported that human blood CD34+ cells isolated by the magnetic cell sorting procedure expressed PAF-R mRNA but did not express membrane PAF-Rs [10]. The high sensitivity of the reverse-transcriptase polymerase chain reaction (RT-PCR) procedure may reveal levels of mRNA expression that could be physiologically irrelevant and that do not lead to the production of PAF-Rs. The in vitro growth of normal CD34+ cells for 3 days in the presence of stimulating cytokines induced the coexpression on CD34+ progenitors of PAF-Rs with the CD33 differentiation marker [10]. These results suggest that PAF-Rs were expressed only by CD34+ cells undergoing several changes in maturational stage, and thus, in antigen expression.
Results of this short clinical study indicate that CD34+ AML blasts (M0, M1, and M2 types) did not express membrane PAF-Rs, even in the presence of the maturation marker CD33. Exposure of cells to PAF led to rapid sequestration of PAF-Rs into their intracellular compartments. This effect is mostly a temporary event, with reexpression of PAF-Rs within 1 hour [11–13]. High amounts of PAF are present in human marrow [14]. To test the hypothesis that bone-marrow-derived PAF might be responsible for the observed PAF-R sequestration inside cells, blasts were washed and incubated up to 3 hours in a PAF-free medium. Under these experimental conditions, no PAF-Rs were detected on the membranes of CD34+ AML blasts, suggesting that their lack of membrane PAF-Rs is not related to PAF-R internalization in response to marrow-derived PAF. Flow cytometry experiments suggest the presence of PAF-Rs inside AML blast cells. Several authors have already reported intracellular PAF binding sites in various cells, such as neutrophils [15,16] and platelets [17]. Normal blood CD34+ cells did not express membrane PAF-Rs [10]. However, their treatment with carbamyl-PAF (a nonmetabolizable PAF agonist) reduced the number of erythroid (BFU-E) and myeloid (colony-forming units-granulocyte-macrophage) colonies in semisolid cultures [18] suggesting the existence of functional intracellular PAF-Rs in normal CD34+ cells. At the present time, we have no direct experimental evidence that intracellular PAF-Rs from CD34+ AML blasts are functional. Indeed, intracellular PAF-Rs are not always functional. For example, when Cys90 or Cys173 is mutated, the resulting PAF-Rs are only found intracellularly and are defective in ligand-binding activity [19]. Clearly, further studies are required to confirm the functionality of intracellular PAF-R from CD34+ AML blasts. Experiments with cell lines reported that the expression of membrane PAF-Rs was upregulated by various agents, including those responsible for cell differentiation [6,7,20]. Thus, HL-60 cells (an M2/M3-type cell line) are a classical model of myeloid maturation, because they differentiate to granulocytes or monocytes when treated with DMSO or vitamin D3, respectively [6,7]. Spontaneous M0/M1 blast cells are not responsive to vitamin D3 [20]. Contrary studies have reported that M2-type AML blasts are efficiently responsive [21] or weakly responsive [22,23] to this molecule. Blast maturity and CD34 expression were reported to determine the effect of a vitamin D analogue [24]. In this study, vitamin D3 (but also DMSO and M-CSF) did not induce AML2 blast differentiation (data not shown). Moreover, these molecules did not induce the expression of membrane PAF-Rs on CD34+ AML2 blasts, strengthening the hypothesis that this receptor might represent a marker of mature cells, and its induction might be a consequence of cell maturation and differentiation.
A previous study reported PAF-R mRNA transcripts in CD10+ tonsillar B lymphocytes [5]. Stimulation of CD10+ tonsillar B lymphocytes with PAF increased their IL-4 mRNA and IL-13 mRNA levels only in a small part of the entire population, suggesting that PAF-Rs were not present on all CD10+ B cells. Our results, using ALL blasts, confirm, first, the presence of membrane PAF-Rs on CD34+CD10+ and CD34-CD10+ B cells, and second, that membrane PAF-Rs were not present on all CD10+ B cells from the same patient. As for AML patients, blasts from ALL patients expressed intracellular PAF-R. As for AML blasts, the functionality of intracellular PAF-Rs in ALL blasts remains an open question that requires further investigation.
In conclusion, blasts from AML0, AML1, and AML2 patients did not express membrane PAF-Rs, suggesting that this receptor represents a marker of mature cells and its membrane induction is a consequence of cell maturation and/or differentiation. Results of this clinical study do not argue in favor of a role for PAF on early lymphopoiesis and myelopoiesis.
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ACKNOWLEDGMENT
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We are grateful to the Ligue Nationale Contre le Cancer (Comité de la Corrèze) for funding our project.
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Received February 8, 2002;
accepted for publication June 6, 2002.
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Abstract
Introduction
