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Expression of Platelet-Activating Factor Receptor Transcript-1 but Not Transcript-2 by Human Bone Marrow Cells

Laboratoire d'Hématologie Expérimentale, Faculté de Médecine, Limoges, France

Key Words. Bone marrow • CD34⁺ progenitors • Platelet-activating factor receptor • Stromal cells • RT-PCR • Bone marrow

Dr. Yves Denizot, Laboratoire d'Hématologie Expérimentale, Faculté de Médecine, 2 rue Dr. Marcland, 87025 Limoges, France.

	Abstract

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The presence of platelet-activating factor receptor (PAF-R) transcripts 1 and 2 was investigated in human bone marrow cells by a reverse transcriptase polymerase chain reaction (RT-PCR) procedure which detected their simultaneous presence. RT-PCR experiments reveal PAF-R transcript 1 (but not 2) in freshly isolated mononuclear marrow cells, CD34⁺ hematopoietic stem/progenitor cells and cultured marrow stromal cells. For these experiments, the 5637 human bladder carcinoma cell line is used as a positive control for the presence of PAF-R transcripts 1 and 2. Flow cytometry experiments confirm the presence of PAF-R on marrow stromal cells and CD34⁺ stem/progenitor cells. In conclusion, the expression of PAF-R transcript 1, which mainly exists in circulating leukocytes, is also found in CD34⁺ stem/progenitor cells and cells of the marrow microenvironment, strengthening the potential role of PAF during marrow hematopoiesis.

	Introduction

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Platelet-activating factor (PAF) is an inflammatory autacoid that acts through PAF receptors (PAF-R) present on the membrane of responsive cells [1]. PAF-R is cloned, and its sequence analysis reveals that it belongs to the superfamily of G protein-coupled receptors [2]. The PAF-R gene produces two different species of mRNA (i.e., transcripts 1 and 2) that generate a unique membrane PAF-R. Their expressions are driven by distinct promoters, and their tissue distributions are different. Thus, PAF-R transcripts 1 and 2 are found in several organs, while circulating leukocytes only express PAF-R transcript 1 [2].

Bone marrow hematopoiesis is regulated by interactions between marrow stromal cells and hematopoietic stem/progenitor cells (CD34⁺ cells). Marrow stromal cells act on CD34⁺ progenitors by cell-to-cell contact and by secreting cytokines that modulate hematopoiesis [3, 4]. CD34 is a surface antigen expressed on hematopoietic progenitors that can be used to select this population that represents 2% and 0.1% of normal marrow and blood mononuclear cells (MNC), respectively [5, 6]. Our laboratory has reported the presence of PAF in the human bone marrow [7] and its potential role during marrow hematopoiesis. Thus, PAF acts on the growth of marrow MNC [8], marrow CD34⁺ cells [9], and marrow stromal cells [10]. PAF modulates macrophage colony-stimulating factor (M-CSF) production by marrow stromal cells [11], suggesting that PAF might influence marrow hematopoiesis through the production of hematopoietic cytokines. In view of the potentially important role of PAF in human marrow hematopoiesis, we have investigated the presence of PAF-R in marrow MNC, CD34⁺ progenitor cells, and marrow stromal cell cultures by polymerase chain reaction on reverse transcripts (RT-PCR) and flow cytometry.

	Materials and Methods

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Cells
Human bone marrow cells were recovered from bone marrow aspirates harvested into heparinized tubes (Vacutainer system, Becton Dickinson; Meylan, France), and they were used for the experiments when the myelogram was considered normal by morphological analysis of the May-Grünwald-Giemsa-stained smears. The procedure was performed on patients undergoing a myelogram as part of a routine procedure. Bone marrow MNC isolated by separation on a Ficoll gradient (400 x g, 20 min) were washed two times with Hank's balanced salt solution (HBSS) (400 x g, 10 min) before use [12]. For bone marrow stromal cell cultures, human bone marrow MNC were seeded in culture flasks in RPMI 1640 with 20% fetal calf serum ([FCS], GIBCO; Cergy Pontoise, France), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in 5% CO₂ in air [13]. Adherent cells were grown to confluence for five to eight weeks with weekly changes of medium and were subcultured after trypsin treatment. For CD34⁺ cell experiments, cells were collected from umbilical cord blood or from frozen peripheral blood stem cells collected from lymphoma patients in complete remission undergoing apheresis for autologous transplantation, as previously reported [9]. CD34⁺ cells were obtained by magnetic cell sorting (MiniMACS, Tebu; Le Perray en Yvelines, France) [9]. The purified population contained 98% and 97% of CD34⁺ cells (mean of two experiments) for cord blood and peripheral blood, respectively. For preparation of blood MNC, human blood was drawn from healthy volunteers using heparin as the anticoagulant. Blood MNC were isolated by separation on a Ficoll gradient (400 x g, 20 min) and washed two times with HBSS (400 x g, 10 min) before use. The 5637 human bladder carcinoma cell line (used as positive control for the presence of PAF-R transcripts 1 and 2) was grown in 10% FCS with antibiotics at 37°C in 5% CO₂ in air.

RT-PCR Analysis of PAF-R Transcripts
Total RNA extracted from bone marrow MNC, blood MNC, blood CD34⁺ cells, bone marrow stromal cells, and 5637 cells was reverse-transcribed as previously described [11]. The complementary DNA (cDNA) was amplified by PCR under the following conditions : 1 min, 94°C; 1 min, 56°C; 1.5 min, 72°C for 35 cycles. PCR reaction was performed in a total volume of 50 µl containing 75 mM of Tris-HCl (pH = 9.0), 20 mM of (NH₄)₂SO₄, 0.01% of Tween 20, 1.5 mM of MgCl₂, 0.4 mM of each of dNTP, 0.3 µM of sense primers, 0.6 µM of antisense primer (0.3 µM for ß-actin), and 0.1 U/ml of DNA polymerase (Eurogentec; Seraing, Belgium). The human PAF-R transcript 1 sense primer was 5'-GACAGCATAGAGGCTGAGGC-3'; the transcript 2 sense primer was 5'-CCTGAGCTCCCCGAGAAGTCA-3'; and the antisense primer was 5'-TAGCCATTAGCAATGACCCC-3', spanning a 225 bp and a 269 bp fragment for transcripts 1 and 2, respectively (the two senses and the antisense primers were used in the same PCR reaction). The constitutive expression of ß-actin [11] in all cell samples represented a positive control of reverse transcription (data not shown). As a negative control, the PCR reaction was performed with all reagents except the cDNA. PCR products were electrophoresed on a 2% agarose gel (GIBCO), visualized by ethidium bromide staining and Southern blotted onto a nylon membrane. To increase the sensitivity, hybridization was carried out using an internal PAF-R oligonucleotide (5'-CACTCTCTT CCCGATTGTTTAC-3') common to transcripts 1 and 2 PCR products that was 3'-end labeled with Digoxigenin-11-dUTP (Boehringer; Meylan, France), according to the manufacturer's instructions. Specific PAF-R PCR products were detected using a chemoluminescent reaction with Lumigen CSPD^® (Boehringer). The identity of PCR products to PAF receptor was also analyzed by cDNA sequencing carried out by Appligene Oncor (Illkirch, France). Finally, the RT-PCR procedure was also performed with 5637 purified mRNA (polyATtract^® System 1000) from Promega (Lyon, France) according to the manufacturer's instructions.

PAF-R Flow Cytometric Analysis
Trypsinated bone marrow stromal cells (5 x 10⁵) were incubated with PAF-R mouse monoclonal antibody (Spi Bio; Massy, France) for 30 min at 4°C. After washing (phosphate-buffered saline with 5% of serum AB), cells were recovered in 50 µl of washing buffer and incubated with fluorescein isothiocyanate (FITC)-labeled goat-anti-mouse monoclonal (GAM) antibodies (Dakopatts; Glostrup, Denmark) for 45 min in the dark at 4°C. After washing, cells were recovered in 500 µl of 4% paraformaldehyde. Cell suspension was then submitted to flow cytometric analysis (XL II, Coulter; Margency, France).

PAF-R was investigated on blood CD34⁺ cells (2 x 10⁵) before and after three days of growth in liquid medium (Iscove's modified Dulbecco's medium with 10% FCS, 10% 5637-conditioned medium as source of CSF and antibiotics) at 37°C in 5% CO₂ in air. Monoclonal antibodies against the following surface molecules were used: phycoerythrin (PE)- and FITC-labeled anti-CD34 antibodies (Becton Dickinson; San Jose, CA), PE-labeled anti-CD33 antibodies (Becton Dickinson), FITC- and PE-labeled anti-CD38 antibodies (Immunotech; Marseille, France, and Coulter Corporation, respectively), FITC- and PE-labeled GAM antibodies, and PAF-R antibodies. For the two-color immunofluorescence flow cytometry analysis, fluorochrome-conjugated monoclonal antibodies were incubated as described above.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Using appropriate primers, a 225 bp and a 269 bp fragment related, respectively, to the PAF-R transcripts 1 and 2 are amplified from the total RNA or the purified mRNA from the 5637 human bladder carcinoma cell line ( Fig. 1). PCR products migrate at a position consistent with their expected sizes, and their sequencing confirms the presence of PAF-R mRNA transcripts 1 and 2 in 5637 cells (data not shown). As shown in Figure 2, blood and marrow MNC, hematopoietic CD34⁺ progenitor cells, and cultured bone marrow stromal cells constitutively express PAF-R transcript 1 but not PAF-R transcript 2. PAF-R transcript 1 is found in 8/8, 8/8, 4/4, 4/4, and 11/11 human blood MNC, marrow MNC, CD34⁺ progenitors from cord blood or peripheral blood, and bone marrow stromal cell cultures, respectively. Our results confirm that leukocytes only express PAF-R transcript 1 [2] and reveal that marrow MNC and CD34⁺ progenitors also express it. We also found that human bone marrow stromal cells only express PAF-R transcript 1, thus confirming and extending a previous result [14].

View larger version (21K): [in this window] [in a new window]   Figure 1. PAF-R transcripts 1 and 2 expression in 5637 cells. RT-PCR products were electrophoresed in a 2% agarose gel and stained with BET. Lane 1: 100 bp DNA size ladder; lanes 2 and 8: PCR blanks for ß-actin and PAF-R, respectively; lanes 3 and 6: ß-actin transcripts using total RNA and purified mRNA, respectively; lane 5: empty; lanes 4 and 7: PAF-R transcripts using total RNA and purified mRNA, respectively; sizes of PCR products are indicated with arrows.

View larger version (75K): [in this window] [in a new window]   Figure 2. PAF-R transcripts 1 and 2 expression in human bone marrow cells. RT-PCR products were electrophoresed in a 2% agarose gel stained with BET (upper part) and hybridized with PAF-R-DIG-labeled probe after Southern blot (lower part). Lanes 1 and 14: 100 bp DNA size ladder; lanes 2 and 13: 5637 cells (positive controls); lanes 3 and 12: PCR blank; lanes 4 and 5: blood CD34+ cells; lanes 6 and 7: blood MNC; lanes 8 and 9: bone marrow MNC; lanes 10 and 11: bone marrow stromal cells. Sizes of PCR products are indicated with arrows.

View larger version (24K): [in this window] [in a new window]   Figure 3. Analysis of PAF-R+ cells among blood CD34+ cells before (A) and after (B) three days of culture in liquid medium. Cells were labeled with PE-conjugated anti-CD34 antibodies and anti-PAF-R antibodies associated with FITC-conjugated GAM as secondary fluorochrome. One representative experiment out of three is shown.

In conclusion, we highlight that PAF-R transcript 1, but not 2, is present in hematopoietic cells from CD34⁺ stem/progenitor cells to mature leukocytes and in bone marrow stromal cells. These results strengthen the potential direct role of PAF during hematopoiesis by acting on the growth of marrow and blood progenitors but also its indirect role through the modulation of cytokine production by cells of the bone marrow microenvironment.

	Acknowledgments

 
V. Desplat was supported by a grant from "La ligue Nationale Contre le Cancer" (Comité de la Corrèze et de l'Indre). A. Besse is the recipient of a grant from the Conseil Régional du Limousin (Bourse PDZR). We thank "La ligue Nationale Contre le Cancer" (Comité de la Corrèze) for funding our project. We thank A. Allegraud for purification of cord blood CD34⁺ cells.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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