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Originally published online as doi:10.1189/jlb.1104684 on March 17, 2005

Published online before print March 17, 2005
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(Journal of Leukocyte Biology. 2005;77:999-1007.)
© 2005 by Society for Leukocyte Biology

Phospholipase D (PLD) gene expression in human neutrophils and HL-60 differentiation

Mauricio Di Fulvio and Julian Gomez-Cambronero1

Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio

1 Correspondence: Department of Physiology and Biophysics, Wright State University, School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435. E-mail: julian.cambronero{at}wright.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human neutrophils exhibit a regulated phospholipase D (PLD) activity that can be measured biochemically in vitro. However, the precise expression pattern of PLD isoforms and their specific biological role(s) are not well understood. Neutrophil mRNA is intrinsically difficult to isolate as a result of the extremely high content of lytic enzymes in the cell’s lysosomal granules. Reverse transcription coupled to polymerase chain reaction indicated that pure populations of human neutrophils had the CD16b+/CD115–/CD20–/CD3{zeta}–/interleukin-5 receptor {alpha}– phenotype. These cells expressed the following splice variants of the PLD1 isoform: PLD1a, PLD1b, PLD1a2, and PLD1b2. As for the PLD2 isoform, neutrophils expressed the PLD2a but not the PLD2b mRNA variant. The relative amount of PLD1/PLD2 transcripts exists in an approximate 4:1 ratio. The expression of PLD isoforms varies during granulocytic differentiation, as demonstrated in the promyelocytic leukemia HL-60 cell line. Further, the pattern of mRNA expression is dependent on the differentiation-inducing agent, 1.25% dimethyl sulfoxide causes a dramatic increase in PLD2a and PLD1b transcripts, and 300 nM all-trans-retinoic acid induced PLD1a expression. These results demonstrate for the first time that human neutrophils express five PLD transcripts and that the PLD genes undergo qualitative changes in transcription regulation during granulocytic differentiation.

Key Words: RT-PCR • ATRA • promyelocytic leukemia • CD markers • monocytes • COS-7 cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The enzyme phospholipase D (PLD) catalyzes the hydrolysis of the terminal diester bond of glycerophospholipids, resulting in the formation of phosphatidic acid (PA) plus a related base [1 , 2 ]. PLD cDNAs have been cloned from a wide variety of species, ranging from bacteria to human. Two different mammalian genes have been isolated from humans, rat, and mouse species [3 4 5 6 7 8 9 10 ]. Human PLD1 and PLD2 genes share ~50% identity and are located in the long arm of chromosome 3 (reverse strand locus 3q26) and in the short arm of chromosome 17 (locus 17p13.1), respectively [5 , 11 , 12 ]. The two mammalian isoforms, PLD1 and PLD2, are regulated differentially by protein kinase C (PKC) and the monomeric G proteins of the adenosine 5'-diphosphate-ribosylation factor (ARF) and Rho families [13 , 14 ].

The human PLD1 gene covers 210 kb genomic DNA defined by 31 exons, 27 of which direct the expression of at least four alternative spliced transcripts: PLD1a (NM_002662), PLD1b, PLD1a2, and PLD1b2 (AJ276230) [15 , 16 ]. PLD1a and PLD1a2 mRNAs express exons 19 (113 bp) and 29 (166 bp), respectively. PLD1b and PLD1b2 mRNAs do not express exon 19. The human PLD2 gene is located in the direct DNA strand of chromosome 17 (p13.1) and is defined by 25 known exons of a genomic region spanning 16.3 kb. The first PLD2 gene exon (112 bp) encodes for the 5'-untranslated region. The initiation codon (A1TG) is located at the second bp of exon 2, whereas the stop codon (TAG2803) is located 568 bp downstream in exon 25. As for PLD2b, this variant is the result of 33 bp alternatively spliced from exon 23 of the originally described PLD2a (NM_002663) [8 , 11 , 12 17 18 ]. Mammalian PLD2, unlike PLD1, is a constitutively active enzyme and shows a weak response to PKC, ARF, and Rho [9 , 12 , 17 18 19 20 21 ].

PLD activity appears to be involved in numerous physiological processes of myeloid cells, such as reduced nicotinamide adenine dinucleotide (NADPH) oxidase activation, phagocytosis, secretion, proliferation, and differentiation [14 ]. Myeloid cells, in particular, the promyelocytic leukemic cell line HL-60, can be induced to differentiate into a mature, neutrophilic-like phenotype by a series of compounds such as dimethyl sulfoxide (DMSO) [22 ], dibutyryl-cyclic adenosine monophosphate (cAMP) [23 ], and all-trans retinoic acid (ATRA) [24 ]. PLD activity is extremely low in resting HL-60 cells, although increases dramatically when HL-60 cells are induced to maturate and differentiate, whichever the inducer used [25 26 27 ]. These findings were related to the induction of PLD1 expression observed during the maturation/differentiation process [25 , 26 , 28 ]. Assigning a role for PLD2 in differentiation was largely ignored in HL-60, as either expression was minimal as compared with PLD1 [28 ], or it was not analyzed [26 ].

Human, terminally differentiated neutrophils express PLD1 and PLD2 at the protein level [15 ]. However, out of the six splice variants of mammalian PLD described, little is known about which particular variant is expressed in what cell type. The aim of the present work was to ascertain the pattern of PLD expression in neutrophils ex vivo and to investigate which isoforms are expressed during neutrophil maturation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Magnetic cell sorter (MACS) separation columns were purchased from Miltenyi Biotec (Bergisch, Gladbach, Germany); PC8, PA, anti-rabbit immunoglobulin G (agarose beads), and tetramethylrhodamine isothiocyanate were from Sigma Chemical Co. (St. Louis, MO); n-[1-3H] butanol (5 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO); 1,2-dioleoyl-sn-glycero-3-phosphatidylbutanol lipid standard was from Avanti Polar Lipids (Alabaster, AL); LK6D silica gel 60 Å thin-layer chromatography plates were from Whatman (Clifton, NJ); Scintiverse II scintillation cocktail and Gel Mount (Biomeda) were purchased from Fisher (Pittsburgh, PA); electrophoresis chemicals were from Bio-Rad Laboratories (Richmond, CA); platinum Pfx polymerase, RNase OUT SuperScriptII reverse transcriptase (RT), random hexamers, and custom primers were from Invitrogen (Carlsbad, CA); deoxy-nucleotide 5'-triphosphates (dNTPs) were from Promega (Madison, WI); the RNeasy mini-kit and the Qiaquick gel purification kit were from Qiagen Inc. (Valencia, CA); alkaline phosphatase-exonuclease I (ExoSAPit) was from USB Corp. (Cleveland, OH).

Primer sequences
The intron-spanning polymerase chain reaction (PCR) primer sequences used for the analysis of peripheral blood cells for selected clusters of differentiation (CD) markers were as follows: CD16b sense 5'-CCA CTC CAG TGT GGC ATC-3', antisense 5'-GCC ACT GCT CTT ATT ACT-3' [29 ]; interleukin-5 receptor {alpha} (IL-5R{alpha}) sense 5'-CCT CCA CTG AAT GTC ACA GCA GAG-3', antisense 5'-TCA AAA CAC AGA ATC CTC CAG GGT-3' [30 ]; CD115 sense 5'-TGC GGC CAG GCT AAA AGG GGA AGA AGA-3', antisense 5'-CCC CGT GTT TTG GAA GGT AGC GTT GTT GGT-3'; CD20 sense 5'-GAA AAA CTC CCC ATC TAC CCA ATA CT-3', antisense 5'-TCC ATG CAA AGG CCA GAT AGA GAT-3'; CD3{zeta} sense 5'-GGG GGA AAG CCG CAG AGA AGG AAG AAC C-3', antisense 5'-GGG AGA ACG AGG AAC CGC CAG GAG ACA G-3'. Four primers were used to analyze PLD1 variants, and five primers were used to analyze PLD2 variants. All primer pairs encompassing PLD (NM_02662 and NM_02663)-expressed sequences were designed to be intron-spanning by using DNAstar PrimerSelect v4.0 software. Those primers were as follows: PLD1-574a/461b sense 5'-AGT CTC TAC AAG CAG CTC CAC AGG CAC CAC-3', antisense 5'-ACC AAT CAG CAG CAG AGC GGA GCA ACT-3'; PLD1-541a2/b2 sense 5'-ACC GGG TAT ATG TCG TGA-3', antisense 5'-CTT CCT AGC CCA GTA TTC TC-3'; PLD1-1252a2 sense 5'-GCA GCA TTG ACA GCA CCT CCA GTT ATT TTA-3', antisense 5'-CTT CCT AGC CCA GTA TTC TCT-3'; PLD2-951a/918b sense 5'-GTC TAC GTG CTT TTG CCC TTA CTC C-3', antisense 5'-TGC CCT TTT CAG CTA TTT CTC ACG-3'; PLD2-881a/848b sense 5'-AGG CCA TTC TGC ACT TTA CTT ACA-3', antisense 5'-TGC CCT TTT CAG CTA TTT CTC AC-3'; PLD2-806a/773b sense 5'-AGC TGG TGA CCT CTC CCT GAC G-3', antisense 5'-GTC TAC GTG CTT TTG CCC TTA CTC C-3'; PLD2-587a/4554b sense 5'-TGG GGA CAG CAT GGC GGG ACT AT-3', antisense 5'-CCA GCG GGG GCA GCA AAG ACT-3'. Custom primers were tested against 10 pg pcDNA-PLD1b or pcDNA-mycPLD2a plasmids (generous gifts from Dr. Sung Ho Ryu, Department of Life Sciences, Postech, South Korea, and Dr. J. David Lambeth, Department of Pathology, Emory University, Atlanta, GA, respectively) as PCR templates. Finally, for reference in the quantitation of mRNA levels of PLD, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer was used: GAPDH sense 5'-GTG AAG GTC GGA GTC AAC GGA TTT-3', antisense 5'-CAC AGT CTT CTG GGT GGC AGT GAT-3'.

Isolation of peripheral blood cells
Neutrophils were isolated as indicated [31 ]. Between 50 and 55 ml peripheral blood was collected from the antecubital vein of healthy individuals (who signed an Institutional Review Board-approved consent form) using sodium citrate as an anticoagulant. The blood was mixed with 15 ml sterile 6% dextran and allowed to settle, and the plasma and buffy coat were removed and centrifuged at 800 g for 5 min. The pellet was resuspended in 35 ml sterile saline and centrifuged again for 15 min at 10°C in a sterile Ficoll-Histopaque discontinuous gradient. Neutrophils were recovered, and contaminating erythrocytes were lysed by a brief (20 s at 4°C) hypotonic shock. Eosinophils were removed from the neutrophil suspension [5x106 cells/ml in Hanks’ balanced salt solution supplemented with HEPES (pH 7.4)] by magnetic cell sorting with the MACS separation columns following the manufacturer’s instructions. The eosinophil population had a >98 ± 2% purity as per morphological observation of Wright-stained cytopreparations. Mononuclear cells (MNCs) containing lymphocytes and monocytes at a 5.5:1 ratio were purified as indicated [32 ], starting from the interface of Ficoll gradients (see above). Approximately 8 x 107 MNCs were washed twice in phosphate-buffered saline at low speed (200 g for 5 min) to reduce platelet contamination and further centrifuged in Percoll discontinuous gradients of known densities. In all cases, purified cell preparations (viability of >96±4%) were collected, washed, resuspended in RPMI media at 2 x 106 cells/ml, and used immediately for total RNA isolation.

Leukemic cells
HL-60 cells were grown at 37°C in a 5% CO2 incubator in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 5 mg/ml gentamicin. Cell density was maintained between 0.1 and 1.0 x 106/ml. HL-60 cells were induced to differentiate by incubation with 1.25% (vol/vol) DMSO for 3 days to achieve the neutrophilic phenotype [differentiation of HL-60 (dHL-60)]. Viability assays were routinely conducted with 0.4% trypan blue stain in cell preparations prior to all analyses. The assessment of cell differentiation was conducted as follows: After 80 h of DMSO incubation, ~75% of HL-60 cells had matured to at least the myelocytic stage and had acquired the ability to release superoxide anion in response to bacterial extracts challenge. To ascertain that DMSO produced the desired neutrophilic phenotype in HL-60 cells, cells were assayed for capacity to reduce nitroblue tetrazolium following treatment with nonviable bacterial extracts. Maturing cells contained formazan deposits as dark, irregularly shaped crystal inclusions in the cytoplasm. Additionally, flow cytometric analysis of surface expression of differentiation-related antigens was performed with fluorescein isothiocyanate-conjugated monoclonal antibodies against CD11b, CD11c, CD13, CD14, CD16, or CD54 [33 ].

Total RNA extraction, RT, PCR, and long PCR
Total RNA was isolated from freshly purified human neutrophils with the RNeasy Protect mini-kit following the manufacturer’s instructions. RNA concentration was determined at 260 nm, and purity was assessed by measuring the 260/280 nm ratio. Only samples within a 1.70–1.95 range were used. First-strand cDNA synthesis was initiated with 0.5–1.0 µg total RNA, 250 ng random hexamers, 500 µM dNTPs, 10 mM dithiothreitol (DTT), 40 units RNase OUT, and 200 units SuperScriptII RT and was incubated at 42°C for 50 min. RT was inactivated at 75°C for 15 min. The PCR reaction mix included 2 µl cDNA, 0.5 units Platinum Pfx polymerase, and 0.4 mM of each dNTP, 2 mM MgSO4, and 50 pmol gene-specific primers in a final volume of 50 µl. The conditions for PCR (Gene Cycler, Bio-Rad) were denaturation at 94°C for 2 min, followed by 40 cycles of 0.5 min at 94°C; annealing, 0.5 min at 55–60°C; and extension, 0.5 min at 68°C. Conditions for long PCR for PLD2 were as follows: denaturation step at 96°C for 2 min, followed by 40 cycles of 0.75 min at 96°C; annealing, 0.75 min at 60°C; and extension, 2.5 min at 68°C.

Quantification of PLD mRNA levels
Quantitation was performed by RT coupled to semiquantitative PCR as published before [34 , 35 ] and adapted for PLD transcripts in human neutrophils. Briefly, the relative expression levels of total PLD1 and PLD2a mRNA isoforms with respect to GAPDH mRNA were determined by reverse transcribing 500 ng total RNA, 500 ng random hexamers, 500 µM dNTPs, 10 mM DTT, 40 units RNase OUT, and 200 units SuperScriptII RT and were incubated at 42°C for 55 min. PCR reaction conditions were: cDNA reaction aliquots equivalent to 30 ng total input RNA (1.2 µl) and 55 pmol PLD1-487 primer for 30 cycles, 50 pmol PLD2-406a primer for 30 cycles, or 37.5 pmol GADPH (555 bp) primer for 24 cycles. The analysis was limited to the products generated only in the exponential phase of the amplification. As a negative control for each set of primers, RT-PCR reactions were performed in the absence of RT and/or RNA/cDNA. After PCR, the content of each independent reaction tube (10 µl) was analyzed by 2% agarose gel electrophoresis. Stained gels were scanned, digitized, and quantitated in a Kodak Gel Logic 200 imaging system.

Real-time PCR
Expression levels of PLD mRNAs were quantified by fluorescent real-time PCR using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA). Real-time PCR reactions were performed in triplicate in a total volume of 50 µl containing 6 µl neutrophil cDNA (equivalent to 30 ng total input RNA), 25 µl 2x IQ Supermix (Bio-Rad), 200 nM of each sense and antisense primer, and 400 nM probe. PLD1 or PLD2 cDNAs were detected using primers and a Texas Red (PLD)-labeled probe (PLD probes were purchased from Applied Biosystems). PCR conditions were as follows: hot start at 95°C, 3 min; denaturation, 95°C, 30 s; annealing, 60°C, 60 s; and extension, 72°C, 60 s during 50 cycles. The relative quantification of PLD1 and PLD2 cDNAs was determined by the comparative cycle threshold (Ct) method. The "cycle threshold" Ct value makes reference to the cycle number arbitrarily chosen from the linear part of the PCR amplification curve, where an increase in fluorescence can be detected above the background.

Statistical analyses
The analysis of multiple intergroup differences in each experiment was conducted by one-way ANOVA followed by Student’s test. A P < 0.05 was used as the criterion of statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of transcripts for CD markers in peripheral blood cells
To study neutrophil PLD mRNA expression, we first tested the ability of our purification protocol to yield pure neutrophil populations. The rationale behind this was that a contamination of other peripheral blood cells (e.g., mononuclear or eosinophils) could make the interpretation of results potentially erroneous. Further, it has been indicated recently that Percoll-based protocols followed by hypotonic lysis may result in neutrophil preparations of insufficient purity for RT-PCR analysis [36 , 37 ]. In our first series of experiments, we investigated the expression of several surface antigen (CDs) transcripts. This was taken as a criterion for cellular identity, as indicated elsewhere [38 39 40 ]. The markers we chose to carry out this study were CD16b for neutrophils, IL-5R{alpha} for eosinophils, CD115 for monocytes, CD20 for B lymphocytes, and CD3{zeta} for T lymphocytes. Results presented in Figure 1 indicate that neutrophil cells purified as indicated in Materials and Methods expressed the neutrophil marker transcript CD16b but not IL-5R{alpha} (Fig. 1A) . The latter marker was expressed in eosinophils but as expected, not in COS-7 or mononculear cells (Fig. 1B) . Neutrophils failed to express CD115, CD20, or CD3{zeta} (Fig. 1C) , markers characteristic of mononuclear cells (Fig. 1D) . These results serve to demonstrate that our cell preparation method renders pure neutrophil populations with the CD16b+/CD115–/CD20–/CD3{zeta}–/IL-5R{alpha}– phenotype.



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  		Figure 1. Detection of transcripts for CD markers in peripheral blood cells. Total RNA was isolated from freshly purified peripheral blood cells, and 500 ng aliquots were reverse-transcribed and subjected to PCR. The figure shows a representative 2% agarose gel electrophoresis of the PCR products stained with ethidium bromide. (A) Purified human neutrophils were screened for mRNA expression of cellular markers [neutrophil CD16b (815 bp) and eosinophil IL-5R{alpha} (542 bp)] using specific sets of primers. The control pane represents a reaction carried over with nuclease-free water in place of RNA. (B) Expression of eosinophil marker (IL-5R{alpha}) in MACS-purified human eosinophils and lack thereof in COS-7 cells, mononuclear cells, and neutrophils. (C) Absence of monocyte CD115 (430 bp), B cell CD20 (502 bp), and T cell CD3{zeta} (490 bp) marker expression in purified human neutrophils. (D) Positive expression of CD115, CD20, and CD3{zeta} mRNAs in purified mononuclear cells (B/T lymphocytes and monocytes).

 
Analysis of PLD1 splice variants in neutrophils
Although the presence of PLD1 and PLD2 isoforms in granulocytes can be detected by Western blotting with specific antibodies [15 , 41 ], the expression of the different variants of PLD1 and PLD2 is not known, as no specific antibodies have been developed against them. Thus, we sought to establish the presence or absence of PLD variants at the mRNA level by RT-PCR using PLD isoform- and variant-specific primers. PLD1 mRNA variants were discriminated by a PLD1-574a/461b primer set that spans the alternatively spliced exon 19 (113 bp) [19 ]. Figure 2A and 2B , shows mRNA expression of PLD1a and PLD1b isoforms of the expected sizes (574 bp and 461 bp, respectively). Two controls for this experiment are presented in Figure 2C . First, PLD1b was amplified using DNA from the plasmid containing the full cDNA sequence of PLD1b (pcDNA-PLD1b). Second, PLD1a and PLD1b were detected in COS-7, a cell line known to normally express low levels of both variants.



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  		Figure 2. Analysis of PLD1a and PLD1b splice variants in neutrophils. (A) PLD1 mRNA isoform expression in neutrophils using a set of primers designed to coamplify PLD1a and PLD1b (PLD1-574a/461b) specifically, which spans the alternatively spliced exon 19 (113 bp). (B) Partial, not to scale, genomic representation of PLD1 exons 18–21. (C) Controls: PLD1a and PLD1b mRNA expression in pcDNA-PLD1b plasmid and COS-7 total RNA.

 
Two additional splice variants of PLD1 (at the C terminus) have been described recently [42 ]. They have been termed PLD1a2 and PLD1b2. Cell transfection and overexpression with these variants have indicated that the C-terminal part of the PLD1 molecule determines the endosomal, intracellular localization of the protein. We have further studied the presence or absence of these two C-terminal splice variants in neutrophils, and the results are presented in Figure 3 . RT-PCR and RT coupled to long PCR experiments were able to detect expression of exon 29 (216 bp spliced out in PLD1a and PLD1b mRNAs) when using the exon 29-specific primer set PLD1-541a2/b2 and the PLD1a2 exon 19- to 29-specific primer PLD1-1252a2, respectively.



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  		Figure 3. Detection of PLD1a2/b2 splice variants in neutrophils. (A) PLD1a2 and PLD1b2 mRNA isoform expression was detected by RT-PCR using PLD1a2/b2 exon 29-specific primers [PLD1-541a2/b2 (541 bp)]. (B) RT coupled to long PCR using PLD1-1252a2 (1252 bp) as a PLD1a2-specific primer. (C) Partial, not to scale, genomic representation of exons 17–31 of the PLD1 gene showing the relative positions of PLD1a2/b2 primers. The alternative exon 29 of the PLD1 gene is also indicated.

 
Analysis of PLD2 splice variants in neutrophils
We next investigated if the two known splice variants of the PLD2 isoform (i.e., PLD2a and PLD2b) were present in neutrophils. For this, we used a battery of specific primers spanning the 33-bp alternatively spliced regions of exon 23. These primers were PLD2-587a/554b, PLD2-951a/918b, PLD2-881a/848b, and PLD2-806a/773b, as well as a PLD2a-specific primer set, PLD2-406a. Figure 4A shows the unequivocal presence of the PLD2a variant. However, PLD2b transcripts could not be detected. This contention is based on three lines of evidence: the absence of proper size bands in Figure 4A , the fact that as a positive control, PLD2-587a/554b did detect PLD2a and PLD2b in human liver (Fig. 4B) , and direct DNA sequence of PLD2-806a/773b. The RT-PCR-generated fragment showed no overlapped sequences at nucleotides 2429–2462 (last 33 bp of exon 23; Fig. 4C ).



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  		Figure 4. Analysis of PLD2 splice variants in neutrophils. (A) Detection of PLD2a mRNA expression by RT-PCR amplification of neutrophil cDNA with the PLD2a-specific primer set (PLD2-406a) and three different sets of primers designed to coamplify PLD2a and PLD2b variants (PLD2-951a/918b, PLD2-881a/848b, and PLD2-806a/773b). (B) Total RNA from neutrophils and human liver cells was reverse-transcribed, and the cDNA was amplified by PCR with the PLD2-587a/554b primer. (C) The complementary strand of PLD2-806a/773b RT-PCR product was sequenced in full. A reverse-strand sequence in a section of interest (nucleotides 2414–2481) is shown (see Results for details).

 
In summary, the results presented in Figures 2 3 4 indicate that human neutrophils express all four known PLD1 variants: a, b, a2, and b2, as well as the a, but not the b, variant of PLD2. These spliced variants would encode for proteins of predicted molecular weights as follows: 120 kDa for PLD1a; 116 kDa for PLD1b; 109.7 kDa for PLD1a2; 105.5 kDa for PLD1b2; and 105 kDa for PLD2a. Commercially available antibodies detect PLD proteins in Western blots of primary cell lysates in those ranges (although sometimes as a smear), as well as other bands of lower relative mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

Quantification of PLD mRNA expression levels in neutrophils
Although PLD1 and PLD2 mRNAs are widely expressed in mammalian tissues, the relative contribution of the two main PLD mRNA isoforms to the total PLD mRNA pool (as well as changes of PLD gene expression) has been studied only in a discrete number of human cells [25 , 43 , 44 ]. Relative amounts of PLD1 and PLD2a mRNAs were measured in neutrophils by RT coupled to semiquantitative PCR. The first two panels of Figure 5 present the establishment of proper conditions for cDNA semiquantitation. First, it was found that the optical density versus RNA concentration was linear between 10 and 75 ng input RNA (Fig. 5A and inset), indicating that PCR efficiency was comparable for the three chosen primers. Second, sigmoidal curves were obtained for the three primers for optical density as a function of the number of cell cycles. For subsequent experiments, a number of cycles were chosen so that the absorbance fell in the linear part of the densitometry curves (Fig. 5B and inset). Once these conditions were established, the measurement of PLD1 and PLD2 was considered semiquantitative. As shown in Figure 5C and inset, neutrophils express more abundant levels of PLD1 mRNA than PLD2a mRNA in an approximate 4:1 PLD1/PLD2a ratio. These semiquantitative results were confirmed and extended by real-time PCR experiments (Fig. 5D and inset).



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  		Figure 5. Quantitation of RNA PLD expression levels. (A) Quantitation conditions: Concentration of RNA. Representative PCR products as a function of input total RNA (1, 25, 50, and 75 ng), separated by 2% agarose gel electrophoresis and stained with ethidium bromide showing the bands of the expected sizes [487 bp (PLD1), 406 bp (PLD2), and 555 bp (GAPDH)]. Shown also is the densitometric analysis (optical density in arbitrary units) as mean ± SEM (n=3). (B) Quantitation conditions: Number of PCR cycles. PCR samples were taken out of the thermocycler at the indicated number of PCR cycles. Also shown is the densitometric analysis (mean±SEM, n=3). (C) Relative contribution of PLD mRNAs in neutrophils. Densitometric analysis (GAPDH ratio) representing the mean ± SEM (n=10) of the gel shown. (D) Representative experiment of the quantitative determination of PLD1 and PLD2 transcripts by real-time PCR. Results are expressed as PCR baseline substracted counted fluorescent units (CFU) versus PCR cycles, where PLD1 and PLD2 levels are represented as Ct (mean±SEM, n=3): CtPLD1 = 32.5 ± 0.4; CtPLD2 = 34.6 ± 0.6, where PLD1 increase relative to PLD2 is 2 – (CtPLD1–CtPLD2) = 4.3 ± 0.6. (Inset) PLD1 and PLD2 relative contribution to the total PLD pool. The relative contribution of PLD2 to the total PLD pool is [2–CtPLD2/(2–CtPLD1+2–CtPLD2)] x 100.

 
Expression of PLD isoforms in neutrophil differentiation
Total PLD activity in HL-60 cells induced to differentiate appear to be highly dependent on the stimuli used [27 , 28 ] and may reflect qualitative differences in cellular populations after differentiation and/or quantitative/qualitative differences in PLD mRNA expression levels. To establish the expression pattern of key white cell markers in DMSO- and ATRA-induced HL-60 cells, the mRNA expression of CD16b (neutrophil cells), CD11b (myeloid cells), and CD14 (monocytic cells; neutrophils, albeit at very low levels) was analyzed. As shown in Figure 6A , HL-60 cells stimulated with DMSO differentiate to a mixed population of cells as judged by the transcript expression for CD14 and CD16b markers. DMSO- and ATRA-induced HL-60 cells expressed transcripts for the myeloid marker CD11b (Fig. 6A and 6B) . However, ATRA stimulation of HL-60 cells did not induce the expression of CD14 (Fig. 6B) . Although DMSO- and ATRA-differentiated HL-60 cells are able to reproduce most of the differentiated phenotypes of the neutrophil in vitro, the expression of white cell markers was different. These results suggest that DMSO and ATRA differentiate HL-60 cells toward different cellular populations and raise the possibility of differential PLD activity as a result of different cellular populations. However, and despite the differences in marker mRNA expression after DMSO and ATRA treatment, total PLD activity did not show significant differences (Fig. 6C) , suggesting that marker mRNA expression and PLD activity may not be related.



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  		Figure 6. Marker mRNA expression during DMSO/ATRA-induced HL-60 differentiation. HL-60 cells were incubated with 1.25% DMSO (A) or 300 nM ATRA (B) for 96 h. Total RNA was extracted at the indicated times, and mRNA expression for CD16b, CD14, and CD11b was determined by RT-PCR. (C) Total PLD activity before and after 96 h treatment with DMSO or ATRA. Results are expressed as percent increase over Control at t0 [CDMSOt0=10,100 counts per minute (cpm)/µg protein; CATRAt0=8500 cpm/µg protein]. (D and E) PLD mRNA variant expression during DMSO (D)- and ATRA (E)-induced HL-60 differentiation. HL-60 cells were incubated with 1.25% DMSO or 300 nM ATRA for 96 h. Total RNA was extracted at the indicated times, and mRNA expression of PLD2a, PLD1a, and PLD1b and was determined by RT-PCR.

 
Hence, the expression pattern of PLD1 and PLD2 mRNA isoforms in HL-60 cells induced to differentiate varies with DMSO and ATRA. As shown in Figure 6 , D and E, DMSO induced a dramatic increase in the mRNA expression levels of PLD2a and PLD1b transcripts, whereas only PLD1a mRNA showed a marked increase in HL-60 cells induced to differentiate with ATRA. These results, when compared with the mature neutrophilic PLD transcriptome, suggest that the absence of differences in total PLD activity between DMSO- and ATRA-induced differentiation in HL-60 cells fails to reflect qualitative and quantitative changes in PLD mRNA expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Data presented here establish for the first time the presence of four splice variants of PLD1 and one of PLD2 in human peripheral blood neutrophils. They also indicate that the level of PLD1 mRNA is higher than the level of PLD2 mRNA. All primers designed and used here showed verifiable PCR products. As such, they could serve for future studies in RT-PCR and for the preparation of labeled probes for Northern blotting to uncover new PLD variants or isoforms (should they be present) in different mammalian tissues.

Human PLD1 and PLD2 genes encode for six PLD variants, PLD1a, PLD1a2, PLD1b, PLD1b2, PLD2a, and PLD2b [5 6 7 8 9 , 11 , 12 , 16 , 42 , 45 ]. RNase protection assays and RT-PCR experiments have suggested the presence of PLD1a, PLD1b, and PLD2 mRNAs in human myeloid cells [25 , 28 ]. We have also shown that human neutrophils express PLD1 and PLD2 at the protein level [15 ]. The data presented here from freshly isolated neutrophils indicate a specific expression pattern of PLD1 isoforms (PLD1a, PLD1b, PLD1a2, and PLD1b2; Fig. 2 ). Lopez et al. [9 ] found PLD2, but not PLD1, transcripts in human peripheral blood leukocytes by Northern blot analysis. This result is intriguing, as PLD1 was detected in neutrophils [15 ], in other white cell-derived cell lines, and in myeloid cells [25 , 26 , 28 , 42 ]. Furthermore, we have detected PLD1 and PLD2 transcripts in freshly isolated CD115+/CD20+/CD3{zeta}+ mononuclear cells and IL-5R{alpha}+ eosinophils/basophils (data not shown). These findings may reflect the fact that PLD transcripts at low levels are usually not detected by Northern blot.

Expression of PLD1a2 and PLD1b2 mRNAs was not reported previously in human neutrophils. As the C termini of PLD1a and PLD1b define precise cellular localization [42 ], our findings of PLD1a2 and PLD1b2 mRNA expression in neutrophils may predict differential endosomal localization of PLD1 isoforms. Moreover, it is likely that isoforms PLD1a2 and PLD1b2 play a role only in neutrophils but not in DMSO/ATRA-differentiated HL-60 cells, as no PLD1a2/b2 mRNAs were detected (data not shown).

As for PLD2, neutrophils expressed PLD2a variant, but not PLD2b (Fig. 3A and 3B) . Extremely low levels of PLD2b mRNA in neutrophils do not seem to explain a lack of detection of the PLD2b mRNA, as direct DNA sequencing of the PLD2-806a/773b RT-PCR products showed no overlapped sequence in the position encompassing the last 33 bp of exon 23 (Fig. 4C) . Furthermore, four different sets of primers designed to coamplify PLD2a and PLD2b mRNAs showed only the band of the "a" size (Fig. 4A and 4B) .

Northern blot experiments substantiated the presence of a small amount of at least one of the two main isoforms of the PLD transcripts in a variety of human, rat, and mouse tissues [7 , 9 , 11 , 12 ]. Notwithstanding, PLD1 mRNA is not detected in any particular abundance in human tissues and seems to have a more restricted expression pattern than a rather heterogeneous PLD2 mRNA expression [7 , 11 , 12 ]. The expression level of PLD transcripts was studied in several cell types [28 , 43 , 46 47 48 49 50 51 52 ]. RNase protection studies demonstrated that myeloid cells express about five times more PLD1 than PLD2 transcripts [25 ]. Peripheral neutrophils express 4.25 ± 0.20 times more PLD1 than PLD2a transcripts, as judged by RT coupled to semiquantitative PCR (Fig. 5A) . The approximate 4:1 ratio appears to reflect the natural levels in neutrophils. In the experimental conditions described here, linearity and no saturation were verified as a function of total input RNA and PCR cycles, respectively. Furthermore, real-time PCR experiments confirmed this finding, showing that ex vivo neutrophils express four to six times more abundant levels of PLD1 mRNA than PLD2a mRNA when compared with ß-tubulin (Fig. 5D) .

Our observations of the divergent expression of PLD isoforms in mature neutrophils versus differentiated HL-60 cells provide an opportunity for further understanding leukemic transformation and the completeness of differentiation induction. PLD activity is involved in several myeloid cell processes during their development and activation, including proliferation of myeloblasts in the bone marrow and secretion, phagocytosis, and NADPH oxidase activation—fundamental functions of differentiated neutrophils. The involvement of PLD in secretory response and reactive oxygen species generation has been suggested in neutrophils and differentiated HL-60 cells [53 54 55 56 57 58 59 ]. Moreover, PLD expression in HL-60 cells is associated with granulocytic differentiation [27 ]. However, total PLD activity in HL-60 cells induced to differentiate appears to be dependent on the stimuli used [27 , 28 ], probably reflecting qualitative differences in cellular population or qualitative/quantitative differences in PLD mRNA expression levels. In differentiated HL-60 cells, increased basal PLD activity was suggested to correlate primarily to PLD1 up-regulation [25 ] or even to PLD1a, PLD1b, and PLD2 isozymes expression [27 , 28 ]. Differentiated HL-60 expresses mainly PLD1a at the protein level, although some signal for PLD1b was detected in Western blots [26 ]. RT-PCR analysis revealed that PLD1 mRNAs as well as total PLD2 transcripts are increased in dibutyryl-cAMP-differentiated HL-60 cells when compared with undifferentiated cells [28 ]. However, ATRA-differentiated HL-60 cells showed a modest increase in PLD1a—even faint bands of PLD1b transcripts were detected [28 ].

As demonstrated in mature neutrophils ex vivo (Fig. 4) , no PLD2b mRNA isoform could be detected at any stage of HL-60 differentiation, regardless of the differentiating agent used. On the contrary, PLD1a2/b2 variants were found only in neutrophils but not in HL-60 (data not shown). As suggested in a previous report [28 ], our results showed increased PLD2a mRNA expression levels when HL-60 cells were induced to differentiate with DMSO or ATRA, although this increase was more dramatic for DMSO-treated cells (Fig. 6D and 6E) . The reason for different PLD2a mRNA expression levels in DMSO- and ATRA-treated HL-60 cells is unknown. However, it may reflect the fact that DMSO-differentiated cells appear to be more heterogeneous than ATRA-differentiated HL-60 cells. The transcript for the monocytic marker CD14 was seen to be expressed only in DMSO-induced HL-60 cells (Fig. 6A) . As neutrophils express CD14 (albeit at low levels), the identification of this mRNA could also be a result of neutrophils. However, no differences in total PLD activity were demonstrated in differentiated HL-60 cells, regardless of the agent used (Fig. 6C) . These results suggest that expression of CD16b, CD11b, and CD14 transcripts is not related to PLD activity, pointing toward qualitative/quantitative differences in PLD mRNA expression after differentiation induced by DMSO or ATRA.

In line with previous results [28 ], only the PLD1a mRNA isoform increased dramatically in HL-60 cells after ATRA induction, and a faint PLD1b band could be detected (Fig. 6E) . However, the opposite situation was seen when DMSO was used as a differentiation agent. Indeed, PLD1b, but not PLD1a, mRNA was detected (Fig. 6E) . The molecular mechanism involved in the differential regulation of alternatively spliced PLD1 transcripts is unknown. It is interesting that PLD2 mRNA expression increased dramatically in HL-60 cells, only when induced to differentiate with DMSO (Fig. 6D and 6E) . Taken together, these results suggest that the PLD genes undergo qualitative/quantitative changes in transcriptional up-regulation during granulocytic dHL-60 cells. Although maturation and dHL-60 cells generated by DMSO and ATRA led to a similar increase in total PLD activity, the differential PLD transcriptome observed in DMSO- and ATRA-induced HL-60 cells may not account for the observed, total PLD activity levels.


   ACKNOWLEDGEMENTS
 
We thank Nick Lehman for the PLD activity experiment and Nick McCray for the real-time PCR experiments. M. D. F. is a recipient of an American Heart Association (Ohio Valley) postdoctoral fellowship (0325364B). This work has been supported by grants from the National Institutes of Health (HL056653) and the American Heart Association 0250417N to J. G-C.

Received November 23, 2004; revised December 7, 2004; accepted February 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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