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Clinical Chemistry 45: 465-471, 1999;
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(Clinical Chemistry. 1999;45:465-471.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Major Interference from Leukocytes in Reverse Transcription-PCR Identified as Neurotoxin Ribonuclease from Eosinophils: Detection of Residual Chronic Myelogenous Leukemia from Cell Lysates by Use of an Eosinophil-depleted Cell Preparation

Mauri M. Hämäläinen1,a, Jarkko U. Eskola2, Jukka Hellman3 and Kari Pulkki4

1 Department of Clinical Chemistry and
2 Joint Biotechnology Laboratory, University of Turku, FIN-20014 Turku, Finland.

3 Centre for Biotechnology, University of Turku and Åbo Akademi University, FIN-20520 Turku, Finland.

4 Central Laboratory, Turku University Central Hospital, FIN-20520 Turku, Finland.
a Address correspondence to this author at: Department of Clinical Chemistry, Turku University Central Hospital, FIN-20520 Turku, Finland. Fax 358 2 2613924; e-mail mauham{at}utu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 105 leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes.

Methods: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque® density gradient.

Results: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by ~90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 105 CD16+ granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control.

Conclusion: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An important clinical application of reverse transcription-PCR (RT-PCR)1 is the analysis of residual leukemia cells after bone marrow transplantation. For the single RT-PCR of a residual disease, mRNA or total RNA from at least 100 000 leukocytes is usually analyzed (1)(2). Extraction of RNA from ribonuclease-rich leukocyte preparations is tedious, irreproducible, and difficult to apply to an automated RT-PCR assay for the clinical laboratory. Previously, we showed that RNA extraction caused 77.7% of the total analytical variation in our Philadelphia chromosome (Ph1 chromosome; bcr/abl translocation) assay from leukocytes of chronic myelogenous leukemia patients (3). Direct application of leukocyte lysate to RT-PCR with the treatment of diethyl pyrocarbonate by the method of Kawasaki (4) was unsuccessful in the assay. A lysate of 101 leukocytes inhibited reverse transcription entirely, whereas the PCR remained almost unaffected. The addition of placental RNase inhibitor (5) to leukocyte lysate did not improve the assay.

Other published methods use a lysate directly from a small number of leukocytes or cultured cells (6)(7)(8)(9), or from boiled cell samples (10). Low numbers of cells can be used without RNA extraction if proper RNase inhibitors are added and if there is universal expression of the mRNA being studied. Heating the sample may partially destroy the mRNA, decreasing the intact transcript of the gene studied to below the detection limit. The purpose of this study was to characterize the RT-inhibitory activity of leukocyte lysate. We found that most of the inhibitory activity is contributed by the eosinophil-derived neurotoxin (EDN), a highly potent ribonuclease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
determination of rt-inhibiting activity
The RT-PCR assay designed to detect the Ph1 chromosome of chronic myelogenous leukemia patients (3) was used to study reverse transcription inhibitors. Briefly, a 5-µL aliquot of leukocyte lysate or diluted chromatography-purified fraction was added to 15 µL of a reverse transcription mixture containing a specific reverse primer, 5'-GACCCTGAGGCTCAAAGTC-3' (from exon 2 of the abl gene), 50 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies), 20 U of RNasin® (Promega Corp.) and 50–100 Ph1 chromosome-positive K562 cells. The tubes were incubated for 30 min at 37 °C. In one experiment, avian myeloblastosis virus (AMV) reverse transcriptase (Finnzymes) instead of M-MLV enzyme was used at 42 °C. After incubation, the tubes were heated for 5 min at 100 °C, and the PCR mixture (80 µL) with the forward primer 5'-TGTGAAACTCCAGACTGTCC-3' (from exon 2 of the bcr gene) and 1 U of AmpliTaq® DNA polymerase (Perkin-Elmer Corp.) were added. The PCR conditions were as follows: 50 s at 95 °C; 1 min at 55 °C; and 2 min at 72 °C for 30 cycles. The 170-bp amplification products were quantified by a sensitive solution hybridization assay using streptavidin-coated microtitration wells and biotin- and europium-labeled probes and by measuring the time-resolved fluorescence of liberated europium (3)(11). We observed that the leukocyte lysate or its chromatography-purified fractions did not inhibit the PCR or hybridization phases of the assay (data not shown). Total RNA extractions were made with the method of Chomczynski and Sacchi (12), using the commercial TRIzol® reagent (Life Technologies).

inhibition of reverse transcription by leukocyte lysate
Leukocytes ( 2 x 106) from a healthy human donor were lysed in 100 µL of sterile water containing 4 units of Inhibit-ACE(TM) RNase inhibitor (5 Prime-3 Prime, Inc.) for 5 min. After centrifugation at 13 000g for 2 min, the supernatant was serially diluted with water to a final concentration of 200 lysed leukocytes/µL. Each dilution (5 µL) was used in RT-PCR assay with 50 Ph1 chromosome-positive K562 cells. Inhibition tests were performed using both M-MLV and AMV reverse transcriptase enzymes.

use of RNase INHIBITORS IN LEUKOCYTE LYSATE
Six commercial RNase inhibitors were tested to find an efficient RNase inhibitor to prevent ribonuclease of leukocyte lysate. Leukocytes (2 x 106) were lysed in 50 µL of water containing 1000 K562 Ph1-positive cells and one of the following human placental RNase inhibitors with 5 mmol/L dithiothreitol: 100 U of RNasin® (Promega), 100 U of RNAguard® (Pharmacia Biotech), 50 U of RNase inhibitor (Perkin-Elmer), or a protein RNase inhibitor (2 U of RNase Block II from Stratagene, 2 U of Inhibit-ACE from 5Prime-3Prime), or 10 mmol/L ribonucleoside-vanadyl complex (New England Biolabs). The centrifuged lysate (5 µL containing 100 K562 cells and 200 000 leukocytes) was used in the RT-PCR assay. Duplicate determinations showed <10% variation.

purification and identification of the rt-inhibiting factor from leukocytes
Leukocyte lysate.
Leukocytes were collected by centrifugation from the buffy coats of discarded whole blood bags after sedimentation of the erythrocytes with 12 g/L dextran. The composition of the leukocytes was as follows: 31.3% lymphocytes, 56.5% neutrophils, 0.9% basophils, 2.6% eosinophils, 4.0% monocytes, and 4.7% large unstained cells. We washed the cells once with phosphate-buffered saline (PBS), and 3 x 109 leukocytes were lysed in 8 mL of 20 mmol/L Tris-HCl buffer, pH 7.4, for 5 min at room temperature. After centrifugation at 5000g for 10 min at 4 °C, the pellet was washed with 6 mL of the same buffer and centrifuged again. The volume of the combined supernatants was 14 mL, and the protein concentration 29.9 g/L, as determined with the folin-phenol reagent (13).

Ion-exchange chromatography.
Leukocyte lysate (13.5 mL) was applied to a 5-mL HiTrap(TM) SP cation-exchange column (Pharmacia Biotech), conditioned with 20 mmol/L Tris-HCl, pH 7.4. The column was washed with 70 mL of the buffer at a flow rate of 2 mL/min, and 5-mL fractions were collected. The proteins adsorbed to the column were eluted with 40 mL of the same buffer containing 2 mol/L NaCl. The ultraviolet absorption of proteins at 280 nm was recorded. The inhibition of reverse transcription was determined from fractions diluted 1:50. Fractions 15 and 16, which contained the RT-inhibiting activity, were concentrated to 1.7 mL using a Centricon(TM) 30 concentrator (Amicon, Inc.). The RT-inhibiting activity was totally retained by the 30 000 molecular weight sieve. The protein concentration of the preparation was 1.25 g/L.

Gel filtration.
A 1-mL aliquot of the active preparation obtained by ion-exchange chromatography was applied to a Superdex® 200 HR 10/30 gel filtration column (Pharmacia Biotech). The column was eluted with 50 mmol/L Tris-HCl buffer containing 9 g/L NaCl, pH 7.4, at a flow rate of 0.6 mL/min, and 1-mL fractions were collected. Fractions 19 and 20, which contained the RT-inhibiting activity, were combined and concentrated to 0.6 mL using a 3000 molecular weight sieve (Centricon 3; Amicon Inc.). The protein concentration of the preparation was 0.31 g/L.

Reversed-phase chromatography.
The concentrated active fraction from gel filtration (0.5 mL) was loaded onto a Vydac C4 (2.1 x 150 mm) reversed-phase (RP) column protected with a C4 guard column (The Separations Group). The elution solvents were as follows: solvent A, 1 g/L trifluoroacetic acid in H2O; solvent B, 0.8 g/L trifluoroacetic in acetonitrile. The solvent gradient used was 2–30% B (0–63 min), 30–60% B (63–95 min), 60–80% B (95–105 min), and 80% B (105–110 min) with a flow rate of 0.15 mL/min. Twenty fractions showing different protein peaks, as determined by their absorbance at 280 nm, were collected, and their RT-inhibiting activity was determined from 1:11 dilutions.

Sequence analysis.
NH2-terminal amino acid sequence analyses of fractions 4 and 5 from the RP chromatography were performed with an Applied Biosystems model 477A protein sequencer equipped with an Applied Biosystems model 120A phenylthiohydantoin amino acid analyzer. The samples were applied to a polybrene-coated and precycled glass fiber filter. Standard cycle parameters provided by the manufacturer were used.

Mass analysis.
A 0.5-µL sample of fraction 5 from the RP chromatography was mixed with 0.5 µL of sinapinic acid (10 g/L) in 600 mL/L acetonitrile and analyzed in a matrix-assisted laser desorption mass spectrometer (MALDI-MSLASERMAT®; Thermo Bioanalysis Ltd.).

determination of edn from leukocyte lysates
EDN [also known as eosinophil protein X (EPX)] was determined using the EPX RIA kit from Pharmacia Diagnostics. The polyclonal antiserum of the kit was raised in rabbits against purified human EPX (14). Lysed leukocytes were centrifuged at 12 000g for 5 min, the supernatant was diluted to contain 106 to 107 lysed cells/mL, and the EDN content of the lysate was determined with the EPX/EDN RIA.

fractionation of leukocytes by ficoll-paque® density gradient
The leukocytes from 10-mL blood samples from four healthy donors were separated from erythrocytes with 12 g/L dextran sedimentation. The leukocytes (2.5–5% eosinophils) were washed with PBS and fractionated to mononuclear cell and granulocyte fractions using Ficoll-Paque density gradient solution (Pharmacia Biotech). The mononuclear cells (1 x 106) were lysed with 500 K562 cells in 50 µL of water containing 80 U of RNasin, and 5 µL of centrifuged lysates was used in the Ph1 chromosome RT-PCR assay. The EDN content of similar lysates without RNasin was determined by the EPX/EDN RIA.

fractionation of leukocytes by cd16 antibodies
Monoclonal anti-CD16 (clone LNK16; HyTest Ltd.) was first biotinylated as described previously (15). Streptavidin-coated microtitration strips (Wallac Co.; cat. no. C122-105) were incubated with the biotinylated anti-CD16 (30 mg/L, 70 µL/well) for 1.5 h at room temperature. The wells were washed three times with PBS, and 200 000 leukocytes (separated from blood with 12 g/L dextran) were added in 50 µL of PBS containing 2 g/L human serum albumin. Leukocytes were incubated for 30 min at room temperature after which unbound cells were washed out three times using the same buffer. Anti-CD16-bound cells were treated with chymopapain (ChymoCell-T; The Boots Company PLC) at 20 kat units/well for 30 min at room temperature to separate the cells from the antibody. The cells were collected, counted, and centrifuged to obtain 1 x 106 cells per tube. The Fc gamma receptor III, CD16, is expressed on neutrophils and basophils but not on eosinophils. The eosinophil-depleted fractions were lysed in 50 µL of autoclaved water, and the centrifuged cell lysates were used for the RT-PCR assay and EDN determination as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
inhibition of reverse transcription by leukocyte lysate
The number of lysed leukocytes needed to inhibit reverse transcription by 50% was ~7000 when M-MLV reverse transcriptase was used and 4000 when the AMV enzyme was used (Fig. 1 ). In both cases ~25 000 lysed leukocytes totally inhibited the reverse transcription reaction. Table 1 shows that the RNase inhibitors were unable to prevent inhibition of reverse transcription when the RT-PCR tube contained a lysate of 200 000 leukocytes. The only positive signal, which was only slightly positive, was obtained when RNase block II was present in the lysate. In addition, ribonucleoside-vanadyl complex without leukocytes showed a 78% inhibition of the assay.



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  		Figure 1. Inhibition by human leukocytes in the Ph1 chromosome RT-PCR of K562 cells.

Ph1-positive K562 cells (500) were lysed with a varying number of leukocytes in 50 µL of water, and 5 µL of the lysate (50 K562 cells) was used for RT-PCR assay. Reverse transcription was performed using either M-MLV or AMV reverse transcriptase. The PCR product was quantified by hybridization with an Eu3+-labeled probe and time-resolved fluorometry.


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  		Table 1. The effect of RNase inhibitors on the analysis of bcr/abl transcript of leukocyte lysate with RT-PCR.1

purification and identification of the rt-inhibiting factor from leukocytes
The three chromatographic steps in the purification of RT-inhibiting factor from leukocytes are shown in Fig. 2 . In the first ion-exchange step (Fig. 2A ), ~20% of the activity was lost with the main protein fraction, which did not adsorb to the column. In the following gel filtration step, the elution volume (19 mL) of the fraction 19 with the highest RT-inhibitory activity indicates a globular protein of Mr 18 000 (Fig. 2B ). The RP chromatography revealed at least 20 fractions that absorbed ultraviolet light at 280 nm (Fig. 2C ). The highest RT-inhibitory activity was in fraction 5. The yield and purification factors in the course of chromatographic procedures are shown in Table 2 . The results are based on the determination of EDN and protein from the chromatographic fractions.



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  		Figure 2. Three-step chromatographic purification of the RT-inhibiting factor from leukocytes.

Dilutions of the fractions (1:50 in A and B; 1:11 in C) were used to measure the inhibition of the RT-PCR assay of 50 Ph1 chromosome-positive K562 cells as described in Fig. 1Up . (A), cation-exchange chromatography of the lysate from 3 x 109 leukocytes with a 5-mL HiTrap SP cation-exchange column. Acidic and neutral proteins were eluted with 20 mmol/L Tris-HCl, pH 7.4, at 2 mL/min and collected in 5-mL fractions. Bound basic proteins were eluted with the same buffer containing 2 mol/L NaCl. (B), gel filtration chromatography of concentrated fractions 15 and 16 (1 mL) from the cation-exchange chromatography shown in Fig. 2AUp , using a Superdex 200 HR 10/30 column. Proteins were eluted with 0.1 mol/L Tris-HCl, pH 7.2, 0.15 mol/L NaCl at 0.6 mL/min and collected in 1-mL fractions. Elution volumes of protein standards (shown as Mr x 103) are shown above the x-axis. (C), RP chromatography of concentrated fractions 19 and 20 (1 mL) from the gel filtration shown in Fig. 2BUp , using Vydac C4 column (2.1 x 150 mm). The elution conditions are given in Materials and Methods. Twenty fractions were collected according to the protein A280 nm chromatogram (inset). EDN was measured by RIA from fractions diluted 1:100. CPS, counts per second.


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  		Table 2. The purification of EDN from leukocytes using a three-step chromatographic procedure.1

sequence analysis
The amino acid sequence of protein from fraction 4 after RP chromatography was as follows: DIPEVVVSLAWDESL, which matches the sequence for human neutrophil 56-amino acid prodefensins (Mr 6306 for prodefensins 1 and 2; Mr 6350 for prodefensin 3) of the SWISS-PROT Protein Sequence Databank (University of Geneva and European Bioinformatics Institute, Geneva, Switzerland). The sequence obtained from fraction 5 was (x = not identified): KPPQFTxAQxFETQH, which matches the sequence for nonsecretory ribonuclease precursor, also known as EDN or EPX. The molecular weight of EDN polypeptide without the 27-amino acid secretory peptide is 15 463. Theoretical pI values of 9.2 (SWISS-PROT Databank) or 8.9 (16) have been calculated for the molecule. The molecular weight of mature granule protein depends on the degree of glycosylation; a molecular weight range of 18 000–25 000 has been reported, with the major band being approximately Mr 18 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (17)(18).

mass analysis
The mass analysis of fraction 5 after RP chromatography showed four peaks (Fig. 3 ): one at m/z 17 444, denoting glycosylated EDN; a double peak of EDN at m/z 8823.2 and 8589.2; and a third peak at m/z 6337.9, denoting a mixture of the 56-amino acid prodefensins 1–3 (19).



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  		Figure 3. Mass analysis of fraction 5 from the RP chromatography (Fig. 2CUp ) step for the purification of RT-inhibiting factor from leukocytes.

Mass analysis was performed on a 0.5-µL sample with a matrix-assisted laser desorption mass spectrometer. Peaks: m/z 17 444, glycosylated EDN; m/z 8589.2 and 8823.6, double peak of EDN; m/z 6337.9, mixture of human neutrophil 56-amino acid prodefensins 1–3.

effect of eosinophil removal
Table 3 shows that 77.4% (range, 57–106%) of the maximum signal was recovered from a mixture of 101 leukocytes and 50 K562 cells in Ph1 chromosome RT-PCR when the CD16+ granulocytes were used, and 52.8% (range, 41–72%) of the signal when the mononuclear cell fraction of the Ficoll-Paque density gradient separation was used. These values are far above the 24.4% signal recovered by the standard total RNA extraction method. The EDN content of cells was approximately one-tenth of that of unfractionated leukocytes. It can be calculated that 101 unfractionated leukocytes (with 2500–5000 eosinophils), the minimum amount for the analysis of residual disease using RT-PCR, contain ~3.6 ng of EDN.


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  		Table 3. The effect of depletion of eosinophils on the analysis of bcr/abl transcript of leukocytes using RT-PCR.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We identified the main RT-inhibiting factor of leukocytes as EDN, using the three-step chromatographic procedure and amino acid sequencing. The binding of EDN to cation-exchange resin at neutral pH enabled a high first-step purification. During this step, ~20% of the activity was lost with neutral and acidic proteins. Most of this RT-inhibiting activity was probably EDN and other cellular ribonucleases (20) that were associated with the main protein fraction and were removed during the column wash.

The purification procedure yielded EDN with some contamination from defensins. Mass analysis showed a molecular weight of 17 444 for EDN, of which the polypeptide body accounts for 15 463. Analyses of urinary ribonuclease Us (21) and of kidney, liver, and spleen nonsecretory ribonucleases (22) have shown that this type of RNase carries a tryptophan-linked mannosyl residue and asparagine-to-N-acetylglucosamine-linked trisaccharides (fucose-[N-acetylglucosamine]2, Mr 570) or pentasaccharides (fucose[N-acetylglucosamine]2[mannose]2, Mr 894) in various proportions. The molecular weight obtained indicates no more than two pentasaccharides or three trisaccharides per EDN molecule. However, the unique amino acid sequences of EDN and defensins confirm their identities. The data in Fig. 2CUp strongly suggest that, of the two proteins, EDN is responsible of the inhibition of reverse transcription.

EDN belongs to the ribonuclease superfamily and has high ribonuclease activity in addition to its reported neurotoxic and helminthotoxic effects (23)(24)(25). Recently, part of the antiretroviral activity of eosinophils has been associated with the ribonuclease activity of EDN (26). It was somewhat surprising to get RNase as a purification product because we took care to exclude the ribonucleases as candidates for reverse transcription inhibitors by testing various ribonuclease inhibitors in leukocyte lysate (Table 1Up ). It had been shown previously that one of the RNase inhibitors tested, Inhibit-ACE, is ineffective against EDN ribonuclease activity (25). The use of heat as a means of inhibiting RNase activity (27)(28) requires that the sample be heated for at least 5 min to inactivate ribonucleases, during which period mRNA is degraded. Our experiments demonstrate that the usual inhibitors are ineffective against high concentrations of RNases in applications such as RT-PCR for the detection of minimal residual disease in leukemia.

Because EDN is expressed almost exclusively in eosinophils [small amounts have also been found in monocytes (20) and neutrophils (29)], we selected eosinophil-free granulocytes with an antibody against the CD16 cell surface marker or mononuclear cells by Ficoll-Paque density gradient and tested the residual RT-inhibiting activity of these leukocyte fractions (Table 3Up ). Selection of eosinophil-free granulocytes with antibodies against CD16 reduced the EDN content of the cells to a concentration where 77% of the RNase-free signal could be recovered. Accordingly, a 53% signal was obtained from the mononuclear cell lysates, whereas only 24% of the signal was obtained when total RNA was extracted from leukocytes that had not been fractionated. The eosinophil-free granulocytes preserved the signal better than the mononuclear cells in spite of their slightly higher EDN content. We suggest that the EDN concentration was below a critical value in both cell fractions, but that the mononuclear cells, probably monocytes, contain other highly active RNases. However, the above values demonstrate that by depleting eosinophils it is possible to avoid RNA extraction during leukocyte sample preparation for RT-PCR. These results open new possibilities for RT-PCR automation in residual disease diagnostics. If eosinophils are selected out during leukocyte separation, the cell lysate can be applied directly to an automated RT-PCR process. We believe that the key point of using leukocyte lysate for RT-PCR without RNA extraction in the method of Kawasaki (4) is the initial selection of mononuclear cells for the assay. It has been observed (30) that to avoid the action of ribonucleases, the extraction of RNA from eosinophil-infiltrated lung requires a modification to the acid guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (12). This supports the results of this study that eosinophil granulocytes are the main source of the ribonuclease activity in leukocytes. The removal of eosinophils from the leukocyte population substantially reduces the risk of mRNA destruction by eosinophil-derived ribonuclease.


   Acknowledgments
 
We thank the Joint Clinical Biochemistry Laboratory of the University of Turku, Turku University Hospital, and Wallac Co. for providing facilities for this study, and Dr. Cornelia Stöckel (Baxter Co., Munich, Germany) for the chymopapain vials.


   Footnotes
 
1 Nonstandard abbreviations: RT, reverse transcription; Ph1 chromosome, Philadelphia chromosome; EDN, eosinophil-derived neurotoxin; M-MLV, Moloney murine leukemia virus; AMV, avian myeloblastosis virus; PBS, phosphate-buffered saline; and RP, reversed-phase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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